Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy

Duncan P. Ryan, Elizabeth A. Gould, Gregory J. Seedorf, Omid Masihzadeh, Steven H. Abman, Sukumar Vijayaraghavan, Wendy B. Macklin, Diego Restrepo and Douglas P. Shepherd ()
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Duncan P. Ryan: Colorado State University
Elizabeth A. Gould: University of Colorado Anschutz Medical Campus
Gregory J. Seedorf: University of Colorado School of Medicine
Omid Masihzadeh: University of Colorado Anschutz Medical Campus
Steven H. Abman: University of Colorado School of Medicine
Sukumar Vijayaraghavan: University of Colorado Anschutz Medical Campus
Wendy B. Macklin: University of Colorado Anschutz Medical Campus
Diego Restrepo: University of Colorado Anschutz Medical Campus
Douglas P. Shepherd: University of Colorado School of Medicine

Nature Communications, 2017, vol. 8, issue 1, 1-10

Abstract: Abstract Optical tissue clearing has revolutionized researchers’ ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.

Date: 2017
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DOI: 10.1038/s41467-017-00514-7

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