Subtraction method for intravital two-photon microscopy: intraparenchymal imaging and quantification of extravasation in mouse brain cortex.

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.