INTRACELLULAR PH MAPPING WITH SNARF-1 AND CONFOCAL MICROSCOPY .1. A QUANTITATIVE TECHNIQUE FOR LIVING TISSUES AND ISOLATED CELLS

A fluorescent pH indicator in conJunction with confocal microscopy, wa s 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 func tion of pH, that is independent of dye concentration, photobleaching o r path length. Cell cultures of rat aortic smooth muscle were loaded w ith 20 muM SNARF-1/AM for 20 min at 37-degrees-C. Intracellular pH lev els were calibrated in situ by treatment of each cell with nigericin ( 20 mum) in solutions of known pH. The cytosolic pH of the majority of cells was uniform, however, pH gradients were evident between the cyto sol and nuclear regions, indicating the ability of this technique to m ap intracellular and intraorganelle pH. Rat C6 glioblastoma spheroids were cultured then loaded with SNARF-1/AM at 10-degrees-C for 90 min. The pH values were calibrated in vitro, using SNARF-I acid in buffered solutions of known pH. Ratio images of the bisected spheroids showed a marked gradient in pH from the outer cells compared with central nec rotic cells. The degree of involvement of acidification in muscle fati gue was investigated by simultaneously determining force generation an d intracellular pH in individual fibres of an intact rat muscle.The in vestigation was performed during a stimulation protocol which induced significant fatigue in the force response of the muscle. The fatigue p rotocol induced little change in cytosolic pH in the fibres. We show t hat the use of SNARF-1, in conjunction with confocal microscopy is a p owerful technique for accurately mapping pH within single cells, multi cellular tissues and intact organs, as well as for accurately recordin g dynamic changes in pH.