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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. [ABSTRACT FROM AUTHOR]

Staining and imaging glial cells in vivo while observing the microvasculature could help understand brain physiology, namely neuronal-glial-vascular communication. Two-photon excitation microscopy provides a means to monitor these interactions at the cellular level in living animals, but the cells of interest must be fluorescent. Injecting dyes intravenously is a rapid and quasi noninvasive method to stain cells in the brain. It necessitates that the dye is soluble in the blood plasma and crosses the blood brain barrier (BBB). We demonstrate here, using two-photon imaging, that sulforhodamine B (SRB) crosses the BBB and stains in vivo, specifically mouse astrocytes. This is confirmed by experiments on primary neurons and astrocytes cultures showing the preferential SRB staining of the latter. SRB is rapidly eliminated from the blood, which allows repeated injections in longitudinal studies. [ABSTRACT FROM AUTHOR]

Until now, the imaging of elastic fibers was restricted to tissue sections using the endofluorescence properties of elastin or histological dyes. Methods to study their morphology in vivo and in situ have been lacking. We present and characterize a new application of a fluorescent dye for two-photon microscopy: sulforhodamine B (SRB), which is shown to specifically stain elastic fibers in vivo. SRB staining of elastic fibers is demonstrated to be better than using elastin endofluorescence for two-photon microscopy. Our imaging method of elastic fibers is shown to be suitable for simultaneous imaging with both other fluorescent intravital dyes and second-harmonic generation (SHG). We illustrate these findings with intravital imaging of elastic and collagen fibers in muscle epimysium and endomysium and in blood vessel walls. We expect SRB staining to become a key method to study elastic fibers in vivo. [ABSTRACT FROM AUTHOR]