Refractive index (RI) compensation maps

Purpose

Use the RI compensation maps window to create maps of light sheet offset positions at different locations in the tissue sample. Find the Offset setting on the SLICE Light Sheet panel panel.

About refractive index compensation

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.

A. Choose the RI compensation map type

Specify the type of refractive index compensation

  • 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 sufficient to obtain clear, crisp images of cleared brain tissue from mouse.

  • X, Y, Z offset maps: BrightSLICE software has the capability of using an interpolation map with offset settings in different XY locations, in addition to Z locations. This takes a significantly longer amount of time to set up and for the large majority of samples, is not necessary.

    The process is similar to setting up a Z Interpolation map in multiple locations in XY.

    For help with setting up an XYZ interpolation map, please contact our Technical Services team by completing the Request Support form.

B. Making a Z interpolation map

In most cases, setting up an RI compensation map for imaging different tissue depths, a "Z Interpolation map", is sufficient for achieving high quality images.

  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 panel.

  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 panel 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 Scan 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. If you are setting up to image an entire sample using the Tissue Scan Workflow, you need to create a Z Interpolation map for each color channel that will be that will be imaged.

    • When you have added data points and verified their effectiveness, remember to save your settings for the color channel in the Multichannel Control panel by clicking Save Blue/Green/Red.

    • Uncheck Use interpolation map in the Tissue Scan workflow panel 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.