Multichannel microspectrofluorometry for topographic and spectral analysis of NAD(P)H fluorescence in single living cells.
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.
D005069:Evaluation Studies as Topic; D005958:Glucosephosphates / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D008832:Microchemistry; D009243:NAD / Q000032:analysis* / Q000378:metabolism; D009249:NADP / Q000032:analysis* / Q000378:metabolism; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D005958:Glucosephosphates; D009243:NAD; D009249:NADP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases