Application of time-resolved infrared spectral photography to chemical kinetics.

We discuss the application of time-resolved infrared spectral photography to the determination of the time-dependent reactant and product concentrations in the simple chain reaction of chlorine atoms, generated by pulsed photolysis, with ethane-Cl(2) mixtures. The technique and experimental results are discussed in terms of the limitations and advantages of the method for general kinetic studies in the microsecond and submicrosecond time domain.

Nondegenerate multichannel four-wave mixing spectraof nitric oxide: two-photon absorption resonance from low-lying excited states.

We show how multichannel nondegenerate four-wave mixing spectroscopy can be applied for fast acquisition of moderate-resolution two-photon resonance spectra of gas-phase samples, specifically, nitric oxide. We present the first results reported for broadband spectra, which can be obtained with a single laser pulse from a number of low-lying vibroelectric states of this molecule, specifically, the A(2)Sigma(nu = 0), A(2)Sigma(3), D(2)Sigma(0), and C(2)II(0). The two-photon resonant susceptibility is normalized to the known nonresonant susceptibility of N(2), permitting comparison with previous data obtained by scanning techniques.

Single-particle velocity and trajectory measurements of microscale inhomogeneities in unstable airflows.

In unstable airflows, it is found that often the seed particles used to make laser Doppler anemometry measurements of fluid velocities follow complicated trajectories in even very small fringe volumes. A technique to obtain maximum particle-trajectory information from the scattered light signal is described. The use of this technique to measure fluid-motion characteristics in regions of unstable flow down to 10-microm scale inhomogeneities is described.

Rapid digitized storage of seed-particle-track images in microscopically inhomogeneous flows.

The light scattered from submicrometer seed particles illuminated by a chopped cw argon laser is imaged on a silicon-intensified-target vidicon. A simple means of rapidly sampling the vidicon output, to store the records of particle-track images on a digital oscilloscope, is described. The digitized records so obtained are suitable for automated processing. Observations of microscale velocity fluctuations, recorded at frame times down to 10 msec, are reported and found to be consistent with earlier results from interferometric laser anemometry. The technique is applicable to turbulent-flow studies over a wide range of velocities and distance scales.

Broadband single-pulse CARS spectra in a fired internal combustion engine.

The first known broadband single-pulse coherent anti-Stokes Raman scattering (CARS) measurements within the cylinder of a firing internal combustion engine are reported. Postcombustion temperature and carbon monoxide concentration are probed with 1-mm(3) spatial resolution and 10-nsec temporal resolution. Space- and time-resolved measurements, as presented here, are shown to be necessary for the study of fluctuating systems such as engines.

Angular distribution of coherent Raman emission in degenerate four-wave mixing with pumping by a single diffraction coupled laser beam: configurations for high spatial resolution.

This paper contains a discussion of the relation between the angular distributions of the coherently emitted radiation and the angular distribution of the incident beams in a degenerate four-wave mixing experiment, when two of the input beams are derived from the same laser source. The objective of the discussion is to obtain beam configurations that permit the use of such spectroscopic techniques when good spatial resolution is required so that the conventional collinear phase matching geometry is inappropriate. Two different configurations are indeed obtained and experimentally demonstrated that have, in the worst direction (longitudinal), spatial resolution of the order of 1 mm, with beam apertures comparable with those used in the standard collinear experiments and without the complicating requirement of two distinct pump beams.

Space- and time-resolved coherent anti-Stokes Raman spectroscopy for combustion diagnostics.

The technique of coherent anti-Stokes Raman spectroscopy can be used to obtain spectra in or near the reaction zone of combustion systems with spatial resolution on the order of 0.1 mm and time resolution on the order of 10 nsec. The latter is achieved by recording the entire spectrum generated during a single laser pulse by a broadband Stokes beam, simultaneously, on a vidicon, whereas the former is achieved by a simplified variant of the crossedbeam phase-matching technique taking advantage of the radiation intensity distribution from a pump laser using an unstable resonator structure.

