WO2009108223A2

WO2009108223A2 - Pathogen detection by simultaneous size/fluorescence measurement

Info

Publication number: WO2009108223A2
Application number: PCT/US2008/083052
Authority: WO
Inventors: Michael Morrell; Gregory Scott Morris; Jien-Ping Jiang
Original assignee: Biovigilant Systems, Inc.
Priority date: 2007-11-09
Filing date: 2008-11-10
Publication date: 2009-09-03
Also published as: WO2009108223A3

Abstract

A method and apparatus for detecting pathogens and particles in a fluid in which particle size and intrinsic fluorescence of a particle is determined. The classification of particles as biologic or inert particles using one or more thresholds is described.

Description

PATHOGEN DETECTION BY SIMULTANEOUS SIZE/FLUORESCENCE

MEASUREMENT

The present invention relates generally to systems and methods for detecting airborne or liquidborne particles, and more particularly to systems and methods for detecting airborne or waterborne particles, classifying the detected particles and distinguishing biologic particles from non-biologic particles. The invention has particular utility in detecting and classifying microbial particles in clean environments, allergens and biological warfare agents and will be described in connection with such utility, although other utilities are contemplated. The monitoring for environmental contamination, including biological particles, is important in a number of industrial and commercial environments such as manufacturing processes for pharmaceutical and hospital, and has become important in public spaces such as airports, banks, postal handling facilities and government offices where there is concern for possible urban terrorist attacks. An urban terrorist attack involving release of biological warfare agents such as bacillus anthracis (anthrax) is presently a realistic concern. Weaponized anthrax spores are extremely dangerous because they can gain passage into the human lungs. A lethal inhalation dose of anthrax spores for humans, LD₅₀ (lethal dose sufficient to kill 50% of the persons exposed) is estimated to be 2,500 to 50,000 spores (see T. V. Inglesby, et al., "Anthrax as a Biological Weapon", JAMA, vol. 281, page 1735, 1999). Some other potential weaponized bio-agents are yersinia pestis (plague), Clostridium botulinum (botulism), and francisella tularensis. In view of this potential threat, there is currently a need for an early warning system to detect such an attack.

In the pharmaceutical, healthcare and food industries a real time detector of environmental microbial levels is useful for public health, quality control and regulatory purposes. For example, parenteral drug manufacturers are required by the Food and Drug Administration to monitor the particulate and microbial levels in their aseptic clean rooms. Conventional microbiological methods require the collection of samples on growth media, and incubation at the correct temperature for the correct period of time (typically days). These methods assume that a viable microorganism is one that will undergo cellular division when placed in or on a growth media. For quantitative tests, growth is demonstrated by a visually detectable colony, a positive result of microorganism growth. There is a significant quantity of published literature that shows substantial limitations of using traditional culture and plate counting methods. For example, the published literature indicates variable results can be obtained depending upon the growth media used, the incubation time and temperature, and the condition of the microorganism prior to attempts to cultivate (e.g., slow growing, stressed, or sub- lethally damaged). In these applications, an instrument that can detect microbial particles, including bacteria, yeasts and molds, in the environment instantaneously and at low concentrations will be a useful tool and have significant advantages over conventional nutrient plate culture methods that require days for microbes to grow and to be detected.

There exist various prior art devices which employ particle size measurement and light induced fluorescence techniques as early warning sensors for bio-terrorist attack release of weaponized bio-agents. Among these devices are Biological Agent Warning Sensor (BAWS) developed by MIT Lincoln Laboratory, fluorescence biological particle detection system of Ho (Jim yew-Wah Ho, US Patent Nos. 5,701,012; 5,895,922; 6,831,279); FLAPS and UV-APS by TSI of Minnesota (Peter P. Hairston; and Frederick R. Quant; US Patent No. 5,999,250), and a fluorescence sensor by Silcott (US Patent No. 6,885,440).

A proposed bio-sensor based on laser-induced fluorescence using a pulsed UV laser is described by T.H. Jeys, et al., Proc. IRIS Active Systems, vol. 1, p.235, 1998. This is capable of detecting an aerosol concentration of five particles per liter of air, but involves expensive and delicate instruments. Other particle counters are manufactured by Met One Instrument, Inc, of Grants Pass, Oregon, Particle Measurement Systems, Inc., of Boulder, Colorado, and Terra Universal Corp., of Anaheim, California.

Various detectors have been designed to detect airborne allergen particles and provide warning to sensitive individuals when the number of particles within an air sample exceeds a predetermined minimum value. These are described in U.S. Patent Nos. 5,646,597, 5,969,622, 5,986,555, 6,008,729, 6,087,947, and 7,053,783, all to Hamburger et al. These detectors all involve direction of a light beam through a sample of environmental air such that part of the beam will be scattered by any particles in the air, a beam blocking device for transmitting only light scattered in a predetermined angular range corresponding to the predetermined allergen size range, and a detector for detecting the transmitted light.

