Light scattering calculations exploring sensitivity of depolarization ratio to shape changes. II. Single rod-shaped vegetative bacteria in air.

In article I of this series, calculations and graphs of the depolarization ratio, D(Theta,lambda)=1-/, for light scattered from an ensemble of single-aerosolized Bacillus spores using the discrete dipole approximation (DDA) (sometimes also called the coupled dipole approximation) were presented. The Sij in these papers denote the appropriate Mueller matrix elements. We compare graphs for different size parameters for both D(Theta,lambda) and the ratio R34(Theta,lambda)=/. The ratio R34(Theta,lambda) was shown previously to be sensitive to diameters of rod-shaped and spherical bacteria suspended in liquids. The present paper isolates the effect of length changes and shows that R34(Theta,lambda) is not very sensitive to these changes, but D(Theta,lambda) is sensitive to length changes when the aspect ratio becomes small enough. In the present article, we extend our analysis to vegetative bacteria which, because of their high percentage of water, generally have a substantially lower index of refraction than spores. The parameters used for the calculations were chosen to simulate values previously measured for log-phase Escherichia coli. Each individual E. coli bacterium appears microscopically approximately like a right-circular cylinder, capped smoothly at each end by a hemisphere of the same diameter. With the present model we focus particular attention on determining the effect, if any, of length changes on the graphs of D(Theta,lambda) and R34(Theta,lambda). We study what happens to these two functions when the diameters of the bacteria remain constant and their basic shape remains that of a capped cylinder, but with total length changed by reducing the length of the cylindrical part of each cell. This approach also allows a test of the model, since the limiting case as the length of the cylindrical part approaches zero is exactly a sphere, which is known to give a value identically equal to zero for D(Theta,lambda) but not for R34(Theta,lambda).

Light scattering calculations exploring sensitivity of depolarization ratio to shape changes. I. Single spores in air.

Calculations of the depolarization ratio, D(Theta, lambda) = 1 - (S22)/(S11), for light scattered from an ensemble or cloud of single aerosolized spores in air were studied using the discrete dipole approximation (DDA), sometimes also called the coupled-dipole approximation. Here S(ij) is the appropriate Mueller matrix element for scattering angle Theta and wavelength lambda. The effect of modest shape changes on D(Theta, lambda) was determined. The shapes compared were prolate ellipsoids versus right circular cylinders joined smoothly to end caps consisting of hemispheres of the same diameter as the cylinder. Using the same models, the graphs of (S34)/(S11) versus angle were compared with those for D(Theta, lambda). The latter shows sensitivity to length in some cases we examined, while (S34)/(S11) does not. Size parameters and optical constants suggested by measurements of Bacillus cereus endospores were used. An ensemble of spores was modeled with prolate spheroids. The results of this model were compared with results of a model using the same size and optical parameters, but for capped cylinders. The two models produced distinguishably different results for the same parameters. In calculations for all the graphs shown, averaging over random orientations was performed. Averaging over size distributions similar to those from experimental measurements was performed where indicated. The results show that measurements of D(Theta, lambda) could be quite useful in characterizing the shape of particles in an unknown aerosol and for distinguishing between two likely shapes, but not to reconstruct the shapes from the graphs alone without additional information.

Sampling and quantitative analysis of clean B. subtilis spores at sub-monolayer coverage by reflectance fourier transform infrared microscopy using gold-coated filter substrates.

A study was conducted to determine the concentration dependency of the mid-infrared (MIR) absorbance of bacterial spores. A range of concentrations of Bacillus subtilis endospores filtered across gold-coated filter membranes were analyzed by Fourier transform infrared (FT-IR) reflectance microscopy. Calibration curves were derived from the peak absorbances associated with Amide A, Amide I, and Amide II vibrational frequencies by automatic baseline fitting to remove most of the scattering contribution. Linear relationships (R2 >or= 0.99) were observed between the concentrations of spores and the baseline-corrected peak absorbance for each frequency studied. Detection limits for our sampled area of 100 x100 microm2 were determined to be 79, 39, and 184 spores (or 7.92 x 10(5), 3.92 x 10(5), and 1.84 x 10(6) spores/cm2) for the Amide A, Amide I, and Amide II peaks, respectively. Absorbance increased linearly above the scattering baseline with particle surface concentration up to 0.9 monolayer (ML) coverage, with the monolayer density calculated to be approximately 1.17 x 10(8) spores/cm2. Scattering as a function of surface concentration, as estimated from extinction values at wavelengths exhibiting low absorbance, becomes nonlinear at a much lower surface concentration. The apparent scattering cross-section per spore decreased monotonically as concentrations increased toward 1.2 ML, while the absolute scattering decreased between 0.9 ML and 1.2 ML coverage. Calculations suggest that transverse spatial coherence effects are the origin of this nonlinearity, while the onset of nonlinearity in the baseline-corrected absorption is probably due to multiple scattering effects, which appear at a high surface concentration. Absorption cross-sections at peaks of the three bands were measured to be (2.15 +/- 0.05) x 10(-9), (1.48 +/- 0.03) x 10(-9), and (0.805 +/- 0.023) x 10(-9) cm2, respectively. These values are smaller by a factor of 2-4 than expected from the literature. The origin of the reduced cross-section is hypothesized to be an electric field effect related to the surface selection rule.

