Abstract:
A method and apparatus for detecting a fluorescent substance tagged to a microparticle are described. The device comprises a single capillary flow carrier system for transporting the microparticle past a selected location, a source of electromagnetic radiation for irradiating the substance tagged to the microparticle, and a detection system for measuring fluorescent light emitted from the substance at the selected location. The method comprises transporting the microparticle to a selected location, irradiating a fluorescent substance tagged to the microparticle, and measuring the fluorescent light emitted from the fluorescent substance at the selected location.

Description:
RELATED APPLICATION 
     This application is a continuation of prior application Serial No. 09/600,021 filed Jul. 7, 2002, now U.S. Pat. No. 6,710,871 issued Mar. 23, 2004, based upon PCT/US98/11958 filed Jun. 9, 1998, and provisional application No. 60/049,292 filed on Jun. 9, 1997 . 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a method and apparatus for detecting microparticles in fluid samples. More particularly, the present invention relates to a method and apparatus which uses a fluid delivery system and laser fluorescence detection system to detect fluorescently tagged microparticles in low concentrations in fluid samples. 
     2. Description of Related Art 
     Detection of microorganisms present at low concentration in fluids is critical to provide microbiological contamination answers faster to better treat patient diseases, to prevent deadly outbreaks, to better manage quality control processes in food, drink, and drug manufacturing plants, and to provide scientists with powerful and easy to use analytical research tools. 
     Testing methods for microorganisms such as  M. tuberculosis, Trichomonas vaginalis, Campylobacter, Salmonella, E. coli , and  Cyclospora  include growth culture methods, PCR methods, fluorescently enhanced microscopic visualizations, ATP bioluminescence techniques, and bactometers. These methods are often slow and expensive, and have limited detection capabilities. 
     Testing devices include epifluorescent microscopes, fluorometers, and flow cytometers. Epifluorescent microscopes are coupled with cooled CCD high-resolution cameras to permit epifluorescent microscopic visualizations of microscopic particles. Fluorometers have limited detection capabilities, and is also not well suited when spectral differentiation in a large population of organisms is required. This is often the case when live versus dead organism differentiation is required. Flow cytometers can be very accurate in detecting and differentiating immuno-fluorescently dead or live labeled particles. However, flow cytometers are expensive and require an experienced technician or scientist to operate it and interpret the data. 
       Cryptosporidium oocysts  and  Giardia  cysts may be detected using an immunofluorescent assay (IFA) procedure. This method uses polyclonal antibodies to stain the cysts which then can be detected by epifluorescent microscopy. This method is extremely labor-intensive, considering the number of particles to be investigated under the epifluorescent microscope by an experienced technician. Flow cytometeters may also be used, but they are very expensive and require an experienced and well-trained technician to operate. Furthermore, flow cytometers still require microscopy confirmation of oocyst identification. 
     Water quality monitoring is vital for managing supplies of unpolluted water for agriculture, industry, and human consumption. Water quality monitoring may be performed using test organisms as indicators of freshwater toxicity, for example, the fathead minnow  Pimephales promelas , the cladoceran  Ceriodaphnia dubia , and the green alga  Selenastrum capricornutum . Test organisms are cultured under standard conditions, and exposed for a period of time to toxicants. Comparison of survival and reproduction rates of test organisms to control organisms provides an indication of water toxicity. 
     Bacteria enzyme activity may be used to assess water quality by using a specially designed enzyme substrate that becomes fluorescent when cleaved. This substrate is cleaved by enzymes in the bacteria and emits fluorescence light when exposed to light of the proper wavelength. The rate of enzyme activity can be measured using a fluorometer, and provides an indirect measurement of the level of toxicant stress on the bacteria. 
     Zooplankton feeding behavior may also be used to assess water quality. Extensive acute toxicity studies have been performed using plankton, in general, and various species of rotifer, in particular. Rotifer feeding and reproduction rates can be used as a rapid toxicity assessment tool. The effect of a wide range of chemicals including xylene, cadmium, copper, mercury, and diazanon on the feeding and reproduction rates of the rotifer  Brachionus calyciflorus  for fresh water and  Brachionus plicatilis  for marine waters has been extensively studied. In the feeding rate method, the rotifers are exposed for several minutes to water containing a toxicant, and then allowed to feed on fluorescently labeled beads. The rotifers are then anesthetized, washed, transferred to a microscope slide, and individually examined using a fluorescent microscope. The feeding rate is estimated by quantifying the intensity of fluorescence of ingested beads in the digestive tract of individual rotifers using an imaging technique. This method requires a trained operator, a camera, and a fluorescent microscope, which makes it slow and expensive. 
     What is needed are methods and apparatus for detecting microparticles such as harmful microorganisms and assessing water quality which is rapid, sensitive, reproducible, substantially automatic, and cost-effective. 
     SUMMARY OF THE INVENTION 
     The present invention is a device for detecting a fluorescent substance tagged to a microparticle. The device comprises a single capillary flow carrier system for transporting the microparticle past a selected location, a source of electromagnetic radiation for irradiating the substance tagged to the microparticle, and a detection system for measuring fluorescent light emitted from the substance at the selected location. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 shows a sample preparation system. 
