BZ-X800 Analyzer BZ-H4A/BZ-H4M/BZ-H4R/BZ-H4C/BZ-H4CM/BZ-H4K

Transcription

1 96M14854 All-in-One Fluorescence Microscope BZ-X800 Analyzer BZ-H4A/BZ-H4M/BZ-H4R/BZ-H4C/BZ-H4CM/BZ-H4K 1 Getting Started 2 Analyzer Names and Functions 3 Loading and Saving Images 4 Processing Images 5 Various Measurements Reference Manual Read this manual before using the system. Keep this manual in a safe place for future reference. 6 Hybrid Cell Count 7 8 Processing the Image Cytometer Data Processing Time-Lapse Data 9 Inserting Text in Images 10 A Other Functions Appendix

2 Introduction This manual describes the installation, handling, and operation procedures and precautions for the BZ-X800 All-in-One Fluorescence Microscope. Before starting any operation, please read this manual carefully in order to fully utilize the BZ-X800 Analysis software. Keep this manual in a safe place for future reference. Symbols This manual uses the following symbols to show important information. Be sure to read these messages carefully. DANGER Indicates a hazardous situation which, if not avoided, will result in death or serious injury. WARNING Indicates a hazardous situation which, if not avoided, could result in death or serious injury. CAUTION Indicates a hazardous situation which, if not avoided, could result in minor or moderate injury. NOTICE Indicates a hazardous situation which, if not avoided, could result in product damage or damages to other properties. Important Indicates cautions and limitations that must be followed during operation. Point Indicates additional information on proper operation. Reference Indicates useful information or tips for better understanding Indicates reference pages in this manual or another manual. General cautions Unauthorized reproduction of this manual in whole or part is prohibited. Please note that the content of this manual is subject to change without notice for the purpose of improvement. Should you find any problem with the content of this manual, such as an unclear point, mistake, or erroneous omission, contact your nearest KEYENCE office. If there are missing or misplaced pages, we will provide a new copy of this manual. The company names and product names used in this manual are registered trademarks or trademarks of the respective companies.

3 Software License Agreement "BZ-X800 Analyzer Application (BZ-H4A), Measurement Application (BZ-H4M), 3D Application (BZ-H4R), Hybrid Cell Count (BZ-H4C), and Macro Cell Count (BZ-H4CM), and Motion Analysis Application (BZ-H4K)" will be provided when you agree with the following license agreement. Make sure to read the following license agreement carefully before using this software. By using this software, it is deemed that the customer agrees with the agreement and that this agreement is entered into. License Agreement "BZ-X800 Analyzer Application (BZ-H4A), Measurement Application (BZ-H4M), 3D Application (BZ-H4R), Hybrid Cell Count (BZ-H4C), and Macro Cell Count (BZ-H4CM), and Motion Analysis Application (BZ-H4K)" (hereinafter referred to as the Software) may be used by customers only who agree with the following Software License Agreement (hereinafter referred to as the Agreement). By using or duplicating all or a part of the software, it is deemed that you agree with the agreement and that the Agreement is entered into. Article 1 (Grant of license) Subject to the terms and conditions of the Agreement, KEYENCE CORPORATION (hereinafter referred to as the Company ) grants customers the non-exclusive right to use the Software. The license of the software shall be a site license. Article 2 (Restriction of reproduction) The Software may be reproduced by the customer only once for backup purposes only. Article 3 (Prohibitions) Following actions shall be prohibited by the customers with regard to the Software. a. Modifications of a part or all of the Software including alterations or additions, provided, however, actions explicitly permitted by the Company shall be exempted such as installing update programs or additional functions. b. Any and all reverse-engineering to analyze the Software including decompiling or disassembling. c. All actions including resale, transfer, redistribution, sub-license, rental, or lease in which the Software and the license key of the Software provided by the Company will be granted to a third party, provided, however, matters permitted by the Company in advance. Article 4 (Copyright) Copyright of the Software, the instruction manuals and other documents shall belong to the Company. Article 5 (Indemnification) The Company shall not be liable for any damages incurred by the customer or by the third party as a result of using the Software. Article 6 (Support) The Company shall provide technical support with regard to questions in relation to the Software based on the Agreement. However, this stipulation shall not guarantee the achievement of the goal which the customer desires by the technical support of the Company. Article 7 (Termination) 1 The agreement shall automatically terminate when the customer stops using the Software by specific methods including discarding the Software and its duplication. 2 The Company shall be entitled to cancel the Agreement without a notice to the customer if the customer infringes any of provisions in the Agreement. In this case, the Software and its duplication shall be returned to the Company or be discarded immediately. 3 If any damage are incurred by the Company which is arisen due to the infringement of the Agreement by the customer, the customer shall indemnify the Company from such damages. Article 8 (Jurisdiction) The Agreement shall be interpreted and governed by the laws of Japan. 96M

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5 Organization of this Manual Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Getting Started Analyzer Names and Functions Loading and Saving Images Processing Images Various Measurements Hybrid Cell Count Processing Image Cytometer data Processing Time-Lapse data This chapter explains the contents of the Analyzer package, the system requirements, and the method of installation. This chapter explains the names and functions of windows, menus, and tools within the Analyzer. This chapter explains the basic procedure for loading images and for saving or printing processed images. This chapter explains image processing, including increasing the resolution of images, applying filters, and displaying them in 3D. This chapter describes how to perform a variety of measurements on the screen, including 2D point-to-point, 2D area, and brightness measurements. This chapter describes the method of extracting areas based on color or brightness in order to quantify target areas. This chapter describes how to analyze much data statistic by well, based on image data obtained by the image cytometer capture. This chapter explains how to create time-lapse motion pictures, insert time stamps on images, measure the brightness variation over time, and select full focus images and best focus images A Chapter 9 Inserting Text in Images This chapter explains how to insert a scale, comments, marks and the time on observation images. Chapter 10 Other Functions This chapter explains various convenient functions including batch saving images, specifying a region of interest, changing the file size, reversing and rotating images, RGB separation, level correction, and changing the display scale factor. Appendix Index The chapter lists reference pages by function names. 3

6 Contents Software License Agreement Organization of this Manual Contents Chapter 1 Getting Started 1-1 Checking the Package Contents Overview of the Analyzer Main Functions and Corresponding Application PC System Environment Installing the Analyzer Software Uninstalling the Analyzer Software Activating the Optional Applications for the Analyzer Activating the Optional Modules for the BZ-X Analyzer Activating the Trial Application Activating the Trial Application Chapter 2 Analyzer Names and Functions 2-1 Names and Functions of the Main Screen Menu Bar Toolbar (Large) Toolbar (Small) Displaying Loaded Images Image Book Chapter 3 Loading and Saving Images 3-1 Loading a Single Image How to load a single image Loading Grouped Images How to load grouped images Window display with grouped images Saving Images Menus related to saving images Printing Images Menus related to printing images

7 Chapter 4 Processing Images 4-1 Haze Reduction for Eliminating Fluorescent Blur Overview of haze reduction Haze reduction procedure Black Balance/White Balance for Adjusting Background Color Overview of black balance Black balance procedure Overview of white balance White balance procedure Overlay for Superimposing Images Overview of overlay Overlay procedure Image Merge for Merging Images Image merge Wide Image Viewer Size Limit for Input Images Operating Wide Image Viewer Saving JPEG, TIFF, and BIG TIFF images with Wide Image Viewer Inserting the Capture Position into a Merged Image Inserting the Capture Position into a Merged Image Full Focus for Creating Omnifocal Images Full focus procedure XYZ Slice for Creating Cross-Sections of Z-Stack Images XYZ slice procedure Max Projection for Displaying the Brightness of Z-Stack Images Max projection procedure D Display of Z-Stack Images Overview of 3D display D display procedure D image procedure Slice function procedure Adjusting the shadow, highlight, and gamma Overview of Lookup Table Lookup Table procedure Adjusting Hue, Saturation, and Luminance Overview of Hue, Saturation, Luminance Hue, Saturation, Luminance procedure Filtering Overview of filters Filtering procedure Calculation, Accumulation, Averaging Overview of image calculation Negative/Positive Inversion Negative/Positive Inversion procedure Gray Conversion Gray conversion procedure Applying Pseudo Color Pseudo color procedure

8 Chapter 5 Various Measurements 5-1 XY Measure XY Measure settings Setting measuring lines Setting display details Setting the measurement results Deleting measuring lines Deleting all measuring lines Area Measurement Setting area measurement Calculating the area of a selected region Measuring the Line Profile Setting the line profile Specifying the line profile Displaying a Histogram Setting the histogram Statistical display Range/Area D Measurement D Measurement method Setting 3D measure Measurement point Displaying Capture Information Displaying capture information Copying Capture Information Copying capture information Chapter 6 Hybrid Cell Count 6-1 Overview of Hybrid Cell Count Overview of hybrid cell count Overview of macro cell count Overview of 3D cell count Overview of time series cell count Counting Fluorescent Images Selecting the image type Specifying the area Adjusting the extraction area shape Displaying and saving measurement results Counting Brightfield Images Selecting the image type Specifying the area Adjusting the extraction area shape Displaying and saving measurement results Counting Phase Contrast Images Selecting the image type Specifying the area Adjusting the extraction area shape Displaying and saving measurement results Counting by Setting a Mask Area

9 Setting the image type and mask area [Hybrid cell count] window when a mask is set Specifying the mask area Adjusting the shape of the mask area Specifying the extraction (1st/2nd) area Adjusting the shape of the extraction (1st/2nd) area Displaying and saving measurement results Macro Cell Count [Macro cell count] window Reading the conditions file Reading the target files Executing macro cell count Saving macro cell count measurement results Re-editing in hybrid cell count D Cell Count Selecting the method of the target area Specifying the extraction area Adjusting the extraction area shape Removing areas by volume and displaying results In the case of counting by setting a target area Chapter 7 Processing the Image Cytometer Data 7-1 Image Cytometer Function Overview of Operation Loading Image Cytometry Image Processing Cell Count Analysis Execution Results Display Performing Image Cytometry Loading Image Cytometry Image Processing Cell Count Analysis Execution Results Display Analyzing Multiple Plate Results Collectively Collective Analysis Operation Displaying the Analyzed Result Results Display Operation

10 Chapter 8 Processing Time-Lapse Data 8-1 Create Movies from Time-Lapse Data Creating movies Inserting a Time Stamp in Images Procedure for inserting time stamp data Setting items in the [Time Lapse Time] dialog box Measuring the Time Series Brightness Variation How to measure the time series brightness variation [Time Series Measure (Specify area/brightness)] dialog box items Creating Fully Focused Images Overview of Full Focus Selecting Images with the Best Focus Function Overview of Best Focus Extracting images using Best Focus Image Stabilizing Function Overview of the Image Stabilizing Function Extracting Still Images from a Video File Extracting still images from a video file Time Series Cell Count Selecting the image type Specifying the extraction area Adjusting the extraction area shape Displaying and saving measurement results Dynamic tracking Overview of Dynamic Tracking Loading time-lapse images Starting Dynamic Tracking Setting the extraction conditions Setting the tracking conditions Tracking modification Confirmation of the results Chapter 9 Inserting Text in Images 9-1 Scale Setting the scale Comment Setting a comment Mark Setting the marker Date and Time Setting the date Setting the time Finalize the inserted object in the image

11 Chapter 10 Other Functions 10-1 Batch Saving Unsaved Images How to batch save unsaved images Specify an Area Specifying an area Changing the File Size How to change the size Changing the file size using the Image Converter Converting BZ-X700 Images to BZ-X800 Images Conversion method Reversing and Rotating Images Reversing images Rotating images Separate the RGB Elements of an image Separating RGB Level Correction Level correction procedure Changing the Display Scale Factor Zoom in Zoom out Arbitrary magnification Appendix A-1 Index A-2 9

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13 Chapter 1 Getting Started This chapter explains the contents of the Analyzer package, the system requirements, and the method of installation. 1-1 Checking the Package Contents Overview of the Analyzer Main Functions and Corresponding Application PC System Environment Installing the Analyzer Software Uninstalling the Analyzer Software Activating the Optional Applications for the Analyzer Activating the Trial Application

14 1-1 Checking the Package Contents The package contains the following items. Before using the system, check that all the accessories are in order. We have thoroughly inspected the package contents before shipment. However, in the event of defective or broken items, please contact your nearest Keyence office. 1 Image Analyzer BZ-X800 Analyzer CD-ROM BZ-X800 Analyzer Reference Manual (this document) Advanced Analysis Application (option) BZ-X800 Analyzer Application BZ-H4A CD-ROM Protection key Measurement Application BZ-H4M License registration form Activation procedure manual D Application BZ-H4R License registration form Activation procedure manual Hybrid Cell Count BZ-H4C License registration form Activation procedure manual Macro Cell Count BZ-H4CM License registration form Activation procedure manual Motion Analysis Application BZ-H4K License registration form Activation procedure manual Reference For operating procedures and further information regarding the advanced analysis applications, refer to "Chapter 4 Processing Images" (page 4-1), "Chapter 5 Various Measurements" (page 5-1), and "Chapter 6 Hybrid Cell Count" (page 6-1). 1-2

15 1-2 Overview of the Analyzer The BZ-X800 Analyzer is an image measurement and analysis software package for the BZ-X800 Series All-in-One Fluorescence Microscope. The section below highlights some of its main features. A wide range of image processing functions Haze reduction (optional) This function applies a no-neighbor deconvolution algorithm to captured images. This enables weak fluorescent signals hidden by fluorescence blurring to be observed clearly, allowing for precise determination of signal localization. 1 Black balance This function adjusts the degree of blackness in the background of fluorescence images to eliminate noise. Overlay This function allows images from different channels to be superimposed to create a single multi-color image. Several other functions are provided in the standard analysis software, including lookup table, and adjustments for hue, saturation, and luminance, and multi-image calculation, accumulation, and averaging. Create high-resolution, wide-area images (optional) Capture neighboring fields-of-view and seamlessly merge them together to create a single high-resolution, wide-area image. Uneven contrast and light intensity between images is eliminated using the shade correction function. Easily obtain fully-focused images of thick samples (optional) Use the motorized Z-axis stage to capture a Z-stack and automatically compile images into a single, fully focused image. View and analyze images in the X, Y, and Z dimensions (optional) With 3D analysis that displays Z-stack images stereoscopically in three dimensions, XYZ slice that captures arbitrary cross-sectional images, and max projection that allows you to see the maximum brightness of each pixel, multidimensional images can be viewed and analyzed with a variety of tools. A wide range of measurement functions (optional) A variety of 2D measurement functions are available, including Hybrid Cell Count for automatically counting and quantifying target areas within an image. By using the image cytometry function that can analyze multiple samples simultaneously, anyone can easily acquire statistical data with high reliability. Multidimensional time-lapse (optional) A variety of tools are available to minimize and eliminate the focal drift and XY movement seen while imaging live cells over prolonged periods of time. 1-3

16 1-3 Main Functions and Corresponding Application This chapter explains the main functions and corresponding application of the Analyzer and advanced applications. 1 Main Functions and Corresponding Application The main functions and the corresponding application are as follows. View menu: Zoom up/down. Command Function Corresponding application Zoom Up Zooms in on the image using the next highest magnification. Standard Zoom Down Zooms out on the image using the next lowest magnification. Standard Arbitrary Magnification Zooms in/out on the image using the specified magnification. Standard Insert menu: Insert a scale, comment, mark, or date and time into the image. Command Function Corresponding application Scale Inserts a scale into the image. Standard Comment Inserts a comment into the image. Standard Mark Inserts a mark into the image. Standard Date Inserts a date into the image. Standard Time Inserts a time into the image. Standard Time Lapse Adds the time-lapse time to the image. BZ-H4A Save inserted object(s) on the image Save inserted object(s) on all images Finalizes the inserted scale and time on the image so that it can no longer be edited. This is required to save the image with the object(s). Finalizes the inserted scale and time on all images in the group so they can no longer be edited. This is required to save the objects on the images. Delete inserted object(s) Deletes the inserted object(s) from the image. Standard BZ-H4A Standard Image Processing menu: Preprocess the image before measurement or adjust the settings to make the image clearer. Command Function Corresponding application Haze Reduction Improves the image quality by removing fluorescence blur. BZ-H4A Overlay Superimposes multiple images to display them as a single image. Standard Image Stitch Stitches multiple images into a single image. (*) BZ-H4A Lookup Table Adjusts the shadow, highlight, and gamma of images. Standard Hue, Saturation, Luminance Adjusts the hue, color saturation, and brightness settings to make the image clearer. Standard Filter Filters the image. Standard Calculation, Accumulation, Averaging Reverse Neg/Pos Performs calculations between multiple images. Inverts the image like a photographic negative by reversing the luminance and RGB values of each pixel in the image. Standard Standard Black Balance Adjust the level of blackness in the background of a fluorescence image. Standard White Balance Adjust the white in an image to achieve the appropriate color. Standard Gray Conversion/ Pseudo Color Gray conversion converts color images to gray scale images made up of black and white tones. Pseudo color converts gray scale images to color images using a specified pseudo color. Standard *The "Advanced Observation Module BZ-H4XD" is necessary to capture data for the image merge. If there is not the advanced observation module, images that can be merged are restricted to 30 images at maximum. 1-4

17 Measure menu: Perform various measurements on the image. Command Function Corresponding application Hybrid Cell Count Automatically extract target areas from an image and quantify their size, brightness, RGB values, etc. View the overall statistics of an entire image, or isolate specific areas. The conditions used to quantify a single image can be applied to up to 400 images using Macro Cell Count. BZ-H4C, BZ-H4CM XY Measure Perform point-and-click 2D measurements. BZ-H4M Area Measurement Measures the area of a specified shape. BZ-H4M Time Series Measure Measures brightness changes in the observed sample over time. BZ-H4A Line Profile Shows the brightness of a specified line drawn on the image. BZ-H4M 1 Histogram Displays a histogram of the image. BZ-H4M Save the measurement on the image Save the measurements on all images Photo Information Display Copy Capture Information Finalizes the measuring line displayed during measurement and the results of the measurement on the image. Finalizes the measuring line displayed during measurement and the results of the measurement on all images on the group. Shows the shooting conditions stored with the image file. Copies the shooting information saved in the image file. BZ-H4M BZ-H4M Standard Standard Image Cytometer menu: Perform various processes for image cytometry. Command Function Corresponding application* Image Cytometry Display statistical data by abstracting the object to many wells and views. BZ-H4C Batch Analysis Process in batch to multiple plates with the same analysis condition. BZ-H4C Result Display Display the result that has been already analyzed. BZ-H4C * To capture data for image cytometry, "Image Cytometer Module BZ-H4XI" and "Advanced Observation Module BZ- H4XD " are necessary. Z-Stack menu: Perform various processes for Z-stack images. Command Function Corresponding application XYZ Slice Displays a vertical cross-section of a Z-stack image group. BZ-H4R Max Projection Extracts and displays information for the brightest pixels in a Z-stack. BZ-H4R 3D Display Builds a 3D image from a group of Z-stack images. BZ-H4R Full Focus Compiles the Z-stack group into a single, fully-focused image. BZ-H4A 3D Cell Count Performs the cell count with 3D. BZ-H4C, BZ-H4R 3D Measure Performs the scale measurement with 3D. BZ-H4R Time Lapse menu: Perform various processes for time-lapse images. Command Function Corresponding application Create a motion picture Create a video (AVI format) from selected time-lapse images. BZ-H4A Play a motion picture Plays video files (AVI format) that you create. BZ-H4A Dynamic Tracking Measures the movement of an object. BZ-H4K Time Series Cell Count Performs the cell count with time series. BZ-H4C, BZ-H4K 1-5

18 Window menu: Changes the way that windows are displayed. Command Function Corresponding application Cascade Windows Cascades multiple windows. Standard Tile Horizontally Displays open windows arranged horizontally. Standard Tile Vertically Displays open windows arranged vertically. Standard Restore All Windows Restores all minimized windows to their original size. Standard 1 Minimize All Windows Minimizes all of the displayed windows. Standard Close All Closes all of the displayed windows. Standard Reference Menu commands that cannot be selected are shown grayed out. The selectable menu commands differ according to the window displayed. The relation between the main functions and required options of the analysis software is as follows. Function Option BZ-H4A BZ-H4M BZ-H4C BZ-H4CM BZ-H4R BZ-H4K Processing Images Stitch Measure Hybrid Cell Count Macro Cell Count Image Cytometry 3D Display 3D Measure 3D Cell Count Dynamic Tracking Time Series Cell Count 1-6

19 1-4 PC System Environment Optimal performance can only be guaranteed if the Analyzer is installed on the PC specified by Keyence for the BZ-X800. Control PC Specifications Item Specifications 1 OS CPU Memory HDD Graphic card Display resolution Windows 10 Pro 64-bit Japanese/English version Intel Xeon Processor E v6 3.0GHz equivalent or higher 16GB or more 20GB or more of free space NVIDIA Quadro P400 2GB equivalent or more 1920 x 1080 or more Important Not all PCs that satisfy the above conditions are guaranteed for operation. Do not install this software on PCs other than the PC specified by KEYENCE or PCs with the equivalent performance (Windows 10 Pro). Note that when the software is installed on PCs other than the above, system files on the PC or other installed applications may not operate. Before operating the BZ-X800 with the specified PC, select "Never" for all power settings in the Power Options Properties of the control PC. 1-7

20 1-5 Installing the Analyzer Software Insert the Analyzer CD-ROM in the drive, and follow the instructions for installation. Point Install this application on the PC specified by Keyence. Use the control PC with Administrator privileges. 1 1 Insert this application CD-ROM into the CD-ROM drive of the PC. The installation software automatically starts. The [InstallShield Wizard] appears. If the installation software does not start automatically, run the Setup.exe file from the CD-ROM drive. 2 Click [Next] button. The [License Agreement] appears. 2 3 Click the option for [I accept the terms in the license agreement], and then click [Next]

21 4 Click the [Install] button. Installation starts. Follow the on-screen instructions and click [Next] when prompted. 4 5 Click [Yes]. The USB key driver will be installed Click the [Finish] button. Installation is completed. The shortcuts for [BZ-X800 Analyzer], [BZ-X800 Image Converter]and [BZ-X800 Wide Image Viewer] are created on the desktop, and [BZ-X800 Analyzer] is displayed in the Windows [Start] menu. 6 Point To activate the advanced applications, "Measurement Application BZ-H4M", "3D Application BZ-H4R", "Hybrid Cell Count BZ-H4C" and "Macro Cell Count BZ-H4CM", acquire the activation key from the optional software activation site. 1-9

22 1-6 Uninstalling the Analyzer Software Delete BZ-X800 Analyzer from [Apps & features] of [Windows Settings] on the Windows operation. 1 Select [Settings] from the [Start] menu. The [Settings] dialog box appears Click [Apps] on the [Windows Settings] window. [Apps & features] dialog box appears. 3 3 Select [BZ-X Analyzer], and click [Uninstall] button. 4 4 Click [Uninstall] button. BZ-X Analyzer is removed. Reference If the [Confirm File Deletion] message of deleting shared files appears, select [Yes to all], and delete everything. If normal uninstallation fails, reinstall the software and try uninstalling it again. 1-10

23 1-7 Activating the Optional Applications for the Analyzer When optional applications for the Analyzer are purchased, license activation is required prior to use. Activating the Optional Modules for the BZ-X Analyzer 1 1 Click [License Information] on the [Others] menu, and click [Activate]. 1 The [Activate License] dialog box is displayed. 2 Click on the URL and access the license activation site. The license activation site is displayed. 3 Check the purchased optional applications, and enter the Authentication Code and the hardware ID displayed on the [Activate License] dialog box. The License Key is displayed. 4 Enter License Key input box for License Key on the [Activate License] dialog box, and check the purchased optional modules. 5 Click [Activate]. A confirmation message is displayed. 5 6 Click [OK]. The license activation is completed. 6 Reference Clicking [Copy to Clipboard] can enter the hardware ID on the license activation site by pasting. 1-11

24 1-8 Activating the Trial Application Trial versions of the Analyzer optional applications can be activated and evaluated for a period of 180 days. 1 Activating the Trial Application 1 1 Click [License Information] on the [Others] menu, and click [Start trial]. 2 2 Click [Open site that issues Trial License Keys] and acquire the license key. 3 3 Enter an address and license key for the trial registration acquired on the license key issue site, and click [OK]. The trial registration is complete and the trial period is displayed. 1-12

25 Chapter 2 Analyzer Names and Functions This chapter explains the names and functions of windows, menus, and tools within the Analyzer. 2-1 Names and Functions of the Main Screen Menu Bar Toolbar (Large) Toolbar (Small) Displaying Loaded Images

26 2-1 Names and Functions of the Main Screen This section explains the names and functions of the main screen of the Analyzer Menu bar Shows the menus for executing commands. Click a menu to display the commands that you can execute. 8 2 Toolbar (Large) Shows icons corresponding to frequently-used menu commands. The selectable icons change according to the type of images being viewed (single image, Z-stack group, or time-lapse group). Click an icon to execute the corresponding command. 3 Toolbar (Small) Shows icons corresponding to common menu commands. The invalid icons according to the state of the data are grayed out. Click an icon to execute the corresponding command. 4 RGB split display Choose whether to display the red, green, or blue components of the image. "10-6 Separate the RGB Elements of an image" (page 10-13) 5 Level correction Adjusts the shadows and highlights of the image. "10-7 Level Correction" (page 10-14) 2-2

27 6 Image book Displays thumbnails of loaded or corrected images. Select a thumbnail to open an image view window in the display area. The appearance of the image view window varies according to the type of observation data; single image (still image), Z-stack image (group image), or time-lapse image (group image). 7 Image display area Displays necessary windows for various menu commands as well as the image view windows. The appearance of the image view window varies according to the type of observation data; single image (still image), Z-stack image (group image), or time-lapse image (group image). 8 Status bar Displays simple help or XYZ and RGB values for a selected point in the image when appropriate. 9 Close button Click this button to exit Analyzer. 10 Maximize button Click this button to display Analyzer at full-screen size. 11 Minimize button Click this button to minimize the Analyzer window

28 2-2 Menu Bar This section explains the names and functions of the menu commands. File menu: Loads and saves observation data. 2 Command Function Refer to Open Opens observation data (single image) and displays the image in the image window. page 3-2 Load a Group Opens a Z-stack, time-lapse or merge group in the group image window. page 3-3 Join Time Lapse Groups Saves two group files of the time lapse together as a single group file. Extract still images from video Extracts still images from movies. page 8-14 Close Save Save As Closes the window displayed in the display area and removes the selected image from the image book (the corresponding data is not deleted). Saves the edited observation data with the original name. You can select whether to embed any inserted items using the Reference [Insert] menu. Saves the edited observation data with a new name. You can select whether to embed any inserted items using the Reference [Insert] menu. page 3-11 page 3-11 Save RGB at once Extracts RGB images and save them simultaneously. page 3-11 Save unsaved images at a time Saves all unsaved images. page 3-11 Print Prints a corrected image or measurement result. page 3-12 Print Preview Displays the print preview on the screen before printing. page 3-12 Exit Exits Analyzer. page 3-11 Edit menu: Restores observation data to its pre-corrected state or copies it. Command Function Refer to Undo Cancels the previous operation and restores the image to its previous state. Undo All Cancels all of the operations performed on the image and restores it to its original state. Copy Copies the selected image and opens the copy in the image book as a new image. Specify Area (Rectangle) Specifies a rectangular area. page 10-3 Specify Area (Ellipse) Specifies an elliptical area. page 10-3 Specify Area Specify Area (Polygon) Specifies a polygonal area. page 10-3 Copy Area Copies the information from the specified area. page 10-6 Paste Area Pastes the information from the specified area. page 10-6 Edit Area Edits the information in the specified area. page 10-5 Cut Image Opens the specified cropped area in the image book as a new image. page 10-5 Resize Changes the size of the selected image. page 10-7 Reverse Horizontal Reverse The image is flipped horizontally. page Vertical Reverse The image is flipped vertically. page Rotate Rotates the image at a predefined angle (0, 90, 180 or 270 degrees) or at a specified angle. page

