Patent Application
20050243307
This invention pertains generally to aerosol analyzers and more specifically to multi-spectral optical analyzers for the real-time detection and classification of biological and non-biological particles.
There is a growing need for the real-time detection and classification of airborne biological and non-biological particles for indoor and outdoor air quality monitoring, as well as, for the early detection of deliberate releases of biological agent aerosols on the battlefield and in urban environments, such as through a terrorist act. Airborne microorganisms can cause diseases and the real-time monitoring of hospitals, manufacturing operations, sewage plants, animal production houses, and recycling or composting plants can help prevent harmful exposure of microorganisms in these environments. Further detection of particle-sized impurities can benefit quality and production, for example, in chip manufacturing processes. There is a further need to monitor the exposure of humans to organic carbon particulates in urban environments. The majority of organic carbon particulates encountered in the environment from such sources as diesel emissions and burning vegetation contain polycyclic aromatic hydrocarbons which are carcinogenic to humans.
The ability to provide a real-time warning of a bio-aerosol attack is a challenging problem. Present state-of-the-art real-time biological point detection involves sensing the auto-fluorescence of biological particulates via the excitation of endogenous fluorophores and by measuring the elastic scattering of particles. There are two primary limitations of the present art. First, is the inability to sense in a reliable manner low levels of cellular and spore type particles in singlet form and protein toxin and viral aggregates that fall below a stated level, e.g. a couple of microns. Second, is the inability to classify biological particles in a manner that produces a low false alarm rate when set for a threat level that corresponds to a low-level attack.
The recent delivery of parcels containing weapons-grade Anthrax, or other biological particles, and the release of these spores into the U.S. postal system demonstrated a spore-type threat delivered primarily in singlet form. Other potential attacks related to terrorist activity could be the release of biological agents into public areas, facilities and government complexes. The dispersal methods employed would determine in what form the biological agent would be packaged. In other words, the dispersal methods employed would determine what size aggregate was generated, or if single cellular or spore-type agents were generated.
For example, with a crop duster or portable crop sprayer, one could assume that a respirable range of aggregates larger than a two to ten (2-10) micron in diameter would be the predominant size generated primarily because of the water droplet diameter that these types of atomizers produce. However, for a covert release in a facility or public area, one could expect a dry powder release or a low output nebulizer could be used that would generate cellular and spore-type agents in single form or viral/protein toxin aggregates that are below 1 micron in size.
U.S. Patent Application Publication No. U.S. 2003/0098422 A1 discloses a method and apparatus for biological particle detection and classification using Mie scattering techniques and auto-fluorescence. Such Application is incorporated by reference in its entirety as if made a part of this present application.
In preparing for all threat scenarios, the ability to detect small viral/protein toxin aggregates and the singlet form of cellular and spore-type agents is required, in addition to, the conventional respirable range aggregate (2-10 micron). A further requirement is the ability to classify biological agents of interest and to separate them from commonly encountered biological particulates such as mold spores, pollens, and other biological cells and spores, as well as, other types of commonly encountered aerosols such as diesel soot and inorganic/organic particulates. Efforts directed at classification of most types of aerosols commonly encountered, as well as the biological agents of interest, will have a direct impact on the false alarm rate of real-time biological agent detection.
The present invention contemplates methods, apparatuses, and systems for detecting and classifying airborne biological and non-biological particles, in real time, based on particle size, density, complex refractive index and auto-fluorescence content. According to the present invention, three physical phenomena are exploited in the detection scheme and involve the interaction of light with an aerosol particle: elastic scattering, absorption, and fluorescence. In addition to these optical phenomena, both a particle's size and density and complex refractive index are determined substantially simultaneously to enhance the particle's detection and identification/classification in real-time.
The present invention is directed to a method for detecting and classifying a single particle comprising illuminating the particle with a light beam having multiple excitation ranges. The particle has measurable and classifiable properties including size and density, complex refractive index, and auto-fluorescence content over different emission ranges. The size of a particle can be determined by measuring the elastic scatter intensity for a specific wavelength(s) and/or by its “time-of-flight” or the time a particle takes to traverse to light beams separated by a known distance upon exiting an accelerating orifice. Density of the particle can be determined by comparing a particle's elastic scatter intensity at one or more wavelengths with the particle's time-of flight. The auto-fluorescence content of the particle is measured by exciting the particle at specific wavelengths and detecting the fluorescence emission from endogenous fluorophores present in the particles of interest. Algorithms applied to classify a particle are based on the relationship of the above parameters to each other.
Further, the present invention is directed to a method for detecting and classifying a particle comprising providing and directing a sample stream containing particles to an optical viewing region and providing a plurality of continuous wave excitation sources, each source emitting a discrete wavelength. A plurality of discrete wavelengths of light from the continuous wave excitation sources is provided to the optical viewing region. Each particle found in the sample stream in the viewing region is illuminated with the excitation sources substantially simultaneously in real-time. Each particle has elastic scattering properties, fluorescence or non-fluorescence emission properties, and dimension and density properties. Light is directed from the viewing region to a plurality of detectors to produce a plurality of signals, and the signals are directed from the detectors to a signal processor to substantially simultaneously measure the elastic scattering properties, the complex refractive index and the fluorescence or non-fluorescence of the particle substantially simultaneously and in substantially real-time. In another preferred embodiment a continuous wave excitation source is used in tandem with a pulsed laser diode with nonlinear crystals to generate second and third harmonic wavelengths.
Still further, the present invention is directed to an apparatus for detecting and classifying a single particle from a sample comprising a plurality of continuous wave excitation sources, each source emitting a discrete wavelength, the wavelengths directed through an optical viewing region and a plurality of detectors to receive the wavelengths directed through the optical viewing region and produce a plurality of signals. A signal processor is in communication with each detector to receive the signal from the detector to substantially simultaneously measure the elastic scattering properties, the complex refractive index and the fluorescence or non-fluorescence of the particle substantially simultaneously and in substantially real time.
a-1f illustrate aerosol sensing configuration block diagrams outlining various embodiments contemplated by the present invention.
a is a schematic representation illustrating a configuration of the present invention having a dual wavelength excitation with two elastic scatter detection channels and one fluorescence detection channel.
b is a schematic representation illustrating a configuration of the present invention having tri-wavelength excitation with three elastic scatter detection channels and two fluorescence detection channel.
c is a schematic representation illustrating a configuration of the present invention having a tri-wavelength excitation with three elastic scatter detection channels and one fluorescence detection channel.
d is a schematic representation illustrating a configuration of the present invention having a dual wavelength excitation with two elastic scatter detection channel.
e is a schematic representation illustrating a configuration of the present invention having a tri-wavelength excitation with three elastic scatter detection channels.
f is a schematic representation illustrating a configuration of the present invention having a single continuous wave excitation and two or three harmonically generated pulsed excitation wavelengths with one fluorescence detection channel and three to four elastic scatter detection channels.
g is a schematic representation illustrating a configuration of the present invention having a single continuous wave excitation and two or three harmonically generated pulsed excitation wavelengths with two fluorescence detection channels and three to four elastic scatter detection channels.
h is a schematic representation illustrating a configuration of the present invention having a single continuous wave excitation and two or three harmonically generated pulsed excitation wavelengths with three fluorescence detection channels and three to four elastic scatter detection channels.
a-f are charted printouts of randomly sampled indoor aerosol particles.
a-f are charted printouts of aerosol waveforms of BG Spores, 0.7 u PSL, and 1.0 u Fluorescent PSL.
a-e illustrate analog signal processing configurations for dual excitation wavelength aerosol sensing.
a is a schematic representation of a single pulse trigger integrator mode of the present invention.
b is a schematic representation of a single pulse trigger integrator mode of the present invention, with pulse duration for long pulse rejection.
c is a schematic representation of a dual trigger pulse integration mode of the present invention, for laser drift correction.
d is a schematic representation of a dual trigger pulse integration mode of the present invention, with pulse duration for laser drift correction, and rejection of extra-long pulses.
e is a schematic representation of a dual trigger pulse integration mode of the present invention, with time-of-flight measurement measured by duration of aerosol travel between two excitation wavelengths and for extra-long pulse rejection.
