TY - JOUR
T1 - Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy
AU - Ryan, Duncan P.
AU - Gould, Elizabeth A.
AU - Seedorf, Gregory J.
AU - Masihzadeh, Omid
AU - Abman, Steven H.
AU - Vijayaraghavan, Sukumar
AU - Macklin, Wendy B.
AU - Restrepo, Diego
AU - Shepherd, Douglas P.
N1 - Funding Information:
We thank Drs Randy Bartels and Mike Taormina for helpful conversations on the use of remote focusing in light-sheet microscopy, Dr Viviana Gradinaru for helpful conversations on tissue clearing and fluorescent labeling, and Drs Ethan Hughes and Radu Moldovan for the use of Zeiss CLARITY and SCALE objectives. G.S. and S.A. acknowledges funding from NIH R01 HL68702. E.G. acknowledges funding from NIH DC014253, DC012280 and NIH DC00566. W.M. acknowledges funding from NIH NS25304. S.V. acknowledges funding from NIH R01 DC008855. O.M. acknowledges funding from Brightfocus Foundation. D.R. acknowledges funding from NSF MRI 1337573, NIH DC014253 and NIH DC00566. D.P.R. and D.P.S. acknowledge startup funding from University of Colorado Denver and NIH AG053690.
PY - 2017/12/1
Y1 - 2017/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-017-00514-7
DO - 10.1038/s41467-017-00514-7
M3 - Article
C2 - 28931809
AN - SCOPUS:85029643770
VL - 8
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 612
ER -