29 View menu: Zoom in/out on the image data. Command Function Refer to Zoom Up Zooms in on the image to the next highest magnification level. page Zoom Down Zooms out on the image to the next lowest magnification level. page Arbitrary Magnification Zooms in/out on the image using the specified magnification. page Insert menu: Insert a scale, comment, mark, or date and time into the image. Command Function Refer to Scale Inserts a scale into the image. page 9-2 Comment Inserts a comment into the image. page 9-4 Mark Inserts a mark into the image. page Date Inserts a date into the image. page 9-10 Time Inserts a time into the image. page 9-12 Time Lapse Adds the time-lapse time to the image. page 8-3 Save inserted object(s) on the image Save inserted object(s) on all images Finalizes the inserted scale and time on the image so that it can no longer be edited. This is required to save the image with the object(s). Finalizes the inserted scale and time on all images in the group so they can no longer be edited. This is required to save the objects on the images. page 9-14 page 9-14 Delete inserted object(s) Deletes all the inserted object(s) from the image. page 9-15 Image Processing menu: Preprocess the image before measurement or adjust the settings to make the image clearer. Command Function Refer to Haze Reduction Improves the image quality by removing fluorescence blur. page 4-2 Overlay Superimposes multiple images to display them as a single image. page 4-9 Image Stitch Stitches multiple images into a single image (with the standard software, up to 30 images can be manually captured and merged). page 4-13 Lookup Table Adjusts the shadow, highlight, and gamma of images. page 4-31 Hue, Saturation, Luminance Adjusts the hue, color saturation, and brightness settings to make the image clearer. page 4-34 Filter Filters the image. page 4-37 Calculation, Accumulation, Averaging Performs calculations between multiple images. page 4-43 Reverse Neg/Pos Inverts the image like a photographic negative by reversing the luminance and RGB values of each pixel in the image. page 4-45 Black Balance Adjust the level of blackness in the background of a fluorescence image. page 4-6 White Balance Adjust the white in an image to achieve the appropriate color. page 4-8 Gray Conversion/Pseudo Color Gray conversion converts color images to gray scale images made up of black and white tones. Pseudo color converts gray scale images to color images using a specified pseudo color. page 4-46, page

30 Measure menu: Perform various measurements on the image. Command Function Refer to Hybrid Cell Count Automatically extract target areas from an image and quantify their size, brightness, RGB values, etc. View the overall statistics of an entire image, or isolate specific areas. The conditions used to quantify a single image can be applied to up to 400 images using Macro Cell Count. page 6-2 XY Measure Perform point-and-click 2D measurements. page 5-2 Area Measurement Measures the area of a specified shape. page 5-17 Time Series Measure Measures brightness changes in the observed sample over time. page Line Profile Shows the brightness of a specified line drawn on the image. page 5-23 Histogram Displays a histogram of the image. page 5-26 Save the measurement on the image Finalizes the measuring line displayed during measurement and the results of the measurement on the image. page 8-15 Photo Information Display Shows the shooting information stored with the image file. page 3-10 Copy Capture Information Copies the shooting information saved in the image file. page 5-35 Image Cytometer menu: Various processes which relate the image cytometer are made. Command Function Refer to Image Cytometry Display statistical data by abstracting the object to many wells and views. page 7-3 Batch Analysis Process in batch to multiple plates with the same analysis condition. page 7-29 Result Display Display the result that has been already analyzed. page 7-31 Z-Stack menu: Performs various processes from the Z-stack images. Command Function Refer to XYZ Slice Displays a vertical cross-section of a Z-stack image group. page 4-23 Max Projection Extracts and displays pixel information for the maximum brightness in a Z-stack image group. page D Display Builds a 3D image from a group of Z-stack images. page 4-26 Full Focus Compiles the Z-stack group into a single, fully-focused image. page D Cell Count Performs the cell count with 3D. page D Measure Performs the scale measurement with 3D. page 5-30 Time Lapse menu: Create videos from time-lapse images. Command Function Refer to Create a motion picture Create a video (AVI format) from selected time-lapse images. page 8-2 Play a motion picture Plays video files that you create. page 8-2 Dynamic Tracking Measures the movement of an object. page 8-23 Time Series Cell Count Performs the cell count with time series. page

31 Window menu: Select how to display windows. Command Function Refer to Cascade Windows Cascades multiple windows. Tile Horizontally Displays open windows arranged horizontally. Tile Vertically Displays open windows arranged vertically. Restore All Windows. Restores all minimized windows to their original size. Minimize All Windows. Minimizes all of the displayed windows. Close All Closes all of the displayed windows. Others menu: Displays the system's version information. Command Function Refer to 2 Version Information Displays information about the Analyzer version. License Information Language Displays the license information of the Analyzer, Measurement Application, 3D Application, Hybrid Cell Count and Macro Cell Count. The license activation is done from this section. Switches display language. Restarting the software is needed to enable switching. Reference Menu commands that cannot be selected are shown grayed out. The selectable menu commands vary depending on the type of image(s) being displayed. 2-7

32 2-3 Toolbar (Large) This section explains the name and function of each icon on the toolbar (large). Click a toolbar icon to execute the corresponding command. 2 Functions of the icons Icon Menu Refer to Common Open Image "3-1 Loading a Single Image" (page 3-2) Load a Group "3-2 Loading Grouped Images" (page 3-3) Image processing Black Balance Overlay "4-2 Black Balance/White Balance for Adjusting Background Color" (page 4-5) "4-3 Overlay for Superimposing Images" (page 4-9) 3D analysis Full Focus "4-7 Full Focus for Creating Omnifocal Images" (page 4-22) 3D Display "4-10 3D Display of Z-Stack Images" (page 4-26) 3D Measure "5-5 3D Measurement" (page 5-30) 3D Cell Count "6-7 3D Cell Count" (page 6-70) 2-8

33 Icon Menu Refer to Measure Image Cytometry "Chapter 7 Processing the Image Cytometer Data" (page 7-1) Batch Analysis "7-4 Analyzing Multiple Plate Results Collectively" (page 7-29) Hybrid Cell Count "Chapter 6 Hybrid Cell Count" (page 6-1) XY Measure "5-1 XY Measure" (page 5-2) Time-lapse Dynamic Tracking "8-9 Dynamic tracking" (page 8-23) 2 Insert Timescale "8-2 Inserting a Time Stamp in Images" (page 8-3) Create Movie "8-1 Create Movies from Time-Lapse Data" (page 8-2) 2-9

34 2-4 Toolbar (Small) This section explains the name and function of each icon on the toolbar (small). Click a toolbar icon to execute the corresponding command. 2 Functions of the icons Icon Menu Refer to Save "3-3 Saving Images" (page 3-11) Undo Undo All Specify area (rectangle) "10-2 Specify an Area" (page 10-3) Specify area (ellipse) "10-2 Specify an Area" (page 10-3) Specify area (polygon) "10-2 Specify an Area" (page 10-3) Cut image " Cut image" (page 10-5) Zoom up display "10-8 Changing the Display Scale Factor" (page 10-15) Zoom down display "10-8 Changing the Display Scale Factor" (page 10-15) 2-10

35 2-5 Displaying Loaded Images The image view window shows the image selected in the image book. The appearance of the image view window varies depending on whether a single image (still image) is selected or a group of images (Z-stack or time-lapse) is selected. Image Book The image book shows thumbnails and file information for registered images. 2 Single image Group of images (Time-lapse) Group of images (Z-stack) Processed image (ex. max projection) Processed image (ex. 3D analysis) 2-11

36 File information The following file information is displayed for each type of image (single, time-lapse, and Z-stack). 1 For single images Color at image selection Explanation Orange Orange Red Green File name Image size (pixels): Width x height. When the image data is not saved, "unsaved data" is displayed in red. The color resolution of the image is displayed 2 2 For time-lapse images Color at image selection Orange Orange Red Green Explanation File name: Folder name and display channel of the time-lapse image are displayed as the file name. Image size (pixels) and number of images: Width x height x number of images. When the image data is not saved or has been processed, "unsaved data" is displayed. The color resolution of the image is displayed. 3 For Z-stack images Color at image selection Orange Orange Red Green Green Explanation File name: Folder name and display channel of the Z-stack image are displayed as the file name. Image size (pixels) and number of images: Width x height x number of images. When the image data is not saved or has been processed, "unsaved data" is displayed. For the main image of a Z-stack image, shows the [Z Pitch] used for creating a 3D image. The color resolution of the image is displayed. Reference For a sub-image of a Z-stack group, the type such as XYZ, Max Projection, or 3D is shown. Thumbnail format The thumbnail for the image is shown in the following formats depending on the image type. Single Large thumbnail with a single border Z-stack (main image) Large thumbnail with multiple borders. The mark is added. Z-stack (sub-image) Time-lapse Small thumbnail with a single border Large thumbnail with multiple borders. The mark is added. Reference Sub-images are displayed only when a Z-stack group image has been processed. 2-12

37 Popup menu Right-click on an image in the image book to display a popup menu of file options. 2 Command Function Open the data folder Opens the folder in Explorer. Window Print Restore all the related windows Minimize all the related windows Close all the related windows Restores all the minimized windows, including sub-images, to their original sizes. Minimizes all the windows including sub-images. Closes all the windows including sub-images. Prints the selected image. Print Preview Save Save As Save RGB at once Undo Undo All Copy Insert pane indicating captured area Displays the print preview for the selected image. Saves the selected image data, overwriting the existing data. Opens the [Save As] dialog box and saves the data with the specified name. Divides the image into RGB images and saves all at once. Restores the image to its previous state. Restores the image to the state when it was loaded. Copies the image data and records the copy in the image book. Displays the capture position of a specified field-of-view on the merged image. Create a motion picture Saves the transition as a movie when the Z-axis position or time changes to the Z- stack image or time lapse image. Close Close the sub image Close All Photo Properties Display Deletes the selected image data including the sub-images. Deletes only the sub-images from the image book. Deletes all of the image data from the image book. Displays the photo condition information for the image. Definition of a sub-image Analyzer divides an observation image into a main image and sub-images. The definition of a sub-image is as follows. Z-stack image Data on which max projection was performed Data on which 3D analysis was performed Data on which XYZ-slice was performed 2-13

38 MEMO

39 Chapter 3 Loading and Saving Images This chapter explains the basic procedure for loading images and for saving or printing processed images. 3-1 Loading a Single Image Loading Grouped Images Saving Images Printing Images

40 3-1 Loading a Single Image This section explains how to load only one still image. How to load a single image The procedure for loading only one still image is as follows. 3 1 Click the (Open Image) icon on the toolbar (Large), or select [Open] from the [File] menu. The [Open] dialog box appears. 2 Select an image file to load (*.jpg, *.tif), and click the [Open] button. The selected image is displayed in the main window and image book. Images that can be loaded are image files (*.jpg, *.tif) captured and saved using BZ-X800. Reference Images can be loaded by dragging and dropping one or more image files to the main window from another application such as Explorer (multiple files can be selected at once). 3-2

41 3-2 Loading Grouped Images This section explains how to load group images (time-lapse, Z-stack, multicolor, and merged images) saved with the Analyzer. How to load grouped images The procedure for loading grouped images for analysis is as follows. 1 Click the [Load a Group] icon on the toolbar (large), or select [Load a Group] from the [File] menu. The [Load a Group] window appears. 3 2 Select a folder containing a Z-stack, time-lapse, multicolor or merged group of images. The current position image for the group image in the folder is displayed. 3 Select the method for loading the group image by clicking one of the [Z-Stack], [Time Lapse], or [Stitch] buttons. 4 Click the [Load] button. The selected group image is displayed in the image display area of the main window and in the image book. 3-3

42 Address Enter the address of the folder to open with [Load a Group]. 2 [Move] button The folder specified in Address opens in the [Load a Group] window. 3 [Update] button Updates the information of the folder tree to the latest. 4 Image processing switch button Selects the type of the image group from [Z-Stack], [Time Lapse], or [Stitch]. 5 Folder tree Lists the folders on your computer in a tree format. 6 [Selection image] slider Selects an image from Z-stack images. Select one image in the Z-axis direction. 7 Z-Stack The image window opens. Set the stack range. A preview of the current position is displayed. "Z-stack settings" (page 3-6) 8 Preview image display area Displays a thumbnail. "Multicolor settings" (page 3-9) 3-4

43 9 Time Lapse The image window opens. Set the time-lapse range. A preview of the current position is displayed. "Time-lapse settings" (page 3-7) 10 Stitching Stitches together the selected images. "Stitching settings" (page 3-8) 11 Photo properties display area Shows the folder name, date, comment and other group image information. "Photo properties display area" (page 3-10) 12 [Full Focus] button Creates the full focus image automatically and load it when loading a time-lapse + Z-stack group or a merged + Z-stack group. 13 [Best Focus] button Opens the [Best focus] dialog box and extracts and loads only images with good focus from the group image. "8-5 Selecting Images with the Best Focus Function" (page 8-10) 3 14 [Reset] button Restores the default settings of the [Load a Group] window. 15 [Load] button Loads the selected data as group data. 16 [Cancel] button Closes the [Load a Group] window. 3-5

44 Window display with grouped images Z-stack settings Menu name Function 1 Upper Specifies the upper limit for the group images to be loaded. You can also use the spin buttons or drag the "upper limit". The default is 1. 2 Upper Set Sets the current position as the upper limit. 3 Lower Specifies the lower limit for the group images to be loaded. You can also use the spin buttons or drag the "lower limit". The default is the number of Z-stack images. 4 Lower Set Sets the "current position" as the lower limit. 5 Current You can also use the spin buttons or drag the "current position". 6 Pitch Displays the interval at which the images were captured. 7 Number of images Displays the number of images from the upper to the lower limit and the total number of images. 3-6

45 Time-lapse settings Menu name Explanation 1 Start Displays the starting capture interval to load. You can also use the spin buttons or drag the timelapse arrow indicator left and right. 2 Start Set Sets the current position as the starting capture interval. 3 End Displays the ending capture interval to load. You can also use the spin buttons or drag the timelapse arrow indicator left and right. 4 End Set Sets the current position as the ending capture interval. 5 Current Shows the current capture interval. You can also use the spin buttons or drag the time-lapse arrow indicator left and right. 6 Interval Displays the time interval at which the time-lapse images were captured. 7 Number of times Displays the number of capture intervals to be loaded and the total number of capture intervals. 8 Stabilize Image Corrects the subtle position difference of the view during the time lapse. 3-7

46 Stitching settings Menu name Function 1 X When the image displayed is a stitched image, the X axis indicates the number of the image displayed in the plus direction. Use the spin buttons to select an image. 2 Y When the image displayed is a stitched image, the Y axis indicates the number of the image displayed in the plus direction. Use the spin buttons to select an image. 3 columns Displays the number of images captured in the X or horizontal direction. 4 rows Displays the number of images captured in the Y or vertical direction. 5 Next Displays the coordinate after the one displayed in the preview image display area. 6 Prev Displays the coordinate before the one displayed in the preview image display area. 3-8

47 Multicolor settings 2, , 3 3 Menu name Function 1 Preview Shows a preview of the image. Shows four images when the overlay is off at a multicolor and a single image for other types of images 2 Checkbox The image window opens. Select CH. Reference If all are deselected, nothing can be loaded. 3 CH numbers Upper left: CH1, upper right: CH2, lower left: CH3, lower right: CH4 4 Color indicators Shows the color specified at the time of observation. 5 Overlay Displays the overlaid image. Point Performs loading image groups to the checked ones on the CH numbers and overlays. For example, remove the checking of each CH number if loading the image groups of overlay images only. 3-9

48 Photo properties display area 3 Menu name Photo properties display Function Shows the date of capture, comments, the type and number of images, and other photo properties. 3-10

49 3-3 Saving Images This chapter explains how to save images edited and processed with the Analyzer. Menus related to saving images 3 Save Saves the edited image with its original name. Reference If you have processed or edited the image, the processed or edited image is saved instead of the original image. Once an image is saved, you cannot restore it to the state before it was processed or edited. If you select [Save inserted object(s) on the image] in the [Insert] menu when you save an image, the comments, marks, date and time and other items inserted in the image are also saved. "9-5 Finalize the inserted object in the image" (page 9-14) Save As Saves the edited image with a new name. In the [Save As] dialog box, specify the location and file name, and click [Save]. Save RGB at once Divides the image into RGB images and saves all at once. Save unsaved images at a time Specify a prefix and location to batch save all unsaved images. Close Closes all open image windows. You can click on the upper right corner of each window. Exit Exits Analyzer. You can also click in the upper right corner of the main window to exit BZ-X Analyzer. 3-11

50 3-4 Printing Images This chapter explains how to print images edited and processed with the Analyzer. Menus related to printing images 3 Print Prints the edited image. Print Preview Displays a preview of the edited image. Print preview example 3-12

51 Chapter 4 Processing Images This chapter explains image processing including increasing the resolution of observation images, applying filters, and displaying them in 3D. 4-1 Haze Reduction for Eliminating Fluorescent Blur Black Balance/White Balance for Adjusting Background Color Overlay for Superimposing Images Image Merge for Merging Images Wide Image Viewer Inserting the Capture Position into a Merged Image Full Focus for Creating Omnifocal Images XYZ Slice for Creating Cross-Sections of Z-Stack Images Max Projection for Displaying the Brightness of Z-Stack Images D Display of Z-Stack Images Adjusting the shadow, highlight, and gamma Adjusting Hue, Saturation, and Luminance Filtering Calculation, Accumulation, Averaging Negative/Positive Inversion Gray Conversion Applying Pseudo Color

52 4-1 Haze Reduction for Eliminating Fluorescent Blur This section explains haze reduction, which uses a no-neighbor deconvolution algorithm to remove fluorescence blurring from images. Point The Analyzer Application, BZ-H4A (optional), is required to use the haze reduction function. Overview of haze reduction This section explains the [Haze Reduction] dialog box that appears when you select the [Haze Reduction] from the [Image Processing] menu. [Haze Reduction] dialog box Original image display area Displays the selected image Z-stack image Slider The slider is not shown for single images. For Z-stack images, use the vertical slider to select an image. Time-lapse images For time-lapse images, use the horizontal slider to select an image. Slider 4-2

53 2 High-quality fluorescence blur removal preview image display area Displays a preview of the result of haze reduction. 3 [Preview 1]/[Preview 2]/[Preview 3]/[Preview 4]/[Preview 5] buttons Click these buttons to display five previews with predefined settings. The preset values are displayed in the blur size, brightness, and reduction rate fields. 4 [Blur size] slider Specifies the extent of image processing. Enter an integer from 3 to [Brightness] slider Adjusts the brightness of the entire image. Enter a value from 0.0 to [Auto] checkbox Select this checkbox to automatically calculate the brightness. 7 [Reduction rate] slider Specify the level of blur removal. Enter a value from 0.00 to [Noise Removal] checkbox Remove subtle noises on the image. 4 9 Magnification Adjusts the display magnification of the image. Adjust the magnification with the button or from the pull-down menu. 10 [Save Conditions] button Saves the blur size, brightness and reduction rate settings as a haze reduction setting file (*.zcf). 11 [Load Conditions] button Loads the saved haze reduction setting file (*.zcf). The blur size, brightness, and reduction rate settings are reflected in the image. 12 [Clear Conditions] button Clear the applied effects. 13 [OK] button Opens the image after haze reduction in the image book as an unsaved image. When the image group of the Z-stack or time-lapse is selected, the process is performed with the same settings on every image. Example of adjusting an image with haze reduction Original image Adjusted image 4-3

54 Haze reduction procedure 1 Select [Haze Reduction] from the [Image processing] menu. The [Haze Reduction] dialog box appears and the unprocessed image is displayed in the original image display area. For Z-stack or time-lapse group images, select the image to display using the slider To use a set of predefined settings, click [Preview 1]/[Preview 2]/[Preview 3]/[Preview 4]/[Preview 5] button. Check the result of the process in the preview image display area. To specify your own settings, set the values for [Blur size], [Brightness], and [Reduction rate] button, check the result of the process in the preview image display area. 3 Click the [OK] button. The adjustments are applied to the image. The image is registered in the image book as a new image, and the adjusted image is displayed in the image window. For Z-stack or time-lapse group images, all the images associated as group images are processed. 4-4

55 4-2 Black Balance/White Balance for Adjusting Background Color This section explains the "black balance" for correcting the blackness of fluorescence observation images, and "white balance" for adjusting the degree of color images. Overview of black balance This section explains the [Black Balance] dialog box that appears when you click the [Black Balance] icon on the toolbar (large). [Black Balance] dialog box Unprocessed image display area Displays the loaded image before adjustment. 2 Processed image display area Displays the image after adjustment. 3 [Black balance area] pull-down menu Selects the size that becomes the base of the black balance process from the following. Large, Middle, Small 4 [Black balance level] slider Adjusts the strength of the black balance process. 5 [Save conditions] button Saves the setting contents as the black balance setting file (*.bxc). 6 [Load conditions] button Loads the saved black balance setting file (*.bxc). The setting contents are reflected. 7 [Clear conditions] button Clear the applied effects. 8 [OK] button Registers it in the image book as an unsaved image after performing the black balance process. 4-5

56 Black balance procedure 1 Click the [Black Balance] button on the toolbar (large). Or select [Black balance] from the [Image processing] menu. The [Black balance] dialog box appears and a rectangular frame is displayed in the unprocessed image display area Select the size of the frame from the [Black balance area] pulldown menu. Select from [Large], [Middle], and [Small]. 3 Drag the frame to the position you want to use as the basis for black balance. The black balance level changes automatically according to the standard. Reference To adjust the black balance of an image, move the rectangular frame to a low-contrast area in the background so that it does not include any fluorescence signals. 4 Move the [Black balance level] slider to right or left to accentuate the effect while checking the preview image in the processed image display area if fine tune is performed. 5 Click the [OK] button. The adjustments are applied to the image. The image is registered in the image book as a new image, and the adjusted image is displayed in the image window. Point To restore the original image, select [Undo] from the [Edit] menu. 4-6

57 Overview of white balance This section explains the [White Balance] dialog box that appears when you select [White Balance] from the [Image Processing] menu. [White Balance] dialog box Unprocessed image display area Displays the loaded image before adjustment. 2 Processed image display area Displays the image after adjustment. 3 [White Balance Area] pull-down menu Selects the size that becomes the base of the white balance process from the following. Large, Middle, Small 4 RGB slider Adjusts the strength of the white balance process. 5 [Save conditions] button Saves the setting contents as the white balance setting file (*.wxc). 6 [Load conditions] button Loads the saved white balance setting file (*.wxc). The setting contents are reflected. 7 [Clear conditions] button Clear the applied effects. 8 [OK] button Registers it in the image book as an unsaved image after performing the white balance process. 4-7

58 White balance procedure 1 Select [White Balance] from the [Image Processing] menu. The [White Balance] dialog box appears and a rectangular frame is displayed in the unprocessed image display area Select the size of the frame from the [White Balance Area] pull-down menu. Select from [Large], [Middle], and [Small]. 3 Drag the frame to the position you want to use as the basis for white balance. The white balance is adjusted automatically according to the standard. 4 Move the [R], [G], and [B] sliders to right or left to accentuate the effect while checking the preview image in the processed image display area if fine tune is performed. 5 Click the [OK] button. The adjustments are applied to the image. The image is registered in the image book as a new image, and the adjusted image is displayed in the image window. Point To restore the original image, select [Undo] from the [Edit] menu. 4-8

59 4-3 Overlay for Superimposing Images This section explains the overlay function for superimposing multiple images to display them as a single image. This function is useful when you need to overlap multiple fluorescence observation images (multi-fluorescent) or brightfield and fluorescent images. Overview of overlay This section explains the [Overlay] dialog box that appears when you click the [Overlay] icon on the toolbar (large). [Overlay] dialog box Preview image display area Displays a preview of the overlay images. Move the images by placing the mouse pointer inside the preview image display area and dragging the images when the pointer changes to. You can check the result of various settings in real time. 2 [Display] checkboxes Select or deselect these checkboxes to show or hide the corresponding images. 3 [Input Image] pull-down menus Select the background base image and the images to overlay on top. 4-9

60 4 [Brightness] slider Adjust the brightness of each image. Specify an integer from 0 to 200. You can also use the slider, spin buttons, or directly enter the values. 5 [Contrast] slider Adjust the contrast of each image. Specify an integer from 0 to 200. You can also use the slider, spin buttons, or directly enter the values. 6 Pseudo color Select from red, green, blue, aqua, violet, yellow, and none. 7 Object of movement Adjusts the position of overlay images. Select from all images or from image 1, 2, 3, and X/Y Shows the position of each overlay image. Specify an integer from -30 to 30. You can also use the spin buttons, or directly enter the values. 9 [Move] buttons Moves the image selected with [Object Movement] up, down, left and right. The current positions of the images are shown in the X and Y boxes. Point When [All] is selected, the current positions of the images are not shown in the X and Y boxes. 10 [Center Display] button Moves the center of the images to the middle of the preview image display area. 11 Magnification Adjust the magnification with the pull-down menu. 12 [Save Conditions] button Saves the settings including brightness, contrast, pseudo color, and X and Y positions as an overlay setting file (*.ocf). 13 [Load Conditions] button Loads the saved overlay setting file (*.ocf). The brightness, contrast, pseudo color, and X and Y position settings are reflected in the image. 14 [Clear conditions] button Clears the brightness, contrast, pseudo color, and X and Y position settings and restores the default settings. 15 [OK] button Opens the overlay image in the image book as an unsaved image. When the image group of the Z-stack or time-lapse is selected, the process is performed with the same settings on every image. 4-10

61 Overlay procedure Click the [Overlay] icon on the toolbar (large). Or select [Overlay] from the [Image Processing] menu. The [Overlay] dialog box appears. 2 Using the [Input Image] pull-down menus, select the background base image and the images to be overlaid on top. Image 1 is the base image. 3 With the [Display] checkboxes, select the images to display. 4 Set the [Brightness], [Contrast], and [Pseudo Color] for each image. 5 To move each image individually, click the corresponding (Object of move buttons) and specify the new position with the X and Y spin buttons. To move all overlay images, click (All button) and specify the new position with. The images move accordingly. Reference You can also move the images by dragging the images in the preview image display area. 6 After setting the conditions, click the [OK] button. The changes are applied to the image. The overlay image is opened in the image book as a new image and is displayed in the image window. 4-11

62 4-4 Image Merge for Merging Images This section explains the image merge function for seamlessly merging images captured with the BZ-X800 Series. Point Image merge is only available for images captured with the BZ or BZ-X Series. The Analyzer Application, BZ-H4A (optional), is required to use the image merge function. Image merge This function takes multiple images and merges them together to form one large, high-resolution image. 4 After image merge Acquiring images for image merge (Manual acquisition) For users who have not purchased the Advanced Observation module BZ-H4XD, images can be acquired for image merge according to the following procedure. The image merge function uses the coordinate positions of the images in order to properly align and merge them. Neighboring images require some overlap in order to be rapidly and seamlessly merged together. Therefore, approximately 30% of the field-of-view must overlap with the surrounding images. The screen moves 70% of the field of view every time you click an XY movement arrow button in the Viewer. The current magnification is used to determine the appropriate travel distance. Since this is automatically adjusted, any magnification can be used. Using the arrow buttons, capture separate images that you would like to merge together. This will ensure the proper overlap is retained to execute the merge. Overlapping image areas Reference For the procedure for acquiring images automatically, refer to BZ-X800 User's Manual "Chapter 10 XY-Stitching and Multi-Point Capture". Up to 30 images can be merged manually. To merge more than 30 images, the Advanced Observation Module BZ- H4XD is required. Click once to move 70% of the field of view. 4-12

63 Overview of image stitch This section explains the [Image Stitch] dialog box that appears when you select the [Image Stitch] from the [Image Processing] menu. [Image Stitch] dialog box [Select CH] Selects the CH to be displayed on the preview area (at merge by multiple CHs only). 2 Preview area 10 Displays the images to use for image stitch. The image can be removed from the merge target by clicking the preview area. 3 Shading correction area Selects the method for correcting shading. 4 [Compression] checkbox Selects whether to save the file with [Compressed] or [Uncompressed]. 5 [Adjust Image Positions (Recommended)] checkbox Aligns the image merge positions automatically based on the source image information. 6 [Base CH] drop-down menu Selects the CH that is set to the standard of the stitch correction position (at merge by multiple CHs only). 7 [Start Stitching] button Starts merging the images selected in the file setting area. 8 [Save All] button Saves the images loaded to the preview area at a time. 9 [Save Preview] button Saves the image with the state of being displayed in the preview area. 10 Copy file path to clipboard The file path can be copied by right-clicking the image on the preview area using a mouse. Use this when recapturing using the Viewer. 11 [OK]/[Cancel] button Presses [OK] loads the merge result to the analysis software. If press [Cancel], the loading is not performed. 4-13

