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Proceedings Paper

Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy
Author(s): Joe Steinman; Margaret Koletar; Bojana Stefanovic; John G. Sled
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Paper Abstract

This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature.

Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo.

In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

Paper Details

Date Published: 14 March 2016
PDF: 7 pages
Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97121V (14 March 2016); doi: 10.1117/12.2211736
Show Author Affiliations
Joe Steinman, The Hospital for Sick Children (Canada)
Univ. of Toronto (Canada)
Margaret Koletar, Sunnybrook Research Institute (Canada)
Bojana Stefanovic, Univ. of Toronto (Canada)
Sunnybrook Research Institute (Canada)
John G. Sled, The Hospital for Sick Children (Canada)
Univ. of Toronto (Canada)

Published in SPIE Proceedings Vol. 9712:
Multiphoton Microscopy in the Biomedical Sciences XVI
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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