In detectors for the determination of biologic particles by fluorescence, detection is typically accomplished by comparing the voltage on a PMT (photomultiplier tube) receiving a particle's fluorescence to a set number, or threshold value, usually a voltage value. If the detected voltage exceeded this threshold, the particle would be counted as a biologic, if it did not then it was assumed that the particle was inert. As an example, the threshold is set at two (2) standard deviations above the noise inherent to the PMT to eliminate false detection of biologies due to electronic noise. As the fluorescence gathering capabilities of detection systems has increased, problems of using a simple voltage threshold have been observed. For example, large plastic particles (effectively considered as non- fluorescing because of their weak fluorescence efficiency) are observed to generate enough emitted light (fluorescence power or intensity) such that they exceeded the voltage threshold set for classifying biologic particles and are detected. As a result large plastic beads would erroneously give a false positive and be counted as a biologic. The same is applicable for other non- biologic particles. One of the reasons for this is that every material has a fluorescence efficiency, such that at a given wavelength absorbed by a particle the amount of fluorescence emitted is dependent on the particle's composition and size. When particles become large enough, the amount of fluorescence emitted can be greater than the threshold value and thus is detected by the PMT, giving a false positive for biologic particles. It is thus desirable to have improved methods to avoid the false detection and classification of non-biologic particles as biologic particles.

For the purpose of detection of microbes in air or liquids such as water, it is of importance to devise an effective system to measure and characterize both particle size and fluorescence generated intrinsically by the microbes. One aspect of the present invention provides a sensor system capable of simultaneously or substantially simultaneously measuring particle size and detecting the presence of intrinsic fluorescence from metabolites and other bio-molecules, on a particle-by-particle basis. Another aspect of the present invention provides methods for differentiating biological particles from inert particles in a fluid by simultaneously measuring particle size and detecting intrinsic fluorescence of a particle. The advantages of this detection scheme and methods over the prior art are several. For one it provides a deterministic particle measurement methodology for characterizing particles rather than relying on statistical models employed in prior art for particle characterization. The deterministic measurement methodology enables more definitive assignment of particle characteristics than the prior art and less reliance on statistical models. It also reduces the possibility of false positives in microbial detection, for example, pollen (larger sizes than microbes) and smoke particles (smaller sizes than microbes) can be excluded from detection. And, it allows detailed analyses of data collected on each individual particle for characterizing the particle, such as intensity of fluorescence signal from a particle as a function of its cross-sectional area or volume, for the purpose of determining the biological status of the particles. Other aspects of the present invention provide improved methods for the detection and/or classification of biologic and non-biologic particles by using a combination of one or more thresholds. In one embodiment of the methods to improve the detection of biologic and non-biologic particles the fluorescence cross-section is used as a threshold for classifying particles as biologic. One advantage of using the fluorescence cross-section based on area or volume of particles as a threshold is that the particle size dependence of the fluorescence power is eliminated, or at least significantly reduced. Another advantage of using the fluorescence cross-section is that thresholds may be set based on families of materials and their equivalent fluorescence efficiency/cross-section. The current invention comprises three main components: (1) a first optical system for measuring an individual particle size; (2) a second optical system to detect a laser- induced intrinsic fluorescence signal from an individual particle; and (3) a data recording format for assigning both particle size and fluorescence intensity to an individual particle, and computer readable program code for differentiating microbes from non- microbes (e.g. inert dust particles).

The optical assembly of the present invention has two optical sub-assemblies: (a) an optical setup to measure the particle size and (b), simultaneous or substantially simultaneously to the particle size measurement, an optical apparatus is used to measure the fluorescence level from the particle being interrogated. As an example, the preferred embodiment of the current invention uses the well-known and often used Mie scattering detection scheme to measure particle size, but applies it in a novel way, enabling the system to make highly accurate measurements of airborne particles with size ranges from 0.5 microns to 20 microns. This capability to make fine distinctions in size is important in order to determine the class of microbe, because different classes of microbes have different size ranges as illustrated in Fig. 1. In one preferred embodiment of the current invention an elliptical mirror is positioned to collected fluorescence emission from the same particle as it is being measured for size. Further features and advantages of the present invention will be seen from the following detailed description, taken in conjunction with the accompanying drawings, wherein:

Fig. l is a plot showing particle size ranges of several airborne inert and microbial particulates;

Fig. 2 is a schematic diagram of an optical system in accordance with the present invention, for performing simultaneous measurements of particle size and fluorescence;

Fig. 3 is a block diagram of an optical system and methods in accordance with the present invention; Fig. 4(a) is a histogram representation of simultaneous measurements of particle size and fluorescence showing particle distribution for microbe-free air;

Fig. 4(b) is a histogram showing simultaneous measurements of particle size and fluorescence for air containing Baker's yeast powder;

Fig. 5 is a histogram representation of simultaneous measurements of 7 micron size fluorescent dye doped particles and fluorescence;

Fig.6 is a block diagram of a method according to one embodiment of the present invention for the detection and classification of biologic and non-biologic particles;

Fig. 7 shows the PMT voltage vs. particle size for fluorescing and non- fluorescing plastic beads; Fig. 8 shows the fluorescence cross-section vs. particle size for fluorescing and non-fluorescing plastic beads;

Fig. 9 shows the voltage threshold and cross-section threshold for 7 μm fluorescing beads;

Fig. 10 shows the voltage threshold and cross-section threshold for 7 μm non- fluorescing beads;

Fig. 11 shows the voltage threshold and cross-section threshold for 10 μm non- fluorescing beads;

Fig. 12 illustrates on a log scale a threshold and particle detection classification scheme according to an embodiment of the present invention. Fig. 13 illustrates the real time 2-d detection of particles by a fluorescence cross- section threshold method according to one embodiment of the present invention;

Fig. 14 is a histogram of the detection of biologies and total particles for the data of Fig. 13; and Fig. 15 is a block diagram of a method according to one embodiment of the present invention for determining and setting the fluorescence cross-section threshold.

