DP2-BSW. User s Manual APPLICATION SOFTWARE

Transcription

1 User s Manual DP2-BSW APPLICATION SOFTWARE This user's manual describes the OLYMPUS DP2-BSW application software to be used with an OLYMPUS Microscope Digital Camera. To ensure safety, obtain optimum performance and familiarize yourself fully with the use of this product, we recommend that you study this manual thoroughly before operation. Together with this manual, please also read the camera instruction manual and the instruction manual of the microscope in order to understand overall operation methods. For more details, please refer to the Online Help of the OLYMPUS DP2-BSW application software. Retain this manual in an easily accessible place near a system for future reference.

2 Any copyrights relating to this manual shall belong to Olympus Corporation. We at Olympus Corporation have tried to make the information contained in this manual as accurate and reliable as possible. Nevertheless, Olympus Corporation disclaims any warranty of any kind, whether expressed or implied, as to any matter whatsoever relating to this manual, including without limitation the merchantability or fitness for any particular purpose. Olympus Corporation will from time to time revise the software described in this manual and reserves the right to make such changes without obligation to notify the purchaser. In no event shall Olympus Corporation be liable for any indirect, special, incidental, or consequential damages arising out of purchase or use of this manual or the information contained herein. No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the prior written permission of Olympus Corporation. Windows, Word, Excel and Access are trademarks of Microsoft Corporation which can be registered in various countries. Adobe and Acrobat are trademarks of Adobe Systems Incorporated which can be registered in various countries. Olympus Corporation All rights reserved Printed in Germany Olympus Corporation, Shinjuku Monolith, 3-1, Nishi Shinjuku 2-chome, Shinjuku-ku, Tokyo, Japan Manual version: October 2007

3 Contents About this manual... 6 Main features of your image analysis program... 7 The user interface: layouts... 8 Arrangement of the components...9 Arrangement of the components (DP-Controller Style)...10 What types of layouts are there?...11 Switch layout...11 Reset layouts...12 Edit layout...12 Tool window display in the different layouts...12 Opening documents Opening images...13 Using the sample images...13 Generating a test image...13 Opening texts...13 Closing documents Which image types are available? How do I recognize what type the image is?...14 Acquiring images Acquiring a single image Preparing the acquisition: Setting the objective used...17 Preparing the acquisition: Setting up an image optimally...18 Carrying out a white balance or a black balance...19 Adjusting the exposure time...20 Set Field Update...22 Selecting the resolution...22 Trimming the image (subarray)...23 Focusing the sample...25 Entering a note...26 Checking the image quality with the line profile...26 Acquiring a time stack

4 Contents File size at acquisition of time stacks...31 Exposure times during acquisition of time stacks...31 Retrospective image calibration...32 Saving images The TIF image format...33 Compression procedure...34 Burning in additional information...34 Creating and viewing multi-dimensional images What is a multi-dimensional image?...35 Creating a multi-channel image...36 Changing the window size and Preview Area...38 Viewing multi-dimensional images...39 Frequently asked questions concerning multi-dimensional images...40 How do I recognize a multi-dimensional image?...40 How do I save a multi-dimensional image?...40 How do I process a multi-dimensional image?...40 How do I add drawing objects to multi-dimensional images?...41 How do I make measurements on multi-dimensional images?...41 How do I get all frames of a multi-dimensional image as individual images?...42 How do I obtain specific frames of a multi-dimensional image?...44 How do I delete frames from a multi-dimensional image? Measuring images Display of measurement results...47 The Measurement toolbar...49 Carrying out measurements...50 How to execute a measurement...50 Example: How to measure a polygon...50 Deleting measurements...51 Saving measurement results...52 Showing measurement results in Excel sheet...52 Editing measurements...52 Changing measurement settings...53 Changing measurement parameters...55 Processing images Applying Filters...58

5 Contents Rotating and mirroring images Commenting on images Adding drawing objects Add drawing object...60 Display/fade out and delete drawing layer...61 Select drawing objects...61 Copy drawing objects...62 Save images with drawing objects...62 Editing drawing objects Insertion of text objects Text-entry mode and layout mode...63 Formatting text objects...63 Printing images The user interface: components Standard and Expert mode...65 Switching the mode...65 The document group Buttons in the document group...66 Creating a new document group...66 Merging two document groups...67 Shifting a document to another document group...68 Floating a document group...69 Toolbars Toolbar overview...71 Changing toolbars...76 Hiding buttons...76 Showing a button again...76 Adding new buttons and commands to a toolbar...76 Setting up a personal toolbar...77 Examples for new or adjusted toolbars...78 Tool windows Making a tool window appear and disappear...79 Docking a tool window...79 Minimize tool windows...80 Description of the tool windows...81 The Image Navigator

6 Contents Adjusting zoom factors for image display...82 The Gallery view...83 Properties...83 Documents...85 The File Explorer...87 Acquisition Process...88 Measurement...88 Channels...89 Switching color channels on/off...89 Changing the image display...90 Saving multi-channel images...91 Stack...91 The Stack Options dialog box...92 Browsing through image stacks...93 Animating image stacks...93 Selecting frames...93 Processing image stacks...94 The Camera Control...94 Buttons in the Camera Control...96 The Camera Settings dialog box...97 Changing automatically generated file names...97 Saving images directly after acquisition...98 Switching the camera to black and white mode...99 Changing the behavior of the live-image Changing the basic unit from meters into inches Line Profile Adjust Display The Histogram group The Display Enhancement group First time configuration of the system Configuring the objective nosepiece Setting the camera adapter's magnification Employing the calibration wizard What are calibration processes used for? Shading Correction White balance Exposure time correction Manual Objective Calibration Step-by-step: Configuring the system Manual Objective Calibration...115

7 Contents Readjusting the configuration When do I have to repeat a calibration process? Starting a single calibration process Switching the calibration process on and off Appendix: Olympus DP20 camera Whitebalance Carrying out a white balance Sensitivity

8 About this manual About this manual The documentation for this image analysis program consists of the online help and this manual. In the manual you will find both an introduction to the product and an explanation of the user interface. By using the extensive step-by-step instructions you can quickly learn the most important procedures for using this software. In the Online help you can find detailed help for all menu commands, all elements in the user interface and all options in the dialog boxes. New users are advised to use the manual to introduce themselves to the product and to use the online help for more detailed questions at a later time. You can summon help at any time by using the F1 key. Additionally, you have the following options to access the online help: The online help can be opened via the F1 key or via the Help menu. Additionally, the button with a question mark is available in every tool window and every dialog box. 6 Operating system Windows XP Windows Vista Your image analysis program runs on the Microsoft Windows XP and Microsoft Windows Vista operating systems. The illustrations of the user interface in this manual were produced with the Microsoft Windows XP operating system. The user interface may have a different appearance when you use the Microsoft Windows Vista operating system. The functionality described is, however, with both operating systems the same.

9 Main features of your image analysis program Main features of your image analysis program Acquiring images p. 16 You can use your image analysis program to acquire high resolution images of a complete sample in a very short period of time. For this purpose the data from the camera which is connected to the microscope is transferred to the computer. Initially you can examine the live-image on the computer so as to adjust it optimally. The live-image will be constantly updated, i.e., when you move the stage to a different position, the live-image will be altered accordingly. You can acquire an image of the sample details currently on display in the live-image, by clicking the Snap button. In this way you create a digital image which is also available after you have switched the live-image off. Saving images p. 33 If you save an acquired image in TIF format, a lot of important image information will be automatically saved, for example regarding the camera used, the resolution, the creation time and so on. You can look at this data at any time if you load the image into your image analysis program You do not need to collect this data separately. Processing images p. 56 You can process the acquired images and optimize the image quality according to your requirements. Various filters are available for this purpose. Additionally, you can rotate and mirror the images. Creating and viewing multi-dimensional images p. 35 Commenting on images p. 26 p. 60 Just as with a snapshot, a multi-dimensional image will also be saved in the TIF file format. Seen from Windows Explorer, you cannot recognize the difference, since in both cases you are dealing with a file with the file name extension, TIF. As opposed to that, a multi-dimensional image always consists of several individual images. These have, for example, been acquired at different times, or in different focus positions. With your image analysis program you can examine and edit multidimensional images and separate the individual images. p. 36 You can also create a multi-dimensional image by combining a series of separate images (e.g. fluorescence images) to create a multi-channel image. p. 29 Additionally, you can define a chronology for a series of separate images and save this as a time stack. You can write a text comment for an image and also make graphic annotations (arrows, lines, circles, text, etc.). 7 Measuring images p. 47 You can make various measurements on images, e.g., the length of a line or the perimeter of an ellipse. The measurement results can be shown in the drawing layer of the image and displayed in a sheet. The measurement results can also be exported.

10 The user interface: layouts The user interface: layouts The user interface is configurable to a high degree and can thus be adapted to the requirements of individual users and tasks. In very many cases the software use follows the usual Microsoft Windows procedures. Users who work a lot with other Windows programs will find the operating steps familiar. The same is true for the application of many keyboard shortcuts, e.g., [Ctrl+C] for copying or [Ctrl+P] for printing. 8 Layout of the user interface The user can choose a "layout" that is suitable for the task in hand. This is an arrangement of the control elements on your screen that is optimal for the task in hand. All layouts are composed of the "document group", "tool window" and "toolbar" components. The document group displays documents (e.g., images). In the tool windows various elements that will help you use and configure the software are available. The toolbars either contain commands which can be carried out directly, or which open a dialog box in which you can define more exact settings.

11 The user interface: layouts Every layout is different in terms of which components are displayed and where they are displayed. These differences derive from the fact that each layout supports the user during the completion of different tasks. You can, however, always change this display and, for example, add additional tool windows. Arrangement of the components The monitor is divided into different fields for the arrangement of the components in the individual layouts. Some areas are always visible and in the same location (e.g., the menu bar and the status bar). Other screen areas are only used by some layouts for display purposes. The following graphic gives you an initial orientation as to which components can be displayed in which areas of the screen. Menu Bar Toolbar area Switch layouts Reset layouts Tool window area Document Group Tool window area 9 Switch to tool window by clicking on the tab Toolbar area Status Bar A detailed description of the components can be found in the chapter "The user interface: components" on page 65.

12 The user interface: layouts Arrangement of the components (DP-Controller Style) When you install the software, two different styles of acquisition are offered. The Standard Style option is preset. You choose the acquisition style you want during the software installation. Changing the acquisition style When you select the DP-Controller Style option, this will result in the tool windows being differently arranged in the layouts. As well as that, the DP Controller toolbar will appear, and other toolbars will no longer be displayed by default. This acquisition style is intended for users who have previously used another image analysis program, and now want to change over to this image analysis program. The software's functionality is identical in both acquisition styles. Its method of operation is also largely the same. This manual describes the software using the Standard Style, since this is preset. You can also change the acquisition style at a later date. To do so, select the Tools > Options command and select the Environment > Layouts entry. Select the acquisition style you want to work with, in the Use Group field. This change will only be effective when you restart your image analysis program. The diagram that follows shows the arrangement of the components in the Acquisition layout, when the acquisition style DP-Controller Style has been selected. 10

13 The user interface: layouts Menu Bar Toolbar area Switch layouts Reset layouts Tool window area Document Group Tool window area Switch to tool window by clicking on the tab Toolbar area Status Bar What types of layouts are there? The user interface consists of two layouts: Acquisition layout, Measure layout. The Acquisition layout The Measure layout Use this layout to acquire images. You can select between different acquisition processes and change the camera settings. In this layout you can make measurements on images. To do so, the Measurement button bar is on display. 11 Switch layout Click the appropriate tab in the menu bar to switch from one layout to another. This is also possible during a live-image. Switch layouts

14 The user interface: layouts Reset layouts If you have changed these layouts during your work, then you can return to the original settings which were supplied with the program. To do so, use the View > Layout > Reset Current Layout command (or click on the Layout button on the top right of the menu bar and select the Reset Current Layout command). Remember, this command only resets the currently-displayed layout, not all layouts. Edit layout If you want to display further toolbars and tool windows in addition to those displayed by default in the layout, then activate the View > Toolbars or View > Tool windows command and select the required entry. You hide a toolbar or a tool window by clicking on the cross on the right upper edge of the title bar. You can redisplay it at any time by using the View command. Tool window display in the different layouts Tool window Display in Acquisition layout Display in Measure layout Not displayed by default Acquisition Process (p. 88) o Adjust Display (p. 102) o Camera Control (p. 94) o Channels (p. 89) o Documents (p. 85) o File Explorer (p. 87) o Gallery (p. 83) o Image Navigator (p. 81) o Properties (p. 83) o 12 Stack (p. 91) o Measurement (p. 88) o Line Profile (p. 101) Task o o

15 Opening documents Opening documents PLEASE NOTE Opening images 1) Use the File > Open > Image... command to load images from a data medium. The Open Image dialog box opens. 2) In the File type list select the Image Formats entry in order to list all the image formats which you can currently open with your image analysis program. 3) Select the All (*.*) entry, in case you want to look at an overview of the contents of a directory, or search for files in other application programs. The program also displays files from other application programs which it cannot read itself. If you try to load these files, the appropriate application program will be opened and the selected document loaded into this program. You can also drag one or more images from Windows Explorer directly onto the document group, in order to open them. Using the sample images Several example images have been automatically installed with your image analysis program. An example image is available for every type of image (see also "Which image types are available?" on page 14). If you would first like to get used to the program, without starting immediately with image acquisition, we recommend that you load these example images. By default, you will find them in the following directory:..\<program directory>\dp2-bsw\images. Generating a test image If you want to get used to the program, then sometimes any image suffices to try out a function. Press [Ctrl+Shift+Alt+T] to generate a color test image. With the [Ctrl+Alt+T] shortcut, you can generate a test image that is made up of 256 gray values. Opening texts Using your image analysis program you can also open and look at texts. Select the File > Open Text... command. The texts must be in TXT or INI file format. Closing documents 13 Closing all documents PLEASE NOTE In the document group, click on the button with the cross to close an opened document. If it has not been saved, you will receive an appropriate warning message. Use the Window > Close All command or the [Ctrl+D] keyboard shortcut to close all opened documents. If you have not yet saved the documents, you will also receive an appropriate message. You close the image analysis program itself in the same way as other Windows programs, by clicking on the cross in the top right corner of the program header.

16 Which image types are available? closing the program closing a document Which image types are available? bit RGB images 16-bit images EXAMPLE 8-bit images You acquire these images by default with your color camera. RGB stands for Red, Green and Blue. A 24-bit true-color image can be described as the superimposition of three 8-bit images that are colored red, green and blue. Use the Channels tool window if you want to look at a true-color image's three different colors separately. This is only possible when a specific check box in the program settings has been selected. Select the Tools > Options... command. Select the Environment > Tool Window entry in the tree view. Select the Channel > Show RGB components check box. Every pixel in a gray-value image can take on one of gray values. However, on the monitor, only 256 gray values can be displayed. Therefore, to be able to display a 16-bit image on your monitor, you must first establish which gray values are to be displayed and how they are to be displayed. As a rule, your image analysis program will automatically determine which are the image's smallest and greatest gray values. This gray value range will be divided into 256 parts and displayed on your monitor. A gray-value image contains gray values between All gray values between will then be displayed on your monitor in black, i.e., with the gray value 0. All gray values from will be displayed with the gray value 1, etc.. The gray values will displayed in white, i.e., with the gray value 255. Every pixel in an 8-bit gray-value image can take on one of 256 gray values. Pseudo Color Tables You can use pseudo color tables to have gray-value images displayed in color on your monitor. Then every gray value will be allotted a hue. Use the Image > Pseudo Color... command, to allocate a pseudo color table to a gray-value image. How do I recognize what type the image is? You can use the Type option in the Properties tool window to find out what type an image is. Alternatively, the Image > Mode command will also supply you with this information.

17 Which image types are available? The entries in the Image > Mode menu show you the image type. For 24- bit true-color images, the RGB Colors and 8 Bit / Channel options are selected (above). For 16-bit gray-value images, the Grayscale and 16 Bit / Channel options are selected (center). When it is an 8-bit grayvalue image, the Grayscale and 8 Bit / Channel menu options are selected. 15

18 Acquiring images Acquiring images Overview of the most important steps you need to take to acquire an image You can use your image analysis program to acquire high resolution images of a complete sample in a very short period of time. Below, you will see an overview of the most important steps you need to take to operate the program correctly. For your first acquisition you should carry out these instructions step for step. Then, when you later make other acquisitions, you will notice that for similar types of sample many of the settings you made for the first acquisition can be adopted without change. Go to the interesting area of the sample. Bring the sample into focus, if necessary with the Focus Indicator. Check the color reproduction. If necessary, carry out a white balance. Check the illumination and adjust it as necessary. Set the objective used in the Microscope Control toolbar. 16 Check the resolution that has been set, possibly set a binning. If necessary, trim the image (subarray). Click the Snap button to make an acquisition.

