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Chapter 20. Separating Spectral Image (Unmixing)

Spectral unmixing using reference data

(1) Select [Image] - [Spectral Unmixing Setting] from the menu bar.

(2) Select [All] from the [Category] list box.

(3) Select reagents to be used for spectral unmixing and click the [Add] button to add them to [Unmixing

Elements] (list on the right pane).

(4) Set pseudo-colors after spectral unmixing.

(5) Click the [Unmix] button to perform spectral unmixing.

Spectral unmixing by creating reference data

(1) Save spectral data as reference data.

Click

at the side of the image frame

and draw ROIs on the image using tools.

Note: Specify simple stain areas only.

(2)

(3)-1

(3)-2

(4)

(5)

Содержание A1

Страница 1: ...1 Confocal Microscope A1 Simple Instruction Manual Ver 4 10 Nikon Corporation This document is subject to change for improvement without prior notice ...

Страница 2: ...t 38 Chapter 14 Capturing Photo Activation Imaging Galvano Scanner Time Series Activation 40 Chapter 15 Image Display Function 43 Chapter 16 Extracting ND2 Files 47 Chapter 17 Exporting ND2 Files 48 Chapter 18 Shutdown 50 Spectrum Imaging 51 Chapter 19 Capturing Spectral Images Spectral Detector 52 Chapter 20 Separating Spectral Image Unmixing 57 Chapter 21 Live Unmixing Spectral Unmixing for Live...

Страница 3: ...oftware is sequentially upgraded and therefore descriptions in this manual may not match the actual equipment in some cases If you have any questions contact us or the dealer from whom you purchased the product This system is highly advanced and there are procedures and conditions specified for its operation Be sure to follow them From the perspective of customer satisfaction we are striving to im...

Страница 4: ...4 Basic Operation A1 Ti E PFS Motorized Stage Piezo Z Stage Intensilight This edition may have unavailable functions depending on model in use and option settings ...

Страница 5: ...ODEL TI HU BC A USB STAGE REMOTE HGFIE TI PS SHUTTER2 5 5 0 3 0 1 This device complie s with Part 15 of the FCC Rules Operatio n is subject to the followin g two conditio ns 1 this device may not cause harmful interfere nce and 2 this device must accept any interfere nce received includin g interfere nce that may cause undesire d operatio n This Class A digital apparat us complie s with Canadia n ...

Страница 6: ...he NIS Elements software Icons for acquisition and analysis For acquisition Use this icon for image acquisition This icon consists of the acquisition function and analysis function For analysis Use this icon for brightness analysis or others This icon consists of only the analysis function Note When not only a confocal microscope is connected but also a camera the Driver selection dialog box opens...

Страница 7: ...e each motorized part Select an item you want to switch from the list Using function buttons enables necessary microscopy to be called at a time Diascopic illumination control State at startup Controlling transmitted light from the microscope Objective switch Condenser switch Fluorescent filter switch When is pressed Optical path switch Controlling transmitted light from the remote control pad Dia...

Страница 8: ...ND filter Necessary Unnecessary No problem if provided Unnecessary Be sure to remove it when acquiring a DIC image using a laser Can be called collectively using the function button on the Ti control pad D filter ND filter Polarizer Slide to put it into optical path Field diaphragm knob Aperture diaphragm open close lever Condenser centering knobs both sides Condenser Condenser lens Eyepiece tube ...

Страница 9: ...to reduce the excitation light and start microscopy ND values are in the range from 1 to 32 The larger the value the darker the excitation light becomes 3 Press the SHUTTER button to close the shutter and finish the fluorescence microscopy LAMP OPEN C HGFIE HG CONTROLLER POWER SHUTTER ND 32 16 8 4 2 1 Refocus switch objective refocusing Escape switch objective retraction Coarse fine motion select ...

Страница 10: ...e the XY stage movement speed and switch to constant speed mode Press this switch while moving the XY stage via the joystick to store the current movement speed as the constant speed To cancel constant speed mode press this switch again 4 Focus knob This knob has the same function as the focus knob supplied with the microscope Turning this knob varies the focus of the microscope Pressing the Z spe...

Страница 11: ...ch focus is maintained with the offset dial on the PFS offset controller and start microscopy Once the PFS operation is started focusing with the focus knob on the microscope is disabled Registering offset amount The focus position adjusted with the offset dial can be registered with the PFS MEMORY button Recalling offset amount Registered focus position can be called with the PFS RECALL button L8...