The methods and systems of the present invention can be used to detect and classify particles in liquids or gases by simultaneously measuring the size and any intrinsic fluorescence from the particles. The methods and systems of the present invention may further be used to differentiate and/or classify biological particles from inert particles.

Fig. 2 is a schematic representation of an optical system for a fluid particle detector system according to a first exemplary embodiment of the invention. This first exemplary embodiment of the system is designed, for example to detect fluid borne particles, such as biologic particles in air or liquid media in industrial applications such as the food and pharmaceutical manufacturing industries and hospitals, as well as clean room applications, and in other civilian applications, for example in buildings or in public transportation areas, to detect harmful levels of other airborne or liquid borne particles that may exist naturally such as mold or bacteria, or which may have been accidentally, inadvertently, naturally, or deliberately released. The systems of the present invention also may be used to detect bio-terrorist agents deliberately released by terrorists or others.

The term "fluid borne particles" as used herein means both airborne particles and liquid borne particles. Liquid borne particles include those in water or other liquid media. Fluid borne particles also includes those in gases. Waterborne particles include those in water and in liquids comprising water.

The term "pathogen" as used herein refers to any airborne or waterborne particles, biological agent, or toxin, which could potentially harm or even kill humans exposed to such particles if present in the air or water, or in other liquids or gases, in sufficient quantities.

The term "biological agent" is defined as any microorganism, pathogen, or infectious substance, toxin, biological toxin, or any naturally occurring, bioengineered or synthesized component of any such micro-organism, pathogen, or infectious substance, whatever its origin or method of production. Such biological agents include, for example, biological toxins, bacteria, viruses, rickettsiae, spores, fungi, and protozoa, as well as others known in the art.

"Biological toxins" are poisonous substances produced or derived from living plants, animals or microorganisms, but also can be produced or altered by chemical means. A toxin, however, generally develops naturally in a host organism (i.e., saxitoxin is produced by marine algae), but genetically altered and/or synthetically manufactured toxins have been produced in a laboratory environment. Compared with microorganisms, toxins have a relatively simple biochemical composition and are not able to reproduce themselves. In many aspects, they are comparable to chemical agents. Such biological toxins are, for example, botulinum and tetanus toxins, staphylococcal enterotoxin B, tricothocene mycotoxins, ricin, saxitoxin, Shiga and Shiga-like toxins, dendrotoxins, erabutoxin b, as well as other known toxins.

The detector system of the present invention is designed to detect particles in a liquid or gas, particular airborne or waterborne particles, and produce outputs indicating, for instance, the number of particles of each size within a range that are detected in a sample, and indicate whether the particles are biologic or non-biologic. The system also may produce an alarm signal or other response if the number of particles exceeds a predetermined value above a normal background level, and/or biological organisms or biological agents and potentially dangerous.

Fig. 2 is a representation of a system 10 for a fluid particle detector system according to an exemplary embodiment of the invention. As shown in Fig. 2, the system 10 includes an excitation source 12 to provide a beam of electromagnetic radiation 14 having a source wavelength. The excitation source is selected to have a wavelength capable of exciting intrinsic fluorescence from metabolites inside microbes. Examples of suitable excitation sources include UV light and visible radiation sources, such as UV light and visible light lasers, LEDs and the like. By way of example, the excitation source 12 preferably operates in a wavelength of about 270 nm to about 410 nm, preferably about 350 nm to about 410 nm. A wavelength of about 270 nm to about 410 nm is chosen based on the premise that microbes comprise three primary metabolites: tryptophan, which normally fluoresces at excitation wavelengths of about 270 nm with a range of about 220 nm - about 300 nm; nicotinamide adenine dinucleotide (NADH) which normally fluoresces at excitation wavelengths of about 340 nm (range about 320 nm - about 420 nm); and riboflavin which normally fluoresces at excitation wavelengths of about 400 nm (range about 320 nm - about 420 nm). In the case of bacterial endospores, dipicolinic acid (DPA) normally fluoresces at excitation wavelengths of about 400nm (range about 320nm-about 420nm). Preferably, however, the excitation source 12 has a wavelength of about 350 to about 410 nm. This wavelength ensures excitation of two of the three aforesaid primary metabolites, NADH, and riboflavin in bio-agents, and DPA, but excludes excitation of interferences such as from diesel engine exhaust and other inert particles such as dust or baby powder. Thus, in a preferred embodiment the present invention makes a judicial selection of the wavelength range of the excitation source 12, which retains the ability of exciting fluorescence from NADH and riboflavin (foregoing the ability to excite tryptophan) while excluding the excitation of interferents such as diesel engine exhaust. This step is taken to reduce false alarms generated by diesel exhaust (which can be excited by short UV wavelengths such as 266 nm light).

