Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-05-25
2003-03-11
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S461100
Reexamination Certificate
active
06532067
ABSTRACT:
FIELD OF INVENTION
This invention pertains generally to fluorescence spectrum analyzers and more specifically to fluorescence spectrum analyzers for measurement of bioaerosols or other single airborne particles.
BACKGROUND OF INVENTION
Bioaerosols, i.e., airborne microorganisms, have both natural and anthropogenic sources. They are found in the workplace and in homes. High concentrations may occur in or around buildings with defective air handling or air-conditioning systems, in houses with domestic animals, in manufacturing operations in which metalworking fluids are used, in dairy or other operations in which animals are confined, in sites of sludge application, in recycling or composting plants, and in sewage plants. Unlike most common atmospheric aerosols, airborne microorganisms can cause diseases, and, along with other biological (e.g., dust mite allergens) and non-biological (e.g., diesel exhaust particles) aerosols can cause allergies and respiratory problems. Bioaerosols are also feared as potential biowarfare and terrorists agents.
Improved methods for measuring aerosols, particularly bioaerosols, are needed. Presently, methods that measure aerosol size distributions in real time provide almost no information about particle types and are not able to identify specific microorganisms. For most allergens, toxins, or microorganisms, culturing the sample or the use of specific protein or nucleic acid recognition molecules is required.
There are presently several methods under development which, while not able to specifically identify bioaerosol particles, can run continuously and give an indication of the presence of biological aerosols. Several fluorescence particle counter devices that measure the elastic scattering and undispersed fluorescence of single aerosol particles as they are drawn through an optical cell have been developed (see Pinnick et al, 1995; Hairston et al, 1997; Seaver et al, 1999 references, below). These devices have shown promise for differentiating between biological and at least some nonbiological aerosols. However, they hold limited promise for classifying biological aerosols. Rather primitive techniques for measuring the fluorescence spectra of single aerosol particles have also been demonstrated (see Nachman et al, 1996; Chen et al, 1996; and Pinnick et al, 1998 references above). However, these techniques are not capable of measuring single particle spectra with a sufficient signal-to-noise ratio to be useful for classifying micrometer-sized biological particles.
In monitoring harmful bioaerosols a rapid response may be necessary in situations where it would be impractical to continuously run a sampler/identifier (a real time monitor might also suggest when to sample for specific harmful bioaerosols). Furthermore, recognition molecules are not always available for all particle types of interest (new bioaerosols may appear). In addition, some studies of bioaerosol dynamics and reactions (evaporation, growth, agglomeration, mixing, etc.) require real-time monitoring capability. Finally, in searching for or studying intermittent sources of bioaerosols, a rapid response may be advantageous. Despite the significant advancement in capabilities of the techniques referred to above, none of these methods are capable of measuring the fluorescence spectra of single micrometer-sized biological particles with a sufficient signal-to-noise ratio to classify them.
Building a point detector that exploits the intrinsic fluorescence of bioaerosol particles for their detection and classification is technically challenging for several reasons. First, particles of interest may exist as a small concentration in a dominant background. Average fluorescence spectra accumulated for a population of aerosol particles may yield little or no information about the few particles of interest (i.e., single-particle spectra are required). Second, fluorescence signals are weak because single particles contain only a few picograms of material, and only a small fraction of the mass of biological particles consists of fluorophors. Third, particles are generally dispersed nonuniformly in the air (their concentration fluctuations follow the Kolmogorov spectrum of atmospheric turbulence), and they must be detected at random times as they are carried rapidly by a stream of air through an optical cell. Fourth, an optimal detector should excite particles in the ultraviolet where most biological particles (and biological molecules) fluoresce efficiently. Ultraviolet laser sources are costly and have relatively low energy output. Fifth, bioaerosols of interest, including individual particles in bioaerosols, may be complex mixtures. Fluorescence from various components of the mixture may limit the usefulness of classification schemes. If fluorescence emission bands were narrow and the number of possible materials in a single particle were small, then it would likely be possible to solve the inverse problem and determine the materials that contributed to the spectrum. However, the intrinsic fluorescence bands from biological materials tend to be spectrally wide; the primary fluorophors in the majority of bioaerosols fall into only a few broad categories (e.g., the aromatic amino acids, tryptophan, tyrosine, and phenylalanine; nicotinamide adenine dinucleotide compounds (NADH); flavins; and chlorophylls); and the number of possible materials is very large. The differences between spectra of bacteria appear to depend on preparation methods (growth media, type and extent of washing of the samples, etc.) more than they depend on intrinsic variations between well-purified bacteria. Therefore, it will not be possible, except with severely restricted classes of bioaerosols, to identify specific bioaerosols based solely on their fluorescence spectra and their size (as determined from elastic scattering). The extent to which it will be possible to characterize naturally occurring and anthropogenically produced bioaerosols (e.g., group them into a few or even a few tens of categories) is yet unknown, however, it is expected that devices according to the invention described herein will provide highly reliable and rapid bioaerosol fluorescence spectra and particle size.
OBJECTS AND SUMMARY OF INVENTION
Accordingly, it is an object of the present invention to provide a device that is capable of reliable and rapid fluorescence spectrum detection, particle sizing, analysis, and classification of biological aerosols. These and other objects are achieved, at least in part, by an Aerosol Fluorescence Spectrum Analyzer (AFSA) that includes an optical element which transfers light from a particle detection volume in a first focal plane to second focal plane; an aerodynamic flow system to move particles to and through the detection volume; a first trigger laser emitting a beam of wavelength &lgr;
1
and focused in a trigger region through which the particles flow on their way to the detection region; a second trigger laser emitting a beam of wavelength &lgr;
2
aimed in a direction approximately orthogonal to the direction of the first particle detecting beam and focused on the trigger region; a first wavelength-selective photodetector sensitive to light scattered from the trigger region and emitting an output signal in response to light of wavelength &lgr;
1
in a predetermined intensity range; a second a wavelength-selective photodetector sensitive to light scattered from the trigger region and emitting an output signal in response to light of wavelength &lgr;
2
in a predetermined intensity range; a pulsed probe laser which emits a pulse of light centered on the particle detection volume triggered by the logically ANDed output signals of the first and second wavelength-selective photodetectors to emit a pulse of light substantially in the first focal plane but downstream from the particle detection volume; a spectral dispersing element positioned in the second focal plane; and a photosensor connected optically to the spectral dispersing element, triggered by the logically ANDed outputs of the first and second
Chang Richard K.
Hill Steven C.
Pan Young-Le
Pinnick Ronald G.
Adams William V.
Evans F. L.
Kelly Mark
The United States of America as represented by the Secretary of
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