     FIG. 2A shows a flow carrier system. 
     FIGS. 2B and 2C show detailed views of two embodiments of the capillary tube. 
     FIG. 3A shows one embodiment of a laser fluorescent measurement setup. 
     FIG. 3B shows another embodiment of a laser fluorescent measurement setup 
     FIG. 3C shows yet another embodiment of a laser fluorescent measurement setup 
     FIG. 4A shows a sample output of a digital processing unit. 
     FIG. 4B shows another sample output of a digital processing unit. 
     FIG. 5A shows the emission spectra for various fluorescent cyanide dyes used to tag microparticles. 
     FIG. 5B shows a flowchart of a signal processing strategy to detect a Cy5 tagged microparticle in the presence of other fluorescently labeled microparticles. 
     FIG. 6A shows a calibration curve obtained with water sample seeded with known amount of 2 μm fluospheres using the device of the present invention. 
     FIG. 6B shows a graph of the dependence of the particle concentration on the particle arrival rate distributions (Poisson statistical model). 
     FIG. 6C shows a graph of the evolution of the integrated detected fluorescence signal versus the SYTO™ 60  E. coli  concentration. 
     FIG. 7A shows normalized bead concentrations versus feeding time according to an analytical model for three different organism concentrations. 
     FIG. 7B shows normalized fluosphere concentrations versus feeding time for a test sample containing 1 ppm of diazanon and a control sample, using a rotifer concentration of 500 rotifers/ml. 
     FIG. 7C shows normalized fluosphere concentrations versus feeding time for a test sample containing 1 ppm of diazanon and a control sample, using a rotifer concentration of 600 rotifers/ml. 
     FIG. 7D shows normalized fluosphere concentrations versus feeding time for a 600 rotifers/ml sample and an 1000 rotifers/ml sample. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     FIG. 1 shows a sample preparation system  100 . Sample preparation system  100  contains a fluid sample  102  suspected of containing microparticles  104 . Fluid sample  102  may be run through a filter or trap to separate out any unwanted or macroscopic particulate matter. In order to prepare a sample to be tested, a fluorescent substance  106  is allowed to react with fluid sample  102  and microparticles  104 . Fluorescent substance  106  may be one or more fluorescent stains, dyes, or reagents designed to stain, tag, or otherwise attach themselves to microparticles  104 . A test sample  108  is obtained by filtering out any free remaining fluorescent substance  106  from fluid sample  102 . Test sample  108  thus contains fluid sample  102  and fluorescent substance  106  attached to microparticles  104 . 
     Fluid sample  102  may be a water sample, urine sample, blood sample, food product sample, or any other fluid to be tested. Fluid sample  102  may contain PCR-amplified samples for detection of viruses such as HIV. Microparticles  104  may be  M. tuberculosis, Trichomonas vaginalis, Campylobacter, Salmonella,    E. coli, Cyclospora, Cryptosporidium  oocysts,  Giardia  cysts, or any other bacterium, virus, fungus, or microorganism that is capable of being tagged. Microparticles  104  may also be CD4 or CD8 cells for monitoring of AIDS patients. Different fluorescent substances  106  may be used with microparticles  104  to allow different types of microorganisms to be detected and distinguished from each other. For example, for bacteria, fluorescent substance  106  can be standard DNA or surface-label-reagent stains. For  Cryptosporidium  oocysts or  Giardia  cysts, fluorescent substance  106  can be a fluorescent dye conjugated with anti-Cryptosporidium or anti-Giardia antibodies, respectively. Fluorescent substance  106  may also be magnetically charged so that it can be affected by a magnetic field. 
     Microparticles  104  may also be microscopic beads containing a fluorescent substance  106 . To measure the concentration of a toxicant in fluid sample  102 , filtro-feeder microorganisms such as rotifers or zooplankton may be added to fluid sample  102  in known quantities. Such filtro-feeder microorganisms have a feeding rate which is a well-known function of toxicant concentration. After a known incubation period, microparticles  104  are added to fluid sample  102 . Microparticles  104  may be fluospheres capable of being ingested by the filtro-feeder organisms, such as latex beads containing a fluorescent dye available from Molecular Probes, Inc., Eugene, Oreg. The fluospheres may have a uniform diameter of 2 μm or have non-uniform sizes. They may have uniform spectro-photometric properties, with a maximum absorption wavelength of 624 nm, and a maximum emission wavelength of 645 nm, or have varying spectro-photometric properties. At known intervals of time, a test sample  108  is drawn from sample preparation system  100 . Test sample  108  is obtained by filtering out any uningested microparticles  104  from fluid sample  102 . Test sample  108  thus contains water sample  102  and organisms  104  with microparticles  104  in their digestive tracts. 
