NIS-Elements C (For CONFOCAL MICROSCOPE A1) Instructions (Ver. 4.40)

Transcription

1 M487E 15.4.NF.17 (1/4) *M487EN17* NIS-Elements C (For CONFOCAL MICROSCOPE A1) Instructions (Ver. 4.40)

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3 Preface Thank you for purchasing the Nikon products. This instruction manual has been prepared for the users of the Camera Settings function of Nikon NIS-Elements. To ensure correct usage, read this manual carefully before operating the product. No part of this manual may be reproduced or transmitted in any form without prior written permission from Nikon. The contents of this manual are subject to change without notice. Although every effort has been made to ensure the accuracy of this manual, errors or inconsistencies may remain. If you note any points that are unclear or incorrect, please contact your nearest Nikon representative. Some of the equipment described in this manual may not be included in the set you have purchased. If you intend to use any other equipment with this product, read the manual for that equipment too. If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. Reference spectrum data of dyes on NIS-Elements are provided from Invitrogen Corporation / Molecular Probes Clontech Laboratories, Inc. Invitrogen Corporation Clontech Spectral data of fluorescence dyes CoralHue Kaede and CoralHue Kusabira-Orange referenced in the NIS-Elements is provided from Amalgaam Co., Ltd. Amalgaam Co., Ltd. Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and other countries. Products and brand names are trademarks or registered trademarks of their respective companies. The TM and marks are not used to identify registered trademarks and trademarks in manual. The images of samples as shown in this document are for reference only, and may appear somewhat different from those actually acquired. i

4 Contents Preface... i Chapter 1 Before You Use Starting and Shutting Down the NIS-Elements C Starting the NIS-Elements C Shutting Down the NIS-Elements C Differences in Window Names Depending on Models Used Setting OS Structure of A1plus Settings Window Combinations of Detection Modes and Functions... 5 Chapter 2 Basic Operations Acquiring the Live Image and Setting the Scan Area... 6 Chapter 3 Acquire Window Functions of Acquire Window Window for Acquiring Various Images Switching Z Stacks Acquisition Directions Timelapse Experiment Notes When Acquiring Images Find Mode Setting for Find Mode Settings Window Multi Position Acquisition Procedure for Multi Position Acquisition Settings Speeding-up of Experiment Sequence Functions of Z Intensity Correction Usage of Functions of Z Intensity Correction Z Intensity Correction Window Piezo Z Stage Movable Range Line Scan XZ Acquiring XZ Image on the Normal Z Stage Acquiring Live Image of the XZ Plane on the Piezo Z Stage Chapter 4 Detection Mode DU Filter and Dye Window Structure of Filter and Dye Window Setting the Optical Path Optical Path Window Selecting the Channel Series Acquisition Window Structure of Acquisition Window Recommended Value Indication/Automatic Application When Acquiring Transmitted Image Only Setting Image Brightness Setting the Pinhole Calculation Settings for Pinhole Size Pinhole Position Alignment HV Linear Correction Auto Gain ii

5 Contents 4.3 2Ex1Em Line Sequence Procedure for 2Ex1Em Line Sequence Settings Ex2Emx2 Line Sequence Procedure for 1Ex2Emx2 Line Sequence Settings Indication When GaAsP DU4 Detector is in Use Chapter 5 Detection Mode SD Filter and Dye Window Structure of Filter and Dye Window Setting the Optical Path Optical Path Window Optical Path Window Switching Tab Detector Tab Binning/Skip Tab Acquisition Window Structure of Acquisition Window Recommended Value Indication/Automatic Application Setting Image Brightness Setting the Pinhole Calculation Settings for Pinhole Size Pinhole Position Alignment Auto Gain Various Views (Spectral Detector-use) Channel View Setting Channel Mixed View Split Channel View Ratio Image View Multi-Range Channel View Color Mode Setting Color Mode Select New Color Window Spectrum Profile Displaying the Spectrum Profile Spectrum Profile Setting Registering Spectrum and the Usage Spectral Unmixing Setting Displaying the Spectral Unmixing Setting Spectral Unmixing Setting Live Unmixing Displaying the Live Unmixing Blind Unmix Displaying the Blind Unmix Image Setting for Blind Unmix Window Chapter 6 Detection Mode VF Filter and Dye Window Structure of Filter and Dye Window Setting the Optical Path iii

6 Contents Optical Path Window Optical Path Window Switching Tab Detector Tab Grating Settings Tab Acquisition Window Structure of Acquisition Window Recommended Value Indication/Automatic Application When Acquiring Transmitted Image Only Setting Image Brightness Setting the Pinhole Calculation Settings for Pinhole Size Pinhole Position Alignment Auto Gain Various Views (Virtual Filter mode-use) Channel View Setting Channel Mixed View Split Channel View Chapter 7 Detection Mode VAAS Filter and Dye Window Structure of Filter and Dye Window Setting the Optical Path Optical Path Window Selecting the Channel Series Acquisition Window Structure of Acquisition Window Recommended Value Indication/Automatic Application Setting Image Brightness HV Linear Correction Auto Gain Various Views (VAAS-use) Displaying the VAAS Live Acquiring the VAAS Live image Displaying the VAAS image Acquiring the VAAS image Channel View Setting Channel Mixed View Split Channel View Chapter 8 Scan Setting Window Galvano Scan Mode Structure of Scan Setting Window Relationships among Scan Area Shape, Resolution, and Scan Speed Scan Settings Fast Galvano Mode Average and Integrate Average Integrate iv

7 Contents 8.2 Resonant Scan Mode Structure of Scan Setting Window Relationships among Scan Area Shape, Resolution, and Scan Speed Scan Settings Average and Integrate Average Integrate Unidirectional and Bidirectional Scan Unidirectional and Bidirectional Scan Motion Scan Settings upon Toggling between Unidirectional and Bidirectional Scan Chapter 9 Navigation Mode How to Display Navigation Mode Structure of Navigation Mode About Scan Areas Galvano Scan Mode Conditions for Setting Scan Areas Scan Area Setting Tools Switching Scan Area Setting Tools Scan Area Zoom Function Scan Area Rotation Function Resonant Scan Mode Conditions for Setting Scan Areas Scan Area Setting Tools Available Switching Scan Area Setting Tools Scan Area Zoom Function Chapter 10 Photo Activation Setting Procedure for Photo Activation Settings Correcting the Photo Activation Position Shift Setting HV Mode in Photo Activation Laser Setting for Photo Activation Usable Laser for Simultaneous Photo Activation Observation Simple ROI Editor Normal Photo Activation Observation (Galvano Mode) Experiment Sequence Setting for Normal Photo Activation Observation Galvano Sequential FRAP Procedure for Galvano Sequential FRAP Settings Setting of Window for FRAP Experiment Sequence Simultaneous Photo Activation Observation (Resonant Mode) Experiment Sequence Setting for Simultaneous Photo Activation Observation (for ROI Photo Activation Area) Experiment Sequence Setting for Simultaneous Photo Activation Observation (for Photo Activation Point and Photo Activation Line) Photo Activation Point Manual Operation Procedure for Photo Activation Point Manual Operation Galvano Shutter Function for Simultaneous Photo Activation Observation Chapter 11 Remote Control Function Structure of NIS-Elements C with the Remote Controller v

8 Contents Operation of Remote Controller and Display in the Window Channel Select Remote Controller Operation and the Corresponding Window for Each Detector Mode Channel Assignment Chapter 12 Using FN1 Microscope Combination with Quadrocular Tube Setting ECLIPSE FN1 Connection Manual Microscope Pad Combination with Digital Imaging Head for A Setting ECLIPSE FN1 Connection FN1 Pad Operating the Z Drive Chapter 13 Using A1+TIRF System Starting the A1+TIRF System Setting ECLIPSE Ti and Laser Connection Optical Configuration Setting Optical Configuration Setting for A Optical Configuration Setting for CCD Camera Procedure of Image Acquisition Merge the Confocal Image and TIRF Image Chapter 14 A1plus Compact GUI Displaying the A1plus Compact GUI Functions of the A1plus Compact GUI Photo Activation Setting Using the A1plus Compact GUI Chapter 15 CLEM Function Basic Operations of CLEM Function Setting of A1 CLEM Window Chapter 16 External Trigger Output Trigger Signal Output Procedure for External Trigger Output Settings External Trigger Output Operation List External Trigger Output Operation List (XYT) External Trigger Output Operation List (XYTZ) External Trigger Output Operation List (Line scan) External Trigger Output Operation List (ROI photo activation) External Trigger Output Operation List (Line photo activation) External Trigger Output Operation List (Point photo activation) External Trigger Output Operation List (Pulse photo activation) External Trigger Output Operation List (Normal Photo activation) External Trigger Output Operation List (Simultaneous photo activation) Chapter 17 Using External Detector Unit Procedure for Settings on the A1plus Settings Window Procedure for Settings on the AUX Settings window Laser Input Port Selection When Multiple Laser Units Are Used vi

9 Contents Chapter 18 Using Data Acquisition Device (NIDAQ) NIDAQ Connection Settings Trigger Signal Input Using NIDAQ Procedure for Setting External Trigger Input for Image Acquisition Procedure for Setting External Trigger Input for Simultaneous Photo Activation Experiment To Acquire Timestamp Information Using NIDAQ Appendix List of Initialization Error Codes vii

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11 1 Before You Use The NIS-Elements C is used as a function of NIS-Elements AR. It cannot be used alone. This section describes the starting/shutdown and structure of the NIS-Elements C window. When a Galvano scanner that does not support the Fast Galvano mode is in use, replace A1plus written in this instruction manual with A1. (For details, see Section 1.1.3, Differences in Window Names Depending on Models Used. 1.1 Starting and Shutting Down the NIS-Elements C This section describes the starting/shutdown of the NIS-Elements C Starting the NIS-Elements C Double-click the NIS-Elements AR icon. Figure NIS-Elements icon The NIS-Elements title window appears. Then, the title window closes and NIS-Elements C starts. Figure Title window Then the Driver selection window appears on the desktop. To use a normal A1 confocal microscope, select Nikon Confocal. * If only one camera is installed, the camera is automatically selected and the Driver selection window is not displayed. * To use the A1+TIRF system, check the [Enable Multi Camera] check box and select both Nikon Confocal and ANDOR. (see Chapter 13) Figure Driver selection window 1

12 Chapter 1 Before You Use As NIS-Elements C starts, the A1plus Settings window opens automatically as well. Figure Initial NIS-Elements window and the A1plus Settings window However, when the NIS-Elements was exited with the A1plus Settings window closed during the previous use, the window does not appear automatically at the next startup because the NIS-Elements reproduces the status at the previous exiting. To manually display the A1plus Settings window, right-click on the gray area (without any setting window displayed) to display a menu as shown below. Select [Acquisition Controls] -> [A1plus Settings] in the menu. Figure Displaying the A1plus Settings window * Other display methods The A1plus Settings window can also be displayed by selecting [View] -> [Acquisition Controls] -> [A1plus Settings] from the menu bar. Figure Displaying the A1plus Settings window Shutting Down the NIS-Elements C The layout of the NIS-Elements C is memorized when it shuts down. 2

13 Chapter 1 Before You Use Differences in Window Names Depending on Models Used If a model that supports the Fast Galvano mode is in use, a name A1plus is displayed at the upper left of each window. For models that do not support the Fast Galvano mode, a name A1 is displayed. Models that support Fast Galvano mode Figure Models that do not support Fast Galvano mode Identification of models Table Window Names of Models That Support or Do Not Support Fast Galvano Mode Window Names of Models That Support Fast Galvano Mode A1plus Settings A1plus Compact GUI A1plus Stimulation A1plus Scan Area Window Names of Models That Do Not Support Fast Galvano Mode A1 Settings A1 Compact GUI A1 Stimulation A1 Scan Area In this instruction manual, window names of models that support Fast Galvano mode are written as A1plus hereinafter. When a model that does not support Fast Galvano mode is in use, replace window name A1plus with A1. Differences depending on laser unit models Indication on the excitation laser indicator and some functions that are displayed on the Optical path window vary depending on laser unit models used. This instruction manual describes operations of the NIS-Elements C using LU-NV windows. (The right figure shows the LU-N4 window configuration without spectral detector.) Avoid using combinations of LU-N4 or LU-N3 laser unit and detection mode (SD or VF) using the spectral detector as much as possible to prevent effects of excitation light. Figure Distinction of laser unit Setting OS If sleep mode is specified in the power supply option of the OS, some devices may not work normally after recovery from the sleep mode. Disable the sleep mode of the OS. 1. Click [Start] -> [Control Panel] -> [Power Options]. 2. Select [Change when the computer sleeps] to display the plan setup editing window. 3. Select [None] from the [Put the computer to sleep:] option. 4. Click the [Save Changes] button to confirm the settings. 3

14 Chapter 1 Before You Use 1.2 Structure of A1plus Settings Window The A1plus Settings window enables to apply various settings, including the laser, adjusting the brightness of the image, the photo activation setting, the scanning method, and scan resolution/speed, to use the Confocal Microscope. (1) (3) (2) (4) (5) (7) (8) (6) Figure A1plus Settings window Table Summary of A1plus Settings window functions Name Function (1) Acquire window display/ nondisplay selection (2) Scan mode selection (3) Acquire window (4) Filter and Dye window (5) Acquisition / Photo Activation window Switches display/nondisplay of the Acquire window. Enables to alternate between the Resonant scan mode and the Galvano scan mode. In the Spectral detector mode and the Virtual Filter mode, only the Galvano scan is effective and you cannot change the scan mode. Enables to display live images, to acquire images (see Chapter 3) or to apply the photo activation settings (see Chapter 10), to use external detector unit (see Chapter 17). The functions available with NIS-Elements are arranged as buttons in this area. Enables to select the channel series to be used and set the optical path. (See Filter and Dye in the chapters concerning detector modes.) The Acquisition window enables to set PMT brightness, laser power, and pinhole size. (See Acquisition in the chapters concerning detector modes.) The Photo Activation window enables to set the desired stimulation laser power. (see Chapter 10) (6) Scan setting window Enables to set a scan method, resolution, scan speed, etc. (see Chapter 8) (7) Remote controller selector icon (8) Optimize button Displayed for the channel group which is currently adjustable with the remote controller. (see Chapter 11) Calculates the recommended value of resolution, zoom magnification, and Z stack step size based on the objective type and the selected excitation wavelength, and the indication/automatic application function can be set in detail. 4

15 Chapter 1 Before You Use 1.3 Combinations of Detection Modes and Functions The following tables lists settable functions in each detection mode. Table Combinations of Detection Modes and Functions Function Detection Mode DU4 SD VF VAAS Galvano Resonant Unidirectional Y Y Y Y Bidirectional Y Y Y Y Unidirectional Y N N Y Bidirectional Y N N Y Channel Series Galvano Resonant Unidirectional Y N N Y Bidirectional Y N N Y Unidirectional Y N N Y Bidirectional Y N N Y Multi position acquisition (Chapter 3) Available in all detection modes (However, no photo activation experiment is available.) Fast Galvano (Only Galvano) (Chapter 8) Y N N Y CLEM (Only Galvano) (Chapter 15) Y N N N HV linear correction Y N N Y Pinhole setting Y Y Y N Using external detector (Chapter 17) Y N N N (Chapter 8) Line skipping Galvano Resonant Unidirectional Y Y Y Y Bidirectional N N N N Unidirectional Y N N Y Bidirectional N N N N Normal photo activation (Chapter 10) Y Y Y Y Simultaneous photo activation (Chapter 10) Y N N Y (Chapter 4) (2Ex1Em/1Ex2Emx2) Particular line sequential Galvano Resonant Unidirectional Y N N N Bidirectional Y N N N Unidirectional Y N N N Bidirectional N N N N Y: Available N: Unavailable * Avoid using combinations of LU-N4 or LU-N3 laser unit and detection mode (SD or VF) using the spectral detector as much as possible to prevent effects of excitation light. 5

16 2 Basic Operations This chapter describes the basic instructions for acquiring live images in the NIS-Elements C. 2.1 Acquiring the Live Image and Setting the Scan Area 1 Selecting the Scan mode Select [Resonant] or [Galvano] for using the Scan mode. * In [SD] and [VF], only the Galvano is effective. If [Resonant] is selected and then detection mode is changed to [SD] or [VF], the scan mode changes automatically to Galvano. Figure Selecting the scan mode 2 Setting the Optical path 1. Display the Optical path window. Click the [Setting] button in the Filter and Dye window. For details of the Optical path settings, see Filter and Dye in the chapters concerning detection modes. Setting button Figure Filter and Dye window Figure Optical path window 6

17 Chapter 2 Basic Operations 2. Select the detection mode (detector). Select [DU4] to use the Standard Detector; [SD] to use the Spectral Detector; [VF] to use the Virtual Filter; [VAAS] to use the VAAS. Detection mode button Figure Selecting the Detection mode (Detector) 3. Activate the automatic mode of Optical path setting. Click the [Auto] button. Setting mode button Figure Selecting the auto mode 4. Select the fluorescence dyes for the channels to be used. For each channel to be used, select a fluorescence dye from the pull-down menu. Once a fluorescence dye is selected, appropriate laser and dichroic mirror are automatically selected. Selects a fluorescence dye from the pull-down menu. Figure Selecting fluorescence dyes 7

18 Chapter 2 Basic Operations 5. Select the channels to be used. Check the check box for each channel to be used. Check the channels to be used. Figure Selecting channels 6. Furthermore, the scan mode that was set in step 1 can be changed. (When [SD] or [VF] is selected, the scan mode is fixed to Galvano.) 7. Select the desired icon to use or disuse the transmitted detector. 8. Click the [OK] button to confirm the Optical path settings. At this time, if a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the manual mode, a confirmation dialog box 1st DM is Invalid! Will you apply? appears. When you apply the selected 1st Dichroic mirror, click the [OK] button. Clicking the [Cancel] button returns the display to the Optical path window. Change the scan mode. Selects the transmitted detector. OK button Figure Selecting the scan mode, transmitted detector and confirming the optional path 8

19 Chapter 2 Basic Operations 3 Applying Scan settings In the Scan setting window, apply various scan settings to acquire the live image. For details of Scan settings, see Chapter 8, Scan Setting Window. Selects the scan method (Unidirectional / Bidirectional). Selects scan magnification. Selects resolution. Selects scan speed. Selects averaging and integration methods. Selects line skipping. Figure Scan setting window 4 Acquiring the live image Click the [Live] button. The live image is acquired and the Live window appears. Figure Acquiring the live image Figure Live window 9

20 Chapter 2 Basic Operations 5 Adjusting the brightness of the live image In the Acquisition window, adjust the brightness of the live image for each channel. See Acquisition window in the chapters concerning detection modes. Figure Acquisition window 6 Setting the scan area Set the scan area for the acquired live image. For details of the scan area, see Chapter 9, Navigation Mode. 1. Switch the Live window to the navigation mode. Click the [Show Scan Area] button in the Live window. Show Scan Area button Figure Switching to navigation mode 10

21 Chapter 2 Basic Operations 2. Select the scan area setting tool to be used. The scan area setting tools differ in their available shapes depending on the scan area selected. Scan area setting tool. Figure Selecting the scan area setting tool 3. Set the scan area with the tool selected. For instructions on selecting and using scan area setting tools, see Scan Area Setting Tools in Section 9.3, About Scan Areas. Scan area Figure Setting the scan area 11

22 Chapter 2 Basic Operations 7 Acquiring the image of the set scan area 1. Right click on the drawn scan area. Right click on scan area Figure Acquiring the live image of scan area Figure Live window after changing the scan area * While working with Frozen image, the live image in the set scan area can also be acquired by clicking the [Live] button. Figure Acquiring the live image 12

23 Chapter 2 Basic Operations Laser InterLocked Indicates the interlock status of the microscope main unit. If the optical path of the microscope main unit is switched to the binocular system, all of the laser shutters close for safety purpose. At this time, the [Laser InterLocked] button blinks and the confocal image acquisition cannot be executed. When you execute the confocal image acquisition again, switch the optical path of the microscope main unit to Confocal, and then click this [Laser InterLocked] button. Laser InterLocked button Figure Laser Interlocked indicator When LU4 or LU4A is in use Note that, if an interlock occurs during image acquisition, the laser shutter operates differently as follows in LU4 and LU4A after the interlock is reset. - LU4: The laser shutter closes when an interlock occurs and opens after the interlock is reset. - LU4A: The laser shutter closes when an interlock occurs but does not open after the interlock is reset. However, the laser shutter opens when the next image acquisition starts. When LU-N4 or LU-N3 is in use When the [Clear Laser Interlock Automatically] check box in the A1plus Settings window is selected in advance, the hardware status is recognized even if an interlock occurs during image acquisition and the interlock is automatically reset. Laser InterLocked button Clear Laser Interlock Automatically check box Figure Automatic release of Laser Interlocked 13

24 Chapter 2 Basic Operations When LU-NV is in use When the [Automatic Laser Interlock release] check box in the LU-NV Configuration window is selected in advance, the hardware status is recognized even if an interlock occurs during image acquisition and the interlock is automatically reset. Automatic Laser Interlock release check box Figure Automatic release of laser Interlocked 14

