Rapid multichannel microfluorometry for metabolic and spectral studies in single living cells

A multichannel microspectrofluorometer has been developed for operation on two modes, a ‘morphological’ mode for the assay of intracellular fluorochromes (e.g. NAD(P)H) in correlation with topography, and a ‘spectral’ mode for wavelength analysis of natural cell fluorescence. This instrument is based on an electron bombardment silicon camera tube (EBS) operated in conjunction with a multiscaling computer. The total NAD(P)H emission from 2 × 30 μm cell strips can be analysed in real time (32.8 msec frame scan) with a signal-to-noise ratio over 100:1. The metabolic changes in cytoplasmic regions are compared with those in regions comprising cytoplasm + nucleus, where the major contribution may be nuclear (cf earlier studies). The observation of a ‘multilocalized’ and asynchronous metabolic response is facilitated with substrates such as glucose-1-phosphate, associated with a longer lag period before the initiation of fluorescence changes. The latter largely occur in the 440–480 nm region. Fluorescence spectra recorded from intracellular regions are nearly super-posable to the spectrum obtained from NAD(P)H crystals.