Application of two dimensional and three dimensional imaging techniques to frog microvessels in-situ loaded with fluorescent indicators

A limitation of the photomultiplier tube (PMT) based quantitation systems in fluorescence microscopy is the lack of spatial information. In this paper, a cooled CCD based imaging system for quantitative imaging in fluorescence microscopy and two applications in single capillaries is presented. Two dimensional (2D) ratio imaging revealed uniform distribution of intracellular ion concentration in the endothelial cells along a capillary. Optical sectioning and image restoration showed that the observed non-homogeneity of indicator loading into the microvessel wall was due to variation in cell thickness.