Multichannel microspectrofluorometry for topographic and spectral analysis of NAD(P)H fluorescence in single living cells

Abstract

Starting from a previously described prototype microspectrofluorometer a more versatile apparatus has been developed with rapid optional operation on a topographic mode for the simultaneous multisite evaluation of NAD(P) reduction-reoxidation transients or on a spectral mode for the analysis of natural and exogenous fluorochromes, in single living cells. On the topographic mode, adetailed kinetic analysis of NAD or NAD P-linked dehydrogenases can be made from 50-100 cell points imultaneously via automatic recording of topographic scans upt to 16 times a second, in correlation with microelectrophoretic intracellular inuection of metabolites (e.g. nearly immediate response to glucose 6-phosphate, 20-25 s delay for 6-phosphogluconate). Rapid shifts from topographic to spectral operation make possible the detection of a change in fluorescence intensity at a specific intracellular site and the immediate verification of its nature (NAD(P)H or exogenous fluorochrome) by spectral observations.