64 Image stitch procedure 1 Select [Image Stitch] from the [Image Processing] menu. The [Image Stitch] dialog box appears. 2 Click either [Load a Group] or [Select Image Files] Select the method for correcting shading. Reference Point 4 Select whether to save the file as [Compressed] or [Uncompressed] for the [Compression] checkbox. 5 To align the image merge positions automatically based on the source image information, select [Adjust Image Positions (Recommended)] checkbox. 6 Click the [Start Stitching] button. Select [Use Image] from the pull-down menu for the mode, click [Open] and select the image for correction. If merging multiple CHs simultaneously, only [Auto] can be selected as the shading correction. If correcting the shading by selecting an image, at image group loading, load only 1 CH or overlay image. " Multicolor settings" (page 3-9) The merged image is displayed as a preview in the [Wide Image Viewer] dialog box. Reference For how to operate the Wide Image Viewer, see "Operating Wide Image Viewer" (page 4-19) 7 7 Click [File] and select [Save as] to save the file. Reference The file is saved as a Wide Image Viewer file (*.ktf). Clicking [OK] in the [Image Stitch] dialog box transfers a compressed image to the Analyzer. 8 Click [OK] in the [Image Stitch] dialog box to load the merged image to the analysis application. Reference If the image resolution exceeds the largest resolution of 4080 x 3072 pixels that can be handled on the analysis application, the image is made small. 4-14

65 Using the best focus function This function will automatically select the best focused image out of the Z-stack captured at each field-of-view in an image merge. 1 Click the [Load a Group] icon on the toolbar (large). Or, select [Load a Group] from the [File] menu. The [Load a Group] window appears Click the [Stitch] button. 3 Check the channel (CH) to merge. 4 Click the [Best Focus] button. The [Image Stitch] progress bar appears. When auto selection of images is complete, the [Image Stitch] dialog box appears, and images with the best focus at each coordinate are loaded in the select file area. 4-15

66 5 Click the [Start Stitching] button. The merged image is displayed as a preview in the [Wide Image Viewer] dialog box Click [File] and select [Save as] to save the file. Reference The file is saved as a Wide Image Viewer file (*.ktf). Clicking [OK] in the [Image Stitch] dialog box transfers the compressed image to the Analyzer. For multi-stained images, use the best focus function for each channel and use the merge function 7 Click [OK] in the [Image Stitch] dialog box to load the merged image to the analysis application. Reference If the image resolution exceeds the largest resolution of 4080 x 3072 pixels that can be handled on the analysis application, the image is made small. Using the full focus function This function will fully-focus each Z-stack group to create one composite image for each field-of-view captured in the image merge. 1 Click the [Load a Group] icon on the toolbar (large). Or, select [Load a Group] from the [File] menu. The [Load a Group] window appears

67 2 Click the [Stitch] button. 3 Check the channel (CH) to merge. 4 Click the [Full Focus] button. The [Executing the Full Focus.] progress bar appears. When full focus is complete, the [Image Stitch] dialog box appears, and the composite fully-focused images for each coordinate are loaded in the select file area. 5 Click the [Start Stitching] button. The merged image is displayed in preview in the [Wide Image Viewer] dialog box Click [File] and select [Save as] to save the file. Reference The file is saved as a Wide Image Viewer file (*.ktf). Clicking [OK] in the [Image Stitch] dialog box transfers the compressed image to the Analyzer. With multiple stains, apply the full focus for each channel before using the merge function. 7 Click [OK] in the [Image Stitch] dialog box to load the merged image to the analysis application. Reference If the image resolution exceeds the largest resolution of 4080 x 3072 pixels that can be handled on the analysis application, the image is made small. 4-17

68 4-5 Wide Image Viewer Size Limit for Input Images The maximum number of pixels that can be processed with the Analyzer is 4080 x 3072 pixels. When merging images, the Wide Image Viewer automatically opens and shows the merged image. Up to x pixels can be displayed on the Wide Image Viewer. 4 <Overview> Images of 4080 x 3072 or less saved using Viewer Directly opened in the Analyzer. ktf file Images can be opened in the Analyzer but the image size will be reduced to 4080 x 3072 pixels or less. The Wide Image Viewer automatically opens with the image Reference When saving files using the Wide Image Viewer, you can select a file format from among ktf, BIGTIFF and TIFF. Point TIFF files and BIGTIFF files exported using the Wide Image Viewer cannot be opened on the "Analyzer". A BIGTIFF file compatible application is required to open BIGTIFF files. 4-18

69 Operating Wide Image Viewer The Wide Image Viewer displays large, high-resolution merged images. 1 1 Click the [File] icon on the tool bar and select [Open]. Select the image to display on the Wide Image Viewer. When merging images through the Analyzer, the Wide Image Viewer automatically opens with the image. 2 Change the [Zoom ratio] of the Overview screen by scrolling the mouse wheel. The square range is enlarged and displayed. Reference Select the zoom ratio from 100%, 50%, 25%, 12.5%, 6.25%, 3.12% and Move the cursor to inside the rectangle, and drag and move within the display range. Reference To view the capture information, click the [View] icon on the tool bar to select [Photo Properties Display]. 4-19

70 Saving JPEG, TIFF, and BIG TIFF images with Wide Image Viewer Resizing and saving images 1 1 Click the [File] icon on the tool bar and select [Shrink/Crop Image]. 4 2 Select either [Shrink Entire Image] or [Crop] for [Shrink/Crop]. For Crop, set a rectangle on the screen. The Ctrl plus mouse wheel enables scaling. 3 Select the file format for [Format] and click [OK]. The save dialog starts up. Then, enter the file name and press the save button. 2 3 Reference If [Shrink Entire Image] is selected for [Shrink/ Crop], the image is automatically reduced and saved with the screen size of 4080 x 3072 pixels or less (one side cannot exceed 4080 pixels). 3 Export in the original scale 1 1 Click the [File] icon on the tool bar and select [Export in the original scale]. 2 Select the file format for [Format] and click [OK]. The save dialog starts up. Then, enter the file name and press the save button. Reference If the size exceeds the following criteria, only BIGTIFF can be selected. 8bit : 50,000 x 50,000 pixel or less 16bit : 45,000 x 45,000 pixel or less 24bit : 37,000 x 37,000 pixel or less 48bit : 26,000 x 26,000 pixel or less 4-20

71 4-6 Inserting the Capture Position into a Merged Image Inserting the Capture Position into a Merged Image 1 Right-click the merged image of [Image book] and select [Insert pane indicating captured area]. 4 2 The [Open] dialog box is displayed. Then, select the image for which to display the capture position, and click [Open]. 3 The [Line Settings] dialog box is displayed. Set the line color and thickness, and click [OK]. 4 The capture area is displayed in as a rectangle within the merged image. To view multiple capture locations, repeat steps 1 and

72 4-7 Full Focus for Creating Omnifocal Images This section explains the full focus function for creating composite images with a large depthof-field by extracting the pixel information from multiple Z-stack images. Point The Analyzer Application, BZ-H4A (optional), is required to use the full focus function. Full focus procedure 1 Load an image group captured using Z-stack photo or multicolor & Z-stack photo. "3-2 Loading Grouped Images" (page 3-3) 4 2 Click the [Full Focus] icon on the toolbar. Or select [Full Focus] from the [Z-Stack] menu. [Image Type Selection] dialog box is displayed. 3 Select the type of images to be full focused. The [Executing the Full Focus.] progress bar is displayed and a composite image is created. When the image has been created, the new image is opened in the image book as a single image. "Full Focus" is displayed in green below the thumbnail. In the top left of the image window, the file name and Full focus are displayed. 4-22

73 4-8 XYZ Slice for Creating Cross-Sections of Z-Stack Images This section explains the XYZ slice function for clipping vertical or horizontal cross-sections of a Z-stack group or multicolor & Z-stack group. You can observe the cross-sectional structure of the sample (target slice), and also the localization of selected cells or cell nuclei. Point The 3D Application, BZ-H4R (optional), is required for using the XYZ slice function. XYZ slice procedure 1 Load the image group captured using Z-stack photo or multicolor & Z-stack photo. "3-2 Loading Grouped Images" (page 3-3) 2 Select [XYZ Slice] from the [Z-Stack] menu. The image slice is displayed in the [XYZ Slice] window. 4 Y Z X Reference To increase the scale of the Z-axis of the XYZ slice, adjust the [Z Pitch] value displayed in the image book. If the Z pitch is 100%, it is displayed with the same reduced scale on the XY direction. 4-23

74 Preview image display area Displays a preview of the image group. 2 X-Y cross-section coordinate Displays the Z-axis coordinate of the current preview image. To change the current location, drag the yellow line, use the mouse wheel, enter a numeric value, or use the spin buttons. This changes the image displayed and the position of the yellow line. 3 X-Z cross-section coordinate Displays the coordinate of the X-Z cross-section preview specified by the horizontal indicator. To change the current location, drag the horizontal indicator, use the mouse wheel, enter a numeric value, or use the spin buttons. This changes the image displayed and the position of the line. 4 Y-Z cross-section coordinate Displays the coordinate of the Y-Z cross-section preview specified by the vertical indicator. To change the current location, drag the vertical indicator, use the mouse wheel, enter a numeric value, or use the spin buttons. This changes the image displayed and the position of the line. 5 Horizontal & vertical indicators Moving the indicators changes the cross-section preview image. 6 X-Z cross-section preview Displays a preview of the cross-section image based on the line specified by the horizontal indicator. The yellow line shows the Z-axis location of the preview image. 7 Y-Z cross-section preview Displays a preview of the cross-section image based on the line specified by the vertical indicator. The yellow line shows the Z-axis location of the preview image. 8 [Show the line] checkbox Sets whether to show or hide the X-Y cross section horizontal/vertical indicator (white) and the X-Z/Y-Z cross section horizontal indicator (yellow). 9 [Overlay the line on captured image.] checkbox Right-clicks the image book to overlay and save the line of horizontal/vertical cursor to the image saved in [Save As]. 4-24

75 4-9 Max Projection for Displaying the Brightness of Z-Stack Images This section explains the max projection function for processing acquired fluorescent images as three-dimensional images with a large depth-of-field. By averaging the intensities of multiple Z-stack images, the entire fluorescent image can be more easily observed. Point The 3D Application, BZ-H4R (optional), is required for using the max projection function. Max projection procedure 1 Load the image group captured using Z-stack photo or multicolor & Z-stack photo. "3-2 Loading Grouped Images" (page 3-3) 2 Select [Max Projection] from the [Z-stack] menu. The [Max Projection] window appears. The loaded Z-stack image is displayed with the XY, YZ and XZ axes superimposed. When superimposed, the image takes the maximum brightness for each coordinate value The X-Y cross-section is displayed as a preview. 2 The Y-Z cross-section is displayed as a preview. 3 The X-Z cross-section is displayed as a preview. Reference To increase the scale of the Z-axis of the max projection, adjust the [Z Pitch] value within the image book. 4-25

76 4-10 3D Display of Z-Stack Images This section explains the "3D Display" for creating 3D images. This is useful for displaying the location and three-dimensional structure of cellular tissue. Point The 3D Application, BZ-H4R (optional), is required for using the realtime 3D display function. Overview of 3D display This section explains the [3D Display] dialog box that appears when you click the [3D Display] icon on the toolbar (large). [3D Display] dialog box Preview image display area Displays a preview of the entire 3D image. This display can be manipulated using a number of different controls. 2 Display setting Switches the preview image between 3D and orthogonal display. 3D display Orthogonal display The 3D display is suitable for observing In the orthogonal display, selected regions of the structure of a sample or the localization the slice or cross-sections of cell tissue can 4-26

77 3 Basic plane The orientation of the XY/YZ/XZ planes from which the image is viewed can be changed. 4 Rotation axis Rotates the current image 90 degrees about the X/Y/Z axes. 5 [RESET] button Resets the 3D display to the initial state. 6 Brightness adjustment Adjusts the brightness, contrast and gamma of the preview image. 7 RED/GREEN/BLUE Turns the red, green and blue channels on or off. If a channel is turned off, that color will not be displayed. 8 Motion picture detailed setting Use this menu to save the 3D preview image as a motion picture file in AVI format. [Save motion picture] only saves the contents selected in this menu. Play Stop Slider (check box) Start Mid End Step angle Play methods Frame rate Plays the selected motion picture. Pauses the current moving 3D image. Selects the specified motion picture at an arbitrary point and displays it as a still image. Plays the motion picture frame-by-frame. Shows whether the Mid points have been set. When you click Set, the current location is marked as the Start. You can jump to this location using the Go to control. When you click Set, the current location is marked as Mid. You can jump to this location using the Go to control. When you click Set, the current location is marked as the End. You can jump to this location using the Go to control. Sets the angle. The smaller the value, the smoother the display. Loop play: Repeats motion picture once it reaches the end. Sequential play: Returns to the start once it reaches the end. Sets the frame rate. The larger the value, the faster the motion picture will play back. 4 9 [Save motion picture] button Saves motion pictures set with [Motion picture detailed setting] as AVI files. 10 [Save still image] button Saves the currently displayed image as a still image file. Reference To increase the scale of the Z-axis of the 3D display, adjust the [Z Pitch] value displayed in the image book. Enter the numerical value, use the up and down arrow buttons, or use the mouse wheel to set the interval value. This adjusts the Z-axis scale. 4-27

78 3D display procedure 1 Load the image group captured using Z-stack photo or multicolor & Z-stack photo. "3-2 Loading Grouped Images" (page 3-3) 2 Click the [3D Display] icon on the toolbar. Or select [3D Display] from the [Z-Stack] menu. The [3D Display] window appears. Using the motion picture detailed setting 4 The Motion picture detailed settings consist of a row of controls, from [Start] (Angle, Magnification) through [Mid], to [End] (Angle, Magnification). These are set using the following procedure Adjust the size and angle of the preview images 3D display, then click [Set] button under [Start]. 2 Set the next size and angle for the 3D display, and then click [Set] button under [Mid]. 3 Finally, set a new size and angle for the 3D display, and then click [Set] button under [End]. 4 Check the selected angles and display sizes (magnification) using the [Go to] buttons. 5 Set [step angle], [Play methods] and [Frame rate]. 6 Click [Play] button to play back the motion picture. 4-28

79 3D image procedure Using the wheel mouse, you can zoom in and out of 3D images, and adjust the X-axis (horizontal) and Y-axis (vertical) of the slice cross-section. 3D image procedure Visual reference Mouse wheel operation Rotate the 3D image (Rotates the image about the center of the observed data) Left click and hold to drag the image. Zoom in and out of the 3D image Use the scroll wheel to zoom in (by moving the wheel down) or zoom out (by moving the wheel up). Displaying the slice cross-section 3D image moving Right click and hold to drag the crosssection indicated by the yellow frame. Clicking the wheel and dragging the image moves the 3D image. 4 Reference If you release the mouse button while dragging, the 3D image continues to rotate in the direction which you move the mouse. To stop the rotation click the preview image display area. The speed at which the 3D image rotates depends on the speed at which you move the mouse. 4-29

80 Slice function procedure If you right click and drag with the mouse, you can display a rectangular cross-section of the X, Y, Z planes of any surface of the object. The plane of the slice is indicated by a yellow frame. 1 Move the mouse pointer to the plane which you want to take a slice of, and then right click. The selected plane is indicated by a yellow frame Right click and hold to drag the cross-section indicated by the yellow frame. 2 3 Release the right-click. This hides the yellow frame and fixes the cross-section slice displayed. Reference You can display a slice cross-section of the X, Y or Z planes. By right clicking and displaying a new yellow frame, you can realign the slice plane

81 4-11 Adjusting the shadow, highlight, and gamma This section explains Lookup table that improves images to be seen easier by the shadow, highlight, and gamma correction. Overview of Lookup Table This section explains the [Lookup Table] dialog box that appears when you select [Lookup Table] from the [Image Processing] menu. [Lookup Table] dialog box Unprocessed image display area Displays the loaded image before adjustment. 2 Processed image display area Displays the image after adjustment. 3 Distribution This item selects whether to show the histogram based on the distribution of brightness, R, G or B pixel values. When you select the [Brightness] for the [Distribution], the brightness is calculated as the following formula: Y = 0.3R + 0.6G + 0.1B And then, histogram is calculated for the converted value. If the picture contains only R, G, or B pixel values because of the pseudo-color, you can select [R], [G], or [B] for the [Distribution] to adjust the brightness easily. 4 [Shadow] slider Adjusts the darkness of the image. Moving the slider to right increases the dark part. 5 [Highlight] slider Adjusts the brightness of the image. Moving the slider to left increases the bright part. 4-31

82 6 [Gamma] slider Adjusts the contrast of bright or dark areas in the image. Moving the slider to the left enhances the contrast of the bright area. Moving the slider to the right enhances the contrast of the dark area. 7 [Auto Shadow & Highlight] button Sets the shadow and highlight value to the right value automatically. 8 [Save Conditions] button Saves the shadow, highlight, and gamma setting contents as the lookup table setting file (*.lxc). 9 [Load Conditions] button Loads the saved lookup table setting file (*.lxc). The set shadow, highlight, and gamma are reflected to the image processing [Clear Conditions] button Clears the set shadow, highlight, and gamma, and returns to the initial setting value. 11 [OK] button Register the image after shadow, highlight, and gamma adjustment in the image book as an unsaved image. When the image group of the Z-stack or time-lapse is selected, the process is performed with the same settings on every image. 4-32

83 Lookup Table procedure 1 Select [Lookup Table] from the [Image Processing] menu. The [Lookup Table] dialog box appears Adjust the [Shadow], [Highlight], and [Gamma] values using the sliders. You can also use the spin buttons or directly enter the values. Click the [Save Conditions] button to save the settings as required. 3 Click the [OK] button. The adjustments are applied to the image. The image is opened in the image book as an unsaved image, and the adjusted image is displayed in the image window. Important Once you click the [OK] button, you cannot restore the default settings by clicking [Clear conditions]. To restore the original image, select [Undo] from the [Edit] menu. 4-33

84 4-12 Adjusting Hue, Saturation, and Luminance This section explains how to adjust the hue, color saturation, and luminance settings to make the image clearer. Overview of Hue, Saturation, Luminance This section explains the [Hue/Saturation/Luminance] dialog box that appears when you select [Hue, Saturation, Luminance] from the [Image Processing] menu. [Hue/Saturation/Luminance] dialog box Unprocessed image display area Displays the loaded image before adjustment. 2 Processed image display area Displays the image after adjustment. 3 Hue circle A color model showing how the color representation changes as a result of adjusting the hue, color saturation, and lightness values. You can visually check the color representation. 4 [Hue] slider Adjusts the hue (i.e. red, yellow, green, blue, and violet) of the image. You can also use the slider, spin buttons or directly enter values. Values between -180 and +180 can be entered for blue, red and green. Moving the slider left or right adjusts the color closer to its complementary color (±180). You can also adjust the hue by dragging the inner circle to rotate it. 4-34

85 5 [Saturation] slider Adjusts the brightness (saturation) of colors. You can also use the slider, spin buttons or directly enter values. Specify a value from -100 to For the inner ring of the hue circle, the rightmost position of the slider represents the current color saturation level (100). The color turns cloudier as you move the slider to the left (-100). 6 [Luminance] slider Adjusts the color contrast (luminance) of the image. You can also use the sliders, spin buttons or directly enter values. The color of the inner ring changes from black to white. You can specify a value from -100 to Moving the slider to the left adjusts the color closer to black (-100), while moving it to the right adjusts it closer to white (+100). 7 Magnification Adjusts the display magnification of the image. Adjust the magnification with the button or from the pull-down menu. 8 [Save Conditions] button Saves the hue, saturation, and luminance settings as a hue, saturation, and luminance setting file (*.sxc). 4 9 [Load Conditions] button Loads the saved hue, saturation, and luminance setting file (*.sxc). The hue, saturation, and luminance settings are reflected in the image. 10 [Clear conditions] button Clears the hue, saturation, and luminance settings you made and restores the default settings. 11 [OK] button Opens the image after hue, saturation, and luminance adjustment in the image book as an unsaved image. When the image group of the Z-stack or time-lapse is selected, the process is performed with the same settings on every image. 4-35

86 Hue, Saturation, Luminance procedure 1 Select [Hue, Saturation, Luminance] from the [Image Processing] menu. The [Hue/Saturation/Luminance] dialog box appears Adjust the [Hue], [Saturation], and [Bright]. You can also use the sliders, spin buttons or directly enter the values. Click the [Save Conditions] button to save the settings as required. 3 Click the [OK] button. The adjustments are applied to the image. The image is opened in the image book as an unsaved image, and the adjusted image is displayed in the image window. Important Once you click the [OK] button, you cannot restore the default settings by clicking [Clear conditions]. To restore the original image, select [Undo] from the [Edit] menu. For group images, select [Undo All] from the [Edit] menu. 4-36

87 4-13 Filtering This section explains the filter function for sharpening edges in images and applying embossing effects. Point When a particular area is selected, the filter is applied only to the selected area. Overview of filters This section explains the [Filter] dialog box that appears when you select [Filter] from the [Image Processing] menu. [Filter] dialog box Unprocessed image display area Displays the loaded image before filtering. 2 Processed image display area Displays the image after filtering. 3 Filter Select one of the following filter types from the pull-down menu. Enhance Edge, Emboss, Color Emboss, Averaging, Sobel, Extract Edge, Roberts, or Median 4 Size Select the pixel matrix size from the following sizes. 3 x 3, 5 x 5, 7 x 7 5 [Magnification] button Adjusts the display magnification of the image. Adjust the magnification with the button or from the pull-down menu. 4-37

88 6 Matrix modifier Set the level of the filtering effect. Divisor : Adjusts the level of filtering effect. The value obtained by multiplying the values in the filter matrix is divided by this value. Bias : Adjusts the brightness of the filtered image. The value obtained by multiplying the values in the filter matrix is divided by the divisor and the bias value is added. 7 [Save Conditions] button Saves the settings as a custom filter setting file (*.fxc). 8 [Load Conditions] button Loads the saved custom filter setting file (*.fxc). The filter settings are reflected in the image. 4 9 [Clear conditions] button Clears the filter settings you made and restores the default settings. 10 [OK] button Opens the image after filtering in the image book as an unsaved image. When the image group of the Z-stack or time-lapse is selected, the process is performed with the same settings on every image. 4-38

89 Filtering procedure View the results of the filtering in the preview image display area. 1 Select [Filter] from the [Image Processing] menu. The [Filter] dialog box appears Select the type of filter from the [Filter] pull-down menu and the size of the matrix from the [Size] pull-down menu. 3 You can also enter values directly in [Divisor] and [Bias] under [Matrix modifier], or use the spin buttons. 4 Click the [OK] button. The adjustments are applied to the image. The image is opened in the image book as an unsaved image, and the adjusted image is displayed in the image window. Important Once you click the [OK] button, you cannot restore the default settings by clicking [Clear conditions]. To restore the original image, select [Undo] from the [Edit] menu. For group images, select [Undo All] from the [Edit] menu. 4-39

90 Filter types With BZ-X Analyzer, you can apply the following types of filters to images. Enhance Edge (weak/medium/strong) Emphasizes the outlines in the image. Original image Processed image 4 Emboss Emphasizes the contrast of the image. Use this filter when bright areas and dark areas exist in the image. Original image Processed image Color emboss Applies the Emboss filter without manipulating the colors in color images. Original image Processed image 4-40

91 Averaging Smoothes out the entire image. Original image Processed image Sobel Emphasizes only the major outlines in the image. Original image Processed image 4 Extract edge Removes the outlines from the image. Original image Processed image 4-41

92 Roberts Emphasizes fine outlines in the image. Original image Processed image Median Removes patchy noise from the image. You can remove noise while maintaining smooth edges. 4 Original image Processed image 4-42

93 4-14 Calculation, Accumulation, Averaging This section explains how to perform calculations on the RGB data of a target image (reference image) and another image of the same format to create a new image based on the calculated results. Overview of image calculation This section explains the [Calculation/Accumulation/Averaging] dialog box that appears when you select [Calculation, Accumulation, Averaging] from the [Image Processing] menu. [Calculation/Accumulation/Averaging] dialog box Reference image Displays the file name of the image used as the reference for calculation, accumulation, and average. A single image selected from the image book is used as the reference image. 2 Calculation image Displays the file name of the image for calculation, accumulation, and average in relation to the reference image. This is a single image with the same bit level. 3 Image Synthesis Selects the synthetic method of images from the followings Add: Adds the calculation image to the reference image. If the calculated results exceed the allowable range for base image data (0-255 for R, G, and B), the results are rounded to values that fall within the range. Multiply: Multiplies the calculation image by the reference image. If the calculated results exceed the allowable range for base image data (0-255 for R, G, and B), the results are rounded to values that fall within the range. Subtract: Subtracts the calculation image from the reference image. If the calculated results exceed the allowable range for base image data (0-255 for R, G, and B), the results are rounded to values that fall within the range. Absolute value of the difference: Calculates the absolute values for the differences of R, G, and B data between the reference image and calculation image. Average: Calculates the average values for the R, G, and B data of the reference image and calculation image. Compare (dark): Compares the R, G, B data for the reference image and calculation image and creates a new image using the R, G, B data with lower values. 4-43

94 Compare (bright): Compares the R, G, B data for the reference image and calculation image and creates a new image using the R, G, B data with higher values. AND: Calculates the logical product (AND) for the R, G, B data of the reference image and calculation image. OR: Calculates the logical sum (OR) for the R, G, B data of the reference image and calculation image. XOR: Calculates the exclusive logical sum (XOR) for the R, G, B data of the reference image and calculation image. 4 Divisor Adjusts the level of the image composition effect. The value obtained by multiplying the value for calculation is divided by this value. 5 Bias Adjusts the brightness of the image after image composition. The value obtained by multiplying the values for calculation is divided by the divisor and the bias value is added. Example of image calculation (Average) 4 Reference image Calculation image New image 4-44

95 4-15 Negative/Positive Inversion This section explains the negative/positive inversion function for inverting the image like a photographic negative by reversing the luminance and RGB values of each pixel in the image. Negative/Positive Inversion procedure 1 Select [Reverse Neg/Pos] from the [Image Processing] menu. The image shown in the image book and image window is inverted like a negative and the original image is overwritten. Reference Important When a particular area is selected, only the selected area is inverted. To restore the original image, select [Undo] from the [Edit] menu. For group images, select [Undo All] from the [Edit] menu. 4 Example of negative/positive inversion Original image Processed image 4-45

96 4-16 Gray Conversion This section explains the gray conversion function for converting an image to gray scale. Gray conversion procedure 1 Select [Gray Conversion/Pseudo Color] from the [Image processing] menu. The [Gray Conversion/Pseudo Color] dialog box appears. 4 2 Select [Gray Conversion] and click the [OK] button. The changes are applied to the image. The image is converted to the grayscale, and overwritten. Reference When a particular area is selected, only the selected area is converted to gray scale. Important To restore the original image, select [Undo] from the [Edit] menu. For group images, select [Undo All] from the [Edit] menu. 4-46

97 4-17 Applying Pseudo Color This section explains "Pseudo color" that converts the image color. Pseudo color procedure 1 Select [Gray Conversion/Pseudo Color] from the [Image processing] menu. The [Gray Conversion/Pseudo Color] dialog box appears. 2 Select [Pseudo Color] and select the color to use from the [Color] pull-down menu. 3 Click the [OK] button. The image is converted to the pseudo color, and overwritten.. 4 Reference When a particular area is selected, only the selected area is converted to pseudo color. Important To restore the original image, select [Undo] from the [Edit] menu. For group images, select [Undo All] from the [Edit] menu. 4-47

98 MEMO

99 Chapter 5 Various Measurements This chapter describes how to perform a variety of 2D measurements. 5-1 XY Measure Area Measurement Measuring the Line Profile Displaying a Histogram D Measurement Displaying Capture Information Copying Capture Information

100 5-1 XY Measure This section explains the XY Measure function for performing measurements including 2D point-to-point, 2D area, and brightness measurements. Point The Measurement Application, BZ-H4M (optional) is required to use the XY Measure. XY Measure settings 1 Select [XY Measure] from the [Measure] menu. The [XY Measure] dialog box appears [XY Measure] button icons Select from the following measurement methods: Between 2-points, Radius, Centers, Count, Angle 1, Angle 2, Perpendicular, Parallel, Segment line, and Freehand line. 2 [Garbage Box] button icon Deletes individual measurement lines. 3 [Delete All] button icon Deletes all measurement lines. 4 Measuring Line Selects the color of measurement lines and measurement points. 6 5 Display Details Sets the thickness of measurement lines and the color of labels. 6 Measurement Result Select whether to display the measurement results in the image window. 5-2