25 3 Acquire Window The Acquire window enables to display the live image, acquire the image or apply the photo activation settings. Additionally, the functions available with NIS-Elements are arranged as buttons in this area. 3.1 Functions of Acquire Window Acquire window display/nondisplay selection (1) (2) (3) (4) (5) (6) (7) (8) Figure Acquire window Table Functions of Acquire window Name (1) Live button Function Enables to display the live image. The Live window opens and displays the live image automatically. The live image is the real-time image that is currently observed with the microscope. Starts/stops live image acquisition in Find mode. (2) Find Mode Find mode is the mode where the live image acquisition is executed by temporarily switching to the high-frame-rate setting in order to ease the detection of the observation object such as a cell. Opens the Find mode settings window. For details of the Find mode, see Section 3.1.2, Find mode. (3) XY button Enables acquiring the captured image of the currently displayed live image. When the captured image is acquired, the Captured window appears separately from the Live window. The captured image is a still image that is acquired by re-scanning the scan area displayed in the current Live window. (4) Fast Piezo Z check box When this check box is selected, images are acquired with priority placed on speed regardless of the specified Z stroke in the Z stack. (This function is enabled only when [Nikon A1 Piezo Z Drive] is selected for the Z Drive.) Clear this check box if stroke takes precedence. 15

26 Chapter 3 Acquire Window Name Function Opens the Capture Z-Series window. Enables to acquire a three-dimensional (X-Y-Z) image. For operations of this window, refer to NIS-Elements AR (Advanced Research) User s Guide. (5) XYZ button Figure Capture Z-Series window The same window can also be opened with the following procedure: - Select [Acquire] on the menu bar and then select [Capture Z-Series] -> [Capture Automatically...] in this order. Opens the Capture Timelapse window. Enables to acquire two-dimensional (X-Y) images in time series. For operations of this window, refer to NIS-Elements AR (Advanced Research) User s Guide. (6) XY Time button Figure Capture Timelapse window The same window can also be opened with the following procedure: - Select [Acquire] on the menu bar and then select [Capture Timelapse] -> [Capture Automatically...] in this order. 16

27 Chapter 3 Acquire Window Name Function Opens the ND Acquisition window. Enables to acquire three-dimensional (X-Y-Z) images in time series. For operations of this window, refer to NIS-Elements AR (Advanced Research) User s Guide. (7) XYZ Time button Figure ND Acquisition window Opens the ND Stimulation window. The photo activation observation can be set. The window to be opened depends on the conditions set in the A1plus Settings window. For operations of this window, see Chapter 10 in this instruction manual. (8) Photo Activation button Normal photo activation observation (Galvano scan mode) Figure Simultaneous photo activation observation (Resonant scan mode) ND Stimulation window The same window can also be opened with the following procedure: - Normal photo activation observation Select [Applications] on the menu bar and then select [Define/Run Sequential Stimulation...] in this order. - Simultaneous photo activation observation Select [Applications] on the menu bar and then select [Define/Run Simultaneous Stimulation...] in this order. 17

28 Chapter 3 Acquire Window Window for Acquiring Various Images The windows shown in (4), (5), and (6) of Table Functions of Acquire window can also be opened with the following procedure: - Select [Applications] on the menu bar and then select [Define/Run ND Acquisition...]. Acquisition switching tab In the ND Acquisition window displayed with the above procedure, click a switching tab to select a function to use. For operations of this window, refer to NIS-Elements AR (Advanced Research) User s Guide. Figure ND Acquisition window Switching Z Stacks Acquisition Directions Z stack image acquisition directions can be switched by [Bottom to Top] or [Top to Bottom]. * When objective piezo is used, strain may be generated in images in particular directions under particular conditions. Figure ND Acquisition window 18

29 Chapter 3 Acquire Window Timelapse Experiment Timestamp in Timelapse Experiment In an experiment of timelapse, timestamp 0 is usually recorded at the beginning of the experiment (when the [Run now] button is clicked), but it is also possible to record timestamp 0 in the first frame. For details, see Options in NIS-Elements AR Help. Figure ND Acquisition window When Using Perform Time Measurement If the Time Measurement is executed with the [Perform Time Measurement] check box selected, a load on processing becomes so high that it may cause the following problems: - When the [Loop] side is set, the time for transition to the next phase may be longer than the time supposed from the frame rate. - When the [Duration] side is set, the number of the frames may be smaller than that supposed from the frame rate. Figure ND Acquisition window 19

30 Chapter 3 Acquire Window Notes When Acquiring Images When Executing Large Image in ND Acquisition window The Large Image is a function to acquire a large image composed of multiple image frames and combine them to form a composite image by using the automatic algorithm, to be used when the target area is larger than the field of view (FOV) of the camera. When this function is executed, the turning action is controlled by the stage. Therefore, it is necessary to execute the calibration before the Large Image function is executed. For operations of Auto calibration, refer to NIS-Elements AR (Advanced Research) User s Guide. Figure ND Acquisition window Figure Auto calibration When Executing the Scan Large Image Function While Both A1 and CCD Camera Are Used When both A1 and CCD camera are used, if the transmitted detector (TD) is in the optical path, it blocks the light from the diascopic illumination. Therefore, the TD must be removed from the optical path before the Scan Large Image setting window is called. * When executing the Scan Large Image function, do not use the optical configuration (O.C.) in which the TD is registered. When the TD is registered in the O.C. even if the TD is removed from the optical path, the TD automatically enters the optical path when an image is acquired. 20

31 Chapter 3 Acquire Window Find Mode By using the Find mode, you can acquire the live image by temporarily switching to the high-frame-rate setting in order to ease the detection of the observation object such as a cell Setting for Find Mode Settings Window (1) (2) (3) (4) (5) (6) (7) (8) Figure Find mode settings window Name Table Summary of Find mode settings window functions Function (1) Change to band scan with aspect: Switches the band scan area by the specified ratio. E.g. If 2x is selected in Scan Size 512 x 512, the band scan area is switched to 512 x 256 in the Find mode. (2) Lower resolution: (3) Line skipping: Changes the scan size. E.g. If 2x is selected in Scan Size 512 x 512, the scan size is changed to 256 x 256 in the Find mode. Changes a line skipping mode to be applied during scanning. E.g. If 2x is selected, the Line skipping is changed to 2 in the Find mode. (4) (5) Turn OFF line & frame averaging Change Galvano to Resonant Changes a setting for Line Average/Frame Average. E.g. Even if the Line Average or Frame Average is set in the normal mode, the live image is acquired by changing the setting to None in the Find mode. Changes the scan mode from Galvano to Resonant. (6) OK button Confirms the settings applied and closes the Find mode settings window. (7) Cancel button Discards the settings applied and closes the Find mode settings window. (8) Apply button Confirms the Find mode settings. If the A1plus Settings window or A1plus Compact GUI window is closed during scan in the Find mode, no GUI menu or button can be selected and the scan cannot be stopped. In that case, press the - (minus) key of the ten-key to stop the scan. 21

32 Chapter 3 Acquire Window 3.2 Multi Position Acquisition You can execute the experiment with multiple points within the same FOV by using the optical configuration (hereinafter referred to as O.C.) where different scan areas are respectively registered. (Galvano scan mode only. Photo activation experiment is not available.) In Multi Position Acquisition, the image acquisition is executed by the ND Sequence Acquisition function of NIS-Elements AR, by using the Lambda series. * About Lambda series: When acquiring multiple excitation lights by emitting multiple lasers, the lasers are not emitted simultaneously but emitted in sequence. By emitting lasers in sequence, the cross talk between channels can be avoided Procedure for Multi Position Acquisition Settings 1 Register the first scan area to O.C. 1. Specify a scan area on the image acquired by Galvano scan mode. * The scan areas usable in the multi position acquisition are the square scan area and the band scan area only. Figure Specify a scan area 2. Register the specified scan area to O.C. Select [Calibration] -> [New Optical Configuration...] from the menu bar to call the wizard. Figure Call the Optical Configuration Wizard 22

33 Chapter 3 Acquire Window 3. Enter the name of O.C. to be registered. 4. Check the setting conditions, and then click the [Finish] button. Enter the name to be registered. Finish button Figure Register the optical configuration 2 Register the second and subsequent scan areas as separate O.C., respectively After that, repeat Step 1 to Step 4 of the multi position. 1 to register the O.C. of each scan area to be acquired in Figure Register the optical configuration 23

34 Chapter 3 Acquire Window 3 Execute the multi position acquisition Register the O.C. of each scan area for each action, and make the experiment setting, respectively. 1. Select [Applications] -> [Define/Run ND Sequence Acquisition...] from the menu bar to open the ND Sequence Acquisition window. Figure Call the ND Sequence Acquisition window Figure ND Sequence Acquisition window 2. Click the first phase and select [ND Acquisition]. Figure ND Sequence Acquisition window 24

35 Chapter 3 Acquire Window 3. Click the [Define...] button to open the experiment setting window. Figure ND Sequence Acquisition window 4. Select the Lambda series tab on experiment setting window and specify the O.C. of the first scan area. 5. When the setting of the experiment sequence of the first scan area is completed, click the [OK] button to close the window. The ND Sequence Acquisition window is resumed. Lambda tab Select the O.C. of the first scan area. OK button Figure Experiment setting window * The Lambda series is used for the multi position acquisition. However, do not set multiple O.C.s in one Lambda series for the purpose of the multi position. If O.C.s with different scan area types and/or sizes are set, displayed size will differ from the original image size, because the image size ratios must be matched within one ND image. 25

36 Chapter 3 Acquire Window 6. Click the next phase and select [ND Acquisition]. 7. Click the [Define...] button to open the experiment setting window. Figure ND Sequence Acquisition window 8. Specify the O.C. of the second scan area. 9. When the setting of the experiment sequence of the second scan area is completed, click the [OK] button to close the window. The ND Sequence Acquisition window is resumed. Select the O.C. of the second scan area. OK button Figure Experiment setting window 10. After that, repeat Step 6 to Step 9 to make the experiment setting for the O.C. of the scan area registered for acquisition within the same FOV. 26

37 Chapter 3 Acquire Window 11. Click the [Run Now] button to execute the multi position acquisition. Figure ND Sequence Acquisition window 27

38 Chapter 3 Acquire Window 3.3 Speeding-up of Experiment Sequence On the Capture Z-Series window, the Capture Timelapse window, the ND Acquisition window, and the ND Stimulation window, check the [Use HW sequencer] check box to let the hardware handle Experiment Sequence. This makes experiment faster. Note that when performing the high speed Experiment Sequence with hardware, the applicable [Z Device] is Ti Piezo ZDrive only. Use HW sequencer Use HW sequencer Figure Capture Z-Series window Figure Capture Timelapse window Use HW sequencer Use HW sequencer Figure ND Stimulation window Figure ND Acquisition window 28

39 Chapter 3 Acquire Window 3.4 Functions of Z Intensity Correction When acquiring the images of a sample at Z drive positions (tomographic images) by Z stack, acquiring images with identical conditions at all Z drive positions makes some of the images to be too bright or too dark depending on the Z drive position to acquire. A solution to this problem is the Z Intensity Correction function. To use the Z Intensity Correction function, first adjust the brightness on the live image by Z drive position you wish to acquire, and then register the optimum brightness setting values (laser power) for each Z drive position. After that, acquire images by using the registered setting values. The brightness of the images for each Z drive position to acquire is automatically controlled, and images are acquired with the optimum brightness at all Z drive positions. * Both visible laser and IR pulse laser are supported. * When Nikon A1 Piezo Z Drive is selected as a Z Device, the sequence where the Z drive is moved after the Lambda series is acquired (where Z series(lambda) is selected as Order of Experiment) cannot be executed. Minimum/recommended number of registrations for the Z Intensity Correction function To use the Z Intensity Correction function, register at least two Z drive positions (Top and Bottom), and to acquire clearer images, it is recommended to register 4 or more positions (Top and Bottom plus two or more intermediate Z drive positions). * A function to load Z drive positions from the ND Acquisition window by clicking the [From ND] button on the Z Intensity Correction window is provided. In that case, only three points Top, Home, and Bottom are loaded. Registration of four or more Z drive positions is recommended by the Z intensity correction function, but there is no operational problem with the setting of one point less than the recommended number. Z drive positions not registered to Z Intensity Correction For the setting values of Z drive positions not registered to Z Intensity Correction are automatically interpolated according to the setting values of the registered Z drive position, and the interpolated setting values are used to acquire images. To check the interpolated setting values, open the Microsoft Excel file output by using the [Export...] button on the Z Intensity Correction window. 29

40 Chapter 3 Acquire Window Usage of Functions of Z Intensity Correction 1 Setting Z Stacks position for image acquisition and Z Device 1. Call the Capture Z-Series window. For setting Z stacks instructions, refer to NIS-Elements AR (Advanced Research) User's Guide. Z stacks Settings Figure Z stacks Settings 2. Select Z Device. Select a Z drive to be used from the pull-down menu. (Piezo is also selectable.) * When Nikon A1 Piezo Z Drive is selected as a Z Device, only laser power can be adjusted. The HV value is not controlled automatically. Select Z drive Figure Selecting Z Device 30

41 Chapter 3 Acquire Window 2 Displaying the Z Intensity Correction window to read Z drive positions 1. Right-click on the gray area (without any setting window displayed) to display a menu. Select [Acquisition Controls] -> [Z Intensity Correction] in the menu to open the Z Intensity Correction window. Figure Displaying the Z Intensity Correction window 2. Clicking the [From ND] button in the Z Intensity Correction window reads the Top, Home, and Bottom Z drive positions. Figure Z Intensity Correction window Figure Z Intensity Correction window 31

42 Chapter 3 Acquire Window 3. To read a Z drive position other than Top, Home, and Bottom, display the Z drive position you want to register and click the [Add New] button. Figure Capture Z-Series window Add New button Figure Z Intensity Correction window Figure Z Intensity Correction window 32

43 Chapter 3 Acquire Window 3 Adjust brightness at the Z drive positions and register them to Z Intensity Correction 1. Double-clicking the Top Z drive position displays the button. Double-click Figure Z Intensity Correction window Figure Z Intensity Correction window 2. Acquiring the live image of Top Z drive position. Click the [Live] button. The live image of Top Z drive position is acquired and the Live window appears. Figure Live image acquisition 33

44 Chapter 3 Acquire Window 3. Adjusting the brightness of the live image. In the Acquisition window, adjust the brightness of the live image for each channel. * When Nikon A1 Piezo Z Drive is selected as a Z Device, only laser power can be adjusted. The HV value is not controlled automatically. Figure Acquisition window 4. Register the adjusted values. Click the [Add New] button in the Z Intensity Correction window. The adjusted values are registered at the Top Z drive position. Add New button Figure Z Intensity Correction window Figure Z Intensity Correction window 34

45 Chapter 3 Acquire Window 5. Repeat steps 1 to 4 for each Z drive position to be registered to move the multiple Z drive positions to be registered next. Figure Z Intensity Correction window 6. After registering all of them, click the [Run Z Corr] button in the Capture Z-Series window to execute the image acquisition. With the registered setting values, the brightness of the image for each Z drive position is automatically controlled and images are acquired with the optimum brightness at all the Z drive positions. The settings registered on the Z Intensity Correction window are exportable to a file by using the [Save...] button, and an exported file is loadable by using the [Load...] button. Figure Image acquisition running 35

46 Chapter 3 Acquire Window Z Intensity Correction Window (1) (7) (2) (8) (3) (4) (5) (6) (9) (10) (11) Figure Z Intensity Correction window Table Functions of Z Intensity Correction window Name (1) Use in ND Multipoint Absolute Relative Function Interpolates registered Z drive positions when acquiring Multipoint regardless of change in Z drive positions. Interpolates Z drive positions with offset according to change in Z drive positions when acquiring Multipoint. (2) Move Z to selected Point Moves the Z drive position to the selected point. (3) Corr. Home Indicates the position to be reference for correction. When [Set as Correction Home] displayed by right-clicking is selected, the selected Z drive position is the reference point for correction. (4) Z-stack range To ND From ND Sends the Top, Home, and Bottom Z drive positions defined in the Z Intensity Correction window to the Capture Z-Series window. Reads the Top, Home, and Bottom Z drive positions defined in the Capture Z-Series window to the Z Intensity Correction window. (5) Use on Live Applies brightness adjustment at each set Z drive position to the live image. (6) Offset Correction Curve Updates the Z drive position interpolation with offset according to the current Z drive position. (7) Add New button Adds Z drive positions to be registered. (8) Remove Registrations button Removes all registrations. Removes the selected item. (9) Load... button Retrieves the saved in a file. (10) Save... button Writes the registrations in a file and saves it. (11) Export... button Writes the registrations in a Microsoft Excel file. The exported file allows the user to check the interpolated values of Z drive position with setting values unregistered. 36

47 Chapter 3 Acquire Window 3.5 Piezo Z Stage Movable Range The following restrictions are provided for the piezo Z stage movable range. Note that the movable range varies depending on the stage type and scan mode. Galvano Scan Mode-use Type Scan Area Z Step (Number of slice images) Unidirectional Scan Bidirectional Scan Restrictions for Movable Range Objective PZT (450 um) * For upright, Ni-E, and FN1 microscopes Objective PZT (200 um) * For upright, Ni-E, and FN1 microscopes Stage PZT (100 um) * For Ti-E microscope Square 1 to to 8000 up to 450 um Band 1 to to 8000 up to 450 um Line 256 to to 1601 up to 450 um Square 1 to to 8000 up to 200 um Band 1 to to 8000 up to 200 um Line 30 to to 1601 up to 80 um Square 1 to to 8000 up to 100 um Band 1 to to 8000 up to 100 um Line 100 to to 1601 up to 40 um Resonant Scan Mode-use Type Scan Area Z Step (Number of slice images) Unidirectional Scan Bidirectional Scan Restrictions for Movable Range Objective PZT (450 um) * For upright, Ni-E, and FN1 microscopes Objective PZT (200 um) * For upright, Ni-E, and FN1 microscopes Stage PZT (100 um) * For Ti-E microscope Square 1 to to 8000 up to 450 um Band 1 to to 8000 up to 450 um Line 256 to to 1601 up to 160 um Square 1 to to 8000 up to 200 um Band 1 to to 8000 up to 200 um Line 230 to to 1601 up to 80 um Square 1 to to 8000 up to 100 um Band 1 to to 8000 up to 100 um Line 258 to to 1601 up to 40 um Fast Piezo Z check box When this check box is selected, images are acquired with priority placed on speed regardless of the specified Z stroke in the Z stack. (This function is enabled only when [Nikon A1 Piezo Z Drive] is selected for the Z Drive.) Clear this check box if stroke takes precedence. Figure Fast Piezo Z 37

48 Chapter 3 Acquire Window 3.6 Line Scan XZ The XZ (cross section) image can be acquired using the line setting tool Acquiring XZ Image on the Normal Z Stage 1. Click the [Live] button. The Live image is acquired and the Live window is displayed. Figure The Acquiring XZ Image 2. Draw the center of the XZ plane with the line setting tool. Figure Live window 38

49 Chapter 3 Acquire Window 3. Click the [XYZ...] button to call the Capture Z-Series window. Figure Calling the Capture Z-Series window 4. Make Z stacks settings and select Z Device. For Ti-E microscope, select [Ti ZDrive] from the pull-down menu. For Ni-E microscope, select [Ni-E ZDrive]. Z stacks Settings Ti ZDrive Figure Selecting Z Device 5. Click the [Run now] button to acquire the XZ image. Figure ND Acquisition of Z-series 39

50 Chapter 3 Acquire Window Acquiring Live Image of the XZ Plane on the Piezo Z Stage The XZ image can be acquired while viewing the XZ cross section on the Live window. 1. Click the [Live] button. The Live image is acquired and the Live window is displayed. Figure Acquiring the live image 2. Draw the center of the XZ plane on the Scan Area window with the straight line setting tool. Figure Live window 3. Acquire the live image of the XZ plane. Click the button to acquire the live image of the XZ plane using the Piezo full range. Figure Live window Figure Live image of the XZ plane 40

51 Chapter 3 Acquire Window Acquiring an XZ image by specifying a Z stack range on the image It is possible to specify the Z stack range while viewing the acquired XZ plane image to acquire the XZ plane image in the range. 1. Click the [Live] button. The Live image is acquired and the Live window is displayed. Figure Acquiring the live image 2. Draw the center of the XZ plane on the Scan Area window with the straight line setting tool. Figure Live window 3. Acquire images of the XZ plane. Click the button to acquire the live image of the XZ plane using the Piezo full range. Click the button to acquire the captured image of the XZ plane using the Piezo full range. Figure Live window Figure Captured image of XZ plane 41

52 Chapter 3 Acquire Window 4. Right-clicking on the acquired XZ plane image displays a menu. Selecting [Define Z-Stack Range] from the menu displays a red line that indicates the Z range on the window. Figure Captured image of XZ plane Figure Captured image of XZ plane 42

53 Chapter 3 Acquire Window 5. Clicking at any Z position draws a red line on the image. Clicking at another Z position confirms the Top and Bottom positions of the Z stack drawing the Z stack range. Top positions of the Z stack Bottom positions of the Z stack Figure Captured image of XZ plane 6. To change the Z stack range, drag the red line. To change the Z stack range width, hold the base point at the red line center with the mouse. Holding a point on the red line other than the base point moves the Z stack range on the image with the range width remaining unchanged. Figure Captured image of XZ plane 43

54 Chapter 3 Acquire Window 7. Right-clicking on the image confirms the displayed Z stack range value, and the value is applied to the ND Acquisition window of the Z-series. (While the XZ live image is being displayed, the live display stops temporarily.) Right-click on the image to confirm Figure Confirming the Z stack range 44

55 Chapter 3 Acquire Window 8. Click the [Run now] button to acquire the XZ image. Figure ND Acquisition of Z-series There are two methods of acquiring XZ image using the piezo Z stage. (1) Select [Nikon A1 Piezo Z Drive] from [Z Device:]. Acquires the Z stack image quickly. (2) Select [Step-by-step Nikon A1 Piezo Z] from [Z Device:]. Acquires the Z stack image when the Z stage stops at each step. Figure ND Acquisition of Z-series 45

