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Journal of Biomedical Optics

Subtraction method for intravital two-photon microscopy: intraparenchymal imaging and quantification of extravasation in mouse brain cortex
Author(s): Pascale Vérant; Raphael Serduc; Boudewijn P. J. van der Sanden; Chantal Rémy; Clement Ricard; Jonathan A. Coles; Jean-Claude Vial
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Paper Abstract

Brain pathologies, including stroke and tumors, are associated with a variable degree of breakdown of the blood-brain barrier (BBB), which can usefully be studied in animal models. We describe a new optical technique for quantifying extravasation in the cortex of the living mouse and for imaging intraparenchymal tissue. Leakiness of the BBB was induced by microbeam x-irradiation. Two fluorescent dyes were simultaneously infused intravenously, one of high molecular weight (fluorescein-labeled dextran, 70 kDa, green fluorescence) and one of low molecular weight (sulforhodamine B, 559 Da, red fluorescence). A two-photon microscope, directed through a cranial window, obtained separate images of the two dyes in the cortex. The gains of the two channels were adjusted so that the signals coming from within the vessels were equal. Subtraction of the image of the fluorescein-dextran from that of the sulforhodamine B gave images in which the vasculature was invisible and the sulforhodamine B in the parenchyma could be imaged with high resolution. Algorithms are presented for rapidly quantifying the extravasation without the need for shape recognition and for calculating the permeability of the BBB. Sulforhodamine B labeled certain intraparenchymal cells; these cells, and other details, were best observed using the subtraction method.

Paper Details

Date Published: 1 January 2008
PDF: 11 pages
J. Biomed. Opt. 13(1) 011002 doi: 10.1117/1.2870083
Published in: Journal of Biomedical Optics Volume 13, Issue 1
Show Author Affiliations
Pascale Vérant, Univ. Joseph Fourier (France)
Raphael Serduc, Univ. Joseph Fourier (France)
Boudewijn P. J. van der Sanden, Institut National de la Santé et de la Recherche Médicale (France)
Chantal Rémy, Univ. Joseph Fourier (France)
Clement Ricard, Univ. Joseph Fourier (France)
Jonathan A. Coles
Jean-Claude Vial, Univ. Joseph Fourier (France)


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