Страница 12: ...power and turning it counterclockwise decreases the power Pressing the dial selects coarse motion or fine motion alternately PMT gain dial Use this dial to adjust the detector sensitivity HV Turning it clockwise increases HV and turning it counterclockwise decreases HV Pressing the dial selects coarse motion or fine motion alternately 4 Adjust the scan speed as needed Scan Speed buttons Use these ...

Страница 13: ...desired microscopy Note To prevent fading close the fluorescent shutter frequently Use the ND filter to look for the sample LAMP OPEN C HGFIE HG CONTROLLER POWER SHUTTER ND 32 16 8 4 2 1 4 3 Switch the optical path to A1 When the Ti remote control pad is available press the A1 function button to switch the optical path to A1 If the Ti remote control pad is not available switch the optical path to ...

Страница 14: ... channel and select a pseudo color 5 If acquiring a transmitted image together with a confocal image click to bring into the optical path Note Before acquiring a transmitted image turn off the light above the microscope Note Because the transmitted light detector is placed in front of transmitted light transmitted images differential interferences DIC cannot be observed visually while putting the ...

Страница 15: ...e a transmitted image together with a confocal image click the TD IN button and check the TD checkbox Note Before acquiring a transmitted image turn off the light above the microscope 4 Select the laser wavelength to be used from Pinhole Select a pinhole size best suited for the objective with Home 5 1 Live Starting scanning Checking the settings 3 1 Selecting a transmitted image 1 Resetting inter...

Страница 16: ...ht click the gray area of the software and select Visualization Control LUTs from the displayed menu to call it 6 To use Auto Gain a function that automatically adjusts the detector sensitivity HV based on the set rate of saturated pixels click the AG button NG is displayed for channels that failed in Auto Gain and the HV values are returned to the previous values Use the Auto Gain setting button ...

Страница 17: ...d with a decimal point In such a case the scan speed is just an approximation and may differ from the actual scan speed 8 Apply Line Average as needed Average is a function to scan the same image multiple times and average it to remove noises Select Line Average Integrate Average in the Scan setting window and select an average scan frequency in Count Note Averaging reduces noise but decreases the...

Страница 18: ...etup dialog set the order of scanning for each channel Setting the order of scanning arbitrarily Set the order of scanning channels using the channel scanning order matrix Using line sequence Select the 1 4 or 4 1 button Apply lasers to each scan line and start scanning Note Be sure to set the TD transmitted image scanning order so that the scanning order comes together with other channels because...

Страница 19: ...want to acquire a transmitted image together with a confocal image click the TD IN button and check the TD checkbox Note Before acquiring a transmitted image turn off the light above the microscope 4 Select the laser wavelength to be used from Pinhole Select a pinhole size best suited for the objective with Home 9 Acquisition 2 Selecting a laser 4 Selecting a pinhole 5 2 Adjusting laser power and ...

Страница 20: ...uto Scale All button to adjust the contrast of the channel automatically to make the image clear Note Turning on and off the Live button scan ON OFF during adjustment minimizes fading Note If the LUTs tab is not displayed right click the gray area of the software and select Visualization Control LUTs from the displayed menu to call it Click the Split Channels button to display channels in division...

Страница 21: ...ng the remote controller during Auto Gain 7 Set the number of pixels to the necessary resolution e g 512 x 512 If the image is dark reduce the scan speed Note Check the pixel dwell for when the resolution is changed Pixel dwell indicates laser application time per pixel The larger the value the brighter the image that can be acquired 8 Apply Line Average as needed Average is a function to scan the...

Страница 22: ...plus Scan Area from the displayed menu to call it This window also opens by clicking the button shown below that is displayed in the Live window or A1plus Settings window 6 3 Select the zoom area Click in the Scan Area window and drag the green frame to reduce the scan area The frame can be moved with the mouse cursor set in the cross arrow state The scan position can be determined by right clicki...

Страница 23: ... The scan area can be rotated by dragging the mouse at the rotation point above the green frame The set rotation angle is shown in Rotation The rotation range is 90 to 90 degrees 6 5 Click the Live button to readjust image acquisition conditions Rotation point Rotation angle ...

Страница 24: ... and select Acquisition Controls A1plus Scan Area from the displayed menu to call it This window also opens by clicking the button shown below that is displayed in the Live window or A1plus Settings window 7 3 Select the area to be scanned 1 ROI scan images the entire window but does not cause photo damage to outside the ROI range Note The Ch series is disabled during ROI scan 1 Click the Edit but...