56 Chapter 3 Acquire Window Acquiring a cross-sectional image in combination with Z Intensity Correction functions (1) Draw the center of the XZ plane on the Scan Area window with the straight line setting tool. (2) Make settings for Z Intensity Correction functions. * For settings of Z Intensity Correction, see 3.4 Functions of Z Intensity Correction. (3) Acquire images of the XZ plane. button: button: Acquire the live image of the XZ plane while performing Z Intensity Correction. Acquire the captured image of the XZ plane while performing Z Intensity Correction. Figure Z Intensity Correction for XZ plane 46

57 4 Detection Mode DU4 This chapter describes the settings for the Standard Detector mode (DU4). 4.1 Filter and Dye Window This window enables to select the desired channel series and set the Optical path Structure of Filter and Dye Window (9) (1) (2) (3) (7) (10) (8) (4) (5) (6) Figure Filter and Dye window (DU4-use) Table Functions of Filter and Dye window (DU4-use) Name Function (1) Detector Indicates the name of the detection mode in current use. (2) Ch series (3) Setting button (4) Status Selects whether to perform scanning by simultaneously firing all lasers for the channels in use or by firing each laser in the specified order. For Ch series selection, see Section 4.1.4, Selecting the Channel Series. Opens the Optical path window. To use, select the detector, the dichroic mirror, the channel, fluorescence dye for each channel, laser and others. Indicates for the settings for each channel (fluorescence dye name, laser wavelength, and wavelength band to be acquired). (5) TD Indicates the status of the motorized transmitted detector. (6) TD IN/OUT button (7) Close mechanical shutter during experiment Sets/removes the motorized transmitted detector in/from the optical path. (IN = Set in the optical path/ OUT = Remove from the optical path) As for the case where the TD IN/OUT button is not displayed, it will be displayed when the motorized transmitted detector is set in the optical path in the Optical path window. If unchecked, the shutter remains open during the ND image acquisition. As the shutter is not opened/closed every image acquisition, the time for the image acquisition can be shortened. * During the interval period, laser power is automatically changed to the minimum but the laser cannot be shut off completely because the shutter is left open. (8) PreScan button (9) Eye Port button Makes settings of the pre-scan mode for correction of the position shift in a zoom change or of the image acquisition area shift in the photo activation by the prior scanning. Changes optical path to eye port. Each function on the A1plus Settings window becomes non-selectable when the Eye Port button is selected. 47

58 Chapter 4 Detection Mode DU4 Name (10) AUX button Function Displays the AUX Settings window to use the external detector unit. For details of the AUX Settings window, see Chapter 17. Optical Configuration Individual data items set in the Standard Detector mode (DU4) can be managed collectively with the Optical Configuration window. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the NIS-Elements AR (Advanced Research) User's Guide Setting the Optical Path Click the [Setting] button of the Filter and Dye window to display the Optical path window. Select the Standard Detector mode (DU4). There are two modes available for Optical path setting, [Auto] and [Manual]. Normally, the auto mode should be used. Setting button Figure Filter and Dye window (DU4-use) Figure Optical path window (for auto mode, DU4-use) 48

59 Chapter 4 Detection Mode DU Optical Path Window (10) (16) (3) (4) (5) (2) (6) (9) (7) (8) (1) (11) (12) (14) (15) (13) (17) (18) Figure Optical path window (for auto mode, DU4-use) Table Functions of Optical path window (DU4-use) (1) Name Detection mode selection button Function Enables to select the Standard Detector mode (DU4). Enables to acquire the 4-channel + TD images. Selects the desired mode for setting the Optical path. (2) Mode selector Activates the auto mode. Once a fluorescence dye is selected, appropriate laser and the dichroic mirror are automatically selected. (3) Sorting fluorescence dye list Activates the manual mode. Enables to set the lasers and the dichroic mirror to be used manually. Sorts the fluorescence dye list according to the selected type. ABC: Displays the list in alphabetical order. Emission: Displays the list in the order of peak wavelength of fluorescence intensity. Excitation: Displays the list in excitation wavelength order. 49

60 Chapter 4 Detection Mode DU4 Name Function Enables selection of an image acquiring method. (4) EXEM 2Ex1Em 1Ex2Emx2 CLEM This menu is effective only in the manual mode. Settings for the 2 excitations 1 emission experiment are enabled. For 2Ex1Em line sequence, see Section 4.3, 2Ex1Em Line Sequence. This menu is effective only in the manual mode. Settings for two sets of the 1 excitation 2 emissions are enabled. For 1Ex2Emx2 line sequence, see Section 4.4, 1Ex2Emx2 Line Sequence. Enables the CLEM function. For details of CLEM, see Chapter 15. Provides the following information: (5) Rainbow chart - Wavelength band for which to acquire images (shown in color and value for each channel) - Spectral profile of fluorescence dye - Excitation laser for fluorescence dye - A color band indicating the wavelengths in the entire band (400 to 750 nm) - Scale of the wavelengths in the entire band (400 to 750 nm) (6) (7) Channel selection check box Channel color setting button Enables to select the channels to be used. Displays the Color Selection window, enables to set the desired color for each channel. Fluorescence In auto mode Selects the fluorescence dye name to be used for each channel. (8) dye selection/input: In manual mode Selects the in-use fluorescence dye name for each channel or enters an arbitrary channel name. (9) (10) (11) Excitation laser select Excitation laser indicator 1st Dichroic mirror select These menus are only effective while in the manual mode. Enables to set the laser wavelength that is set with the software configuration, regardless of the setting of the Filter cube display/select. Displays the current setting for the laser. The currently set laser icon is displayed in a large size, and the optical path is indicated. This menu is only effective while in the manual mode. Enables to manually select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combination of the selected laser wavelength and the 1st Dichroic mirror,! appears following the 1st Dichroic mirror name. Selects the scanner unit to be used. (12) Scan mode select Galvano Galvano scan mode allows high-quality imaging of up to pixels. Resonant Resonant scan mode allows high-speed imaging of 420 frames per second. 50

61 Chapter 4 Detection Mode DU4 Name Function (13) Transmitted Detector selection button Brings the transmitted detector into the Optical path, to enable the ability. Brings the transmitted detector out of the Optical path, to disable the ability. (14) (15) (16) Filter cube display/select Ch series selection ND filter installation icon This menu is effective only in the manual mode. The filter cube to be mounted on the detector can be selected regardless of the excitation laser. Selects whether to perform scanning by simultaneously firing all lasers for the channels in use or by firing each laser in the specified order. For Ch series selection, see Section 4.1.4, Selecting the Channel Series. An icon is displayed on the line of laser with ND filter installed. * This icon indicates whether ND filter is installed or not, and does not indicate insertion or removal of ND filter (IN = Insert in the optical path, OUT = Remove from the optical path). Insertion or removal of ND filter is performed on the Acquisition window. (17) OK button Confirms the Optical path settings applied and closes the Optical path window. (18) Cancel button Discards the Optical path settings applied and closes the Optical path window. 51

62 Chapter 4 Detection Mode DU Selecting the Channel Series The [Ch series] menu enables to select whether to perform scanning by simultaneously firing all lasers for the channels to be used or by firing each laser in the specified order. There are four options for channel series, None, Line 1->4, Line 4->1, and Custom, either of which can be selected from the pull-down menu. * Channel series can be applied to the image acquisition phase in the photo activation experiment sequence (but cannot be applied to the stimulation phase). Figure Selecting the channel series (Filter and Dye window) Figure Selecting the channel series (Optical path window) Table Functions of channel series Name None Line 1->4 Line 4->1 Custom Function Performs scanning by simultaneously firing all lasers for the channels to be used. Performs scanning by sequentially firing the lasers for the channels to be used (Ch1 -> Ch2 -> Ch3 -> Ch4). Performs scanning by sequentially firing the lasers for the channels to be used (Ch4 -> Ch3 -> Ch2 -> Ch1). Performs scanning by firing the lasers in desired order for the channels to be used. * When [Line 1->4] or [Line 4->1] is selected, the lasers are fired sequentially for each scan line. This scan method is called the line sequence. Ch1 Ch2 Ch3 The laser for Ch1 scans the 1st line. The laser for Ch2 scans the 1st line. The laser for Ch3 scans the 1st line. A series of the same scanning procedure is repeated for each subsequent channel. Ch1 The laser for Ch1 scans the 2nd line. Ch4 The laser for Ch4 scans the 1st line. Figure Scanning motion in line sequence (Line 1->4 is selected) 52

63 Chapter 4 Detection Mode DU4 Custom Selecting Custom from the [Ch series] menu displays the current channel scan order and the [Custom] button. Clicking the [Custom] button opens the Line Channel Series Setup window to allow setting the scan order for each channel. Current channel scan order Custom button Figure Channel series (Custom) Channel name (1) Scan order Fluorescence wavelength (4) (2) (3) Figure Line Channel Series Setup window (5) (6) Name Table Functions of channel series Function (1) Scan order matrix for each channel Allows the user to set desired order of channels to be scanned. Each laser can also be fired simultaneously to multiple channels by a single scan. (Example: Firing lasers simultaneously for Ch1 and Ch2 by the first scan.) However, one channel cannot be set twice or more times. (Selecting two or more check boxes in the same vertical line of the matrix is prohibited.) (2) 1->4 button Set the channel to be scanned for each laser in order of Ch1, Ch2, Ch3, and Ch4. (3) 4->1 button Set the channel to be scanned for each laser in order of Ch4, Ch3, Ch2, and Ch1. (4) TD check box Allows the user to set the TD scan order if the transmitted detector (TD) is in the optical path. Be sure to set the TD scan order so that the scan order comes together with other channels because single TD scan is disabled. (Example: Laser is fired to Ch3 and TD by the second scan.) (5) OK button Confirms the settings applied and closes the window. (6) Cancel button Discards the settings applied and closes the window. 53

64 Chapter 4 Detection Mode DU4 4.2 Acquisition Window The Acquisition window enables to set PMT brightness (detection sensitivity), laser power, and pinhole size Structure of Acquisition Window (1) (5) (6) (7) (2) (11) (8) (3) (4) Displays laser power value (13) (9) (10) (12) (14) (15) (16) (17) Figure Acquisition window (DU4-use) Table Functions of Acquisition window (DU4-use) (1) Name Acquisition/Photo Activation window switching Function Switches between the Acquisition and Photo Activation windows. For the Photo Activation window, see Chapter 10. Displays the laser power value (integer obtained after A/D conversion divided by 10) of the current channel by clicking this button. (2) Laser power monitor button During the image acquisition, the laser power cannot be measured and this button is grayed out. * When a laser unit of the LU-N series is in use, the value displayed in the monitor does not increase over a certain value with the increase in the laser power value, but this is not a problem. (3) Brightness adjustment for each channel For each of the channels (Ch1 to Ch4), use the HV, Offset, Laser, and ND filter IN/OUT controls to adjust the brightness of the live image. (4) Channel selection Selects the channels (Ch1 to Ch4, and/or TD) to acquire the desired images. Do this by adding a check mark. When fluorescence is excited, all excitation lights are detected by the transmitted detector (TD). If the BA filter for 405 laser is installed in the TD, excitation light is not detected by the TD. When acquiring the transmitted image (TD image) only, see Section , When Acquiring Transmitted Image Only. 54

65 Chapter 4 Detection Mode DU4 Name Function (5) Select All Channels button Selects all channels for acquiring images. (6) Fluorescence dye name indication The fluorescence dye name specified in the Optical path window is indicated. (7) Channel color Displays the channel color specified in the Optical path window. (8) Laser wavelength indication Displays the currently selected laser wavelength. Selects whether the laser is emitted or not. * When LU-NV is in use, this button is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. (9) Laser ON/OFF button ON status OFF status The laser is emitted. The AOTF shutter closes and the laser power value becomes 0. When switched from OFF to ON, the laser power value set in the previous ON status is applied. (10) Pinhole (11) HV Linear Correction Adjusts the pinhole size. For pinhole size, see Section 4.2.3, Setting the Pinhole. Enables or disables HV Linear Correction. For HV Linear Correction, see Section 4.2.4, HV Linear Correction. (12) (13) Brightness adjustment for transmitted detector Remote controller selector icon For the transmitted detector, use the HV and Offset controls to adjust the brightness of the live image. Displayed for the channel group which is currently adjustable with the remote controller, see Chapter 11. (14) AG button (15) Auto Gain setting button (16) Optimize button Automatically adjusts the HV value (HV gain) of the currently selected channel to the optimum values. For Auto Gain, see Section 4.2.5, Auto Gain. Sets the ratio of saturation pixels used for automatic HV gain correction. The window for range of the ratio of saturation pixels settings appears when this button is clicked. For setting for ratio of saturation pixels, see Setting for ratio of saturation pixels in the Section 4.2.5, Auto Gain. Displays the XYZ Size Setup window. In the XYZ Size Setup window, the calculation method of the recommended values of the resolution, zoom magnification, and Z stack step size can be set. For the XYZ Size Setup window, see Section , Recommended Value Indication/Automatic Application in the next page. (17) External Port button Makes the external detector unit available. For details of using the external detector unit, see Chapter

66 Chapter 4 Detection Mode DU Recommended Value Indication/Automatic Application By the function of the recommended value indication/automatic application, the recommended values of the appropriate resolution, zoom magnification, and Z stack step size are calculated based on the objective type and the selected excitation wavelength. Using the calculated recommended values enables the image acquisition clearer and with less damage to the sample. Recommended Value Automatic Application To automatically apply the recommended values to the parameters, set the [Nyquist XY] button of the Scan Area window to ON. Nyquist XY button Figure Scan Area window Indicates the recommended value of the resolution. Indicates the recommended value of the scan magnification. Figure Location of Recommended Value Indication * When the laser or objective in use is changed, the recommended values are recalculated, and newly indicated and automatically applied. 56

67 Chapter 4 Detection Mode DU4 Recommended Value Settings Detailed settings of the recommended values are made in the XYZ Size Setup window that is displayed by clicking the [Optimize] button of the Acquisition window. If the [Nyquist XY] button of the Scan Area window is ON, the recommended values are automatically applied to the parameters. Or if the [Nyquist XY] button is OFF, the recommended values of the scan size and zoom are indicated in the Scan setting window. (1) (2) (3) (4) (5) (6) Figure XYZ Size Setup window Table Functions of XYZ Size Setup window Name (1) Zoom Preference Function When the [Nyquist XY] button is ON, keeps the scan size and applied the recommended value of the zoom. (2) Resolution Preference When the [Nyquist XY] button is ON, keeps the zoom and applied the recommended value of the scan size. (3) Use Cropping Fits the scan size in detail by using Crop Scan. Sets the Z step size calculation method. (4) Suggested Step (Z) Recommend (Z~1/3 FWHM) Half Overlap (Z=1/2 FWHM) Minimum Overlap (Z=FWHM) Perfect Voxel (Z=X=Y) Approximately one third of the thickness of optical section (FWHM value). One half of the thickness of optical section (FWHM value). The thickness of optical section (FWHM value). Value same as the pixel size. (5) OK button Confirms the XYZ Size Setup applied and closes the XYZ Size Setup window. (6) Cancel button Discards the XYZ Size Setup applied and closes the XYZ Size Setup window. 57

68 Chapter 4 Detection Mode DU When Acquiring Transmitted Image Only By using the TD channel, you can acquire the image with the TD channel only. 1. Display the Optical path window. Select a channel for the laser power control and deselect other unnecessary channel s check box. * Even when the multiple channels are selected in the Optical path window, the image with the TD channel only can be acquired by deselecting the unnecessary check box in the Acquisition window. In this case, the laser power value of the channel that is deselected at last is used for acquiring the TD image. Figure Optical path window (DU4-use) 2. Set the transmitted detector into the optical path and then click the [OK] button to close the Optical path window. Figure Optical path window (DU4-use) 3. Use the HV and Offset controls of the transmitted detector to adjust the brightness of the live image. 4. Use a channel that remains to On to control the laser power to set the laser power value. 5. Deselect the unnecessary check boxes other than TD. Deselect this check box at last. Set the laser power value. Adjust the transmitted detector. Figure Acquisition window (DU4-use) 6. Acquire the image. 58

69 Chapter 4 Detection Mode DU Setting Image Brightness For the live images of each channel, adjust HV, Offset, Laser and ND filter IN/OUT to obtain clear images. (1) (2) (3) (6) (4) (5) Figure Setting the live image brightness (DU4-use) Table Brightness adjustment functions for the live image (DU4-use) Name (1) HV (2) Offset (3) Laser (*) (4) HV (TD) (5) Offset (TD) Function Sets the voltage to be applied to the PMT. Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value. Sets the BL offset value of the PMT. Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value. Sets the laser power value. Slider bar: Slides to the right or left to set the laser power value. Arrow buttons: Click either arrow button to increase or decrease the laser power value stepwise. Direct entry in laser power value display field: Type the desired setting value. Sets the voltage to be applied to the transmitted detector. Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value. Sets the offset value of the transmitted detector. Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value. (6) ND filter IN/OUT button (*) Inserts/removes the ND filter in/from the optical path. (IN = Insert in the optical path/ OUT = Remove from the optical path) This button is displayed only for lasers that can control insertion/removal of ND filter. * When LU-NV is in use, this function is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. 59

70 Chapter 4 Detection Mode DU4 PMT Overload If too much gain is applied with reference to the illumination intensity, the gain is automatically shut down to protect PMT and the following PMT Overload dialog box is displayed. Figure PMT Overload dialog box 60

71 Chapter 4 Detection Mode DU Setting the Pinhole (1) (2) (7) (5) (6) (3) (4) Figure Setting the Pinhole (DU4-use) Table Pinhole setting functions (DU4-use) Name (1) Pinhole size setting (2) Pinhole button Function Sets a pinhole size for A1 system. Slider bar: Slides to the right or left to set the pinhole size. (Unit: A.U.) Arrow buttons: Click either arrow button to increase or decrease the pinhole size stepwise. Direct entry in pinhole size display field: Type the desired setting value. Displays the A.U. Calculation Settings window to calculate the pinhole size. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) Changes the pinhole to the predetermined home position. (3) Home The value of the home position can be changed in the A.U. Calculation Settings window. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) (4) Pinhole size Indicates pinhole size of A1 system. (Unit: um) (5) thickness of optical section Indicates the FWHM (full width at half maximum) of z airy disk. (6) Optical Resolution The actual size of 1 pixel square calculated from the optical information (for objectives and scan parameters) and the size acquired from an image. (7) Reference excitation wavelength for the pinhole size calculation Selects the excitation wavelength as the reference of the automatic calculation of the pinhole size from the laser wavelengths, or enter it manually in the A.U. Calculation Settings window. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) 61

72 Chapter 4 Detection Mode DU Calculation Settings for Pinhole Size This section describes the setting window for calculating the pinhole size. Click the [Pinhole] button in Acquisition window, the A.U. Calculation Settings window appears. (Usually, the [Recommend] is selected to enable automatic calculation. [Recommend] calculates the A.U. value by using the Nikon-recommended EM and NA values.) (1) (2) (3) (4) (5) (6) (7) Figure A.U. Calculation Settings window Table A.U. Calculation Settings window (1) Name Select calculation method Function Recommend Sets parameters automatically. (Nikon recommended) User Setting Allows the user to manually set parameters. (2) EM:emission wavelength[um] Selected Laser wavelength Manual Calculates parameters by using the laser wavelength selected in the pinhole combo box of the Acquisition window as the emission wavelength (EM value). The wavelength displayed in the combo box is to be the laser wavelength set in the Optical Setting window. Allows the user to manually set parameters. (The parameter is calculated with the input value as the emission wavelength (EM value).) Enter the value directly from the keyboard. Sets refractive index of the objective. (3) NA:Numerical Aperture NA_obj(NA of objective lens) Min(NA_obj, NA_sample) Regardless of whether or not the objective NA value exceeds the refractive index of the sample, executes calculation by using the objective NA as the calculation parameter. When the objective NA value does not exceed the refractive index of the sample, executes calculation by using the objective NA as the calculation parameter. When the objective NA value exceeds the refractive index of the sample, executes calculation by using the sample refractive index. (4) Airy Home Position Sets a home position of pinhole. Direct entry in Airy Home Position value display field: Type the desired setting value. (5) Keep A.U. check box When checked, the pinhole size is fixed by the A.U. when the selected wavelength or objective is changed. (However changes by the um.) When unchecked, the pinhole size is fixed by the um. (However changes by the A.U.) (6) OK button (7) Cancel button Confirms the A.U. Calculation Settings applied and closes the A.U. Calculation Settings window. Discards the A.U. Calculation Settings applied and closes the A.U. Calculation Settings window. 62

73 Chapter 4 Detection Mode DU Pinhole Position Alignment This section describes the setting window for aligning the pinhole position. The pinhole position can be aligned by manually shifting the condensing lens in the A1plus Pinhole Alignment window. As shown below, right-click on the gray area (without any setting window displayed) to display a menu. Then select [Acquisition Controls] -> [A1plus Pinhole Alignment] in the menu to open the Scan Area window. Figure How to display the A1plus Pinhole Alignment window (1) (2) (3) (4) Figure A1plus Pinhole Alignment window Table A1plus Pinhole Alignment window Name (1) Coarse/Fine button Function The condensing lens position can be shifted by the shift amount set in the Coarse/Fine field by using these buttons. (2) Coarse/Fine field Shift amount of the condensing lens position can be set in these fields. (3) Lens Position X/Y The X and Y coordinates of the current condensing lens position is displayed. (4) Reset button The condensing lens potion is reset to the default position set at the factory. 63

