Tissue scan workflow

Purpose

Use the Tissue Scan Workflow to image all or part of your cleared, fluorescently labeled specimen and stitch/compile the image tiles into high resolution 3D image volumes. The workflow walks you through the process of defining image acquisition settings and conducting the scan to image your specimen.

How tissue scanning works

The SLICE system captures a series of individual image tiles from the entire specimen or a selected portion of it, then processes and combines the tiles into a 3D image volume according to your specifications. The tissue scan workflow enables you to easily customize image-volume acquisition settings and preview results before launching the scan to acquire an image of the entire sample.

Before you start

  • Determine the illumination settings in the SLICE Light Sheet panel that provide good illumination of the tissue with minimal or negligent photobleaching. To do this, you will primarily adjust the Emission slider for each color channel to be imaged.

  • Use the Camera Histogram panel and Camera Settings to optimize visualization.

  • (Optional) Move around and view the specimen using the Software Joystick. You may want to place markers on structures/regions of interest.

  • To keep the live image in focus, you may need to adjust the Offset slider for the active light sheet(s) in the SLICE Light Sheet panel as you move the stage to different Z locations within the sample.

  • (Recommended) Prepare for image-volume acquisition by placing markers to define the scan region. Click a marker in the marker toolbar, then click close to, but outside the left, top, right, and bottom (in X/Y) of the specimen or the portion of the specimen that you want to image.

    If you plan to designate the scan/imaging area by image-volume size (in mm) or number of image tiles, move the specimen so that the top left of the desired area for image capture is in the current field of view.

Tissue-scanning procedure

Click Tissue scan workflow on the Acquire ribbon.

The workflow steps are displayed in the Tissue Scanning dialog box that opens.

Commands available in every step of the workflow:

Start over with a new workflow; the settings will revert to those specified in the previous completed workflow.

Prev step Next Step Advance in the workflow or revisit a previous step. Alternatively, you can click the steps listed at the top of the workflow to jump to that step.

View information in the User Guide on completing the current step in the workflow.

  1. Setup

    Choose how you want to define the XY area to be imaged, whether to include a prescan for the presence of tissue, and which color channels to include.

    • Scan type

      Decide on a method for defining the XY area of the specimen to scan for imaging. Choose one of the following (In step 2, you will define the scan area as per the chosen method):

      • Scan extents of placed markers: (Recommended) Place markers to define the area to be scanned and imaged.

      • Scan defined volume: Define a volume size in mm3 to be scanned and imaged.

        The scan will begin at the current field of view and proceed toward the right and below.

      • Scan X by Y image stacks: Define an area size as a rectangular grid of fields of view to be scanned and imaged.

        The scan will begin at the current field of view and proceed toward the right and below. When the scan is complete, the stage will move back to its starting position.

    • Pre-Scan: Determine where the specimen is present or absent so that "empty" areas will not be imaged.

      Check the box to Exclude sites with no tissue. When checked, BrightSLICE software pre-scans the specified area to identify whether the specimen is present or not. During image acquisition, areas with no specimen are skipped; excluding empty scan sites saves time and storage space.

    • Other Setup

      Multichannel Setup Click Multichannel Setup to set or confirm the color channels you want to acquire in the Multichannel Setup window.

      1. The window opens on the Multichannel Acquires 1-3 tab. Additional color-channels are displayed on the Multichannel Acquires 4-6 tab of the window.

      2. Check the box to Acquire the channels for multichannel image stacks a stack at a time.

      3. Check or clear the boxes to Acquire channel 1 (2 or 3) on the Multichannel Acquires tabs to indicate which color-channels to capture in the scan.

      Status Dialog Setup Open the Status Dialog Setup window to specify how frequently the image acquisition status will update during the scan. Be aware that updating the status will add time to the image acquisition process.

  2. XY settings

    Specify the XY dimensions of the region to be imaged and the desired overlap between individual image tiles.

    1. Specify the region to be scanned and imaged. The information requested depends on which Scan type was selected in the previous step.