Страница 25: ... commits the ROI being edited 3 Click the Finish button to close the ROI Editor The ROI drawn in the Live window is hidden and the scan position is determined The drawn ROI is displayed in the Scan Area window with a light blue frame Note If a ROI is drawn on Frozen images click the Live button to start ROI scanning ...

Страница 26: ...In the Scan Area window drag the light blue frame to reduce its size The frame can be moved with the mouse cursor set in the cross arrow state When the size or position of the frame is changed the light blue frame turns to red The scan position can be determined by right clicking Note If a crop is drawn on Frozen images click the Live button to start crop scanning 7 4 Readjust image acquisition co...

Страница 27: ... the range for capturing Z series images 1 Click the XYZ button to open the Capture Z Series dialog box Note The Z image acquisition mode is switched according to whether or not Fast Piezo Z is checked Checked Speed priority mode Images are acquired giving a higher priority to speed regardless of the specified Z stroke This setting is enabled only when Nikon A1 Piezo Z Drive is selected as Z drive...

Страница 28: ...lane in the cube increases 5 Click the Live button and move the focus knob fine motion mode of the microscope while checking the image Click the Bottom button to determine the bottom position Note Move the focus knob in the direction where the value of the plane in the cube decreases 6 Determine Step 7 Select Ti ZDrive for Z Device 8 Check the Save to File checkbox as needed and acquire images whi...

Страница 29: ... application time per pixel The larger the value the brighter the image that can be acquired 2 Apply Line Average as needed Average is a function to scan the same image multiple times and average it to remove noises Select Line Average Integrate Average in the Scan setting window and select an average scan frequency in Count Note Averaging reduces noise but decreases the frame rate number of image...

Страница 30: ... Correction dialog box Right click the gray area of the software and select Acquisition Controls Z Intensity Correction from the displayed menu to call it 9 3 Specify the Z position and set brightness at that position 1 Click to register the Z points as Z items Clicking this button also registers three points top home and bottom automatically To register additional points click while checking the ...

Страница 31: ...this checkbox is checked changing the Z position displays the Live image with the set HV and laser power After you finish checking uncheck the checkbox 3 After the Z position is moved to where you want to adjust brightness adjust HV and laser power 4 To register the adjusted HV and laser power click After you finish adjusting brightness at each Z position you can close Z Intensity Correction 5 Aft...

Страница 32: ...age data size 10 2 Save the three dimensional image of a desired angle as an image 1 Drag the white frame of the three dimensional image with the mouse and adjust it to a desired angle Note Ctrl mouse left click drag Cut cross section Mouse right click drag Move three dimensional image display position Turning mouse scroll wheel Zoom 2 Select Edit Create View Snapshot 8bit RGB from the menu bar to...

Страница 33: ...g direction with the Presets button 3 Click the Settings button to open the Movie Maker settings dialog box Set Movie duration playback time and Frames per second playback speed and click the OK button Note Frames per Second fps is the number of images displayed per second Moving picture becomes smoother as the number of images increases Use 3 to 10 fps as a guideline ...

Страница 34: ...e button to create a movie 5 Select the acquired rotation image and select File Save As from the menu bar to save the image in the AVI file format Note We recommend that the avi image file be saved with No Compression Note Select Use Original Acq Time when saving the file 5 ...

Страница 35: ... or button at the side of the image frame to create an image Show Slices View button Click this button to display XZ and YZ cross sections of any position Clicking displays the selected position Drag the cross with the mouse Show Maximum Intensity Projection button Clicking this button detects the maximum brightness pixel from all frames to build an image This button is useful when capturing a thi...

Страница 36: ...me interval and Duration duration time Note Select No Delay for the Interval to acquire images at the highest speed Note Two or more phases can be created Selecting two or more phases allows variable time lapse time lapse that changes the interval during the process 3 Check the Save to File checkbox to acquire images while saving them Note We recommend acquiring time series images while saving the...

Страница 37: ... application time per pixel The larger the value the brighter the image that can be acquired 2 Apply Line Average as needed Average is a function to scan the same image multiple times and average it to remove noises Select Line Average Integrate Average in the Scan setting window and select an average scan frequency in Count Note Averaging reduces noise but decreases the frame rate number of image...