19 Acquiring images PLEASE NOTE Successful image acquisition requires correct configuration of your image analysis program. As a rule, the configuration takes place immediately after the software has been installed. However, you will need to repeat certain configurations should you have altered settings in your system. Should your acquisitions not lead to the results you expected, even though you have checked all of the settings mentioned above, please check your system configuration. More detailed information can be found in the chapter "First time configuration of the system" on page 104. Acquiring a single image 1) Switch to the Acquisition layout and activate the Camera Control tool window. 2) Click on the Live button. The live-image will be displayed. A new document with the title "Live (active)" will be created. 3) Go to the area of the image that interests you and adjust the image optimally (e.g., exposure time and focusing). To do this, use the possibilities offered you in the Camera Control tool window. Further information can be found in the chapter "Preparing the acquisition: Setting up an image optimally" on page 18. 4) In the Camera Control tool window, click the Snap button. See also "The Camera Control" on page 94. The image will be acquired. Normally, your image-analysis program supplies the name "Image+<serial number>". If you would like to change these settings, see the chapter "Changing automatically generated file names" on page 97. 5) Save the image. Further information is available in "Saving images" on page 33. Preparing the acquisition: Setting the objective used It is imperative that you inform your image analysis program which objective you intend to use for the acquisition. This step is necessary to ensure that the images are correctly calibrated. Only on correctly calibrated images will the scale bar show the correct scale. Especially for making measurements, correctly calibrated images are an essential prerequisite. PLEASE NOTE When your system has been correctly configured, every objective present will have its own button on the Microscope Control toolbar. You can see the magnification on the color ring with which the objective is identified. As well as that, a tooltip with the name of the objective will appear when you move your pointer over it. To inform your image analysis program which objective is to be used, simply click the respective button. This button will then retain its pressed appearance, so you can recognize which objective is in use at any time. If you have forgotten to inform the image analysis program which objective has been used for the acquisition, the images will be calibrated wrongly. In this case you can calibrate the images retrospectively. Further information can be found in the chapter "Retrospective image calibration" on page

20 Acquiring images Preparing the acquisition: Setting up an image optimally You can only acquire a high quality single image if the display in the live-image has been optimally set up before the acquisition takes place. To do this you should make the adjustments described below, to the settings in the Camera Control tool window. PLEASE NOTE For the description of the Camera Control, a sample camera was used. Since certain settings are, however, camera specific, (e. g., the available resolutions), this tool window can have a different appearance when another camera is connected to the image analysis program. By using the Maximize Live to screen button, you can have the live-image displayed at the maximum possible size. The entire available place on your monitor's screen will then be taken up by the image. Use this view to have a good look at the sample. You leave this view by pressing the [Esc] key. In the Camera Control tool window you make Carry out a white balance settings that are important for the acquisition. Shift Metering Maximize live-image, display line profile Regions Switch live mode on/off Acquire image Select exposure time mode and set exposure time Set Field Update Select resolution If necessary employ binning. 18 Use Focus Indicator Trim the image (subarray) Compose notes Camera dialog box access Settings

21 Acquiring images Acquisition style DP-Controller PLEASE NOTE This is how you carry out a white balance If you chose the DP-Controller Style acquisition style when you installed the software, the Camera Control tool window will not be located by default on the right hand side of your screen, but in the lower area of the screen instead (see explanation "Arrangement of the components (DP-Controller Style)" on page 10). This is, however, for the operation and for the explanation that follows, of no importance. Carrying out a white balance or a black balance This is only possible in the live-image. These two buttons can be found at the top of the Camera Control tool window. They are only active when you are in the live mode. Depending on the type of acquisition you want to make, you carry out either a white balance or a black balance. It doesn't make sense to first make a white balance, then a black balance for one and the same sample. As a rule, you will carry out a white balance for your image, since the black balance is only used with fluorescence images. If the colors are not correctly reproduced, you must carry out a white balance before you make an acquisition. When using white balance, the individual color channels are scaled in such a way that the white or neutral gray area of the image displayed on the monitor is displayed correctly as white or gray. If you subsequently acquire a whole series of similar samples, as a rule, you then don't need to carry out another white balance before each acquisition, unless you change the lamp voltage. Inversely, a white balance is always necessary when you change lamps or the lamp voltage. Use the line profile to check whether or not a white balance is necessary (see "Checking the image quality with the line profile" on page 26). The white balance must be made once for every objective you use. Then, when you later change your objective you will not need to make another white balance. Carrying out a white balance 1) In the Camera Control tool window click the White Balance on ROI button. 2) In the image window, draw a frame with the left mouse button depressed. The area within this frame should be as uniformly white or gray as possible. 3) When you are satisfied with the frame's position, click the right mouse button and select Confirm Input. Alternatively you can doubleclick in the frame. 19 Carrying out a black balance The settings for the individual color channels will be optimized in the set area. You can see the results immediately in the image. With fluorescence acquisitions, the background, that is everything that does not belong to the excited sample, should be displayed in a uniform black. Therefore, you should carry out a black balance whenever you want to acquire fluorescence images and observe that the background is not uniformly displayed in black. This is how you carry out a black balance 1) In the Camera Control tool window click the Black Balance on ROI button. The mouse pointer will appear within the image. 2) With the left mouse button depressed draw a frame. The area within this frame should be as uniformly black as possible.

22 Acquiring images 3) When you are satisfied with the frame's position, click the right mouse button and select Confirm Input. Alternatively you can doubleclick in the frame. The settings for the individual color channels will be optimized in the set area. You can see the results immediately in the image. Adjusting the exposure time A correctly set exposure time is of absolute priority for the quality of your acquisitions. An automatic exposure time mode, which will already produce satisfactory results for a great variety of acquisitions, is preset. Depending on the properties of your sample, however, it can become necessary to switch to the manual exposure time mode (e.g., samples with a strongly reflecting surface). If you want to change the exposure time mode, click the option you want in the Exposure group (either Manual or Automatic). Manual exposure time mode In the manual exposure time mode you can select the exposure yourself. In the manual exposure time mode you can set the exposure time yourself. This mode can be recommended when you want to make a variety of different acquisitions of a sample to be able to compare their brightness. This is how you set the exposure time manually 1) If it is not already selected, click the Manual option. You will find this option in the Camera Control tool window, in the Exposure group. 2) Set the exposure time. To do this you have the following options: Use the slide controls. Click the plus and minus buttons. Use the arrow key at the right hand end of the edit field. Enter an exposure time in the edit field, then press the [Enter] key. 20 The exposure time will be adjusted accordingly. 3) If you wish, your image analysis program will supply you with a benchmark, with the help of which you will be able to set a suitable exposure time. To obtain this, click the One time Auto Exposure button. The value set will be that which the automatic exposure time mode supplies. You can then adjust this value as you wish. Automatic exposure time mode In the automatic exposure time mode your image analysis program automatically defines the exposure time for each single acquisition, so that the mean image brightness always remains the same. This mode can be recommended when your acquisitions are always made with similar samples, and therefore require similar exposure times.

23 Acquiring images In automatic exposure time mode the exposure time is constantly checked and updated automatically. This is how you set the exposure time automatically 1) If it is not already selected, click the Automatic option. You will find this option in the Camera Control tool window, in the Exposure group. Your image analysis program will then calculate the exposure time automatically. PLEASE NOTE In the camera settings, maximum exposure times that limit the upwards search for the correct exposure times, are defined. You can see these values in the Camera Settings dialog box, and can also change them there. To do this, make the Camera Settings dialog box appear, and go to the <camera name> entry. Then you will see the Maximum exposure time group. Region This is how you configure the automatic exposure time There are several ways in which you can determine how the automatic exposure time will be determined. Click the Lock Current Exposure Time button to have the currently calculated exposure time used for the complete duration of the live-mode. This will deactivate the automatic exposure time. That means that even when you move to a completely different place on your sample, the exposure time will not be adjusted accordingly. The Lock Current Exposure Time button will remain active (also for later live-images), until you switch it off again. If you want to, you can determine that the exposure time will only be measured on a part of the live-image, you do this in the Region field. Select an entry from the picklist. 21 If you have selected a spot, you have to define the Region of Interest (ROI) on which the exposure time is to be measured, in the live-image. Should you want to position the spot in the center of the image, you should use the Center Spot on Image button for this.

24 Acquiring images Adjustment PLEASE NOTE If you want to, you can select a value from the Adjustment picklist. With this value you can correct the automatic exposure time that has been calculated by your image analysis program. This can become necessary when you also want to use the automatic exposure time calculation for samples in which the intensity in the image is not homogeneously distributed. That is, for example, the case with fluorescence images. To correct the automatic exposure time, switch to the live-mode and select a value from the Adjustment list. The largest possible correction value is 4, the smallest -4. Should your images be overexposed or appear to be too bright, when you use the automatic exposure time, select a negative correction value. Should your images be underexposed when you use the automatic exposure time, select a positive correction value. Test the effect of the exposure time correction in the live-image. Set Field Update The Field Update check box switches between a simple and an enhanced frame rate. The capacity of the CPU in your PC determines the extend to which the frame rate can be enhanced. By default the enhanced frame rate is set. This is easily recognizable by the fact that the Field Update check box is selected. This means that the live-image will be quickly updated, even if the image information changes quickly. However, this setting will require a considerable amount of your processor's capacity. Clear the Field Update check box, if an enhanced frame rate is not especially important for you. This will avoid blocking more of your processor's capacity than necessary. The Field Update check box will not be active if a 1280 X 960 resolution (binning) has been set for the live-image. Selecting the resolution Your camera can acquire images with a variety of different resolutions. Which resolutions are possible, depends on the camera being used. For example, the following resolutions can be available: 22 By default, the highest resolution will always be selected. This has the advantage that all of the data the camera has acquired will be read out, and therefore the image will contain the most information. The disadvantage is, that a high resolution reduces the speed at which the live-image is acquired. Should you, therefore, wish to move around quickly within the sample, it can make sense to set a lower resolution. This is also known as binning. With binning, only a part of the available pixels will be read out from the CCD chip. Then, less data will be transferred from the camera into the image analysis program, and the live-image will be updated more quickly. However, when you use binning, the display of your image

25 Acquiring images will suffer from a loss of quality. Which resolution is the best for your purpose, will depend on the nature of your sample, and the amount of storage capacity you have available. Changing the resolution If you want to change the resolution, the following possibilities are available to you: For the live-image: Select the value you want from the Live resolution picklist. You can set a different (lower) resolution for the live-image than that which you use for the acquisition. That makes sense, for example, when you wish to have a live-image that reacts quickly, but do not want to suffer a loss of quality in the image portrayal when you acquire it. When the live-image is updated, while a binning is being used, it may cause temporary distortions or color shifts. These will disappear as soon as you remain stationary at one point in the sample. For the image to be acquired (Snapshot): Select the value you want from the Snap resolution picklist. If you acquire images with a lower resolution, it will provide you with files that are smaller. An image that -with the highest resolution- needs approx. 14 MB of storage capacity, needs -with the lowest resolution- less than 1 MB. After the image has been saved, its size will be shown in the Size on Disk field in the Documents group in the Properties tool window. For both the live-image and the acquired image: You can link the resolution of the live-image to the resolution that is used for the snapshot. Then you only need to set a new resolution in one place. To do this, click the button with the chain icon. The picklist in the Live resolution field will then immediately become inactive. The value shown will, however, be updated if you set another value in the Snap resolution field. PLEASE NOTE Trimming the image (subarray) If you are only interested in a part of the image on display, you can determine that only the data pertaining to this part of the image will be read out of the camera's chip (Partial Readout). You can define which part that is in the live-image. This function is not available for every camera model. With a subarray you limit the amount of data that is exported from the camera to your image analysis program. You trim the image already before the acquisition. This will make the live-image quicker, and reduce the size of the acquired image's file. This is how you set up a subarray 1) First you define which part of the image you are interested in. To do so, click the Set ROI for Subarray button. The last previously defined ROI (Region of Interest) will be displayed in the image window. 2) Move the ROI to the place in the image that interests you. To do this, click once in the ROI, then with the left mouse button depressed, drag it to the place you want it. 3) Then adjust its size. To do this, drag it by its handles. Confirm the ROI either by doubleclicking in it, or by selecting the Confirm Input command in the context menu. 23

26 Acquiring images By doing so, you switch on the subarray. The live-image will then only show the image segment you have defined. In the illustration to the left, you can see that a ROI for a subarray has been defined. The illustration on the right shows the result after the subarray has been switched on. 4) Then determine whether the subarray is only to be employed for displaying the live-image, or also for the acquisition. If it is also to be employed for the acquired image, clear the Live only check box. By default, the subarray will only be employed for the live-image, that is why the Live only check box is preselected. The display will be updated. 24 Subarray and Binning PLEASE NOTE Switching off a subarray 1) Click the Switch Subarray on and off button to switch the subarray method off. The complete live-image will be displayed again. In the same way as binning (decreasing the resolution), the definition of a subarray will increase the speed of reaction in the live-image, and reduce the size of the file. In this respect the choice of a subarray is an alternative to a binning. As opposed to binning, a subarray displays the part of the live-image that has been defined without a loss in quality. You can also combine a binning with a subarray. In this way you will increase the speed in the live-image still further, and also reduce the size of your files even more. Should you not have set a subarray before you acquired an image but are only interested in a part of this image, you can trim it afterwards by using the Image > Crop command. Further information is available in the online help.

27 Acquiring images The Focus Indicator shows in red the sharpest focusing that has already been achieved. The focusing sharpness that is currently being achieved will be shown in blue. In this illustration you see a very badly focused image. This illustration shows an image that has been focused better, but is still not sufficiently focused. Focusing the sample To help you bring the sample into focus, the Camera Control tool window puts the Focus Indicator at your disposal. Unlike the human eye, this calculates the sharpness of the focus on a mathematical basis. The Focus Indicator enables you to control the focus settings during the live-image. To begin with, you define a rectangular area of the image that is to be analyzed for this calculation. The maximum contrast serves as a monitor for the sharpness in this area. The Focus Indicator has a display that indicates the relative degree of sharpness by means of two superimposed bars. One of these bars is red, the other blue. The red bar shows the sharpest focusing that has already been achieved. The blue bar shows the sharpness of focus that is being achieved with the current focusing. If you start a live-image with an activated Focus Indicator or activate it during a liveimage, it will in each case show the mean sharpness (50%). This is because there is no absolute measurement for the sharpness in an image and because the Focus Indicator only gives relative values which are related to the initial situation that was present when the live-image was started. This is how you bring the sample into focus by using the Focus Indicator 1) Go to the place in the sample that interests you and switch to the live mode. 2) Click the Switch focus indicator on/off button to switch the Focus Indicator on. The sharpness monitor in the Focus Indicator will appear. The sharpness monitor begins with a value of 50%. A yellow rectangle will appear in the image. Only the pixels that are within this rectangle will be taken into account for the measurement of the sharpness of your image. 3) Change the position and size of the rectangle, so that an area of the sample that especially interests you will be measured. 4) Turn the precision focusing knob on your microscope to change the focusing. While doing this, watch the display in the Focus Indicator. The red bar shows the sharpest focusing that has already been achieved. The blue bar shows the sharpness of focus that is being achieved with the current focusing. red bar red-blue bar 25

28 Acquiring images This illustration shows an image that has been very well focused. blue bar PLEASE NOTE PLEASE NOTE 5) Change the focusing on your microscope until the display is as completely blue as possible. 6) Click the Reset focus indicator button, to set the sharpness monitor's bars at the relative value of 90%. Utilize this function if you start working with a very blurry image and if the sharpness monitor's bar reaches the limit to the right while focusing. By resetting to 90%, the display gains more room for additional focusing. 7) When you are satisfied with how sharply the image has been focused, switch the Focus Indicator off. You can additionally use the line profile to check the focusing (see "Checking the image quality with the line profile" on page 26). A binning that has been set does not influence the functionality of the Focus Indicator. Entering a note In the Camera Control tool window you can enter a note in the Note field. This will, from then on, be saved with all of the images you acquire. Then you will no longer need to copy or continually enter, a note that you wish to add to a great many images. When you close your program this note will be deleted, and you can enter another note the next time you start your program. It can happen that you want to add this note to most of the images, but not to one particular image. In such cases you can delete the note in the Properties tool window, in the Document group. 26 Checking the image quality with the line profile You can have a line profile displayed during the live-image. Then a white line will appear. Your image analysis program measures the intensity in the live-image along this line, and shows it in the Line Profile window. The distance along which the line profile will be measured is plotted along the X axis. Along the Y axis, the intensity is plotted. With a color camera, the red, green and blue color channels will be measured separately. With a monochrome camera, or in the black/white mode of a color camera, you will see only one intensity profile.

