Alain W. Wouters

MICROSPECTROFLUOROMETRIC APPROACH TO THE STUDY OF FREE/BOUND NAD(P)H RATIO AS METABOLIC INDICATOR IN VARIOUS CELL TYPES

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 (δIf) due to microinjection of glucose‐6‐phosphate (G6P) into EL2 ascites cells. Above 510 nm, δIF is identical to the spectrum of free NADH. Below 510 nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured δIF level. The difference between δIf 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 δIF 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.

Speed of sound and temperature in the ocean by Brillouin scattering

A method is described to measure the speed of sound and the temperature in the sea as functions of depth. Backscattered laser light is analyzed with an interferometric spectrometer. The speed of sound at very short acoustic wavelengths is obtained directly from the wavelength shift of the Brillouin scattered light, and the temperature is deduced from the speed of sound together with auxiliary information on depth and salinity. Experiments are described.

Direct method of decoding multiple images.

In certain applications, especially x-ray and gamma-ray photography, pinhole cameras provide one of the only methods of producing sharp images. To increase the image quality, the use of an array of multiple pinholes is found valuable. The resulting blurred image must then be decoded. A particularly direct method of decoding using the original array of pinholes is possible, providing theautocorrelation function of the array is sharply peaked. Preliminary results with representative arrays are discussed.

Microspectrofluorometric Procedures and Their Applications in Biological Systems

The study of intracellular enzyme-substrate reactions has preoccupied biochemists since the latter part of the 19th century. In due time most of the metabolic and biosynthetic pathways were identified and reconstructed from cell extracts and homogenates. These findings represent the “skeleton” of actual dynamic processes in the living cell, which involve the simultaneous operation or interactions of cell organelles and compartments. Recent progress on the photodetection of intracellular pigments and fluorochromes, as well as the latest developments in microscopic-optical designs, allow the in situ kinetic study of such phenomena using optical techniques such as microspectrofluorometry.(1–11) The versatility of these methods is dependent upon the choice of endogenous or exogenous probes, making it possible to evaluate a number of functional parameters.

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.

INTERFEROMETRIC STUDIES OF SPECTRAL LINES IN THE SOLAR CORONA.

A photographic Fabry-Perot interferometer was used to measure the breadth and wavelength of the Fexiv spectral line at 5303 Å in the solar corona, during the eclipse of 7 March, 1970, in Mexico. The observations were consistent with a possible large-scale vortex structure in a bright streamer, in which cooler gases revolved about a hotter core.

The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observation within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.