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Evident FV4000-BX63L Operation Manual

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FV4000-BX63L
Operation Manual
Ver.4.1
2025/2
(cellSens-FV ver3.2.1)

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Summary of Contents for Evident FV4000-BX63L

  • Page 1 FV4000-BX63L Operation Manual Ver.4.1 2025/2 (cellSens-FV ver3.2.1)

  • Page 2: Table Of Contents

    ■Contents ―Image Acquisition― Starting the system Controller Observation via eyepieces XY Image Acquisition Virtual channel scan XYZ image Acquisition BrightZ XYT Image Acquisition Exiting the system 2D view and operation 3D view and operation Creating the movie Processing Projection Series Analysis 1D Profile Saving Images with the photon numbers Export...
  • Page 3 ―Motorized Stage― Option: Motorized Stage Multi Area Time Lapse Acquisition Multi Area Time Lapse Map Image(1) Multi Area Time Lapse Map Image(2) Overlap section in stitching Stitched image Acquisition using Map Image Stitching Focus Map Setting of well plate Calibration of the well plate Map Image display area for well plate Registration of Image Acquisition Area...
  • Page 4 ―Advanced― Stimulation Super resolution FV-OSR Append Image Extraction Merge Channel Colocalization 3D Deconvolution TruAI Noise Reduction BATCH ~batch processing for multiple images Stitching after projection Sequence Manager New Dye registration ー Spectral Imaging ― Lambda scan Unmixing (１）Blind Unmixing Unmixing（２）Normal Unmixing Unmixing（3）Spectral Image Unmixing ーAppendix―...

  • Page 5: Image Acquisition

    Image Acquisition...

  • Page 6: Starting The System

    ② Log on Windows with your own user ID. ④Laser Combiner Wait until desktop appears. Rotate start key 【Factory Default ID & password】 Factory User ID：evident Password：evident Switch on the ③ Switch on the central power. main power Laser controller ④...

  • Page 7: Controller

    Controller See the next page for observation via eyepieces Brightness adjustment for focusing nob. Transmitted Fluorescence × × FL shutter FL mirror...

  • Page 8: Observation Via Eyepieces

    Observation via eyepieces Change light path to binocular 100% ★Fluorescence ★DIC(Transmitted light) ＊ Select the objective lens on the TPC. ＊Select the objective lens on the TPC ① Click “Ocular” in [Ocular] Tool window. ① Click “Ocular” in [Ocular] Tool window. ②...

  • Page 9: Xy Image Acquisition

    XY Image Acquisition(1) ① Select “Standard” in [Detector Setting] tool window. ① ⑧ Assign the FL probe and Ch ⑦ ② Click ② ③ Click ④ Double click the FL probes to observe。 ⑤ When registering the FL probes, TD channel is registered automatically.
  • Page 10 XY Image Acquisition（２） Setting the scanner in [LSM Imaging] ⑩ Select the type of scanner and mode. ⑪ Set “Scan Size” and “Aspect Ratio”. ⑩ ⑫ Set “Zoom” and “Rotation”. Clicking “Optimize”, Zoom changes to make pixel size to ½ of optical resolution. ⑬...

  • Page 11: Virtual Channel Scan

    Virtual Channel Scan acquiring multiple CH image whose channel number is lager than the number of detector ① Select “Standard” in [Detector Setting] tool window. Assigning the detector to channel ⑤ ④ ② Press the button on [Detector Setting] Tool Window. ③...

  • Page 12: Xyz Image Acquisition

    XYZ Image Acquisition(Start/End) ＊Before starting the following procedure, adjust for XY imaging. ① Reset: Reset to initial values. Setting Z series ② ① Select “ON” in [Z] on [Series] Tool Window. ② Select “Start/End” in [Motor] on [Z section]. Press the button, current position is set as 0.00.
  • Page 13 XYZ Image Acquisition(Range) ＊Before starting the following procedure, adjust for XY imaging. ① Reset: Reset to initial values. Setting the Z-series ② ① Select “ON” in [Z] in [Series]tool window. ② Select [Range] in [Motor] on [▼Z Section] ③ Register the Range ④...