101 [Measurement result] dialog box Displayed during each XY Measure operation, it shows each measurement result in the table. You can also display the results of using different measurement methods in one table No. The reference number associated with the object. 2 Measure The type of measurement being performed. 3 Result The numeric value of the measurement results. 5 Reference A total of 999 individual measurements can be performed, including between [Between 2-points] (the distance between two points), [Radius], [Angle], [Centers] (the distances between the centers of two circles), [Perpendicular] (the length of vertical lines) and [Parallel] (the distance between parallel lines). The count function can measure up to 999 measurement points. 4 Decimal Position Set the number of decimal places displayed after the decimal point (0 to 3) using the spin buttons. 5 [Result Output] button Click to save the measurement results as a tab delimited text file. 6 [Calibration] checkbox When the checkbox is selected, the results display can be switched between pixels and µm based on the reference length set in the [Calibration Setting] dialog box. 7 [Set] button Click to display the [Calibration Setting] dialog box. In [Reference length], enter a value for one pixel (µm units). [Calibration Setting] dialog box 5-3

102 Measuring the distance between two points Measures the distance between two specified points. 1 Click the [Between 2-points] button icon in the [XY Measure] dialog box. 2 Click the start point of the distance you want to measure. 3 2 The start point, along with a reference number, is displayed. To delete the start point, right click. In addition, the [Measurement Result] dialog box appears. 3 Click the end point. A straight line appears between the two specified points, and the distance between them is displayed in the [Measurement Result] dialog box. The No. in the [Measurement Result] dialog box is the reference number for the center of the two points. 5 Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 4 To measure the distance between two new points, repeat Steps 2 and 3 above. 5-4

103 Measuring the radius of a circle Measures the radius of a circle drawn through three specified points. 1 Click the [Radius] button icon in the [XY Measure] dialog box Click on one point on the circumference of the circle to measure. The point selected is marked with an x. 3 Click on a second point on the circumference of the circle. The second point is also marked with an x. 4 4 Click on a third point on the circumference of the circle. The third point is marked with an x. A circle passing through the three points, as well as the center of the circle, is displayed. Reference If you right click before selecting the third point, the previously-selected points are deleted. In addition, the [Measurement Result] dialog box appears and the radius of the circle is displayed. The No. in the [Measurement Result] dialog box is the reference number for the center of the circle. 5 Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5 To measure the radius of a new circle, repeat Steps 2 to 4 above. 5-5

104 Measuring the distance between circle centers Measures the distance between the centers of two specified circles. 1 Click the [Centers] icon in the [XY Measure] dialog box. 3 2 Following the same procedures used to measure the radius of a circle, click on three points on the circumference of the circle that will serve as the start point. The first circle is displayed In the same manner, click on three points on the circumference of the circle that will serve as the end point. A line appears joining the centers of the two circles. In addition, the [Measurement Result] dialog box appears and the distance between the centers of the two circles is displayed. "Measuring the radius of a circle" (page 5-5) The No. in the [Measurement Result] dialog box is the reference number for the center of the line joining the two circles. Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5-6

105 Point count Counts the number of specified points. 1 Click the [Count] button icon in the [XY Measure] dialog box. The [Measurement result] dialog box appears. 2 Click on the points to count. Each time you click, the point selected is marked with an x, and the measurement results are displayed in [Count] at the bottom left of the [Measurement Result] dialog box. For this observation, five points are indicated. Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. 2 "9-5 Finalize the inserted object in the image" (page 9-14)

106 Angle 1 (3-point angle) measurement Measures the interior angle of an angle comprised of three specified points. 1 Click the [Angle 1] button icon in the [XY Measure] dialog box. 2 Click on the image to specify the first point forming the angle. 2 3 Move the mouse, and click on another point. The [Measurement Result] dialog box appears Click on a third point, which will be the vertex of the angle. The interior angle of the angle determined by the three points is displayed in the [Measurement Result] dialog box. The No. in the [Measurement Result] dialog box is the reference number for the third point. 5 Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5-8

107 Angle 2 (4-point angle) measurement Measures the interior angle created at the point where two specified straight lines intersect. 1 Click the [Angle 2] button icon in the [XY Measure] dialog box Click the two points on the line forming the angle in the image. Select two points (the first and second points) to create the first line forming the angle you want to measure. 3 Click a further two points (the third and fourth points) to form a second straight line. The [Measurement Result] dialog box appears and the angle at the vertex where the two lines cross is displayed. The No. in the [Measurement Result] dialog box is the reference number for the second and fourth points of the lines forming the angle. Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5 Reference About the measurement results values Using this type of measurement, angles are calculated in the following way. The line created by the first two points specified is Line A and the second line specified is Line B. The angle is determined by rotating Line A in a clockwise direction until it exactly matches Line B. However, note that the results will vary depending on the order in which the lines were selected. Line A (Initially selected line) Line B (Subsequently selected line) Line B (Subsequently selected line) Line A (Initially selected line) 5-9

108 Measuring the length of a perpendicular line Measures the length of a line drawn perpendicular to a reference line. 1 Click the [Perpendicular] button icon in the [XY Measure] dialog box. 4, 5 2 Click the location through which you want to draw the reference line. The reference line is drawn through the points selected. In addition, the [Measurement Result] dialog box appears. 3 Set the angle of the dotted line, and then click. This is the reference line for the measurement. Right click to redraw the reference line Click on the location you want to measure. The location selected is marked with an x, and the length of the perpendicular line from that point to the reference line is displayed in the [Measurement Result] dialog box. The No. in the [Measurement Result] dialog box indicates the reference number associated with the point clicked in Step 4. Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5 To measure the length of a new perpendicular line from the specified reference line, repeat Step 4 above. To stop measuring perpendicular lines, double click in Step

109 Measuring the distance between parallel lines Measures the distance between a reference line and a line drawn parallel to it Click the [Parallel] button icon in the [XY Measure] dialog box. 2 Click the location through which you want to draw the reference line. The reference line is drawn through the points selected. 3 Set the angle of the reference line, and then click. This is the reference line for the measurement. Right click to redraw the reference line. The [Measurement Result] dialog box appears. 3 4 Click on the location you want to measure. The location selected is marked with an x, and the distance between the parallel line passing through this point and the reference line is shown in the [Measurement Result] dialog box. The No. in the [Measurement Result] dialog box indicates the reference number associated with the point clicked in Step 4. 5 Reference To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5 To measure the distance between another parallel line and the same reference line, repeat Step 4 above. To stop measuring parallel lines, double click in Step

110 Measuring the distance between segment lines Measures the total length of a segment line connecting specified points. 1 Click the [Segment Line] button icon in the [XY Measure] dialog box. 2, 3 2 Click on the start point of the segment line you want to measure. The point selected is marked with an x. The [Measurement Result] dialog box appears. 3 Click on the points necessary to construct the segment line. To specify the end point of the line, double click. 5 Each point is marked with an x, and the length of each line segment is displayed in the [Measurement Result] dialog box. The No. in the [Measurement Result] dialog box is the reference number for the segment lines. Reference Double clicking before you specify the end point of the line allows you to return to the previously-selected point. To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5-12

111 Measuring freehand lines Measures the length of a freehand line connecting specified points. 1 Click the [Freehand Line] button icon in the [XY Measure] dialog box. 2, 3 2 Click on the start point of the freehand line you want to measure. The point selected is marked with an x. The [Measurement Result] dialog box appears. 3 Trace the freehand line you want to measure. Click again to specify the end point of the line. The start and end points are marked with an x, and the length of the freehand line is calculated. The No. in the [Measurement Result] dialog box indicates the reference number displayed on the freehand line. Reference Right clicking before you specify the end point by clicking allows you to cancel the start point. To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. 5 "9-5 Finalize the inserted object in the image" (page 9-14) 5-13

112 Setting measuring lines Set the color of measurement lines and measurement points. Setting the color of measuring lines Select the color to use from the [Measurement Line] pull down list in the [Measuring Line] menu. Setting the color of measurement points Select the color to use from the [Measurement Point] pull down list in the [Measuring Line] menu. 5 Setting display details Set the thickness of measuring lines and the color of labels (showing the reference number associated with each element). Setting the color of labels Select the label color from the [Label] pull down list in the [Display Details] menu. Setting the thickness of measurement lines and points Select [Thin] or [Thick] in [Display Details]. The settings will also affect measuring lines that are already displayed. 5-14

113 Setting the measurement results The measurement results of measured items are displayed in the image window. Display/hide measurement results Select whether to display the measurement results in the image window. Setting the measurement results display (including font type) 1 Double click or right click the [Measurement Result] displayed in the image window. The [Measurement Result] dialog box appears Set the following items, as necessary: 1 Font Select the font. 2 Font size Select the font size. 3 Style Select the font style. 4 Font color Select the color from the pull down menu. 5 Background To set a transparent background, check [Transparent]. To set another background color, first uncheck [Transparent]. Next, select a color from the Background Color pull down menu. 6 Display Set the number of results to be displayed by directly entering a value or using the spin buttons. Up to 20 results can be displayed in the image window. When there are more than [Number] items, the [Number] items with the highest reference number are displayed. 5 3 Click [OK] button to finish. 5-15

114 Deleting measuring lines Deletes individual measuring lines currently displayed. 1 Click the [Garbage box] button icon in the main [XY Measure] dialog box. 2 Click on the points to delete. The measuring line and measurement result are deleted. The reference numbers for items measured after the one you have deleted are moved up. 5 Deleting all measuring lines Deletes all of the currently displayed measuring lines. 1 Click the [Delete All] button icon in the [XY Measure] dialog box. A message confirming whether you want to delete all items is displayed. 2 Click the [OK] button. All measuring lines and their results are deleted

115 5-2 Area Measurement This section explains the area measurement function for measuring the area or brightness of a region specified by a polygon, circle, or freehand line. Point The Analyzer Application, BZ-H4M (optional), is required to use the area measurement function. Setting area measurement 1 Select [Area Measurement] from the [Measure] menu. The [Area Measurement] dialog box appears. 1 [Area Measurement] button icons 1 Select from the following measurement methods: Polygon, Circle draw, Freehand line [Run] button icon Measures the area of the specified region [Garbage Box] button icon Deletes the measured region. 6 4 [Delete All] button icon Deletes all measured regions Measuring Line Sets the color of measuring lines and points. 6 Display Details Sets the thickness of measuring lines and the color of labels. 7 Measurement Result Select whether to display the measurement results in the image window. 8 [Area and region check] checkbox When the checkbox is selected, the region for measurement is displayed in the color of the measuring lines when you click it. 5-17

116 [Measurement Result] dialog box Displayed during the main measurement operations, it shows the results of each measurement in a table. You can also display multiple types of main measurement results in one table No. The reference number associated with the object. 5 2 Measurement item The type of area measurement of each object and the measurement items. 3 Measurement result Displays the measurement results of each object. The following data is displayed. Color image No., area, as well as the maximum, minimum, average brightness and integrated value for each of the red, blue and green channels Monochrome image No., area, maximum brightness, minimum brightness, average brightness, and integrated value 4 Total area The total area of the region measured. 5 Number of decimals Set the number of decimal places displayed after the decimal point (0 to 3) using the spin buttons. 6 [Result output] button Click to save the measurement results as a tab delimited text file. 7 [Calibration] checkbox When the checkbox is selected, the results display can be switched between pixels and µm based on the reference length set in the [Calibration setting] dialog box. 5-18

117 8 [Set] button Click to display the [Calibration Setting] dialog box. In [Reference Length], enter a value for one pixel (µm units). [Calibration setting] dialog box Point The value of the area measured includes the measuring line. As a visual aid, each region measured on the image is marked with a reference number. When a region is deleted, the reference numbers of all regions created after the one deleted are moved up. Where the measurement lines of the specified polygon or circle overlap, the area is divided and then measured, as illustrated in the following diagram. area 1 area 2 area

118 Calculating the area of a selected region Measuring the area of a polygon 1 Click the [Polygon] button icon in the [Area Measurement] dialog box Click on one of the vertices of the region (polygon) you want to measure. 3 Click on the other vertices in order, to outline the desired area. Reference Right clicking returns you to the previouslyselected vertex. 5 4 Double click when selecting the last vertex of the area you want to measure. The polygon you will measure is displayed on the image. Reference To create multiple regions, repeat Steps 2 to 4 above. 5 Click the [Run] button icon in the [Area Measurement] dialog box, and then click on the area you want to measure. The [Measurement Result] dialog box appears and the measurement results are displayed. The No. in the [Measurement Result] dialog box is the reference number for the center of each region. Reference If the [Area and region check] checkbox is selected, clicking on the polygon area displays it in the same color as the measuring line. This allows you to more easily check the extent of the region being measured. To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5-20

119 Measuring the area of a circle 1 Click the [Circle Draw] button icon in the [Area Measurement] dialog box. 2 Click on one point on the circumference of the circle to measure. 2 3 The point selected is marked with an x. 3 Click on a second point on the circumference of the circle. The second point is also marked with an x. Reference If you right click before selecting a third point, the previously-selected point is deleted. 4 4 Click on a third point on the circumference of the circle. The third point is marked with an x. A circle passing through the three points, as well as the center of the circle, is displayed. Reference To create multiple regions (circles), repeat Steps 2 to 4 above. 5 5 Click the [Run] button icon in the [Area Measurement] dialog box, and then click on the area you want to measure. The [Measurement Result] dialog box appears and the measurement results are displayed. The No. in the [Measurement Result] dialog box is the reference number for the center of each region. Reference If the [Area and region check] checkbox is selected, clicking on the polygon area displays it in the same color as the measuring line. This allows you to more easily check the extent of the region being measured. To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5-21

120 Measuring the area enclosed by a freehand line 1 Click the [Freehand Line] button icon in the [Area Measurement] dialog box. 2 2 Click on the start point of the region (freehand line) you want to measure. A freehand line is drawn. 3 Trace an outline to enclose the region you want to measure. 3 4 Double click when the end of the freehand line reaches the start point. The measurement region enclosed by the freehand line is displayed on the image. 5 Reference To create multiple regions, repeat Steps 2 to 4 above. 5 Click the [Run] button icon in the [Area Measurement] dialog box, and then click on the area you want to measure. The [Measurement Result] dialog box appears and the measurement results are displayed. The No. in the [Measurement Result] dialog box is the reference number for the center of each region. Reference If the [Area and region check] checkbox is selected, clicking on the polygon area displays it in the same color as the measuring line. This allows you to more easily check the extent of the region being measured. To finalize the measurement results in the image, select [Save inserted object(s) on the image] from the [Measure] menu. "9-5 Finalize the inserted object in the image" (page 9-14) 5-22

121 5-3 Measuring the Line Profile This section explains the line profile function for drawing a line on the image and measuring the brightness intensity of this line. Point The Analyzer Application, BZ-H4M (optional), is required to use the line profile function. Setting the line profile 1 Select [Line Profile] from the [Measure] menu. The [Line Profile] dialog box appears. When you draw a line on the image, the line profile is displayed. [Line Profile] dialog box (example) * The following line profile is of a color image Line profile Displays the brightness on the line as coordinates. The X-axis indicates the distance from the start point of the line (number of pixels) and the Y-axis indicates RGB brightness. 2 Cursor Drag left or right to specify a point from the brightness of the line profile. The corresponding point (x mark) on the measuring line moves according to the cursor. 3 [Between 2-Points] button icon Specify a line for measuring the line profile. 4 [Freehand Line] button icon Specify a freehand line for measuring the line profile. 5-23

122 5 White background Sets the background of the line profile to white. 6 Black background Sets the background of the line profile to black. 7 [2] checkbox Changes the number of line profiles. Selecting the check box [2] can display up to 2 profiles. 8 Position Indicates the position (X/Y coordinate) of the point on the measuring line. Line profiles can be drawn (up to 2 line profiles), and the position and brightness can be displayed on each profile of the first or second. 9 RGB-brightness Values for RGB brightness on the measuring line. (A single brightness value is displayed for monochrome images.) 5 10 [Color] check boxes Display/hide each color in the profile of a color image. When unchecked, the profile for that color disappears. (This box is not displayed with monochrome images.) 11 Change display order Changes the order in which the color profiles are displayed. Click a color name and click to display the profile in front. Click and it is displayed behind. (This button is not displayed with monochrome images.) 12 Display details Changes the color and thickness of measuring lines. 13 [Result output] button Click to save the measurement results as a tab delimited text file. The following items are output to the file. The coordinate values of the point on the measuring line and the RGB brightness values 5-24

123 Specifying the line profile Measuring the brightness of a line 1 Select [Line Profile] from the [Measure] menu. The [Line Profile] dialog box appears Click the [Between 2-Points] button icon at bottom left. 3 Click on the start point and end point of the part you want to measure on the line. A measuring line is drawn on the image, and the brightness on the measuring line (line profile) is displayed in the [Line Profile] dialog box. 5 4 Moving the mouse pointer over the line profile display area displays the cursor (light blue). 3 5 Move the cursor (light blue) to the location you want to measure. The point (x mark) corresponding to the X-coordinate (distance from the start point of the measuring line) is displayed on the measuring line of the image. The location of this point is shown in the [X =] and [Y =] area of the [Line Profile] dialog box, and its brightness is shown in the [Rbrightness], [G-brightness] and [B-brightness] area. (A single brightness value is displayed with monochrome images.) 5 5 Selecting the check box [2] can draw the line profiles (max.: 2 profiles). If changing the first line profile, uncheck the box [2] and draw line again. 5-25

124 5-4 Displaying a Histogram This section explains the histogram function for displaying the distribution of brightness in the displayed image. Point The Analyzer Application, BZ-H4M (optional), is required to use the histogram function. Setting the histogram 1 Select [Histogram] from the [Measure] menu. The [Histogram] dialog box opens, and a histogram showing the distribution of brightness in the image is displayed. Color image Monochrome image 5-26

125 1 Brightness distribution histogram A histogram showing the distribution of brightness in the image. The X-axis represents the brightness, and the Y-axis represents the corresponding pixel count for each brightness value. 2 [Zoom] button Enlarges the histogram. 3 [Reset] button Returns an enlarged histogram to the default magnification. 4 White background Sets the background of the histogram to white. 5 Black background Sets the background of the histogram to black. 6 Statistics Displays statistical data about the distribution of brightness. 7 Range/Area Sets the range of brightness specific to the histogram, and displays information on that range of brightness. 5 8 Data display box Displays statistical and range/area data. 9 [Color] check boxes Selects whether to display the distribution of each color in a color image. If this box is unchecked, the corresponding color is not displayed in the histogram. (This box is not displayed for monochrome images.) 10 Change display order Changes the order in which the color distributions are displayed in the histogram. Click a color name and click to display the histogram in front. Click and it is displayed one place behind. (This box is not displayed for monochrome images.) 11 Number of decimals Set the number of decimal places displayed after the decimal point (0 to 3) using the spin buttons. 12 [Result Output] button Click to save the measurement results as a tab delimited text file. The following items are output to the file. Number of data, maximum value, minimum value, average value, distribution, standard deviation, lower range, upper range, area, range/area expressed as a percentage, item name, data count for each brightness value 5-27

126 Statistical display Displays statistical data about the distribution of brightness. 1 Select [Histogram] from the [Measure] menu. The [Histogram] dialog box opens, and a histogram showing the distribution of brightness in the image is displayed. 2 2 In the [Histogram] dialog box, select [Statistics]. The statistical data appears in the data display area. The following information is displayed. 5 Item Data Count Max. value Min. value Ave. value Distribution Std. Deviation Explanation Shows the total number of data in the image. Shows the maximum brightness in the image. Shows the minimum brightness in the image. Shows the average brightness in the image. Represents the variation in the data. This value is calculated by taking the sum of the squares of the difference between each data and the average value, and then dividing by (Number of data-1). The standard deviation in brightness in the image. 5-28

127 Range/Area Sets the brightness range, and displays information on that brightness range. 2 1 Select [Histogram] from the [Measure] menu. The [Histogram] dialog box opens, and a histogram showing the distribution of brightness in the image is displayed. 2 In the [Histogram] dialog box, select [Range/Area]. Two measuring lines appear on the histogram. 3 3 Drag the lines to select the brightness range you want to measure. Information about the brightness range appears in the data display area. The following information is displayed. 5 Item X1 X2 Area Explanation Shows the brightness value for the smallest value in the range specified (the X-coordinate of the left-hand measurement line). Shows the brightness value for the largest value in the range specified (the X-coordinate of the right-hand measurement line). Shows the area of the region covered by the specified brightness values. % Shows the proportion of the area specified compared to the total area. 5-29

128 5-5 3D Measurement This part explains "3D Measurement" that performs the measurement with 3D to the Z-stack image. Point The 3D Application, BZ-H4R (optional), is required for using the 3D Measure function. 3D Measurement method 1 Load an image group captured using Z-stack photo. "3-2 Loading Grouped Images" (page 3-3) 2 Click the [3D Measure] icon in the toolbar (large). Alternately, select 3D Measure] from the [Z-Stack] menu. The screen as the same as the XZY slice appears. 5 " XYZ slice procedure" (page 4-23) [3D Measure] dialog box and [Measurement Result] dialog box appear. 3 Select the measurement tool, and perform the measurement. The measurement tool can be selected as follows. Item Between 2-Points Between 2-Ellipses Segment Line Segment Line2 Area Count Garbage Box Delete All Description Measures the distance between two points specified arbitrarily. Measures the distance between the ellipse specified arbitrarily. An ellipse is specified by 5 points. Measures the total length of the segment lines produced by linking arbitrary points. Doubleclicking sets the end point. Measures the total length of the segment lines produced by linking arbitrary ellipses. An ellipse is specified by 5 points. Double-clicking sets the end point. Measures the area of the region specified by the polygon. Double-clicking sets the end point. Measures the number of arbitrary points. Deletes the displayed measuring lines individually. Deletes all displayed measuring lines. The measurement result is displayed on the [Measurement Result] dialog box. 5-30

129 4 Confirm the measurement result. If checking [Calibration] checkbox, the measurement value is displayed with µm etc. If unchecking [Calibration] checkbox, the measurement value is displayed with pixel. To confirm the calibration, click the [Set] button. Point The measurement value cannot be changed from this screen. If changing the calibration, end the 3D measurement, and change the calibration from the screen of 2D scale measurement etc. "5-1 XY Measure" (page 5-2) The changed calibration is not reflected until the 3D measurement screen is regenerated

130 Setting 3D measure You operate the following way in [3D Measure] window. 5 XZ cross section is shown. If mouse wheeling is moved on this display, the Y position is moved. XY cross section is shown. If mouse wheeling is moved on this display, the Z position is moved. YZ cross section is shown. If mouse wheeling is moved on this display, the X position is moved. z y x 5-32

131 Measurement point The measurement points are measured by clicking the inside of the XY cross section, XZ cross section, and YZ cross section. Clicks the measurement points with moving the cross section position by mouse wheel. You cannot change the cross section during the ellipse distance before specifying a ellipse. You cannot change the cross section during area measurement. To zoom the image, click the measurement point with checking [Zoom and place] checkbox. 5 Clicking displays the zoom window of the click position. The color or thickness of the measurement line, as well as the color of the measurement point/label can be changed from [Display Settings]. The measurement result is displayed in [Measurement Result] window. Displaying/non-displaying the measurement result window can be changed by [Display]/[Non-Display] of the measurement results. 5-33

132 5-6 Displaying Capture Information Displaying capture information Displays information including the lens used for captured images, camera settings, filter settings, image processing settings, etc. 1 Click [Photo Information Display] from the [Measure] menu. Reference The information can also be displayed by right-clicking the image in the [Image book] and clicking [Photo Information Display]

133 5-7 Copying Capture Information Copying capture information 1 Click [Copy Capture Information] from the [Measure] menu. 2 Click [Select a file] and select both the [Source image] file and [Destination image] file. 3 Clicking [Copy] saves the capture information from the source file to the destination file

134 MEMO

135 Chapter 6 Hybrid Cell Count This section describes "Hybrid Cell Count", which is used to extract the intensity and color from fluorescent, brightfield, and phase contrast images. 6-1 Overview of Hybrid Cell Count Counting Fluorescent Images Counting Brightfield Images Counting Phase Contrast Images Counting by Setting a Mask Area Macro Cell Count D Cell Count

136 6-1 Overview of Hybrid Cell Count Hybrid cell count consists of the following two functions. Hybrid cell count: Performs processing on a single image. Macro cell count: Performs batch processing on multiple images. 3D Cell Count: Perform the 3D cell count to the Z stack images. Time Series Cell Count: Measure time series changes of the cell count value to the time-lapse images. This section gives an overview of cell count functions. Point The "Hybrid Cell Count BZ-H4C" is required in order to use hybrid cell count. The "Macro Cell Count BZ-H4CM" is required in order to use macro cell count. The "Macro Cell Count BZ-H4C", and the "3D Application BZ-H4R" are required in order to use 3D cell count. The "Macro Cell Count BZ-H4C", and the "Motion Analysis Application BZ-H4K" are required in order to use time series cell count

137 Overview of hybrid cell count Hybrid cell count extracts the selected measurement area using the specified method, and displays the results. Hybrid cell count can be used on fluorescent images, brightfield images, or phase contrast images. A mask area can also be specified. The procedure for hybrid cell count varies as follows depending on whether or not a mask is specified. If a mask is not specified 1 Select the type of image 2 Extract the measurement area 3 Adjust the shape of the extracted area 4 Check the measurement results If a mask is specified (single extraction) 1 Select the type of image 2 Specify the number of extractions 3 Create the mask area 4 Adjust the shape of the mask area 5 Extract the measurement area 6 Adjust the shape of the extracted area 7 Check the measurement results 6 If a mask is specified (double extraction) 1 Select the type of image 2 Specify the number of extractions 3 Create the mask area 4 Adjust the shape of the mask area 5 Extract the measurement area (1st) 6 Adjust the shape of the extracted area (1st) 7 Extract the measurement area (2nd) 8 Adjust the shape of the extracted area (2nd) 9 Check the measurement results Point The images that can be read by hybrid cell count are within 4080 x 3072 pixels (one side is 4080 pixels or less). 6-3

138 Overview of macro cell count Macro cell count is a function that batch executes the same processing on multiple files. The details of the processing executed in hybrid cell count need to be saved in a conditions file in advance. "Save conditions" (page 6-41) If the specified conditions are not suitable for the images being processed, the following actions can be performed. It can be edited again in hybrid cell count after executing macro cell count. The positions of the specified areas can be moved in the macro cell count preview screen. If macro cell count is executed on merged images, the region where the images overlap can be excluded from the measurement. Point The images that can be read by macro cell count are within 4080 x 3072 pixels (one side is 4080 pixels or less). Overview of 3D cell count 6 The 3D cell count function performs a cell count in 3D to a Z stack image. Measure results in 3D such as the volume or surface area of the object. 3D Cell Count can be applied to fluorescence images. Overview of time series cell count The time series cell count function measures time series changes of cell quantification to time-lapse images. It can be applied to fluorescence images, bright field images, and phase contrast images. The time series cell count is explained in the chapter of the time-lapse. "8-8 Time Series Cell Count" (page 8-15) 6-4

139 6-2 Counting Fluorescent Images This chapter describes how to quantify fluorescent images. Selecting the image type The procedure for selecting the image type is as follows. 1 Click the [Hybrid Cell Count] icon in the toolbar (large). Alternately, select [Hybrid Cell Count] from the [Measure] menu. The [Hybrid cell count] dialog box appears Click [Fluorescence] in [Select image type]. Reference To specify a mask, specify it in [Specify target area]. "6-5 Counting by Setting a Mask Area" (page 6-48) 3 Click the [Start] button. Hybrid cell count starts and the [Hybrid cell count] window appears. 6-5

140 Specifying the area The area to extract can be specified by any of brightness extraction (simple mode), brightness extraction (detail mode), brightness extraction (manual pick), colocalization, or cell separation methods. [Hybrid cell count] window when specifying the area Hybrid cell count operating procedure Displays the operating procedure and the current stage of hybrid cell count. 2 Change zoom Adjusts the zoom for displaying images. Select the [Zoom] check box and adjust the zoom using the buttons or from the pull-down menu. 3 RGB Select Color When a fluorescent image or brightfield image is selected, the image can be displayed in each of the individual color components of red, green, and blue. This is the same as the RGB split display in the main screen. "2-1 Names and Functions of the Main Screen" (page 2-2) Reference If you switch to separated display from brightness extraction (simple mode), the threshold value is obtained automatically. This is not available for colocalization. 4 Level correction Adjusts the shadows and highlights in the brightness at the step of specifying the area. This is the same as the level correction in the main screen. "2-1 Names and Functions of the Main Screen" (page 2-2) Reference This is not displayed for colocalization. 6-6