74 Chapter 4 Detection Mode DU4 For memorizing the pinhole position (the condensing lens position) NIS-Elements can memorize the pinhole position that is adjusted in the A1plus Pinhole Alignment window. Select [Edit] -> [Options] -> [User rights] in the menu bar to display the window and then select the [Privileges] tab. Select the [Modify Shared Confocal Alignment] check box in the [Privileges] area so that NIS-Elements memorizes the NIS-Elements C pinhole position when the software ends. 64

75 Chapter 4 Detection Mode DU HV Linear Correction When HV changes, Gain changes as shown in the graph captioned Without HV Linear Correction. As HV increases, the gain variation (the variation of image brightness) is gradual initially, and it becomes steep beyond a certain point. The gain variation can be automatically corrected to be linear with HV variation by the function called HV Linear Correction. With this correction, gain varies at the same rate as the HV adjustment. Gain Gain Without HV Linear Correction HV With HV Linear Correction HV Figure Gain vs. HV To enable HV Linear Correction, check the [HV Linear Correction] check box. Figure HV Linear Correction When HV Linear Correction is enabled or disabled, HV is reset to 0 V once. 65

76 Chapter 4 Detection Mode DU Auto Gain Auto Gain is a function to automatically correct the value of HV gain to set the optimum image brightness. Automatic HV gain correction is performed within the predetermined range of the ratio of saturation pixels. Both Galvano mode and Resonant mode are supported. Automatic HV gain correction is performed only when channels are selected. For a TD, automatic adjustment is performed when it is selected. After execution of Auto Gain, in the window indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed by channel. For a channel on which Auto Gain failed, x is indicated and the HV value returns to its original value. In 2Ex1Em or 1Ex2Emx2 line sequence, Auto Gain cannot be executed. When setting the line scan, Auto Gain cannot be executed. During execution of Auto Gain, do not perform manual adjustments in the Acquisition window and adjustments by the remote controller. Auto Gain button Auto Gain setting button Auto Gain does not execute on unselected channels. Figure Execution of Auto Gain (DU4-use) Channel name Auto Gain status... Failed... Completed If checked, the window is automatically closed when Auto Gain is completed. Figure Auto Gain progress 66

77 Chapter 4 Detection Mode DU4 Setting for ratio of saturation pixels Set the maximum and minimum value for the Ratio of saturation pixels used for automatic HV gain correction. Click the [Auto Gain Setting] button to display the Auto gain setup window. Set the maximum and minimum value for the ratio of saturation pixels in Auto gain setup window. Figure Displaying the Auto gain setup window (1) (2) (3) (4) (5) (6) (7) Figure Setting for ratio of saturation pixels Table Setting for ratio of saturation pixels (1) Name Target Maximal Intensity Function Specifies the application ratio of the setting of the ratio of saturation pixels. Sets the percentage (%) of the maximum value to be applied. (2) Advanced Settings If checked, advanced settings of the ratio of saturation pixels are enabled. (3) Overillumination Tolerance (Area) Minimum Maximum Sets the minimum value for Ratio of saturation pixels. Sets the maximum value for Ratio of saturation pixels. (4) Perform in Find Mode (fast) If checked, execution in the Find mode is enabled. (5) Perform Auto Offset If checked, the offset value is set automatically. (6) OK button (7) Cancel button Confirms the settings of Auto gain setup applied and closes the Auto gain setup window. Discards the settings of Auto gain setup applied and closes the Auto gain setup window. 67

78 Chapter 4 Detection Mode DU Ex1Em Line Sequence This section describes the settings for 2Ex1Em line sequence to make the 2 excitations 1 emission experiment. 2Ex1Em (2 Excitations 1 Emission) means an experiment made when using a fluorescence dye whose fluorescence intensity varies with the wavelength of excitation lasers. The excitation lasers are changed, but the wavelength of acquired fluorescence is identical Procedure for 2Ex1Em Line Sequence Settings 1 Selecting the Scan mode Select [Resonant] or [Galvano] for using the Scan mode. Figure Selecting the scan mode 2 Setting the Optical path 1. Click the [Setting] button in the Filter and Dye window. For details of the Optical path settings, see Section 4.1, Filter and Dye Window. Setting button Figure Filter and Dye window 2. Select the Standard detector mode (DU4) of Optical path setting. Standard Detector [DU4] Figure Selecting the Detection mode (Detector) 68

79 Chapter 4 Detection Mode DU4 3. Activate the Manual mode of Optical path setting. Click the [Manual] button. A pull-down menu to select an image acquiring method appears. Manual button Pull-down menu Figure Selecting the Manual mode 4. Select the [2Ex1Em] from the pull-down menu. When the [2Ex1Em] is selected, [Line 1->4] is selected automatically in Ch Series. (Channel series is not selectable.) Selects 2Ex1Em line sequence. [Line 1->4] is selected automatically. Figure Selecting 2Ex1Em line sequence 69

80 Chapter 4 Detection Mode DU4 5. Select a PMT channel. Select a PMT channel to be used from the pull-down menu. Selects a PMT channel from the pull-down menu. Figure Selecting PMT channel 6. Select the excitation lasers. Select the wavelength for excitation lasers from the pull-down menu. Selects the excitation lasers from the pull-down menu. Figure Selecting wavelength for excitation lasers 7. Set the channel color. When click the [Channel color setting] button, displays the Color Selection window, enables to set the desired color for each channel. Enter any desired channel name for each channel. Channel color setting button Figure Enter the channel name Selecting channels color and input channels name 70

81 Chapter 4 Detection Mode DU4 8. Select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected,! appears following the 1st Dichroic mirror name. Selects the 1st Dichroic mirror. Figure Optical path settings 9. Select the transmitted detector icon to use or disuse and the filter cubes to be mounted on the detector, clicking the [OK] button to confirm the Optical path settings. Selects the transmitted detector icon to use or disuse. Selects the filter cubes. OK button Figure Optical path settings 71

82 Chapter 4 Detection Mode DU4 3 Applying Scan settings In the Scan setting window, apply various scan settings to acquire the live image. For details of Scan settings, see Chapter 8, Scan Setting Window. Selects the scan method (Unidirectional/Bidirectional). Selects scan magnification. Selects resolution. Selects scan speed. Selects averaging and integration methods. Figure Scan setting window 4 Acquiring the live image Click the [Live] button. The live image is acquired and the Live window appears. Figure Acquiring the live image Figure Live image 5 Adjusting the brightness of the live image 72

83 Chapter 4 Detection Mode DU4 In the Acquisition window, adjust a PMT for the excitation lasers. Set the HV value, the Offset value, and the Laser power value. For details of the acquisition settings, see Section 4.2, Acquisition Window. * In 2Ex1Em line sequence, two excitation lasers are controlled by a single PMT. Therefore, if the HV value or Offset value of one channel is changed, the same value is set for the other channel in an interlocked manner. (The Laser power value is not interlocked, so set it for each channel.) Figure Acquisition window 6 Acquiring the captured image Click the [XY] button. The captured image is acquired and displayed in the image window. Figure Acquiring the Captured image Figure Captured image 73

84 Chapter 4 Detection Mode DU Ex2Emx2 Line Sequence This section describes the settings for the 1Ex2Emx2 line sequence to make an experiment with the settings for two sets of 1 excitation 2 emissions Procedure for 1Ex2Emx2 Line Sequence Settings 1 Selecting the Scan mode Select [Resonant] or [Galvano] for using the Scan mode. Figure Selecting the scan mode 2 Setting the Optical path 1. Click the [Setting] button in the Filter and Dye window. For details of the Optical path settings, see Section 4.1, Filter and Dye Window. Setting button Figure Filter and Dye window 2. Select the Standard detector mode (DU4) of Optical path setting. Standard Detector [DU4] Figure Selecting the Detection mode (Detector) 74

85 Chapter 4 Detection Mode DU4 3. Activate the Manual mode of Optical path setting. Click the [Manual] button. A pull-down menu to select an image acquiring method appears. Manual button Pull-down menu Figure Selecting the Manual mode 4. Select [1Ex2Emx2] from the pull-down menu. When [1Ex2Emx2] is selected, [Line 1->4] is selected automatically in Ch Series. (Channel series is not selectable.) Selects 1Ex2Emx2 line sequence. [Line 1->4] is selected automatically. Figure Selecting 1Ex2Emx2 line sequence 75

86 Chapter 4 Detection Mode DU4 5. Select a PMT channel. Select a PMT channel to be used from the pull-down menu. Make PMT setting respectively for Em #1 and Em #2. (Identical PMT numbers cannot be set.) Selects a PMT channel from the pull-down menu. Figure Selecting PMT channel 6. Select the excitation lasers. Select the wavelength for excitation lasers from the pull-down menu. Make excitation laser setting respectively for Ex1 and Ex2. Selects the excitation lasers from the pull-down menu. Figure Selecting wavelength for excitation lasers 7. Set the channel color. When click the [Channel color setting] button, displays the Color Selection window, enables to set the desired color for each channel. Channel color setting button Figure Selecting channels color 76

87 Chapter 4 Detection Mode DU4 8. Select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected,! appears following the 1st Dichroic mirror name. Selects the 1st Dichroic mirror. Figure Optical path settings 9. Select the transmitted detector icon to use or disuse and the filter cubes to be mounted on the detector, clicking the [OK] button to confirm the Optical path settings. Selects the transmitted detector icon to use or disuse. Selects the filter cubes. OK button Figure Optical path settings 77

88 Chapter 4 Detection Mode DU4 3 Applying Scan settings In the Scan setting window, apply various scan settings to acquire the live image. For details of Scan settings, see Chapter 8, Scan Setting Window. Selects the scan method (Unidirectional/Bidirectional). Selects scan magnification. Selects resolution. Selects scan speed. Selects averaging and integration methods. Figure Scan setting window 4 Acquiring the live image Click the [Live] button. The live image is acquired and the Live window appears. Figure Acquiring the live image Figure Live image 78

89 Chapter 4 Detection Mode DU4 5 Adjusting the brightness of the live image In the Acquisition window, adjust a PMT for the excitation lasers. Set the HV value, the Offset value, and the Laser power value. For details of the acquisition settings, see Section 4.2, Acquisition Window. * In the 1Ex2Emx2 line sequence, each excitation laser is controlled by the two PMTs. For example, if the HV value or Offset value of Ch1 is changed, the same value is set for Ch3 in an interlocked manner. In the combination of Ch1 and Ch2 or Ch3 and Ch4 where the same laser is used, the laser power interlocks. If the laser power of one channel is changed, the same value is set for the other channel. PMT set for Em #1 interlocks PMT set for Em #2 interlocks Figure Interlocks in HV and offset value Laser set for Ex1 interlocks Laser set for Ex2 interlocks Figure Laser power interlocks 79

90 Chapter 4 Detection Mode DU4 6 Acquiring the captured image Click the [XY] button. The captured image is acquired and displayed in the image window. Figure Acquiring the Captured image Figure Captured image 80

91 Chapter 4 Detection Mode DU4 4.5 Indication When GaAsP DU4 Detector is in Use When a DU4 detector incorporating the GaAsP device is in use, GaAsP or (G) is indicated in the relevant channel displayed in the Filter and Dye window, Optical path window, Acquisition window, and A1plus Compact GUI. Figure Filter and Dye window and Acquisition window Figure A1plus Compact GUI Figure Optical path window 81

92 5 Detection Mode SD This chapter describes the settings for the Spectral Detector mode (SD). 5.1 Filter and Dye Window This window enables to set the Optical path Structure of Filter and Dye Window (9) (1) (3) (2) (10) (8) (7) (6) (4) (5) Figure Filter and Dye window (SD-use) Table Functions of Filter and Dye window (SD-use) Name Function (1) Detector Indicates the name of the detection mode in current use. (2) Setting button (3) Status Opens the Optical path window. In the Optical path window, sets the detector to use, the excitation laser, fluorescence dye for each excitation laser, the dichroic mirror and others. Indicates for the settings for each excitation laser (fluorescence dye name, laser wavelength, and wavelength band to be acquired). (4) TD Indicates the status of the motorized transmitted detector. (5) TD IN/OUT button (6) (7) Spectral Detector setting information Close mechanical shutter during experiment (8) PreScan button (9) Eye Port button (10) AUX button Sets/removes the motorized transmitted detector in/from the optical path. (IN = Set in the optical path/ OUT = Remove from the optical path) As for the case where the TD IN/OUT button is not displayed, it will be displayed when the motorized transmitted detector is set in the optical path in the Optical path window. Displays the information set on the Spectral Detector. If unchecked, the shutter remains open during the ND image acquisition. As the shutter is not opened/closed every image acquisition, the time for the image acquisition can be shortened. * During the interval period, laser power is automatically changed to the minimum but the laser cannot be shut off completely because the shutter is left open. Makes settings of the pre-scan mode for correction of the position shift in a zoom change or of the image acquisition area shift in the photo activation by the prior scanning. Changes optical path to eye port. Each function on the A1plus Settings window becomes non-selectable when the Eye Port button is selected. Displays the AUX Settings window to use the external detector unit. For details of the AUX Settings window, see Chapter

93 Chapter 5 Detection Mode SD Optical Configuration Individual data items set in the Spectral Detector mode (SD) can be managed collectively with the Optical Configuration window. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the NIS-Elements AR (Advanced Research) User's Guide Setting the Optical Path Click the [Setting] button of the Filter and Dye window to display the Optical path window. Select the Spectral Detector mode (SD). There are two modes available for Optical path setting, [Auto] and [Manual]. Normally, the auto mode should be used. Setting button Figure Filter and Dye window Figure Optical path window (for auto mode, SD-use) 83

94 Chapter 5 Detection Mode SD Optical Path Window (3) (11) Switching Tab (4) (8) (2) (5) (7) (6) (1) (9) (10) (12) (13) Figure Table Optical path window (for auto mode, SD-use) Functions of Optical path window (SD-use) (1) Name Detection mode selection button Function Enabled to select the Spectral Detector mode (SD). Enables to acquire the 32-channel + TD spectral images simultaneously. Selects the desired mode for setting the Optical path. (2) Mode selector Activates the auto mode. Once the fluorescence dye to be used is selected, the appropriate laser and the dichroic mirror, and the acquired wavelength range and resolution are automatically selected. (3) Excitation laser indicator Activates the manual mode. Enables to set all of the laser, the dichroic mirror, acquired wavelength range, and resolution to be used manually. Displays the current setting for the laser. The currently set laser icon is displayed in a large size, and the optical path is indicated. 84

95 Chapter 5 Detection Mode SD Name Function (4) (5) (6) (7) Sorting fluorescence dye list Excitation laser selection check box Fluorescence dye selection/input: Excitation laser wavelength select This function is effective only in the auto mode. Sorts the fluorescence dye list according to the selected type. ABC: Displays the list in alphabetical order. Emission: Displays the list in the order of peak wavelength of fluorescence intensity. Excitation: Displays the list in excitation wavelength order. Enables to select the excitation lasers to be used. This function is effective only in the auto mode. Selects the in-use fluorescence dye name for each channel or enters an arbitrary channel name. These menus are only effective while in the manual mode. Enables to set the laser wavelength that is set with the software configuration, regardless of the setting of the Filter cube display/select. Provides the following information: (8) Rainbow chart - Wavelength band for which to acquire images (shown in color and value for each excitation laser) - Spectral profile of fluorescence dye - Excitation laser for fluorescence dye - A color band indicating the wavelengths in the entire band (400 to 750 nm) - Scale of the wavelengths in the entire band (400 to 750 nm) (9) (10) (11) 1st Dichroic mirror select Transmitted detector selection button ND filter installation icon This menu is only effective while in the manual mode. Enables to manually select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combination of the selected laser wavelength and the 1st Dichroic mirror,! appears following the 1st Dichroic mirror name. Brings the transmitted detector into the Optical path, to enable the ability. Brings the transmitted detector out of the Optical path, to disable the ability. An icon is displayed on the line of laser with ND filter installed. * This icon indicates whether ND filter is installed or not, and does not indicate insertion or removal of ND filter (IN = Insert in the optical path, OUT = Remove from the optical path). Insertion or removal of ND filter is performed on the Acquisition window. (12) OK button Confirms the Optical path settings applied and closes the Optical path window. (13) Cancel button Discards the Optical path settings applied and closes the Optical path window. 85

96 Chapter 5 Detection Mode SD Optical Path Window Switching Tab The tab for switching between [Detector] and [Binning/Skip] is displayed on the right top of the Optical path window Detector Tab Rainbow chart (1) (2) (3) (4) Excitation laser selection check box (7) (8) Excitation laser wavelength select (5) (6) Figure Optical path window (for manual mode, Detector tab) Table Functions of Detector tab Name (1) Resolution (2) Channels Function Selects a wavelength resolution. (Enabled in the manual mode only.) Selectable from 2.5, 6, or 10nm. Selects the number of channels (number of PMTs). (Enabled in the manual mode only.) Up to 32 channels can be selected in the wavelength range of 400nm to 750nm. (3) Binning Displays the number of channel binning currently set. (4) Wavelength range setting bar Sets a wavelength range in a wavelength range from 400nm to 750nm. (Enabled in the manual mode only.) Sets a range by shifting the wavelength range setting bar to the right or left or by enlarging or reducing it. (Linked with the above setting of the number of channels.) * A part of the wavelength range may be displayed in black depending on the setting conditions. In the wavelength range displayed in black, no wavelength range can be set. (5) Start (6) All (7) End (8) Enlarge button Displays the start wavelength of the wavelength range currently selected. Enabled to enlarge or reduce the range in units of wavelength resolution with the right or left button in the manual mode. Enabled to shift to the right or left within the currently selected wavelength range without the wavelength interval being changed. (Enabled in the manual mode only.) Displays the end wavelength of the wavelength range currently selected. In the manual mode, the range in units of wavelength resolution can be enlarged or reduced using the right and left buttons. Enlarges the rainbow chart. The display is switched in three levels. 86

97 Chapter 5 Detection Mode SD Binning/Skip Tab With the inter-channel binning, the dark image can be brightened. (Enabled in the manual mode only.) Further, channels within the set wavelength range can be arbitrarily skipped. Since masked channel data is not acquired, the data volume can be reduced. Set this tab after the setting of the [Detector] tab is confirmed. (1) Wavelength indication (2) (3) Enlarge button (1) Figure Optical path window (Binning/Skip tab) Table Functions of Binning/Skip tab (1) Binning Name Function Sets the number of channels to be combined into one channel. Two to four channels can be set. When Binning is set, the number of channels set with the [Detector] tab is automatically re-set to the closest number of channels that can be divided by the binning value. (2) (3) PMT All on/off button PMT skip selection check box Resets all PMT skips that have been set. Leaves one channel and skips all of other PMTs. Sets skip in each channel. If this box is clicked, (black) is displayed and skip is set. Channel data with skip set is not acquired during scan. * If the setting of the [Detector] tab is changed, the setting with the [Binning/Skip] tab is cancelled. 87

98 Chapter 5 Detection Mode SD 5.2 Acquisition Window The Acquisition window enables to set PMT brightness (detection sensitivity), laser power, and pinhole size Structure of Acquisition Window (1) (3) (4) (2) (5) Displays laser power value (10) (6) (7) (8) (11) (12) (13) (9) Figure Acquisition window (SD-use) Table Functions of Acquisition window (SD-use) Name Function (1) Acquisition/Photo Activation window switching (2) Laser power monitor button Switches between the Acquisition and Photo Activation windows. For the Photo Activation window, see Chapter 10. Displays the laser power value (integer obtained after A/D conversion divided by 10) of the current excitation laser by clicking this button. During the image acquisition, the laser power cannot be measured and this button is grayed out. (3) Excitation laser color Displays the excitation laser color specified in the Optical path window. (4) Laser wavelength indication The currently selected laser wavelength is indicated. Selects whether the laser is emitted or not. * When LU-NV is in use, this button is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. (5) Laser ON/OFF button ON status The laser is emitted. The AOTF shutter closes and the laser power value becomes 0. OFF status (6) Si HV Adjusts HV of the Spectral Detector. When switched from OFF to ON, the laser power value set in the previous ON status is applied. (7) Pinhole Adjusts the pinhole size. For pinhole size, see Section 5.2.3, Setting the Pinhole. 88

99 Chapter 5 Detection Mode SD (8) Name Brightness adjustment for transmitted detector Function For the transmitted detector, use the HV and Offset controls to adjust the brightness of the live image. (9) TD channel selection Enables to acquire TD images by checking the check box. When fluorescence is excited, all excitation lights are detected by the transmitted detector (TD). If the BA filter for 405 laser is installed in the TD, excitation light is not detected by the TD. (10) Remote controller selector icon Displayed for the excitation laser group which is currently adjustable with the remote controller, see Chapter 11. (11) AG button (12) Auto Gain setting button (13) Optimize button Automatically adjusts the Si HV value (Si HV gain) of the currently selected excitation laser to the optimum values. For Auto Gain, see Section 5.2.4, Auto Gain. Sets the ratio of saturation pixels used for automatic Si HV gain correction. The window for range of the ratio of saturation pixels setting appears when this button is clicked. For setting for ratio of saturation pixels, see Setting for ratio of saturation pixels in the Section 5.2.4, Auto Gain. Displays the XYZ Size Setup window. In the XYZ Size Setup window, the calculation method of the recommended values of the resolution, zoom magnification, and Z stack step size can be set. For the XYZ Size Setup window, see Section , Recommended Value Indication/Automatic Application in the next page. 89

100 Chapter 5 Detection Mode SD Recommended Value Indication/Automatic Application By the function of the recommended value indication/automatic application, the recommended values of the appropriate resolution, zoom magnification, and Z stack step size are calculated based on the objective type and the selected excitation wavelength. Using the calculated recommended values enables the image acquisition clearer and with less damage to the sample. Recommended Value Automatic Application To automatically apply the recommended values to the parameters, set the [Nyquist XY] button of the Scan Area window to ON. Nyquist XY button Figure Scan Area window Indicates the recommended value of the resolution. Indicates the recommended value of the scan magnification. Figure Location of Recommended Value Indication * When the laser or objective in use is changed, the recommended values are recalculated, and newly indicated and automatically applied. 90