The present invention relates to enhanced methods, apparatuses and systems for the detection and classification of biological and non-biological particulates in a real-time manner. There are three physical phenomena that are exploited in the detection scheme and involve the interaction of light with an aerosol particle: elastic scattering, absorption, and fluorescence. In addition to these optical phenomena, both a particle's size and density are determined substantially simultaneously. When considering scattering and absorption, the interaction of light with an aerosol particle is indicated by the complex refractive index of the particle material with respect to the medium in which the particle is suspended. The real and imaginary parts of the complex refractive index relate to the particle's refractive index and aerosol absorption index, respectively. This is defined as m=n−ik, where m is the complex refractive index, n is the refractive index and k is the aerosol absorption index. The complex refractive index is a function of the excitation wavelength and is dependent on the chemical composition of the particle. The real part of the complex refractive index provides information concerning the particle's size. A particle's absorptive properties can be measured indirectly by measuring the elastic scattering at two or more excitation wavelengths.
Model calculations of the influence of the complex refractive index on the scattering properties of a particle can be based on the Mie theory of light scattering assuming the particle is not too irregular in shape. The illumination method described herein involves excitation of individual aerosol particles excited one at a time. Using Mie theory, the sensor's response can be modeled and predicted. Discrete particulate counters measure the amount of light scattered into a given angular range from a single particle as it traverses a beam of light. The response R for a given scattering geometry is defined by this scattered light normalized to the excitation beam. For a linearly polarized light beam irradiating a particle from one direction the response is given by
where I1 and I2 are the scattered light intensities polarized parallel and perpendicular relative to the plane of oscillation of the electric vector of the incoming radiation, m is the complex refractive index and x is the dimensionless particle size parameter defined by x=πDp/λ, with Dp being the actual particle diameter and λ the wavelength of the irradiation (1). G(φ,θ) is a geometrical factor related to the specific optical design θ1 and θ2, together with φ1, and φ2, are truncation angles limiting the solid angle in which the scattered light is collected. By looking at this scattering response at two or more wavelengths information concerning the absorptive properties of a particle can be indirectly measured, as well as, its size. This technique can be used as a biological indicator through the appropriate selection of one or more excitation wavelengths that correspond to a peak absorption band for endogenous fluorophores or chromophores. Prediction of the degree of absorption by different types of aerosol particles can be achieved by looking at certain fluorophores or chromophores present in the particle using the following equation
where I0 is the overall intensity of the laser beam, ε is the molar decadic absorption coefficient of the fluorophore or chromophore, C is the fluorophore/chromophore concentration in the particle, and R is the radius of the particle.
With this technique applied properly, a very sensitive response to the absorptive properties of the particle can be observed. The angular collection geometry and the excitation wavelengths used play a major role in the sensor's response. For example, one could observe an almost two orders of magnitude difference in the scattering intensity when comparing a 1 μm non-absorbing particle with a refractive index of 1.4 to a 1 μm absorbing particle with a refractive index of 1.4-0.5 i when excited at 630 nm and collecting backward scatter at 150° to 170°. If an angular collection geometry of 10° to 30° was employed, the intensity difference for the above particle types would be less than a factor of two.
The proper selection of the excitation wavelengths would also permit one to measure auto-florescence due to the presence of endogenous fluorophores within the biological particle. By employing two excitation wavelengths in the following ranges: 266-280 nm and 400-415 nm one can measure spectrally dispersed auto-fluorescence from, for example, the following endogenous fluorophores: aromatic amino acids, NADH, flavins, and chlorophylls.
According to one embodiment, the present invention contemplates the use of a third excitation wavelength that is a nonabsorbing or minimally absorbing wavelength for biological particles and falls into the visible to near-IR part of the electromagnetic spectrum, i.e. one that does not interfere with the fluorescence emission spectrum of the above fluorophores.
Using the three laser excitation approach of the present invention, with excitation in the following ranges 266-300 nm, 350-430 nm, and 600 nm 1.5 μm, one can measure the elastic scattering and absorptive properties of individual particles, as well as, the fluorescence emission of most of the endogenous fluorophores found in biological particles.