Страница 38: ...ot displayed on the software right click the gray area of the software and select Analysis Controls Time Measurement from the displayed menu to call Time Measurement 13 3 Set a ROI Region of Interest on the image 1 Click the Define button to open the Define ROI window 2 Set a ROI on the image 3 Click the Finish button to finish the setting 1 2 3 ...

Страница 39: ... Export button to save the export data as text data Note If the data cannot be saved as text data select Edit Options Data export Global Settings from the menu bar and check Export text files into folder and then specify the save destination folder 13 5 How to Use Time Measurement Select the ROI to be displayed on the graph Click the ROI to be displayed Two or more ROIs can be selected by clicking...

Страница 40: ...ries 14 2 Set the area where photo activation is to be performed 1 Click at the side of the image frame and select Simple ROI Editor Draw a ROI on the image using tools on the tool bar Note Using allows point activation 2 Right click on the ROI and select Use as Stimulation ROI from the displayed menu and then select Stimulation Group Note ROIs can be divided into up to three groups A group can co...

Страница 41: ...asers can be used for activation 4 Move the laser bar to select the laser power for activation 5 Select Scan Speed for activation Note Consider that Scan Speed is the time required for a single activation When 1 Sec Frame is selected the time for a single activation is one second 6 When there are Stimulation Groups 2 and 3 repeat Steps 2 to 5 14 4 Set time series for photo activation imaging 1 Cli...

Страница 42: ...ation Duration Set the duration time of image acquisition or photo activation When Loops is set duration is automatically determined Loops Set the number of image acquisition or photo activation execution times 3 Check the Save to File checkbox to acquire images while saving them Note Images are saved in nd2 file format Note We recommend acquiring time series images while saving them 4 Click to re...

Страница 43: ...s at the same time Reclicking this button restores the previous display 15 2 Multidimensional image display switch 1 Clicking an item on the scale bar allows you to view each series image Clicking or shifts the image to the next one 2 Use to play back the image Reclicking this button stops playback Use to adjust the playback speed Use for real time playback at the speed of actual image acquisition...

Страница 44: ...apse image 1 Right click on a time lapse image and select Show ND Information from the displayed menu to display the acquisition information on the image 2 Right click on the displayed acquisition information and select ND Info Properties from the displayed menu to open the Label Properties dialog box 3 In the Text Box tab you can add or delete information to be displayed and change the display fo...

Страница 45: ... desired file format 15 6 Reading image acquisition conditions Capturing conditions such as information of the Optical path window and the frequency of averaging at the time of acquisition can be read from the acquired image 1 Click to open an image nd2 whose acquisition conditions are to be read 2 Right click on the image and select Reuse Camera Setting from the displayed menu 3 The setting condi...

Страница 46: ...LUT Display of the LUTs varies with the number of channels Up to three channels All channels are displayed simultaneously Vertical display or horizontal display can be selected with Four channels or more A single channel is displayed Display channel can be selected from the pull down menu 3 Clicking the Reset LUTs button returns the display without contrast adjustment 4 Clicking the Pixel Saturati...

Страница 47: ...n be cropped 1 Click to open the nd2 file 2 Select File Import Export Crop ND Document from the menu bar 3 Specify the dimension and range of files you want to crop 4 Click the Crop button to create a new nd2 file Time axis Only arbitrary time axis range is selectable Z axis Only arbitrary Z axis range is selectable Wavelength Only arbitrary channels are selectable ...

Страница 48: ...NIS Elements To view such images by other software they must be exported in TIF file format 1 Click to open the nd2 file 2 Select File Import Export Export ND Document from the menu bar to open the ND Export window 3 Specify the save destination folder file name and file format Save destination folder File format File name ...

Страница 49: ... as an 8 bit file Combination of Channel TIF Compatibility Options and Insert Overlay Depth 12 bit to 16 bit 12 bit to 8 bit Mono image 12 bit 16 bit 8 bit Color image 12 bit RGB 16 bit RGB 8 bit RGB Channel merged Multi tif 12 bit Multi tif 16 bit Multi tif 8 bit Channel merged Insert overlay 12 bit RGB 16 bit RGB 8 bit RGB Image Image on a single channel Merged Overlapping images Light yellow Ou...