29 Acquiring images Line profile for a correctly exposed and focussed 24-bit truecolor image. Displayed Intensity (0-255) Stretch along which the line profile will be measured. This is how you switch on the line profile 1) In the Camera Control tool window, click the Line Profile button. As soon as the line profile is displayed, the button will appear clicked. You can recognize this status by the button's yellow background. Then a white line will appear in the live-image. 2) If you want to, you can move the line to whichever place on the live-image you wish. Simply shift the line, or the starting point or the end point of the line, with your left mouse button pressed. 3) Use the line profile to check the quality of your images. Every change made in the camera control will have an immediate effect on the line profile. You can check the following settings with the line profile: In the illustration on the left, you can see the line profile for an image that shows a very marked shading. The line profile drops sharply at the edges since much less light falls on the sample there. Is my image shading corrected? Shading occurs when the image is not homogeneously illuminated. when shading occurs you can see this immediately in the line profile. Move to a position on the slide where there is no sample and no scratches or grime, and defocus. Measure the line profile diagonally across the whole image. The line profile should now be horizontal. If the intensity decreases to the left and right, carry out another shading correction. 27 In the illustration on the right, you see a line profile after a shading correction has been made. All 3 lines run horizontally.

30 Acquiring images In the illustration on the left, you can see the line profile before a white balance has been carried out. The line for the red channel is cut off at the top (see arrow), because the image has a red color tinge. In the illustration on the right, you see a line profile after a white balance has been made. All 3 lines can be clearly seen. In the illustration on the left, you can see the line profile for an image that is overexposed, on the right for an underexposed image. If this were a correctly exposed image the line would be more in the middle of the Line Profile window. Do I need to carry out a white balance? At places where the sample is colorless, all three color channels of a color camera must be superimposed. Should this not be the case, you will have to carry out a white balance. Is my image correctly exposed? An underexposed image can be detected by a flat line profile in which no high Y-values exist. If the image is underexposed the line profile will be cut off at the top. 28 In the illustration on the left, you can see the line profile for an image that is correctly focused, on the right for a blurred image. The lines are serrated when the image is correctly focused. With a blurred image, the lines are flat. Is my image correctly focused? Your image is correctly focused when the line profile shows the most details. A flat line indicates an insufficiently focused image.

31 Acquiring images Acquiring a time stack Overview of the most important steps you need to take to acquire an image In a time stack all of the frames were acquired at different times. A time stack shows you how the section of the sample changed with time. To begin with, for the acquisition of a time stack make the same settings in the live-image as you do for the acquisition of a single image. Then you must define the time sequence in which the images are to be acquired. Change to the Acquisition tool window. This tool window is the one from where you can start the acquisition. To do so, click on the Start t-stack button. Focus the sample in the live-image and set a suitable exposure time. Check the color reproduction. If necessary, carry out a white balance. Set the objective used in the Microscope Control toolbar. Check the settings by acquiring a test image (Snap button). Change to the Acquisition Process tool window. 29 Configure the acquisition in the Options group. Click the Start t-stack button. View and If save necessary, time stack define the a subarray. Stack tool window.

32 Acquiring images Saving time stacks Even though your time stack consists of several images, it is saved as a single file in TIF format. When you acquire time stacks, files can quickly become very large. Thus when you want to acquire time stacks with more than 20 images, you have to increase the maximum permitted file size. To do so, open the Options dialog box by using the Tools > Options... command and select the Images > Memory Management option in the tree view. Enter the new maximum permitted file size in the Maximum image size field. This is how you acquire a time stack 1) First, acquire a single image to check the image quality. In the Camera Control tool window, click on the Snap button. 2) Change to the Acquisition Process tool window. The acquisition process t-stack is preselected. 3) Select the required settings for the acquisition in the Options group. Select the required settings in the Options group. Define the required values by clicking on the small arrow keys. Alternatively, enter a value using the keyboard and press the [Enter] key. 30 Recording time Interval PLEASE NOTE Start delay Total frames Define the duration of the acquisition in the Recording time field. By default the unit of time is set to seconds. You can, however, easily alter this unit by clicking on the black arrow to the right of the time unit and then selecting a different unit. You can choose between milliseconds (ms), seconds (s), minutes (min) and hours (h). Use the Interval field to set the number of images you want to have acquired per time unit. If you enter the value "0" here, the camera will acquire the images as quickly as possible. In this case, no value will be displayed in the Total frames field. Define in the Start delay field whether the first acquisition should take place immediately after clicking on the Start t-stack button, or whether an acquisition delay should take place. In the Total frames field you can see the total number of the images to be acquired. In the process, the first image, which represents the beginning of the acquisition, is also counted. For this reason, with an acquisition time of 60 seconds and an interval of 10 seconds, not six images will be acquired, but instead seven: the first at the start of acquisition, the second after 10 seconds, the third after 20 seconds, etc. 4) Click on the Start t-stack button. If you have not defined a start delay, the acquisition will start immediately. A new document will be set up. This displays the first image.

33 Acquiring images The Start t-stack button changes into the Stop t-stack button. A click on this button will stop the acquisition process. The images of the time stack acquired until this moment will be preserved. The task bar is displayed in the status bar on the bottom left of the screen. It gives you information about how much longer the acquisition will take. As soon as the next image has been acquired, this is displayed. Following acquisition of the final image the Stop t-stack button changes into the Start t-stack button and the task list is hidden. The last image of the acquired time stack remains displayed. 5) Look at the time stack. Open the Stack tool window (View > Tool Windows command) and have a look at the images you acquired. You can also have the time stack played back by clicking on the Play button in this tool window. To do so you may have to reduce or increase the play duration in the Stack Options dialog box. See also "The Stack Options dialog box" on page 92. PLEASE NOTE 6) Save the time stack in TIF file format. When other programs are running on your PC, for instance a virus scanning program, it can interfere with the performance when a time stack is being acquired. PLEASE NOTE File size at acquisition of time stacks When you acquire time stacks, files can quickly become very large. Thus when you want to acquire time stacks with more than 20 images, you have to increase the maximum permitted file size. The default setting is 350 MB. To do so, select the Tools > Options > Images > Memory Management menu commands. Enter the new maximum permitted file size in the Maximum image size field. For technical reasons, only images with a maximum file size of 2 GB (2048 MB) can currently be created. Before acquisition check the available capacity on your hard disk. Exposure times during acquisition of time stacks The exposure time always remains the same during the acquisition of a time stack. 31

34 Acquiring images Retrospective image calibration The correct image calibration is extremely important for many functions. For instance, only when the image calibration is correct, does the scale bar for an image show correct values. Measuring images only produces good results if the images are correctly calibrated. Images which you have acquired using your image analysis program are automatically correctly calibrated when these two conditions have been met: You have correctly configured the system and You have, before the acquisition, selected the objective you are going to use, in the Microscope Control toolbar. A retrospective image calibration can be necessary if, for example, you load images which were created using another application program, or if you have acquired an image using your image analysis program, without previously selecting the objective used in the software. The image you want to calibrate must contain a distance that you know the exact length of. 1) Load the image. 2) Use the Image > Calibrate Image... command. 32 3) Select an appropriate unit. 4) Enter the length of the known segment in the Length field. 5) Select the Fixed check box. 6) Select the appropriate X/Y ratio for the camera with which the image is to be acquired. If your camera has square pixels, enter the value "1" in the X/Y-ratio filter. 7) Click the Arbitrary button. 8) Use your mouse pointer to determine the beginning and end point of the known segment, and set each of these with a click of the left mouse button. 9) Click the right mouse button and select Confirm Input. 10) Click the OK button to confirm the calibration. 11) Check the new calibration using the scale bar. If the scale bar is not already displayed, switch it on by using the [Shift+F4] keyboard shortcut.

35 Saving images Display of three images which have not yet been saved. Saving images You should always save important images immediately following acquisition. You can recognize images that have not yet been saved by the asterisk icon following the image name (in the Documents tool window and in the image title). Furthermore, you will be asked when ending the program if you would like to save unsaved images. You can also configure your image analysis program in such a way that all images are saved automatically after image acquisition. Further information can be found in the chapter "Saving images directly after acquisition" on page The TIF image format Images you have acquired with your image analysis program always contain a certain amount of additional data. This includes, for example, image calibration, measurement results or drawing objects. This supplementary information will only be retained if the images are saved in the TIF format. For this reason we recommend you to save all images in TIF format. This format is preset by default. 16-bit images can only be saved in the TIF file format.

36 Saving images Compression procedure For TIF format you can define various compression procedures, so as to reduce the size of the image. A more exact description can be found in the online help. If you want to load the images with another application program, you should not compress the images when saving them, since loading compressed TIF images is a problem for most application programs. Burning in additional information You can burn additional image information into the image, e.g., drawing objects or measurement results. By this, they become an integral part of the image and will always be shown. All of the pixels that lie beneath the additional information displayed on the image (e.g., beneath the scale bar) will be overwritten by the burning in. In this way, image information will, of course, be lost. To burn in the additional information, use the Image > Burn In Info command. PLEASE NOTE Before you burn in additional image information, please consider the possible consequences. For example, when executing the command Burn In Info, an image can be converted automatically into another image type. This may involve a loss of data. Therefore, save important images in TIF format before you burn in additional information. Then, select the Burn In Info command and save the image again, using a different name. You can find further information about the burning in of additional information in the online help. 34

37 Creating and viewing multi-dimensional images Creating and viewing multi-dimensional images What is a multi-dimensional image? You can combine a series of separate images into a multi-dimensional image. The separate images of a multi-dimensional image can be 8-bit or 16-bit gray-value images or also 24-bit true-color images. All separate images generally show this same sample, but differ either in the color information, the acquisition time or the Z position of the stage. There are the following types of multi-dimensional images: Multi-channel image Above you can see a multi-channel image, taken from fluorescence microscopy. The multichannel image was created by combining the three images shown below. A multi-channel image shows, as a rule, a sample that has been excited with several different fluorochromes. The multi-channel image is made up of a combination of the individual fluorescence images. Red Green Blue 35 PLEASE NOTE Time stack The Channels tool window shows the individual color channels that make up a multichannel image. RGB images are not multi-channel images. Although you can also display the red, blue and green color channels here, it is always the same image, the 24 bits of which are displayed as three separate 8-bit color channels. A multi-channel image, on the other hand, consists of an arbitrary number (minimum: 2) of individually acquired images which are retrospectively combined to form one image. In a time stack all of the frames were acquired at different times. A time stack shows you how the section of the sample changed with time.

38 Creating and viewing multi-dimensional images PLEASE NOTE Z-stack In the Stack tool window you can also play back a time stack as a film. You can find instructions for acquiring a time stack in the chapter "Acquiring a time stack" on page 29. A Z-stack contains images which belong to different focus positions, i.e., the stage was located in a different Z position for the acquisition of each image. In the Stack tool window you can also play back a Z-stack as a film. 36 Creating a multi-channel image You can combine a series of gray-value images into a multi-channel image. These can be either 8-bit gray-value images or 16-bit gray-value images. The prerequisite therefore is, that all of the separate images have the same bit depth. The image size and image calibration will be adopted from the first image. Switching the camera mode You can switch your camera from color to black and white mode. More detailed information can be found in the chapter "Switching the camera to black and white mode" on page 99. This is how you create a multi-channel image 1) Load the gray-value images that you want to combine into a multi channel fluorescence image. 2) Activate the first image. 3) Use the Image > Combine Channels... command. The Combine Channels dialog box will open. In the Available Images sheet, the active image will be automatically entered as the first color channel.

39 Creating and viewing multi-dimensional images 4) If necessary, change the options for the preview and the preview area. Further information can be found in the chapter "Changing the window size and Preview Area" on page 38. 5) Click once in the Name cell. Enter a name for the channel, or accept the suggested name "Channel 1". You can increase the width of the row so that the description will fit into it. 6) By default, the first channel will be assigned the color red. To change the active color click this color field. Select one of the colors from the palette on the Standard tab, or activate the Custom tab to define a color of your choice. 7) In the next free row, click the Images cell. You will be presented with a picklist containing all of the images that you can combine with the active image. 37 8) Select the next image which is to be entered as a color channel. By default it has the name "Channel 2" and is green. If required, change the name and the color 9) Repeat steps 6 and 7 to enter the third image as a color channel. By default it has the name "Channel 3" and is blue. The sheet could now look something like this:

40 Creating and viewing multi-dimensional images 10) If required you can change the pixel shift of the new channel. This is important when the image structures do not lie exactly on top of each other, for example, because the stage was moved slightly between the separate image acquisitions. For the pixel shift use the arrow keys for example. These move the image by one pixel per click in the direction of the arrow. You can also even correct the pixel shift retrospectively. To do so, use the Image > Geometry > Shift Channel command. Further information is available in the online help. 11) Click the OK button to create your multi-channel image. A new image document with the default name "Unnamed" will be created. PLEASE NOTE 12) Use the File > Save as... command to save your new multi-channel image. Always use the TIF file format when saving a multi-channel image. Further information about the settings that are possible in the Combine Channels dialog box, can be found in the Online Help. 38 Dialog box size Preview Area You can define any section of the image as a preview area. To do this, shift the white selection rectangle to the required location in the image. Only this position will be displayed in the dialog box. Changing the window size and Preview Area You can drag the dialog box to any size you want. Doubleclick the dialog box's header to enlarge the dialog box to full-screen size. Clicking the header again will return the dialog box to its original size. As a rule, only one segment of the source image can be displayed in the dialog box. In both the image document and the image navigator, the segment of the image on display will be indicated by a white frame. The areas of the image not on display will be shown shaded. Use the zoom buttons in the dialog box to make the image segment on display larger or smaller. You can also use the mouse directly in the image window, to change the position and size of the preview area.

41 Creating and viewing multi-dimensional images Preview options You can choose between a number of preview options. Original & Preview displays the same image segment twice in the dialog box. The first one shown is the source image. The second one shown is the resulting image with the channel settings that have been chosen. Only Preview displays only the image segment with the resulting image in the dialog box. Preview in Image displays the chosen channel settings directly in the image window. The dialog box no longer contains a preview window. Then, only the controls will be shown. No Preview does not show a preview at all. With this option you can define the channels and create them immediately. Viewing multi-dimensional images In order to have the multi-dimensional images displayed, open the Channels and Stack tool windows ( View > Tool Windows command). Another possibility for looking at all the separate images of a multi-dimensional image is offered by the Tile View toolbar. Use the Tile View button in this toolbar to display all the separate images of a multidimensional image on the monitor. To display a particular separate image in the image window, select the image and use the Single View button in the Image Tile toolbar. The selected frame will then be displayed in the document window. A time stack that consists of 30 individual images will be displayed as shown when the Tile View button is activated. In the example shown the first image is selected. 39

42 Creating and viewing multi-dimensional images Frequently asked questions concerning multi-dimensional images How do I recognize a multi-dimensional image? You will recognize a multi-dimensional image by its stack-icon, which is displayed on the tab, before the document name. Additionally, in the Properties tool window, you can use the Frame Count option to find out how many separate images are contained in any given image. A multi-dimensional image contains at least two separate images. In the example shown the multi-dimensional image consists of 80 individual images. How do I save a multi-dimensional image? Multi-dimensional images can only be saved in the TIF file format. Otherwise they loose a great deal of their image information during saving. 40 How do I process a multi-dimensional image? Image processing functions (e.g. the use of a filter or the rotation or mirroring of an image) either have an effect on the entire image or on a selection of individual images. Before you access a function for processing images, select the separate images you wish to process in the Channels resp. Stack tool window. The frames you have selected will then be highlighted in color in the respective tool window (see illustration). Select the Target > Selected Frames and Channels option in the processing function's dialog box, to determine that the function only affects the selected frames. Select the Target > All Frames and Channels option to make it possible to process all of the separate images.