141 5 Image display area Displays the image that is being processed. The content that is displayed differs depending on the settings in the display details area. 6 Extraction method icons When a fluorescent image or brightfield image is selected, the methods for selecting the extraction area are displayed as icons. When these are clicked, the content displayed in the extraction selection area changes. Reference These are not displayed when a phase contrast image is selected. 7 Extraction selection area Specifies the extraction area. The content that is displayed differs depending on the extraction method. 8 [Specify area] button Click this button to display the [Specify area] dialog box which is used to specify the extraction area. Reference This is not displayed when a mask is set. 9 Display details area Specifies in detail the content to display for the image being processed. 10 [Next] button Advances to the adjust area shape step

142 Setting fields in the [Specify area] dialog box The setting fields in the [Specify area] dialog box that is displayed when the [Specify area] button is clicked are as follows [Area specified] button icons The extraction area is specified using the following methods. [Rectangle]: Drag a rectangle to extract. [Ellipse]: Drag an ellipse to extract. [Polygon]: Enclose the area to measure by sequentially clicking each vertex point and double click to indicate the final vertex point. To specify multiple areas, repeat this procedure. [Free line]: Drag the mouse around the perimeter of the area to extract. Reference If you right-click while specifying the vertices of the polygon, the last point specified is cleared. If the measurement lines overlap while specifying the area using polygons or circles, the regions are combined as shown in the following diagram. Area 1 Area 2 2 [Delete] button icon After clicking this button, move the mouse pointer inside a configured extraction area to delete the extraction area. 3 [Del All] button icon Click this button to delete all of the extraction areas. 4 [Copy] button Click this button while a rectangle or ellipse object is selected to create a copy of the selected object. 6-8

143 5 [Edit] button Click this button while a rectangle or ellipse object is selected to display the [Edit] dialog box which allows you to edit the position and size of the object. The display units in the [Edit] dialog box can be converted from pixels to microns by selecting the [Calibration] check box. Setting fields in the display details area The setting fields in the display details area are as follows [Display Results] check box When this check box is selected, the extraction results are displayed in the image display area. 6 2 Transparency The transparency of the extraction area is controlled by using the slider, [ ][ ] buttons, or by directly entering a numeric value. The extraction becomes darker as the value gets closer to zero, and gets lighter as it gets closer to Extract Color Specifies the display color for the extracted area. 4 Specified Area Specify the display color of the specified area. 5 Highlight When a fluorescent image or brightfield image is selected, specifies the display color of highlights. Reference This is not displayed for brightness extraction (simple mode) or cell separation. 6-9

144 Specifying the area using brightness extraction (simple mode) This section gives the procedure for selecting the extraction area by specifying the brightness. 1 Click the [Brightness] icon in the [Hybrid cell count] window and select [Simple mode]. The extraction selection area switches to [Extract by Brightness (Simple)] mode Adjust the threshold value by using the slider, [ ][ ] buttons, or by directly entering a numeric value. Regions that are brighter than the specified threshold value are extracted. Reference To return to the threshold value when the hybrid cell count was started, click the [Auto] button. 3 To configure the diffusion filter, select the [Smooth Edges] check box. 4 To configure elimination of brightness variation, select the [Uniform Brightness] check box. 3 5 If setting the specifying area, click the [Specify area] button and specify an area. "Setting fields in the [Specify area] dialog box" (page 6-8) 6 Click the [Next] button. Advance to the adjust area shape step. 6-10

145 Specifying the area using brightness extraction (detail mode) This section gives the procedure for selecting the extraction area by specifying the brightness in a histogram. 1 Click the [Brightness] icon in the [Hybrid cell count] window and select [Detail mode]. The extraction selection area switches to [Extract by Brightness (Advanced)] mode Drag the mouse over the extraction range in the histogram in the [Extract by Brightness (Advanced)] area. The cells in the dragged range are displayed highlighted in the image display area. 3 4 Reference In the histogram, the X axis represents the brightness and the Y axis represents the number of pixels that have each brightness value. Once the extraction range has been selected by dragging, it can be adjusted by using the [ ][ ] buttons or by directly entering numeric values in [Range]. Clicking the [Auto] button sets the values to the optimal brightness range for extraction. 3 To limit the extraction size, select the [Specify Size Limits] check box. 4 Click the [Add area] button. If the [Specify Size Limits] check box was selected in step 3, the [Extraction size limits] dialog box is displayed. Proceed to Step 5. If the [Specify Size Limits] check box was not selected in Step 3, cells in the highlight color change to the extraction color, and are fixed as the extraction area. Proceed to Step

146 5 When the [Extraction size limits] dialog box is displayed, click a cell in the highlight color. The clicked cell changes to the extraction color, and the lower and upper limits on the extraction area of the cell are displayed in [Size of extracted area]. If you want to adjust the extraction size, use the [ ][ ] buttons or directly enter a numeric value. If you then click another cell, the maximum value and minimum value of area from among the multiple clicked cells are displayed. The cells within the range between that maximum value and minimum value also change to the extracted color. Reference To display the previous value, click the [Last value] button. To undo your last action, click the [Undo] button. To clear the settings, click the [Clear] button. 6 6 Click the [OK] button. You are returned to the [Hybrid cell count] window. Reference To extract multiple areas, repeat Steps 2 to 6. To undo your last action, click the [Undo] button. To return to the initial state, click the [Reset] button. 6 7 If setting the specifying area, click the [Specify area] button and specify an area.. "Setting fields in the [Specify area] dialog box" (page 6-8) 8 If setting the filter, click the [Filter settings] button. The [Filter settings] dialog box is displayed. Reference The [Filter settings] dialog box always starts in the new state with the content not depending on the values from the previous time it was displayed. For example, if you set the parameter to 10 in noise removal and then close the dialog box, the parameter returns to zero when you open the dialog box again. At this time, the image displayed in the image display area is the image with the parameter of 10 applied. If the parameter is set to 10 in this state, another parameter of 10 is applied again to the current image to which the parameter of 10 has already been configured. 6-12

147 In the [Filter] pull-down menu, select the filter type from [Uniform Brightness], [Noise Elimination], [Noise removal (strong)], and [Haze reduction]. Reference When the [Preview extraction results] check box is selected, the extraction results with the filter applied are displayed in the image display area. If the filter is already configured, click the [Undo all] button to discard all of the settings and close the [Filter settings] dialog box. 10 Adjust each of the parameters by using the slider, [ ][ ] buttons, or by directly entering a numeric value Click the [OK] button. You are returned to the [Hybrid cell count] window. 12 Click the [Next] button. Advance to the adjust area shape step

148 Specifying the area using brightness extraction (manual pick) This section gives the procedure for specifying a point or area to use as the brightness reference in order to extract areas with the same brightness range. Point The process of specifying the area using brightness extraction (manual pick) cannot be reproduced using macro cell count. To specify by dragging The following is the procedure for extracting the cell with the largest area from within the area specified by dragging. 1 Click the [Brightness] icon in the [Hybrid cell count] window and select [Manual pick]. The extraction selection area switches to [Extract by Brightness (Manual)] mode Click the [Drag] tab. 3 Drag the mouse over a rectangular region in the image display area to specify the measurement area. The area you selected by dragging changes to green, and the cell with the largest area within the extraction area is displayed in the highlight color. Reference To clear the extraction area, move the mouse pointer outside the extraction area and click or right-click. 4 Select the [Bright] or [Dark] radio button in [Extraction Setting]. [Bright]: Extracts the bright regions. [Dark]: Extracts the dark regions. 5 Adjust the threshold value by using the slider, [ ][ ] buttons, or by directly entering a numeric value. Reference To return to the threshold value when the hybrid cell count was started, click the [Auto] button. 6-14

149 6 To fill holes in the image, select the [Fill holes] check box. If there are any holes in the extracted image, all of the holes in the image are filled. 7 Select the outline strength from [Coarse], [Medium], and [Fine] in the [Edge Outline] pull-down menu Reference 8 Click the [Add area] button. The weaker the outline, the stronger the effect of the diffusion filter works. The affected cells change to the extraction color. Reference To undo your last action, click the [Undo] button. Note that the [Undo] button becomes inactive if you go back from the adjust area shape step to the specify area step. To return to the initial state, click the [Reset] button. 9 Click the [Next] button. Advance to the adjust area shape step

150 To specify by clicking The following is the procedure for clicking a point within the area you want to extract. This extracts the area within the range specified in the brightness range using the selected point as a reference. 1 Click the [Brightness] icon in the [Hybrid cell count] window and select [Manual pick]. The extraction selection area switches to [Extract by Brightness (Manual)] mode Click the [Click] tab. 3 Click part of the area you want to extract in the image display area. The area within the specified brightness range centered on the clicked point is displayed highlighted. 4 Adjust the brightness range by using the slider, [ ][ ] buttons, or by directly entering a numeric value. 5 Select the outline strength from [Coarse], [Medium], and [Fine] in the [Edge Outline] pull-down menu. 6 Click the [Add area] button. The affected area changes to the extraction color. Reference To extract multiple areas, repeat Steps 3 to 6. To undo your last action, click the [Undo] button. Note that the [Undo] button becomes inactive if you go back from the adjust area shape step to the specify area step. To return to the initial state, click the [Reset] button. 7 Click the [Next] button. Advance to the adjust area shape step. 6-16

151 Specifying the area using colocalization This section gives the procedure for selecting the extraction area from a scattergram by selecting 2 colors to analyze. 1 Click the [Colocalization] icon in the [Hybrid cell count] window. The extraction selection area switches to [Colocalization] mode. 2 Select the combination of colors you want to analyze from [R+G], [G+B], and [R+B] in [Color Pair]. 2 3 Click the [Settings] button. The [Brightness Range] dialog box is displayed Scattergram Displays a scattergram of the colors you selected in Step 2. 2 [Delete] button After clicking this button, click the mouse pointer inside a configured extraction area in the scattergram to delete the extraction area. 3 [Del all] button Click this button to delete all of the extraction areas configured in the scattergram. 4 [Save image] button Click this button to save an image of the scattergram in a bmp format file. 5 [Output ranges] button Click this button to save the state of the scattergram in a CSV format file. 6 4 Drag the mouse over the areas you want to extract in the scattergram. The area selected by dragging is displayed highlighted in the image display area. When you finish dragging and release the mouse button, the highlight color changes to the extraction color. Reference Multiple extraction areas can be selected at once. 5 Click the [OK] button. You are returned to the [Hybrid cell count] window. 6-17

152 6 Click the [Specify area] button and specify the area. "Setting fields in the [Specify area] dialog box" (page 6-8) 7 Click the [Next] button. Advance to the adjust area shape step. Specifying the area using cell separation This section gives the procedure for selecting the extraction area by separating the cells within the specified area according to a given degree of separation. 1 Click the [Cell separation] icon in the [Hybrid cell count] window. The extraction selection area switches to [Cell separation] mode Set the degree of separation by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The larger the degree of separation, the more finely the areas of the cells are separated. 3 Adjust the threshold value by using the [Brightness Threshold] slider, [ ][ ] buttons, or by directly entering a numeric value. Reference To return to the threshold value when the hybrid cell count was started, click the [Auto] button. 4 To eliminate variations in brightness, select the [Uniform Brightness] check box. 5 If setting the specifying area, click the [Specify area] button and specify an area. "Setting fields in the [Specify area] dialog box" (page 6-8) 6 Click the [Next] button. Advance to the adjust area shape step. 6-18

153 Adjusting the extraction area shape This section describes how to adjust the extraction area by using standard methods or advanced methods. [Hybrid cell count] window when adjusting the area Hybrid cell count operating procedure Displays the operating procedure and the current stage of hybrid cell count. 2 Change zoom Adjusts the zoom for displaying images. Select the [Zoom] check box and adjust the zoom using the buttons or from the pull-down menu. 3 Image display area Displays the image that is being processed. The content that is displayed differs depending on the settings in the display details area. 4 Adjust Ext. Area This area is used to adjust the shape of the extraction area. The shape adjustment methods can be switched between the [Normal] and [Details] tabs. 5 Display details area Specifies in detail the content to display for the image being processed. 6 [Back] button Returns to the specify area step. 7 [Next] button Advances to the check results step. 6-19

154 Setting fields in the [Normal] tab of the shape adjustment area 1 [Remove unwanted areas] button Specifies and removes unwanted areas. 2 [Fill] button 1 This fills any holes of less than a given area in cells in either the entire screen or the specified area. 3 [Fill Cracks] button 2 This fills any missing parts in cells in either the entire screen or the specified area. 4 [Separate areas] button This separates any cells in either the entire screen or the specified area according to a given degree of separation. This is more effective when fill processing is performed in advance. 5 [Undo] button This command will undo the most recent command. Note that the [Undo] button becomes inactive if you go back from the check result step to the adjust area shape step. 6 [Reset] button This resets all of the shape adjustment processing. 6 Setting fields in the [Details] tab of the shape adjustment area [Select on histogram] button Removes the area specified in the histogram. 2 [Select on image] button Removes the area specified in the image display area. 3 [Correct area] button icons These buttons execute various types of area corrections. 4 [Undo] button This command will undo the most recent command. Note that the [Undo] button becomes inactive if you go back from the check result step to the adjust area shape step. 5 [Reset] button This resets all of the shape adjustment processing

155 Setting fields in the display details area [Display Results] check box When this check box is selected, the extraction results are displayed in the image display area. 2 Transparency The transparency of the extraction area is controlled by using the slider, [ ][ ] buttons, or by directly entering a numeric value. The extraction becomes darker as the value gets closer to zero, and gets lighter as it gets closer to Extract Color Specifies the display color for the extracted area. 4 Specified Area Specify the display color of the specified area. 6 5 Highlight Specifies the display color for highlights. 6 Details Click this button to display the [Details] dialog box which is used to select the following items. Display color mode ([Monochrome], [Random Clr]) Background color ([OFF], [Black], [White]) Color and thickness of outlines ([OFF], [Thin], [Thick]) Reference The colors and fonts of labels cannot be selected while adjusting the shape of the extraction area. 6-21

156 Standard methods for adjusting the shape Remove unwanted areas Specifies and removes unwanted areas. 1 Click the [Remove unwanted areas] button in the [Normal] tab in the shape adjustment area. The [Remove areas] dialog box appears [Area] tab Select to specify the unwanted areas by area. 2 Area Click or drag the mouse over the areas you want to remove in the image display area. The maximum area of the cells within or that touch the specified frame is displayed, and areas where the area is less than this are displayed highlighted as removal areas. The area can be adjusted by using the slider, [ ][ ] buttons, or by directly entering a numeric value. 3 [Remove areas at screen edges] button Click to remove cells that touch the edge of the screen. 4 [Shape (circle)] tab Select to specify the cells to remove by shape (circle). 5 Histogram area Specify the range of areas to leave by dragging the mouse over the histogram. Areas other than the specified range are displayed highlighted as areas to remove. 6 Upper and lower limits Displays the upper limit and lower limit of the range specified in the histogram. The upper limit and lower limit can be adjusted by using the slider, [ ][ ] buttons, or by directly entering a numeric value

157 2 Click the [Area] tab or [Shape (circle)] tab. 3 Specify unwanted areas. If you selected the [Area] tab in Step 2: Click or drag the mouse over the areas you want to remove in the image display area. If you selected the [Shape (circle)] tab in Step 2: Drag the mouse over the area you want to leave in the histogram. Fill 4 Click the [OK] button. The specified areas are removed and you are returned to the [Hybrid cell count] window. This fills any holes of less than a given area in cells in either the entire screen or the specified area. 2 1 Click the [Fill] button in the [Normal] tab in the shape adjustment area. The [Fill holes] dialog box appears. 2 To specify the area of holes to fill: Select the [Manual] radio button and specify the area. To fill all holes: Select the [Auto] radio button. The affected area is displayed highlighted. 6 Reference When the [Manual] radio button is selected, you can also specify holes by clicking or dragging the mouse over any holes in the image display area. 3 3 Click the [OK] button. The hole fill processing is executed and you are returned to the [Hybrid cell count] window. 6-23

158 Fill cracks This fills any missing parts in cells in the specified area. 1 Click the [Fill Cracks] button in the [Normal] tab in the shape adjustment area. The [Fill cracks] dialog box appears. 2 Adjust the degree of filling by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The affected area is displayed highlighted. 2 3 Specify the target for filling missing parts by using the [Target] slider, [ ][ ] buttons, or by directly entering a numeric value. 3 4 Click the [OK] button. Fill missing parts processing is carried out and the target cells change to the extraction color. You are returned to the [Hybrid cell count] window. 4 6 Separate areas This separates any cells in either the entire screen or the specified area according to a given degree of separation. This is more effective when fill processing is performed in advance. 1 Click the [Separate areas] button in the [Normal] tab in the shape adjustment area. The [Separate] dialog box appears. 2 2 Specify the degree of separation by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The cells within the area are separated by color according to the specified degree of separation. Reference The larger the degree of separation, the more finely the areas of the cells are separated. 3 3 Specify the separation target by using the [Target] slider, [ ][ ] buttons, or by directly entering a numeric value. 4 4 Click the [OK] button. Separation processing is carried out and each respective color will change to the extraction color. You are returned to the [Hybrid cell count] window. 6-24

159 Advanced methods for adjusting the shape Select on histogram Click the [Select on histogram] button in the [Details] tab in the shape adjustment area. The [Select on histogram] dialog box is displayed. 1 Item Select the item to display in the histogram from [Area], [Perimeter], [Major axis], [Minor axis (width)], [Ratio of major to minor axis], and [Circularity]. 2 Histogram area Specify the range of areas to leave by dragging the mouse over the histogram. Areas other than the specified range are displayed highlighted as areas to remove. 3 Upper and lower limits Displays the upper limit and lower limit of the range specified in the histogram. The upper limit and lower limit can be adjusted by using the slider, [ ][ ] buttons, or by directly entering a numeric value. 4 [Lower] and [Upper] buttons Selects the upper limit or lower limit of the area to remove. For example, click the [Upper] button and then click any cell in the image display area to remove areas above that cell. 5 [Remove areas at screen edges] button Remove cells that touch the edge of the screen. 6 2 Specify unwanted areas. 3 Click the [OK] button. The specified areas are removed and you are returned to the [Hybrid cell count] window. 6-25

160 Select on image Removes the specified area from the count. 1 Click the [Select on image] button in the [Details] tab in the shape adjustment area. The [Select on image] message is displayed. 2 Select the areas you want to remove by clicking or dragging the mouse over the image display area. The selected area is displayed highlighted as an area to remove. Reference Click a selected area again to clear the selection. 3 Click the [OK] button. The specified areas are removed and you are returned to the [Hybrid cell count] window

161 Fill cracks This fills any missing parts of cells in either the entire screen or the specified area. 1 Click the [Fill cracks] button in the [Details] tab in the shape adjustment area. The [Fill cracks] dialog box appears. 2 Adjust the degree of filling missing parts by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The affected cells are displayed highlighted To batch specify the areas to process: Specify the target for filling missing parts by using the [Target] slider, [ ][ ] buttons, or by directly entering a numeric value. Proceed to Step 6. To individually specify the areas to process: Click the [Specify individually] button. Proceed to Step 4. 4 Select the areas to process by clicking or dragging the mouse over the image display area while the [Specify individually] message is displayed. The affected cells change to the extraction color. Reference Click a selected area again to clear the selection. Multiple areas can be selected at once. 6 5 Click the [OK] button in the [Specify individually] message. The specified areas are set and you are returned to the [Fill cracks] dialog box. 5 Reference At this time, the [Specify individually] button in the [Fill cracks] dialog box becomes displayed as the [Select All] button. Click the [Select All] button to clear the selected areas. 6 Click the [OK] button in the [Fill cracks] dialog box. The missing part fill processing is executed and you are returned to the [Hybrid cell count] window. 6-27

162 Deburring This deburrs any protrusions from cells in either the entire screen or the specified area. 1 Click the [Deburring] button in the [Details] tab in the shape adjustment area. The [Deburring] dialog box appears. 2 Adjust the degree of deburr by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The affected area is displayed highlighted To batch specify the areas to process: Specify the deburr target by using the [Target] slider, [ ][ ] buttons, or by directly entering a numeric value. Proceed to Step 6. To individually specify the areas to process: Click the [Specify individually] button. Proceed to Step Select the areas to process by clicking or dragging the mouse over the image display area while the [Specify individually] message is displayed. The affected cells change to the extraction color. Reference Click a selected area again to clear the selection. Multiple areas can be selected at once. 5 Click the [OK] button in the [Specify individually] message. The specified areas are set and you are returned to the [Deburring] dialog box. 5 Reference At this time, the [Specify individually] button in the [Deburring] dialog box becomes displayed as the [Select All] button. Click the [Select All] button to clear the selected areas. 6 Click the [OK] button in the [Deburring] dialog box. The deburr processing is executed and you are returned to the [Hybrid cell count] window. 6-28

163 Fill holes This fills any holes of less than a given area in cells in either the entire screen or the specified area. 1 Click the [Fill holes] button in the [Details] tab in the shape adjustment area. The [Fill holes] dialog box appears. 2 2 To specify the area of holes to fill: Select the [Manual] radio button and specify the area. To fill all holes: Select the [Auto] radio button. The affected area is displayed highlighted. Reference When the [Manual] radio button is selected, you can also specify holes by clicking or dragging the mouse over any holes in the image display area. 3 3 Click the [OK] button. The hole fill processing is executed and you are returned to the [Hybrid cell count] window

164 Separate This separates any cells in either the entire screen or the specified area according to a given degree of separation. This is more effective when fill processing is performed in advance. 1 Click the [Separate] button in the [Details] tab in the shape adjustment area. The [Separate] dialog box appears. 2 2 Adjust the degree of separation by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The cells within the area are separated by color according to the specified degree of separation. Reference The larger the degree of separation, the more finely the areas of the cells are separated To batch specify the areas to process: Specify the target for parts to be separated by using the [Target] slider, [ ][ ] buttons, or by directly entering a numeric value. Proceed to Step 6. To individually specify the areas to process: Click the [Specify individually] button. Proceed to Step 4. 4 Select the areas to process by clicking or dragging the mouse over the image display area while the [Specify individually] message is displayed. The affected cells change to the extraction color. Reference Click a selected area again to clear the selection. Multiple areas can be selected at once. 5 Click the [OK] button in the [Specify individually] message. The specified areas are set and you are returned to the [Separate] dialog box. 5 Reference At this time, the [Specify individually] button in the [Separate] dialog box becomes displayed as the [Select All] button. Click the [Select All] button to clear the selected areas. 6 Click the [OK] button in the [Separate] dialog box. Separation processing is carried out and each respective color will change to the extraction color. You are returned to the [Hybrid cell count] window. 6-30

165 Merge This combines cells in either the entire screen or the specified area. 1 Click the [Merge] button in the [Details] tab in the shape adjustment area. The [Merge] dialog box appears. 2 2 Specify the degree of conjoining by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The affected area is displayed highlighted. If setting an area for process individually: proceed to Step 3. If setting an area for process collectively: proceed to Step 6. 3 Click the [Specify individually] button. 3 4 Select the area for process by clicking or dragging in the image display area with the [Specify individually] message displayed. The affected cells change to the extraction color. Reference Click a selected area again to clear the selection. Multiple areas can be selected at once. 5 Click the [OK] button in the [Specify individually] message. The specified areas are set and you are returned to the [Merge] dialog box. 6 5 Reference At this time, the [Specify individually] button in the [Merge] dialog box becomes displayed as the [Select All] button. Click the [Select All] button to clear the selected areas. 6 Click the [OK] button in the [Merge] dialog box. The conjoin processing is executed and you are returned to the [Hybrid cell count] window. Invert This inverts the extraction area. 1 Click [Invert] in the [Details] tab in the shape adjustment area. The extraction area is inverted. 6-31

166 Expand This expands cells in either the entire screen or the specified area. 1 Click the [Expand] button in the [Details] tab in the shape adjustment area. The [Expand] dialog box appears. 2 2 Adjust the degree of expansion by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The affected area is displayed highlighted. If setting an area for process individually: proceed to Step 3. If setting an area for process collectively: proceed to Step 6. 3 Click the [Specify individually] button. 3 4 Select the area for process by clicking or dragging in the image display area with the [Specify individually] message displayed. The affected cells change to the extraction color. 6 Reference Click a selected area again to clear the selection. Multiple areas can be selected at once. 5 Click the [OK] button in the [Specify individually] message. The specified areas are set and you are returned to the [Expand] dialog box. 5 Reference At this time, the [Specify individually] button in the [Expand] dialog box becomes displayed as the [Select All] button. Click the [Select All] button to clear the selected areas. 6 Click the [OK] button in the [Expand] dialog box. The expand processing is executed and you are returned to the [Hybrid cell count] window. 6-32

167 Shrink This shrinks cells in either the entire screen or the specified area. 1 Click the [Shrink] button in the [Details] tab in the shape adjustment area. The [Shrink] dialog box appears. 2 2 Adjust the degree of shrinkage by using the [Value] slider, [ ][ ] buttons, or by directly entering a numeric value. The affected area is displayed highlighted. If setting an area for process individually: proceed to Step 3. If setting an area for process collectively: proceed to Step 6. 3 Click the [Specify individually] button. 3 4 Select the area for process by clicking or dragging in the image display area with the [Specify individually] message displayed. The affected cells change to the extraction color. Reference Click a selected area again to clear the selection. Multiple areas can be selected at once. 5 Click the [OK] button in the [Specify individually] message. The specified areas are set and you are returned to the [Shrink] dialog box. 6 5 Reference At this time, the [Specify individually] button in the [Shrink] dialog box becomes displayed as the [Select All] button. Click the [Select All] button to clear the selected areas. 6 Click the [OK] button in the [Shrink] dialog box. The shrink processing is executed and you are returned to the [Hybrid cell count] window. 6-33

168 Fine Edit This allows manual editing of the specified area. Point Processing after editing the area cannot be reproduced using macro cell count Click the [Fine Edit] button in the [Details] tab in the shape adjustment area. The [Fine Edit] dialog box appears. 1 Editing Method This specifies the method for editing the area from [Add] and [Delete]. 2 Edit button icons The area can be edited using the following methods. [Line]: Adds or deletes an area in a straight line from the starting point to the ending point of dragging the mouse. [Free line]: Adds or deletes an area along a free path following the mouse from the starting point to the ending point of dragging the mouse. [Fill]: Adds or deletes a rectangular area with opposing corners at the starting point and ending point of dragging the mouse. 3 [Undo] button icon This command will undo the most recent command. 4 Size Specifies the line width for the [Line] button icon and [Free line] button icon. 2 Select the [Add] radio button or [Delete] radio button. 3 Click the [Line] button icon, [Free line] button icon, or [Fill] button icon, and edit the area in the image display area. 4 Click the [OK] button. Edit area processing is carried out and the affected areas change to the extraction color. You are returned to the [Hybrid cell count] window. 6-34

169 Displaying and saving measurement results In hybrid cell count, the measurement results can be displayed using the following methods. Measurement results list display Displays the measurement items and measurement results. Histogram display Displays the measurement results in a histogram. Furthermore, the measurement results can be saved using the following methods. Save display image Saves the image in the image display area. Save conditions Saves the specified area and adjust area shape settings as a conditions file. This chapter describes each of the methods for displaying and saving measurement results

170 [Hybrid cell count] window when checking the results Hybrid cell count operating procedure display area Displays the operating procedure and the current stage of hybrid cell count Change zoom Adjusts the zoom for displaying images. Select the [Zoom] check box and adjust the zoom using the buttons or from the pull-down menu. 3 Image display area Displays the image that is being processed. The content that is displayed differs depending on the settings in the display details area. 4 Results View area Specifies how the measurement results are displayed. 5 Display details area Specifies in detail the content to display for the image being processed. 6 [Back] button Returns to the adjust area shape step. 7 [Exit] button Exits the hybrid cell count. 6-36

171 Setting fields in the display details area [Display Results] check box When this check box is selected, the extraction results are displayed in the image display area. 2 Transparency The transparency of the extraction area is controlled by using the slider, [ ][ ] buttons, or by directly entering a numeric value. The extraction area becomes darker as the value gets closer to zero, and gets lighter as it gets closer to Extract color Specifies the display color for the extracted area. 4 [Display Labels] check box When this check box is selected, the labels are displayed. 6 5 Highlight Specifies the display color for highlights. 6 [Details] button Click this button to display the [Details] dialog box which is used to select the following items. Display color mode ([Monochrome], [Random Clr]) Background color ([OFF], [Black], [White]) Color and thickness of outlines ([OFF], [Thin], [Thick]) Label color and font 6-37