  • Page 14: Brightz

    Bright Z scanning while correcting the brightness against the Z position This function cannot be used with Virtual Z ② ＊Before setting the following, adjust XY image setting and Z stack condition. ① Select [Tool Window]＞[BrightZ] . ② Select “ON” on ”During Z-series” in When this is on, Bright Z is available while changing focus manually.

  • Page 15: Xyt Image Acquisition

    XYT Image Acquisition ＊Before starting the following procedure, make adjustments for XY imaging.(refer to page 8-9) ④ Setting Time Series ⑤ ① Select ”ON” in [Time] on [Series ] Tool ③ Window. ② Set the interval to acquire the image in [Interval] and [Cycle] on [Time Lapse].

  • Page 16: Exiting The System

    Exiting the system Exiting the software and PC ① Close the software. ② Shut down the Windows. Touch Panel Controller（TPC) ③Touch Turning OFF the power the “OFF” Touch Panel Controller（TPC) ④TPC main ③ Tap the “OFF” on display of TPC. switch ④...

  • Page 17: View And Operation

    ２D view and operation Drawing scale bar and ROI • A straight line can be drawn by pressing Shift key when left-clicking. Scale bar Playing animation of the series images • These button are used to play animation of the series images. Set the range Frame rate play...

  • Page 18: View And Operation

    3D view and operation(1) Opening the file and displaying 3D image Open and activate the Z series image. Select [Volume] tab to display 3D image. Drag the mouse on the 3D image in the direction you want to rotate. ■Zooming 3D image Mouse wheel :Zooms toward the center of the 3D image displayed.
  • Page 19 3D view and operation（２） Clipping ■Clipping/Cut Displays the yellow frame in the image constructed in 3D. Dragging this frame with the mouse will display only the area visible from the frame. ■Clip and cut Show “clipping” and “cut” at the same time. Clip and Cut changing the cross collection XY/YZ/XZ by pressing these button.

  • Page 20: Creating The Movie

    Creating the movie ＊Opening the file and displaying 3D image ① Press the button and select ① [Volume setting] in the [Tool Window] menu. ② Select [movie] tab in [Volume setting] tool window. ③ Select “Key Frame” in [Movie Item] ④...

  • Page 21: Processing Projection

    Projection(1) ① Click to show projection image。 ② File > Save As > Projection Image OR ① right-click on the image > Save As > Projection Image. ③ Save the image (.OIR is the extension only available)
  • Page 22 Projection(2) ② ① ① Press the button to switch to “Viewer mode”. ② Select [Analysis] in the [Tool Window] menu. ③ Select “Single process” and “Post processing” as the illustration below. ③ ④ Select the [Processing] tab and resister “projection” in [Processing Item]. ⑤...

  • Page 23: Series Analysis

    Drawing a Intensity Graph ：Series Analysis (Specifying ROI on the series image) ② ① ③ ④ ⑤ ⑥ ⑦ ① Select [Analysis] in the Tool window menu. ② Select the ROI tool in [Analysis ROI], and specify the area you want to measure on the item.

  • Page 24: Profile

    Drawing average intensity profile： 1D profile （the arbitrary line viewing the series image ） ① ② ③ ④ ⑤ ⑥ ⑦ ① Select [Analysis] in the Tool window menu. ② Select the ROI tool in [Analysis ROI], and specify the area you want to measure on the item.

  • Page 25: Saving Images With The Photon Numbers

    Saving images with the photon numbers ① From the File menu, select SaveAs/PhotonImage, or right-click on the image and select SaveAs/Photon. ② New OIR file that reflects the photon numbers will be created. *"_photon" will be displayed at the end of the file name and will be saved.

  • Page 26: Export

    Exporting the image the image can be exported in the file format which can be used by other software. A. Exporting a single image ① Right-click on the image to be exported. Select [Export] in the menu displayed. The [Export] dialog box appears. ②...

  • Page 27: Reload And Saving Observation Methods

    Reloading and saving Observation Method ① Save/ load the observation method ① Select the [Observation Method] in [Tool Window] menu. ＊Each function :to load the observation condition selected in the list :to save the current condition and add the list :to update the current conditions :to delete the condition from the list :to sort the list...