Improved dispersion of bacterial endospores for quantitative infrared sampling on gold coated porous alumina membranes.

An improved method for qualitative and quantitative sampling of bacterial endospores using Fourier transform infrared (FT-IR) microscopy on gold-coated porous alumina membranes is presented. Bacillus subtilis endospores were filtered onto gold-coated alumina membranes serving as substrates. Studies in the mid-infrared (MIR) region revealed the characteristic bacterial absorption spectrum at low surface concentration, while scanning electron microscopy (SEM) images of the same samples provided precise calculation of the surface concentration of the bacterial endospores. Under the conditions of study, the average concentration of endospores was determined to be 1356 +/- 35 spores in a 100 x 100 mum(2) area, with a relative standard deviation of 0.0260. Examination of ten random spots on multiple substrates with FT-IR microscopy apertured to the same area gave an average relative standard deviation of 0.0482 in the signal strength of the amide A band at 3278 cm(-1). An extinction cross-section in reflection of sigma(ext) = (7.8 +/- 0.6) x 10(-9) cm(2)/endospore was calculated for the amide A band at the frequency of its peak absorbance, 3278 cm(-1). The absorption cross-section of the amide A band in reflection is estimated to be sigma(abs) approximately (2.10 +/- 0.12) x 10(-9) cm(2)/endospore.

In situ measurement of the infrared absorption and extinction of chemical and biologically derived aerosols using flow-through photoacoustics.

In an effort to establish a more reliable set of optical cross sections for a variety of chemical and biological aerosol simulants, we have developed a flow-through photoacoustic system that is capable of measuring absolute, mass-normalized extinction and absorption cross sections. By employing a flow-through design we avoid issues associated with closed aerosol photoacoustic systems and improve sensitivity. Although the results shown here were obtained for the tunable CO2 laser waveband region, i.e., 9.20-10.80 microm, application to other wavelengths is easily achievable. The aerosols considered are categorized as biological, chemical, and inorganic in origin, i.e., Bacillus atrophaeus endospores, dimethicone silicone oil (SF-96 grade 50), and kaolin clay powder (alumina and silicate), respectively. Results compare well with spectral extinction measured previously by Fourier-transform infrared spectroscopy. Comparisons with Mie theory calculations based on previously published complex indices of refraction and measured size distributions are also presented.

Two-dimensional multiwavelength fluorescence spectra of dipicolinic acid and calcium dipicolinate.

Dipicolinic acid (DPA) and the Ca2+ complex of DPA (CaDPA) are major chemical components of bacterial spores. With fluorescence being considered for the detection and identification of spores, it is important to understand the optical properties of the major components of the spores. We report in some detail on the room-temperature fluorescence excitation and emission spectra of DPA and its calcium ion complex and provide a comparison of the excitation-emission spectrum in a dry, wet paste and aqueous form. DPA solutions have weak, if any, fluorescence, with increased fluorescence when the DPA is dry. After exposure to a broad source UV light of the DPA, wet or dry, we observe a large increase in fluorescence with a maximum intensity emission peak at around 440 nm for excitation light with a wavelength of around 360 nm. There is a slight blueshift in the absorption spectra of UV-exposed DPA from the unexposed DPA solution. CaDPA in solution shows a slight fluorescence with increased fluorescence in the dry form, and a substantial increase of fluorescence was observed after UV exposure with an emission peak of around 410 nm for excitation around 305 nm. The detailed excitation-emission spectra are necessary for better interpretation of the fluorescence spectra of bacterial spores where DPA is a major chemical component.

Rapid optically based measurements of diameter and length for spherical or rod-shaped bacteria in vivo.

The application in light scattering of the Mueller matrix ratio (S34)/(S11) for determining average particle size is extended to a large size parameter range for spherical or randomly oriented rod-shaped particles such as micro-organisms. It is shown that combining the graph of this ratio with a Coulter counter measurement of particle volume gives results in agreement with microscopic measurements. Thus this combination provides a method to measure particle diameter and width simultaneously in real time for elongated particles such as bacteria, which are measured in vivo with this method. An approximate empirical formula is developed to estimate the motion of the extrema in the graph of the oscillating matrix ratio as size changes occur. This formula is also shown to be consistent with wavelength changes.

Two-dimensional angular optical scattering patterns of microdroplets in the mid infrared with strong and weak absorption.

Two-dimensional angular optical scattering (TAOS) patterns of droplets composed of a mixture of H2O and D2O are detected in the mid infrared. First, a lens is used in the Abbé sine condition to collect a small solid angle of light, where the scattering pattern matches well numerical simulations based on Mie theory. Next, TAOS patterns from droplets spanning a large (approximately 27pi sr) solid angle are captured simultaneously at two wavelengths. The effects of absorption are evident in the patterns and are discernible without the need for curve matching by Mie theory.