Burt V. Bronk

Silver metal induced surface enhanced Raman of bacteria

Bacteria treated with sodium borohydride serve as a nucleating substrate for reduction of silver ions that forms a rough silver metal coating surrounding the microorganism. Corroding away the silver reveals that the general shape and size of the bacteria remain intact. Intense Surface Enhanced Raman spectra of the coated bacteria are measured. The Raman spectra of four different bacteria: Escherichia coli, Acinetobacter calcoaceticus RAG-1, Pseudomonas aeruginosa YS-7, and Bacillus megaterium are all quite similar, suggesting that the spectra are selective and sensitive to a specific molecular species, which dominates the spectra and which is present in all (these) bacteria.

Internal and scattered electric fields in the discrete dipole approximation

The calculated scattering matrix elements and interior electric fields for a dielectric sphere based on the discrete dipole approximation (DDA) are compared with the exact Mie solution for homogeneous and composite spheres. For homogeneous spheres the macroscopic average field produced at each DDA dipole site by the incident field combined with the field from all DDA sites is found to be approximated by the factor (n12+2)/3 multiplied by the Mie macroscopic field, where n1 is the refractive index. This holds to surprising accuracy, considering the finite wavelength and the small number of dipoles used in the DDA approximation. The approximate relation is most accurate near the center of the sphere and least accurate at the interface. The relation also holds for electric fields within composite spheres, with poorer agreement near each interface, where the refractive index changes. The dependence of this relation on parameters of the model is examined.

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.

Real-time detection and characterization of individual flowing airborne biological particles: fluorescence spectra and elastic scattering measurements

Real-time methods which is reagentless and could detect and partially characterize bioaerosols are of current interest. We present a technique for real-time measurement of UV-excited fluorescence spectra and two-dimensional angular optical scattering (TAOS) from individual flowing biological aerosol particles. The fluorescence spectra have been observed from more than 20 samples including Bacillus subtilis, Escherichia coli, Erwinia herbicola, allergens, dust, and smoke. The S/N and resolution of the spectra are sufficient for observing small lineshape differences among the same type of bioaerosol prepared under different conditions. The additional information from TAOS regarding particle size, shape, and granularity has the potential of aiding in distinguishing bacterial aerosols from other aerosols, such as diesel and cigarette smoke.

Surface-enhanced Raman spectroscopy of bacteria coated by silver

We present a novel method to measure Raman spectra from whole bacteria cells by using Surface Enhanced Raman Scattering (SERS). We deposit a silver coat on Escherichia coli and Bacillus megaterium bacteria and measure strongly enhanced (greater than 400,000 fold) and highly reproducible Raman spectra. The spectra are rich but not overly congested, as the surface enhancement is selective to the precise chemical nature of the biochemical molecules, and their proximity to the silver particulate matter. The main bands we observe can be associated with peptides and polysaccharides in the cell- wall and its membrane. The spectra from E. coli (a Gram- negative bacterium) and B. megaterium (a Gram-positive bacterium) are similar in their general form, but differ in detail. The spectrum from a commercial yeast extract is vastly different. This approach can be extended to probe the internal chemical environment within bacteria and applied to the identification of micro-organisms also applied to studying other biochemical problems and phenomena, such as biomineralization, heavy metal toxicity, cell-wall structure and others.

Concentration, size, and excitation power effects on fluorescence from microdroplets and microparticles containing tryptophan and bacteria

Our group has been developing a system for single-particle fluorescence detection of aerosolized agents. This paper describes the most recent steps in the evolution of this system. The effects of fluorophore concentrations, droplet size, and excitation power have also been investigated with microdroplets containing tryptophan in water to determine the effects of these parameters on our previous results. The vibrating orifice droplet generator was chosen for this study base don its ability to generate particles of well- known and reproducible size. The power levels required to reach saturation and photodegradation were determined. In addition, the collection of fluorescence emission was optimized through the use of a UV achromatic photographic lens. This arrangement permitted collection of images of the droplet stream. Finally, the use of a dual-beam, conditional firing scheme facilitated the collection of improved signal- to-noise single-shot spectra from individual biological particles.

Physical perturbation for fluorescent characterization of microorganism particles

The motivation for using response to physical perturbation to classify microparticles came from our previous experiments with Dipicolinic Acid (DPA). DPA as a calcium complex is a major component of bacterial spores, constituting more than 5% of their dry weight. It is not commonly found in other natural products and therefore its presence is indicative of the presence of bacterial spores. Previous schemes utilizing the presence of DPA to detect these spores have relied on fluorescence which occurs when lanthanide metals (e.g., terbium) are added to a solution where the presence of DPA is to be determined. We have recently demonstrated that changes in the fluorescence of DPA can be stimulated without the addition of such reagents. Thus after exposure to UV light, a substantial increase of fluorescence emitted by DPA solutions with a peak at 410 nm occurs for excitation light with wavelength less than approximately 305 nm.

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).

Fluorescence of dipicolinic acid as a possible component of the observed UV emission spectra of bacterial spores

Dipicolinic acid (DPA) and the Ca2+ complex of DPA (CaDPA) are well-known and are major chemical components of bacterial spores. DPAs native fluorescence is very weak and is thought to be completely masked by the fluorescence of tryptophan when this compound is presented. Thus fluorescence related to DPA in spores is assumed by many authors to be completely absent. AWe show that the fluorescence of CaDPA is substantial for excitation between about 290 nm and 310 nm with emission peaking near 400 nm. This emission is at the long wavelength tail for emission form tryptophan. We examine whether the emission of CaDPA could contribute to the total emission spectrum when bacterial spores are present in an aerosol, for excitation wavelength in the neighborhood of 310 nm. In this report we present measurements of fluorescence excitation and emission for CaDPA and compare them with that of DPA and tryptophan.

Two-dimensional angular optical scattering patterns of aerosol particles in the mid-infrared: measurements designed to obtain particle absorption

Real-time and in-situ detection and discrimination of aerosol particles, especially bio-aerosols, continues to be an important challenge. The technique labeled TAOS (Two-dimensional Angular Optical Scattering) characterizes particles based upon the angular distribution of elastically scattered light. The detected angular distribution of light, labeled the TAOS pattern, depends upon the particle’s shape, size, surface features, and its complex refractive index. Thus, the absorptive properties of a particle affect the TAOS pattern. Furthermore, we expect to use this change in the TAOS pattern, which occurs when the particle absorption band includes the input wavelength, to characterize the strength of the absorption. Thus, by illuminating a particle in the mid-infrared wavelength range, high frequency vibrational modes that are unique to the aerosol can be reached and quantified. Spherical aerosol particles (in the diameter range of 50-60 micrometers) were generated via a droplet generator and illuminated by an Interband Cascade (IC) laser designed to emit in the 3-5 micrometers wavelength range. The TAOS pattern of the elastically scattered light was detected with an InSb-focal-plane-array infrared camera.