  • Page 28: Configuration

    Configuration ■Changing the objective lens ① ＊Exchange the objective lens to be used. ① Select [Configuration] in [Tool] menu. The [Configuration] dialog box appears. ② Select [Microscope] tab. ② ③ Select [Objective Lens]. ④ Select the name of the mounted ③...
  • Page 29 Mortorized Stage *Option...

  • Page 30: Option: Motorized Stage

    ◆only for motorized stage ◆ Option : Motorized stage ◼ Multi Area Time Lapse Position４ Position１ Position３ Position２ Register multiple areas and repeatedly acquire • images . It is also possible to set the interval. • Time lapse data of multiple area can be acquired •...

  • Page 31: Multi Area Time Lapse Acquisition

    ◆Only for Multi Area Time Lapse Motorized stage◆ Imaging Acquisition ＊Before starting the following procedure, ① make adjustments for XY imaging. ① Press the button in [Live] Window. ② Select the [Map] sub pane. Registering the group of MATL ② ③...

  • Page 32: Multi Area Time Lapse Map Image(1)

    ◆Only for motorized Multi Area Time lapse stage ◆ using Map Image (1) ＊Before starting the following procedure, ① make adjustments for XY imaging. ① Press the button in [Live Window], the sub pane appears. ② Select [Map] tab. ② Create the map ③...

  • Page 33: Multi Area Time Lapse Map Image(2)

    ◆Only for Multi Area Time lapse motorized stage◆ using Map Image(2) Registering the multiple area ① Set the higher magnification objective lens. double-clicking on the map image, ① the stage is moved to at the center of the map. ② Press the to register the position and acquiring parameters.

  • Page 34: Overlap Section In Stitching

    Overlap section at tiling imaging ① Select [configuration] in [Tools] to open the dialog on the left. ② Select [XY stage] in the [Preference] tub. ③ Enter the number directly in “Overlap section at tiling imaging” ＊example↓ Objective lens 10X or less:20%～30% 10X or more:15%～20％...

  • Page 35: Stitched Image Acquisition Using Map Image

    Acquiring the stitched image using Map Image(1) ① Before starting the following procedure, make adjustment for XY imaging. ② Setting the acquiring area. ＊3 types A. Register the Column×Row ② 1. Press the in area registration. The [Define Matrix] dialog box is appears.
  • Page 36 Acquiring the stitched image using Map Image(2) ③ Check the registered area List Processing to stitch after acquiring whether it is ticked to “Enable” at the image. the area which you want acquire images. ① Open the image. ② Press the button.

  • Page 37: Stitching

    Stitching ① Open the data you acquired. How to open the image after closing the image. 1)Select File>Open>MATL 2)Select the folder. And open「matl.omp2info] 3)After open the file, subpanel is opened. And select the Map tub automatically. ② Click on the top left of the image in the Map tub to select all image.

  • Page 38: Focus Map

    Adjusting the tilt in Z direction ～Focus Map～ ① Before starting the following ③ ④ ⑤ ⑥ procedure, make adjustment for XY imaging. ② Setting the acquiring area (more than 2X2).（p.39） ③ Click “FocusMap” button . The 3 red focus points will be displayed in MATL ROI.

  • Page 39: Setting Of Well Plate

    Setting of the Well Plate ■Setting ② ① Select [Tools]＞[Configuration]. ③ ② Select the [Preference] tub . ③ Select the [Plate] on the left list. ③ Select the appropriate well. ※Corning is preset. ④ Press the button. Register plate in Custom plate setting ④...

  • Page 40: Calibration Of The Well Plate

    Calibration of the well plate ＊Before starting the following procedure, ① make adjustment for XY imaging. Set the Map mode ① Select [Live] window. And select the button to display the tool on the left side of the LIVE image. ②...

  • Page 41: Map Image Display Area For Well Plate

    Map Image display area for well plate(1) When selecting plate type in [Plate], it is displayed for microplate. Pattern diagram for well plate ① ① Line is alphabet , column is number. ：well in the stage operation range click →selected double click →move to center of the well ：well where the stage located ：well selected...
  • Page 42 Map Image display area for well plate(2) Copy and Paste of the scan setting of the one well Target row and column can se selected by clicking on row/column nuber. • All the wells can be selected by context short cut key. •...