In the system 10 illustrated in Fig. 2, environmental air (or a liquid sample) is drawn into the system through a nozzle 16 for particle sampling. Nozzle 16 has an opening 18 in its middle section (the opening forming a sample cell) to allow the laser beam to pass through the particle stream. The sample cell may be a gas (e.g., air) sample cell or liquid (e.g., water) sample cell. Portions of the excitation source passing through the sample area are scattered at various angles by various sized particles in the sample stream, and unscattered portions of the beam of light remain unscattered passing through the sample area. Directly downstream from the laser beam is a Mie scattering particle- size detector 20. Mie scattering particle-size detector 20 includes a beam blocker 22 in front of collimator lens 24, and a condenser lens 26 for focusing a portion of the light beam 14 scattered by particles in the sample stream onto a particle detector 28. Off axis from the laser beam 14, an elliptical mirror 30 is placed at the particle- sampling region in such a way that the intersection of the incoming particle stream and the laser beam is at one of the two foci of the ellipsoid, while a fluorescence detector 32 (in this case a photo-multiplier tube (PMT)) occupies the other focus. This design utilizes the fact that a point source of light emanating from one of the two foci of an ellipsoid will be focused onto the other. In this optical design, the elliptical mirror 30 concentrates the fluorescence signal from microbial particles and focuses it onto the fluorescence detector 32. An optical filter 34 is placed in front of the fluorescence detector to block scattered excitation laser light and pass the induced fluorescence. In one embodiment, the optical filter is a high pass filter that blocks scattered light below about 450 nm.

The beam blocking device 22 on the opposite side of the same cell from the radiation source 12 blocks at least a portion of the unscattered excitation source and can also be used for limiting the range of particles measured to a predetermined size range. The beam blocker 22 is designed to absorb, stop and/or contain non-scattered elements of the beam of electromagnetic radiation 14, e.g. the laser beam, and may comprise light absorbent materials, such as vinyl, fluoroelastomers, metallic materials or the like, and/or geometries designed to collect and contain the radiation attached to a front surface of, for example, an optical element. Other features and considerations for the beam blocker 22 are disclosed in some of the earlier US Patents to Hamburger et al. listed above, and in PCT Application Serial No. PCT/US2006027638, the disclosures of which that are not inconsistent with the disclosure herein are incorporated herein by reference.

The particle detector 20 may comprise, for example, a photodiode for sizing the particles, for example, as described in the earlier US Patent to Hamburger et al., listed above, the disclosure of which that is not inconsistent with the disclosure herein is incorporated herein by reference.

The system of the present invention also further comprise a processing unit for processing particle size distribution and particle fluorescence at a given time and displaying this information on an output device. The data may for example be displayed as a histogram. The device and methods of the present invention may further comprise computer readable program code for integrating detected particle size and detected intrinsic fluorescence, and for differentiating between biologic and non-biologic particles.

The present invention's use of Mie scattering also facilitates the placement of optical components for the detection of light induced fluorescence to concurrently examine individual particles for the presence of the metabolites NADH, riboflavin and other bio-molecules (e.g. dipicolinic acid), which are necessary intermediates for metabolism of living organisms, and therefore exist in microbes such as bacteria, fungi (e.g. yeasts and molds), and spores. If these chemical compounds exist in a bio-aerosol, they are excited by the photon energy excitation source and subsequently emit auto- fluorescence light which may be detected by an instrument based on the detection scheme outlined above. While this detection scheme is not capable of identifying the genus or species of microbes, and viruses may be too small and lack the metabolism for detection, this detection scheme's ability to simultaneously or substantially simultaneously determine for each particle the size of the particle and if it is biologic or inert indicates to the user the presence or absence of microbial contamination.

Referring to Fig. 3, the functionality of one embodiment of the simultaneous particle sizing and fluorescence measurement methods of the present invention is depicted in the graphic presentation of the measurement results from such as an instrument. The principle of operation is as follows: an instrument continuously monitors the environmental air (or liquid) to measure the size of each individual airborne (or liquid borne) particle in real time and to concurrently determine whether that particle emits fluorescence or not at the excitation wavelength, and to further measure the intensity of the fluorescence. A threshold (or set of thresholds) is set for the fluorescence signal. The fluorescence signal threshold can be fluorescence signal intensity, fluorescence intensity as a function of particle cross-sectional area or a function of particle volume. For example, if the fluorescence signal is below (or above in some instances) the set level, the particle is marked inert and/or the data is discarded. While, if the fluorescence signal threshold exceeds the set level (or is below in some instances), the particle is marked biological and/or the data is recorded. The combined data of particle size and fluorescence signal strength can be used to determine the presence or absence of microbes on a particle-by-particle basis. The particle size and fluorescence intensity data from an individual particle may also be used to estimate relative abundance of biochemical compounds inside the biological particles.

In other embodiments of the present invention, particles are classified as either inert or biological based on particle size and/or fluorescence intensity measurements. For example, the size of a particle, based for example of radius, diameter or volume, can be used to determine whether a particle falls in the size range of biological particles, and fluorescence intensity is used to confirm that the particle is a biologic. In other aspects of the present invention, the size information of a particle is used to classify whether a particle is a microorganism. Mie scattering methods are suitable for detecting and classifying particles in gases or liquids from about 0.3 to about 50 μm. In some methods of the present invention, the size of particles is detected and it is determined if the particles fall within a predetermined size range. This can be done by setting lower and/or upper scatter thresholds (also referred to as particle thresholds), so that data from particles with sizes above and/or below the thresholds are discarded. A preferred size range for the detection of particles in the methods of the present invention is preferably about 0.5 to about 20 μm, preferably about 0.5 to about 15 μm, or preferably about 0.5 to about 10 μm. Particle size and fluorescence intensity data from an individual particle can also be used to differentiate between pollen and allergens from microbes. In the methods of the present invention, the particle size and fluorescence signal data from an individual particle can further be used to estimate the relative abundance of biochemical compounds inside of biological particles. Figs. 4(a) and 4(b) illustrate the functionality of a detector in accordance with the present invention. They show environmental airborne particle data measured using this detection scheme. In each graph, the upper part depicts in logarithmic scale the particle size histogram of the cumulative number of particles counted during the sampling period versus particle size (from 1 micron to 13 microns); solid bars represent inert particles whereas striped bars indicate the presence of microbes. The lower part of the graph is a real-time snap shot of the number of particles detected within a 1 second sampling period: each spike represents one single particle and its height corresponds to the particle size. In Fig. 4(a), the test was done for clean air, so there were only inert particles, free from microbes. In a second test, Baker's yeast powder (Sacchawmyces cerevisiae) was released into the air. The presence of the microbe was detected and shown by the striped bars in the histogram in Fig. 4(b).