      • Marker extent settings displays when Scan extents of placed markers was chosen as the Scan type in the Setup step. The Region Definition window opens with instructions to place markers on the left, top, right, and bottom of the area to be scanned and imaged.

        • Click to place markers following the onscreen instructions. When all four markers are placed, a blue box appears around the area you have specified.

          • If you placed markers on the right/left and top/bottom of the specimen before starting the workflow, you can quickly jump to their locations in the Macro View window by clicking Go to, then clicking the marker in the Macro View window. Once the marker is in the current field of view in the Main window, click on or near the original marker to place a new marker outside the edge of the tissue.

          • Use the Software Joystick or the field move arrows on the Move ribbon to navigate to the appropriate portion of your specimen and click to place the markers. if you want to scan and image the entire specimen, be sure to locate the farthest edges of the tissue by moving the stage in X, Y, and Z orientations as needed to find it periphery. Place markers outside the edge of tissue.

          • When the four markers have been placed, the area designated for the scan will be indicated with a shaded box.

        • Use Previous Scan Area Use the scan area used in the previous scan.

          Note that the previous scan area is available for the current software session only. If you close and re-open BrightSLICE or switch to a different user profile, the scan area is not retained.

        • Reset Remove any markers that were placed; you will be prompted to start over and place markers to designate the area to be scanned and imaged.

      • Set the Scan volume settings (displays when Scan defined volume was chosen as the Scan type in the Setup step):

        • Position the stage so that the top left field of view to be imaged is displayed in the live-camera feed. Move the stage if necessary using the Software Joystickor the field move arrows on the Move ribbon. The scan will begin at the current field of view and proceed toward the right and below.

        • Type the Width and Height settings (in mm) into the open boxes.

        • Use Previous Scan Area Use the scan area used in the previous scan.

          Note that the previous scan area is available for the current software session only. If you close and re-open BrightSLICE or switch to a different user profile, the scan area is not retained.

      • Choose Grid settings (displays when Scan X by Y image stacks was chosen as the Scan type in the Setup step):

        • Position the stage so that the top left field of view to be imaged is displayed in the live-camera feed. Move the stage if necessary using the Software Joystickor the field move arrows on the Move ribbon. The scan will begin at the current field of view and proceed toward the right in the X orientation and below in Y.
        • Type in the number of fields of view (X, then Y) in a rectangular grid to include in the scan.

    2. (optional) Click Open Macroview in the Tools section to open the Macro View window.

      (This button is not shown if you chose Scan X by Y image stacks as the Scan type.)

      In the Macro View window, the selected scan area is represented by a blue box and a dashed white line shows the field of view displayed in the main BrightSLICE window. Any markers that were placed will be displayed but the live camera feed is not displayed.

    3. Set the image-tile overlap for image acquisition by typing the desired value into the Overlap percentage box.

      Overlap percentage: Type in the desired amount; we recommend:

      • 10% when there are well-defined structures across the entire imaging area; this helps enable accurate stitching.

      • 15% or more when the imaging area has homogeneous regions that do not include easily distinguishable structures.

      The display lists the number of pixels that will be trimmed or that represent the percent overlap (calculated from the Overlap percentage entered). These values represent the trim/overlap prior to stitching, the final values are likely to vary somewhat from these estimates.

      BrightSLICE assembles image tiles captured from each field of view into composite images/image stacks according to the position of the microscope stage when the image tile was captured.

      If Stitching XYZ is selected (as recommended) when you stitch/compile the image tiles, BrightSLICE registers visible features in the individual image tiles and montages them into a single image stack. At least 10% overlap is required to facilitate this process. We recommend including 10–15% overlap.

      • Once the individual image tiles are registered and montaged, the overlap is trimmed down the center. The amount trimmed from each image tile is likely to be slightly more or less than half the overlap, depending on how much the stitching process repositioned the image tiles.

      • Stitching adjusts for minor discrepancies in stage position that can be caused by factors including thermal drift, sample movement inside the cuvette, and stage-movement inaccuracy.