172 Display measurement results Measure results list 1 Click the [Measure Result] button in the results view area. The [Measure Result] dialog box appears. Click the [Save Results] button to save the measurement results in a CSV format file. When a row is selected, the selected area is displayed highlighted in the image display area Count, total (area), and area ratio (specified area) Displays the number, total (area), and area ratio of the measured areas. 2 Measurement items and measurement results Displays the measurement items and measurement results. The items that are displayed initially are as follows. Item Description No. Area Perimeter Major axis Minor axis (width) Brightness (integration) Brightness (maximum) Brightness (minimum) Brightness (mean) Displays the label associated with the measurement area. Displays the area of the measurement area. Displays the perimeter of the measurement area. Displays the maximum length between any two points that lie internal circumference of the measurement area. Displays the distance between two parallel lines that encompass the particle and are parallel to the line along the major axis. Displays the value of multiplying the area of the measurement area by the mean brightness before extraction. This is effective for finding the amount of expression during fluorescence observation. Displays the maximum brightness value in the measurement area. Displays the minimum brightness value in the measurement area. Displays the mean brightness value in the measurement area. 3 Measurement result statistics Displays statistics (average, standard deviation, maximum, minimum, total) for each of the measurement items. 6-38

173 4 [Measurement items] button Click this button to bring up the [Measurement item setting] dialog box. The measurement items to add to the display in the list of measurement results can be selected from the following. Item Feret diameter (X) Feret diameter (Y) R (integration) R (maximum) R (minimum) R (mean) G (integration) G (maximum) G (minimum) G (mean) B (integration) B (maximum) B (minimum) B (mean) Description Displays the length along the X axis of a rectangle circumscribing the measurement area. Displays the length along the Y axis of a rectangle circumscribing the measurement area. Displays the value of multiplying the area of the measurement area by the intensity of the R component before extraction. This is effective for finding the amount of expression of the R component when a fluorescent image is selected. Displays the maximum intensity of the R component in the measurement area. Displays the minimum intensity of the R component in the measurement area. Displays the mean intensity of the R component in the measurement area. Displays the value of multiplying the area of the measurement area by the intensity of the G component before extraction. This is effective for finding the amount of expression of the G component when a fluorescent image is selected. Displays the maximum intensity of the G component in the measurement area. Displays the minimum intensity of the G component in the measurement area. Displays the mean intensity of the G component in the measurement area. Displays the value of multiplying the area of the measurement area by the intensity of the B component before extraction. This is effective for finding the amount of expression of the B component when a fluorescent image is selected. Displays the maximum intensity of the B component in the measurement area. Displays the minimum intensity of the B component in the measurement area. Displays the mean intensity of the B component in the measurement area. 6 5 Decimal places Set the number of decimal places to display (0 to 3) using the [ ][ ] buttons. 6 [Save Results] button Click this button to save the measurement results in a CSV format file. 7 Number of rows selected Displays the number of rows selected. 6-39

174 Histogram 1 Click the [Histogram] button in the results view area. The [Histogram] dialog box appears X-axis Displays the measurement value. 2 Y-axis Displays the number of areas counted for each measurement value. 3 Measurement item Select the measurement item to display in the histogram from the pull-down menu. The measurement results are displayed in the histogram separately for each measurement item such as area, perimeter, Feret diameter (X/Y), major axis, minor axis (width), and brightness integrated value. The items that can be selected and the content of the items are the same as the [Measurement items] list of measurement items. 4 Data section Set the number of sections to split the X axis into using the [ ][ ] buttons. 5 Number of areas counted When a particular location is clicked or dragged over with the mouse in the histogram, the number of areas counted in the clicked or dragged location is displayed. 6 Decimal places Set the number of decimal places (0 to 3) to display in values displayed as decimals using the [ ][ ] buttons. 7 [Save Results] button Click this button to save the measurement results in a CSV format file. 6-40

175 Save measurement results Save display image This saves the image after extraction. 1 Click the [Save this image] button in the results view area. The [Save As] dialog box is displayed. 2 Specify the [File name], [Save as type], and [Comment]. Reference If you specify JPEG in [Save as type], you can set the quality of the JPEG image. Save conditions 3 Click the [Save] button. This saves the specify area and adjust shape settings as a conditions file (*.mcd). Conditions files are used by macro cell count to perform processing using the same conditions on different images. "6-6 Macro Cell Count" (page 6-59) 1 Click the [Save conditions] button in the results view area. The [Save As] dialog box is displayed. 6 2 Specify the [File name] and then click the [Save] button. 6-41

176 6-3 Counting Brightfield Images This chapter describes how to quantify brightfield images. Selecting the image type The procedure for selecting the image type is as follows. 1 Click the [Hybrid Cell Count] icon in the toolbar (large). Alternately, select [Hybrid Cell Count] from the [Measure] menu. The [Hybrid cell count] dialog box appears Click [Bright field] in [Select image type]. Reference To specify a mask, specify it in [Specify target area]. "6-5 Counting by Setting a Mask Area" (page 6-48) 3 Click the [Start] button. Hybrid cell count starts and the [Hybrid cell count] window appears. 6-42

177 Specifying the area The area to extract can be specified by any of brightness extraction (simple mode), brightness extraction (detail mode), brightness extraction (manual pick), or hue methods. [Hybrid cell count] window when specifying the area The basic display content is the same as when a fluorescent image is selected. "[Hybrid cell count] window when specifying the area" (page 6-6) Specifying the area using brightness extraction (simple mode, detail mode, manual pick) The basic operations are the same as when a fluorescent image is selected. However, the following points differ from when a fluorescent image is selected. Regions that are darker than the specified threshold value are extracted. (When a fluorescent image is selected, regions that are brighter than the specified threshold value are extracted.) "Specifying the area using brightness extraction (simple mode)" (page 6-10), "Specifying the area using brightness extraction (detail mode)" (page 6-11), "Specifying the area using brightness extraction (manual pick)" (page 6-14) Specifying the area using hue This section gives the procedure for setting the range of tolerance for the color to extract by using the color of a point clicked in the image display area as a reference. 6 1 Click the [Hue] icon in the [Hybrid cell count] window. The extraction selection area switches to [Color] mode. 2 Move the mouse pointer over and click the color you want to extract in the image display area. Areas with the same color as the clicked color are displayed highlighted. 3 Adjust the tolerance by using the slider, [ ][ ] buttons, or by directly entering a numeric value. 3 Reference As the tolerance value becomes smaller, the range of colors that are extracted becomes more limited. 6-43

178 Low Color tolerance High Click the [Add area] button. The affected area changes to the extraction color. Reference To extract multiple areas, repeat Steps 2 to 4. This allows parameters to be set individually for each arbitrary color information. To undo your last action, click the [Undo] button. Note that the [Undo] button becomes inactive if go back from the adjust area shape step to the specify area step. To clear the settings, click the [Reset] button. 5 To configure the diffusion filter, select the [Smooth Edges] check box. 6 To configure elimination of brightness variation, select the [Uniform Brightness] check box. 7 If setting the specifying area, click the [Specify area] button and specify an area. "Setting fields in the [Specify area] dialog box" (page 6-8) 8 Click the [Next] button. Advances to the adjust area shape step. 6-44

179 Adjusting the extraction area shape The operations are the same as when a fluorescent image is selected. "Adjusting the extraction area shape" (page 6-19) Displaying and saving measurement results The operations are the same as when a fluorescent image is selected. "Displaying and saving measurement results" (page 6-35)

180 6-4 Counting Phase Contrast Images This chapter describes how to count phase contrast images. Selecting the image type The procedure for selecting the image type is as follows. 1 Click the [Hybrid Cell Count] icon in the toolbar (large). Alternately, select [Hybrid Cell Count] from the [Measure] menu. The [Hybrid cell count] dialog box appears Click [Phase contrast] in [Select image type]. Reference To specify a mask, specify it in [Specify target area]. "6-5 Counting by Setting a Mask Area" (page 6-48) 3 Click the [Start] button. Hybrid cell count starts and the [Hybrid cell count] window appears. 6-46

181 Specifying the area [Hybrid cell count] window when specifying the area The basic display content is the same as when a fluorescent image is selected. "[Hybrid cell count] window when specifying the area" (page 6-6) Specified area 1 Adjust the threshold value by using the slider, [ ][ ] buttons, or by directly entering a numeric value in [Threshold] in the extraction selection area. 1 Reference To return to the threshold value when the hybrid cell count was started, click the [Auto] button. 2 2 To fill holes in the extraction area, select the degree of filling from [OFF], [Weak], and [Strong] in the [Fill Cracks] pull-down menu To separate the cells within the specified area, select the [Separate] check box and adjust the degree of separation by using the slider, [ ][ ] buttons, or by directly entering a numeric value. The cells within the area are separated by color according to the specified degree of separation. 6 Reference The larger the degree of separation, the more finely the areas of the cells are separated. If the threshold is changed while the [Separate] check box is selected, the separation settings are cleared. 4 If setting the specifying area, click [Specify area]. The [Specify area] dialog box is displayed. 5 Specify the area and click the [OK] button. "Setting fields in the [Specify area] dialog box" (page 6-8) 6 Click the [Next] button. Advance to the adjust area shape step. Adjusting the extraction area shape The operations are the same as when a fluorescent image is selected. "Adjusting the extraction area shape" (page 6-19) Displaying and saving measurement results The operations are the same as when a fluorescent image is selected. "Displaying and saving measurement results" (page 6-35) 6-47

182 6-5 Counting by Setting a Mask Area This section explains how to count by setting a mask area in order to analyze overlapping images with different wavelengths and images with different colors. Setting the image type and mask area The procedures for selecting the image type and for setting the mask area are as follows. 1 Click the [Hybrid Cell Count] icon in the toolbar (large). Alternately, select [Hybrid Cell Count] from the [Measure] menu. The [Hybrid cell count] dialog box appears Click [Fluorescence], [Bright field], or [Phase contrast] in [Select image type]. 3 Select the method for setting the mask in [Specify target area]. [OFF]: No mask set. [Single extraction]: Extract by setting a mask and specifying a single color. Select this to extract a single type of cell after setting the mask. [Double extraction]: Extract by setting a mask and specifying two colors. Select this to extract two types of cells after setting the mask. 4 Click the [Start] button. Hybrid cell count starts and the [Hybrid cell count] window appears. Reference If you selected [Fluorescence] in Step 2, the [Separated Color Display] dialog box is displayed before the hybrid cell count starts. Select the color to display separately and click the [OK] button. 6-48

183 [Hybrid cell count] window when a mask is set The following sections in the [Hybrid cell count] window when a mask is set differ from the case where a mask is not set. Hybrid cell count operating procedure Display details area in the check results screen All other areas and buttons are the same as when a mask is not set. The content of the hybrid cell count operating procedure and display details area are as follows. Hybrid cell count operating procedure 6 Display details area 6-49

184 Hybrid cell count operating procedure For one color extraction For two color extraction Specify mask area Specify the mask area. 6 2 Format shape of mask area Adjust the shape of the mask area. 3 Specify extraction area Specify the extraction area. 4 Read another image Read another image when specifying the extraction area. This can be clicked and another file can be read. 5 Format shape of extraction area Adjust the shape of the extraction area. 6 Check results Check the measurement results. 6-50

185 Setting fields in the display details area (check results screen) [Display Results] check box When this check box is selected, the extraction results are displayed in the image display area. 2 Transparency The transparency of the extraction area is controlled by using the slider, [ ][ ] buttons, or by directly entering a numeric value. The extraction area becomes darker as the value gets closer to zero, and gets lighter as it gets closer to Extract Color Specifies the display color for the extracted area. In the case of two color extraction, two buttons for specifying the color are displayed allowing you to specify the colors for each extraction. 6 4 Target area Specifies the display color of the mask area. 5 [Display Labels] check box When this check box is selected, labels are displayed in the image display area. 6 Highlight Specifies the display color for highlights. 7 Details Click this button to display the [Details] dialog box which is used to select the following items. Display color mode ([Monochrome], [Random Clr]) Background color ([OFF], [Black], [White]) Color and thickness of outlines ([OFF], [Thin], [Thick]) Reference The colors and fonts of labels cannot be selected while adjusting the shape of the area. 6-51

186 Specifying the mask area The mask area can be specified by any of brightness extraction (simple mode), brightness extraction (detail mode), brightness extraction (manual pick), colocalization, cell separation, hue, or phase contrast methods. The basic operations are the same as specifying the extraction area in the case where a mask is not set. Reference When a fluorescent image is selected, the [Separated Color Display] dialog box is displayed before you advance to the specify mask area step. Select the color to display separately and click the [OK] button. For details on specifying the area in fluorescent images, "Specifying the area" (page 6-6), for details on specifying the area in brightfield images, "Specifying the area" (page 6-43), for details on specifying the area in phase contrast images, "Specifying the area" (page 6-47) Adjusting the shape of the mask area The basic operations are the same as adjusting the shape of the area in the case where a mask is not set. Reference When a fluorescent image is selected, the [Separated Color Display] dialog box is displayed when you click the [Next] button before you advance to the specify extraction area step. Select the color to display separately and click the [OK] button. "Adjusting the extraction area shape" (page 6-19)

187 Specifying the extraction (1st/2nd) area The operations are the same as specifying the area in the case where a mask is not set. In the case of two color extraction, repeat the same procedure a second time. For details on specifying the area in fluorescent images, "Specifying the area" (page 6-6), for details on specifying the area in brightfield images, "Specifying the area" (page 6-43), for details on specifying the area in phase contrast images, "Specifying the area" (page 6-47) The operation procedure differs only for the case of reading another image when specifying the extraction area. Operation procedure when loading another image Click the button in the hybrid cell count operating procedure Reference If the button is displayed grayed out, you cannot load another image. A message is displayed confirming whether or not to discard the current extraction processing. 2 Click the [OK] button. The [Open] dialog box is displayed. 3 Specify the file and click the [Open] button. The [Hybrid cell count] dialog box appears

188 4 Select the image type and click the [Apply] button. A new image is displayed in the image display area in the [Hybrid cell count] window. Reference If you open an image with a different size from the mask image, the area that matches the size of the smaller of the images is displayed in the image display area with the top left of both images aligned

189 Adjusting the shape of the extraction (1st/2nd) area The operations are the same as adjusting the shape of the area in the case where a mask is not set. In the case of two color extraction, repeat the same procedure a second time. "Adjusting the extraction area shape" (page 6-19) Displaying and saving measurement results The measurement results when a mask is set can be displayed using the following methods the same as when a mask is not set. Measurement results list display Displays the measurement items and measurement results. Histogram display Displays the measurement results in a histogram. Similarly, the measurement results can be saved using the following methods. Save display image Saves the image in the image display area. Save conditions Saves the specify area and adjust area shape settings as a conditions file

190 Display measurement results Measure results list Click the [Measure Result] button in the results view area. The [Measure Result] dialog box appears. Click the [Save Results] button to save the measurement results in a CSV format file [Normal] and [Target Area] tabs The [Target Area] tab is displayed when a mask is set. The content of the [Normal] tab is the same when a mask is not set. "Display measurement results" (page 6-38) 6-56

191 2 Measure results list (when the [Target Area] tab is selected) Displays a list of measurement results. The display items vary depending on whether one color extraction or two color extraction was executed. For one color extraction Item Description No. Area (target area) Count Area (integration) Area ratio Displays the label associated with the mask area. Displays the area of the mask area. Displays the number of extraction areas in the mask area. Displays the area integrated value of the extraction areas in the mask area. Displays the ratio of the total area of the extracted areas to the area of the mask area. For two color extraction Item Description No. Count (1st) Count (2nd) Area (1st integration) Area (2nd integration) Area ratio (1st) Area ratio (2nd) Area (target area) Count Area (integration) Area ratio Displays the label associated with the mask area. Displays the number of 1st extraction areas in the mask area. Displays the number of 2nd extraction areas in the mask area. Displays the area integrated value of 1st extraction areas in the mask area. Displays the area integrated value of 2nd extraction areas in the mask area. Displays the ratio of the total area of the 1st extracted areas to the area of the mask area. Displays the ratio of the total area of the 2nd extracted areas to the area of the mask area. Displays the area of the mask area. Displays the total number of areas extracted 1st and 2nd in the mask area. Displays the total area integrated value of the areas extracted 1st and 2nd in the mask area. Displays the ratio of the total area of the 1st and 2nd extracted areas to the area of the mask area. 6 3 [Measurement] button Clicking this button displays the [Measurement item setting] dialog box. You can select measurement items to display additionally in the measurement results list from the following. Item Perimeter (target area) Major axis (target area) Minor axis (target area width) Description This shows the outer perimeter of the target area. This shows the maximum length of the distance between 2 points on the circumference of the target area. This shows the distance between 2 lines that are parallel to the major axis and just encompass the target area. 6-57

192 6 Item Feret diameter (target area X) Feret diameter (target area Y) Brightness (target area integration) Brightness (target area maximum) Brightness (target area minimum) Brightness (target area mean) R (target area integration) R (target area maximum) R (target area minimum) R (target area mean) G (target area integration) G (target area maximum) G (target area minimum) G (target area mean) B (target area integration) B (target area maximum) B (target area minimum) B (target area mean) Description This shows the length parallel with the X axis of rectangle circumscribed to the target area. This shows the length parallel with the Y axis of rectangle circumscribed to the target area. This shows the value of multiplying the area of the mask area by the mean brightness before extraction. This is effective when acquiring the amount of expression of fluorescence observation. This shows the maximum brightness within the target area. This shows the minimum brightness within the target area. This shows the mean brightness of the target area. This shows the value of multiplying the area of the mask area by the intensity of the R component before extraction. This is effective when acquiring the amount of expression of the R component during fluorescent image selection. This shows the maximum intensity of the R component within the target area. This shows the minimum intensity of the R component within the target area. This shows the mean intensity of the R component within the target area. This shows the value of multiplying the area of the mask area by the intensity of the G component before extraction. This is effective when acquiring the amount of expression of the G component during fluorescent image selection. This shows the maximum intensity of the G component within the target area. This shows the minimum intensity of the G component within the target area. This shows the mean intensity of the G component within the target area. This shows the value of multiplying the area of the mask area by the intensity of the B component before extraction. This is effective when acquiring the amount of expression of the B component during fluorescent image selection. This shows the maximum intensity of the B component within the target area. This shows the minimum intensity of the B component within the target area. This shows the mean intensity of the B component within the target area. 4 Decimal places Set the number of decimal places to display (0 to 3) using the [ ][ ] buttons. 5 [Save Results] button Click this button to save the measurement results in a CSV format file. 6 Number of rows selected Displays the number of rows currently selected. Histogram The display content is the same as the histogram in the case where a mask is not set. "Histogram" (page 6-40) Save measurement results Save display image The operation procedures are the same as saving display images when a mask is not set. "Save display image" (page 6-41) Save conditions The operation procedures are the same as saving conditions when a mask is not set. "Save conditions" (page 6-41) 6-58

193 6-6 Macro Cell Count This section describes "Macro Cell Count", which measures multiple files using the same conditions. The conditions file needs to have been saved using hybrid cell count in advance. Point The process of specifying the area using brightness extraction (manual pick) and the process of editing the area using advanced methods for adjusting the area cannot be reproduced using macro cell count. "Save conditions" (page 6-41) [Macro cell count] window This section describes the [Macro cell count] window that is displayed when macro cell count is started Read conditions file area Specifies and loads the conditions file. You can also check the content of the conditions file in detail. 2 Target files list area Used to add and delete files to execute the macro cell count on. The list also displays the status and processing results of the files. 3 Execute area Executes the macro cell count, displays a preview of the execution results, and displays the measurement results. 4 Save area Saves the execution results. 5 Undo area Selects the file after executing macro cell count and edits it again in hybrid cell count. The file after editing is configured as the macro cell count conditions file. 6-59

194 Details on the target files list area [Add] and [Batch load] buttons Reads the files to execute the macro cell count on. 6 2 [Delete] and [Delete all] buttons Deletes target files. 3 List of target files Displays a list of the target files that have been read. The items that are displayed are as follows. Item Description Name Status Count Area (integration) Brightness (integration) Position correction value (X) Position correction value (Y) Displays the filename. If another file was read using the mask function, the filename of the image for the mask area and the filename of the image for extracting areas are displayed. Displays the status of the file. The items that are displayed are as follows. [Standby]: Macro cell count can be executed. [XX-Complete]: Macro cell count has finished executing. The "XX" indicates the number of times macro cell count has been executed. For example, if the 2nd macro cell count execution has finished, [02-Complete] is displayed. [Error]: Executing macro cell count exited with an error. Blank: No file is selected (before executing macro cell count). Displays the count results after executing the macro cell count. If the mask area was set to [Single extraction] or [Double extraction], the count results for each extraction area are displayed. Displays the area integrated value after executing the macro cell count. If the mask area was set to [Single extraction] or [Double extraction], the area of the mask area is also displayed. Displays the brightness integrated value after executing the macro cell count. Displays the position of the extraction area. The position registered in the conditions file is zero pixels. Displays the position of the extraction area. The position registered in the conditions file is zero pixels. Reference After the macro cell count has finished executing, the target files are sorted in ascending or descending order each time an item is clicked. 6-60

195 4 Statistics results display Displays the statistics results after executing the macro cell count. The display items vary as follows depending on whether processing with a mask set was specified as the conditions file. If the mask area setting was [OFF] Count, area (integration), brightness (integration), position correction value (X), position correction value (Y) If the mask area setting was [Single extraction] Count (mask), count (1st), area (mask), area (1st accumulation), area ratio (1st), and brightness (1st accumulation) If the mask area setting was [Double extraction] Count (mask), count (1st), count (2nd), area (mask), area (1st accumulation), area (2nd accumulation), area ratio (1st), and area ratio (2nd) 5 [Select all] and [Clear all] buttons Selects or clears all of the check boxes in the target file list. When a check box is selected, macro cell count is executed on the file. 6 Decimal places Set the number of decimal places to display (0 to 3) using the [ ][ ] buttons

196 Reading the conditions file The procedure for reading the macro cell count conditions file is as follows. Reference The conditions file needs to have been saved using hybrid cell count in advance. "Save conditions" (page 6-41) 1 1 Click the [Open] button in the read conditions file area. The [Open] dialog box is displayed Specify the conditions file (*.mcd) and click the [Open] button. The filename and a thumbnail of the conditions file are displayed in the read conditions file area. Reference If a conditions file with different mask settings is read when a conditions file and target files have already been read, all of the files in the target file list are discarded. 3 To check the details of the read conditions file, click the [details] button. The [Details] dialog box appears. 6-62

197 Reading the target files The procedure for reading the target files to execute macro cell count on is as follows. 1 1 Click the [Add] button or the [Batch load] button in the target file list area. If you click the [Add] button, the [Open] dialog box is displayed, and select the image to be added. If you click the [Batch load] button, the [Open] dialog box is displayed, and select the group capture information file (*.gci). Reference You can also open files by dragging them into the target file list. 2 Select the file or folder and click the [OK] button. The read files are displayed in the target file list. Reference If you have specified a conditions file where you set a mask and performed hybrid cell count on multiple images, read all of the images that are used. In this case, these images are treated as a single file. If a merged image exists in the folder that is batch read, all of the images in the folder are read

198 Executing macro cell count The procedure for executing macro cell count is as follows. 1 Select the check box for the target files in the target file list. 1 Reference If you click the [Select all] button, the check boxes are selected for all of the files. 2 To use image stitching mode, select the [Image stitching mode] check box in the execute area Reference Merged images captured using the BZ-X800 contain duplicate areas around the top, bottom, left, and right. As a result, if the entire images were extracted and measured, the duplicate parts would be measured multiple times. When the [Image stitching mode] check box is selected, the duplicate areas around the top, bottom, left, and right are excluded from measurement. 3 Click the [Run] button. The macro cell count is executed. When it finishes, the [Preview] dialog box is displayed allowing you to check the execution results. Reference If you batch process multiple files, the processing results for the file that is displayed topmost in the target file list are displayed in the [Preview] dialog box. If the [Image stitching mode] check box was selected, duplicate areas are deleted in the preview for display. 6-64

199 Image display area Displays the macro cell count result image. 2 [Display Results] check box When this check box is selected, the extraction results are displayed in the image display area. 3 Transparency The transparency of the extraction area is controlled by using the slider, [ ][ ] buttons, or by directly entering a numeric value. The extraction area becomes darker as the value gets closer to zero, and gets lighter as it gets closer to Extract Color Specifies the display color for the extracted area. 5 Specified Area (when mask is not set) Specifies the display color of the specified area. Mask (when mask is set) Specifies the display color of the mask area. 6 [Display Labels] check box When this check box is selected, labels associated with the area are displayed. 7 [Details] button Click this button to display the [Details] dialog box which is used to configure the following items. Display color mode ([Monochrome], [Random Clr]) Background color ([OFF], [Black], [White]) Color and thickness of outlines ([OFF], [Thin], [Thick]) Label color and font

200 8 Change zoom Adjusts the zoom for displaying images. Select the [Zoom] check box and adjust the zoom using the buttons or from the pull-down menu. 9 Adjust the position of the specified area (when the area was specified without setting a mask) If you click the [OFF] button to adjust the position of the specified area, the specified area is displayed in the image display area, allowing you to move the position of the specified area by dragging. 6 Display overlay (when a mask is set and multiple images are used) Select the display mode from [All], [Target area only], [1st extraction area only], and [2nd extraction area only]. Reference If the region is moved after executing macro cell count, the results of the macro cell count are discarded. Reference Nothing is displayed if only a single image is used in the mask setting. 4 Click the [Close] button in the [Preview] dialog box. Reference If you want to display the [Preview] dialog box again, click the [Preview] button in the execute and save area. The [Macro cell count] window can be controlled even while the [Preview] dialog box is displayed. 5 To view the measurement results, click the [Measure Result] button. The [Measure Result] dialog box appears. "Display measurement results" (page 6-56) 6-66

201 Saving macro cell count measurement results The procedure for saving the macro cell count measurement results is as follows Click the [Save] button in the execute and save area. The [Save settings] dialog box is displayed. 1 Save folder Specifies the folder to save the files in. 2 Prefix Specifies the character string to be attached at the beginning of the file name. 3 [Statistics results] check box If this check box is selected, the batch processed statistics results are saved in a single CSV format file. 4 [Image] check box When this check box is selected, the result images for all of the files are saved. You should set the save format and comments. Reference 5 [Measure Result] check box If JPEG is specified as the save format, the compression ratio is fixed at 100. If this check box is selected, the measurement results for all of the files are saved in separate CSV format files. If a mask is set, select the [Target area] check box. 6 2 Set the save folder, prefix, and save method, then click the [Save] button. Files where the [Status] is [Complete] in the target file list are saved in the specified location. 6-67

202 Re-editing in hybrid cell count The procedure for using hybrid cell count to re-edit files that have finished being processed by macro cell count is as follows After the macro cell count processing has finished, select the rows of the files to re-edit from the target file list. 2 Click the [Edit] button. The [Hybrid cell count] window is displayed. 3 Specify an area accordingly. 4 Click the [Next] button. A message is displayed to confirm formatting the shape of the area according to the settings in the conditions file. 5 Click the [OK] button. 6 Adjust the shape of the area further as necessary. 5 7 Click the [Next] button. Advances to the check results step. 8 Check the measurement results. 6-68

203 9 Once you have finished editing, click the [Finish Editing] button. A message is displayed confirming whether or not to save the details of the processing in a conditions file and return to the [Macro cell count] window Click the [OK] button. The [Save As] dialog box is displayed Specify the save folder and filename for the conditions file, and then click the [Save] button. The [Macro cell count] window is displayed with the saved conditions file loaded. Read the target files as necessary and execute macro cell count. "Reading the target files" (page 6-63) "Executing macro cell count" (page 6-64) 6-69

204 6-7 3D Cell Count This part explains "3D Cell Count" which performs the cell count in 3D to a Z-stack. Point The "Macro Cell Count BZ-H4C", and the "3D Application BZ-H4R" are required in order to use 3D cell count. The cell count condition is decided for each image of Z slices. Then, according to the 3D connection, the volume is measured. The object is also separated according to the 3D connection. The objects that are separated in a certain Z slice, but are connected in another Z slice, are regarded as one object. Selecting the method of the target area 6 1 Click the [3D Cell Count] icon in the toolbar (large). Alternately, select [3D Cell Count] from the [Z-Stack] menu. The [3D Cell Count] dialog box appears. 2 Select the type of the target area. 2 3 Click the [Start] button. The [3D Cell Count] window appears. Reference The 3D cell count can be executed only to fluorescence images. Therefore, it is different from the hybrid cell count, so the selections of the brightfield image or contrast image are not displayed

205 Specifying the extraction area Set the extraction conditions. Reference Extraction Method is the same as the hybrid cell count. However, the object cannot be specified by clicking individually on the screen, such as the manual pick. " Specifying the area" (page 6-6) 6 2 Change the Z-axis location, and confirm the same extraction settings for other Z slices. 6-71