Fig. 5 shows a data set obtained when 7 micron fluorescent dye doped plastic beads were disseminated into a detector capable of simultaneous particle size and fluorescence measurement scheme. The striped bars show the presence of fluorescence from those particles with a size distribution in the 7 micron size range.

In the methods of the present invention it has been found that improvements in the detection and classification of particles as biologic or non-biologic particles can be obtained using a combination of one or more fluorescence thresholds and scatter thresholds. These thresholds are used in the methods of the present invention to determine the classification of detected particles. The scatter (or particle) threshold has been described above and can be used to set lower and/or upper limits for the size range of particles that are detected. The fluorescence thresholds can be based on the fluorescence signal intensity, fluorescence intensity as a function of particle cross- sectional area or surface area, or a function of particle volume, or some function or combination of these. For example, a lower fluorescence intensity threshold can be set for the fluorescence signal intensity. If the fluorescence signal intensity is below the set level the particle is marked inert, counted as an inert particle or the data can be discarded. As used herein fluorescence intensity and fluorescence power are used interchangeably. The particle size information of a particle can be used to determine the surface area, cross-section area of a particle or the volume of a particle. For example, the volume of a particle may be derived by first determining the diameter of a particle and calculating the volume based on this diameter. The fluorescence intensity from an individual particle can also be normalized to the size, diameter, surface area or volume of the particle and used to differentiate and/or classify between inert particles and microbes, or for example, between pollen and allergens and microbes.

Regardless of the size of particles, so long as they were made of the same material, the particles will all have the same fluorescence efficiency (FE). Thus for a particular particle, the amount of fluorescence emitted by the particle (its fluorescence power) is a function of the amount of light absorbed by the particle multiplied by its fluorescence efficiency.

Fluorescence Power (FP) = Amount of light absorbed x FE

Since the amount of light absorbed is a function of the surface area or volume of the particle, i.e. is a function of the amount of light interacting with the surface or volume, the emitted fluorescence power is dependent of the surface area and/or volume of the particle. For any particle, the fluorescence power is a function of the surface area and/or volume of the particle. Assuming that particles are approximately spherical, the fluorescence power (or fluorescence intensity) is a function of the radius squared (R²), where R is the radius of the particle. (The surface area of a sphere is S=4πR .) Further, as the emitted fluorescence power is also dependent on and a function of the particle volume, it is also a function of radius cubed (R³). (The volume of a sphere is V=4/3 πR³.) The dependence of the fluorescence power as a function of particle volume is particularly applicable when the particles are small and the entire particle absorbs light. Thus for small particles the fluorescence power is dependent on the entire volume of the particle. In the methods of the present invention it has been found that particle volume can be used to approximate all particle sizes and types. The fluorescence power of a particle can be normalized to the particle size by dividing by the radius squared or cubed of the particle to yield the fluorescence cross- section for a detected particle. The fluorescence cross-section of a particle with a radius R is a function of the fluorescence power divided by the radius cubed. Alternatively, the fluorescence cross-section is a function of the fluorescence power divided by the radius squared. A particle's fluorescence cross-section is then compared to a volumetric threshold (also referred to as a cross-section threshold or cross-sectional threshold or cross-sectional fluorescence threshold or fluorescence cross-sectional threshold) to classify the particle as biologic or non-biologic. Embodiments of the present invention provide methods for using the fluorescence cross-section of particles to set thresholds for the detection and/or classification of biologic particles and non-biologic particles. Methods of the invention include, but are not limited to, using either the radius squared or the radius cubed as a method for determining the cross-sectional threshold for the detection and classification of biologic and non-biologic particles. In some embodiments of the present invention, the cross-sectional fluorescence thresholds used are either a function of the fluorescence intensity divided by radius squared or radius cubed. The fluorescence cross-sectional threshold is also referred to as the volumetric threshold. One advantage of the methods of the present invention is that by using the fluorescence cross-section of a particle the size dependence of the fluorescence intensity (power) is eliminated, or at least significantly reduced. Further, fluorescence cross- section can be used to set thresholds for the detection and classification of a particle as biologic, significantly reducing the false classification of non-biologic particles as biologies (false positives). Another advantage of using the fluorescence cross-section is that thresholds can be set based on families of materials and their equivalent fluorescence efficiency or cross-section, again improving detection and classification of particles. Preferred embodiments of the present invention provide methods for detection of biologies and non-biologies particle using the fluorescence cross-section of a particle based on particle volume or surface area and detection thresholds based on fluorescence cross-sections.

