Refractive index (RI) compensation maps

Purpose

Use the RI compensation maps window to create maps of offset positions at different locations in the tissue sample. The Offset setting can be found on the SLICE Light Sheet controls window.

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.

 

More information coming soon

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