Therefore, according to the present invention, by dedicating three detection channels for the measuring the elastic scatter at each of the excitation wavelengths, and dedicating another three detection channels to the detection of the fluorescence emission of aromatic amino acids (300-400 nm), NADH and flavins (420-600 nm) and chlorophyll (600-700 nm) one is able to generate a seven dimensional or greater feature space (particle size, three elastic scattering channels, and three fluorescence channels) and a significantly enhanced classification scheme can be developed that is an improvement over all known detection methods.
According to the present invention, by providing separation of the one of the excitation beams from the other two, or separating all three from each other, the duration time for an individual aerosol particle can be measured as it is accelerated through each of the beams. Assuming a low Reynolds value (no turbulence), a particle's density can be determined from the time it takes the individual aerosol particle to traverse the beams. With the assumption that the velocities of a particle passing through two beams can be expressed as v1,=b1,/Δt, and v2=b2-Δt2 the Stokes equation can be written in the following form:
where v is the velocity, b1, and b2 are the widths of the beams, m is the mass of the particle (for spheres, m=4/3R3p), Δv=v1−v2, Δt is the time the particle takes to traverse two beams, μ is the viscosity of air and a is the radius of the a particle. From this equation, the density p can be calculated for spherical particles.
According to a further embodiment of the present invention, by determining a particle's density and size, as well as measuring the elastic scattering at three wavelengths and fluorescence at three wavelengths ranges, an eight dimensional or greater feature space can be created providing a further means for classifying biological and non-biological particles.
Described herein are seven aerosol sensing configurations which are variations of two or more excitation wavelengths and two or more detection channels. In all seven configurations aerosol is drawn into an optical viewing region at 0.5 to 30 liters per minute and particles are illuminated one at a time with two or more light beams.
Various light collection geometries can be employed with different parameters applied for the different physical phenomena employed. For fluorescence, collection of fluorescence orthogonal to the direction of the light beam is recommended to minimize the effects of stray light on the fluorescence signal(s). For elastic scattering and absorbance collecting near forward scatter and back scatter separately, backscatter alone, side angle scatter alone, and side scatter and back scatter together are optimal collection geometries for separating the scattering component of an aerosol from the absorptive component.
a illustrates a configuration whereby two excitation sources are employed and three detection channels: two for elastic scatter at two different wavelengths and one for fluorescence detection. Aerosol is drawn into the sensor cell 200 through an aerosol nozzle (not shown) and is introduced into an optical viewing region 201. Two excitation sources 100, 105 are employed. The excitation sources 100, 105 can be either a continuous source or modulated at 20 MHz or greater frequency and can be a laser, light emitting diode or some other light emitting device. Excitation source 100 is a longer wavelength than excitation source 105 in one of the two wavelength ranges 350-430 nm and 600-1500 nm. Excitation source 105 is a shorter wavelength than excitation source 100 and emits in one of two wavelength ranges: 266-300 nm or 350-430 nm. Light emitted from these sources are collimated using an aspheric lens 110a and then spatially filtered by focusing the collimated light using another aspheric lens 110b onto a pin hole aperture 115 and then re-collimated using another aspheric lens 110c. Collimated light from both sources can then be introduced to a narrow bandpass filters 120, 125 for removal of unwanted wavelengths emitted from sources 100 and 105 or from auto-fluorescence produced from the optical elements. Collimated light from both excitation sources 100, 105 are introduced to dichroic mirrors 130 and 135 positioned at 45 degrees relative to the collimated light. Dichroic mirrors 130 and 135 provide the means for alignment of the two collimated sources onto the same optical train and also provide the primary means for optical alignment. The two collimated beams can be aligned to fall along the same path or can be separated along the plane orthogonal to the aerosol inlet nozzle so that particle time-of-flight and density measurements can be performed. Dichroic mirrors 130 and 135 also provide additional optical filtering by removing unwanted wavelengths emitted from sources 100 and 105 or from auto-fluorescence of the optical elements. Light exiting dichroic mirrors 130 and 135 is then introduced to a series a beam shaping optics creating a sheet of light at the aerosol nozzle region that is from about 5 to about 300 μ in thickness and a depth of field and beam width that is at least two times (2×) larger than the diameter of the inlet (e.g. aerosol nozzle). In one embodiment, a spherical lens 140 and a cylindrical lens 145 are used to generate the above geometry. In one preferred embodiment of the present invention, a spherical lens 140 and a Powell lens 145 are used.