206 Adjusting the extraction area shape Remove unnecessary areas or fill holes accordingly by adjustment tool. Reference As for the adjustment tools, both [Normal] and [Details] tabs are the same as the adjustment tools of the hybrid cell count. However, modification cannot be made individually on the screen, which can be done in the case of such as [Fine Edit]. " Adjusting the extraction area shape" (page 6-19) 2 Change the Z-axis location, and confirm the same extraction settings for other Z slices. 3 Click the [Next] button. The extraction process is performed to all Z-stack images. Also, the process detects the three dimensional structure as a whole. Reference In the diagram displayed, the object is separated by Z slices. However, the object is regarded as one whole specimen by detecting the three dimensional structure. To separate in 3D, adjust separation parameters so that the object is separated on all Z slices. Separating by adjustment 6-72

207 Removing areas by volume and displaying results Object removal can be specified by volume because three dimensional structure is detected. Histogram or 3D measurement results can be displayed. Removing unwanted areas 1 Click the [Remove unwanted areas] button. The [Remove particles] dialog box appears. 6 2 Click the particles to be removed on the [3D Cell Count] window. The value in [Volume] is reflected on the [Remove particles] dialog box. 3 Click the [OK] button. Remove the particles that is equal or less than the specified volume from the result. 6-73

208 Displaying and saving measurement results Measure results list 1 Click the [Measure Result] button in the results view area. The [Measure Result] dialog box appears. " Measure results list" (page 6-38) For the original item of the 3D cell count, refer to item of the 3D cell count" (page 6-74) "Original Histogram 1 Click the [Histogram] button in the results view area. The [Histogram] dialog box appears. Main dispaly item on the [Histogram] dialog box is the same as the hybrid cell count. " Histogram" (page 6-40) For the original item of the 3D cell count, refer to item of the 3D cell count" (page 6-74) "Original 6 Save measurement results 1 Click the [Save this images] button in the results view area. The [Save As] dialog box is displayed. Main saved item on the [Save As] dialog box is the same as the hybrid cell count. " Save display image" (page 6-41) For the original item of the 3D cell count, refer to item of the 3D cell count" (page 6-74) "Original Original item of the 3D cell count The measurement items which can be output by 3D cell count include the followings in addition to the normal hybrid cell count. Item Description Volume Surface area Displays the volume of the measurement area. Displays the surface area of the measurement area. Note that if there is a hollow inside the measurement area, it becomes the sum of the surface area of both the front and back side. (It becomes the sum of the area of both A and B in the following diagram) B A Feret diameter (Z) Displays the length parallel with the Z axis of rectangle circumscribed to the measurement area. 6-74

209 Display the result with 3D 1 Click the [3D Display] button. The extraction results be displayed with 3D. The operation method is the same as the 3D display of the normal Z-stack images. "4-10 3D Display of Z-Stack Images" (page 4-26) Click the [Details] button to display the [Details] dialog box. If selecting [Thin] or [Thick] for outline display by the [Details] button of [Display details] of the measurement results, an outline is drawn in the 3D display, and the pattern like contour lines can be confirmed. If the outline display is [OFF], the contour lines are not drawn. If changing the display settings, close the 3D display, and click the [3D display] button again

210 In the case of counting by setting a target area If the measurement is performed by setting a target area, select [Single extraction] or [Double extraction] on the screen where the 3D cell count is called. If the counting is performed by setting the target area, it is the same as the target area settings of the normal hybrid cell count. "6-5 Counting by Setting a Mask Area" (page 6-48) 6 Another one can be selected for the image of the target area settings from that of the extraction settings at measurement using the target area. Clicking the [Specify Ext. Area] button can select the image for setting the extraction area. It is different from the normal hybrid cell count, so as for the image specified at this time, select the group capture information file (*.gci) with the same settings as the image which is currently processed. Point The group capture information file (*.gci) which can be specified is only the one with the same condition as the image which is currently processed. When the Z-stack image is loaded to the analysis software, if the upper/lower limit of the Z-stack is changed, or the size or the number of bits for the image is changed in the analysis software, the group capture information file (*.gci) generated at capturing cannot be loaded. After condition is changed, if the changed group capture information file (*.gci) is saved from [Save As] by the analysis software, the file can be loaded. 6-76

211 Chapter 7 Processing the Image Cytometer Data This section explains how to analyze much data statistic by each well, based on image data obtained by the image cytometer capture. Point To use the image cytometer function, the extensibility application "Hybrid cell count BZ-H4C" (option) is necessary. 7-1 Image Cytometer Function Overview of Operation Performing Image Cytometry Analyzing Multiple Plate Results Collectively Displaying the Analyzed Result

212 7-1 Image Cytometer Function This is the function for analyzing images of multiple samples simultaneously. Since statistical data by well can be acquired to multiple wells, reliable results can be acquired effectively. The extracting condition for the target can be set intuitively by easy operation and looking at images. The time-series change of a living cell can be analyzed by combination with the time lapse function. 7 The heat map of the extraction result The histogram of the extraction result 7-2

213 7-2 Overview of Operation The image cytometry proceeds with the following flow. Loading Image Cytometry Image Processing Cell Count Analysis Execution Results Display Loading Image Cytometry Selects how to load image data. Selects whether or not to perform the process as a single image, merged image, Z-stack image, and time lapse image. 7 "Loading Image Cytometry" (page 7-6) 7-3

214 Image Processing Selects the image processing to be applied as preprocessing of the cell count. "Image Processing" (page 7-12) Cell Count Selects the condition to extract the object. 7 "Cell Count" (page 7-14) 7-4

215 Analysis Execution Selects the target well and view to execute the analysis. "Analysis Execution" (page 7-17) Results Display Display the result of analysis. The heat map or histogram for grasping the distribution of extraction object can be confirmed. 7 "Results Display" (page 7-20) 7-5

216 7-3 Performing Image Cytometry Loading Image Cytometry Loading Image Cytometry Image Processing Cell Count Analysis Execution Results Display Selects how to load image data. Selects whether or not to perform the process as a single image, merged image, Z-stack image and time lapse image. Starting Image Cytometry 1 Click the [Image Cytometry] icon on the toolbar (Large). Or, select [Image Cytometry] from the [Image Cytometer] menu. 7 2 Select the folder where the image Cytometer data exists. 3 Select [Single], [Time lapse], or [Merge] according to the type of the loading image. 7-6

217 Loading Single Images Select "single" as the type of the loading image. Single/Merge Select "single" if all target images are handled in the same manner. 7 If the target images include a well edge, the well edge is mistakenly regarded as an extraction object when the images are processed in the same manner by "single." If it is the case, you can detect only the object to be extracted after merging to exclude the position outside the well. Single Merge Reference If the merged image exceeds the maximum resolution of 4080 x 3072, the cell count is executed after the image is made small to within the range. The resolution need not be made small for analysis when process is performed by single. To merge them, the view must be all connected. 7-7

218 2 Select the well to be displayed on the stage map. 3 Select the view to be displayed on the well map. 4 Confirm the image corresponding to the selected well and view. The analysis object is displayed within the frame of the orange color. 5 Click [Load] loads the target image to move to the next process. Loading Time Lapse Images Images captured by time lapse capture can select "time lapse." In this case, moving slider can confirm the time-series change of the object

219 Loading Stitched Images If loading stitched images, select the merging settings before processing the images of the image cytometer. 1 Single Stitch Select [Stitching] and click [Load]. Point 2 Clicking [Single] or [Stitching] can switch whether the loaded image is single image or merged image. Perform settings for merging on the image merge screen, and click [Start Stitching]. For more information about the image merge, see " Overview of image stitch" (page 4-13). 3 The merging result is displayed on the Wide Image Viewer screen, so finish it. The message to ask for file saving is displayed at finishing. Save it as needed. There is no problem even if the file is not saved to proceed with image cytometry. 4 On the image merge screen, click [OK]. 7-9

220 Loading Z-Stack Images If the image data is acquired by using the Z-stack, the following screen appears Z-stack information Selects the Z-stack image to be displayed on the screen [Full Focus] button Performs loading by generating full focus images from the Z-stack images. 3 [Best Focus] button Performs loading by extracting the most focused image from the Z-stack image. 4 [Load] button Loads the Z-stack position displayed currently. 7-10

221 Loading the Multi-Color The preview image is divided into 4 at loading the multi-color image. Note that the image is not divided and displayed if the loading CH is only one, or the check box of the overlay is selected. p Loads the ones where the check box of each CH or check box of the overlay is selected. The loaded CH can be used as the original image in the next image processing or cell count

222 Image Processing Loading Image Cytometry Image Processing Cell Count Analysis Execution Results Display Overview of Image Processing Selects the image processing to be applied as preprocessing of the cell count. Select processes such as the haze reduction or edge emphasis, to stabilize the cell count results by applying it beforehand. The image processing defined here is applied to all views and wells, then executes the cell count. Before selecting the image processing Selecting the image processing After selecting the image processing The Image Processing Screen [Load] button Loads the analysis condition file (*.ica) from here if applying the same image processing with the existing analysis condition file. 2 [Detail Settings] button Selects the image processing to be applied from here. 3 [Back] button Returns the process of image cytometry to the former step. You cannot return more from the step of image processing. If changing the displayed image to the image of another well and view, the process of image cytometry must be suspended to return to the beginning. 4 [Next] button Proceeds with the process of image cytometry to the next step. 7-12

223 Operation of Image Processing 1 Click [Detail Settings]. The detailed settings screen of image processing is displayed Select image processing to be applied. 3 Set the contents of the process. 4 Click the [OK] button. The setting contents are displayed in the image processing list. 4 Reference They are applied in order from the top. The applied order can be changed with the arrows of up and down. Unchecking the check box disables the image processing temporarily. Edits the parameter of the image processing. Changes the order of the image processing. Deletes the image processing. Reference 5 5 For more information about the image processing, see chapter 4. "Chapter 4 Processing Images" (page 4-1) Click the [OK] button. Returns to the image processing screen, and the screen is updated. 6 Confirm the effect by looking at the image after the image processing. 7 Click the [Next] button

224 Cell Count Loading Image Cytometry Image Processing Cell Count Analysis Execution Results Display Overview of Cell Count Selects the extracting condition for the object. The cell count will be performed to all views and wells based on the extracting condition defined here for the steps thereafter. Before cell count After cell count 7 Specifying the area 7-14 Adjusting the area Confirming the result

225 Cell Count Screen [Load] button Loads the analysis condition file (*.ica) from here if applying the same cell count with the existing analysis condition file. 2 [Detail Settings] button Selects the extracting condition to be applied from here. 3 [Clear] button Clear the extracting condition, and return it to the initial state. 4 7 [Back] button Returns the process of image cytometry to the former step. 5 [Next] button Proceeds with the process of image cytometry to the next step. 7-15

226 Operation of Cell Count 1 Click [Detail Settings] button. The [Hybrid cell count] dialog box appears. 2 Select the type of the image and mask, and click [Start] button. The hybrid cell count starts, and the [Hybrid cell count] window appears. Confirm the specification, adjustment, and result of the extraction area thereafter. For more information, see "6-1 Overview of Hybrid Cell Count" (page 6-2). If the process is finished, the cell count screen is updated. 3 Confirm the result by looking at the image after the image processing. 4 Click the [Next] button

227 Analysis Execution Loading Image Cytometry Image Processing Cell Count Analysis Execution Results Display Overview of Analysis Execution Selects the target well and view to execute the analysis. Click the [Next] button here to proceed, and the image processing on the former processing is applied to the all selected views and wells, then the process of extracting objects by cell count is performed collectively. Screen for Executing Analysis [Select all] button Selects all wells and views. 2 [Clear] button Clears the selecting. 3 Stage map The selected well for analysis is displayed in blue. The right-click selects and clears the object. The left-click displays the image at the position. 7-17

228 4 Well map The selected view for analysis is displayed in blue. The right-click selects and clears the object. The left-click displays the image at the position. View selection Multiple fields of view to be targeted can be selected by right-drag. If right-drag is done by setting the selected view to the starting point, the selection is cleared. If the well display is too small, the scaling can be done by mouse wheel. For wells, by right-drag as well, multiple wells can be selected and cleared simultaneously. 7 5 [Save merged image] check box Selects the check box if saving merged images by image data, and specifies a place for saving from [Save settings] (this is displayed only if the merge is selected at the loading step). 6 [Back] button Returns the process of image cytometry to the former step. 7 [Next] button Executes the analysis processing in an actual manner. 7-18

229 Analysis Execution Operation Select the well for analysis on the stage map. 2 Select the view for analysis on the well map. 3 Click [Next]. The screen processing and cell count are executed collectively to the target well and view

230 Results Display Loading Image Cytometry Image Processing Analysis Execution Cell Count Results Display Overview of Results Display Display the result of analysis. The heat map or histogram for the distribution of extraction object can be confirmed. The analysis result can be output by CSV. Also, the analysis condition can be saved to analyze other well plates with the same condition. View heat map Well heat map Well time-series Confirmation of Statistical Results Stage map Selects the well for output. The right-click selects and clears the object. The left-click displays the image at the position. 2 Well map Selects the view for output. The right-click selects and clear the object. The left-click displays the image at the position. 7-20

231 3 Preview screen The selected image of the well and view is displayed. The original image of [Cell count] is displayed. It can be displayed in another window with zooming up. If it is a time lapse image, a slider corresponding to the time is displayed. The following operation can be made in this window. Zoom Selecting the check box can confirm the details since the displayed image is zoomed up by mouse wheel. Extraction result Selecting the check box can display the extracted result on the image. Label Selecting the check box can display the extraction number on the extraction result. 4 Output unit Specifies whether to display the result by well unit or by view unit. In the case of display by view unit, the well for output is only the well selected as the preview position. 7 Output unit: well Output unit: view 5 Measurement value Selects the measurement value displayed on the heat map, graph area, and statistical value tab. Item Number of extraction Area Perimeter Major axis Minor axis Brightness R G B Feret diameter X Feret diameter Y Number of masks This shows the number of extracted objects. This shows the area for extraction. This shows the outer perimeter. Description This shows the maximum length of the distance between arbitrary 2 points on the circumference. This shows the distance between 2 lines that are parallel to the major axis and just encompass the target area. This shows the brightness for extraction. This shows the pixel value of R component. This shows the pixel value of G component. This shows the pixel value of B component. This shows the length parallel with the X axis of rectangle circumscribed to the extracted area. This shows the length parallel with the Y axis of rectangle circumscribed to the extracted area. This shows the number of mask areas where the numbers of count (1st) and count (2nd) are extracted. 7-21

232 6 Heat map This indicates the distribution of the measurement values. The color is displayed denser as the value is larger. Pressing [Show details] displays the heat map of the view in the well. Show details Clicking [Change] can change the gradient display of the heat map. 7 7 Graph area Selects the type of graph to display the measurement value. Histogram Bar graph Time-series Individual histogram Displays the histogram with the numbers of wells and views to the measurement value. Selecting an area on the histogram can confirm the number of data of the section. Displays the measurement value by well or by view. Displays the time-series change of the measurement value to the time lapse data. Displays the individual data for extraction. Histogram Bar graph Time-series 7-22

233 8 Data area Displays the measurement value. Statistic Individual Mask Statistic tab Displays the measurement value by well unit or by view unit. Displays the individual measurement value by extraction object. Displays a measurement value to a mask. Aggregation object By clicking the accumulation, maximum, minimum, average, standard deviation, the heat map or graph area displays corresponding values. Maximum (area) with variation Minimum (area) without variation

234 Confirmation Operation of Statistical Results Select the well for output on the stage map. 2 Select the view for output on the well map. 7 3 Select the well or view as the output unit. 4 Select the value to be confirmed as the measurement value. 5 Select the type of the graph. 6 Select the aggregation object in the statistic tab of the data area. 7 Confirm the heat map or graph. 7-24

235 Confirmation of Individual Data Individual histogram Selects this if confirming individual data by extraction object. 2 Individual measurement value Selects the measurement value to be displayed in the graph area. For more information about the items, see " Display measurement results" (page 6-38). 3 Data area (individual tab) Displays each measurement value about the extracted object. Since the number of data is huge, the maximum display here is 10,000 lines. If saving the measurement results, the results of more than 10,000 lines can be output there. Therefore, refer to the saved results if they cannot be displayed to the full. 7 Preview position: displays only the data of the well/view displayed in the preview image. All output objects: display all data for output. 7-25

236 Confirmation Operation of Individual Data Select the well for output on the stage map. 2 Select the view for output on the well map. 3 Select the individual histogram as the graph type. 7 4 Select the measurement value to be confirmed as the individual measurement value. 5 Select [Preview Position] or [All Output Targets] on the individual tab of the data area. 6 Confirm the graph. 7-26

237 Saving Results Screen of Saving Results [Save Conditions] button The conditions of image cytometry can be saved as the analysis condition file (*.ica). The saved condition files can be used at the following condition specification. Image processing settings Cell count settings Condition specification at collective analysis 2 [Save Result] button The CSV files of the analysis results, heat maps, graphs, and individual extracted images can be saved. 7 Save settings screen Place to be saved Specifies the place where files are saved. 2 Prefix Specifies character strings put on the front of the file names. 7-27

238 3 Display state Specifies the output of the CSV, graph, and heat map of statistical results. 4 Measurement results for output Measurement results Outputs measurement results to CSV. If using masks, the output of measurement results of masks can be selected as well. You can select the method of saving files individually by view unit or saving them by merging. Image Saves images of the extracted results. 5 Data for restoration Saves data to restore analysis results to the analysis result file (*.icr). If selecting [Preview images] check box, preview images can be displayed when confirming results from the analysis result files

239 7-4 Analyzing Multiple Plate Results Collectively The analysis of multiple well plates with the same condition can be made by saving the analyzed result to a well plate beforehand. Collective Analysis Operation 1 Click [Batch Analysis] on the toolbar (Large). Or, Select [Batch Analysis] from the [Image Cytometer] menu Select the analysis condition file (*.ica). 3 Specify capture data (*.ici) for analysis. Clicking the [Edit Analysis Settings] button can change analysis conditions. 4 If saving the merged images as image data, select the [Save Stitching Image] check box, and specify a place to be saved from [Saved File]. 5 Click the [Execute analysis] button, and perform analysis collectively to targeted multiple capture data. The screen of the results display is almost the same as one at normal case, however, the well plate selection or well plate heat map can be confirmed. 7-29

240 6 7 6 Select an output well plate. 7 Select an appropriate one as the output unit from [Plate], [Well], or [Field of view]. If selecting [Plate], the result by plate unit to a well plate is displayed

241 7-5 Displaying the Analyzed Result If saving data for restoration at saving the result of image cytometry, the analysis result can be displayed. The result can be confirmed soon without waiting time for the analysis. Results Display Operation 1 Select [Results Display] from the image cytometer menu. 2 Select the analysis result file (*.icr). 3 The image cytometry screen is displayed, and the analysis results can be confirmed

242 MEMO

243 Chapter 8 Processing Time-Lapse Data This chapter explains how to create time-lapse videos, insert the time when the image was captured, measure the time series brightness variation, and select full focus images and best focus images. 8-1 Create Movies from Time-Lapse Data Inserting a Time Stamp in Images Measuring the Time Series Brightness Variation Creating Fully Focused Images Selecting Images with the Best Focus Function Image Stabilizing Function Extracting Still Images from a Video File Time Series Cell Count Dynamic tracking

244 8-1 Create Movies from Time-Lapse Data This section explains how to create video files (AVI format) from time-lapse group images. Point The advanced Analyzer Application, BZ-H4A (optional), is required to create movies. Creating movies 1 Click the [Load a Group] icon on the toolbar (large), or select [Load a Group] from the [File] menu, and load a time-lapse group of images. 2 Click the [Create Movie] icon on the toolbar (large). Or select [Create a motion picture] from the [Time Lapse] menu. The [Save As] dialog box appears. 8 3 Set the [Frame Rate] (between 1.0 to 15.0 fps), enter the file name, and then click [Save]. An AVI video file is created. When movie output is complete, the [Output Completed] dialog box appears. 3 4 Click the [Play] button to check the movie. You can also play saved movie files by selecting [Play a motion picture] from the [Time Lapse] menu. 4 Important To play movies, an application that can play AVI files such as Windows Media Player must be installed in the PC. 8-2

245 8-2 Inserting a Time Stamp in Images This section explains how to insert a time stamp in time-lapse group images. Point The advanced Analyzer Application, BZ-H4A (optional), is required to insert a time stamp. Procedure for inserting time stamp data 1 Click the [Load a Group] icon on the toolbar (large), or select [Load a Group] from the [File] menu, and load a time-lapse group image. 2 Click the [Insert Timescale] icon on the toolbar (large). Or select [Time Lapse] from the [Insert] menu. The [Time Lapse Time] dialog box appears. 3 Set the various items in the [Time Lapse Time] dialog box. 4 When you have finished with the settings, click the [OK] button. The capture time is inserted on each image of the time-lapse group. To move the position of the time, drag it with the mouse pointer, which changes to while it is placed on the time. Reference Editing and deleting the time-lapse time To edit time-lapse time inserted in an image, double click the time-lapse time to display the [Time-lapse time] dialog box and edit the time. Right click on the time-lapse time and you can edit or delete the time with either [Editing of Time Lapse Time] or [Deletion of Time Lapse Time] in the popup menu. 8 To finalize the time stamp in the image, select [Save inserted object(s) on all images] from the [Insert] menu. 8-3

246 Setting items in the [Time Lapse Time] dialog box Time Set the starting capture time. You can display a minus time by selecting [-] (minus) from the pull-down menu. 2 Interval Set the interval between image capture. 3 Clear Sets the time to zero. 4 Reset Resets the starting time to the time stamp of the first image. Set the capture interval of the time-lapse group as the interval. 5 Time format Select the format of the time to display. You can select from the following options. 8 6 Font Select the font. 7 Font size Set the font size. 8 Style Set the font style. 9 Font color Set the font color. 10 Background Set the background color. To set a transparent background, check [Transparent]. 11 Preview The time is shown inserted in the image. 8-4

247 8-3 Measuring the Time Series Brightness Variation This section explains how to measure the brightness variation over the duration of a timelapse group. Point The advanced Analyzer Application, BZ-H4A (optional), is required to measure the time series variation. How to measure the time series brightness variation 1 Select [Time Series Measure] from the [Measure] menu. The [Time series measure (Specify area)] dialog box and [Time Series Measure (Brightness)] dialog box appears [Time Series Measure (Specify area)] dialog box [Time Series Measure (Brightness)] dialog box 2 Click the [Specify Measurement Area] button icon in the [Time Series Measure (Specify area)] dialog box to set the area you want to measure. For this example, the [Freehand Line] button icon is selected. 3 Specify an area to measure. Place the mouse pointer on the image, and when it changes to, draw a freehand line. 4 Click the [Run] button icon in the [Time Series Measure (Specify area)] dialog box, and move the mouse pointer inside the specified area in the image. The mouse pointer changes to. 8-5

248 5 Click with the mouse. A measurement number is displayed inside the set area. In the [Time Series Measure (Brightness)] dialog box, the brightness variation of the selected area is shown for all images in the group

249 [Time Series Measure (Specify area/brightness)] dialog box items The setting items in the [Time series measure (Specify area)] dialog box and [Time Series Measure (Brightness)] dialog box are as follows. [Time series measure (Specify area)] dialog box items [Specify Measurement Area] icon Select from the following measurement methods: Polygon, Circle draw, Freehand line 2 [Run] icon Click the icon and move the mouse pointer inside the set measurement area. The mouse pointer changes to. Clicking displays a measurement number inside the area. 3 [Garbage Box] icon Click the button and move the mouse pointer inside the set measurement area. The mouse pointer changes to, and the area is filled with the color of the measurement line. If clicking with the state of filling the target area, the measurement result is deleted. If clicking with the state without the measurement result (with the state deleted), the measurement area is deleted. 4 [Delete All] icon Deletes all of the measurement settings. 5 Measuring line Selects the color of measurement lines and measurement points. 8 6 Display details Select the color of labels and the thickness of measuring lines. 7 [Area and region check] checkbox When the checkbox is selected, the region for measurement is displayed in the color of the measuring lines when you click it. 8-7

250 [Time Series Measure (Brightness)] dialog box setting items Average value/maximum value Displays the average value and maximum value of the brightness in the specified area. Select a value from the pull-down menu. 8 2 Display color for the brightness of each measurement number Displays the display color for the brightness of each measurement number. Clicking a color switches between display/hide. 3 Brightness of specified points Moving the mouse pointer over the graph displays a light blue vertical cursor. The brightness of the specified time axis (frame number) is displayed at the position of the cursor. 4 Brightness, R-brightness, G-brightness, B-brightness Select the type of brightness to display. You can also select the brightness for each RGB value. 5 White background, Black background Sets the background of the graph to white or black. 6 Number of decimals Set the number of decimal places (0 to 3) using the spin buttons for displaying the brightness of the specified time axis. 7 [Result output] button Saves the measurement results as a tab delimited text file. 8-8

251 8-4 Creating Fully Focused Images This section explains how to create fully-focused images when a Z-stack is captured during each time-lapse interval. Overview of Full Focus This will create a new time-lapse group of fully-focused images [Full Focus] button Uses the Z-stack to create a fully-focused image for each capture interval, and creates a new time lapse group of focused images. 8-9

252 8-5 Selecting Images with the Best Focus Function This section explains how to extract images from a Z-stack and time-lapse group that have the best focus for creating a new time-lapse group. Overview of Best Focus Extracts the best-focused image from each Z-stack to create a new time-lapse group Select [Best Focus] button Extracts a single in-focus image from each Z-stack captured during the time-lapse experiment, and creates a new time-lapse group of the best-focused images. 8-10

253 Extracting images using Best Focus 1 Click [Load a Group] on the toolbar (large). Or select [Load a Group] from the [File] menu. The [Load a Group] window appears Select the folder containing a Z-stack or time-lapse group image from the folder tree and click the [Best Focus] button. The [Select Best Focus] window appears. 8-11

254 Select images manually 3 Move the Z-stack slider at the right of the image display area (lower level), and select an image with the best focus. 4 Click the [Select] button under the image. The selected image is displayed in the selected image display area (upper level). 5 Repeat Steps 3 and 4 to select the best focus images for all of the Z-stack groups Click the [Load] button. A time-lapse group is created from the best focus images. Select images automatically Click [Auto Select] button. Best focus images are selected automatically from all the Z- stack groups. 4 Click the [Load] button. A time-lapse group is created from the best focus images. 8-12

255 8-6 Image Stabilizing Function This function will correct for deviations in the captured field-of-view due to vibrations or temperature fluctuations. Overview of the Image Stabilizing Function 8 1 [Stabilize Images] checkbox Selecting the [Stabilize Images] checkbox and clicking the [Load] button corrects the deviations in the field-ofview between images and creates a new time-lapse group. 1 Reference When the Image Stabilizing Function has been used, a white frame is displayed on the images. 8-13

256 8-7 Extracting Still Images from a Video File Extracting still images from a video file To extract still images from the motion picture file, perform the following procedure. 1 Select [Extract still images from video] from the [File] menu, and open the [Extract Still Images] dialog box. 8 2 Click [Browse] and specify the [AVI file name] to extract and the [Destination folder]. 3 Select either [TIFF] or [JPEG] for [Format]. When selecting JPEG, set the [JPEG quality] using the [ ]/ [ ] buttons. 4 Click [Extract]. After extraction, click [Close]. The extracted images are saved in the destination folder. 8-14

257 8-8 Time Series Cell Count This section explains "Time-lapse count" that performs time series cell count to time-lapse images. The time series change of the extracted object can be confirmed. Point The "Motion Analysis Application BZ-H4K", and the "Macro Cell Count BZ-H4C" are required in order to use time series cell count. Selecting the image type 1 Select [Time Series Cell Count] from the [Time Lapse] menu. The [Time Series Cell Count] dialog box appears Select the image type from [Fluorescence], [Brightfield], or [Phase Contrast]. 4 3 To specify a mask, specify it in [Specify target area]. 4 Click the [Start] button. Time series cell count starts and the [Time Series Cell Count] window appears

258 Specifying the extraction area Set the extraction conditions. 8 Reference Extraction Method is the same as the hybrid cell count. However, the object cannot be specified by clicking individually on the screen, such as the manual pick. " Specifying the area" (page 6-6) 2 Move the time slider, and confirm the same extraction settings for other time points. Play with advancing the time automatically. Move the display image by one frame. Change the capture time interval. The extraction is performed within the area surrounded by time slider with blue color. Position of displaying image Position of extraction start Position of extraction end 8-16