        If stage-movement inaccuracy occurs, a red warning message is displayed on the bottom right of the main BrightSLICE window .

      If you do not employ stitching (Stitching XYZ is NOT selected), the pixels trimmed from each image tile will match the values displayed in the workflow.

    4. Blending (not recommended at this step): Minimizes the appearance of tiling by averaging the pixel intensities where image tiles meet/overlap.

      1. Check the box to Enable blending

      2. Confirm or specify the Blend width in pixels; to change the blend width, enter a different number of pixels in the box.

        We recommend 5 px blend-width.

  3. Z settings

    Specify the top and bottom of the region to be imaged.

    It is helpful to open the Z-meter from the Workspace ribbon when setting the top and bottom of the area to be scanned and imaged.

    1. Move the stage to approximately center the area to be imaged in XY using either:

      The X,Y control arrows of the Software Joystick

      The Macro View window: click Go to, then click the center of the area to be imaged to jump to that location.

    2. Choose how you want to define the Z dimension for imaging, then specify the top and bottom of the region to be imaged as follows.

      • User measured—visually set the top and bottom of the region to be scanned and imaged.

        1. Navigate to the top or bottom of the area to be imaged and adjust the light sheet offset if needed to see a clear, crisp structure. Click Set in the workflow window. The Z location will be recorded.

        2. Repeat for the other Z-dimension (top or bottom).

        3. Enter the desired Distance between planes in microns (Z step). With the 5x illumination objective, the Z resolution of the SLICE system is ~8 µm, we recommend choosing a value that is approximately half that value, i.e., 4–5 µm to capture detailed structures in the specimen.

          The number of focal planes in stack is calculated and displayed based on your selections.

      • Manual input: Enter the requested information in Stack options:

        • Desired height: total stack height in microns of the image volume you want to acquire.

        • Distance between planes: the desired distance between planes in microns (Z step). With the 5X illumination objective, the Z resolution of the SLICE system is ~8 µm, we recommend choosing a value that is approximately half that value, i.e., 4–5 µm to capture detailed structures in the specimen.

          The number of focal planes in stack is calculated and displayed based on the stack options that you select.

    3. Refractive Index Compensation: leave unchecked in this step.

  4. Sheet selection

    Choose whether to use the current illumination settings for imaging (recommended) or enable split between light sheets to illuminate the left side of the tissue with the left light sheet and the right side with the right light sheet.

    Set the light-sheet illumination settings for imaging the specimen by choosing either:

    • Use current channel settings: (Recommended) Use the SLICE Light Sheet settings saved for the enabled color channels in the Multichannel Control panel.

      For most sample, we recommend using both light sheets together.

    • Enable split between light sheets feature: Specify settings to use the left light sheet to illuminate the right side of the sample and the right light for the left side of the sample—this information cannot be saved in the Multichannel Control panel. If this option is selected, the input settings override any settings for the color channel stored in the Multichannel Control panel. Note that when split mode is selected, the SLICE Light Sheet dialog will reflect this with a green ball on the Enable Split button and the green ball indicating that either Enable Left or Enable Right light sheet is active, depending on the position of the stage.

      • The threshold is the location in the X dimension where the illumination will switch from the left to the right side. You can type a coordinate into the box or choose one of the buttons to set the threshold:

      • Center of region of interest The center of the current region of interest is used as the threshold.

      • Center of tissue You will set the left and right extents of the region to be imaged and the threshold is set at the center of the markers.

      • Center of current live image The threshold is the center of the current field of view.

    Choosing to use one or both light sheets for imaging

    You can illuminate the sample with one or both light sheets (left and right) throughout the tissue scanning/imaging process. The approaches have a trade-off between minimizing imaging artifacts and achieving maximum resolution. Illuminating with both light sheets together helps reduce striping or banding artifacts that represent shadows cast by opaque material in the sample; this is common in light-sheet microscopy of cleared tissue and leads to signal loss in the horizontal direction. The SLICE system can mitigate such permanent signal loss by providing complementary illumination from opposite directions and can correct striping-pattern artifacts using a destriping algorithm. While differences in scattering and attenuation between the two light sheets may slightly degrade resolution, in well-cleared samples, the reduction in striping artifacts generally outweighs this minimal change in resolution.