101 Chapter 5 Detection Mode SD Recommended Value Settings Detailed settings of the recommended values are made in the XYZ Size Setup window that is displayed by clicking the [Optimize] button of the Acquisition window. If the [Nyquist XY] button of the Scan Area window is ON, the recommended values are automatically applied to the parameters. Or if the [Nyquist XY] button is OFF, the recommended values of the scan size and zoom are indicated in the Scan setting window. (1) (2) (3) (4) (5) (6) Figure XYZ Size Setup window Table Functions of XYZ Size Setup window Name (1) Zoom Preference Function When the [Nyquist XY] button is ON, keeps the scan size and applied the recommended value of the zoom. (2) Resolution Preference When the [Nyquist XY] button is ON, keeps the zoom and applied the recommended value of the scan size. (3) Use Cropping Fits the scan size in detail by using Crop Scan. Sets the Z step size calculation method. (4) Suggested Step (Z) Recommend (Z~1/3 FWHM) Half Overlap (Z=1/2 FWHM) Minimum Overlap (Z=FWHM) Perfect Voxel (Z=X=Y) Approximately one third of the thickness of optical section (FWHM value). One half of the thickness of optical section (FWHM value). The thickness of optical section (FWHM value). Value same as the pixel size. (5) OK button Confirms the XYZ Size Setup applied and closes the XYZ Size Setup window. (6) Cancel button Discards the XYZ Size Setup applied and closes the XYZ Size Setup window. 91

102 Chapter 5 Detection Mode SD Setting Image Brightness For each excitation laser, adjust HV, Offset, Laser and ND filter IN/OUT to obtain clear images. (5) (1) (4) (2) (3) Figure Setting the live image brightness (SD-use) Table Brightness adjustment functions for the live image (SD-use) Name (1) Laser (*) (2) HV (3) Offset (4) Si HV (5) ND filter IN/OUT button (*) Function Sets the laser power value. Slider bar: Slides to the right or left to set the laser power value. Arrow buttons: Click either arrow button to increase or decrease the laser power value stepwise. Direct entry in laser power value display field: Type the desired setting value. Sets the voltage to be applied to the transmitted detector. Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value. Sets the offset value of the transmitted detector. Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value. Adjusts HV of the Spectral Detector. (Applied to all excitation lasers.) Slider bar: Slides to the right or left to set the Si HV value. Arrow buttons: Click either arrow button to increase or decrease the Si HV value stepwise. Direct entry in Si HV value display field: Type the desired setting value. Inserts/removes the ND filter in/from the optical path. (IN = Insert in the optical path/ OUT = Remove from the optical path) This button is displayed only for lasers that can control insertion/removal of ND filter. * When LU-NV is in use, this function is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. PMT Overload If too much gain is applied with reference to the illumination intensity, the gain is automatically shut down to protect PMT and the following PMT Overload dialog box is displayed. Figure PMT Overload dialog box 92

103 Chapter 5 Detection Mode SD Setting the Pinhole (1) (2) (7) (5) (6) (3) (4) Figure Setting the Pinhole (SD-use) Table Pinhole setting functions (SD-use) Name (1) Pinhole size setting (2) Pinhole button Function Sets a pinhole size for A1 system. Slider bar: Slides to the right or left to set the pinhole size. (Unit: A.U.) Arrow buttons: Click either arrow button to increase or decrease the pinhole size stepwise. Direct entry in pinhole size display field: Type the desired setting value. Displays the A.U. Calculation Settings window to calculate the pinhole size. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) Changes the pinhole to the predetermined home position. (3) Home The value of the home position can be changed in the A.U. Calculation Settings window. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) (4) Pinhole size Indicates pinhole size of A1 system. (Unit: um) (5) thickness of optical section Indicates the FWHM (full width at half maximum) of z airy disk. (6) Optical Resolution The actual size of 1 pixel square calculated from the optical information (for objectives and scan parameters) and the size acquired from an image. (7) Reference excitation wavelength for the pinhole size calculation Selects the excitation wavelength as the reference of the automatic calculation of the pinhole size from the laser wavelengths, or enter it manually in the A.U. Calculation Settings window. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) 93

104 Chapter 5 Detection Mode SD Calculation Settings for Pinhole Size This section describes the setting window for calculating the pinhole size. Click the [Pinhole] button in Acquisition window, the A.U. Calculation Settings window appears. (Usually, the [Recommend] is selected to enable automatic calculation. [Recommend] calculates the A.U. value by using the Nikon-recommended EM and NA values.) (1) (2) (3) (4) (5) (6) (7) Figure Table A.U. Calculation Settings window A.U. Calculation Settings window (1) Name Select calculation method (2) EM:emission wavelength[um] Function Recommend Sets parameters automatically. (Nikon recommended) User Setting Selected Laser wavelength Manual Allows the user to manually set parameters. Calculates parameters by using the laser wavelength selected in the pinhole combo box of the Acquisition window as the emission wavelength (EM value). The wavelength displayed in the combo box is to be the laser wavelength set in the Optical Setting window. Allows the user to manually set parameters. (The parameter is calculated with the input value as the emission wavelength (EM value).) Enter the value directly from the keyboard. Sets refractive index of the objective. (3) NA:Numerical Aperture NA_obj(NA of objective lens) Min(NA_obj, NA_sample) Regardless of whether or not the objective NA value exceeds the refractive index of the sample, executes calculation by using the objective NA as the calculation parameter. When the objective NA value does not exceed the refractive index of the sample, executes calculation by using the objective NA as the calculation parameter. When the objective NA value exceeds the refractive index of the sample, executes calculation by using the sample refractive index. (4) (5) Airy Home Position Keep A.U. check box (6) OK button (7) Cancel button Sets a home position of pinhole. Direct entry in Airy Home Position value display field: Type the desired setting value. When checked, the pinhole size is fixed by the A.U. when the selected wavelength or objective is changed. (However changes by the um.) When unchecked, the pinhole size is fixed by the um. (However changes by the A.U.) Confirms the A.U. Calculation Settings applied and closes the A.U. Calculation Settings window. Discards the A.U. Calculation Settings applied and closes the A.U. Calculation Settings window. 94

105 Chapter 5 Detection Mode SD Pinhole Position Alignment This section describes the setting window for aligning the pinhole position. The pinhole position can be aligned by manually shifting the condensing lens in the A1plus Pinhole Alignment window. As shown below, right-click on the gray area (without any setting window displayed) to display a menu. Then select [Acquisition Controls] -> [A1plus Pinhole Alignment] in the menu to open the Scan Area window. Figure How to display the A1plus Pinhole Alignment window (1) (2) (3) (4) Figure A1plus Pinhole Alignment window Table A1plus Pinhole Alignment window Name (1) Coarse/Fine button Function The condensing lens position can be shifted by the shift amount set in the Coarse/Fine field by using these buttons. (2) Coarse/Fine field Shift amount of the condensing lens position can be set in these fields. (3) Lens Position X/Y The X and Y coordinates of the current condensing lens position is displayed. (4) Reset button The condensing lens potion is reset to the default position set at the factory. 95

106 Chapter 5 Detection Mode SD For memorizing the pinhole position (the condensing lens position) NIS-Elements can memorize the pinhole position that is adjusted in the A1plus Pinhole Alignment window. Select [Edit] -> [Options] -> [User rights] in the menu bar to display the window and then select the [Privileges] tab. Select the [Modify Shared Confocal Alignment] check box in the [Privileges] area so that NIS-Elements memorizes the NIS-Elements C pinhole position when the software ends. 96

107 Chapter 5 Detection Mode SD Auto Gain Auto Gain is a function to automatically correct the value of Si HV gain to set the optimum image brightness. Automatic Si HV gain correction is performed within the predetermined range of the ratio of saturation pixels. Automatic Si HV gain correction is performed only Si HV. For a TD, automatic adjustment is performed when it is selected. After execution of Auto Gain, in the window indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed. If Auto Gain failed, x is indicated and the Si HV value returns to its original value. When setting the line scan, Auto Gain cannot be executed. During execution of Auto Gain, do not perform manual adjustments in the Acquisition window and adjustments by the remote controller. Auto Gain button Auto Gain setting button Figure Execution of Auto Gain (SD-use) Auto Gain status... Failed... Completed If checked, the window is automatically closed when Auto Gain is completed. Figure Auto Gain progress 97

108 Chapter 5 Detection Mode SD Setting for ratio of saturation pixels Set the maximum and minimum value for the Ratio of saturation pixels used for automatic Si HV gain correction. Click the [Auto Gain Setting] button to display the Auto gain setup window. Set the maximum and minimum value for the ratio of saturation pixels in Auto gain setup window. Figure Displaying the Auto gain setup window (1) (2) (3) (4) (5) (6) (7) Figure Setting for ratio of saturation pixels Table Setting for ratio of saturation pixels (1) Name Target Maximal Intensity Function Specifies the application ratio of the setting of the ratio of saturation pixels. Sets the percentage (%) of the maximum value to be applied. (2) Advanced Settings If checked, advanced settings of the ratio of saturation pixels are enabled. (3) Overillumination Tolerance (Area) Minimum Maximum Sets the minimum value for Ratio of saturation pixels. Sets the maximum value for Ratio of saturation pixels. (4) Perform in Find Mode (fast) If checked, execution in the Find mode is enabled. (5) Perform Auto Offset If checked, the offset value is set automatically. (6) OK button (7) Cancel button Confirms the settings of Auto gain setup applied and closes the Auto gain setup window. Discards the settings of Auto gain setup applied and closes the Auto gain setup window. 98

109 Chapter 5 Detection Mode SD 5.3 Various Views (Spectral Detector-use) This section describes various spectral views Channel View Setting Channel Mixed View From multiple channels acquired with the Spectral Detector (SD), selected channels are mixed and displayed. 1. Open the Live window. Figure Live window 2. Select desired channels. While pressing the [Ctrl] key, click desired channels. To select a range, select the channel as the start point first, then while pressing the [Shift] key, click the channel as the end point. For selection of channels in multiple ranges, see Section , Multi-Range Channel View. Figure Channel view bar 99

110 Chapter 5 Detection Mode SD Split Channel View Selected channels are split into respective channels and displayed. 1. Click the [Split Components] button. All image mixing all channels, respective channel images, TD image, Ratio image, Custom image are displayed. Split Components button Figure Live window Figure Split channel view * For switching from Split channel view to Channel mixed view, click the [Split Components] button again. 100

111 Chapter 5 Detection Mode SD 2. Right-click on the [Custom] button and a menu appears. Select [Properties...] on the menu. The Custom window appears to allow you to change the channels for the Custom View. Custom button Figure Split channel view (Custom image) 101

112 Chapter 5 Detection Mode SD Ratio Image View The Ratio image view is displayed. Right-click on the window to display a menu. Selecting [Ratio View] from the menu changes the window to the Ratio image. Figure Displaying the Ratio image view Figure Ratio image view 102

113 Chapter 5 Detection Mode SD Multi-Range Channel View Mouse operation for displaying multi-range channels is as follows: First range Second range Transmitted button Figure Multi-range channel view 1. Set and display the First range. Click the channel at the left end of the First range. 2. While pressing the [Shift] key, click the channel at the right end of the First range. 3. Select the Second range. While pressing the [Ctrl] key, click the channel at the left end of the Second range. 4. While pressing the [Ctrl] + [Shift] key, click the channel at the left end of the Second range. 5. Click the [Transmitted] button. While pressing the [Ctrl] key, click the [Transmitted] button. Then, the TD image and the images of the selected channels are mixed and displayed. Figure Channel mixed view 103

114 Chapter 5 Detection Mode SD Color Mode Setting Color Mode The color mode switching method and channel color assignment are shown below. Select the desired color mode from three modes; True Color, Custom Color, Grouped Color and Gray Scale and switch the display. To set the color mode, be sure to turn ON the [Treat as Spectral] button. (If it is turned OFF, spectral information hidden.) True Color button Custom Color button Grouped Color button Gray Scale button Treat as Spectral button Figure Frozen window * Settings of Custom Color, Grouped Color, Gray Scale To configure detailed settings, use the LUTs window. To Displaying the LUTs window is shown below. Click the [Show LUTs window] button or right-click on the gray area (without any setting window displayed) to display a menu as shown below. Select [Visualization Controls] -> [LUTs] in the menu to open the LUTs window. Show LUTs window button Figure Displaying the LUTs window 104

115 Chapter 5 Detection Mode SD Displaying the True Color Image Images of all channel data are displayed using the wavelength colors corresponding to the wavelength range provided during data acquisition. Colors that are approximately same as those viewed by bare eyes are displayed. Click the [True Color] button to display the True color image. True Color button Figure True Color image 105

116 Chapter 5 Detection Mode SD Displaying the Custom Color Image Custom Colors are assigned to respective channel data and images are displayed using multiple channel data. Custom Color assignment uses the LUTs window. Click the [Custom Color] button to display the Custom color image. Custom Color button Figure Custom Color button Custom Color Setting Click on the [Reference color] button, then opens the Select New Color window. For the Select New Color window, see Section , Select New Color Window. Reference color button Click + or - button to increase/decrease the Reference color button. Figure Custom Color setting window * In Custom Color mode, channels between [Reference color] buttons are color-interpolated and displayed. 106

117 Chapter 5 Detection Mode SD Displaying the Grouped Color Image With image acquired using the Spectral Detector, channels in a specified range can be grouped and colors can be assigned by group. Grouped Color assignment uses the LUTs window. Click the [Grouped Color] button to display the Grouped color image. Grouped Color button Figure Grouped Color button Grouped Color Setting Click on the [Reference color] button, then opens the Select New Color window. For the Select New Color window, see Section , Select New Color Window. Reference color button To change an area, pick the border between adjoining groups by the mouse and move to the right or left. Click + or - button to increase/decrease the Reference color button. Figure Grouped Color setting window * In Grouped Color mode, the area is split by the number set in [Reference color] button. Channels in each area are all displayed with the same color. 107

118 Chapter 5 Detection Mode SD * In the channel bar of image window, can change an area too. Click the group to change, then pick the border between adjoining groups by the mouse and move to the right or left. Figure Grouped Color mode 108

119 Chapter 5 Detection Mode SD Displaying the Gray Scale Image Each channel is displayed with Gray Scale (Monochrome 256 gradations). Gray Scale assignment uses the LUTs window. Click the [Gray Scale] button to display the Gray Scale image. Gray Scale button Figure Gray Scale button Figure Gray Scale image Gray Scale Setting Selecting other pseudo color Figure Gray Scale setting window * The pseudo color menu also allows changing the displayed color settings. 109

120 Chapter 5 Detection Mode SD Select New Color Window In this window, colors to be assigned to channels are selected. Click the [Reference color] button on Custom Color or Grouped Color settings to display this window. 1. In the Select New Color window, select the desired tab from three [Color palette] tabs. Color palette tab [Palette] Select a color from red, green, blue, yellow, purple, cyan, and white. Colors; yellow, purple, and cyan, support color weakness. Figure Select new color window (Palette) 2. Select the color to be assigned. In the [Hue] and [Wavelength] tabs, a numeric value can be directly entered or the bar displayed in the color range can be moved to the right or left for selection. [Hue] A hue is set. A hue in a range of 0 to 240 can be set. A numeric value can be directly entered or the bar displayed in the color range can be moved to the right or left for selection. Figure Select new color window (Hue) 3. The selected color is displayed in [Sample]. [Wavelength] A color is set using a wavelength in the wavelength range. A wavelength is specified with a numeric value or bar to select a wavelength color. Selected color Figure Select new color window (Wavelength) 110

121 Chapter 5 Detection Mode SD Spectrum Profile Brightness of the ROI area specified in the spectral image can be decomposed and displayed for each 32 channels Displaying the Spectrum Profile 1. Specify the ROI area in the spectral image. (If two or more ROI areas are selected, graphs are displayed for the colors of the ROI selected areas on the profile graph.) Specify the ROI area with ROI drawing tool Figure Specify the ROI area (Spectral image) 2. As shown below, right-click on the gray area (without any setting window displayed) to display a menu. Select [Visualization Controls] -> [Spectrum Profile] in the menu to open Spectrum Profile. Figure Displaying the Spectrum Profile 111

122 Chapter 5 Detection Mode SD Figure Spectrum Profile (all ROI areas are displayed) 3. To display the ROI on the graph, remove the check mark from [Display All ROI] and select the desired ROI from the ROI list. ROI area selection Information of all ROI areas is displayed. Figure Spectrum Profile (displayed for each ROI area) X-axis: 32-channel spectral colors displayed. Y-axis: ROI brightness value or background brightness value displayed. 112

123 Chapter 5 Detection Mode SD Spectrum Profile Setting (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) Window switching tabs Figure Spectrum Profile * If any fluorescence dye is added in [Add spectra] the ideal line of fluorescence dye reaction is displayed on the graph and can be used as an indicator about whether the fluorescence dye is correctly reacting. Name Table Summary of Spectrum Profile graph functions Function (1) Unmix Separates the wavelength information of a spectral image and displays an Unmixing image. For Unmix, see Section 5.3.4, Spectral Unmixing Setting. (2) Manage Stored Spectra Displays the item registered in Stored. (3) Export Exports numeric data to Microsoft Excel. (4) Display All ROI Displays all of the active ROIs. (5) Vertical Scale Absolute Vertical Scale Normalized Scale to cursor Free cursor Cursor to maximum Cursor to minimum Enlarges the window assuming that the brightness minimum to maximum displayed in the graph as 100%. Displays the brightness of each ROI in the Y-axis direction as a relative value to 100%. (Normalizing correction) Calculates the aberration of the curve so that the cross point between bar graphs will be Y:1 and displays a relative graph. Displays a cursor that can be moved to any position. When the cursor is picked with the mouse, brightness of the pixel at the cursor position can be checked as information. Moves the cursor to the maximum value of the specified ROI s brightness. Moves the cursor to the minimum value (0 or larger) of the specified ROI s brightness. 113

124 Chapter 5 Detection Mode SD Name Function (6) Options Opens the Options window for Spectrum Profile. (7) Find Spectra Automatically detects spectra. Specifies the number of classifications (2 to 4) for spectra to automatically separate the wavelength or use Auto Search for separation without specifying the number of classifications. (8) Define Opens the Simple ROI Editor tool. (9) Clear (10) Store Spectrum Move Up Clears the ROI area specified in the image. (Before clearing, a confirmation message is displayed.) Stores the user-defined spectrum (wavelength information). The spectrum defined here is also added to the fluorescence dye selection list in the Optical path window. Brings the selected spectra to one line above. (11) Move down Add spectra Brings the selected spectra to one line below. Adds a spectrum as an indicator. Remove spectra Removes a spectrum as an indicator. 114

125 Chapter 5 Detection Mode SD Registering Spectrum and the Usage The spectrum (wavelength information) defined in the Spectrum Profile window by the user is available as fluorescence dye on the Optical path window. 1. Click the [Store Spectrum] button to display the Store Spectrum window. Store Spectrum button Figure Spectrum Profile 2. Enter a fluorescence dye name in the [Spectrum Name:] field and click the [OK] button. Enter a fluorescence dye name OK button Figure Store Spectrum 3. Display the Optical path window. Setting button Figure Filter and Dye window 115

126 Chapter 5 Detection Mode SD 4. Select the registered fluorescence dye name from the fluorescence dye selection menu of the selected channel. The window to select the excitation laser wavelength to be used appears. Figure Optical path window 5. Select the excitation laser wavelength to be used for this fluorescence dye and click the [OK] button. Figure Selecting excitation laser The fluorescence dye name and the excitation laser wavelength of the selected channel are updated. Figure Optical path window * When changing the excitation laser wavelength defined for each fluorescence dye If you click the fluorescence dye name while pressing the [Ctrl] key with the fluorescence dye (for which you want to change the excitation laser wavelength) set for the channel, the excitation laser selection window (described in step 5) appears. Confirm that the displayed fluorescence dye name is that for which you want to change the excitation laser wavelength, and then re-set the excitation laser wavelength. 116

127 Chapter 5 Detection Mode SD Spectral Unmixing Setting Separate the wavelength information of a spectral image and display an Unmixing image. If wavelengths overlap (because multiple fluorescence dyes are in use) and differences are hard to identify, wavelength information can be separated and displayed Displaying the Spectral Unmixing Setting 1. Specify the wavelength to be separated in the spectral image or on the Frozen window using the ROI area. Specify the ROI area with ROI drawing tool Figure Specifying the ROI area (Spectral image) 2. Open the Spectral Unmixing Setting window. Select [Image] -> [Spectral Unmixing Setting...] on the menu bar. Figure Displaying Spectral Unmixing Setting window 117

128 Chapter 5 Detection Mode SD 3. If [ROIs] is selected from [Category:] in [Source Elements], [Elements:] displays the elements (ROIs) of the target to be separated. ROIs Elements Figure Spectral Unmixing Setting 4. Using the [Add] button, add the elements of the target to be separated from [Elements:] in [Source Elements] to [Unmixing Elements]. Figure Spectral Unmixing Setting 118

129 Chapter 5 Detection Mode SD 5. Click the [Unmix] button to open the unmixed image window separately from the Frozen window. Figure Spectral Unmixing Setting Figure Spectral Unmixing view * In addition to specifying using the ROI area, wavelength information can be separated by specifying the fluorescence dye in use. However, noise provided upon image acquisition may appear. * If the image as a result of normal Unmix looks ambiguous, performing Unmix with the [Fast Unmix] check box selected may lead to a good result. 119