43 Creating and viewing multi-dimensional images If a filter is only to be used on specific images in an image stack, then the individual images first have to be selected in the Stack tool window. The required filter can be subsequently selected in the Process menu and configured in the Filter:<filter name> dialog box. In this window you define that the filter is only to be used on the selected individual images (Target > Selected Frames and Channels). An image processing function does not change the source image's dimensions. The resulting image is, therefore, comprised of the same number of individual images as the source image. How do I add drawing objects to multi-dimensional images? With the buttons in the Drawing toolbar you can also quickly add drawing objects to multi-dimensional images. The procedure is the same as when you add a drawing object to an individual image. Further information is available in "Adding drawing objects" on page 60. The inserted drawing objects are automatically displayed in all frames of the multidimensional image. It is not possible to display drawing objects on some frames only, or to use different drawing objects for each frame. This is because all drawing objects are written on a special drawing layer. Try and picture the drawing layer like a transparency which is placed over all the frames. If you only want to add a drawing object to a specific image in a multi-dimensional image, you must separate the image. Then you can add the drawing objects. However, the image will then no longer be a multi-dimensional image, but a single image. 41 How do I make measurements on multi-dimensional images? In the Measure layout the Measurement toolbar is available by default for measuring images. Additionally, you can also open the Measurement tool window. The procedure for measuring multi-dimensional images is the same as for measuring an individual image. The difference is that you must previously select the image that you want to measure in the Channels or Stack tool window. For a time stack for example, this can be the first or the tenth image. Doubleclick on the selected image so that it is displayed in the document group. Measure it exactly as if you were measuring an individual image. Further information is available in "Carrying out measurements" on page 50. The measurement results are automatically displayed in all frames of the multidimensional image.

44 Creating and viewing multi-dimensional images The illustration shows a time stack in which an ellipse has been measured. If you access the Tile View toolbar and change into the Tile View view, you will see that the measurement is displayed on all images in the time stack. It is not possible to display measurement results on some frames only, or to carry out different measurements for each frame. This is because all measurement results are written on a special drawing layer. Try and picture the drawing layer like a transparency which is placed over all the frames. If you want to measure several or all of the frames of a multi-dimensional image, you have to separate the multi-dimensional image and measure the individual images. If it is important for you to document on which image the measurement was carried out, you can add a text object with an appropriate comment (using the Drawing toolbar). 42 Possibility 1 How do I get all frames of a multi-dimensional image as individual images? 1) Select the required multi-dimensional image. 2) Open the required tool window (View > Tool Windows command). It depends on the type of multi-dimensional image, which of the two is the correct one: In the case of multi-channel images: Activate the Channels tool window. In the case of time stacks or Z-stacks: Activate the Stacks tool window. 3) Select the uppermost entry (e.g. the one with the stack symbol for a Z-stack), rightclick and select the Separate command from the context menu. All frames of the multi-dimensional image will be created as individual images.

45 Creating and viewing multi-dimensional images To obtain all the images in a multi-dimensional image stack, select the top entry in the Channels or Stacks. tool window Thus all images in the image stack will automatically be selected. You can see this by the orange colored marking. Possibility 2 1) Select the required multi-dimensional image. If you are not certain whether all of the images you want have been selected, you can check this first in the Stack or the Channels tool window. Only the images that have been selected will be separated. 2) Activate the Image > Separate command and select the criterion according to which you would like to separate the multi-dimensional image. Options which are not available are displayed in gray. 43 The multi-dimensional image is separated. Each image is displayed as an individual image in its own separate document.

46 Creating and viewing multi-dimensional images How do I obtain specific frames of a multi-dimensional image? 1) Select the required multi-dimensional image. 2) Open the required tool window (View > Tool Windows command). 3) Mark the required images in the Channels or Stack tool window, click the right mouse key and select the Separate command in the context menu. The standard Windows conventions, which you probably know from the Windows operating system, are valid for image selection. Selecting single, non consecutive documents: Selecting a series of documents: Selecting all documents and clearing some documents: Selecting all documents Hold the [Ctrl] key depressed and click on the required documents. Hold the [Shift] key depressed and click on the first and last file that you want to select. Hold the [Shift] key depressed and click on the first and last file that you want to select. Then hold the [Ctrl] key depressed and click the documents that are to be cleared. Use the [Ctrl+A] keyboard shortcut. (To do so, the Stack, respectively the Channels tool window must be active, with the toolbar title highlighted in orange). The multi-dimensional image is separated. Each image is displayed as an individual image in its own separate document. 44

47 Creating and viewing multi-dimensional images In the illustration the first, fifth, eighth and eleventh images of a time stack are selected. The Separate command creates a separate document for each of these selected images. How do I delete frames from a multi-dimensional image? You cannot delete frames from a multi-dimensional image (e.g., by selecting the image and pressing the [Del] key). You can however select the required images in the Stack or Channels tool window and then create a new multi-dimensional image which only consists of the required images. 45

48 Creating and viewing multi-dimensional images In the illustration the first, fifth, eighth and eleventh images of a time stack are selected. The Extract command creates a new time stack which consists of the selected images only. This is how you create a new multi-dimensional image consisting of selected frames 1) Select the required multi-dimensional image. 2) Open the Stack tool window (View > Tool Windows command). 3) Mark the required images in this tool window, click the right mouse key and select the Extract command in the context menu. The new multi-dimensional image is created. 46

49 Measuring images Measuring images PLEASE NOTE Your image analysis program offers a wide range of measurement functions. They enable you to quickly count objects and measure segments and areas. All the results are saved together with the images and can also be exported to a sheet. All of the measurement functions are also available in the live-image. You can both make measurements on frames and on a particular image from a multidimensional image stack. See also "How do I make measurements on multi-dimensional images?" on page 41. Calibrated images are a definite prerequisite for measuring! Images which you have acquired using your image analysis program are automatically correctly calibrated if you have selected the objective used in the Microscope Control toolbar prior to image acquisition. Change to the Measure layout to make measurements on images. Here, the Measurement button bar is on display. Additionally, you can open the Measurement tool window. In this tool window you have fast access to all measurement functions and settings. The tool window also contains a list of the measurement values which shows all measured values for the active image. Display of measurement results The measurement results will be shown on the image in a special data layer. Onscreen, the image and the drawing layer are shown together. The data of each, however, will be stored separately in the TIF file format. (Provided you save the image in the TIF format. For saving in other file formats see "Saving measurement results" on page 52). Try to picture the data layer as a transparency which is placed over the image. When you measure an image, the image itself will not be changed by having the measurement results displayed on it. The measurement results will be simultaneously displayed in the Measurement tool window. There, you can compare the measurement results especially easily, and to do so, sort them in different ways. The Measurement tool window should always be visible when you carry out measurements. To do so, click the Enable/Disable Auto Hide button to fix the tool window on the screen. Then you can check the measurement results immediately in the list of the measurement values, once the measurement has been completed. The Enable/Disable Auto Hide button is located on the right edge of the tool window's title bar. 47

50 Measuring images You see an image on which a number of different measurements have been made. These measurements are displayed on the image. At the same time the Measurement tool window displays the measurement results. Point Circle Polygon Ellipse Polyline Line Rectangle 48 Sort measurement results By default the completed measurements are sorted in alphabetical order in the Type column. You can, however, also change this order at any time. To do this, click on the Type column header in the list view. The measurements are then sorted in reverse alphabetical order. Clicking again returns the table to the alphabetical order. You can also change the sort criterion and sort the measurements, for example, according to the area measured. To do so, click on the Area column header. The measurements are then sorted in decreasing order. This means that the measurement with the highest value is right at the top. A further click on the column header sorts the results in increasing order. Measurements which do not have the required value (e.g., a pixel measurement) are located right at the end (or right at the beginning) of the series.

51 Measuring images The illustration shows the results of several measurements in the standard alphabetical sorting order. This illustration shows the sorting order following a change to the Area sort criterion. The measurement results are now sorted according to the area measured. The Measurement toolbar The Measurement toolbar contains the buttons activating the measurement functions. Here, you can also find the buttons for selecting, exporting, and deleting measurements and for changing the measurement settings. Measurement Options Select Measurements Export Measurements Delete Measurement Closed Polygon Ellipse 2 Points Circle Rectangle Polyline Arbitrary Line Point Origin Select Measurement Objects 49

52 Measuring images Carrying out measurements Begin a measurement by clicking on the button with the measurement you want. In the measurement mode your mouse pointer will take on the shape of a cross. The measurement mode remains active even when the first measurement has been completed. You can therefore immediately carry out further measurements with this measurement function, even on several images. Click the measurement function button once again in order to deactivate the measurement mode. pressed not pressed Should you want to select the measurement object after you have made the measurement, first click the Select Measurement Objects button, then click the measurement object. You can only select measurement objects in the image when this button has been pressed (recognizable by its yellow background). How to execute a measurement In effect, all measurements are made according to the same principles. Only the way in which you define the measurement object and how you complete the measurement differs from measurement object to measurement object. 1) Begin the measurement by clicking the button for the measurement you want to make, in the Measurement tool window. 2) Then carry out the measurement you want to make, on the image. You will need to make one or more clicks with the left mouse button to do this. 3) To complete the measurement, it is, in many cases, sufficient to simply release the left mouse button. Some measurements, for example of a Polyline, require you to press the right mouse button. The measurement results will be displayed. 50 Example: How to measure a polygon 1) Click the Closed Polygon button. 2) Position the mouse pointer on a point on the object's edge and click the left mouse button. 3) Set additional points on the edge of the object. In this way you can compose the contour from numerous straight segments. 4) Click with the right mouse button to close the contour. The polygon will then be shown in the image. The results will be listed in the Measurement tool window. You can find a description of all the settings options in this dialog box in the online help.

53 Measuring images Deleting measurements The selection of a measurement object always takes place in both the image and the list simultaneously. You can choose where you want to select a measurement object. You delete measurements either by selecting the required measurements and pressing the [Del] key, or by clicking on the Delete Measurement button in the Measurement tool window. You can select the measurements you want to delete either in the list of the measurement values or in the image. To be able to select the measurements in the image, you must first press the Select Measurement Objects button. 51 Delete all measurements PLEASE NOTE If you have carried out a lot of measurements and want to delete them all quickly, do the following: 1) Activate the Measurement tool window. 2) Use the [Ctrl+A] shortcut. All measurements will be selected. 3) Press the [Del] key. All measurements will be deleted. The deletion of measurements cannot be reversed.

54 Measuring images Saving measurement results If you save images in which you have carried out measurements in TIF file format, then the measurements will also be saved at the same time. When you load the image, the measurements will automatically be displayed in the image, and the list containing all of the measurement results will also be displayed. You can then supplement, change or hide, your measurements. PLEASE NOTE When you save images on which you have carried out measurements, in another file format, e. g., BMP or JPG, the measurements will be burnt into the image. These cannot be supplemented, changed or hidden, when you load these images again later. Showing measurement results in Excel sheet You can export the results shown in the Measurement tool window into an Excel sheet. 1) Click on the Export Measurements button. The standard dialog box for saving documents will open. 2) Select the drive and directory in which you want to save this document in the Save in list. In the File Type list, only the "Excel-Sheet (*.xls)" will be available. 3) Enter the name of the measurement sheet in the File name field. 4) Click the Save button, to have the measurement values saved as an Excel sheet. The measurement results will be exported the way they are currently displayed in the Measurement tool window. If MS-Excel has been installed on your computer, the exported measurement sheet will be immediately opened by default. You can change this behavior in the Tools > Options > Measurement > General dialog box. 52 Editing measurements PLEASE NOTE You can edit existing measurements. For this purpose you must select the measurement object to be edited. To do so, click the Select Measurement Object button in the Measurement tool window and click on the required measurement object. Alternatively, you can also click on the required measurement in the list of measurements. Now you can: shift a measurement object, change the size of a measurement object, change the form of a measurement object, change the position of the measurement label. The form of the mouse pointer shows you whether you are shifting a measurement object or changing its form: This cursor shows that you are shifting the measurement object. This cursor shows that you are changing the measurement object. This cursor shows you are changing the form of the measurement object.

55 Measuring images Shift measurement object Change size or form Change position In the example shown the measurement label has been moved to optimize the view. To shift the measurement object, press the left mouse button and move the mouse. To change the size or form of a measurement object, move the mouse pointer over a handle. By dragging the marker with the mouse button depressed, you can adjust the object's size as wished. To change the position of the measurement label, select the measurement object and position the pointer over the measurement label handle. Then pull the measurement label to a new position. PLEASE NOTE Update the measurement results In the case of a pixel measurement the cross which shows the position of the pixel and the handle for the measurement label are very close to each other. Ensure here that you know whether you are moving the pixel or the measurement label. During the editing of the measurement, the measurement values displayed in the image will be constantly updated. The measurement values in the Measurement tool window, will not be updated until you have completed the editing. Changing measurement settings The default settings for measuring will be sufficient for most measurements. Nevertheless, you may need to alter settings. You can, for example, choose another color for displaying the measurement label. To do so, activate the Options dialog box using the Measurement Options button. Alternatively, access the Tools > Options menu command and select the Measurement entry. The Options dialog box offers four entries for the measurement settings: 53

56 Measuring images 54 In the General group you can change general settings for measuring, for example, whether or not an exported sheet is to be opened directly after being exported to Microsoft Excel. The prerequisite for this is that MS-Excel has been installed on your computer. The settings of the Origin group relate to measurements in which the coordinates origin is important. For example, where a point measurement is concerned. The measured point position is shown based on the origin of the coordinate system, which is in the upper left corner of the image, by default. You can change the pixel position here. In the Label group you find various settings which relate to display, form and position of the measurement label. In the Results group you can control the display of the measurement results, for example the number of decimal places. Up to 7 places behind the decimal point are possible. In the online help you can find a detailed description of all the setting options in this window.

57 Measuring images Changing measurement parameters If you measure a circle in the image, for example, certain measurement parameters are used automatically for the measurement. For the three possible types of measurement objects (2-D objects, lines and pixels) one or more measurement parameter is set by default: for a pixel, the X and Y position, for a line or polyline, the length, for a circle, ellipse, rectangle or a closed polygon, the area and the perimeter. Here you see the list of the measurement results. For each measurement object the default defined measurement parameters are displayed. You can change these default settings and display further measurement parameters. You can also fade out parameters which are of no interest to you. To do this, activate the Select Measurements dialog box by clicking on the Select Measurements button in the Measurement tool window. When measuring certain objects, such as a circles, numerous values are measured simultaneously. In this case, the value that comes first in the Select Measurements dialog box will be displayed in the image. In this window you can also change the order of the measurement parameters, so that for example in the case of a circle, the area is no longer displayed in the image but the perimeter. You can find a description of all the settings options in this dialog box in the online help. 55

58 Processing images Processing images The Process menu offers you several functions for processing images, with them you can change the contrast or the colors of an image. Every dialog box that is opened when you use a function for processing images is made up in the same way. Example of a dialog box which is opened when an image processing function is activated. 56 Select Processing Function Dialog box size Preview Area As you can see in the illustration above, a selection menu appears when you click the Select Processing Function button. The menu provides you with access to all other image processing functions. You do not therefore need to leave this dialog box if you want to activate another filter. However, you cannot carry out several image processing functions one after another. Because when you select another image processing function, you change the function and a previously selected function has no effect on the original image. You can drag the dialog box to any size you want. Doubleclick the dialog box's header to enlarge the dialog box to full-screen size. Clicking the header again will return the dialog box to its original size. As a rule, only one segment of the source image can be displayed in the dialog box. In both the image document and the image navigator, the segment of the image on display will be indicated by a white frame. The areas of the image not on display will be shown shaded.