  5. RI Compensation

    Specify the type of refractive index compensation to include: none, Z-orientation (recommended), or X, Y, Z RI compensation.

    Choose from the Refractive index (RI) compensation options:

    • No offset maps: No RI compensation is applied.

      Click Next Step. You will advance to step 7.

    • Z offset maps (Recommended): The same RI compensation is applied at specific Z positions across all XY positions in the sample. In our experience, this is is sufficient to obtain clear, crisp images of cleared brain tissue from mouse.

      Once selected, click Next Step. You will define the points for the Z-offset map in the next step of the workflow.

    • X, Y, Z offset maps: RI compensation can be defined and applied differently in X, Y, and Z orientations. This mode takes the longest to set up, but may be beneficial for some samples.

      • Bounds of map: If selected, indicate the region the offset map will cover using one of the following:

        Use region of interest The region defined for the scan/imaging area in earlier step of the workflow is used for the offset map. This is the easiest way to define the bounds of the offset map.

        Specify with markers You will be prompted to indicate the X,Y, Z offset map bounds by placing markers onscreen

      • Domain Spacing: Type in values that indicate

    RI compensation: what it is and how it works

    Refractive index (RI) compensation adjusts the offset of the light sheet(s) to mitigate optical distortions that occur when light passes through materials with different RIs and causes blurring artifacts. In deep tissue light sheet imaging, image quality can suffer at greater tissue depths because the light sheet illumination plane is slightly deflected so that it no longer perfectly aligns with the imaging plane of the SLICE microscope. BrightSLICE software addresses this problem through RI compensation, which offsets the light sheet to match the shifted image plane. This can restore image sharpness, even deep within the tissue. For cleared samples with a uniform RI in the XY orientation, we recommend choosing Z offset maps to compensate; this typically enables sharp focus throughout a 1-cm thick cleared mouse brain. For more complex tissues that exhibit heterogeneous RI values in the XY orientaiton as well as in the Z orientation, BrightSLICE software provides the option to create an X, Y, Z offset map. When enabled, this option performs precise, location-specific adjustments, helping to ensure high-quality imaging even in more complex samples.

    In order to use RI compensation, you optimize the Offset value for the light sheet(s) that will be used for image acquisition at different areas in the specimen to create data points; this is repeated for each color channel included in the tissue scan. For a Z offset map, it is sufficient to make data points at the same X-Y location in multiple Z planes; for an X, Y, Z offset map, data points must be created at different locations in all three orientations. These RI data points are used to create a grid of light-sheet offset values that form the RI compensation map. When RI compensation is enabled and you direct BrightSLICE software to Use interpolation map, the light-sheet offset for any location in the specimen is interpolated from the nearest points in the grid to adjust the light sheet(s) to compensate for RI differences as the sample is scanned and imaged.

  6. RI Map Points

    Optimize the light sheet offsets for each color channel and save as data points to create an RI compensation map. You will also save your imagining settings for each color channel and enable interpolation from the RI compensation map you create.

    Z offset map: Follow these instructions if you chose Z offset maps in the previous step

    1. Setup

      1. You may need to uncheck Use interpolation map in the workflow dialog or in the RI Compensation Maps panel.

      2. Select one of the color channels that will be included in the tissue scan from the Channel drop-down menu.

        You will create an RI compensation map for each color channel separately.

      3. (If necessary) In the RI Compensation Maps panel, click Delete All to remove any existing data points in the map.

      4. Illuminate the sample with the color channel selected in the previous step; use either the left or right light sheet using controls in the SLICE Light Sheet controls.

    2. Move to a field of view in the tissue with clear, defined structures and optimize visualization of the tissue:

    3. Add a data point for the bottom of the tissue to the RI compensation map as follows:

      1. Move to a Z-position near the bottom of the tissue, but away from the edge of the tissue. If needed, move to a position in XY with complex, non-uniform details to focus on.