130 Chapter 5 Detection Mode SD * Specifying the background color of ROI. As shown below, specify the ROI area in the part to be designated as the background color. Right-click the mouse on the created ROI area to display a menu. From the menu, select [Use as Background ROI]. Figure Changing the setting of the ROI area Figure Spectral Unmixing view 120

131 Chapter 5 Detection Mode SD Spectral Unmixing Setting (1) (4) (5) (6) (7) (8) (9) (10) (11) (12) (3) (13) (14) (2) Figure Spectral Unmixing Setting Name Table Summary of Spectral Unmixing Setting functions Function (1) Category: (2) Add> Displays the category of the ROI, user-registered wavelength information, fluorescence dye, etc. Selects the elements of the target to be separated from [Elements:] and adds to [Unmixing Elements]. (3) Elements: Selects the elements of the target to be separated. (4) Fast Unmix (5) Add Remainder Channel If check is turned ON, the calculation algorithm is simplified and higher-speed separation is performed compared with normal Unmix. * Since Live Unmixing uses high-speed calculations unlike normal Unmix, Fast Unmix is not applied even if this check box is selected. This function enables calculation of remainder data in the Unmixing calculation. When selected, the remainder data is shown as an image in the Unmixing calculation result. When deselected, the remainder data is not shown. (6) Open Retrieves the setting information saved in an XML file. (7) Save Writes the setting information in an XML file and saves it. (8) (9) Move the Element one line Up Move the Element one line down Brings the selected Element to one line above. Brings the selected Element to one line below. 121

132 Chapter 5 Detection Mode SD Name Function (10) Remove the Element Removes the selected Element. (11) Remove all Removes all Elements. (12) Color The graph color and post-unmix image can be set to any color. (13) Remove Spectra (14) Rename Spectrum Enabled only when [User Defined] or [From Blind Unmixing] is selected for [Category:] area. Removes the items selected in [Elements:] area. Enabled only when [User Defined] or [From Blind Unmixing] is selected for [Category:] area. Changes the names of items selected in [Elements:] area. Note <Remainder data> The Remainder data is used as a quality standard for the data produced by the Unmix calculation. The Remainder data is represented as an absolute value for the total of differences between measurement data (b) and the total of Unmixed data (a). This data is added as one channel data to Unmixed data. 122

133 Chapter 5 Detection Mode SD Live Unmixing Live observation is available in the state where spectral images are separated for each wavelength Displaying the Live Unmixing 1. Specify the wavelength to be separated in the spectral image using the ROI area. Specify the ROI area with ROI drawing tool Figure Specify the ROI area (Spectral image) 2. Open the Spectral Unmixing Setting window. Select [Image] -> [Spectral Unmixing Setting...] on the menu bar. Figure Displaying the Spectral Unmixing Setting window 123

134 Chapter 5 Detection Mode SD 3. If [ROIs] is selected from [Category:] in [Source Elements], [Elements:] displays the elements (ROIs) of the target to be separated. ROIs Elements Figure Spectral Unmixing Setting 4. Using the [Add] button, add the elements of the target to be separated from [Elements:] in [Source Elements] to [Unmixing Elements]. Figure Spectral Unmixing Setting 124

135 Chapter 5 Detection Mode SD 5. Click the [Close] button to confirm the wavelength you want to separate. (If you click the [Unmix] button instead, normal Unmix image starts to be captured.) Figure Spectral Unmixing Setting * Fast Unmix Since Live Unmixing uses high-speed calculations unlike normal Unmix, Fast Unmix is not applied even if the [Fast Unmix] check box is selected. * In addition to specifying using the ROI area, wavelength information can be separated by specifying the fluorescence dye in use. However, noise provided upon image acquisition may appear. 6. Click the [Live Unmixing] button on the horizontal toolbar. If the wavelength to be separated is not specified, the message of Invalid unmixing definition no unmixing elements defined. appears. Live Unmixing button Figure Live Unmixing 125

136 Chapter 5 Detection Mode SD * Alternative method of switching to Live Unmixing. As shown in the figure on the right, select [Image] -> [Live Unmixing] on the menu bar. Figure Switching to Live Unmixing 7. Click the [Live] button, the live image is switched to the Unmix live image. Figure Acquiring the Unmix live image Figure Live Unmixing * Click the [Live Unmixing] button or select [Image] -> [Live Unmixing] on the menu bar again to return to the regular live image. 126

137 Chapter 5 Detection Mode SD Blind Unmix Automatically search for typical spectra and display an Unmix image separated by the spectral wavelength information. If wavelengths overlap (because multiple fluorescence dyes are in use) and differences are hard to identify, wavelength information can be separated and displayed. Blind Unmix allows automatic separation by specifying the number of classifications or separation without specifying the number of classifications by using [Auto Search.] Displaying the Blind Unmix Image 1. Open the Blind Unmix window while the acquired spectral image is displayed. Select [Image] -> [Blind Unmixing...] on the menu bar. 2. To specify the number of classifications, select one from [2] to [4] radio button in the Number of Classifications pane. Select [Auto Search] radio button when not specifying the number of classifications. 3. Click the [Find] button to execute the Blind Unmix. On completion of Blind Unmix, an image window opens for the image unmixed with the detected spectra. Figure Blind Unmix window Figure Spectral Unmixing view 127

138 Chapter 5 Detection Mode SD Setting for Blind Unmix Window (1) (2) (3) (4) Figure Blind Unmix window Name Table Summary of Blind Unmix window functions Function Allows you to select the number of classifications for automatic separation of spectral wavelength information. (1) Number of Classifications Number of Classifications [2] to [4] Auto Search Automatically separate the spectral wavelength information by the specified number of classifications. Automatically separate the spectral wavelength information without specifying the number of classifications. (2) Background (3) Remainder Allows you to set the threshold for elimination of the background offset noise. 0 to 4095 is specifiable. For a 16-bit spectral image, 0 to is specifiable. (Specifying the maximum value causes all to be regarded as background offset noise and no spectral wavelength information to be detected.) This function enables calculation of remainder data in the Unmixing calculation. When selected, the remainder data is shown as an image in the Unmixing calculation result. When deselected, the remainder data is not shown. (4) Find Starts automatic detection of the spectral wavelength information. 128

139 6 Detection Mode VF This chapter describes the settings for the Virtual Filter mode (VF). The Virtual Filter is a function that provides up to four binning groups for up to 32 channels spectral data and adjusts brightness of each group. 6.1 Filter and Dye Window This window enables to set the Optical path Structure of Filter and Dye Window (8) (1) (2) (6) (9) (7) (3) (4) (5) Figure Filter and Dye window (VF-use) Table Functions of Filter and Dye window (VF-use) Name Function (1) Detector Indicates the name of the detection mode in current use. (2) Setting button Opens the Optical path window. To use, select the detector, the dichroic mirror, the channel, fluorescence dye for each channel, laser and others. (3) Status Indicates for the settings for each channel (fluorescence dye name, laser wavelength, and wavelength band to be acquired). (4) TD Indicates the status of the motorized transmitted detector. (5) TD IN/OUT button Sets/removes the motorized transmitted detector in/from the optical path. (IN = Set in the optical path/ OUT = Remove from the optical path) As for the case where the TD IN/OUT button is not displayed, it will be displayed when the motorized transmitted detector is set in the optical path in the Optical path window. (6) Close mechanical shutter during experiment If unchecked, the shutter remains open during the ND image acquisition. As the shutter is not opened/closed every image acquisition, the time for the image acquisition can be shortened. * During the interval period, laser power is automatically changed to the minimum but the laser cannot be shut off completely because the shutter is left open. (7) PreScan button Makes settings of the pre-scan mode for correction of the position shift in a zoom change or of the image acquisition area shift in the photo activation by the prior scanning. 129

140 Chapter 6 Detection Mode VF Name (8) Eye Port button (9) AUX button Function Changes optical path to eye port. Each function on the A1plus Settings window becomes non-selectable when the Eye Port button is selected. Displays the AUX Settings window to use the external detector unit. For details of the AUX Settings window, see Chapter 17. Optical Configuration Individual data items set in the Virtual Filter mode (VF) can be managed collectively with the Optical Configuration window. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the NIS-Elements AR (Advanced Research) User's Guide Setting the Optical Path Click the [Setting] button of the Filter and Dye window to display the Optical path window. Select the Virtual Filter mode (VF). There are two modes available for Optical path setting, [Auto] and [Manual]. Normally, the auto mode should be used. Setting button Figure Filter and Dye window (VF-use) Figure Optical path window (for auto mode, VF-use) 130

141 Chapter 6 Detection Mode VF Optical Path Window (3) (13) Switching Tab (4) (9) (2) (5) (10) (1) (11) (12) (14) (15) (6) (7) (8) Figure Table Optical path window (for manual mode, VF-use) Functions of Optical path window (VF-use) (1) Name Detection mode selection button Function Enabled to select the Virtual Filter mode (VF). Binning is performed for the spectral data of the concurrent 32 channels to group it into up to four groups, enabling acquisition of an image of light of a specified wavelength range. Selects the desired mode for setting the Optical path. (2) Mode selector (3) (4) Excitation laser indicator Sorting fluorescence dye list Activates the auto mode. Once the fluorescence dye to be used is selected, the appropriate laser, the dichroic mirror, and the wavelength range acquired from the virtual channel are automatically selected. Activates the manual mode. Enables to set all of the laser, the dichroic mirror, and the wavelength range acquired from the virtual channel to be used manually. Displays the current setting for the laser. The currently set laser icon is displayed in a large size, and the optical path is indicated. Sorts the fluorescence dye list according to the selected type. ABC: Displays the list in alphabetical order. Emission: Displays the list in the order of peak wavelength of fluorescence intensity. Excitation: Displays the list in excitation wavelength order. 131

142 Chapter 6 Detection Mode VF (5) (6) Name Channel selection check box Channel color setting button Function Enables to select the channels to be used. (Up to 4 channel.) Displays the Color Selection window, enables to set the desired color for each channel. Selects the fluorescence dye name to be used for each channel. (7) Fluorescence dye selection/input: In auto mode * When the auto mode is used in the Virtual Filter mode, set the fluorescence dye name in the order of the fluorescence wavelength (short wavelength -> long wavelength). Also when the manual mode is used, set the laser selection in the order of the excitation laser. Note that if the setting is not made in the order of the fluorescence wavelength, the order is automatically changed. In manual mode Selects the in-use fluorescence dye name for each channel or enters an arbitrary channel name. (8) Excitation laser select These menus are only effective while in the manual mode. Enables to set the laser wavelength that is set with the software configuration, regardless of the setting of the Filter cube display/select. Provides the following information: (9) Rainbow chart - Wavelength band for which to acquire images (shown in color and value for each channel) - Spectral profile of fluorescence dye - Excitation laser for fluorescence dye - A color band indicating the wavelengths in the entire band (400 to 750 nm) - Scale of the wavelengths in the entire band (400 to 750 nm) (10) (11) (12) (13) Acquisition range for each virtual channel slider bar 1st Dichroic mirror select Transmitted detector selection button ND filter installation icon Specifies the laser wavelength range to be acquired for each virtual channel. * When shifting the slider bar in Auto mode, the Mode selector changes to manual mode. This menu is only effective while in the manual mode. Enables to manually select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combination of the selected laser wavelength and the 1st Dichroic mirror,! appears following the 1st Dichroic mirror name. Brings the transmitted detector into the Optical path, to enable the ability. Brings the transmitted detector out of the Optical path, to disable the ability. An icon is displayed on the line of laser with ND filter installed. * This icon indicates whether ND filter is installed or not, and does not indicate insertion or removal of ND filter (IN = Insert in the optical path, OUT = Remove from the optical path). Insertion or removal of ND filter is performed on the Acquisition window. (14) OK button Confirms the Optical path settings applied and closes the Optical path window. (15) Cancel button Discards the Optical path settings applied and closes the Optical path window. 132

143 Chapter 6 Detection Mode VF Optical Path Window Switching Tab By selecting the manual mode at setting mode, the tab for switching between [Detector] and [Grating Settings] is displayed on the right top of the Optical path window Detector Tab Rainbow chart (1) (2) (3) Channel selection check box Channel color setting button Fluorescence dye selection/input: Excitation laser select (4) Figure Optical path window (Detector tab) Table Functions of Detector tab Name Function (1) Resolution Displays the wavelength resolution currently set. (2) Channels Displays the number of channels (number of PMTs) currently set. (3) Binning The number of channel binning is fixed to 1. (4) Acquisition range for each virtual channel slider bar Specifies the laser wavelength range to be acquired for each virtual channel. The wavelength range of each channel can be overlapped with each other. The Grating range (gray area displayed in the rainbow chart) can be set. 133

144 Chapter 6 Detection Mode VF Grating Settings Tab The [Grating Settings] tab is displayed only when the manual mode is selected at setting mode. Set the range for grating and set the wavelength range for the channels selected within the range. Rainbow chart (1) (2) (3) (4) (7) (5) (6) (8) Figure Optical path window (Grating Settings tab) Table Functions of Grating Settings tab Name Function (1) Resolution (2) Channels Selects a wavelength resolution. Selectable from 2.5, 6, or 10nm. Once the Resolution is selected, number of channels (number of PMTs) is automatically selected. To change the number of channels to 32, drag the edge of the Grating range setting bar. (3) Binning The number of channel binning is fixed to 1. (4) Grating range setting bar Sets a wavelength range in a wavelength range from 400nm to 750nm. The range depends on the grating resolution. It is shiftable horizontally but the width of the bar cannot be reduced. * A part of the wavelength range may be displayed in black depending on the setting conditions. In the wavelength range displayed in black, no wavelength range can be set. (5) Start Displays the start wavelength of the Grating range currently selected. The right and left buttons cannot be used when the Virtual Filter mode is selected. (6) All Enabled to shift to the right or left within the currently selected Grating range. (7) End (8) Enlarge button Displays the end wavelength of the Grating range currently selected. The right and left buttons cannot be used when the Virtual Filter mode is selected. Enlarges the rainbow chart. The display is switched in three levels. 134

145 Chapter 6 Detection Mode VF 6.2 Acquisition Window The Acquisition window enables to set PMT brightness (detection sensitivity), laser power, and pinhole size Structure of Acquisition Window (1) (5) (6) (7) (2) (8) (3) (4) Displays laser power value (13) (9) (10) (11) (12) (14) (15) (16) Figure Acquisition window (VF-use) Table Functions of Acquisition window (VF-use) (1) Name Acquisition/Photo Activation window switching Function Switches between the Acquisition and Photo Activation windows. For the Photo Activation window, see Chapter 10. (2) Laser power monitor button Displays the laser power value (integer obtained after A/D conversion divided by 10) of the current channel by clicking this button. During the image acquisition, the laser power cannot be measured and this button is grayed out. (3) Brightness adjustment for each channel For each of the virtual channels, use the Gain Laser and ND filter IN/OUT controls to adjust the brightness of the live image. (4) Channel selection Selects the virtual channels (Ch1 to Ch4, and/or TD) to acquire the desired images. Do this by adding a check mark. When fluorescence is excited, all excitation lights are detected by the transmitted detector (TD). If the BA filter for 405 laser is installed in the TD, excitation light is not detected by the TD. When acquiring the transmitted image (TD image) only, see Section , When Acquiring Transmitted Image Only. (5) Select All Channels button Selects all channels for acquiring images. 135

146 Chapter 6 Detection Mode VF (6) Name Fluorescence dye name indication Function The fluorescence dye name specified in the Optical path window is indicated. (7) Channel color Displays the channel color specified in the Optical path window. (8) Laser wavelength indication The currently selected laser wavelength is indicated. Selects whether the laser is emitted or not. * When LU-NV is in use, this button is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. (9) Laser ON/OFF button ON status OFF status The laser is emitted. The AOTF shutter closes and the laser power value becomes 0. When switched from OFF to ON, the laser power value set in the previous ON status is applied. (10) Si HV Adjusts HV of the Spectral detector. (11) Pinhole Adjusts the pinhole size. For pinhole size, see Section 6.2.3, Setting the Pinhole. (12) (13) Brightness adjustment for transmitted detector Remote controller selector icon For the transmitted detector, use the HV and Offset controls to adjust the brightness of the live image. Displayed for the channel group which is currently adjustable with the remote controller, see Chapter 11. (14) AG button (15) Auto Gain setting button (16) Optimize button Automatically adjusts the Si HV value (Si HV gain) of the currently selected channel to the optimum values. For Auto Gain, see Section 6.2.4, Auto Gain. Sets the ratio of saturation pixels used for automatic Si HV gain correction. The window for range of the ratio of saturation pixels settings appears when this button is clicked. For setting for ratio of saturation pixels, see "Setting for ratio of saturation pixels" in the Section 6.2.4, "Auto Gain. Displays the XYZ Size Setup window. In the XYZ Size Setup window, the calculation method of the recommended values of the resolution, zoom magnification, and Z stack step size can be set. For the XYZ Size Setup window, see Section , Recommended Value Indication/Automatic Application in the next page. 136

147 Chapter 6 Detection Mode VF Recommended Value Indication/Automatic Application By the function of the recommended value indication/automatic application, the recommended values of the appropriate resolution, zoom magnification, and Z stack step size are calculated based on the objective type and the selected excitation wavelength. Using the calculated recommended values enables the image acquisition clearer and with less damage to the sample. Recommended Value Automatic Application To automatically apply the recommended values to the parameters, set the [Nyquist XY] button of the Scan Area window to ON. Nyquist XY button Figure Scan Area window Indicates the recommended value of the resolution. Indicates the recommended value of the scan magnification. Figure Location of Recommended Value Indication * When the laser or objective in use is changed, the recommended values are recalculated, and newly indicated and automatically applied. 137

148 Chapter 6 Detection Mode VF Recommended Value Settings Detailed settings of the recommended values are made in the XYZ Size Setup window that is displayed by clicking the [Optimize] button of the Acquisition window. If the [Nyquist XY] button of the Scan Area window is ON, the recommended values are automatically applied to the parameters. Or if the [Nyquist XY] button is OFF, the recommended values of the scan size and zoom are indicated in the Scan setting window. (1) (2) (3) (4) (5) (6) Figure XYZ Size Setup window Table Functions of XYZ Size Setup window Name (1) Zoom Preference Function When the [Nyquist XY] button is ON, keeps the scan size and applied the recommended value of the zoom. (2) Resolution Preference When the [Nyquist XY] button is ON, keeps the zoom and applied the recommended value of the scan size. (3) Use Cropping Fits the scan size in detail by using Crop Scan. Sets the Z step size calculation method. (4) Suggested Step (Z) Recommend (Z~1/3 FWHM) Half Overlap (Z=1/2 FWHM) Minimum Overlap (Z=FWHM) Perfect Voxel (Z=X=Y) Approximately one third of the thickness of optical section (FWHM value). One half of the thickness of optical section (FWHM value). The thickness of optical section (FWHM value). Value same as the pixel size. (5) OK button Confirms the XYZ Size Setup applied and closes the XYZ Size Setup window. (6) Cancel button Discards the XYZ Size Setup applied and closes the XYZ Size Setup window. 138

149 Chapter 6 Detection Mode VF When Acquiring Transmitted Image Only By using the TD channel, you can acquire the image with the TD channel only. 1. Display the Optical path window. Select a channel for the laser power control and deselect other unnecessary channel s check box. * Even when the multiple channels are selected in the Optical path window, the image with the TD channel only can be acquired by deselecting the unnecessary check box in the Acquisition window. In this case, the laser power value of the channel that is deselected at last is used for acquiring the TD image. Figure Optical path window (VF -use) 2. Set the transmitted detector into the optical path and then click the [OK] button to close the Optical path window. Figure Optical path window (VF -use) 3. Use the HV and Offset controls of the transmitted detector to adjust the brightness of the live image. 4. Use a channel that remains to On to control the laser power to set the laser power value. 5. Deselect the unnecessary check boxes other than TD. Deselect this check box at last. Set the laser power value. Adjust the transmitted detector. Figure Acquisition window (VF -use) 6. Acquire the image. 139

150 Chapter 6 Detection Mode VF Setting Image Brightness For the live images of each Virtual channel, adjust Gain, Laser, ND filter IN/OUT, Si HV, HV (TD), and Offset (TD) to obtain clear images. (6) (1) (2) (3) (4) (5) Figure Setting the live image brightness (VF-use) Table Brightness adjustment functions for the live image (VF-use) Name (1) Gain (2) Laser (*) (3) Si HV (4) HV (5) Offset Function Sets the PMT Gain. Slider bar: Slides to the right or left to set the gain value. Arrow buttons: Click either arrow button to increase or decrease the gain value stepwise. Direct entry in gain value display field: Type the desired setting value. Sets the laser power value. Slider bar: Slides to the right or left to set the laser power value. Arrow buttons: Click either arrow button to increase or decrease the laser power value stepwise. Direct entry in laser power value display field: Type the desired setting value. Adjusts HV of the Spectral detector. (Applied to all Virtual channel groups.) Slider bar: Slides to the right or left to set the Si HV value. Arrow buttons: Click either arrow button to increase or decrease the Si HV value stepwise. Direct entry in Si HV value display field: Type the desired setting value. Sets the voltage to be applied to the transmitted detector. Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value. Sets the offset value of the transmitted detector. Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value. (6) ND filter IN/OUT button (*) Inserts/removes the ND filter in/from the optical path. (IN = Insert in the optical path/ OUT = Remove from the optical path) This button is displayed only for lasers that can control insertion/removal of ND filter. * When LU-NV is in use, this function is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. 140