  • Page 43: Registration Of Image Acquisition Area

    Registration of Image Acquisition area ① Select the [LSM Imaging] in [Area Registration] on [Map] tab. ① ② Register the Image acquisition area. ② A.Specify any area 1. Press the button to resister an area with the stage position at the time the button is pressed.

  • Page 44: Advanced

    Advanced...
  • Page 45 Stimulation Condition setting ① ① Select the [LSM Stimulation] in [Tool Window] ② menu. ④ ② Select the ROI and resister the stimulation area on the Image. ⑤ ※Recommendation Tornado scan Laser ND Filter=None A:trnado scan at a constant angular velocity with a circle. L:trnado scan at a constant linear velocity with a circle.

  • Page 46: Stimulation

    Stimulation ① ② ③ Drawing the graph of brightness with LIVE image ① Select the[Live Graph] in the [Tool window] menu. ② Specifying the place checked the intensity by drawing the ROI in [Analysis ROI]. ③ Ticking these checkboxes displays the intensity profile.

  • Page 47: Super Resolution Fv-Osr

    ※OSR Super Resolution:FV-OSR license The resolution can be improved to around 120 nm by using FV-OSR. ① Adjusting focus and set Laser Intensity(%), Sensitivity(HV), Gain and Offset on [PMT setting] Tool Window. ② Select the [Super-resolution] in the [Tool Window]. ③...

  • Page 48: Append Image

    Append Images ① Select the [Analysis] in the [Tool window] menu. ① ② Select the [Single process] and [Post processing] in the [Analysis] tool window. ② ③ ④ ③ Open the [Processing] tab. ④ Select “Append Images” in the [▼Edit Images].

  • Page 49: Extraction

    Extraction ① Select the [Analysis] in the [Tool window] ① menu. ② ② Select the [Single process] and [Post processing] in the [Analysis] tool window. ③ ④ ③ Open the [Processing] tab. ④ Select “Extraction” in the [▼Edit Images]. ⑤ Click the [Input], and select the image for image processing.

  • Page 50: Merge Channel

    Merge Channels ① Select the [Analysis] in the [Tool window] ① menu. ② ② Select the [Single process] and [Post processing] in the [Analysis] tool window. ③ ④ ③ Open the [Processing] tab. ④ Select “Merge Channels” in the [▼Edit Images].

  • Page 51: Colocalization

    Colocalization ～cellSens～ ④ ⑧ ⑤ ⑦ ⑥ ① ⑨ ① Open the Image on FV software and Right-click to select [Send to cellSens]. ② cellSens starts up. ③ Select the [Colocalization] .(Measure>Colocalization) ④ Select the two color channel on which you want to perform the measurement colocalization. ⑤...

  • Page 52: Deconvolution

    ※Option : cellSens 3D Deconvolution 3D Deconvolution ～cellSens~ ④ ⑥ ⑦ ① ⑤ ⑧ ① Open the acquired Image (Z stack) and double click on the image. Select the [Send to CellSens]. ② cellSens is displayed. ③ Select the [Process]>[Deconvolution]>[Constrained Iterative…]. ④...

  • Page 53: Truai Noise Reduction

    ※FV40S- TruAI Noise Reduction AINR option High S/N image will be expected by using AI noise reduction. ① Open the images. ② Show Analysis tool window by Tool Window > Analysis. ③ Select [Single process] and [Post processing] then select [TruAI Noise Reduction] in Processing Item.

  • Page 54: Batch ~Batch Processing For Multiple Images

    BATCH： Batch processing for multiple images ① ■Available condition →Select the [Single process] and [Post ② Processing] in the [Analysis] tool window. ① Select the [Single process] and [Post processing]. ③ ② Select the [Batch] tab. ⑤ ③ Press the [Add] to display the dialog box. ④...

  • Page 55: Stitching After Projection

    Stitching after projection : For large volumes of data, time can be reduced. ① ① Select the [Analysis] in the [Tool window] menu. And select the [Single process] and ② [Post processing]. ② Select the [BATCH] tab. ③ Press the [Add] to display the dialog box. ④...