The two light beams generated from the beam shaping elements 140 and 145 are then introduced into the optical viewing region 201. Particles are illuminated, one at a time, in this region 201 with an aerosol migration time of 100 to 2000 nanoseconds. Light exiting this region in the forward direction is collected using a light trap 215.
In the embodiment illustrated in
Two collector lenses 150, 152 are used in this embodiment. One is used for collecting elastically scattered light emitted by the particle from both of the excitation wavelengths and one is used for collecting fluorescence emission from the illuminated particle. Elastically scattered light at the two excitation wavelengths is then introduced to a narrow bandpass filter element 165. The filter element 165 is comprised of two halves with one region filtering all wavelengths but that desired from source 100 and the other region filtering all wavelengths but that desired from source 105. For the fluorescence channel, filter element 160 is used to filter out all wavelengths except a wavelength range that corresponds to a fluorescence emission for a certain fluorophore or group of fluorophores commonly encountered in a biological or non-biological particle.
Elastically scattered light that has been filtered using filter element 165 is then introduced to a light receiving element (light detector) 210. The light receiving element is a detector array having two or more detector elements such as a photomultiplier tube array, silicon photodiode array or avalanche photodiode array. Fluorescence emission that has been filtered using filter element 160 is introduced to a single receiving element (light detector) 205 such as a photomultiplier tube, avalanche photodiode, or a silicon photodiode that has a similar sensitivity as a photomultiplier tube or avalanche photodiode. Signals from both light receiving elements 205 and 210 are then introduced to a preamplifier circuits 300 whereby a 100-2000 nanosecond current pulse is converted first to an analog voltage and then to a digital signal using an analog-to-digital converter 305. The signals from all three channels are then introduced to a signal processor 320 for analysis. The signal processor 320 can be a microcontroller, digital signal processor, field programmable gate array or a microcomputer, as would be readily understood by one skilled in the field of signal processing.
The preamp circuits 300 can be configured to serve analog signal processing functions. For each of the aerosol sensor configurations illustrated in
a-5h illustrate some of the contemplated analog signal processing configurations. Using these approaches aerosol events are triggered by monitoring one or two elastic scatter channels followed by the integration and/or peak detection of the analog signal generated from the elastic scatter channels, integrating the signals generated from the fluorescent detection channels during the trigger period, measuring the pulse duration during the trigger period, and measuring of the time-of-flight period between two elastic scatter channels if two of the light beams are purposely separated from each other by a known distance.
More specifically,
b is a schematic diagram that illustrates another analog signal processing configuration for a dual wavelength excitation scheme as illustrated in
c is a schematic diagram that illustrates another analog signal processing configuration, contemplated by the present invention, for a dual wavelength excitation scheme as illustrated in
d is a schematic diagram that illustrates another analog signal processing configuration as contemplated by the present invention, for a dual wavelength excitation scheme as illustrated in
e is a schematic diagram that illustrates another analog signal processing configuration contemplated by the present invention for a dual wavelength excitation scheme as illustrated in
For purposes of illustration
c is a schematic diagram that illustrates three excitation sources with three elastic scatter detection channels and one fluorescence detection channel. The excitation sources 100, 105, 107 can be either a continuous source or modulated at 20 MHz or greater frequency and can be a laser, light emitting diode or some other light emitting device. Excitation source 100 is a longer wavelength than excitation source 105, 107 in the wavelength range of 600-1500 nm. Excitation source 105 is a shorter wavelength than excitation source 100 and emits in the range of 350-430 nm. Excitation source 107 is a shorter wavelength than excitation source 100, 105 and emits in the range of 266-300 nm. Narrow bandpass filters 120,125,180 are used for removal of unwanted wavelengths emitted from sources 100,105,107 or from auto-fluorescence produced from the optical elements. The same beam shaping optics approach as illustrated in
d is a schematic diagram that illustrates two excitation sources with two elastic scatter detection channels and no fluorescence detection channel. The excitation sources 100, 105, can be either a continuous source or modulated at 20 MHz or greater frequency and can be a laser, light emitting diode or some other light emitting device. Excitation source 100 is a longer wavelength than excitation source 105 in the wavelength range of 600-1500 nm. Excitation source 105 is a shorter wavelength than excitation source 100 and emits either in the range of of 266-300 nm or 350-430 nm. Narrow bandpass filters 120, 125 are used for removal of unwanted wavelengths emitted from sources 100, 105 or from auto-fluorescence produced from the optical elements. The same beam shaping optics approach as illustrated in
