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, Douglas P. Shepherd

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


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.

Original languageEnglish (US)
Article number612
JournalNature communications
Issue number1
StatePublished - Dec 1 2017
Externally publishedYes

ASJC Scopus subject areas

  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology
  • General Physics and Astronomy


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