Stephen Holler

Real-time measurement of fluorescence spectra from single airborne biological particles

Improved real-time methods for characterizing airborne biological particles are needed. Here we review our efforts in developing techniques for measuring the laser-induced fluorescence (total and spectrally dispersed) of individual airborne particles, and describe our present system, which can measure fluorescence spectra of single micrometer-sized bioaerosol particles with good signal-to-noise ratios. We demonstrate the capability of this system by showing measured spectra of a variety of airborne particles generated in the laboratory from road dust, ammonium sulfate, Bacillus subtilis and other bacteria prepared under various conditions, allergens, cigarette smoke, and chicken-house dust. These spectra illustrate the capability of the system to distinguish between some biological and nonbiological aerosols, and among several types of laboratory-generated biological aerosols. We suggest improvements needed to make our system field portable.

Single-shot fluorescence spectra of individual micrometer-sized bioaerosols illuminated by a 351- or a 266-nm ultraviolet laser

Reproducible fluorescence spectra of individual 2- to 5-microm -diameter biological aerosol particles excited with a single shot from a Q -switched laser (266 or 351 nm) have been obtained with highly improved signal-to-noise ratios. Critical to the advance are crossed diode-laser trigger beams, which precisely define the sample volume, and a reflecting objective, which minimizes chromatic aberration and has a large N.A. for collecting fluorescence. Several allergens (red oak, meadow oat pollen, paper mulberry pollen, and puffball spores) have different fluorescence spectra. Bacillus subtilis fluorescence spectrum deteriorates at high 266-nm incident intensity. Dry riboflavin particles illuminated with a 351-nm light exhibit a new 420-nm fluorescence peak that grows nonlinearly with laser pulse energy.

Two-dimensional angular optical scattering for the characterization of airborne microparticles

Two-dimensional angular optical scattering (TAOS) is recorded for several particle shapes and configurations. A lens is used to collect a large solid angle of the light and transform the angular profile into a planar distribution according to the Abbé sine condition. Qualitative agreement is found between experiment and theory for the TAOS from spheroids having the same aspect ratio but different sizes. A distinctive irregular island structure is observed in the TAOS from clusters of Bacillus subtilis spores and polystyrene latex spheres. The density per solid angle of these islands is found to increase with cluster diameter.

High-speed, high-sensitivity aerosol fluorescence spectrum detection using a 32-anode photomultiplier tube detector

We employ a 32-anode photomultiplier tube (PMT) in a fluorescence detection system and demonstrate its ability to record broad fluorescence spectra at frame rates in excess of 1.4×103u200aHz, which is 56× faster than the frame rate of an intensified charge coupled device detector. The multi-anode PMT has single-photon detectable sensitivity. A new data acquisition and processing system for the multi-anode PMT, together with the system-controlling software, has been developed. The performance characteristics of the fluorescence detection system, including the data rate capability, dynamic range, signal-to-noise ratio, and crosstalk among the different anodes, are reported. The 32-anode PMT and acquisition system are suitable for a real-time, field-portable, multichannel optical analyzer.

Spontaneous emission spectra from microdroplets

The emission spectrum from a fluorescent microdroplet is compared with theory for the first time. The spectrum of oriented surfactant molecules on a levitated microdroplet is found to be in good agreement with semiclassical theory of the interaction of whispering gallery modes with excited molecules. The orientation of these surface entities is estimated through this comparison.

Two-dimensional angular optical scattering patterns as droplets evolve into clusters

Remarkable changes in the two-dimensional angular optical scattering (TAOS) pattern are observed when an acoustically levitated water droplet with inclusions evolves into a dry aggregate. The emergence of polystyrene latex sphere inclusions near the droplet surface has a clear effect on the TAOS. A speckle pattern in the TAOS replaces the continuous contours associated with Mie scattering from a smooth droplet. Once the contours break up, this speckle pattern persists as the levitated droplet evaporates, making the transition from a droplet with inclusions to a dried aggregate. The initial emergence of the speckle pattern is observed for an inclusion volume fraction near 8%.

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.

Classification of single-particle two-dimensional angular optical scattering patterns and heuristic scatterer reconstruction

Two-dimensional angular optical scattering (TAOS) is an experimental technique by which the azimuth- and polar-angle-dependent intensity patterns of monochromatic light scattered by isolated material particles are collected within a limited aperture. TAOS patterns are analyzed with the ultimate goal of estimating the class of shapes and the set of complex refractive indices to which the scatterer may belong. The implemented heuristic reconstruction procedure integrates experimental techniques and methods of data analysis. It consists of three stages: (1) TAOS patterns are classified by cluster analysis and classification is validated by principal components analysis, (2) prior information about the shape and refractive index of the particles is acquired from analytical electron microscopy and drives the computation of TAOS patterns by a T-matrix code, and (3) cluster analysis is repeated by including one computed pattern at a time and ranking it according to distance to a cluster. As a result, some simulated scatterers are found, the computed patterns of which merge with clusters of experimental patterns. Whereas the reconstruction of each individual scatterer from the available TAOS pattern would not have been feasible, possible scatterer classes are identified. The investigated material is tire dust from laboratory abrasion tests of aerodynamic size <10 μm.

Optical scattering (TAOS) by tire debris particles: preliminary results.

Tire debris particles from low severity laboratory wear tests have been investigated by the TAOS optical scattering facility at Yale University. The incident wavelength is 532 nm. After the TAOS event some particle samples have been imaged by a scanning electron microscope and microanalyzed. The TAOS intensity patterns recorded within a solid angle in the backward sector have been processed by cluster analysis and compared with the patterns computed by a T-matrix code. Preliminary agreement has been found between TAOS data and the particle models (size, shape, refractive index). The purpose of the investigation is to obtain signatures of the material, based on its TAOS pattern.

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.