One method of the present invention is illustrated in Fig. 6. In this method, using an optical system such as shown in Fig. 2, a 405 nm laser diode excitation light source interacts with a particle which absorbs the light, scatters (elastically) the light and/or emits fluorescence. The scattered light and fluorescence are detected by a photodiode and PMT respectfully. The voltage signal from these detectors is sent to a data acquisition system (DAQ). From these signals the fluorescence power and particle size are calculated, from which the fluorescence cross-section is calculated. If the value is above the pre-determined threshold fluorescence cross-section the particle is recorded as a biologic particle, if it is below the fluorescence cross-section it is recorded as an inert particle. In the methods of the present invention, the fluorescence cross-section can be calculated from the voltage of the PMT and the voltage of the photodiode, as the PMT voltage is a measure of and is proportional to the fluorophore content of a particle, while the voltage of the photodiode is a measure of and is proportional to particle size. Typically the detection systems useful with the fluorescence cross-section and threshold methods of the present invention are optical systems. Suitable detection systems for measuring the particle size and/or fluorescence of particles in air or a liquid media include those described herein and those described in US Patent Application Ser. Nos. 11/193,204, and 11/457,988, the disclosures of which that are not inconsistent with the disclosure herein are incorporated herein by reference.

Examples of the threshold methods of the present invention

The following examples are intended to illustrate certain embodiments of the present invention, but are not to be construed as limiting and do not exemplify the full scope of the invention.

All data was collected using an optical detection system that simultaneously can detect particle size and fluorescence as described herein and illustrated in Fig. 2. In this detection system light scattered and fluorescence emitted by a sample particle is simultaneously detected by a photodiode to determine particle size and by a PMT detector to detect any fluorescence emitted by particles in the sample stream, from which the biologic or non-biologic nature of the a particle may be detected and classified.

In the following examples plastic beads dyed with a fluorescent dye are used to represent biologic particles and non-dyed plastic beads to represent non-biologic particles. These polystyrene-latex (PS) beads are, for example, available from Duke Scientific, Inc. (Palo Alto, CA).

An example of the measured photomultiplier tube (PMT) voltage for fluorescing and non-fluorescing plastic beads measured with a detection system according to the present invention is shown in Fig. 7. Fig. 7 shows the PMT voltage vs. particle size for two different kinds of plastic beads: (1) the squares shown in the upper curve represent beads doped with green fluorescent dye (used to simulate biological particles) and (2) the circles shown in the lower curve are plastic beads with no dye (these represent, for example, an interferent (non-biologic or inert particle) and should not generate a biological signal). Also shown in Figure 7 is a threshold voltage for the PMT of a typical detection system, shown by the horizontal range line set at 40 mV. The PMT voltage is a measure of and is proportional to the fluorophore content of a particle. As Figure 7 illustrates all the fluorescing beads (square symbols in upper curve) exceed the threshold value of 40 mV and thus would be considered biological particles. The 7 μm the plastic bead without dye (lower curve, black circle) is starting to generate enough fluorescence as to be seen by the PMT (7-8 mV) but still does not exceed the threshold value of 40 mV for being counted as a biologic particle. Thus, even with a simple PMT voltage based threshold for determining biologies from non-biologic particles the 7 μm plastic bead in this example would not be counted as a biologic. The case is different for 10 μm and large plastic beads. These generate sufficient fluorescence to exceed a PMT threshold voltage set at 40 mV and would incorrectly be counted as biologic particles and cause a false positive count.

When the same data for the fluorescing (filled squares) and non-fluorescing (solid circles) plastic beads is plotted as PMT voltage (the fluorescence intensity being proportional to the PMT voltage) divided by particle volume, in other words the fluorescent cross-section as a function of radius cubed, the separation between the fluorescing (filled square symbols ) and non-fluorescing plastic beads (filled circle symbols) increases significantly as shown in Fig. 8, and providing better differentiation of the particles. AU the green dyed beads (fluorescing) (upper curve, solid squares), with the exception of the 7 μm green beads, show a value of about 100 mV/μm³ for the fluorescence cross-section regardless of the size of the bead. The 7 μm green beads appear below the 100 mV/μm³ fluorescence cross-section line shown in Fig. 8 because the 7 μm beads are saturating the PMT as seen in Fig. 7. The non-dyed plastic beads (lower curve, solid circles) also show a fairly constant value for the fluorescence cross- section of about 0.08mV/ μm . However, because they have a much lower fluorescence efficiency, about 3 orders of magnitude lower than the dyed beads, as shown in Fig. 8, their fluorescence cross-section is very different to that of the dyed beads.

By setting a volumetric threshold (fluorescence cross-sectional threshold) just above that of the non-fluorescing plastic beads (or non-biologic particles), dotted line in graph at 0.15 mV/μm³, the classification of non-fluorescing particles as biologies can be prevented, or greatly reduced, thus reducing false biological counts as would be observed if only a threshold voltage was used.

Example - the detection of 7 μm plastic beads using a threshold voltage method and a fluorescence cross-section threshold method

Figs. 9 and 10 show a fluorescence cross-sectional threshold method and a voltage threshold method for the detection of fluorescing and non-fluorescing 7 μm plastic beads. (The voltage threshold is also referred to as a fluorescence threshold.) In Fig. 9, the top of the graph shows data for fluorescing 7 μm plastic beads using the voltage threshold method where the voltage threshold was set to 40 mV. The bottom graph of Fig. 9 shows data for fluorescing 7 μm plastic beads using a fluorescence cross- sectional threshold method of the present invention. In both graphs the total particle count, open squares (upper graph) or solid squares (lower graph), and the portion of those that are considered biological, solid squares (upper graph) or solid circles (lower graph), are shown. Each distribution is fitted to a Gaussian response, solid or dotted lines, to eliminate the effects from any contaminants, such as room air, that may skew the distributions. To illustrate differences between the cross-sectional fluorescence threshold method and the voltage threshold method the ratio of the biological particles to the total particle count from the Gaussian fits as a percent biological were 86% for the voltage threshold and 87% for the cross-sectional fluorescence threshold method. It is observed that the particle distribution for both the threshold voltage and cross-section method are nearly identical for the 7 μm fluorescing beads. In this case, since the 7 μm fluorescing plastic beads are strongly fluorescing, very little difference between the two threshold methods is observed.

