Under excitation at 365 nm, the cell fluorescence is mainly due to bound and free NAD(P)H, plus a small contribution from flavins. Resolution is first attempted in the simplest case. i.e. the increase spectrum (AI,) due to microinjection of glucose-6-phosphate (G6P) into EL2 ascites cells. Above 510nm, AI, is identical to the spectrum of free NADH. Below 510nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured AI, level. The difference between AI, and the free NADH spectrum (maximum at 475 nm) yields a spectrum suggestive of bound NADH with maximum at 450 nm. Thus, with free and bound NADH, the entire AI, can be reconstructed, with some assumptions as to the relative quantum yields of the two components. This seems to leave no place for a flavin component.The questions raised by the lack of such a component are answered using a new microspectrofluorometer, which allows correlated monitoring of NAD(P)H and flavins with excitations at 365 and 436 nm, respectively. As detected by excitation at 436 nm, injections of G6P, malate, ADP, and treatments with azide, cyanide O i partial anaerobiosis, all indeed show a redox change of flavins, in the sense of decreased emission. It is understandable, however, that such a change which is not very large even using 436 nm excitation should remain undetected when flavins are excited at 365 nm, i.e. using the tail of their excitation spectrum.In contrast to the increased AI, spectrum recorded in response to injected substrate, the initial spectrum ( I , ) of the cell prior to a metabolic perturbation reveals a third component, even with 365 nm excitation. The position and reactivity of this component shows flavin-like properties. The structural resolution attainable makes it possible to obtain the evaluation of free vs. bound NAD(P)H and flavin fluorochromes in the mitochondria1 and cytosolic compartments of the intact cell.

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SUMMARYTo gain a better understanding of the internalization of rhodamines, vital staining of living cells in situ by two different rhodamines, R110 and R123, was studied by microfluorometry. These dyes differ strongly in their lipophilic properties because of differences in charge distribution. Microspectrofluorometry was used to study the fluorescence emission spectra of R110-loaded cells to determine reliable loading conditions. Cell uptake and cell efflux studies of R110 were performed by numerical microfluorescence imaging. A slower uptake was observed for R110 (14 hr) vs R123 (2 hr), but the R110 efflux was much more rapid (30 min) than that of R123 ( Ͼ 24 hr). Although it appeared in the R110 and R123 co-localization study that R110 was able to accumulate in mitochondria, labeling with R110 was lower than with R123. Our results indicate that, rhodamine 110 in its acid cationic form is able to cross the plasma and mitochondrial membrane and to accumulate in cell compartments as does the cationic rhodamine 123. However, because of its acido-basic properties, R110 should be able to decrease the pH of cell compartments, depending on their ability to regulate pH. In such a model, mitochondrial pH should be more greatly decreased than cytosolic pH, leading to a lower mitochondrial accumulation of R110 than of R123. Surprisingly, these effects, which should affect the energetic state of mitochondria, do not influence cell growth, because no cytotoxic effect was observed.( J Histochem Cytochem 45:403-412, 1997 )

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