Страница 50: ...off the power to the controller 18 4 Turn off the power to the microscope 1 Turn off the power to the microscope main body 2 Turn off the power to the mercury lamp for visual fluorescence microscopy 3 Turn off the power to the halogen lamp for visual diascopic microscopy 4 Turn off the power to the piezo Z stage 5 Turn off the power to the motorized stage 18 5 Shut down the PC Turn the key ...

Страница 51: ...51 Spectrum Imaging A1 Ti E PFS Motorized Stage Piezo Z Stage Intensilight This edition may have unavailable functions depending on model in use and option settings ...

Страница 52: ...trol pad to select the desired microscopy Note To prevent fading close the fluorescent shutter frequently Use the ND filter to look for the sample LAMP OPEN C HGFIE HG CONTROLLER POWER SHUTTER ND 32 16 8 4 2 1 19 3 Switch the optical path to A1 When the Ti remote control pad is available press the A1 function button to switch the optical path to A1 If the Ti remote control pad is not available swi...

Страница 53: ...0 nm and a value for Channels number of channels to be used from 1 to 32 Set the wavelength band to be acquired by shifting the bar 6 Select BS20 80 for 1st DM 7 If acquiring a transmitted image together with a spectral image click to bring into the optical path Note Before acquiring a transmitted image turn off the light above the microscope Note Because the transmitted light detector is placed i...

Страница 54: ... reflected laser light are covered with a mask plate and channel data is not acquired Selecting a channel skip box facilitates subsequent spectrum analysis The volume of data is reduced a little 9 Click the OK button to set the optical path 19 6 Determine image acquisition conditions 8 2 8 1 9 7 Acquisition 3 1 Live Starting scanning 1 Selecting a transmitted image 2 Selecting a pinhole 3 2 Laser ...

Страница 55: ...ast of the channel automatically to make the image clear Note Turning on and off the Live button scan ON OFF during adjustment minimizes fading Note If the LUTs tab is not displayed right click the gray area of the software and select Visualization Control LUTs from the displayed menu to call it 4 To use Auto Gain a function that automatically adjusts the detector sensitivity HV based on the set r...

Страница 56: ... Line Average as needed Average is a function to scan the same image multiple times and average it to remove noises Select Line Average Integrate Average in the Scan setting window and select an average scan frequency in Count Note Averaging reduces noise but decreases the frame rate number of images acquirable per second 7 Click the XY button to acquire an image 19 7 Save an image Make the image ...

Страница 57: ...ame and draw ROIs on the image using tools Note Specify areas that include only shades of the same color up to the number of pigments 2 areas or more 2 Select Image Spectral Unmixing Setting from the menu bar 3 Select ROIs from the Category list box 4 Select ROIs to be used for spectral unmixing and click the Add button to add them to Unmixing Elements list on the right pane 5 Set pseudo colors af...

Страница 58: ...used for spectral unmixing and click the Add button to add them to Unmixing Elements list on the right pane 4 Set pseudo colors after spectral unmixing 5 Click the Unmix button to perform spectral unmixing 3 Spectral unmixing by creating reference data 1 Save spectral data as reference data Click at the side of the image frame and draw ROIs on the image using tools Note Specify simple stain areas ...

Страница 59: ...ofile is not displayed on the software right click the gray area of the software and select Visualization Controls Spectrum Profile from the displayed menu to call Spectrum Profile 3 Select the spectrum graph of a ROI which you want to save and then click 4 Enter a reagent name in the Spectrum Name box and then click the OK button to save the spectral data ...

Страница 60: ...ctral unmixing and click the Add button to add them to Unmixing Elements list on the right pane 8 Set pseudo colors after spectral unmixing 9 Click the Unmix button to perform spectral unmixing How to use Spectrum Profile Displays the Y axis of graph as relative values Displays the Y axis of graph as brightness values Inserts a spectrum graph originated by the fluorescence manufacturer as referenc...

Страница 61: ...ry by following the procedure for Separating Spectral Image 3 Click the Cancel or Close button to close the Spectral Unmixing Setting window 21 2 Perform spectral unmixing for live image 1 Click the Live button and adjust Laser laser power and Si HV detector sensitivity while checking the image Note The Si HV setting is common to all lasers Make fine adjustments with the laser power setting 2 Clic...

Страница 62: ...Chapter 21 Live Unmixing Spectral Unmixing for Live Image A1 62 Live Unmixing ...