e is a schematic diagram that illustrates three excitation sources with three elastic scatter detection channels and no fluorescence detection channels. The excitation sources 100, 105, 107 can be either a continuous source or modulated at 20 MHz or greater frequency and can be a laser, light emitting diode or some other light emitting device. Excitation source 100 is a longer wavelength than excitation source 105, 107 in the wavelength range of 600-1500 nm. Excitation source 105 is a shorter wavelength than excitation source 100 and emits in the range of 350-430 nm. Excitation source 107 is a shorter wavelength than excitation source 100, 105 and emits in the range of 266-300 nm. Narrow bandpass filters 120,125,180 are used for removal of unwanted wavelengths emitted from sources 100,105,107 or from auto-fluorescence produced from the optical elements. The same beam shaping optics approach as illustrated in
f is a schematic diagram that illustrates two excitation sources with three to four elastic scatter detection channels and one fluorescence detection channel. Excitation source 103 can be either a continuous source or modulated at 20 MHz or greater frequency and can be a laser, light emitting diode or some other light emitting device. Excitation source 112 is a pulsed laser diode. Nonlinear crystals 113, 114 are used to generate second and third harmonic frequencies. In this approach excitation source 112 is fired when the system detects the presence of a particle. Excitation source 103 is required to have a wavelength equal to or longer than excitation source 112 and narrow bandpass filter 122 filters any unwanted wavelengths. One example is to use a 1500 nm laser diode for excitation source 103 and a 1064 nm laser diode for excitation source 112. Second and third harmonic generation of a 1064 nm source would produce harmonics at 532 nm and 266 nm, respectively. The same beam shaping optics approach as illustrated in
g is a schematic diagram that illustrates two excitation sources with three to four elastic scatter detection channels and two fluorescence detection channels. Excitation source 103 can be either a continuous source or modulated at 20 MHz or greater frequency and can be a laser, light emitting diode or some other light emitting device. Excitation source 112 is a pulsed laser diode. Nonlinear crystals 113, 114 are used to generate second and third harmonic frequencies. In this approach excitation source 112 is fired when the system detects the presence of a particle. Excitation source 103 is required to have a wavelength equal to or longer than excitation source 112 and narrow bandpass filter 122 filters any unwanted wavelengths. One example is to use a 1500 nm laser diode for excitation source 103 and a 1064 nm laser diode for excitation source 112. Second and third harmonic generation of a 1064 nm source would produce harmonics at 532 nm and 266 nm, respectively. The same beam shaping optics approach as illustrated in
It is contemplated that the present particle detection and classification invention is highly useful when incorporated into various environments requiring immediate particle detection and classification. Such environments include the indoor and out-of-doors environments. Therefore, the present invention may be incorporated into any open environment, or any closed environment such as buildings, vehicles, or any other enclosed structure. It is understood that “vehicles” include both manned and unmanned enclosed spaced or objects including cars, truck, tanks, boats, airplanes, space stations including all military and non-military type applications.
The following Examples summarize the preferred sensor configurations of the present invention. Note that for each Example, both time-of-flight and particle density can be measured substantially simultaneously with the appropriate/same detection channels
SystemVariation 1:
System Variation 2
System Variation 3
System Variation 4
System Variation 5
System Variation 6
System Version 7
The foregoing disclosure of the preferred embodiments of the present invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many variations and modifications of the embodiments described herein will be apparent to one of ordinary skill in the art in light of the above disclosure. The scope of the invention is to be defined only by the claims appended hereto, and by their equivalents.
Further, in describing representative embodiments of the present invention, the specification may have presented the method and/or process of the present invention as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process of the present invention should not be limited to the performance of their steps in the order presented, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the present invention.
This application claims the benefit of U.S. Provisional Patent Application No. 60/446,042, filed Apr. 29, 2003.