Raman spectra of heme a, cytochrome oxidase-ligand complexes, and alkaline denatured oxidase.

We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.

A phase-shift fluorometer using a laser and a transverse electrooptic modulator for subnanosecond lifetime measurements.

We described a simple phase-shift fluorometer using continuous laser excitation. The laser enables the use of a transverse mode electrooptic modulator with a half-wave retardation voltage of about 200 V (in contrast to many kilovolts of longitudinal modulators) at frequencies up to 100 MHz. The modulated fluorescence signal is detected, after passing through a double monochromator, by a photomultiplier tube feeding a radio frequency (RF) tuned amplifier. THE RF phase is then determined by phase-sensitive detection using a double balanced mixer with the reference obtained from a PIN photodiode-turned amplifier combination which detects light split off from the main exciting beam. The laser and double monochromator allow the observation of modulated Raman solvent and Rayleigh scatterin, which are convenient for determining the zero reference phase.

A moving spot high resolution scanning microspectrometer using focused laser illumination: illustrations with acriflavin stained sephadex beads and human fibroblast nuclei.

We describe a high resolution moving spot scanning microspectrometer, capable of absorption or fluorescence detection, using focused laser illumination which is moved over the sample by rotating the laser beam direction prior to focusing. This rotation is achieved by reflecting the beam from mirrors mounted on bending mode piezoelectric transducers which, when bent by an applied voltage, cause the mirrors to rotate. The images of optically thin samples are analyzed by considering the convolution of the focused spot intensity distribution with the absorbance of a uniformly stained spherical particle. This analysis is verified experimentally with data from acriflavin stained Sephadex beads. Data from acriflavin-Feulgen stained human fibroblasts indicate that the efficiency of this type of nuclear staining is about 2 to 3 dye molecules incorporated per 100 nucleotide pairs. Quantitative data on fading of acriflavin fluorescence in stained fibroblasts indicate that fading is negligible in the time required to record the microscope images.

Reverse transcriptase activity per virion for avian myeloblastosis virus and Rauscher murine leukemia virus.

We have measured reverse transcriptase enzyme activity per virus particle for samples of avian myeloblastosis virus (BAI strain) and murine leukemia virus (RAUSCHER) USing the synthetic template poly(rC)-oligo(dG). Absolute virus concentrations were determined directly by laser beat frequency spectroscopy. Enzyme activity per virion was determined from the slope of the activity plotted as a function of virus concentration. With this reverse transcriptase assay, the minimum activity (expressed as picomoles of dGTP incorporated/virion per hour) is estimated at (28.1 +/- 4.2) X 10(-7) for avian myeloblastosis virus and (1.1 +/- 0.2) X 10(-7) for murine leukemia virus. The sensitivity of this assay, which is determined by the level of incorporated radioactivity measurable above background, is 2.5 X 10(-4) virions for avian myeloblastosis virus (with dGTP specific activity of 8.9 Ci/mmol) and 88 X 10(-4) virions for murine leukemia virus (with dGTP specific activity of 6.52 CI/mmol). These results show that although reverse transcriptase assays can obviously be used to measure relative virus concentrations of equally purified samples of the same virus, they can be very misleading when used to compare the concentrations of different virus species.

Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy.

We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.

Electrophoretic mobilities of RNA tumor viruses. Studies by Doppler-shifted light scattering spectroscopy.

We have used laser beat frequency light scattering spectroscopy to measure, at several pH values, the electrophoretic mobilities of purified avian myeloblastosis (AMV), murine leukemia (MuLV), murine mammary tumor (MuMTV), and feline leukemia (FeLV) viruses. The mobilities of these viruses are similar at pH greater than or equal to7 (-2.7 to -3.2 X 10(-4) (cm/sec)/(V/cm). The isoelectric points of MuLV and AMV are apparently less than pH 3, whereas for FeLV the data could be interpreted to indicate an isoelectric point between 3 and 5. Using a Debye-Hückel model to describe the interaction between electrolytes and virus, we show that our values for the mobility of MuMTV, obtained in ionic strength 0.005, are consistent with the values of Sarkar et al. ((1973), Cancer Res. 33, 2283), obtained in ionic strength of 0.10. This model is then used to calculate surface charge densities. In terms of the density of charged groups, the RNA tumor virus envelope is not very different from the erythrocyte membrane.