Страница 63: ...te control pad to select the desired microscopy Note To prevent fading close the fluorescent shutter frequently Use the ND filter to look for the sample LAMP OPEN C HGFIE HG CONTROLLER POWER SHUTTER ND 32 16 8 4 2 1 22 3 Switch the optical path to A1 When the Ti remote control pad is available press the A1 function button to switch the optical path to A1 If the Ti remote control pad is not availab...

Страница 64: ...setting for the confocal system required for acquiring images Check the settings 1 Click to open the Optical path window 2 Click the VF button to use the spectral detector in the virtual filter mode 3 Click the Manual button to set the optical path in the manual mode 4 Click the Grating Settings tab 1 3 2 4 ...

Страница 65: ...red by shifting the bar 6 Click the Detector tab and make other settings 7 Select the laser to be used 8 Set the channel detecting range by extending reducing the bar 9 Select combination of lasers to be used for 1st DM 10 If acquiring a transmitted image together with a fluorescent image click to bring into the optical path 11 Click the OK button to set the optical path 7 8 5 1 5 2 6 11 9 10 ...

Страница 66: ...efore acquiring a transmitted image turn off the light above the microscope 3 Select the laser wavelength to be used from the pull down menu of Pinhole Select a pinhole size best suited for the objective with Home 6 1 Selecting resolution 6 3 Selecting scan speed 6 2 Laser application time per pixel 8 Acquisition 4 1 Live Starting scanning 4 2 Adjusting laser power and gain 2 2 Selecting a transmi...

Страница 67: ...LUTs tab is not displayed right click the gray area of the software and select Visualization Control LUTs from the displayed menu to call it 5 To use Auto Gain a function that automatically adjusts the detector sensitivity HV based on the set rate of saturated pixels click the AG button NG is displayed for channels that failed in Auto Gain and the HV values are returned to the previous values Use ...

Страница 68: ...ply Line Average as needed Average is a function to scan the same image multiple times and average it to remove noises Select Line Average Integrate Average in the Scan setting window and select an average scan frequency in Count Note Averaging reduces noise but decreases the frame rate number of images acquirable per second 8 Click the XY button to acquire an image 22 7 Save an image Make the ima...

Страница 69: ...69 Motorized Stage This edition does not include some functions depending on model and option settings ...

Страница 70: ...on information Checking the Include Z checkbox also registers the Z position The Z position of the microscope or the offset value tracking focus position of the perfect focus system can be registered Note If the checkboxes in the Point Name column are not displayed click 5 Repeat Steps 3 to 4 to register points as needed Coarse Fine Y X X Y Ex Fine Coarse Fine Ex Fine Z Speed XY Speed Constant Spe...

Страница 71: ...e per pixel The larger the value the brighter the image that can be acquired 2 Apply Line Average as needed Average is a function to scan the same image multiple times and average it to remove noises Select Line Average Integrate Average in the Scan setting window and select an average scan frequency in Count Note Averaging reduces noise but decreases the frame rate number of images acquirable per...

Страница 72: ...move the focus knob fine motion mode of the microscope while checking the image and then click the Top button to determine the top position Note Move the focus knob in the direction where the value of the plane in the cube increases 4 Click the Live button and move the focus knob fine motion mode of the microscope while checking the image Click the Bottom button to determine the bottom position No...

Страница 73: ...an range Set the position on the large image at which the current field of view is to be set in Fields Placement Around the current position Scanning is performed around the current position Current position is at top left corner Scanning is performed with the current position set at the top left corner When selecting Left top right and bottom limits Select the top bottom right and left margins Se...

Страница 74: ... image overlap correction Overlap Sets the percentage of image overlap correction No overlap Stitches images without image overlap correction Stitching via Selects how the images are stitched Image registration Enables image overlap correction position correction when stitching images 4 Select whether or not to use shading correction Automatic postprocessing Enables automatic post processing of ac...

Страница 75: ...ocus Set the number of images after which the focus is readjusted Focus manually Adjusts display magnification so that the display area can fit the macro screen Note To use Use Focus Surface for focusing set the Focus Surface in accordance with the following procedure 1 Select Devices Focus Surface Setup from the menu bar to open the Focus Surface tab of the XYZ Overview window Display the XYZ Ove...

Страница 76: ...This option is enabled when the Save large image to file checkbox is checked Save to Auto capture folder Saves large images to the auto capture folder Filename Specify the save destination folder and file name of large images By default the file name is set to Large Image To alter the file name change the Large Image part Format Selects the file format of large images from nd2 and tif When selecti...