59 Processing images In all image processing functions you can define any section of the image as a preview area. To do this, shift the selection rectangle to the required location in the image. Only this position will be displayed in the dialog box. Preview options Use the zoom buttons in the dialog box to make the image segment on display larger or smaller. You can also use the mouse directly in the image window, to change the position and size of the preview area. Please note that you can also change the size of the dialog box. The size of the preview area changes at the same time. When you carry out a filter operation, you can select one of several preview options. The image itself, will only be changed when you confirm the filter operation with the OK button in the Filter: <filter name> dialog box. If you quit the preview using the Cancel button, the image remains unchanged. PLEASE NOTE Original & Preview displays the same image segment twice in the dialog box. The first one shown is the source image. The second is the image that results when the current parameters are used. The image segment you have selected will be indicated in the image window by a white frame. The rest of the image will be shown shaded. Only Preview displays only the image segment with the resulting image in the dialog box. Preview in Image shows the filter results directly in the image window. The dialog box no longer contains a preview window. Then, only the controls will be shown. No Preview does not show a preview at all. You can configure and directly apply the filter when you use this option. When you process a large image, the calculation takes some time. In this case, definitely use the default defined Original & Preview preview function to test the effect of the image processing in one image segment. Even processing an image segment, can, depending on the amount of data, take a long time. Watch the status indicator. It will show you when the processing operation has been completed. 57

60 Processing images Applying Filters You can find all available filters in the Process menu. This menu is only displayed when an image is loaded. 1) Load the selected image. 2) Select the required filter in the Process menu. The Filter:<filter name> dialog box will appear. 3) Select an outstanding image segment for the image preview. 4) Define the required parameters in the dialog box. The parameters are different, depending on the filter selected. You can find exact information about the parameters for each filter in the online help. 5) Click OK, to accept the current parameters and to execute the selected image processing function. Please note that the original image will be changed. No new image document will be created. You can, however use the Edit > Undo command to restore the source image. 6) Alternatively click the Default, button to return the parameter to a sensible initial value. Or click the Cancel button to cancel the operation and return to the way the image was originally displayed. Rotating and mirroring images Use the commands in the Image > Geometry menu to rotate or mirror the active image. 58 Rotate images Mirror images When you rotate it through 90, 180 and 270 an image's length/width ratio will not be changed. You can also enter other angles. To do this, select the Rotate... command and enter the degrees in the Rotate dialog box in the Angle field. In the process, the length/ width ratio will change. The result is that then there are image segments for which no image information is available. These sections will be filled out in white. You can mirror images on a selectable axis. To do this, activate the Mirror... command and select the required axis in the Mirror dialog box. The mirroring occurs without loss, i.e., all of the image's pixels will be retained, only their position changes. If you mirror twice along the same axis the source image will be restored.

61 Processing images The Mirror dialog box. On the left you can see the current display of the selected image segment. On the right you can see the preview. It shows how the selected image segment will be mirrored if you click the OK button. PLEASE NOTE Drawing objects are not rotated or mirrored. The image calibration is retained when the image is mirrored and rotated. 59

62 Commenting on images Commenting on images Adding drawing objects PLEASE NOTE The Drawing toolbar. The Select Drawing Objects button is active. You can add drawing objects to acquired images, for example, to draw attention to important image details. Added drawing objects do not overwrite any image information since they are written on a special drawing layer. To add drawing objects use the Drawing toolbar. If this toolbar is not displayed, access it by using the View > Toolbars > Drawing command. To work with drawing objects, you have to be in edit-object mode. To do this, click the Select Drawing Objects button on the Drawing toolbar. 60 Add drawing object 1) To switch to the edit-object mode, click the Select Drawing Objects button. 2) Click the desired button in the Drawing toolbar to insert a drawing object. The following drawing objects are available: Insert a line Insert an arrow Add rectangle (or square, if you hold down the [Shift] key), Add ellipse (or add circle, if you hold down the [Shift] key). 3) Add the drawing object by clicking on the required segment of the image. You add a line and an arrow by defining the starting and finishing point. You draw a rectangle or an ellipse to the required size using the mouse. When it has been drawn, the object will automatically be selected. You can then move it to the required position using the mouse. 4) You can then change the appearance of the drawing object. To do this, use the buttons in the Drawing toolbar or activate the Drawing Object Properties dialog box with a double click. 5) If you want to use the buttons in the Drawing toolbar, select the Line Color, Line Width and Line Style buttons, to set the line's characteristics, the Fill Color, Line Color and Line Width buttons, to set the fill color and the line color and width, for an ellipse or a rectangle. 6) If you want to change the properties of a drawing object in the Drawing Object Properties dialog box, doubleclick on a drawing object. The Drawing Object Properties dialog box will open.

63 Commenting on images 7) Carry out the required settings here. You can find a description of this dialog box in the online help. You can only define a fill pattern in this dialog box, since there is no icon for it in the Drawing toolbar. The Handle Drawings toolbar. Display/fade out and delete drawing layer All data applying to drawing objects will be written into a special drawing layer. Try to picture the drawing layer as a transparency which is placed over the image. Editing the drawing layer does not affect the image. You can make the drawing layer disappear temporarily or delete it, at any time. To do so, use the Handle Drawings toolbar. If this toolbar is not displayed, use the View > Toolbars > Handle Drawings command to have it displayed. If you have deleted the drawing layer by accident, you can undo the deletion by using the [Ctrl+Z] keyboard shortcut (as long as the image remains opened). Clear selection Select drawing objects Use one of the following methods to select a collection of drawing objects. Leftclick an object. While keeping the [Shift] key depressed, click other objects you want to edit. Objects you have selected will be indicated by white or gray markers. While keeping the left mouse button depressed, drag a rectangle to encompass several objects. All objects that are completely inside this rectangle will be selected. All the existing selections will be deleted. To select all of the drawing objects in a document, use the [Ctrl + A] shortcut. To clear the selection, click on any position in the background of the document with the left mouse button. 61

64 Commenting on images Copy drawing objects You can copy selected drawing objects onto the clipboard and then add them to other images. 1) Select the drawing object you want to copy. 2) Use the [Ctrl + C] shortcut to copy the selected drawing objects onto the clipboard. 3) Activate the image onto which you want to paste the drawing objects. 4) Use the [Ctrl + V] shortcut to paste the drawing objects into the position you have selected. The drawing objects will be inserted in the same position as in the source image. PLEASE NOTE Save images with drawing objects If you have inserted drawing objects onto an image, you will have to use the TIF format when you save this image. Otherwise the drawing objects will be burnt into the image. Editing drawing objects The Draw menu provides many possibilities for grouping, positioning and arranging drawing objects. The Draw menu offers you a wide range of functions for grouping, positioning and arranging drawing objects. To open the menu click the Draw button. You will find it on the Drawing toolbar. 62 A detailed description of all functions can be found in the online help.

65 Commenting on images Insertion of text objects 1) Click the Text button in the Drawing toolbar. 2) Leftclick the position where you want the top left hand corner of the text object you want to insert, to be. 3) Enter the required text. You can write as many lines of text as you want. However, only the lines that are within the text object will be visible. All the lines that do not fit within the text object remain hidden from view. The software determines the default font for a new text object. You can subsequently change it as you wish. 4) Click once outside of the text object, to leave the text-entry mode. The text object will be displayed. If you want to change the text once again, doubleclick on the text object. Text-entry mode and layout mode Immediately after you have inserted a text object you will be in the text-entry mode. In the text-entry mode you can write as many lines of text as you want and edit them as required. You recognize the text-entry mode by the fact that the mouse pointer has the form of the text cursor (narrow black line). However, if you want to change the size, position or background color of a text object, you must be in layout mode. To switch from the layout mode into the text-entry mode, doubleclick the text object. PLEASE NOTE Formatting text objects 1) Select the text that you want to format. 2) Use the Text Color button to set the color for the selected text. 3) Use the Fill Color, Line Color and Line Width buttons to set the fill color, and the line color and width, for the text object. 4) Use the Formatting Font toolbar to change the font type and font size. Select more than one text object and you can change the font characteristics (type, size and color), background colors and text frames, for all of these objects simultaneously. 63

66 Printing images Printing images To print an image, proceed as follows: 1) Select the File > Print... command. Alternatively, use the File > Print Preview command, to open a print out preview, then click the Print button. The Print Image dialog box will open. 64 When printing an image the page layout defined on the tab File > Page Setup > Single Image is used automatically. 2) Select the required settings. Use the Printer group to change your printer's settings for the printout. Please note that these settings are only valid until you close your image analysis program. The default operating system settings will be adopted again at the next start of your image analysis program. Select the Print to file check box to first save the complete document that you want to print out in a print file. In the Frame width field, enter the width of the frame which is to be drawn around each image. The unit in use is 1/4 point. For no frames, enter 0. In the Number of copies field, enter the number of printed copies of the image that are to be printed. Click the Page Setup...button to open the Page Setup dialog box, if you want to change the page layout, e.g. make new entries in the header or footer, or change the number of images per page. Further information is available in the online help. 3) Click the OK button to start the print out.

67 The user interface: components The user interface: components Standard and Expert mode The components of the user interface are to a great extent configurable, so that they can be easily adapted to meet the requirements of individual users and tasks. Whether you are in the Standard or Expert mode is the decisive factor as to which possibilities you have to change the appearance of the user interface. The Standard mode is set by default. Tool windows and the document group cannot be dragged out of the user interface. The Expert mode provides you with many possibilities for changing the user interface. In the Expert mode you can, for instance, drag all of the tool windows, just as you do a normal dialog box, from their position on the right or left edge of the user interface, to any position on your screen. As well as that, the document group can be positioned anywhere on your screen. Switching the mode Select the Tools > Options... command to change the mode. Select the Environment entry in the tree view and select the desired option in the User interface group. The document group The document group contains all opened documents. These may be images or texts. Here for example, you can find all of the images you have acquired during a work session, and have not yet closed. The live-image and the resulting images, e.g., following filtering, are also displayed here. By default, there is only one document group. However, you can easily add new document groups. An individual tab will be set up in the document group for each image or text document. The document created last will always be shown at the end of the line. You can easily change this order by clicking on the document's name and dragging it to the left. In this example, there are four images loaded in the document group. The image "RGB.tif" is on display. 65 Click on the name of a loaded image to display it in the document group. The name of the image on display has an orange background. In front of the image name, an icon will show you what type of image it is.

68 The user interface: components An icon in front of the image name, shows you what type of image it is. 24-bit image multi-dimensional 24-bit image gray-value image multi-dimensional gray-value image Closing documents Buttons in the document group In the Standard mode there are three buttons at the top right of the document group. The buttons with an Arrow are at first inactive, when you start the program. Only when you have opened so many documents that all of the tabs can no longer be displayed in the document group will they become activated: Then you can click one of the two arrows to make the list of tabs scroll to the left or the right. That will enable you to see the tabs that were previously not shown. Click on the button with the cross to close a document that is open (image or text). If it has not been saved, you will receive an appropriate warning message. close the program close a document PLEASE NOTE You close the image analysis program itself in the same way as other Windows programs, by clicking on the cross in the top right corner of the program header. If you have documents which have still not been saved, an appropriate enquiry will appear. In the Expert mode the document group contains a fourth button that looks like a hand. Drag this button to another place on the user interface or to a second monitor, to close the document group. See also p PLEASE NOTE Possibility 1 Creating a new document group The setting up of a second document group makes sense when you want to compare two images on the monitor. Then load an image into one document group and the other image into the newly-set-up document group. To be able to set up a new document group, you will need to have loaded at least two documents. There are two ways of setting up a new document group: via the Window menu, or interactively with the docking aid. 1) Select the Window > Split/Unsplit > Document Group command. The entry in brackets next to the Document Group indicates in which position the new document group will be set up, for example, to the right of, or below, the active document group. The document group will be created.

69 The user interface: components On the left you see a document group with three loaded documents. On the right you see what happens when a new document group has been set up. Possibility 2 1) Click on the title of the document that you want to move. 2) While keeping the left mouse button depressed, move your mouse to the center of the document group. " The mouse pointer will change shape. " Orange triangles will appear in the document group. 3) Move your mouse up near to these triangles. " A dashed frame will show you where, in each case, the new document group can be set up. 4) Move your mouse to the position you decide on, then release the mouse button. " The new document group will then be set up in that position. 67 If you want to set up a new document group interactively with the docking aid, move you mouse pointer near to one of the orange triangles that have appeared. Then a dashed frame will show you the places where it is possible to set up your new group. Merging two document groups Here too you have a choice of two possibilities. Possibility 1 Select the Window > Split/Unsplit > Document Group command. " The two document groups will be merged.

70 The user interface: components Possibility 2 PLEASE NOTE Select the document you want to move, and drag it with the left mouse key depressed onto the other document group. The two document groups will be merged. By using either of the possibilities described above you will merge the documents permanently. As an alternative, you can also dynamically switch between the display of one or more document groups. If you want to do this, go to the Window menu, and use the Window > Toggle Document Layout command. This command is only available when at least two document groups are present. Every time you use this command, all of the document groups that are present will either be merged or separated. Instead of using the Window > Toggle Document Layout command, you can also use the button with the same name, in the Window toolbar. See the illustration on p. 72. Shifting a document to another document group You can shift documents into another document group. 1) Select the image you want to move by leftclicking on the tab with the image name. 2) Drag the document into the document group you want it to be in. A docking aid is displayed. 3) Release the left mouse button. The document is then located in the other document group and will be displayed. 1 start 2 action 3 result 68 PLEASE NOTE If you keep the [Shift] key depressed while you move the document into the other document group, you will create a new document group in the process.

71 The user interface: components 1 start 2 action (with Shift-key pressed) 3 result NOTE Floating a document group The document group is normally docked at a defined position in the user interface. You can, however, change this position, provided that you are in the Expert mode. In the Standard mode the corresponding commands are not available. It makes sense, for example, to float the document group so as to drag it onto the other screen if your system is equipped with two monitors. 1) Click the button with the hand or click the document's header and move your mouse a little upwards or downwards. The mouse pointer changes into a folder icon. Alternatively, you can also use the Window > Float/Unfloat Document Group command. 2) With the left mouse button depressed, move the folder icon to any position outside the document group. A dashed frame shows you where the document group will be moved to if you release the mouse button. 3) Release the left mouse button to confirm the current position of the document group. 4) To dock the document group once again, click on the hand icon again and drag the document icon back to its original position. 69

72 The user interface: components Here you see a screen arrangement in which the document group has been dragged out of the user interface. It is displayed as its own window, to the right of the user interface. image analysis program document group 70

73 The user interface: components Toolbars The toolbars illustrated below are available. Use the View > Toolbars command to make specific toolbars appear or disappear. You can display an explanation of the function for each button in the toolbar, by moving the mouse pointer over the button. A small tooltip then appears: Manual Exposure At the same time, you can see a complete explanation of the function in the status bar. PLEASE NOTE Toolbars are by default located ("docked") at a specific place on the user interface. In this mode the toolbar header will not be displayed. You can, however, remove the toolbar from its predefined position at any time, and place it anywhere you want on your monitor. Or you can dock the toolbar at another position, e.g., on the left hand, or right hand, edge of the screen. Whether a toolbar is to be displayed or not, and where it is to be displayed, will be saved separately for each layout. You can, therefore, decide, for instance, that you want to show the View toolbar in the Acquisition layout, but not in the others. Toolbar overview Standard Window With the Standard toolbar you can administer files (create, open, save, print) and cut or copy and paste text and image segments. You can also redo and undo commands and access the online help. 71 With the Window toolbar you can manage document groups (split and unsplit document groups, float and unfloat the active document group, toggle between several document groups (in the expert mode), and synchronize image documents). Use the Synchronize image windows button to synchronize images. When using the synchronization process, you go to one image document and select the image segment that interests you, and that you want to have displayed on your monitor. Your image analysis program will then automatically display the same segment in the other synchronized image documents. The synchronization process is especially useful when you want to compare several images. Further information is available in the online help.

74 The user interface: components In the left image you can see four document groups, each with one image. The Float/Unfloat Document Group button This button will only be on display when you are in the Expert mode (see p. 65). With it you can change the position of document groups and, for example, drag them onto another monitor. Further information can be found in the chapter "Floating a document group" on page 69. The Toggle Document Layout button This button is only available when several document groups have been opened. A click on the button combines all documents which have been opened in different document groups, to form one document group. In this way more space is provided on the screen for the image display. Another click on the button displays the individual document groups again. The right image shows the view following a click the Toggle Document Layout button. Now the images from the document groups have been combined to form one document group, recognizable by the four tabs. In this way each image can be displayed larger. 72 Microscope Control It is possible to synchronize image documents. When using the synchronization process, you go to one image document and select the image segment that interests you, and that you want to have displayed on your monitor. Your image analysis program will then automatically display the same segment in the other synchronized image documents. The synchronization process is especially useful when you want to compare several images. Another click on this button deactivates the synchronization. You can find further information about the synchronization of images in the online help. Using the Microscope Control toolbar you inform the image analysis program which objective you will use when acquiring an image. To do this, click before the acquisition on the button of the objective used. This information is very important for the correct calibration. The data regarding which buttons are to be displayed in this toolbar will be read from your hardware configuration. Thus you are only offered buttons for the objectives which are configured in the Nosepiece Properties dialog box. Therefore, if your microscope only has three objectives, the toolbar will only contain three buttons to choose objectives from.

