Multisite topographic microfluorometry of intracellular and exogenous fluorochromes.

Abstract. Microspectro fluorometry combined with microinjection of metabolites allows the monitoring of pathways regulating the energy metabolism, biosynthetic or metabolizing activity of the intact living cell. The topographic scan of ? 100–150 adjacent regions within a cell or the spectral scan of fluorescence from a single cell region is completed within 60 ms. The in situ activity of intracellular enzymes and substrate concentration vs rate relationships are derived from the kinetic analysis of transients (e.g. NAD(P) ? NAD(P)H) elicited by sequential injections with increasing doses of substrate (e.g. glucose-6-P, 6-phosphogluconate). A method of approximation is used to predict the non-linear behavior of whole biochemical systems (e.g. the sum of reactions involved in NAD(P) reduction-reoxidation transients). Approximation to power law kinetics is validated by prediction of system behavior over a 10–100 fold variation in the input concentration of substrate. The NAD-dependent glycolytic chain (faster rise kinetics) and NADPH-dependent shunt pathway (slower rise kinetics) are triggered optionally, by enhanced demand for degradation, synthesis or metabo-lization of exogenous compounds (hydrocarbons, aflatoxins). Compartmentation of NAD(P) pathways is suggested by differences in the reoxidation of cytoplasmic vs nuclear NAD(P)H. The intercellular transfer of molecules can be evaluated by microinjection of fluorescent tracers. The difference spectra ΔI_F due to microinjection of substrate are resolved in terms of bound vs free NAD(P)H (with differentiation of other endogenous and exogenous fluorochromes). On the basis of the above, a tentative scheme of intra- and intercellular dynamics can be developed.