This is not the case for non-fluorescing 7 μm plastic beads as illustrated in Fig. 10. In this case, as shown in Fig. 7, the non-fluorescing 7 μm plastic beads are just below the 40 m V voltage threshold. In Fig. 10 the total counts are shown as open squares in the upper graph and solid squares in the lower graph, and the biologic counts as solid squares in upper graph and solid circles in the lower graph. In this case, the voltage threshold method, top graph in Fig. 10, shows some biological counts (solid squares) due to the fact the some of the beads are fluorescing a little stronger than others and the curve is shifted to the larger particle size of the distribution where the fluorescence is also larger. In the bottom graph, the curve for biologic particle is shown as the solid circles and the total counts as solid squares. To help illustrate the improved differentiation of non-biologic particles by the cross-sectional threshold method of the present invention the ratio of those particles being classified as biologic to the total particle count from the Gaussian fits as a percent biological were 22% for the voltage threshold and only 4% for the cross-sectional fluorescence threshold method. The cross- sectional threshold method is able to reduce the percentage counts for biological particles by a factor of about 5 to 6. For the non-fluorescing 7 μm plastic beads representing non- biologic particles, the cross-sectional threshold method is able to better reject false biologies detection by reducing the percent biologic count. Example - the detection of 1 Q μm plastic beads using a threshold voltage method and a fluorescence cross-section threshold method

Fig.l 1 shows data for 10 μm non-fluorescing plastic beads, the top graph showing data for a voltage threshold method and the bottom graph data for a cross- sectional threshold method. Total particle counts are shown by open (upper curve) or closed squares (lower curve) and biological particles as solid squares (upper curve) or circles (lower curve). In Fig. 7, these beads had a sufficiently high PMT voltage that was above the threshold voltage so they would have been wrongly counted as biologic particles. The advantage of the fluorescence cross-section method of the present invention is immediately apparent from Fig. 11. Each distribution is fitted to a Gaussian response, the solid or dotted lines, to eliminate the effects from any contaminants, such as room air, that may skew the distributions. To illustrate the improved differentiation of the cross-section method of the present invention and the threshold voltage method the ratio of the biological particles to total particles from the Gaussian fits as a percent biological were 77% for the voltage threshold and 1% for the cross-sectional fluorescence threshold method. As shown by the top graph of Fig. 11, the threshold voltage method makes the 10 μm non-fluorescing plastic beads appear as nearly 80% biologic particles, whereas the cross-sectional threshold method rejects the fluorescence of the large plastic beads and only about 1% of the counts are classified as biologic.

This data illustrates the advantages of the methods of the present invention for reducing or eliminating false positive counts for particle in biological detection systems. In some embodiments, fluorescence cross-sectional threshold methods are used to reduce or eliminate false positive counts of inert particles by biological particle detection systems. The method is partially applicable for large particles. In one embodiment, fluorescence cross-section methods as described herein eliminate or reduce significantly the excessive biologic count observed for large particle sizes. In one embodiment, fluorescence cross-section methods as described herein reduces false biologic count observed for large particle sizes. While the examples illustrate the methods of the present invention for dye doped

(fluorescing) and non-doped (non-fluorescing) plastic beads, the methods are equally applicable to other interferants, such as, for example, paper dust, and dyes, and to biologic particles. The method of using a volumetric threshold is a very powerful tool to find biologies in otherwise cluttered sets of data. Fig. 12 illustrates a threshold method of the present invention. While the cross- sectional (also referred to as the volumetric threshold) may be used as the only threshold for the detection and classification of biologic and non-biologic particles in the methods of the present invention, a combination of one or more thresholds chosen from the voltage threshold (also referred to as the fluorescence threshold), particle threshold (also referred to a the scatter threshold) and cross-sectional threshold are preferred. As illustrated in Fig. 12 for a preferred embodiment of the detection and classification methods of the present invention a voltage threshold (also referred to as the fluorescence threshold), a particle threshold (also referred to a the scatter threshold) and a cross- sectional threshold are used to provided improved detection and classification of biologic and non-biologic particles. Fig. 12 shows the region where particles would be classified as biologic (fluorescent) particles (gray shading) and non-biologic (non-fluorescent) particles (combined cross-hatch shading regions). Fig. 12 also shows the region where the volumetric threshold methods of the present invention provides improved particle classification (cross-hatch shading next to the diagonally dashed line).