259 Adjusting the extraction area shape Remove unnecessary areas or fill holes accordingly by adjustment tool. Reference As for the adjustment tools, both [Normal] and [Details] tabs are the same as the adjustment tools of the hybrid cell count. However, modification cannot be made individually on the screen, which can be done in the case of such as [Fine Edit]. " Adjusting the extraction area shape" (page 6-19) 8 2 Move the time slider, and confirm the same extraction settings for other time points. 3 Click the [Next] button. The extraction process is performed to all time-lapse images. 8-17

260 Displaying and saving measurement results [Time Series Cell Count] window for checking results Setting fields in the display details area The item of display details is the same as the hybrid cell count. " Setting fields in the display details area" (page 6-37)

261 Display measurement results 1 Click the [Measure Result] button. The [Measure Result] dialog box appears. The [Time] and [Normal] tabs are displayed in the dialog box. The [Target area] tab also appears at target area settings. [Time] tab The measurement value which can be selected is the same as the measurement value of the hybrid cell count. " Display measurement results" (page 6-38) Point As for the measurement value, there may have integration or max, min, and mean in each extracted block. Pay attention to the difference with the mean or max and min displayed in the time series tab. 8 Brightness (integration) : 4000 Brightness (maximum) : 200 Brightness (minimum) : 20 Brightness (mean) : 40 Brightness (integration) : 2000 Brightness (maximum) : 100 Brightness (minimum) : 30 Brightness (mean) : 20 Brightness (integration) : 6000 Brightness (maximum) : 150 Brightness (minimum) : 10 Brightness (mean) : 30 For example, suppose the extraction result at certain time became as above. If selecting "Brightness (integration)" as the measurement value in the time series tab, the mean, standard deviation, max, min, and total of "Brightness (integration)" at the time are displayed. If the values of the brightness (integration) are 4000, 2000, and 6000, the result becomes as follows. Mean -> 4000 Standard deviation -> 1633 Max -> 4000 Min -> 2000 Total -> If the target area is set, [Target area] other than [Normal] becomes selectable. 8-19

262 If clicking the [Target area] button, the measurement value of the target area becomes selectable. The measurement value which can be selected is the same as the target area measurement value of the hybrid cell count. " Display measurement results" (page 6-56) [Normal] tab Displays the measurement value list of each extracted block at certain time in the [Normal] tab. It is the same as the measurement result item of the hybrid cell count. " Display measurement results" (page 6-38) The displayed time can be changed by moving the time slider. 8 [Target Area] tab The displayed time can be changed by moving the time slider. " Display measurement results" (page 6-56) 8-20

263 Histogram 1 Click the [Histogram] button in the [Time Series Cell Count] window. Displays the histogram of the measurement result at certain time. It is the same as the histogram display of the hybrid cell count. " Histogram" (page 6-40) The displayed time can be changed by moving the time slider. Time series graph 1 Click the [Time Series Graph] button in the [Time Series Cell Count] window. Displays the time series graph of the measurement value. 8 The measurement value which can be selected is the same as the measurement value of the hybrid cell count. " Display measurement results" (page 6-38) [Average], [Standard Deviation], [Max], [Min], and [Total] can be selected as the statistic value. It is the same meaning as the item of the [Time] tab in the [Measurement Result] dialog box. 8-21

264 Save measurement results 1 Click the [Save] button in the [Time Series Cell Count] window. The save settings are the same as the savings of the measurement result of the macro cell count. " Saving macro cell count measurement results" (page 6-67)

265 8-9 Dynamic tracking This section explains dynamic tracking and analysis by measuring an object's movement throughout time lapse images. Point The "Motion Analysis Application BZ-H4K" is required in order to use dynamic tracking. Overview of Dynamic Tracking Time 1 Time 2 Dynamic tracking result Extraction result Extraction result Extract the object for performing the dynamic tracking. The extraction method is the same as hybrid cell count. The dynamic Tracking is conducted by tracking the extraction area movement over time

266 Loading time-lapse images 1 Click the [Load a Group] icon on the toolbar (large), or select [Load a group] from the [File] menu, and load a time-lapse group of images. Point If checking the [Stabilize Image] checkbox, the object movement may be corrected and thus the measurement of the movement distance etc. may be incorrect. Therefore, the [Stabilize Image] checkbox is unchecked. If checking the [Stabilize Image] checkbox and loading the time-lapse images, the white frame is displayed around the loaded grouped images. If performing the dynamic tracking for the loaded image while stabilizing the field of view, crop the portion outside the white frame by specifying the area and call the dynamic tracking

267 Starting Dynamic Tracking 1 Click the [Dynamic Tracking] icon in the toolbar (large). Alternately, Select [Dynamic Tracking] from the [Time Lapse] menu. The [Dynamic Tracking] dialog box appears. 2 Select the image type from [Fluorescence], [Bright field], or [Phase Contrast]. 2 3 Click the [Start] button

268 Setting the extraction conditions Set the extracting outline condition for the object. Set a brightness threshold of brightness. To extract only the outline of the object, click [Outline]. The [Extract outline] dialog box appears. 8 Selecting [Outline] in the [Extract outline] dialog box performs the dynamic tracking to the outline of the object. Selecting [Outline + image] in the [Extract outline] dialog box performs the dynamic tracking to the image and outline of the object which are blended by the value specified by [Ratio]. 2 Perform the adjustment process to the extraction result. As for the adjustment tools, both [Normal] and [Details] tabs are the same as the adjustment tools of the hybrid cell count. However, modification cannot be made individually on the screen, which can be done in the case of such as [Fine Edit]. " Adjusting the extraction area shape" (page 6-19) 8-26

269 Change the display settings of the extraction result accordingly. Selecting the [Display Results] checking switches to display/non-display the extraction result. Click the [Details] button to display the [Details] dialog box. You can change the the detailed display conditions in the [Details] dialog box. 1 Display Tracking Results Sets the color of tracking result and display trajectory. Color Selects the color of display result. Display trajectory Sets the ON/OFF of [Display trajectory]. [Frames] box/slider Sets how many frames ahead from the position of the loci are displayed. 2 Display Extraction Results You can specify the following settings as "Display trajectory". Mode: [Monochrome], [Random Clr] Background: [OFF], [White] and [Black] Transparency Color (extract color) Highlight 3 Outline You can specify the following settings as "Outline". Outline color Outline display: [OFF], [Thin], and [Thick]

270 Setting the tracking conditions 1 Click the tracking object. Track the object in the range surrounded by the solid line. The object is searched in the range surrounded by the dotted line in the next frame. If the movement is fast, make the dotted area larger. Only the extracted objects can be set as the tracking object. Point If multiple objects are clicked, then multiple objects can be tracked simultaneously. 2 Click the [Start tracking] button. 8 Reference Click if tracking the time axis reversely. The mark of [Start tracking] become from. If the object is tracked, the locus is displayed. 8-28

271 Tracking modification Adding the tracking object The object for tracking can be added during ongoing time. Also, other conditions such as extraction condition settings can be changed during ongoing time Change the time slider to change the image. The place where the tracking condition is changed 8 The area which has been tracked Position currently displayed The area which has not been tracked Play the image automatically. Display the next or previous image. Display the next or previous condition changing point. The track setting of the current position is cleared. The track settings of all positions are cleared. 2 Click the tracking object. 8-29

272 Modifying mistaken detection If the tracking detects the object mistakenly, it can be modified. The object which is tracked The object which has been detected mistakenly 1 Overlap the mouse cursor to the object detected mistakenly. The frame for the tracking settings is displayed. 8 2 Drag the frame to set the right object again. 3 Click the [Start tracking] button. The tracking is started again. 8-30

273 End of the tracking The tracking can be ended. 1 Right-click in the state that the tracking setting frame is displayed. 2 Select the [Exit]. Reference If selecting [Delete], the tracking settings are deleted

274 Confirmation of the results The results of the dynamic tracking can be confirmed from the lower right icon. Item Description Displays the measurement results. 8 Displays the time series graph. Displays the time movement graph. Save the image of tracked locus. Changes the capture interval. 8-32

275 Display measurement results [Time] tab Displays the results of measurement items with time series. If there are multiple tracking objects, the measurement value for each tracking object is placed side by side. Measurement value of the tracking "No.1" Measurement value of the tracking "No.2" Measurement value of the tracking "No.3"... As for the measurement items, the following items can be selected. Item Description Position X Position Y Relative Distance Relative Distance X Relative Distance Y Movement amount Movement amount X Movement amount Y Speed X Speed Y Speed Acceleration X Acceleration Y Acceleration Feret diameter (X) Feret diameter (Y) Area Perimeter Major axis Minor axis (width) Brightness (integration) X component of the position set as the origin point on the top-left of the image. Y component of the position set as the origin point on the top-left of the image. The distance between the tracking start position and the current position. The X component of the position set as the origin point for the tracking start position. The Y component of the position set as the origin point for the tracking start position. The movement amount from the front frame. The X component of the movement amount from the front frame. The Y component of the movement amount from the front frame. X component of speed. Y component of speed. Amount of speed. X component of acceleration. Y component of acceleration. Amount of acceleration. The length along the X axis of a rectangle circumscribing the target item. The length along the Y axis of a rectangle circumscribing the target item. The area of the object. The length of the outer perimeter of the object. The maximum length between any two points that lie on the inner perimeter of the target item. The distance between two lines that are parallel to the major axis and just encompass the target area. The value of multiplying the area of the extracted part by the mean brightness before extraction. This is effective for finding the amount of expression of the fluorescence observation

276 8 Item Brightness (maximum) Brightness (minimum) Brightness (mean) R (integration) R (maximum) R (minimum) R (mean) G (integration) G (maximum) G (minimum) G (mean) B (integration) B (maximum) B (minimum) B (mean) [Individual] tab Description The maximum value of brightness within the extracted area. The minimum value of brightness within the extracted area. The mean value of brightness within the extracted area. The value of multiplying the area of the extracted part by the intensity of R component before extraction. This is effective when finding the amount of expression of R component in the fluorescence observation. The maximum intensity of the R component within the extracted area. The minimum intensity of the R component within the extracted area. The average intensity of the R component within the extracted area. The value of multiplying the area of the extracted part by the intensity of G component before extraction. This is effective when finding the amount of expression of G component in the fluorescence observation. The maximum intensity of the G component within the extracted area. The minimum intensity of the G component within the extracted area. The average intensity of the G component within the extracted area. The value of multiplying the area of the extracted part by the intensity of B component before extraction. This is effective when finding the amount of expression of B component in the fluorescence observation. The maximum intensity of the B component within the extracted area. The minimum intensity of the B component within the extracted area. The average intensity of the B component within the extracted area. Displays the measurement value to the individual tracking object. Displays the measurement value of the tracking object at the time currently displayed. If changing the display position by the time slider, the measurement value changes. 8-34

277 Time series graph Changes at each time of the measurement value can be confirmed. If selecting [Only Specified], only the result of the tracking object selected on the [Individual] tab in the [Measurement Result] window. You can also change the selection by clicking the preview area

278 Chemotaxis graph Display the loci of the tracked object as a graph. Selecting [Set the tracking start position in the center] displays as a graph the loci on the relative position with setting the tracking start position as the origin point. If selecting [Only Specified], only the result of the tracking object selected on the [Individual] tab in the [Measurement Result] window. You can also change the selection by clicking the preview area

279 Chapter 9 Inserting Text in Images This chapter explains how to insert a scale, comment, mark, or time into an observation image. 9-1 Scale Comment Mark Date and Time Finalize the inserted object in the image

280 9-1 Scale This section explains how to display a scale on a captured image. Setting the scale 1 Select [Scale] from the [Insert] menu. The [Scale] dialog box appears Shape Select the shape of the scale. 2 Edge Type Select the type of bar when [Bar] or [XY-Bar] is selected for [Type]. 3 Scale Width Set the scale length. Select the length from the pull-down list or enter any value in the box. The length can also be changed by dragging the end of the scale on the image. Checking [Overlay Digit] displays the value of the scale length on the image. Set the number of decimal places for [Decimal Position]. 4 Color Select the color of the scale. 9 5 Width Select the line width. 6 Font Select the color of the displayed characters. Clicking [Setting] shows options for font and sizing. 7 Background Select the background color for the displayed characters. Checking [Transparent] makes the background transparent. 8 Calibration Clicking [Settings] can set the calibration value. 2 Set the shape of the scale with the [Scale] dialog box. 3 When you have finished with the settings, click the [OK] button. The scale is displayed on the image. Until the scale is finalized in the image, you can move and edit it. "9-5 Finalize the inserted object in the image" (page 9-14) 9-2

281 Moving the scale Move the displayed scale. 1 When the mouse is placed on the scale displayed on the image, the mouse pointer changes to. Put the mouse pointer on the center position in the case of mesh. 2 Drag the scale to where you want it. Editing the scale Edit the displayed scale. 1 Double click the scale. You can also right click on the scale and select [Editing of Scale] from the pop-up menu. The [Scale] dialog box appears. Right-click on the crossing position of the scale in the case of mesh. 2 Edit the scale with the [Scale] dialog box. "Setting the scale" (page 9-2) 9 Reference The length of the scale can be changed by dragging the intersection in the case of on the end of the scale bar or mesh. Deleting the scale Delete the displayed scale. 1 Right click on the scale and select [Deletion of Scale] from the pop-up menu. The scale is deleted from the image. 9-3

282 9-2 Comment This section explains how to enter a comment in an image. Setting a comment 1 Select [Comment] from the [Insert] menu. The [Enter comment] dialog box appears Comment Enter the comment to display. 2 Font Select the font. 3 Font size Set the font size. 4 Style Set the font style. 5 Font color Set the font color. 9 6 Background Set the background color. To set a transparent background, check [Transparent]. 7 Preview The comment is shown inserted in the image. 2 Set each item. The set items can be confirmed with [Preview]. 3 Click [OK] button. The comment is displayed on the image. Until the comment is finalized in the image, you can move and edit it. "9-5 Finalize the inserted object in the image" (page 9-14) 9-4

283 Moving the comment Move the displayed comment. 1 When the mouse is placed on the comment displayed on the image, the mouse pointer changes to. 2 Drag the comment to where you want it. Editing the comment Edit the displayed comment. 1 Double click the comment. You can also right click on the comment and select [Editing of Comment] from the pop-up menu. The [Enter Comment] dialog box appears. 2 Edit the comment with the [Enter Comment] dialog box. "Setting a comment" (page 9-4) Deleting the comment Delete the displayed comment. 1 Right click on the comment and select [Deletion of Comment] from the pop-up menu. The comment is deleted from the image

284 9-3 Mark Enter arrows and other figures on the image. This allows you to draw attention to specific target areas. Setting the marker 1 Select [Mark] from the [Insert] menu. The [Save Mark] dialog box appears Mark list Select a mark from this list. 2 Drawing color Set the color of the mark. 3 Preview The mark is shown inserted in the image. 2 2 Select the color of the mark from the [Drawing color] pull-down menu Select a mark from the mark list. Here, "11Filled DOWN arrow mark" is selected. 9-6

285 4 4 Place the mouse pointer on the position to mark and click. The "11Filled DOWN arrow mark" mark appears where you clicked. 5 Click the [Close] button to finish. You can enter a comment next to the mark. "Setting a comment" (page 9-4) Until the mark is finalized in the image, you can move and edit it. "9-5 Finalize the inserted object in the image" (page 9-14) Resizing the mark You can change the size of the displayed marker. 1 Place the mouse pointer on the mark. A rectangular frame with handles appears around the mark. 2 Place the mouse pointer on a handle at the corner or center of the displayed rectangle. The mouse pointer changes to,,,,or, depending on where it is placed. 3 Drag the handle to change the size of the mark

286 Rotating the mark Rotate the displayed mark. 1 Place the mouse pointer on the mark. A rectangular frame with handles appears around the mark. 1 2 Move the mouse pointer to where it changes to Drag the marker to rotate it

287 Moving the mark Move the displayed mark. 1 Place the mouse pointer on the mark. A rectangular frame with handles appears around the mark. 2 When the mouse pointer changes to, drag the mark to where you want it. Editing the mark Edit the displayed marker. 1 Double click the marker. You can also right click on the mark and select [Editing of Mark] from the pop-up menu. The [Select Mark] dialog box appears. 2 Set the mark with the [Select Mark] dialog box. "Setting the marker" (page 9-6) 9 Deleting the mark Delete the displayed mark. 1 Right click on the mark and select [Deletion of Mark] from the pop-up menu. The marker is deleted from the image. 9-9

288 9-4 Date and Time This section explains how to enter the date and time in an image. Setting the date 1 Select [Date] from the [Insert] menu. The [Date] dialog box appears Date Enter the date to display. 2 Date format Select the format of the date to display. 3 Font Select the font. 4 Font size Set the font size. 5 Style Set the font style. 9 6 Font color Set the font color. 7 Background Set the background color. To set a transparent background, check [Transparent]. 8 Preview 2 Set each item. The date is shown inserted in the image. The set items can be confirmed with [Preview]. 3 Click the [OK] button. The date is displayed on the image. Until the date is finalized in the image, you can move and edit it. "9-5 Finalize the inserted object in the image" (page 9-14) 9-10

289 Moving the date Move the displayed date. 1 When the mouse is placed on the date displayed on the image, the mouse pointer changes to. 2 Drag the date to where you want it. Editing the date Edit the displayed date. 1 Double click the date. You can also right click on the date and select [Editing of Date] from the pop-up menu. The [Date] dialog box appears. 2 Edit the date with the [Date] dialog box. "Setting the date" (page 9-10) Deleting the date Delete the displayed date. 1 Right click on the date and select [Deletion of Date] from the pop-up menu. The date is deleted from the image

290 Setting the time 1 Select [Time] from the [Insert] menu. The [Time] dialog box appears Time Enter the time to display. 2 Time format Select the format of the time to display. 3 Font Select the font. 4 Font size Set the font size. 5 Style Set the font style. 6 Font color Set the font color. 7 Background Set the background color. To set a transparent background, check [Transparent]. 8 Preview The time is shown inserted in the image. 9 2 Set each item. The set items can be confirmed with [Preview]. 3 Click the [OK] button. The time is displayed on the image. Until the time is finalized in the image, you can move and edit it. "9-5 Finalize the inserted object in the image" (page 9-14) 9-12

291 Moving the time Move the displayed time. 1 When the mouse is placed on the time displayed on the image, the mouse pointer changes to. 2 Drag the time to where you want it. Editing the time Edit the displayed time. 1 Double click the time. You can also right click on the time and select [Editing of Time] from the pop-up menu. The [Time] dialog box appears. 2 Edit the time with the [Time] dialog box. "Setting the time" (page 9-12) Deleting the time Delete the displayed time. 1 Right click on the time and select [Deletion of Time] from the pop-up menu. The time is deleted from the image

292 9-5 Finalize the inserted object in the image This section explains how to save inserted scales and comments as part of the image. How to finalize the inserted object in the image This step must be performed in order to save the inserted objects on the image. Point The finalized comments and scales are saved as part of the image. Therefore, once the fonts and colors have been finalized in the image, you cannot change or delete them. 1 Confirm that the scales and comments are inserted as desired into the displayed image. 2 Select [Save inserted object(s) on the image] from the [Insert] menu. When the image is saved, the scales and comments become part of the image. 2 9 How to finalize all inserted objects in images Finalize the scales and comments inserted in every image within a group. 1 Confirm that the scales and comments are inserted as desired into the displayed group image. 2 Select [Save inserted object(s) on the image] from the [Insert] menu. Now when the group is saved, the scales and comments become part of the images

293 How to delete inserted objects Delete all the inserted scales and comments. 1 Select [Delete inserted object(s)] from the [Insert] menu. All the scales and comments are deleted from the image. Point Scales and comments cannot be deleted after performing [Save the inserted object(s) on the image]. 1 How to finalize the measured object in the image This step must be performed in order to save the measured objects on the image. Point The finalized area measurements are saved as part of the image. Therefore, once the fonts and colors have been finalized in the image, you cannot change or delete them. 1 Confirm that the area measurements are inserted as desired into the displayed image. 2 2 Select [Save the measurement on the image.] from the [Measure] menu. When the image is saved, the measurements become part of the image

294 MEMO

295 Chapter 10 Other Functions This chapter explains various convenient functions including batch saving images, specifying an area, changing the file size, reversing and rotating images, RGB separation, level correction, and changing the display scale factor Batch Saving Unsaved Images Specify an Area Changing the File Size Converting BZ-X700 Images to BZ-X800 Images Reversing and Rotating Images Separate the RGB Elements of an image Level Correction Changing the Display Scale Factor

296 10-1 Batch Saving Unsaved Images This section explains how to batch save unsaved images that have been processed. How to batch save unsaved images All images the display as "unsaved data" in the image book can be batch saved at once. Point Group images are not batch saved. 1 Select [Save unsaved images at a time] from the [File] menu. The [save unsaved images at a time] dialog box appears. 2 Set prefixes for the destination folder and file names, select the file type, and enter desired comments Save destination Specify a folder for saving images. 2 Prefix Designate the prefix for the file names. 3 File type From the pull-down menu, select the format for saving images. 4 Comment You can also save a comment when you save images. 3 Click the [Save] button. The images are saved in the specified folder, and the image file names in the image book and image window change to the specified file name. 10-2

297 10-2 Specify an Area This section explains how to specify an area on the image to copy or cut the area specifying information. Specifying an area 1 Click the [Specify Area] icon on the toolbar (small). Or from the [Edit] menu, select [Specify Area], then any of [Specify Area (Rectangle)], [Specify Area (Ellipse)] or [Specify Area (Polygon)]. 2 Drag over an area to specify it. 10 Reference With a specified area, select [Copy area] in [Specify area] in the [Edit] menu. You can paste the content of the specified area into another image (However, this does not support a polygon). With a specified area, click [Cut image] on the toolbar (small). The cut image is opened in the image book as a new image. How to clear the area 1 Click the [Specify Area] icon on the toolbar (Small) with the state of area selected. The state of selecting area is cleared. Reference Clicking the icon in selection clears the state of specifying the area as well. 10-3

298 Moving the area or adjusting the size You can move or adjust the shape or size of the specified area (However, this does not support a polygon). 1 Place the mouse pointer over one of the handles on the rectangular outline. The mouse pointer changes to a hand mark ( mark ( ) depending on its position. ) or a resize 2 To adjust the position, click in the area to display the hand mark, and drag it to where you want it. To adjust the shape or size, click on one of its corners or sides to display a resize mark and drag it

299 Cut image Specify and cut an area in an image, and open it as a new image in the image book. 1 Specify the area of the image to cut. "10-2 Specify an Area" (page 10-3) 2 Click the [Cut image] icon on the toolbar (small). Or from the [Edit] menu, select [Specify Area], and then [Cut Image]. The specified area is cut from the image and displayed in a separate window. Editing the specified area Edit the position and size of the specified area (However, this does not support a polygon). 1 From the [Edit] menu, select [Specify area] then [Edit area]. 2 Enter a value to edit the area. 10 Reference X Y Width Height Set the starting coordinates for the set area. Set the size of the area. When the [Calibration] checkbox is selected, the values for width and height change from pixels to microns. 10-5

300 Copying and pasting the specified area location The size and location of the selected area can be copied and pasted to another image (However, this does not support a polygon). 1 With an area set, from the [Edit] menu select [Specify Area] then [Copy Area]. 2 Select an image for the copy destination of the area specifying information. 2 3 From the [Edit] menu, select [Specify Area] then [Paste Area]. The copied area's size and location is pasted on the image

301 10-3 Changing the File Size The section explains how to increase and reduce the image (file) size. How to change the size 1 Select [Resize] from the [Edit] menu. The [Resize] dialog box appears. 2 Set the resize ratio. Reference The resize ratio cannot exceed 4,080 x 3,072 pixels (for group images: 1,360 x 1,024 pixels). 3 Click the [OK] button. The image is displayed at the resize ratio set. Saving the resized image overwrites the original image

302 Changing the file size using the Image Converter 1 Double click the Image Converter icon. 2 Select the [Resize] checkbox for [Choose operation]. 3 When reducing the size, select [Shrink] from the pull-down menu, and adjust the reduction ratio by using the [ ]/[ ] buttons or directly entering a value. To crop the image, select [Crop] from the pull-down menu, and adjust the width and height by using the [ ]/[ ] buttons or directly entering a value Click [Browse] for Source/Destination Folder and select the [Source Folder] and [Destination Folder] for the image to be converted. Selecting the [Include Sub-folder] checkbox also converts any images contained in subfolders within the source folder. 5 Select either [TIFF] or [JPEG] for the [Format] checkbox. Set the [JPEG Quality] by using the [ ]/[ ] buttons or directly entering a value. 6 Click [Execute] and click [Exit]. 10-8

303 10-4 Converting BZ-X700 Images to BZ-X800 Images This converts images captured using the BZ-X700 into the BZ-X800 format. If opening a group image of the merged one or Z stack etc. captured by BZ-X700 in the BZ-X800 analysis software, change it with this function. Conversion method 1 Double click the Image Converter icon. 2 Select the [Convert] checkbox for [Choose operation]. 3 Click [Browse] for Source/Destination Folder and select the [Source Folder] and [Destination Folder] for the image to be converted. Selecting the [Include Sub-folder] checkbox also converts any images contained in subfolders within the source folder. 4 Select either [TIFF] or [JPEG] for the [Format] checkbox. Set the [JPEG quality] by using the [ ]/[ ] buttons or directly entering a value. 5 Click [Execute] and click [Exit]

304 10-5 Reversing and Rotating Images This section explains how to reverse and rotate images. Reversing images Horizontal reverse Example of horizontal inversion 1 From the [Edit] menu, select [Reverse], then select [Horizontal Reverse]. The image is inverted horizontally. 10 Vertical reverse Example of vertical inversion 1 From the [Edit] menu, select [Reverse], then select [Vertical Reverse]. The image is inverted vertically

305 Rotating images 1 Select [Rotate] from the [Edit] menu. The [Rotate by specifying an angle] dialog box appears Angle setting area Click a radio button to select a rotation angle. To specify an arbitrary angle, enter a value. If selecting [Arbitrary angle], enter the number value of the rotation angle, or perform dragging on the preview area to specify an angle. 2 Preview area Shows the image rotated at the specified angle as a preview. 3 [OK] button Click to finish the rotation settings Select a rotation angle or specify an arbitrary angle in the [Rotate by specifying an angle] dialog box. The image is rotated at the specified angle. You can check the rotated image in the preview area

306 Example of rotating an image by 90 degrees 3 Click the [OK] button. The image is rotated by the set angle

307 10-6 Separate the RGB Elements of an image This section explains how to separate the different [R], [G], and [B] elements of an image. Separating RGB 1 In the [Split display] icon on the toolbar (small), uncheck the [R], [G], and [B] checkboxes. The selected color element is separated from the image. 2 Select [Copy] from the [Edit] menu. Or click the [Apply] icon on the toolbar (small). The separated image is opened in the image book

308 10-7 Level Correction This section explains the Level Correction function for adjusting the brightness levels of the shadows and highlights in the image. Level correction procedure Automatic correction The procedure for automatic level correction is as follows. 1 Load an image for level correction Click [Level correction] in [Auto] button on the toolbar (small). The brightness levels of the shadows and highlights in the image are adjusted automatically. 3 Click the [Apply] button. The corrected image is opened in the image book. Manual correction The procedure for manual level correction is as follows Load an image for level correction. 2 Drag the edges of the histogram in [Level correction] on the toolbar (small) to adjust the shadows and highlights. Adjusting shadows Adjusting highlights Shadows : Pixels to the left of the shadow adjustment position are adjusted as the lowest tones (black). Highlights : Pixels to the right of the highlight adjustment position are adjusted as the highest tones. 3 Click the [Apply] button. The corrected image is opened in the image book

309 10-8 Changing the Display Scale Factor This section explains how to enlarge and reduce an image. Zoom in 1 With the window of the image you want to enlarge active, click the [Zoom up display] icon on the toolbar (small). Or select [Zoom Up] from the [View] menu. Original image Zoom up display Zoom out 1 With the window of the image you want to reduce active, click the [Zoom down display] icon on the toolbar (small). Or select [Zoom Down] from the [View] menu. 10 Original image Zoom down display 10-15

310 Arbitrary magnification 1 Right click on an image. The [Magnification] pop-up menu appears. 2 Select the magnification. Set a value from 0.1 to 5.0. You can also set the magnification by selecting [Arbitrary magnification] from the [View] menu. Reference You can perform scaling by keeping on pressing the Ctrl key of the keyboard with moving the mouse wheel as well. You can change magnification by using the [Zoom up display] icon and [Zoom down display] icon on the toolbar (Small) as well. The size of 3D display, XYZ slice, and max projection images cannot be changed