        • Adjust the Offset for the active light sheet in the SLICE Light Sheet controls to optimize the appearance of details in the tissue.

        • Using the other light sheet, adjust the Offset to see the same details clearly.

        • Toggle between the left and right light sheets, adjusting the Offset of the active light sheet. The goal is to clearly see the same structural details in the tissue, using either the right or the left light sheet for illumination.

      2. Add the data point for the bottom of the tissue to the RI compensation map using one of the following methods:

        • In the RI Compensation Maps panel, click Add Z.

        • In the Tissue Scanning Workflow panel, click Add Data Point.

        After adding the data point, it will be listed in the RI Compensation Maps panel.

    4. Add a data point for the top of the tissue to the RI compensation map.

      Move to a position near the top of the tissue, again finding a defined structure to focus on, and repeat the process to refine the offsets of the left and right light sheets.

    5. Check the effectiveness of RI compensation with the two data points as follows:

      1. Check the box to Use interpolation map; do this either in the Tissue Scanning workflow window or in the RI Compensation Maps panel. When you move the stage in the Z direction, BrightSLICE will change the light sheet offset based on linear interpolation between the RI compensation data points.

      2. With both light sheets enabled, navigate through the tissue in the Z direction. Verify that the details in the tissue remain crisp and clear throughout all Z positions. If not, you can add additional data points.

      3. Save your settings for the color channel in the Multichannel Control panel; click Save Blue/Green/Red. Current settings from the following panels will be saved:

    6. Repeat for the other color channels that will be used for the tissue scan.

      • Be sure to uncheck Use interpolation map in the workflow dialog or in the RI Compensation Maps panel before starting with the next color channel.

      • Click Delete All to remove data points added for the previous color channel before adding data points for the next color channel.

      • When steps c–e are complete, remember to save your settings for the color channel in the Multichannel Control panel by clicking Save Blue/Green/Red.

    X,Y, Z offset map: instructions in progress

  7. Sample acquire (optional, but recommended)

    Acquiring an image of a large image can be time consuming; we recommend that you conduct a sample image acquisition to test the settings before starting a full tissue scan. You can scan a sample of the specimen to help you evaluate the effects of your settings on image acquisition in this optional workflow step.

    • Check the box to Acquire sample image.

    • To skip this step, do not check the box and click Next Step to continue the workflow.

    To conduct a sample image acquisition, check the Acquire sample image checkbox and follow the steps delineated in the sample Acquire section of the workflow window:

    1. Position the specimen so that the area of interest for the sample acquire is onscreen.

    2. Select a scan size from the dropdown menu; 2 x 2 grid is often a sufficient area for the sample.
      Type in the number of Z-planes you want to include; 100 planes is typically more than enough for the preview.

    3. Start Scan Click to start the sample scan. A progress window will be displayed while images are captured.

    4. When scanning is complete, the sample image appears in the Main window and the Tissue Scan Preview dialog box opens.

      Review the sample-image preview and indicate how you want to proceed:

      • Compile into single image file: Check the box to compile your sample tissue-scan image tiles into a single image stack, then click Start Processing.

      • Postpone stitching Sample image-tile files are acquired and saved, but are not stitched into a single image stack.

        If desired, you can stitch your sample tissue scan using the Image stitcher (go to Image > Post-Processing).

      • Delete Image Deletes the sample preview image/image tiles.

    5. If needed, modify tissue scan settings and repeat the Sample Acquire in this workflow step to optimize image acquisition.

  8. Filter setup

    In this step, you can choose to apply background subtraction and/or flatfield correction and specify the filter parameters.

    We recommend skipping this step in most cases and instead, applying filters during post-processing (see options on the Image ribbon).

    Background subtraction estimates and removes the background illumination from images using an adaptive, local adjustment of image contrast. This filter can reduce tiling artifacts when the image tiles are processed to create a single image.

    • Check/uncheck the On/Off boxes to indicate which color channels you want to include in the filtering operation.