Страница 77: ...77 High Speed Imaging A1 Ti E PFS Motorized Stage Piezo Z Stage Intensilight This edition may have unavailable functions depending on model in use and option settings ...

Страница 78: ...onant 25 3 Determine image acquisition conditions 1 Click to select bidirectional scan scan speed typical 30 frames second 8 Acquisition 2 Selecting lasers 4 Selecting a pinhole 5 2 Adjusting laser power and HV 3 2 Selecting a transmitted image Checking the settings 3 1 Selecting a transmitted image 5 1 Live Starting scan Selecting a scan mode Laser application time per pixel 1 Selecting bidirecti...

Страница 79: ...call it This window also opens by clicking the button shown below that is displayed in the Live window or A1plus Settings window Scan Area Setting X Resolution Y Resolution Scan Speed and Unit Square scan area 512 512 30 Frame sec Band scan area X Y 1 1 2 512 256 60 Frame sec Band scan area X Y 1 1 4 512 128 120 Frame sec Band scan area X Y 1 1 8 512 64 230 Frame sec Band scan area X Y 1 1 16 512 ...

Страница 80: ...r sensitivity while checking the image Note Use Offset 0 as the standard setting Note Using the Pixel Saturation Indication button in the LUTs tab during adjustment makes it easy to adjust the sensitivity Note If the displayed image is dark click the Auto Scale All button to adjust the contrast of the channel automatically to make the image clear Note Turning on and off the Live button scan ON OFF...

Страница 81: ...Auto Gain is disabled when line scan is set Do not make a manual adjustment in the Acquisition window and do not make an adjustment using the remote controller during Auto Gain 7 Apply Line Average as needed Average is a function to scan the same image multiple times and average it to remove noises Select Line Average Integrate Average in the Scan setting window and select an average scan frequenc...

Страница 82: ...eps 25 1 to 25 3 in Chapter 25 Capturing High Speed Images 26 2 Open the ND Acquisition dialog box 1 Click the XYZ Time button to open the ND Acquisition dialog box 26 3 Determine the Z acquisition range of the ZT series 1 Without perfect focus system 1 Click the Z tab 2 Click the Defined top bottom button 3 Click the Reset button 1 2 1 3 1 1 ...

Страница 83: ...n mode of the microscope while checking the image Click the Bottom button to determine the bottom position Note Move the focus knob in the direction where the value of the plane in the cube decreases 6 Determine Step 7 Specify A1 Piezo Z Drive for Z Device Note The drive range of Piezo ZDrive is 100 μm at ordinary temperature Set the total range to 100 μm or less Note The drive range of Piezo ZDri...

Страница 84: ...SE Figure Ti E Front Operation Panel and Offset Controller 2 Click the Z tab 3 Click the Asymmetric mode defined by range button 4 Turn the offset dial in the direction where the value in the cube decreases and determine the position of the sample bottom near the objective 5 Click 6 Enter 0 in Below 7 Enter a value of the sample thickness in Above 8 Determine Step 9 Specify A1 Piezo Z Drive for Z ...

Страница 85: ...k the Time tab 2 Determine Interval time interval and Duration duration time 3 Check the Save to File checkbox to acquire images while saving them Note Images are saved in nd2 file format Note We recommend acquiring time series images while saving them 26 5 Click the Run now button to acquire ZT series images 3 1 2 ...

Страница 86: ...ormed 1 Click at the side of the image frame and select Simple ROI Editor Draw a ROI on the image using tools on the tool bar Note Using allows point activation 2 Right click on the ROI and select Use as Stimulation ROI from the displayed menu and then select Stimulation Group Note ROIs can be divided into up to three groups A group can contain two or more ROIs Different activation conditions can ...

Страница 87: ...To use 488 nm lasers however the filter for 488 nm simultaneous activation needs to be installed 4 Move the laser bar to select the laser power for activation 5 Select Scan Speed for activation Note Consider that Scan Speed is the time required for a single activation When 1 Sec Frame is selected the time for a single activation is one second 6 When there are Stimulation Groups 2 and 3 repeat Step...

Страница 88: ...Loops Set the number of photo activation execution times 3 Set the time series time settings Duration Set the duration time of image acquisition When Loops is set duration is automatically determined Loops Set the number of image acquisition execution times 4 Check the Save to File checkbox to acquire images while saving them Note Images are saved in nd2 file format Note We recommend acquiring tim...

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