Quantitation of RNA tumor viruses by spectroscopy of density gradient gels.

We have developed a system for virus particle quantitation based on the measurement of the optical absorbance of stained viruses which first have been banded at their buoyant density in an equilibrum 24 to 53% (wt/wt) sucrose density gradient, then fixed in position in the gradient by photopolymerizing an acrylamide-riboflavin mixture in the sucrose, and finally stained and destained. Using plasma from mice infected with leukemia virus (Rauscher) or chickens infected with avian myeloblastosis virus (BAI strain) or suitable controls, we have shown that this technique specifically detects RNA tumor viruses. By using virus stock solutions for which the absolute concentrations were determined by laser beat frequency spectroscopy, we have calibrated the absorbance of the viral bands in terms of virus particle concentration. Using 0.8-ml gradients gels (4 by 45 mm) we can detect as low as 2 x 10(7) viral particles with Coomassie blue staining and 6 x 10(6) viral particles with a more sensitive staining procedure using amido black.

Hydrodynamic diameters of RNA tumor viruses. Studies by laser beat frequency light scattering spectroscopy of avian myeloblastosis and Rauscher murine leukemia viruses.

The diffusion constants of avian myeloblastosis virus (AMV) and murine leukemia virus (MuLV) (Rauscher) suspensions in buffer and in 30% sucrose were determined by laser beat frequency light scattering spectroscopy at a series of temperatures ranging rom 5 to 25 degrees. By the use of the Stokes-Einstein equation, the following hydrodynamic diameters are calculated at 20 degrees: MuLV, 154 plus or minus 3 nm in sucrose and 145 plus or minus 7 nm in buffer; AMV, 144 plus or minus 3 nm in sucrose and 138 plus or minus 4 nm in buffer. While the diameters measured in buffer were temperature independent, the diameters measured in sucrose decreased by about 20% as the temperature was raised from 5 to 25 degrees. The concentration of virus particles in the suspensions ranged from 10 7 to 10 9 particles/ml. The absolute particle concentrations are estimated within plus or minus 30% by determining the dilution needed to reach a concentration sufficiently low that the particle number fluctuation contribution was comparable to that of the interference scattering. Particle weights of 3.9 x 10 8 daltons for MuLV and 4.0 x 10 8 daltons for AMV were calculated from the diffusion constants and from our own experimentally determined sedimentation coefficients. From these particle weights and the hydrodynamic diameters of the viruses, we calculated the per cent of the hydrodynamic volume of the viruses which could be freely penetrated by water, viz., 57% for AMV and 69% for MuLV.

Comparison of the resonance Raman spectra of carbon monoxy and oxy hemoglobin and myoglobin: similarities and differences in heme electron distribution.

With 441.6-nm excitation, which is near the Soret band, we observe that the resonance Raman spectra of hemoproteins contain not only the bands between 650 and 1700 cm-1 which arise from vibrations of the conjugated macrocycle, but also bands below 650 cm-1, some of which involve vibrations of the iron pyrrole-nitrogen bonds. The spectra of the oxygen and carbon monoxide complexes of both myoglobin and hemoglobin are sufficiently similar to those of low spin met derivatives, that the electronic distribution on the heme for both ligands can be interpreted as that of a low spin ferriheme. This agrees with an earlier interpretation, by others, of comparative optical absorption spectra and, as pointed out previously, would imply that in the complex the ligands are bound as O2- and CO-. However, band frequencies and relative intensities differ somewhat between the carbon monoxide and oxygen complexes of the same protein, which indicates differences between the details of the pi-electron distributions in the corresponding complexes.