Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

Abstract

A fluorescent pH indicator in conjunction with confocal microscopy, was used to map intracellular pH in a variety of cells and tissues with high spatial resolution. The new pH-sensitive fluorescent probe SNARF-1 was excited with the 488 nm band of the argon ion laser of a Bio-Rad MRC-500 confocal microscope. Ratio images were created with pixel-by-pixel division, with the intensity of these images representing a function of pH, that is independent of dye concentration, photobleaching or path length. Cell cultures of rat aortic smooth muscle were loaded with 20 μm{cyrillic} SNARF-1/AM for 20 min at 37°C. Intracellular pH levels were calibrated in situ by treatment of each cell with nigericin ..