    • For the color channels enabled (box is checked), indicate a Diameter (µm) that corresponds to the physical scale of the foreground objects you want to preserve in that channel.

      • If this parameter is set too low (small), the centers of larger objects may be removed. If it is set too high, the filtering may not have the desired impact on image quality.

      • This setting may be different for the different color channels.

      • In most cases, this value should be larger than the size of the objects you want to visualize.

    Flatfield correction: Mitigates uneven illumination artifacts in fluorescence imaging using intelligent, image-data based correction algorithms—we recommend using it for fluorescence imaging

    • Check/uncheck the On/Off box for each color channel to indicate whether to apply flatfield correction to that color channel.

  9. Start scanning

    Choose where to store images after acquisition and start the tissue scan.

    1. Indicate image file handling choices:

      Image file location

      Choose a location to save the scan image: Click Browse... and, in the Save As window that opens:

      • Navigate to a storage location on the hard drive of the computer that runs the SLICE microscope and type in a file name for your tissue-scanning results.
      • Select the file format you prefer from the Save as type dropdown menu and click Save. The file path for the scan will be displayed in the workflow.

      Enable background tile copy to remote location: Check the box to copy each acquired image tile a more remote location of your choice.

      Delete source tiles after copying to remote location: Individual image tiles are saved initially in a folder on the computer that runs the SLICE microscope. Check the box to delete these individual files once they have been copied to a remote location (this will free up disk space).

    2. Select options for the scan by clicking or clearing the check boxes.

      Note that, in most cases, we recommend that you choose only Postpone stitching. We recommend that you process the image tiles using the Image Stitcher and Image Filters available on the Image ribbon.

      • Keep image open: Displays the final image in the Image Organizer when the scan is complete.

      • Remove temporary files: Individual image tiles are saved in a folder until they are compiled into the final image montage at the end of the slide scan. Check Remove temporary files to delete the individual files once the image has been compiled (this will minimize required disk space).

      • Preview: Acquires images from each field of view selected for the slide scan, then compiles them into a preview image for review. The preview image opens in the Main window as does the Slide Scan Compile with Preview dialog box.

        (see description of the Preview dialog box in Step 7.c., above.)

      • Postpone stitching: (Recommended) Image-tile files are acquired and saved, but are not stitched (compiled) into the full tissue-scan image.

        Use the Image Stitcher to process your image tiles into a single image of the entire tissue scan area. You an also use the sophisticated Image Filters available in BrightSLICE software. Go to Image > Post-Processing to find and use these tools.

      • Flatfield correction: Mitigates uneven illumination artifacts in fluorescence imaging using intelligent, image-data based correction algorithms—we recommend using it for fluorescence imaging

      • Adv. Stitching XY and Adv. Stitching XYZ: Advanced stitching uses the image-tile overlap to visually align individual image tiles in the X, Y, and Z or X, Y orientations. It results in excellent image-tile alignment and clean stitched images—we recommend using it in most cases.

      • Background subtraction estimates and removes the background illumination from images using an adaptive, local adjustment of image contrast. This filter can reduce tiling artifacts when the image tiles are processed to create a single image.

      • Compression ratio: Use the slider to adjust the compression and reduce the size of the image files. The current ratio is displayed.

        • In most cases, a compression ratio of 10:1 or 20:1 is appropriate.

      • Enable Blending:Blends the edges of each field of view smoothly. Blending involves a slight loss of image detail, in general we recommend stitching instead. If you choose to employ blending, generally, 5-10 pixel Blend width is sufficient.

      • X,Y save resolution: Choose a resolution from the dropdown menu. Reducing the resolution combines signal from adjacent pixels in order to reduce the size of the stitched image. Note that reducing the resolution can negatively impact image quality. The balance between image size and quality needs to be determined based on your experimental needs for the image data.

    3. The Scan Summary lists the settings used for the tissue scan.

      Review the settings before beginning the scan.

      Check the estimated required and available disk space. If they are not sufficient, take steps to create more local disk space and/or remote storage space.

    4. Start scanning Click to begin the tissue scan to image the sample.