  • Page 56: Sequence Manager

    Sequence Manager （making protocol of acquiring） ① ↑ delete the selected task ↑ ②、③ ↑Append the task Loop ④(drag) Making the protcol ① ① Select the [Sequence Manager] in ② [Tool Window] menu. ② Press the button in [Add task] to add the task.

  • Page 57: New Dye Registration

    New registration of Dye ① Select the [Dye Editor] in Tools. ② Press the to display the dialog box. :edit based on the data of the "User Dye" selected in [User Dyes] ③ Enter each items. ○Excitation peak/fFuorescence peak ：Airy Disk ○Automatic emission priority ：refer to next page ④...
  • Page 58 New registration of Dye In case of Alexa488（preset） ・one color→500-600 ・488/640→500-580 ・488/568→500-540 Emission Priority ■set automatically V（405）：430-470 C（440）：460-500 B（488）：500-600 500-580 500-540 E（514）：530-630 ：530-580 Y（561）：570-670 ：570-620 O（594）：610-710 R（640）：650-750 ■selecting Ex＋ automatically selects the main laser wavelength. ■If you enter something other than the above, Select "Custome"...

  • Page 59: ー Spectral Imaging

    Spectral Imaging...

  • Page 60: Lambda Scan

    Lambda series(1) （Ch setting） ＊Before starting the following procedure, make adjustments for imaging. Changing to Lambda mode ① Select “Lambda” in [Mode] in [Detector setting] Tool Window Select Channel ② Press the button. The [Dye & Detector Select] dialog box appears.
  • Page 61 Lambda series(2) （LightPath setting） ⑤ Select [LightPath] in Tool Window. ⑥ Select “LSMScanner” tab at bottom of LightPath tool window. ⑦ Select DMs to guide fluorescent light to detectors. DMs are selectable by clicking mirror icon. A. Selecting Excitation DM B.
  • Page 62 Lambda series(3) （Scan setting） Setting the wavelength ⑦ Set “Band Width” and “Step size” in [Spectral setting]. Bandwidth Recommend：15nm or so. When the image is dark, enlarge bandwidth. Step Size Recommend :5nm or so. Small step size leads to accurate spectrum. ⑨...

  • Page 63: Unmixing

    Processing: Unmixing⑴ Blind Unmixing ～by setting the number of dyes~ ① Press the button to switch ② ③ to “Viewer mode”. ② Select [Analysis] in [Tool Window] ④ menu. [Analysis] Tool Window appears. ④ ③ Select [Single process] mode, press the [Post Processing] button.

  • Page 64: Unmixing（２）Normal Unmixing

    Processing :Unmixing⑵ Normal Unmixing ～by specifying dye data~ ① Press the button to switch ② ③ to “Viewer mode”. ② Select [Analysis] in [Tool Window] ④ menu. [Analysis] Tool Window appears. ④ ③ Select [Single process] mode, press the [Post Processing] button. ⑤...

  • Page 65: Unmixing（3）Spectral Image Unmixing

    Processing : Unmixing⑶ Spectral Image Unmixing ～by specifying dyes~ ① Press the button to switch ② ③ to “Viewer mode”. ② Select [Analysis] in [Tool Window] ④ menu. [Analysis] Tool Window appears. ④ ③ Select [Single process] mode, press the [Post Processing] button.
  • Page 66 Appendix...

  • Page 67: Laser Power Correction (Lpm)

    Laser power correction（LPM） Setting of Laser power correction ① Select [Tools] > [Configuration] > [Preference] tab >[LPM] . ② a. Laser power correction can be used when checked. -Auto: Laser power correction works during scanning. There will be delay before scan starts. -Manual：Laser power correction will not work automatically.

  • Page 68: Font Size / Reset The Layout / Trouble Shooting

    ■Font size setting 1. Open the [Configuration] ＞ [Preference] 2. Select the ”Font size” in [Software] . 3. Font size can be adjusted in3 types. 4. Restarting the software. Font size below cannot be changed Launch display ‒ Title of the software/tool window ‒...

  • Page 69: How To Create Log File

    ■How to create Log file ※When trouble occurred, create log file and make a note when it happened. ※When software is frozen, create log file with remaining software. ※Log file can be created only when logging in with Administrator. 1. Click Start on windows and launch OLYMPUS cellSens FV >...

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