151 Chapter 6 Detection Mode VF PMT Overload If too much gain is applied with reference to the illumination intensity, the gain is automatically shut down to protect PMT and the following PMT Overload dialog box is displayed. Figure PMT Overload dialog box 141

152 Chapter 6 Detection Mode VF Setting the Pinhole (1) (2) (7) (5) (6) (3) (4) Figure Setting the Pinhole (VF-use) Table Pinhole setting functions (VF-use) Name (1) Pinhole size setting (2) Pinhole button Function Sets a pinhole size for A1 system. Slider bar: Slides to the right or left to set the pinhole size. (Unit: A.U.) Arrow buttons: Click either arrow button to increase or decrease the pinhole size stepwise. Direct entry in pinhole size display field: Type the desired setting value. Displays the A.U. Calculation Settings window to calculate the pinhole size. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) Changes the pinhole to the predetermined home position. (3) Home The value of the home position can be changed in the A.U. Calculation Settings window. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) (4) Pinhole size Indicates pinhole size of A1 system. (Unit: um) (5) thickness of optical section Indicates the FWHM (full width at half maximum) of z airy disk. (6) Optical Resolution The actual size of 1 pixel square calculated from the optical information (for objectives and scan parameters) and the size acquired from an image. (7) Reference excitation wavelength for the pinhole size calculation Selects the excitation wavelength as the reference of the automatic calculation of the pinhole size from the laser wavelengths, or enter it manually in the A.U. Calculation Settings window. (For A.U. Calculation Settings, see Section , Calculation Settings for Pinhole Size. ) 142

153 Chapter 6 Detection Mode VF Calculation Settings for Pinhole Size This section describes the setting window for calculating the pinhole size. Click the [Pinhole] button in Acquisition window, the A.U. Calculation Settings window appears. (Usually, the [Recommend] is selected to enable automatic calculation. [Recommend] calculates the A.U. value by using the Nikon-recommended EM and NA values.) (1) (2) (3) (4) (5) (6) (7) Figure A.U. Calculation Settings window Table A.U. Calculation Settings window (1) Name Select calculation method Function Recommend Sets parameters automatically. (Nikon recommended) User Setting Allows the user to manually set parameters. (2) EM:emission wavelength[um] Selected Laser wavelength Manual Calculates parameters by using the laser wavelength selected in the pinhole combo box of the Acquisition window as the emission wavelength (EM value). The wavelength displayed in the combo box is to be the laser wavelength set in the Optical Setting window. Allows the user to manually set parameters. (The parameter is calculated with the input value as the emission wavelength (EM value).) Enter the value directly from the keyboard. Sets refractive index of the objective. (3) NA:Numerical Aperture NA_obj(NA of objective lens) Min(NA_obj, NA_sample) Regardless of whether or not the objective NA value exceeds the refractive index of the sample, executes calculation by using the objective NA as the calculation parameter. When the objective NA value does not exceed the refractive index of the sample, executes calculation by using the objective NA as the calculation parameter. When the objective NA value exceeds the refractive index of the sample, executes calculation by using the sample refractive index. (4) (5) Airy Home Position Keep A.U. check box Sets a home position of pinhole. Direct entry in Airy Home Position value display field: Type the desired setting value. When checked, the pinhole size is fixed by the A.U. when the selected wavelength or objective is changed. (However changes by the um.) When unchecked, the pinhole size is fixed by the um. (However changes by the A.U.) (6) OK button (7) Cancel button Confirms the A.U. Calculation Settings applied and closes the A.U. Calculation Settings window. Discards the A.U. Calculation Settings applied and closes the A.U. Calculation Settings window. 143

154 Chapter 6 Detection Mode VF Pinhole Position Alignment This section describes the setting window for aligning the pinhole position. The pinhole position can be aligned by manually shifting the condensing lens in the A1plus Pinhole Alignment window. As shown below, right-click on the gray area (without any setting window displayed) to display a menu. Then select [Acquisition Controls] -> [A1plus Pinhole Alignment] in the menu to open the Scan Area window. Figure How to display the A1plus Pinhole Alignment window (1) (2) (3) (4) Figure A1plus Pinhole Alignment window Table A1plus Pinhole Alignment window Name (1) Coarse/Fine button Function The condensing lens position can be shifted by the shift amount set in the Coarse/Fine field by using these buttons. (2) Coarse/Fine field Shift amount of the condensing lens position can be set in these fields. (3) Lens Position X/Y The X and Y coordinates of the current condensing lens position is displayed. (4) Reset button The condensing lens potion is reset to the default position set at the factory. 144

155 Chapter 6 Detection Mode VF For memorizing the pinhole position (the condensing lens position) NIS-Elements can memorize the pinhole position that is adjusted in the A1plus Pinhole Alignment window. Select [Edit] -> [Options] -> [User rights] in the menu bar to display the window and then select the [Privileges] tab. Select the [Modify Shared Confocal Alignment] check box in the [Privileges] area so that NIS-Elements memorizes the NIS-Elements C pinhole position when the software ends. 145

156 Chapter 6 Detection Mode VF Auto Gain Auto Gain is a function to automatically correct the value of Si HV gain to set the optimum image brightness. Automatic Si HV gain correction is performed within the predetermined range of the ratio of saturation pixels. Automatic Si HV gain correction is performed only Si HV. For a TD, automatic adjustment is performed when it is selected. After execution of Auto Gain, in the window indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed. If Auto Gain failed, x is indicated and the Si HV value returns to its original value. When setting the line scan, Auto Gain cannot be executed. During execution of Auto Gain, do not perform manual adjustments in the Acquisition window and adjustments by the remote controller. Auto Gain button Auto Gain setting button Figure Execution of Auto Gain (VF-use) Auto Gain status... Failed... Completed If checked, the window is automatically closed when Auto Gain is completed. Figure Auto Gain progress 146

157 Chapter 6 Detection Mode VF Setting for ratio of saturation pixels Set the maximum and minimum value for the Ratio of saturation pixels used for automatic Si HV gain correction. Click the [Auto Gain Setting] button to display the Auto gain setup window. Set the maximum and minimum value for the ratio of saturation pixels in Auto gain setup window. Figure Displaying the Auto gain setup window (1) (2) (3) (4) (5) (6) (7) Figure Setting for ratio of saturation pixels Table Setting for ratio of saturation pixels (1) Name Target Maximal Intensity Function Specifies the application ratio of the setting of the ratio of saturation pixels. Sets the percentage (%) of the maximum value to be applied. (2) Advanced Settings If checked, advanced settings of the ratio of saturation pixels are enabled. (3) Overillumination Tolerance (Area) Minimum Maximum Sets the minimum value for Ratio of saturation pixels. Sets the maximum value for Ratio of saturation pixels. (4) Perform in Find Mode (fast) If checked, execution in the Find mode is enabled. (5) Perform Auto Offset If checked, the offset value is set automatically. (6) OK button (7) Cancel button Confirms the settings of Auto gain setup applied and closes the Auto gain setup window. Discards the settings of Auto gain setup applied and closes the Auto gain setup window. 147

158 Chapter 6 Detection Mode VF 6.3 Various Views (Virtual Filter mode-use) This section describes various Virtual Filter mode (VF) views Channel View Setting Channel Mixed View Images acquired in the Virtual Filter mode (VF) are displayed in the method suitable to the purpose. All image The [All] tab is selected, all the virtual channels are mixed to display. All tab Figure All image 148

159 Chapter 6 Detection Mode VF Each channel image To display the image of each virtual channel, select the tab corresponding to the channel. Virtual channel tabs Figure Each channel image TD tab Figure TD image 149

160 Chapter 6 Detection Mode VF Custom image Custom image displays a mixed image of selected multiple channels. To change channels to be mixed, re-select channels. Right-click on the [Custom] tab and a menu appears. Select [Properties...] on the menu. The Custom window appears to allow you to change the channels for the Custom View. Custom tab Figure Selecting channels (Custom image) 150

161 Chapter 6 Detection Mode VF Ratio image The Ratio image view is displayed. Right-click on the window to display a menu. Selecting [Ratio View] from the menu changes the window to the Ratio image. Figure Displaying the Ratio image view Figure Ratio image 151

162 Chapter 6 Detection Mode VF * You can change the combination of channels to be displayed in the Ratio View. Right-click on the window and a menu appears. Select [Ratio Properties...] on the menu. The Ratio Properties window appears to allow you to change the channels for the Ratio View. Figure Ratio Properties window 152

163 Chapter 6 Detection Mode VF Split Channel View Virtual channels are split into respective channels and displayed. Click the [Split Components] button. All image mixing all channels, respective channel images, TD image, Ratio image, Custom image are displayed. Split Components button Figure Live window Figure Split channel view * For switching from Split channel view to Channel mixed view, click the [Split Components] button again. 153

164 7 Detection Mode VAAS This chapter describes the settings for the VAAS mode. VAAS (Virtual Adaptable Aperture System) concurrently detects fluorescent light beams passing through two pinholes of different sizes and performs deconvolution. With this function, flare light can be eliminated while the brightness of the image is maintained with the larger pinhole. 7.1 Filter and Dye Window This window enables to set the Optical path Structure of Filter and Dye Window (9) (1) (2) (3) (7) (10) (8) (4) (5) (6) Figure Filter and Dye window (VAAS-use) Table Functions of Filter and Dye window (VAAS-use) Name Function (1) Detector Indicates the name of the detection mode in current use. (2) Ch series (3) Setting button (4) Status Selects whether to perform scanning by simultaneously firing all lasers for the channels in use or by firing each laser in the specified order. For Ch series selection, see Section 7.1.4, Selecting the Channel Series. Opens the Optical path window. To use, select the detector, the dichroic mirror, the channel, fluorescence dye for each channel, laser and others. Indicates for the settings for each channel (fluorescence dye name, laser wavelength, and wavelength band to be acquired). (5) TD Indicates the status of the motorized transmitted detector. (6) TD IN/OUT button Sets/removes the motorized transmitted detector in/from the optical path. (IN = Set in the optical path/ OUT = Remove from the optical path) As for the case where the TD IN/OUT button is not displayed, it will be displayed when the motorized transmitted detector is set in the optical path in the Optical path window. (7) Close mechanical shutter during experiment If unchecked, the shutter remains open during the ND image acquisition. As the shutter is not opened/closed every image acquisition, the time for the image acquisition can be shortened. * During the interval period, laser power is automatically changed to the minimum but the laser cannot be shut off completely because the shutter is left open. (8) PreScan button Makes settings of the pre-scan mode for correction of the position shift in a zoom change or of the image acquisition area shift in the photo activation by the prior scanning. 154

165 Chapter 7 Detection Mode VAAS Name (9) Eye Port button (10) AUX button Function Changes optical path to eye port. Each function on the A1plus Settings window becomes non-selectable when the Eye Port button is selected. Displays the AUX Settings window to use the external detector unit. For details of the AUX Settings window, see Chapter 17. Optical Configuration Individual data items set in the VAAS mode can be managed collectively with the Optical Configuration window. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the NIS-Elements AR (Advanced Research) User's Guide Setting the Optical Path Click the [Setting] button of the Filter and Dye window to display the Optical path window. Select the VAAS mode (VAAS). There are two modes available for Optical path setting, [Auto] and [Manual]. Normally, the auto mode should be used. Setting button Figure Filter and Dye window (VAAS-use) Figure Optical path window (for auto mode, VAAS-use) 155

166 Chapter 7 Detection Mode VAAS Optical Path Window (3) (15) (4) (9) (2) (5) (8) (6) (7) (1) (10) (11) (13) (14) (12) (16) (17) Figure Table Optical path window (for auto mode, VAAS-use) Functions of Optical path window (VAAS-use) (1) Name Detection mode selection button (2) Mode selector (3) (4) Excitation laser indicator Sorting fluorescence dye list Function Enabled to select the VAAS mode. In the Galvano scan mode, up to 9 images can be acquired with 4 channels (an In focus image and a Near focus image for each channel) + TD. In the Resonant scan mode, up to 5 images can be acquired with 2 channels (an In focus image and a Near focus image for each channel) + TD. Selects the desired mode for setting the Optical path. Activates the auto mode. Once a fluorescence dye is selected, appropriate laser and the dichroic mirror are automatically selected. Activates the manual mode. Enables to set the lasers and the dichroic mirror to be used manually. Displays the current setting for the laser. The currently set laser icon is displayed in a large size, and the optical path is indicated. Sorts the fluorescence dye list according to the selected type. ABC: Displays the list in alphabetical order. Emission: Displays the list in the order of peak wavelength of fluorescence intensity. Excitation: Displays the list in excitation wavelength order. 156

167 Chapter 7 Detection Mode VAAS (5) (6) Name Channel selection check box Channel color setting button Enables to select the channels to be used. Function Displays the Color Selection window, enables to set the desired color for each channel. (7) (8) Fluorescence dye selection/input: Excitation laser select (9) Rainbow chart (10) 1st Dichroic mirror select (11) Scan mode select In auto mode In manual mode Selects the fluorescence dye name to be used for each channel. Selects the in-use fluorescence dye name for each channel or enters an arbitrary channel name. These fields are only effective while in the manual mode. Enables to set the laser wavelength that is set with the software configuration, regardless of the setting of the Filter cube display/select. Provides the following information: - Wavelength band for which to acquire images (shown in color and value for each channel) - Spectral profile of fluorescence dye - Excitation laser for fluorescence dye - A color band indicating the wavelengths in the entire band (400 to 750 nm) - Scale of the wavelengths in the entire band (400 to 750 nm) This menu is only effective while in the manual mode. Enables to manually select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combination of the selected laser wavelength and the 1st Dichroic mirror,! appears following the 1st Dichroic mirror name. Selects the scanner unit to be used. Galvano Resonant Galvano scan mode allows high-quality imaging of up to pixels. Resonant scan mode allows high-speed imaging of 420 frames per second. (12) (13) (14) (15) Transmitted detector selection button Filter cube display/select Ch series selection ND filter installation icon Brings the transmitted detector into the Optical path, to enable the ability. Brings the transmitted detector out of the Optical path, to disable the ability. In manual mode only, the filter cube to be mounted on the detector can be selected regardless of the excitation laser. Selects whether to perform scanning by simultaneously firing all lasers for the channels in use or by firing each laser in the specified order. For Ch series selection, see Section 7.1.4, Selecting the Channel Series. An icon is displayed on the line of laser with ND filter installed. * This icon indicates whether ND filter is installed or not, and does not indicate insertion or removal of ND filter (IN = Insert in the optical path, OUT = Remove from the optical path). Insertion or removal of ND filter is performed on the Acquisition window. (16) OK button Confirms the Optical path settings applied and closes the Optical path window. (17) Cancel button Discards the Optical path settings applied and closes the Optical path window. 157

168 Chapter 7 Detection Mode VAAS Selecting the Channel Series The [Ch series] menu enables to select whether to perform scanning by simultaneously firing all lasers for the channels to be used or by firing each laser in the specified order. There are four options for channel series, None, Line 1->4, Line 4->1, and Custom, either of which can be selected from the pull-down menu. * Channel series can be applied to the image acquisition phase in the photo activation experiment sequence (but cannot be applied to the stimulation phase). Figure Selecting the channel series (Filter and Dye window) Figure Selecting the channel series (Optical path window) Table Functions of channel series Name None Line 1->4 Line 4->1 Custom Function Performs scanning by simultaneously firing all lasers for the channels to be used. Performs scanning by sequentially firing the lasers for the channels to be used (Ch1 -> Ch2 -> Ch3 -> Ch4). Performs scanning by sequentially firing the lasers for the channels to be used (Ch4 -> Ch3 -> Ch2 -> Ch1). Performs scanning by firing the lasers in desired order for the channels to be used. * When [Line 1->4] or [Line 4->1] is selected, the lasers are fired sequentially for each scan line. This scan method is called the line sequence. Ch1 Ch2 Ch3 The laser for Ch1 scans the 1st line. The laser for Ch2 scans the 1st line. The laser for Ch3 scans the 1st line. A series of the same scanning procedure is repeated for each subsequent channel. Ch1 The laser for Ch1 scans the 2nd line. Ch4 The laser for Ch4 scans the 1st line. Figure Scanning motion in line sequence (Line 1->4 is selected) 158

169 Chapter 7 Detection Mode VAAS Custom Selecting Custom from the [Ch series] menu displays the current channel scan order and the [Custom] button. Clicking the [Custom] button opens the Line Channel Series Setup window to allow setting the scan order for each channel. Current channel scan order Custom button Figure Channel series (Custom) Channel name (1) Scan order Fluorescence wavelength (4) Name (2) (3) Figure Table Line Channel Series Setup window Functions of channel series Function (5) (6) (1) Scan order matrix for each channel Allows the user to set desired order of channels to be scanned. Each laser can also be fired simultaneously to multiple channels by a single scan. (Example: Firing lasers simultaneously for Ch1 and Ch2 by the first scan.) However, one channel cannot be set twice or more times. (Selecting two or more check boxes in the same vertical line of the matrix is prohibited.) (2) 1->4 button Set the channel to be scanned for each laser in order of Ch1, Ch2, Ch3, and Ch4. (3) 4->1 button Set the channel to be scanned for each laser in order of Ch4, Ch3, Ch2, and Ch1. (4) TD check box Allows the user to set the TD scan order if the transmitted detector (TD) is in the optical path. Be sure to set the TD scan order so that the scan order comes together with other channels because single TD scan is disabled. (Example: Laser is fired to Ch3 and TD by the second scan.) (5) OK button Confirms the settings applied and closes the window. (6) Cancel button Discards the settings applied and closes the window. 159

170 Chapter 7 Detection Mode VAAS 7.2 Acquisition Window The Acquisition window enables to set PMT brightness (detection sensitivity) and laser power Structure of Acquisition Window (1) (7) (8) (9) (10) (2) (3) (11) (12) (4) (5) (6) (13) Displays laser power value (17) (14) (15) (16) Figure Acquisition window (VAAS-use) Table Functions of Acquisition window (VAAS-use) (1) (2) (3) Name Acquisition/Photo Activation window switching Laser power monitor button In focus/near focus adjustment window switching tabs (4) Channel selection Function Switches between the Acquisition and Photo Activation windows. For the Photo Activation window, see Chapter 10. Displays the laser power value (integer obtained after A/D conversion divided by 10) of the current channel by clicking this button. During the image acquisition, the laser power cannot be measured and this button is grayed out. * When a laser unit of the LU-N series is in use, the value displayed in the monitor does not increase over a certain value with the increase in the laser power value, but this is not a problem. Used to switch between the In focus and Near focus PMT adjustment windows. To acquire the VAAS image, PMT of an In focus image (image of the focus area) and a Near focus image (image of the non focus area) can be adjusted respectively. In focus tab Near focus tab The light that passes through the normal pinhole is detected. (Normally-acquired confocal image) The light that does not pass through the normal pinhole is detected. Selects the channels (Ch1 to Ch4, and/or TD) to acquire the desired images. Do this by adding a check mark. When fluorescence is excited, all excitation lights are detected by the transmitted detector (TD). If the BA filter for 405 laser is installed in the TD, excitation light is not detected by the TD. 160

171 Chapter 7 Detection Mode VAAS (5) Name Brightness adjustment for each channel Function For each of the channels (Ch1 to Ch4), use the HV, Offset, Laser, and ND filter IN/OUT controls to adjust the brightness of the live image. Selects whether the laser is emitted or not. * When LU-NV is in use, this button is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. (6) Laser ON/OFF button ON status OFF status The laser is emitted. The AOTF shutter closes and the laser power value becomes 0. When switched from OFF to ON, the laser power value set in the previous ON status is applied. (7) (8) Select All Channels button Fluorescence dye name indication Selects all channels for acquiring images. The fluorescence dye name specified in the Optical path window is indicated. (9) Channel color Displays the channel color specified in the Optical path window. (10) HV Linear Correction (11) In-Near link check box (12) (13) Laser wavelength indication Remote controller selector icon (14) AG button (15) Auto Gain setting button (16) Optimize button (17) Brightness adjustment for transmitted detector Enables or disables HV Linear Correction. For HV Linear Correction, see Section 7.2.3, HV Linear Correction. The In-Near link check box is checked, the PMT adjustment for In focus and Near focus are linked. When you adjust either PMT, the other PMT is also adjusted automatically. Displays the currently selected laser wavelength. Displayed for the channel group which is currently adjustable with the remote controller, see Chapter 11. Automatically adjusts the HV value (HV gain) of the currently selected channel to the optimum values. It is not applicable when laser irradiation is OFF, even if a channel is selected. For Auto Gain, see Section 7.2.4, Auto Gain. Sets the ratio of saturation pixels used for automatic HV gain correction. The window for range of the ratio of saturation pixels settings appears when this button is clicked. For setting for ratio of saturation pixels, see Setting for ratio of saturation pixels in the Section 7.2.4, Auto Gain. Displays the XYZ Size Setup window. In the XYZ Size Setup window, the calculation method of the recommended values of the resolution, zoom magnification, and Z stack step size can be set. For the XYZ Size Setup window, see Section , Recommended Value Indication/Automatic Application in the next page. For the transmitted detector, use the HV and Offset controls to adjust the brightness of the live image. Pinhole size in the VAAS mode In the VAAS mode, the pinhole size is fixed and cannot be changed. The pinhole size ratio is as shown below: Size of the In focus pinhole < Size of the Near focus pinhole 161

172 Chapter 7 Detection Mode VAAS Recommended Value Indication/Automatic Application By the function of the recommended value indication/automatic application, the recommended values of the appropriate resolution, zoom magnification, and Z stack step size are calculated based on the objective type and the selected excitation wavelength. Using the calculated recommended values enables the image acquisition clearer and with less damage to the sample. Recommended Value Automatic Application To automatically apply the recommended values to the parameters, set the [Nyquist XY] button of the Scan Area window to ON. Nyquist XY button Figure Scan Area window Indicates the recommended value of the resolution. Indicates the recommended value of the scan magnification. Figure Location of Recommended Value Indication * When the laser or objective in use is changed, the recommended values are recalculated, and newly indicated and automatically applied. 162