75 The user interface: components View Drawing With the Tile View toolbar you can quickly activate and deactivate toolbars and tool windows if you do not want to use the View >Toolbars or View >Tool Windows menu. The buttons in the Drawing toolbar are only active when you have loaded an image. It makes a variety of drawing functions (Line, Rectangle, Ellipse) available to you, as well as options for color selection and line styles. See also "Commenting on images" on page 60. To draw a circle, click the Ellipse button and hold the [Shift] key depressed while you draw the shape. To draw a square, click on the Rectangle button and hold the [Shift] key depressed while you draw the shape. Formatting Font Handle Drawings The buttons of the Formatting Font toolbar are only active when you have clicked the Text button, in the Drawing toolbar. In this case you can format the text entered here. Toolbox Also, all the elements that you draw using the Drawing toolbar are not written directly into the image, but rather into a separate drawing layer. In this way, no image information is overwritten. With the buttons in the Handle Drawings toolbar you can hide and delete these drawing objects. In the Toolbox toolbar you find various functions which support you when you view images: In the Pan Mode your mouse pointer will take on the shape of a hand. If you click on the left mouse button, the mouse pointer becomes a fist. With it you can grab an image and shift it within the image document. This is only possible if the image is not fully displayed within the document group. In the Zoom Mode you can enlarge an image in small steps by clicking the left mouse button. A click on the right mouse button reduces the image's size once again. With the Magnifying Glass button, you can view image details with a magnifying glass. If you click on this button, the mouse pointer becomes a magnifying glass. Move the magnifying glass onto the image detail that you wish to see enlarged, then click the left mouse button. The section will then be shown in four times higher magnification than the rest of the image. With the Select Drawing Objects button you can select objects. These can all be objects that you have drawn into the image with the Drawing toolbar (texts, ellipses, lines, etc.). Click on the object to be selected. You can select several objects by holding the [Shift] key depressed while you select them. 73

76 The user interface: components Zoom With the Rectangular Marquee button you can draw a rectangular frame with the mouse and thus select an interesting image segment. You can copy, cut or delete this image segment. To do so, use the commands of the context menu. If you delete the segment, a white area appears in its place. Click on the selected segment again to deactivate the selection. The Rectangular Marquee button must have been selected when you do so. You cannot deactivate the selection if you have switched to another mode in the meantime (e.g., Zoom mode). With the Crop Image button you select the square image segment that you want to keep. All of the image information outside the rectangle will be discarded. Use this button, for example, to cut borders that you do not need off an image. Before the crop is carried out, you must confirm your selection with the Confirm Input button. Alternatively, you can cancel your selection with the Abort Input button. Use the Confirm Input and Abort Input buttons to confirm or discard the selection of a segment that you have defined on the image. You may have very different reasons for selecting an image segment. For example, because you want to cut or copy an image segment. Or because you want to define the size of a subarray. In any case you must click on one of the buttons to confirm or discard your selection, since your image analysis program will wait for input here. Tile View The buttons in the Zoom toolbar relate only to images. If a text document is displayed they are inactive. You can zoom an image up and down step-by-step, zoom up to the original size (button Actual Pixels) and zoom to a size whereby the image can still be completely displayed in the image document (button Fit to Window). 74 Use this toolbar to gain an overview of all of the frames making up a multi-dimensional image. To do so, click the Tile View button. In the tile view all of the individual images making up the multi-dimensional image that is active will be displayed, if they belong to the selected dimension. Click on one of the frames so as to activate this image. The active image is marked with a colored frame. When you then switch to the single view, the image you have activated will be displayed on your monitor.

77 The user interface: components Measurement Selecting a dimension You can choose the dimension that is to be displayed in the tile view from the list. The following abbreviations will be used in it: C = Color channel. Shows the individual color channels that make up a multi-channel image. T = Time. Shows all of the frames that make up a time stack. Z = Z stack. Shows all of the frames that make up a Z stack. If the active image has several dimensions (e.g. a time stack that has several color channels) the list will also show combined entries. See also "Creating and viewing multi-dimensional images" on page 35. Devices This toolbar is also part of the Measurement tool window. Therefore, when you measure images and have the Measurement tool window on display, you do not need to also make the Measurement toolbar appear. In other cases it may be a good idea to work with the Measurement toolbar only. For instance, if you need as much of the monitor space for image display as possible and if it is okay for you that the measurement results can only be seen in the image. You will find a detailed description on how to measure images in the chapter "Measuring images" on page 47. The Measurement toolbar that is part of the Measurement tool window has some additional buttons with which you can access the measurement parameters and measurement options. See also "The Measurement toolbar" on page 49. Camera Control This toolbar enables you to you quickly access and change the calibration and configuration data of your system. Clicking the Calibrations, Optical Path or Device Settings button will open the respective dialog box where you can change your system configuration. The Devices toolbar is not shown by default. You can have it displayed when you need to change your system configuration on a regular basis. Further information can be found in the chapter "First time configuration of the system" on page Change Language The Camera Control toolbar contains the most important settings you need to configure how single images are to be acquired. It contains some of the functions offered in the Camera Control tool window (switch live-image on/off, acquire image, change exposure mode and set exposure time, carry out white balance, open Camera Control tool window). Use this toolbar if you do not need all the functions offered in the Camera Control tool window. The Change Language toolbar is only available if your image analysis program has been installed on a system on which Asian fonts are installed. With it, you can change the language your image analysis program uses. English and Japanese are available. Clicking on one of the buttons will switch the language. Some software controls, e.g., the menus, will be immediately shown in the selected language. However,

78 The user interface: components DP Controller please note that you must restart your image analysis program to completely switch all controls to the new language. You will receive an appropriate message when clicking on one of the buttons. This toolbar will be displayed by default, when during the installation you selected the DP-Controller Style as your acquisition style (see illustration p. 10). This acquisition style is intended for users who have previously used another image analysis program, and now want to change over to this image analysis program. Changing toolbars When delivered, certain toolbars are displayed in each layout. These contain the most frequently used buttons. This standard configuration can be easily adjusted as required. To do this, you have the following options: You can hide or show buttons. You can add new buttons to the existing toolbars. You can set up a completely new toolbar and equip it with the buttons you require for your work. 76 Here you can see the three steps necessary to hide a button (using the example of the Toggle Document Layout button, which is to be removed from the Window toolbar). Hiding buttons 1) To make it easy for you to add or remove buttons, all toolbars have a small arrow either on their right, or on their lower edge. Click on this arrow and select the Add or Remove Buttons command. You will then be offered all of the toolbars docked in that area of the screen. 2) Select the toolbar you want to change. 3) Click on the field in front of the button which you want to hide. The check will disappear and the button will no longer be displayed on the toolbar. Showing a button again 1) Repeat steps 1 and 2 from the section "Hiding buttons" and select the check in front of the button that you want to show again. Adding new buttons and commands to a toolbar If you need even more buttons for your work, you can select them and add them to a toolbar of your choice.

79 The user interface: components In the example shown, the Align Top button is to be added to the Drawing toolbar. 1) Click on the small arrow on the right or lower edge of the toolbar and select the Add or Remove Buttons > Customize command. Alternatively, you can select the Tools > Customize... or View > Toolbars > Customize... command. The Customize dialog box opens, the Commands tab is active. 2) In the Categories list, select the command group from which you want to add a new command as a button. 3) In the Command list select the required command and drag the command onto the required toolbar. If a command is matched to an icon, this will be added to the toolbar. In other cases see the command text. See also "Examples for new or adjusted toolbars" on page 78. The view will be updated. 4) Close the Customize dialog box. 5) When you end the program you will be asked if you want to save the configuration. Answer this question with Yes and give the configuration a name. The new configuration will be loaded automatically at the next program start. This is carried out by dragging the icon out of the Customize dialog box into the required toolbar. 77 Setting up a personal toolbar You can also create a personal toolbar and add all buttons or commands to it that you consider necessary. 1) Select the View > Toolbars > Customize... command. 2) Select the Toolbars tab in the Customize dialog box. 3) Click on the New button and give the new toolbar a name. The new toolbar will appear in the top left-hand area of your screen. Since it does not yet contain any buttons, it can easily be overlooked. Look out for a toolbar that's empty, like the one shown on the left.

80 The user interface: components 4) Go to the Commands tab. 5) Select the command group in the Categories list from which you want to add commands as buttons to your toolbar. 6) Select the required commands in the Commands list and drag them to your personal toolbar. Your toolbar will be set up. You can also fill the toolbar by pressing the [ALT] key and dragging the required buttons from the other toolbars into your toolbar. The buttons from the other toolbars will be moved out of position. If you also press the [Ctrl] key while dragging the buttons, they will be copied. 7) When you end the program you will be asked if you want to save the configuration. Answer this question with Yes and give the configuration a name. The new configuration with the personal toolbar will be loaded automatically during the next program start. Your personal toolbar can also be hidden or displayed like other toolbars. You can see examples of customized toolbars. These can contain command texts or icons, or both. Examples for new or adjusted toolbars 78

81 The user interface: components Tool windows Tool windows combine functions into groups. These may be very different functions. For example, in the Properties tool window you will find all the information available on the active document. You will find the tool windows by default docked on the left and right of the document group. To save space, several tool windows may lie on top of each other. They are then arranged as tabs. In this case, activate the required tool window by clicking the corresponding tab below the window. Here you can see 3 tool windows lying on top of each other. The Documents tool window is currently displayed. Click on a tab to display one of the two other tool windows. Which tool windows are shown as a standard depends on the layout you have chosen. You can adjust the default settings at any time so as to display or hide single tool windows for a layout. To do so use the View > Tool Windows command. Making a tool window appear and disappear 79 Manually displayed or hidden tool windows will be saved automatically and are available again the next time you start the program. A return to the original layout (View > Layout > Reset Current Layout button) will have the result that only the tool windows that are defined by default will be displayed. Docking a tool window You can remove a tool window which is grouped as a tab with other tool windows at any time from the common window, and then for example assign it to another group of tool windows.

82 The user interface: components PLEASE NOTE In this example, the Acquisition Process, Adjust Display and Camera Control tool windows have been faded out with Enable/ Disable Auto Hide. To do this, you must be in Expert mode (default setting), see p. 65. Do the following: 1) Drag the tool window over another tool window, e.g., on the right hand border of the user interface. The docking aid above the tool window now appears as illustrated on the left. 2) Move the mouse pointer to the center of the docking aid then release the mouse button to have the tool window docked as a tab over the tool window you have already docked there. If you press the [Ctrl] key while shifting the tool window, the docking aid will be faded out. You can then move the tool window, as a freely movable window, into any position you wish. Minimize tool windows The Enable/Disable Auto Hide button is available in every tool window. A click on the button minimizes the tool window, on the edge of the screen a mouseover zone appears with the name of the tool window. As soon as the mouse moves across this zone, the tool window is displayed and you can define the settings as usual. If several tool windows are arranged over each other, all of them will be minimized. As the mouse pointer has been positioned over this button, the Camera Control tool window is currently on display. 80 You can show the other two tool windows by positioning the pointer over the button. The advantage is that the tool window can then be displayed over the whole area that otherwise would be shared by several tool windows. In this way the display is very clear.

83 The user interface: components Showing a tool window again A renewed click on the Enable/Disable Auto Hide button will make the tool window appear in its original size and position once more. Alternatively, you can also use the View > Layout > Reset Current Layout command to do this. Description of the tool windows Depending on the selected layout different tool windows are displayed by default. Also the position where the tool window is shown may vary depending on the layout. These differences derive from the fact that each layout supports the user during the completion of different tasks. The layouts show a sensible selection of tool windows on display. You can, however, always change this display and add additional tool windows. To do so, use the View > Tool Windows command and select the tool window you want to have shown. The Image Navigator The image navigator is used to control which image segment will be displayed in the image document. It shows a thumbnail of the active image. On the thumbnail is the red navigation frame. As the image is always shown completely after being acquired or opened, the navigation frame initially has the same size as the image itself. In the case of a higher zoom factor, the navigation frame becomes accordingly smaller. It shows the section of the image that is currently on display in the image document. If you shift the navigation frame in the Image Navigator tool window to another image segment, the display in the image document will be updated. When loading an image, the navigation frame includes the entire image. navigation frame 81

84 The user interface: components When viewing an image with a high zoom factor the navigator frame only includes a part of the image. Only this image segment is shown in the document group. navigation frame Adjusting zoom factors for image display Your image analysis program offers several possibilities for adjusting the zoom factor at which an image is displayed. 1) In the image navigator, draw the frame to a different size using the left mouse button. As soon as you release the mouse button, the zoom factor with which the selected image section is then displayed in the document group, will be shown. The zoom factor is automatically adjusted, so that the section within the frame fills the image document exactly. 2) Enter the image's zoom factor directly in the edit field in the Image Navigator tool window and press [Enter], or use the slide control. change size of navigation frame 82 enter zoom factor and press [Enter] change position of the slide control 3) Use the buttons in the Zoom toolbar to set the size at which the image is shown in the image window. 4) Use the commands of the context menu to adjust the zoom factor.

85 The user interface: components PLEASE NOTE The current zoom factor is also displayed on the bottom right in the status bar. The Gallery view The gallery view permits a rapid overview of all the opened documents. Doubleclick on a thumbnail to display it in the document group. This is important when you have opened many images and are looking for a specific image. The thumbnails then facilitate fast orientation. The gallery view permits fast orientation through all opened documents (images and texts). You can limit the types of document displayed. To do so, position your mouse pointer on a free area within the gallery, rightclick, then select the Images Only command. Then only images will be displayed in the gallery view. Open the context menu again, and select the Show All entry, to once more have all of the loaded documents displayed. Should you like to have the gallery view on display in any of the layouts, use the View > Tool Windows > Gallery command. If you like to, you can minimize it by using the Enable/Disable Auto Hide button (on the right part of the gallery's title bar). Properties Fields always displayed Information in the Properties tool window in the Document group. On the left you can see the information when a text document has been loaded, on the right when an image which has been acquired with your image analysis program has been loaded. In the Properties tool window you see information about the displayed document. The details in this information may vary depending on whether you are dealing with an image or text document and whether the image was acquired with this software or not. The Documents group is displayed for every type of document. It contains important information regarding the loaded document. Depending on the document opened, different information is displayed. 83 Entering a note on an image How to open the Note dialog box. If you want to write a note on a document, doubleclick on the [...] button in the Note field (in the Properties tool window). Then, enter your comment in the Note dialog box.

86 The user interface: components The text in the Note field of the Camera Control is automatically copied into the Note field of the image properties. Here you can add, edit or delete it. In the Camera Control tool window, you can enter a note in the Note field, too. If you enter a comment here, it will immediately be saved with all images which you acquire. Then you will no longer need to copy or continually enter, a note that you wish to add to a great many images. As opposed to that, the comment that you enter in the Properties tool window is only valid for the image for which you are currently editing or looking at the properties. If you have entered a note in the Camera Control tool window, you can edit it here. Copy note contents 84 Additional information with images You can print a comment along with the image. Further information is available in the online help. With all images that you have acquired with your image analysis software you will find comprehensive additional information. This information is saved with the image if you save it in TIF format and then redisplayed when the image is loaded.

87 The user interface: components Information shown in the Properties tool window when an image that was acquired with your image analysis program is active. Further information is available in the online help. Documents The Documents tool window provides a fast overview of all the opened documents. 85 You can choose between a tree view display (left illustration) and a list display (right illustration). The Documents tool window provides you with an overview of all opened documents. These may also be documents which have not yet been saved. Use this tool window if you have opened a lot of documents and require a fast overview.