Another aspect of the present invention relates to methods for the real time detection of biologic particles. In these embodiments, the cross-section threshold methods as described herein are used as a method to detect and differentiate different material types in real time. This is illustrated in Fig. 13. Fig. 13 shows a simulated real time display of particles being injected and measured simultaneously by fluorescence and particle size detectors using an optical detection system as described herein. The data from the detectors is sent via a data acquisition interface to a computer processor where the data is processed by methods described herein and displayed on a monitor or other user interface. In Fig. 13 the incoming particles are shown as a 2D histogram, where the x-axis is the particle size (μm) and the y-axis is the fluorescent cross-section (in this case as the log of the fluorescent cross section), and the z-axis is the counts (contour lines representing more counts). The example graph clearly demonstrates that two different types of fluorescing materials are being measured, one having a small size, around 1 μm, with a large fluorescence cross-section of approximately 10 mV/μm , and a second material of with larger size particle of about 4 μm but with a much weaker fluorescence cross-section of approx 0.1 mV/μm³.

Without the use of the fluorescence cross-section methods of the present invention the user would instead see a histogram graph of counts vs. particle size as illustrated in Fig. 14 for the particle mixture of the data of Fig. 13. Although the presence of two types of equally sized particles might possibly be apparent in the histogram in Fig. 14 (squares represent total particles and circles biologies), the advantage of the 2D cross-sectional histogram is clearly seen. Fig. 13 illustrates that the cross-sectional methods of the present invention provides improved differentiation and additional depth, clearly showing that the two particles observed are of a vastly different composition or material type and size.

Another embodiment of the methods of the present invention is shown in Fig. 15 for calculating and setting the cross-sectional threshold for classifying inert and biologic particles. In this example, 15 μm PS beads are used to represent inert particles (interferents) that produce false positives because they generate enough fluorescence power to produce measureable fluorescence intensity. Other beads of different sizes or materials may also be used, and would be chosen to provide the best representation of inert particles that would be generating false positive for a particular detection system. One criteria for the selection of the interferent is that it has weak enough fluorescence to just be detected and thus discriminated. The beads representing interferents (inert particles) are sampled though a detection system, for example, a detection system as described herein or any other detection systems known in the art for the measurement of particle size and fluorescence from particles. As shown in Step 2, for each particle detected the log of fluorescence voltage divided by size voltage cubed is calculated. The voltages are a measure of and are proportional to the fluorophore content and size of a particle. The data generated can be plotted, for example, in Step 3 as a histogram. From the data generated a cross-sectional threshold value can be determined by the method of Step 4 or 5. In the first embodiment, the cross-section threshold can be determined by fitting a Gaussian to the data and setting the threshold at the mean plus 2 standard deviations (about 97%). Alternatively, the cross-sectional threshold can be set such that at least 90% of the counts for 15 μm beads fall below the threshold and will not be counted as biological particles. In preferred embodiments the threshold is chosen so that at least 80% of the counts from the calibration particles fall below the threshold, alternatively, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% of the counts from the calibration particles fall below the threshold.

It should be emphasized that the above-described embodiments of the present invention, particularly, any "preferred" embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the invention. Many variations and modifications may be made to the above-described embodiments of the invention without departing substantially from the spirit and principles of the invention. All such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.

Claims

Claims:

I . A method of differentiating biological particles from inert particles in a fluid which comprises simultaneously measuring a particle size and detecting intrinsic fluorescence from that particle.

2. The method of claim 1, wherein fluorescence intensity is measured and assigned a value, and including the step of classifying the particle as either inert or biological based on particle size and fluorescence intensity.

3. The method of claim 2, wherein size information of the particle is used to classify whether that particle is a microorganism.

4. The method of claim 2, wherein classification of the particle is derived from determining a fluorescence cross-section for the particle.

5. The method of claim 4, wherein volume of the particle is derived by first determining the diameter of the particle, and calculating its volume based on said diameter.

6. The method of claim 2, wherein particle size and fluorescence intensity data from an individual particle is used to differentiate between inert particles and microbes.

7. The method of claim 1, further comprising using computer readable program code for integrating detected particle size and detected intrinsic fluorescence.

8. The method of claim 2, wherein particle size and fluorescence intensity value from an individual particle is normalized by its size, surface area or volume and used to differentiate between inert particles and biologic particles.

9. The method of claim 2, wherein particle size and fluorescence intensity value from an individual particle is normalized by its size, surface area or volume and used to differentiate between pollen and allergens from microbes.

10. The method of claim 1 , wherein the fluid comprises air or water.

I I. A method for detecting and classifying a particle in a liquid or gas comprising illuminating the particle with a light source, and simultaneously measuring a size of the particle and any intrinsic fluorescence from the particle.

12. The method of claim 11 , further comprising using computer readable program code for integrating detected particle size and detected intrinsic fluorescence.

13. The method of claim 1 1, comprising the step of measuring fluorescence intensity.

14. The method of claim 13, comprising the step of comparing particle size information and fluorescence intensity to classify the particle as inert or biologic in origin.

15. The method of claim 1 1, comprising the step of classifying the particle based on its diameter or volume and fluorescence intensity.

16. The method of claim 11, comprising the step of classifying the particle based on its fluorescence intensity normalized by its diameter or volume.

17. The method of claim 11, comprising determining and setting one or more thresholds.

18. The method of claim 17, wherein one or more thresholds are selected from the group comprising of fluorescence voltage thresholds, volumetric thresholds, and scatter thresholds.

19. The method of claim 11 , comprising the step of setting a cross-sectional threshold.

20. The method of claim 19, further comprising setting the threshold at a value of at least 90% of the counts from interferent particles fall below the threshold.

21. The method of claim 19, wherein the threshold is set at mean plus two (2) standard deviations of the distribution from interferent particles.

22. The method of claim 19, wherein the cross-sectional threshold is a function of cross-sectional area of particles or the volume of particles.