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Nat Commun. 2017 Sep 20;8(1):612. doi: 10.1038/s41467-017-00514-7.

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

Author information

1
Department of Physics, Colorado State University, Fort Collins, CO, 80523, USA.
2
Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO,, 80045, USA.
3
Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO,, 80045, USA.
4
Pediatric Heart Lung Center, University of Colorado Anschutz Medical Campus, Aurora, CO,, 80045, USA.
5
Department of Ophthalmology, University of Colorado Anschutz Medical Campus, Aurora, CO,, 80045, USA.
6
Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA.
7
Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO,, 80045, USA. douglas.shepherd@ucdenver.edu.
8
Pediatric Heart Lung Center, University of Colorado Anschutz Medical Campus, Aurora, CO,, 80045, USA. douglas.shepherd@ucdenver.edu.
9
Department of Physics, University of Colorado Denver, Denver, CO, 80217, USA. douglas.shepherd@ucdenver.edu.

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.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.

PMID:
28931809
PMCID:
PMC5606987
DOI:
10.1038/s41467-017-00514-7
[Indexed for MEDLINE]
Free PMC Article

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