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Mammalian cells in culture (BHK-21, PtK2, Friend, human glia, and glioma cells) have been observed by reflection contrast microscopy . Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle . In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation . Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength . The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm .Refractive indices were found to be higher (1 .38-1 .40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1 .354-1 .368) . In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1 .353 and 1 .368 were found . This was thought to result from a microfilamentous network within the cortical cytoplasm.Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium .KEY WORDS quantitative microscopy reflection contrast microscopy -cortical cytoplasm -interference microscopy " refractive indexThe shape of cells in culture is a result of the subtle interaction between the macromolecular cytoskeletal organization (29), the cytoplasmic membrane, and regions of substrate adhesion . The cytoskeleton is composed of structures that maintain tension as well as flexion (28) . Tensile properties are provided by actin-like filament bundles (22), fine J . CELL BIOLOGY

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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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Single islet cells in monolayer cultures of neonatal rat pancreas were microinjected with fluorescein and scanned topographically by microfluorometry. Fluorescein spread from an injected islet cell directly into neighboring islet cells, and, in the presence of 16.7 millimolar glucose, significantly more islet cells communicated with the injected cell than in glucose-free medium. Islet cells were also microinjected with glycolytic substrates and activators that produced transient changes in cellular levels of reduced pyridine nucleotides-nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate [NAD(P)H]. Changes in NAD(P)H fluorescence were observed in islet cells incubated first for 18 hours in very low glucose concentrations and then in a glucose-free medium and injected with glycolytic substrates and activators; however, little change of fluorescence occurred in adjacent islet cells. In contrast, after adding 16.7 millimolar glucose to the medium, injection of glycolytic substrates and activators produced transient changes in NAD(P)H fluorescence in the injected cell and in neighboring cells.

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