Victor Legallais

A versatile time-sharing multichannel spectrophotometer, reflectometer, and fluorometer☆

A simple turbine-driven multi-wavelength time-sharing apparatus affords flexibility and versatility in optical measurements and serves as a fluorometer, reflectometer, and spectrophotometer for four wavelength channels. As a reflectance fluorometer, nearly simultaneous readout of tissue reflectance and NADH or flavoprotein fluorescence can be obtained. As a transmission spectrophotometer and reflectometer, hemoproteins such as myoglobin, catalase, and cytochrome a + a3 with its copper component may be measured. This instrument offers the advantages of high speed of time-sharing, and simplicity, compactness, and flexibility not found in previous designs. In addition, it causes minimal acoustic and electrical disturbance to the experimental system.

A Rapid and Sensitive Recording Spectrophotometer for the Visible and Ultraviolet Region. I. Description and Performance

A double‐beam recording spectrophotometer has been developed for rapidly obtaining spectra of labile intermediates in biochemical reactions. Light from a monochromator is split into two beams by a chopping mirror and then the ratio of light intensities in two optical paths is measured. This ratio is expressed as percent absorption or converted electronically into units of optical density. Results are recorded on a linear wavelength scale at a maximum rate of 6 mμ per second. A servo system corrects the nonlinearity of the wavelength scale of the quartz monochromator. The noise level corresponds to a change of optical density of 10−4 at 400 mμ with a spectral interval of 3 mμ. The over‐all accuracy on standard solutions (National Bureau of Standards) is about 2 percent. The air‐against‐air zero absorption line varies only 0.004 in optical density from 210 to 650 mμ.

Differential Microfluorimeter for the Localization of Reduced Pyridine Nucleotide in Living Cells

This paper describes a differential microfluorimeter suitable for quantitative measurement and localization of the reduced pyridine nucleotide of the intact cell by means of its fluorescence emission band at 443 mμ. Applications of the method to different types of biological material are described.

Low temperature trapping method for cytochrome oxidase oxygen intermediates.

Abstract The trapping of intermediates in the cytochrome oxidase-oxygen reaction is made possible by a three-step procedure: 1) Oxygenation of the ferrous cytochrome oxidase-carbon monoxide compound at a temperature sufficiently low that no ligand exchange with oxygen occurs (−20 to −30°C); 2) flash photolysis at a temperature sufficiently low that the subsequent reaction with oxygen can readily be measured with a multichannel spectrophotometer; 3) rapid cooling of the reaction in progress to a sufficiently low temperature that no further reaction takes place and detailed optical or electron paramagnetic resonance spectroscopy can be performed. Since the freezing point of the mitochondrial suspensions during oxygenation can be in the range of −20°C, only nondeleterious concentrations of ethylene glycol are employed. The reaction kinetics are observed in the solid state in small-diameter circular tubes suitable for rapid trapping. Efficient optical coupling to the sample tube for transmission spectroscopy and efficient laser photolysis are similarly provided by fiber optics coupling. The method is suitable for studying in detail three compounds of cytochrome oxidase and oxygen formed in the temperature range from −125°C upwards. In addition, the electron transfer processes of the respiratory chain are accurately delineated.

Compensation for Light Source Noise in a Sensitive Dual Wavelength Spectrophotometer

Double beam (dual wavelength) spectrophotometers generally employ alternating, rather than simultaneous, illumination of a single sample with wavelengths λ1 and λ2 to secure large aperture viewing without optical cross‐talk. Although this technique allows the use of phase sensitive detection, which greatly reduces the effect of noise entering the system between light switch and demodulator, it also impairs the systems immunity to lamp noise, as compared to a true differential system, in which λ1 and λ2 simultaneously illuminate the sample. To improve the noise rejection, we have devised a method of compensation which uses a second photomultiplier to monitor the time shared light beam before it enters the sample. The resulting signal is normalized and subtracted from the measure signal in a differential amplifier over a bandwidth including frequencies considerably above the switching frequency fc. Details of design are presented together with experimental data showing that greatly improved performance can...

A Variable Capacitor for Measurements of Pressure and Mechanical Displacements; A Theoretical Analysis and Its Experimental Evaluation

A variable capacitor is described for measuring (1) small displacements, (2) small volume changes, and (3) pressure differences. The capacitor consists of a deflectable diaphragm and a fixed electrode. The diaphragm is metallic, plane‐parallel, clamped at the edges, and at ground potential; the electrode, at an a.c. potential, has a plane surface parallel to the undeflected plate across an air gap. For use in displacement measurements, the diaphragms center is deflected by a point contact from a mechanical link to the observed system, or by a uniform pressure load from a fluid link to the system. The fluid link is used also when measuring volume changes and pressure differences. The plate deflection results in a change in the air gap, and thus generates a capacitance signal. This signal is measured by electrical methods.A theoretical analysis of this variable capacitor is presented; sensitivity and alinearity factors for the three uses of the device are derived. The experimental performance shows reasona...

Rapid and Sensitive Spectrophotometry. II. A Stopped‐Flow Attachment for a Stabilized Quartz Spectrophotometer

The stabilization of the light sources and the photocell circuits of a quartz spectrophotometer and the design of suitable stopped flow apparatuses are described. The latter have been designed for 1.6‐ and 10‐mm optical paths and have been shown to give rapid mixing (<0.1 sec). Errors of optical density measurement are diminished by rectangular observation tubes, and errors of extinction coefficients are minimized by a narrow spectral interval (less than 2 mμ from 230–600 mμ). The error of optical density caused by the photoelectric circuits is about 1×10−4 for a response time (10–90 percent) of 1 sec. The application of this apparatus to the measurement of rapid chemical reactions in the visible and ultraviolet regions is described.

Combined Fluorometer and Double‐Beam Spectrophotometer for Reflectance Measurements

An apparatus suitable for recordings of extracellular oxygen tension in terms of the degree of hemoglobin dissociation and intracellular oxidation‐reduction state in terms of the fluorescence level of mitochondrial and cytoplasmic reduced pyridine nucleotide has been constructed. A time sharing sequence permitting light and dark intervals for reflectance fluorometry and two flashes of different wavelengths for reflectance spectrophotometry operates at a repetition frequency of 60 cps. Fixed filters are used in fluorometry for excitation and emission and continuously variable interference filters are used in spectrophotometry.

A dual-wavelength spectrophotometer and fluorometer using interference filters

Abstract A special dual-wavelength instrument employing interference filters, together with a high-intensity excitation and sensitive readout fluorometer, has been constructed for the simultaneous observation of fluorescence and absorbancy changes in suspensions of mitochondria. The apparatus is appropriate for measuring these parameters in a 1 cc volume of a suspension of rat liver mitochondria at a concentration of approximately 1 mg of protein per milliliter. The sensitivities are adequate to determine the fluorescence/absorbancy ratios to an accuracy of 2%, as indicated by experimental trials. The apparatus permits an identification and characterization of flavoprotein signals from mitochondrial suspensions.

Sensitive dual wavelength densitometer with suppression of the support introduced background optical density

Abstract An instrument for the direct photometric scanning of zones is described. The method involves the alternative flashing of two beams of light through the support. The wavelengths used are the peak of absorption and a selected reference at one of the sides of the absorption curve of the colored compound in the zone. This technique allows an inherent suppression of the background optical density of the support while maintaining full sensitivity and specificity. This results in a performance increase of about two orders of magnitude when sensitivity or precision are compared with single wavelength operating instruments.