88 The user interface: components Display document Selecting several documents Additionally, you can easily administer the opened documents in the Documents tool window: Doubleclick on an entry in the document structure to display the document in the document group. The standard Windows conventions, which you probably know from the Windows operating system, are valid for document selection. Commands such as for example, "Save", are valid for all selected documents. Selecting single, non consecutive documents: Selecting a series of documents: Selecting all documents and clearing some documents: Selecting all documents Hold the [Ctrl] key depressed and click on the required documents. Hold the [Shift] key depressed and click on the first and last file that you want to select. Hold the [Shift] key depressed and click on the first and last file that you want to select. Then hold the [Ctrl] key depressed and click the documents that are to be cleared. Use the [Ctrl] + A keyboard shortcut (For this the Documents tool window must be active, i.e., its header displayed in orange). 86 Saving all documents Display of 6 documents in the Documents tool window. By the missing file endings and the asterisks you can see that the image "Image_03" has not yet been saved. All opened documents can be easily saved by changing to the tree view if required, selecting the Opened documents entry and by clicking on the Save button in the Standard toolbar. With this command all opened documents will be saved. For the documents that were not previously saved, a dialog box, in which you can define their storage location and file names, will appear. You can recognize a document that has not been saved by its missing file name extension, and when it's an image, by the little asterisk behind the file's name. Closing a document You close single documents by selecting the document, clicking the right mouse button, and selecting the Close Document command in the context menu. If you have not yet saved the documents, you will receive an appropriate message. If you want to close all documents of one particular type, access the tree view and select either the Images or Text entry. Then press the right mouse button and select the Close Document command in the context menu. All documents of this type will be closed. Here too, you will receive a warning message in the case of unsaved documents.

89 The user interface: components The File Explorer This tool window is not on display by default. If required, display it by using the View > Tool Windows > File Explorer command. Use the File Explorer to quickly load images which are located on your hard disk or on external storage media. Similarly to Windows Explorer, the File Explorer helps you to navigate quickly through complex directory structures, until you reach the required image. This will be loaded into the active document group when you doubleclick on the image, or drag it into the document group. The file explorer is divided in two: In the upper area you see the directory tree, in the lower, the documents that are contained in the selected directory are shown. PLEASE NOTE As opposed to the Documents tool window, you can use the File Explorer to physically delete files from your hard disk. To do so, select the file and select the Delete command in the context menu. You will receive a warning message before the image is deleted. 87

90 The user interface: components Acquisition Process Use this tool window to configure how a time stack is to be acquired. Clicking the Start t-stack button will start the acquisition. You can find further information under "Acquiring a time stack" on page 29. Measurement Use the Measurement tool window when you want to measure images. The toolbar in the Measurement tool window contains numerous important preferences you can use when you are interactively measuring. This tool window is at the same time the measurement display and contains all of the values that have been measured on the active image. To enable you to see the list of the measurements properly, the Measurement tool window should be docked on the lower edge of the user interface. Further information on measurements can be found in the chapter "Measuring images" on page 47. The Measurement tool window displays the measurement results. 88

91 The user interface: components Channels PLEASE NOTE Appearance of the Channels tool window when a multi-channel image is loaded. Use the Channels tool window to stipulate how a multi-channel image is to be displayed on your monitor, or to change this. A multi-channel image shows, as a rule, a sample that has been excited with several different fluorochromes. The multi-channel image is made up of a combination of the individual fluorescence images. You will find an illustration of a multi-channel image on p. 35. RGB images are not multi-channel images. Although you can also display the red, blue and green color channels here, it is always the same image, the 24 bits of which are displayed as three separate 8-bit color channels. A multi-channel image, on the other hand, consists of an arbitrary number (minimum: 2) of individually acquired images which are retrospectively combined to form one image. Switching color channels on/off You can set which color channels, resp. combination of color channels, will be displayed on your monitor. The tool window lists all of the color channels that are in the active image. All of the color channels that are at the moment displayed on your monitor will be indicated by an eye icon. 89

92 The user interface: components Display of a multi-channel image when only two color channels are selected: In the upper image the red and green channels are selected and in the lower image the red and blue one. To have a color channel switched off, click its eye icon. Click an empty cell without an eye icon, to have the corresponding color channel switched back on. Click the first row, to have all color channels displayed. On your monitor the colors of the individual channels will be superimposed. For this reason, image segments which are shown both in the red and in the green color channel are displayed in yellow. 90 Changing the image display The individual images that make up a multi-channel image are gray-value images. Every individual image can be displayed in color. Open the menu next to the Choose Color Mapping button, to be shown a selection of different color tables you can use to color the separate images. Select the Show Fluorescence Color menu entry, to have every separate image displayed in its fluorescence color. The active fluorescence color will be displayed for every channel, in the color field next to the channel's name. To change the active color click this color field. Select the Show Grayscale menu entry, to have every separate image displayed as a gray-value image. In a superimposed image the gray values will be added together. The superimposed image can, therefore, be over exposed in those segments where all of the individual images are light. Select the Show Pseudo Color Table menu entry, to load the currently set pseudo color table for the multi-channel image. You can use pseudo color tables to have gray-value images displayed in color on your monitor. Then every gray value will be allotted a hue. All of the separate images belonging to the multi-channel image and the superimposed image will be displayed in colors from the same palette.

93 The user interface: components In this display mode, click the field to the right of the superimposed image to open the Pseudo Color dialog box. In this dialog box you can change the active palette for the multi-channel image, or define a new one. PLEASE NOTE You can switch from any one of the display modes to another at any time. The settings for the fluorescence colors will not change when you assign a pseudo color table. Saving multi-channel images When saving a multi-channel image you also save the information about how the image was displayed. This refers to which color channels had been selected (only these will be displayed again by default when reloading the image), which color mapping had been selected (Choose Color Mapping button). Stack The Stack tool window defines the display of an image stack on the monitor. You can browse through the image stack or animate it. An image stack can be a time stack, a Z-stack or also an AVI video. In a time stack all of the frames were acquired at different times. A time stack shows you how the section of the sample changed with time. A Z-stack contains frames acquired at different focus positions. 91

94 The user interface: components The Stack tool window displays how many frames make up an image stack (see arrow). Depending on whether it is a time stack (left) or a Z-stack (right), the display is slightly different. The Stack Options dialog box You can still change the appearance of the Stack tool window in the Stack Options dialog box. Change play back time 92 Display the selected images Setting play back time By default a time stack is played back at the rate at which it was acquired. Frequently however, this rate is unsuitable for later examination (e.g., when images were acquired every 30 minutes). In this case you can adjust the rate. In the Frame Rate group, you determine how many frames are to be displayed per second. The unit is 1/s (frames per second).

95 The user interface: components Display the selected images In the Stack Options dialog box you define, amongst other things, whether the images which are selected are to be displayed in the lower section of the tool window. If the Show "selection summary" in tool window check box is selected the entry "Time: 1-5; 9-11/61" indicates that a time stack with a total of 61 images has been loaded. The frames 1-5 and 9-11 have been selected. You can find a detailed description of the additional setting options in the Stack Options dialog box in the online help. Browsing through image stacks The Stack tool window lists all of the frames contained in the active image stack. The frame that is at that moment displayed will be identified by an eye icon. You have several possible ways of selecting which frame is to be displayed on your monitor. Click an empty field next to the thumbnail, to have the corresponding image displayed on your monitor. Move the slide control in the tool window. The eye icon will always show you which frame is currently on display. Animating image stacks Use the toolbar in the Stack tool window to animate an image stack. You will then always animate only the images in this stack that have been selected. Selecting frames The Stack tool window lists all of the frames contained in the active image stack. To select a frame, simply click the thumbnail. It will then be highlighted in color in the tool window. Should you want to select more than one frame, use the following commands for this purpose: Selecting several, non consecutive, images: Hold the [Ctrl] key depressed and click on the required documents. Selecting a series of images: Hold the [Shift] key depressed and click on the first and last file that you want to select. 93 Selecting all images and clearing some of them: Selecting all images Hold the [Shift] key depressed and click on the first and last image that you want to select. Then hold the [Ctrl] key depressed and click the images that are to be cleared. Click once on the topmost entry in the list, or use the [Ctrl] + A keyboard shortcut.

96 The user interface: components Processing image stacks Image processing functions (e.g., the use of a filter) either have an effect on the entire image or only on a selection of frames. If you only want to use the function on selected frames, then do the following: 1) Select individual images that you want to edit in the Stacks tool window. 2) Select the Target > Selected Frames and Channels option in the processing function's dialog box, to determine that the function only affects the selected frames. PLEASE NOTE When you save an image stack its current appearance on the monitor will also be saved. This is to say, that when you load an image stack, the frame that was selected at the time the stack was saved will then be the one displayed on your monitor. 94 The Camera Control In the Acquisition layout, this tool window is available by default. Here you make important settings regarding image acquisition. Further information is available in "Acquiring images" on page 16. PLEASE NOTE For the description of the Camera Control, a sample camera was used. Since certain settings are, however, camera specific, (e. g., the available resolutions), this tool window can have a different appearance when another camera is connected to the image analysis program. Live-image display and image acquisition is started with a click on the Live and Snap buttons. Using these buttons you can: Switching the live-image on and off without acquiring an image Switching to the live-image and acquiring an image

97 The user interface: components In the Camera Control tool window you find the Live and Snap buttons, with which you can activate and deactivate the live-image. 95 PLEASE NOTE Switching the live-image on and off without acquiring an image 1) Click on the Live button. The temporary document Live (active)* will be set up in the document group. In this document the live-image is displayed. The Live button changes itself into Stop Live. 2) Click on the Stop Live button. The live-image will be deactivated. You can change the behavior of the live-image. Further information can be found in the chapter "Changing the behavior of the live-image" on page 100.

98 The user interface: components Acquiring an image immediately Switching to the live-image and acquiring an image 1) Click on the Live button. The temporary document Live (active)* will be set up in the document group. In this document the live-image is displayed. 2) Click the Snap button. The live-image will be switched off and a new image document will be created. This image has the default name which is set in the Camera Settings > Acquisition Document Name dialog box in the Prefix for images field, together with a serial number. You can rename and save the image. If you have not already saved it when you end your software, you will be asked if you want to do so. When you have optimally set up your sample on the microscope you can acquire an image immediately, without having first viewed it in the live-image. To do so, click the Snap button. Buttons in the Camera Control At the top of the Camera Control tool window you will find several buttons. Some of these are only active in the live-image. Shift Metering Regions Line Profile Maximize Live to Screen Black Balance on ROI White Balance on ROI 96 Shift Metering Regions You see a live-image that has been switched on. In the Camera Control, the line profile and the Focus Indicator have been switched on, as well as the automatic exposure time, determined on the basis of an image segment. This button must have been pressed when you want to move one of the regions that you use to make measurements in the image, in the Camera Control. Metering Regions autom. exposure time (dashed square, here on spot 30%) Focus Indicator (yellow square) Line profile (white line) This button will always become automatically pressed (recognizable by its yellow background) as soon as you have set a Metering Region. When it has this status, you can move the Metering Regions you have chosen, at any time.

99 The user interface: components If, however, you have, in the meantime, inserted drawing objects or have carried out measurements (Drawings toolbar, respectively Measurement) toolbar/ tool window), this button will no longer be pressed. Then you will have to press the button once more, before you can move Metering Regions again. Further information is available in the online help. The Camera Settings dialog box This dialog box groups many settings that influence how your image analysis program acquires images. Please find a description of some frequently used settings below. A description of all settings can be found in the online help. Changing automatically generated file names Normally, your image-analysis program supplies the name "Image+<serial number>". You can change this default setting. 1) In the Camera Control tool window, click the Settings button. The Camera Settings dialog box appears. 2) Change to the Acquisition > Document Name entry. 3) Enter a new name in the Prefix field. The new name will also be displayed in the Preview field, plus a consecutive No. 4) Should you want not only to give it a new name, but also, e.g., add the acquisition date to the image name, then you will have to click the Customize button. Numerous possibilities for making changes will then be made available to you in the Customize Image Name dialog box. Further information is available in the online help. 5) If you want to start the numbering from a certain value, change the value in the Start field, e.g., to 50. Then the first image begins with the set number, e.g., 50. All additional images will be counted up by a further value. 6) Define in the Digits field how many figures the numbering should consist of, e.g., "3" for the number "001". 97

100 The user interface: components PLEASE NOTE 7) Select the Automatic Reset check box when you want to be sure that images that belong together will be consecutively, serially numbered without any breaks. You can then no longer change the numbering of the images manually. A detailed description of this function can be found in the online help. You can also change the file name by clicking in the Acquisition Process tool window on the More Options... button. Then a dialog box, which has the name of the selected acquisition process, opens. Change here to the Document Name tab and define the required settings. It makes no difference for the image analysis program in which position you change the file name since both dialog boxes use the same settings and thus always display the same data. 98 Saving images directly after acquisition By default, when you make an image acquisition, a new image document will be created and displayed in the document group. You can rename and save this image. You can have your images saved directly after acquisition. 1) To do this, make the Camera Settings dialog box appear, and go to the Acquisition > Save entry. 2) Select the Save captured images automatically check box. Then you will be able to edit the other fields in this dialog box. 3) Select the Close after save check box to have the image document close immediately after the image has been saved. The images will then be saved and closed immediately after their acquisition, and not displayed in the document group. Use this possibility when you want to acquire a large number of images one after the other, which you do not want to view more precisely until a later time. 4) In the Filetype list, select the file format in which you want the images to be saved. For the different image file formats TIF, JPEG and JPEG 2000 there are also additional settings that should be taken into account when saving images. Use the Options... button to obtain them. Further information is available in the online help. 5) If you want to change the path listed below, click on the [...] button and browse to the directory you want to save the files in. From now on your images will be saved automatically in this path.

101 The user interface: components NOTE 6) Select the Create Subdirectory check box to have images that belong together automatically saved in their own separate directory. By default, all of the images that you acquire in one day will be saved in the same directory. On the following day, a new directory will be automatically opened. 7) Click the Customize... button if you want to change the name that has been suggested for the subdirectory. 8) Check to see if the name shown in the Preview field for the current subdirectory is what you want it to be. Switching the camera to black and white mode By default, you acquire 24-bit true-color images with your camera. However, you can switch your camera to a mode in which 8-bit gray-value images are acquired. That is, for example, necessary when you make fluorescence acquisitions, and want to subsequently combine them into a multi-channel image. This is how you switch the camera 1) To do this, make the Camera Settings dialog box appear, and go to the <camera name>general entry. 2) Select the 8 bit Gray Scale entry in the Image type picklist. The live-image display will change immediately and your camera will acquire 8-bit gray-value images. 99

102 The user interface: components 100 The settings that apply to the live-image's behavior are shown here in their default status. Changing the behavior of the live-image You can change the behavior of the live-image if you want to. 1) To do this, make the Camera Settings dialog box appear, and go to the Acquisition > General entry. 2) Select the option you want in the Live group. By default, the One live document only option is selected. This means that a live-image that has been stopped will be overwritten, as soon as you return to the live-image again. As well as that, the Close document when live is stopped check box is also selected. This goes one step further: The live document will be closed when you exit the live mode.

103 The user interface: components 3) If you want to you can change the live-image's behavior. When you do this, please make use of the information about the various options in the online help. 4) Close the dialog box by clicking the OK button. Changing the basic unit from meters into inches You can change the basic unit for the X/Y-calibration that is to be used for the image acquisition, from meters into inches. In this case, all of the acquired images will automatically be calibrated in the basic unit "inches". All of the measurement values will then also be given in inches. 1) Make the Camera Settings dialog box appear, and go to the Acquisition > General entry. 2) From the Basic Unit list, select the Inches [in] entry. All of the images that you subsequently acquire with your software will then be automatically calibrated in the new basic unit "inches". Line Profile Line profile for a correctly exposed and focussed 24-bit truecolor image. You can have a line profile displayed during the live-image. To do so, in the Camera Control tool window, click the Line Profile button. As soon as you make the line profile appear, you will see a white line in the live-image. Your image analysis program measures the intensity of the live-image along this line. The segment along which the line profile will be measured is plotted along the X axis. Along the Y axis, the intensity. You can position the line wherever you want. Use the line profile to check the quality of your images. Further information can be found in the chapter "Checking the image quality with the line profile" on page

104 The user interface: components Adjust Display The Adjust Display tool window offers you a variety of possibilities for changing the appearance of the current image. PLEASE NOTE This function does not alter any image data. You only change how the image will appear on your monitor. The settings made for the image display will be saved together with the image if you save the image in the TIF format. When reloading the image it will be shown with the same settings. The tool window consists of the Histogram and Display Enhancement groups. 102 PLEASE NOTE The Histogram group The Histogram group shows the intensity histogram of the active image. In the histogram, respectively the intensity distribution, the number of pixels is plotted against the intensity. Therefore, it shows how many pixels are present in the image and their intensity. With a true-color image the histogram shows three curves, namely the intensity distribution for the red, green and blue color channels. The histogram is also shown when you are in live-mode. In this case it is called an online histogram. It shows the intensity distribution during live-image display. The online histogram will be continually updated. If you change the settings, e.g., reduce or increase the exposure time, you will see that the online histogram changes. The online histogram is therefore a good control of whether or not the image is well illuminated. In an image that is overexposed the intensity distribution will be cut off on the right-hand side. If the image is underexposed, the distribution will be shifted strongly to the left. Use the toolbar above the histogram to change the appearance of the histogram, e.g., the size. Further information is available in the online help. The group below the histogram is only for 16-bit images relevant. In it you determine how a 16-bit image will appear on your monitor. Further information is available in the online help.

