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.

Procedure for Quick RI Compensation Map Setup

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

  1. Enable the color channel you want to map in the Multichannel Control panel.

  2. In the RI Compensation Maps panel, do the following:

    1. Uncheck the box to Use Interpolation Map.

    2. Select Z as the Interpolation map type.

    3. For the Channel to view, click the channel number (12 or 3) that corresponds to the channel selected in the Multichannel Control panel (step 1).

    4. Click Delete Z or Delete All if there are Z values displayed in the white box.

  3. Adjust the Offset for each light sheet to obtain crsip, clear images and click Add Z:

    1. Adjust left sheet offset: In the SLICE Light Sheet panel, disable the Right light sheet and enable the Left light sheet.

      Adjust the Offset in the Left Sheet Controls pane until the image becomes crisp and clear.

    2. Adjust right sheet Offset: Disable the Left light sheet and enable the Right light sheet.

      Adjust the Offset for the right light sheet to obtain a crisp, clear image.

    3. Verify that the light sheets are coplanar: Toggle between left, right, and both light sheets and adjust the Offset(s) as needed to produce a clear, uniform image with all illumination options.

    4. Click Add Z in the RI Compensation Maps panel. This adds one RI compensation point to the map for the color channel.

  4. Move along the Z-axis using the joystick until the image loses focus and repeat step 3.

    1. Repeat the Offset adjustments in the SLICE Light Sheet panel at the new, blurry Z position for both light sheets (steps 3.a–c).

    2. Once the field of view is in focus with all illumination options, click Add Z in the RI Compensation Maps panel to add another point to the map.

  5. Move around the sample in the Z-orientation and add more points as needed following the steps above, then check the box to Use Interpolation Map.

    Based on the Z map created and interpolating between the RI compensation points, BrightSLICE software will auto-adjust the light sheet Offset to mitigate effects of RI differences in the microscopy sample.

X Y Z Interpolation map setup

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.