173 Chapter 7 Detection Mode VAAS Recommended Value Settings Detailed settings of the recommended values are made in the XYZ Size Setup window that is displayed by clicking the [Optimize] button of the Acquisition window. If the [Nyquist XY] button of the Scan Area window is ON, the recommended values are automatically applied to the parameters. Or if the [Nyquist XY] button is OFF, the recommended values of the scan size and zoom are indicated in the Scan setting window. (1) (2) (3) (4) (5) (6) Figure XYZ Size Setup window Table Functions of XYZ Size Setup window Name (1) Zoom Preference Function When the [Nyquist XY] button is ON, keeps the scan size and applied the recommended value of the zoom. (2) Resolution Preference When the [Nyquist XY] button is ON, keeps the zoom and applied the recommended value of the scan size. (3) Use Cropping Fits the scan size in detail by using Crop Scan. Sets the Z step size calculation method. (4) Suggested Step (Z) Recommend (Z~1/3 FWHM) Half Overlap (Z=1/2 FWHM) Minimum Overlap (Z=FWHM) Perfect Voxel (Z=X=Y) Approximately one third of the thickness of optical section (FWHM value). One half of the thickness of optical section (FWHM value). The thickness of optical section (FWHM value). Value same as the pixel size. (5) OK button Confirms the XYZ Size Setup applied and closes the XYZ Size Setup window. (6) Cancel button Discards the XYZ Size Setup applied and closes the XYZ Size Setup window. 163

174 Chapter 7 Detection Mode VAAS Setting Image Brightness To acquire a clear image, adjust HV, Offset, Laser, ND filter IN/OUT for an In focus and a Near focus images of each channel and adjust HV and Offset for TD. (1) (2) (3) (6) (4) (5) Figure Setting the live image brightness (VAAS-use) Table Brightness adjustment functions for the live image (VAAS-use) Name (1) HV (2) Offset (3) Laser (*) (4) HV (TD) (5) Offset (TD) Function Sets the voltage to be applied to the PMT. Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value. Sets the BL offset value of the PMT. Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value. Sets the laser power value. Slider bar: Slides to the right or left to set the laser power value. Arrow buttons: Click either arrow button to increase or decrease the laser power value stepwise. Direct entry in laser power value display field: Type the desired setting value. Sets the voltage to be applied to the transmitted detector. Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value. Sets the offset value of the transmitted detector. Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value. (6) ND filter IN/OUT button (*) Inserts /removes the ND filter in/from the optical path. (IN = Insert in the optical path/ OUT = Remove from the optical path) This button is displayed only for lasers that can control insertion/removal of ND filter. * When LU-NV is in use, this function is grayed out and is disabled while the button on the front panel of the laser unit is OFF or blinking. 164

175 Chapter 7 Detection Mode VAAS PMT Overload If too much gain is applied with reference to the illumination intensity, the gain is automatically shut down to protect PMT and the following PMT Overload dialog box is displayed. Figure PMT Overload dialog box Pinhole Position Alignment This section describes the setting window for aligning the pinhole position. The pinhole position can be aligned by manually shifting the condensing lens in the A1plus Pinhole Alignment window. As shown below, right-click on the gray area (without any setting window displayed) to display a menu. Then select [Acquisition Controls] -> [A1plus Pinhole Alignment] in the menu to open the Scan Area window. Figure How to display the A1plus Pinhole Alignment window (1) (2) (3) (4) Figure A1plus Pinhole Alignment window Table A1plus Pinhole Alignment window Name (1) Coarse/Fine button Function The condensing lens position can be shifted by the shift amount set in the Coarse/Fine field by using these buttons. (2) Coarse/Fine field Shift amount of the condensing lens position can be set in these fields. (3) Lens Position X/Y The X and Y coordinates of the current condensing lens position is displayed. (4) Reset button The condensing lens potion is reset to the default position set at the factory. 165

176 Chapter 7 Detection Mode VAAS For memorizing the pinhole position (the condensing lens position) NIS-Elements can memorize the pinhole position that is adjusted in the A1plus Pinhole Alignment window. Select [Edit] -> [Options] -> [User rights] in the menu bar to display the window and then select the [Privileges] tab. Select the [Modify Shared Confocal Alignment] check box in the [Privileges] area so that NIS-Elements memorizes the NIS-Elements C pinhole position when the software ends. 166

177 Chapter 7 Detection Mode VAAS HV Linear Correction When HV changes, Gain changes as shown in the graph captioned Without HV Linear Correction. As HV increases, the gain variation (the variation of image brightness) is gradual initially, and it becomes steep beyond a certain point. The gain variation can be automatically corrected to be linear with HV variation by the function called HV Linear Correction. With this correction, gain varies at the same rate as the HV adjustment. Gain Gain Without HV Linear Correction HV With HV Linear Correction HV Figure Gain vs. HV To enable HV Linear Correction, check the [HV Linear Correction] check box. Figure HV Liner Correction When HV Linear Correction is enabled or disabled, HV is reset to 0 V once. 167

178 Chapter 7 Detection Mode VAAS Auto Gain Auto Gain is a function to automatically correct the value of HV gain to set the optimum image brightness. Automatic HV gain correction is performed within the predetermined range of the ratio of saturation pixels. Both Galvano mode and Resonant mode are supported. Automatic HV gain correction is performed only when channels are selected. For a TD, automatic adjustment is performed when it is selected. After execution of Auto Gain, in the window indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed by channel. For a channel on which Auto Gain failed, x is indicated and the HV value returns to its original value. When setting the line scan, Auto Gain cannot be executed. During execution of Auto Gain, do not perform manual adjustments in the Acquisition window and adjustments by the remote controller. When [In-Near link] check box is selected, the progress status is shown only for the In focus channel. Auto Gain button Auto Gain setting button Auto Gain does not execute on unselected channels. Figure Execution of Auto Gain (VAAS-use) Channel name Auto Gain status... Failed... Completed If checked, the window is automatically closed when Auto Gain is completed. Figure Auto Gain progress 168

179 Chapter 7 Detection Mode VAAS Setting for ratio of saturation pixels Set the maximum and minimum value for the Ratio of saturation pixels used for automatic HV gain correction. Click the [Auto Gain Setting] button to display the Auto gain setup window. Set the maximum and minimum value for the ratio of saturation pixels in Auto gain setup window. Figure Displaying the Auto gain setup window (1) (2) (3) (4) (5) (6) (7) Figure Setting for ratio of saturation pixels Table Setting for ratio of saturation pixels (1) Name Target Maximal Intensity Function Specifies the application ratio of the setting of the ratio of saturation pixels. Sets the percentage (%) of the maximum value to be applied. (2) Advanced Settings If checked, advanced settings of the ratio of saturation pixels are enabled. (3) Overillumination Tolerance (Area) Minimum Maximum Sets the minimum value for Ratio of saturation pixels. Sets the maximum value for Ratio of saturation pixels. (4) Perform in Find Mode (fast) If checked, execution in the Find mode is enabled. (5) Perform Auto Offset If checked, the offset value is set automatically. (6) OK button (7) Cancel button Confirms the settings of Auto gain setup applied and closes the Auto gain setup window. Discards the settings of Auto gain setup applied and closes the Auto gain setup window. 169

180 Chapter 7 Detection Mode VAAS 7.3 Various Views (VAAS-use) This section describes various VAAS mode views Displaying the VAAS Live You can adjust parameters of channels during live observation to display a VAAS live image while calculating it Acquiring the VAAS Live image 1. Select [Image] on the menu bar and then select [Calculate VAAS Live...]. The VAAS Live Settings window appears. Figure VAAS Live Settings 2. In the VAAS Live Settings window, click the [Apply] button to set to ON. Figure VAAS Live Settings window 170

181 Chapter 7 Detection Mode VAAS 3. Adjust parameters of each channel to display a VAAS live image while calculating it. Parameters for each channel Figure VAAS Live Settings window Figure VAAS Live image 171

182 Chapter 7 Detection Mode VAAS Displaying the VAAS image Acquiring the VAAS image To acquire the VAAS image, you have to set Optical path, PMT brightness (detector's sensitivity), and laser power before setting calculation method of the VAAS image. 1. Select [Image] on the menu bar and then select [Calculate VAAS...]. The VAAS Processing Settings window and the VAAS image window appear. Figure VAAS Processing Settings Figure VAAS Processing Settings window and VAAS image window 172

183 Chapter 7 Detection Mode VAAS 2. Adjust parameters of each channel in the VAAS Processing Settings window. Click the [OK] button to calculate and display the VAAS image. OK button Parameters for each channel Figure VAAS Processing Settings window Figure VAAS Processing Settings window and VAAS image window Figure VAAS image 173

184 Chapter 7 Detection Mode VAAS Channel View Setting Channel Mixed View Images acquired in the VAAS mode are displayed in the method suitable to the purpose. All image The [All] tab is selected, all the virtual channels are mixed to display. All tab Figure All image 174

185 Chapter 7 Detection Mode VAAS Each channel image To display the image of each channel, select the tab corresponding to the channel. Each channel tabs Figure Each channel image TD tab Figure TD image 175

186 Chapter 7 Detection Mode VAAS Custom image Custom image displays a mixed image of selected multiple channels. To change channels to be mixed, re-select channels. Right-click on the [Custom] tab and a menu appears. Select [Properties...] on the menu. The Custom window appears to allow you to change the channels for the Custom View. Custom tab Figure Selecting channels (Custom image) 176

187 Chapter 7 Detection Mode VAAS Ratio image The Ratio image view is displayed. Right-click on the window to display a menu. Selecting [Ratio View] from the menu changes the window to the Ratio image. Figure Displaying the Ratio image view Figure Ratio image 177

188 Chapter 7 Detection Mode VAAS * You can change the combination of channels to be displayed in the Ratio View. Right-click on the window and a menu appears. Select [Ratio Properties...] on the menu. The Ratio Properties window appears to allow you to change the channels for the Ratio View. Figure Ratio Properties window 178

189 Chapter 7 Detection Mode VAAS Split Channel View VAAS image is split into respective channels and displayed. Click the [Split Components] button. All image mixing all channels, VAAS In-focus and Near-focus images of respective channel, Custom image, TD image, and Ratio image are displayed. Split Components button Figure VAAS image Figure Split channel view * For switching from Split channel view to Channel mixed view, click the [Split Components] button again. 179

190 8 Scan Setting Window This window enables to set scanning conditions, such as resolution, scan speed, and magnification. The setting items in the Scan setting window vary, for example depending on the selected scan mode and scan area. 8.1 Galvano Scan Mode In the Galvano scan mode, high-quality imaging of up to 4096 x 4096 pixels is available Structure of Scan Setting Window (1) Scan magnification (4) (1) (5) (2) (3) (6) Figure Scan setting window in Galvano scan mode Table Name Summary of functions in Scan setting window (Galvano scan mode) Function Sets conditions for scanning. For details of scan settings, see Section 8.1.3, Scan Settings. (1) Scan setting Scan Direction Scan Size Fast button Selecting Scan Speed Scan magnification Selects Unidirectional or Bidirectional scan. Selects a resolution. (Unit=pixel) This button is enabled only for models that support the Fast Galvano mode. Turning on this button makes the Galvano scanner processing speed higher than the normal high-speed mode. * For speeding-up of the Galvano scanner, see Section 8.1.4, Fast Galvano Mode. Selects a scan speed. Sets a scan magnification. (2) Line Average/ Integrate Provides options for scanning a given line or area a number of times to display an image of the averaged or integrated values. For details of averaging and integration, see Section 8.1.5, Average and Integrate. (3) Line skipping Selects a line skipping mode to be applied during scanning. (4) (5) (6) Scan Zoom Reset button Switch button for the previous setting Scan Area window button Sets the scan magnification to Returns to the previous setting. Displays the Scan Area window. For details of Scan Area window, see Chapter

191 Chapter 8 Scan Setting Window Relationships among Scan Area Shape, Resolution, and Scan Speed This section describes the relationship of the resolution and scan speed in the Scan setting window, and the relationship of the scan area shape set in the navigation mode versus the resolution and scan speed for the Galvano scan mode. Relationship of Resolution and Scan Speed Once a Scan Size (resolution) is set, the software automatically generates a list (see Table 8.1-2) of the scan speeds available with that resolution, making them selectable from the Scan Speed pull-down menu. For example, suppose you select the Galvano scan mode, set the resolution for the Square scan area to X = 512 and Y = 512 pixels, and select the Unidirectional scan. Then, values listed in the Scan Speed pull-down menu are: 0.031, 0.042, 0.063, 0.125, 0.25, 0.5, or 1. * When the Band scan area is selected in the Galvano scan mode, scan speed values listed in the Scan Speed pull-down menu are guideline, which may not be the actual scan speed. Retention of Scan Settings for Different Scan Areas For each scan area shape, the latest Scan settings are retained. Once a scan area is selected in the navigation mode, the navigation mode displays the scan area that has been set at last, and the Scan setting window displays the set values of Scan settings. Automatic Change of Scan Settings with Change in the Band Scan Area Shape If the ratio of X and Y lengths of the Band scan area is changed: Resolution: Does not change Scan speed: Changes based on the new ratio of X and Y lengths, in a manner that gives the same pixel dwell. 181

192 Chapter 8 Scan Setting Window Table Combinations of resolution and scan speed (Galvano scan mode with the Square scan area) Resolution Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan Uni-scan 0.25 Uni-scan Uni-scan Uni-scan 0.5 Uni-scan Uni-scan Uni-scan Uni-scan 1 Uni-scan Uni-scan Uni-scan Scan Speed 2 Uni-scan Uni-scan Uni-scan 4 Uni-scan Uni-scan BBi ii- --ssccaann Uni-scan/ Bi-scan BBi ii- --ssccaann Uni-scan/ Bi-scan Bi-scan 5 UUnni ii- --ssccaann 8 Uni-scan Uni-scan Bi-scan 9 UUnni ii- --ssccaann 10 BBi ii- --ssccaann 16 Uni-scan Bi-scan 19 BBi ii- --ssccaann 32 Bi-scan : Using these combinations of resolution and scan speed, only the unidirectional scan.is possible. : Using these combinations of resolution and scan speed, only the bidirectional scan is possible. : When the zoom magnification is 4X or higher, using these combinations of resolution and scan speed, both the unidirectional and bidirectional scan are possible. : When the Fast Galvano mode is ON, these combinations of resolution and scan speed is possible. 182

193 Chapter 8 Scan Setting Window Scan Settings This section describes the Scan settings for Galvano scan mode. Unidirectional scan Bidirectional scan (8) (7) (1) (2) (5) (6) (3) (4) Figure Scan settings in Galvano scan mode Table Functions of Scan settings in Galvano scan mode Name (1) Scan Direction (2) Scan Size Function Toggles between Unidirectional and Bidirectional scan. Bidirectional scan is only selectable if the Square scan area, Band scan area, or Line scan area is set. By default, Unidirectional scan is selected. Sets the scan resolution in the X-direction. (Setting unit: Pixel) The resolution in the Y-direction is automatically calculated from the X to Y ratio of the scan area. Pull-down menu: Selects the desired resolution from this list. [ ] and [ ] buttons: Click these to select resolutions one after another. This button is enabled only for models that support the Fast Galvano mode. (3) Fast button Turning on this button makes the Galvano scanner processing speed higher than the normal high-speed mode. (4) (5) Selecting Scan Speed Line Average/ Integrate * For speeding-up of the Galvano scanner, see Section 8.1.4, Fast Galvano Mode. Sets the scan speed. (Setting unit: Frame/sec or lines/sec in line scan mode) Pull-down menu: Selects the desired scan speed from this list. [ ] and [ ] buttons: Click these to select scan speeds one after another. Provides options for scanning a given line or area a number of times to display an image of the averaged or integrated values. For details of Average and Integrate, see Section 8.1.5, Average and Integrate. (6) Line skipping Sets the line skipping mode to be applied during scanning. Line skipping increases scan speed. (Pixel dwell does not change.) Pull-down menu: Selects None (no line skipping), 2, 4, 8, or 16. E.g., If 2 is selected, scanning runs on the 1st line, skips the 2nd line, runs on the 3rd line, etc. 183

194 Chapter 8 Scan Setting Window (7) Name Scan magnification Function Sets scan magnification. Slider bar: Slides to the right or left to set the scan magnification. Arrow buttons: Click either arrow button to increase or decrease the scan magnification stepwise. Direct entry in scan magnification display field: Type the desired setting value. (8) Pixel Dwell Indicates the laser irradiation time per pixel. This value is automatically confirmed from scan resolution and speed. Correcting the Image Shifting when Setting Bidirectional Scan Image shifting correction when Bidirectional scan is selected in Galvano scan mode is shown below. When Bidirectional scan is selected from Scan Direction, the [Direction mismatch adjustment] button appears for shift correction. Click this button to display the Align bidirectional scanner window. [1] Select Bidirectional scan. [2] Click the [Direction mismatch adjustment] button for Bidirectional scan. Figure Correcting the image shifting for Bidirectional scan (Galvano scan mode) [3] Correct the image mismatch caused by Bidirectional scan. Figure Align bidirectional scanner window Table Item Correcting the image shifting for Bidirectional scan (Galvano scan mode) Description Image shift correction range -150µs to 150µs Image shift correction action Sets the correction value. Slider bar: Slides to the right or left to set the correction value. Arrow buttons: Click either arrow button to increase or decrease the correction value in steps of 0.1µs. Direct entry in correction value display field: Type the correction value. 184

195 Chapter 8 Scan Setting Window Fast Galvano Mode If you use a model that supports the Fast Galvano mode, the scan speed of the Galvano scanner can be higher than the normal high-speed mode. If you use the multi photon confocal microscope system A1 MP (A1 Multi photon), see Chapter 11 in NIS-Elements C (For Multi Photon Confocal Microscope A1 MP) Instructions (Ver. 4.30). Turning on the Fast button makes the scan speed ultra-high. Fast button Figure Fast Galvano mode Determining the relevant model For models that support the Fast Galvano mode, A1plus is displayed in various windows. * In the examples below, A1 Settings and A1 Scan Area are displayed for models that do not support the Fast Galvano mode. Figure Identification of models 185

196 Chapter 8 Scan Setting Window Notes on the use of Fast Galvano mode Note the following restrictions when using the Fast Galvano mode. Only 256 and 512 are available for Scan Size. When Scan Area rotation has been set, turning on the [Fast] button resets the rotation angle. The Fast Galvano mode cannot be used together with the Spectral Detector mode (SD) or Virtual Filter mode (VF). (If the detector is changed to [SD] or [VF] while the Fast Galvano mode is turned on, the Fast Galvano mode is automatically turned off.) When Piezo setting has been made, turning on the [Fast] button resets the Piezo setting. If correction of image deviation is greatly changed in the bidirectional scan, it takes several seconds until the correction is applied, but this is no problem. Wait until the correction is applied. Table Details of Fast Galvano mode Available Scan Size Scan Speed Available Detector Scan Zoom Scan area rotation function CROP scan area ROI scan area Free line scan AUX Mode CLEM Mode Use of Piezo Photo activation experiment Unidirectional 9 fps 5 fps Bidirectional 19 fps 10 fps DU4/VAAS x8 to x1000 Unavailable Unavailable Unavailable Unavailable Unavailable Unavailable Unavailable Unavailable 186

197 Chapter 8 Scan Setting Window Average and Integrate This section describes the averaging and integration settings for Galvano scan mode. Figure Average and Integrate (Galvano scan mode) Table Average and Integrate options and functions (Galvano scan mode) Item Function None Average Integrate Disables both averaging and integration. Scans a given scan line a set number of times and acquires an image of the averaged values. Scans a given scan line a set number of times and acquires an image of the integrated values. Scan lines A scan line is an X-direction line in the scan area, with a height of one pixel. For example, a scan area with the Y-direction resolution of 256 pixels has 256 scan lines. Scan line Figure Scan lines 187

198 Chapter 8 Scan Setting Window Average To use Average, select the Count to define the number of scans required for averaging. Figure Average (Galvano scan mode) Integrate To use Integrate, select the Count to define the number of scans required for integration. Figure Integrate (Galvano scan mode) 188

199 Chapter 8 Scan Setting Window 8.2 Resonant Scan Mode In the Resonant scan mode, high-speed imaging of up to 420 frames per second is available. The high-speed imaging is unavailable in the Spectral Detector mode and the Virtual Filter mode Structure of Scan Setting Window (1) Scan magnification (1) (5) (2) (3) (6) (4) Figure Scan setting window in Resonant scan mode Table Name Summary of functions in Scan setting window (Resonant scan mode) Function Sets conditions for scanning. For details of scan settings, see Section 8.2.3, Scan Settings. (1) Scan setting Scan Direction Scan Size Scan Speed Scan magnification Selects Unidirectional or Bidirectional scan. Displays a resolution (fixed), in pixels. Displays the scan speed. (Unit: Frame/sec or lines/sec in line scan mode) Sets a scan magnification. (2) Line Average/ Integrate Provides options for scanning a given line or area a number of times to display an image of the averaged or integrated values. For details of Average and Integrate, see Section 8.2.4, Average and Integrate. (3) Line skipping Selects a line skipping mode to be applied during scanning. (4) (5) (6) Scan Zoom Reset button Switch button for the previous setting Scan Area window button Sets the scan magnification to 1. Returns to the previous setting. Displays the Scan Area window. For details of Scan Area window, see Chapter