105 The user interface: components Brightness Contrast Gamma The Display Enhancement group Use this slide control in this group to adjust the Brightness, Contrast and Gamma values for the image currently displayed on your monitor. You will see all changes immediately on the whole image. Click the Default button to cancel the contrast settings and return to the way the image was originally displayed. Move the Brightness slide control to the right (left) to make the image brighter (darker). Move the Contrast slide control to change the contrast values of the image. Values between 50 and 100 increase the image contrast. Values between 0 and 50 decrease the image contrast. At a setting of 50 the image contrast remains unchanged. Move the Gamma slide to the left, to make the image altogether darker. At the same time the contrast of bright image areas will be increased. Move the Gamma slide control to the right, to make the image altogether brighter. At the same time the contrast of dark image areas will be increased. 103

106 First time configuration of the system First time configuration of the system Overview After successfully installing the image analysis program and starting it for the first time, you will be asked to configure your system. This step is necessary to ensure that your image analysis system will run correctly. A complete system configuration is only necessary when the software has been installed for the first time on a PC and is started for the first time. When you make changes in the configuration of your microscope at a later date, only single components will have to be reconfigured (see "Readjusting the configuration" on page 118). Even when you update the software, the calibration data can be retained. You will receive a corresponding query while you are updating it. A complete configuration of the whole system requires approximately 20 minutes of your time. Your image analysis program will guide you through all the necessary steps in the correct sequence. Just follow the instructions it gives you. On the following pages you will find some basic information regarding the system configuration. If you prefer to start without further ado, refer to the chapter "Step-bystep: Configuring the system" on page 107. This configuration encompasses three steps: Configuring the objective nosepiece Your image analysis program has to know which objectives are being used and at which position in the nosepiece each one is fitted. Only then will the buttons on the Microscope Control toolbar be correctly defined. And only with this information can the images be correctly calibrated with regard to lengths (important for making measurements on the images). 104 Buttons in the Microscope Control toolbar. NOTE The color coding of the buttons corresponds to that of the objectives. This simplifies your orientation. Additionally, the objective's description and magnification will be shown in a tooltip when you move your mouse over these buttons. When acquiring the first images after concluding the configuration, you will notice that information regarding the objective used has been integrated in the image information. You can see it in the Microscope group of the Properties tool window (see the figure on p. 84). Setting the camera adapter's magnification The magnification of the camera adapter is also important for the image calibration. Most camera adapters have a magnification of This value is preset by default. If your camera adapter has another magnification, please change the proposed value. The magnification is imprinted on your camera adapter. Employing the calibration wizard Your image analysis program offers you a wizard for the three calibration processes. After you have installed the software for the first time, you will have to run all processes in sequence. When you have to reemploy one of the calibration processes later on, you may select only this process.

107 First time configuration of the system This dialog box will show you the status of all calibration processes. On the left you can see an incomplete system calibration, on the right, a finalized calibration. Using the Acquire > Calibrations... command, you can look for the status of the calibration at any time. To be able to work optimally with the system, all calibration processes should have the Calibrated status. The following icons will be used to indicate if a calibration is necessary: This calibration process has been carried out successfully. This icon can mean two things: Either, a calibration process that is recommended but not essential, has not yet been carried out. In this case, the status shown is Recommended. Or, calibration data exists, but the calibration is switched off. In this case, the status shown is Disabled. To switch the calibration on again, click the Edit... button. The left illustration shows an image where the online shading correction has not been activated. In comparison to the image on the right, one can notice the inhomogoneities in the illumination. What are calibration processes used for? Shading Correction Every optical system with a camera and microscope creates, even when the device is very carefully set up, image inhomogeneities in the illumination of the sample. These are called shading. 105

108 First time configuration of the system Dark correction image Flatfield correction image For a shading correction, the image errors will first be determined in order to be able to make corrections to the acquired image. To determine which flaws there are in the image, two types of correction images will be acquired: The Dark correction image is an acquisition during which no light falls on the camera. Here, defects resulting from noise or defective pixels on the camera chip can be corrected by the correction image. The Dark correction image is characteristic for each camera and need only be acquired once for every available camera resolution. It is valid for all objectives. The illumination of the complete optical system without an object, (or with a reference sample in the reflected light mode), will be shown on the Flatfield correction image. In addition to the camera characteristics, the microscope's optical characteristics, especially the objective being used, are incorporated in the correction image. Correspondingly, an individual correction image must be made for every objective and for every one of your camera's resolution settings. You have to acquire new illumination correction images every time you make changes in your optical system. Examples for such changes are cleaning the microscope, exchanging objectives or the camera or changing the illumination. White balance The White Balance calibration process takes care of the correct color reproduction even for your first images. You can later make the exact setting for each acquired image in the Camera Control with the White Balance on ROI button. This procedure is described on p. 19. Exposure time correction When you change the objective, the mean image brightness changes too. When the exposure time correction has been activated, after an objective has been changed, the exposure time will be automatically adjusted, in such a way that the mean image brightness remains approximately the same. You must carry out the Exposure Correction calibration process again when exchanging objectives or adding new objectives. Manual Objective Calibration 106 All of the images that you acquire with your image analysis program are automatically X/Y-calibrated. Use the Manual Objective Calibration calibration process if this calibration is not precise enough for you. To be able to carry out the calibration process, you will need a calibration standard. You will find a step-by-step instruction on p. 115.

109 First time configuration of the system Step-by-step: Configuring the system 1) Start the image-analysis software. The following request will be shown: 2) Confirm the request configuration with OK. The Device Settings dialog box appears: 3) Select the Manual Nosepiece entry. 4) Click the Properties... button. The Nosepiece Properties dialog box will open. No entries for the objectives of your microscope have been made, as of yet: 107 5) For position 1 select the smallest magnification available on your system from the Magnification picklist. The magnification is imprinted on the objective. 6) Select the exact designation of the objective from the Type picklist. This list contains all available Olympus objectives with the selected magnification. The correct numerical aperture will be automatically added.

110 First time configuration of the system NOTE If you do not use Olympus objectives, select an objective with the same magnification. Check and eventually change the value of the numerical aperture. The illustration shows the configuration of an objective nosepiece fitted with 4 objectives. If you don`t know the exact designation of your objectives' or you don`t work with Olympus microscopes: Set the correct magnification and select an objective with properties similar to the one you use. Check whether the automatically set Numerical Aperture corresponds to the numerical aperture imprinted on your objective. If this is not the case, change the value by placing the cursor in the NA field and entering the new value. 7) You may change the description of the objective in the Description field, if you wish. This description will be shown as tooltip when you place the cursor on this objective's symbol in the Microscope Control toolbar. 8) Should the objective not use "Air" as the immersion medium, select the immersion medium used from the picklist. 9) Should you want to save the working distance together with the image information, enter the value here. The Working Distance is, as a rule, printed on the objective. 10) Enter all objectives installed on your system. The dialog box now shows all objectives which are currently available on your microscope ) Confirm the objective configuration with OK. The Nosepiece Properties dialog box will close. The configuration of the objectives is now complete. The Device Settings dialog box appears again:

111 First time configuration of the system 12) Mark the Camera Adapter entry and click the Properties... button. The Camera Adapter Properties dialog box will open: 13) Check whether the magnification of your camera adapter conforms to the set value. If not, select the appropriate value from the picklist. The magnification is imprinted on your camera adapter. 14) Close the Camera Adapter Properties and Device Settings dialog boxes by clicking OK. The configuration of the camera adapter is now complete. Your image analysis program will now ask you to continue with the calibration of your system. You will see the following message: ) Confirm the message with OK. The Calibrations- <camera name> dialog box appears:

112 First time configuration of the system This dialog box shows the status of all calibration processes prior to the calibration. 16) Click on the Start Wizard button. The Calibration Wizard dialog box will be opened: ) Click the Next button to start the acquisition of correction images. A dialog box informing you which images you have to acquire, will be opened:

113 First time configuration of the system 18) Click Next. Your image analysis program will switch into live mode. The Shading Correction - Dark dialog box will be opened: 19) You will be asked to close the light path selector knob on your microscope, so that no light can fall on the camera. Then click Next. Your image analysis program will now acquire the dark correction images. Subsequently the Objective Specific Calibrations dialog box will be opened: 111

114 First time configuration of the system In this dialog box, an objective for which no correction images have yet been acquired, will be shown by a yellow warning icon in front of its name. When the first installation is made, all of the objectives are therefore marked in this way. Flatfield correction images have to be acquired separately for every objective and for every resolution. This means that when you have 4 objectives and 4 resolutions you will need to acquire 16 correction images. 20) Adopt the settings in this dialog box without any changes, and click the Next button. You will see the following message: 112 The wizard will inform you when you have to bring which objective into the light path. The resolution will be automatically changed by the system. NOTE 21) Open the light path selector knob as the wizard requests you to. 22) Then, bring the objective with the smallest magnification into the light path and click Next. 23) Click Next. The correction images for this objective will be acquired. This may take a short time. Should you get a warning informing you that the correction image cannot be acquired because the image is overexposed, please cancel the acquisition.insert a ND filter into the light path and repeat the correction image acquisition. Please do not reduce the lamp voltage, nor use any LBD filter in order to reduce the illumination intensity. This would lead to correction images with incorrect values. The following message will be shown:

115 First time configuration of the system In this dialog box you can determine if the correction images you have acquired are to be used immediately. 24) Bring the next objective into the light path. 25) Click OK and then Next in the Objective Specific Calibration dialog box. The correction image will be acquired. 26) Repeat the last two steps for all additional objectives. The Objective Specific Calibrations dialog box will inform you that the correction image have successfully been acquired. 27) Select whether you want to use the correction image from now on. This activates the shading correction.. The shading correction should, as a rule, always be switched on. This is also the default setting. This is visualized by the selected Flatfield entry in the Shading Correction picklist. If you want to disable the shading correction, you have to select the entry Off from the Shading Correction picklist. 28) Click the Finish button to finalize the calibration and return to the Calibrations dialog box. You have now successfully performed all calibrations. You will see the following message: 113

116 First time configuration of the system 29) Click the Finish button. The status of the system calibration now looks like this: ) Click Close. Now you can acquire images with your image analysis program. Further information is available in "Acquiring images" on page 16.

117 First time configuration of the system Manual Objective Calibration In the Calibrate objective list, all of the objectives that you have registered with your software are listed. 1) Lay a calibration standard under your light microscope. 2) If you use a magnification changer, set it at the magnification level 1 and select the magnification 1x in the Microscope Control toolbar. 3) Use the Acquire > Calibrations... command. 4) Select the Manual Objective Calibration calibration process and click the Calibrate... button. The software wizard will lead you through the individual steps for the calibration. The dialog box shows you the objective's name. 5) Select the check box in front of each of the objectives that you want to calibrate, then click the Next > button. 115 Your software will automatically switch to the live mode. 6) Bring the objective you want to calibrate into the light path. 7) Focus on the calibration standard, then click the Next > button.

118 First time configuration of the system First, select the unit you want. With a 1 mm long calibration standard that is graduated into 100 scale divisions, select e.g., the unit µm. For a manual objective calibration you require a calibration standard. 8) Select an appropriate unit. 9) In the Length field, you enter the distance on the calibration standard that you want to use for the calibration. With a calibration standard that is 1 mm long, you enter, for example, 800 in this field, when you want to use the reference distance between the scale divisions 10 and 90 for the calibration. 10) Click the Draw arbitrary calibration line button. Your mouse pointer will appear in the image window. 11) Determine the starting and end point of the calibration reference distance with your mouse pointer, in this case a distance of 800 µm. You define these two points by clicking them with your left mouse button ) Click the right mouse button and select the Confirm Input command in the context menu. 13) Click the Next > button if you want to calibrate further objectives, resp. the Finish button when you don't have any more objectives to calibrate.

119 First time configuration of the system When the last objective calibration has been carried out, the Manual Objective Calibration dialog box will open. In it, the actual objective magnification that was the outcome of the manual objective calibration will be shown for each of the manually calibrated objectives. 14) Close the Manual Objective Calibration dialog box with OK. In the Calibrations dialog box the Manual Objective Calibration calibration process will then have the status "Calibrated". 15) Close the Calibrations dialog box with OK. When you then acquire an image, the X/Y-calibration of this image will be calculated with the new magnification values. Please note that before you make the image acquisition, you will have to select the objective you want to use, in the Microscope Control toolbar. When you use a magnification changer, you will also have to select the magnification level. 117

120 Readjusting the configuration Readjusting the configuration When working with the system in daily use, you will have to reconfigure some components. In most cases, this will be the nosepiece, for example when changing objectives. Usually you will then have to repeat one or more calibration processes. In this case, don't, as described above, carry out all of the processes, one after the other, but rather choose only the processes you need. When do I have to repeat a calibration process? When the camera has been released and remounted, or inadvertently rotated Perform a shading correction When objectives have been exchanged, or a new objective has been added Configure the objective nosepiece Employ exposure time correction Perform a shading correction Carry out a white balance When an objective has been temporarily removed and then remounted Perform a shading correction When a lamp has been changed, or the voltage altered Perform a shading correction Carry out a white balance When the microscope has been cleaned Perform a shading correction When the condenser has been centered 118 Perform a shading correction Carry out a white balance

121 Readjusting the configuration To start a particular calibration process, select the desired process and click the Calibrate... button. Starting a single calibration process 1) Use the Acquire > Calibrations... command. 2) Select the process you want, for example Whitebalance, and click the Calibrate... button. The wizard will guide you through the steps of the calibration. When you select a calibration process in the Calibrations dialog box, then click the Edit... button, you will see a dialog box with the results that have been calculated. Switching the calibration process on and off You can view the Shading Correction and Exposure Correction calibration processes, and switch them on and off. 1) To do so, use the Acquire > Calibrations... command. 2) Select the process you want, and click the Edit... button. 119 You see the correction values that have been calculated.

122 Readjusting the configuration PLEASE NOTE 3) Switching the calibration process you want on or off. To do this for the exposure correction, select the Enable correction check box. For the shading correction, select the corresponding entry in the picklist. With the exposure correction, the correction values can be edited. Changes should, however, only be made by experienced users. 4) Close the dialog box with OK. 120

123 Appendix: Olympus DP20 camera Appendix: Olympus DP20 camera In this section you'll find information about functions of the Camera Control tool window which are specific to the Olympus DP20 camera. The remaining functions are fully described in the "Acquiring images" chapter. This is what the Camera Control tool window of your image analysis program possibly looks like, when it is connected to the Olympus DP20 camera. DP20 specific functions Whitebalance The white balance functions of the Olympus DP20 camera differ from the one described in the "Acquiring images" chapter. Carry out a new white balance each time you start working with the camera and as soon as the ambient light situation changes. If the colors are not correctly reproduced, you must carry out a white balance before you make an acquisition. When using white balance, the individual color channels are scaled in such a way that the white or neutral gray area of the image displayed on the monitor is displayed correctly as white or gray. If you subsequently acquire a whole series of similar samples and do not change the lamp voltage, then you generally do not need to carry out a white balance before each acquisition. Inversely, a white balance is always necessary when you change lamps or lamp voltage. Use the line profile to check whether or not a white balance is necessary. When working with the Olympus DP20 camera, you can perform a white balance automatically or manually (this again can be achieved in two different ways - either via the One Touch functionality or using predefined color temperatures). 121