     FIG. 2A shows a flow carrier system  200 . Flow carrier system  200  is a fluid delivery system which introduces test sample  108  into a capillary tube  202 . Capillary tube  202  may have very thin walls and excellent optical properties. Capillary tube  202  may have an internal diameter configured to admit microparticles  104  one at a time. A section of capillary tube  202  defines a test volume  204 . Capillary tube  202  may be at least partially coupled to an optical table  206 , which serves to hold capillary tube  202  in place. Multiple capillary tubes  202  may be arranged in parallel to obtain higher throughputs. 
     Flow carrier system  200  may include a pump system  216  coupled to capillary tube  202 . Pump system  216  may be a syringe  208  which contains test sample  108 , and injects test sample  108  through capillary tube  202  and test volume  204 . After passing through capillary tube  202 , sample  108  may pass into a dump  212 . In this manner, microparticles  104  in test sample  102  may be passed one at a time through capillary tube  202 . Pump system  216  may further include a syringe pump  214  coupled to syringe  208 . Syringe pump  214  is configured for precise control of flow of test sample  108  through capillary tube  202 . Pump system  216  may also be a peristaltic pump. 
     FIGS. 2B and 2C show detailed views of two embodiments of capillary tube  202 . FIG. 2B shows microparticles  104  flowing through capillary tube  202  towards test volume  204 . FIG. 2C shows capillary tube  202  with a magnetic element  220  positioned in a concentric fashion around capillary tube  202 . Magnetic element  220  may be a continuous ring, or be comprised of one or more separate elements. Magnetic element  220  may be used in conjunction with microparticles  104  and fluorescent substance  106  which are magnetically charged. This configuration may assist in substantially focusing microparticles  104  tagged with fluorescent substance  106  to the center of capillary tube  202  as they flow through test volume  204 , thus improving detection of microparticles  104 . 
     FIG. 3A shows a laser fluorescent measurement setup  300 . A laser  302  generates a laser beam  304 . Laser beam  304  may be focused through one or more lenses  306  onto test volume  204 . The wavelength and beam size of laser  302  is selected according to the absorption wavelength of fluorescent substance  106  and the size of capillary tube  202 . 
     When test sample  108  is passed through test volume  204 , any fluorescent substance  106  present in test sample  108  is exposed to laser beam  302 . Alternatively, a standing test sample  108  in capillary tube  202  may be moved relative to laser beam  302  to expose test sample  108 . A collecting lens  310  collects and images fluorescence light emitted by fluorescent substance  106  onto a photo-multiplier  312 . A set of interference filters  314  may be placed in front of photo-multiplier  312  to filter out the resonant light from the fluorescence light. A photodiode  316  may be placed on the opposite side of test chamber  308  to collect the resonant light. Output from photo-multiplier  312  may be sent to a first digital processing unit  318  to analyze fluorescence peaks. Output from photodiode  316  may be sent to a second digital processing unit  320  to analyze Mie scattering peaks. 
     FIG. 3B shows another laser fluorescent measurement setup  300 . In this case, the fluorescence emissions pass through a diffraction grating  313  and are imaged onto a multiple detector array  315 . The focal length and aperture of collecting lens  310 , the dispersion characteristics of grating  313 , and the size and separation of the multiple detectors in array  315  are optimized to detect at least two or three fluorescent emission bands specific to the emission spectrum of fluorescent substance  106  as well as resonant light. A set of interference filters  317  may be used to single out the fluorescence emission of fluorescent substance  106  used to tag microparticles  104 . By reading the fluorescence emission at multiple spectral locations using multiple interferential filters  317  with specific transmission characteristics, the particular fluorescent substance  106  used can be detected and distinguished. The contribution of the total fluorescence signal to each detector will provide the data needed to differentiate the particular fluorescent substance  106  from the fluorescence emissions of non-tagged particles. Output from multiple detector array  315  may be fed to a digital processing unit  318 , which processes and digitizes the multiple signals delivered by multiple detector array  315 . 
     FIG. 3C shows another embodiment of a laser fluorescent measurement setup  300  A plurality of lasers  302  generates a plurality of laser beams  304 . Laser beams  304  may be focused through one or more lenses  306  onto test volume  204 . The size of laser beam  304  may be matched to the size of capillary tube  202 . The wavelengths of lasers  302  are tuned to specific absorption bands of fluorescent substance  106 . This multiple laser and detection system may assist in reducing false positive and negative results associated with a single laser system. 
     FIG. 4A shows a sample output from digital processing unit  318 . The voltage signal coming out of photo-multiplier  312  is digitized and transferred to a computer where it can be manipulated and analyzed. The voltage signal may be digitized at a frequency of up to 3000 Hz with 8-bit precision. 
     Every time fluorescent substance  106  passes through test volume  204 , a fluorescence peak  402  is created. A threshold value  404  may be selected according to the baseline signal level and its variance. The number of fluorescence peaks  402  detected above threshold value  404 , along with the size of voltage spikes, give a measurement of the amount of fluorescent substance  106 . In the case of toxicant concentration, comparing this data with the data for an uncontaminated control sample permits determination of the toxicant concentration in water sample  102 . 
     FIG. 4B shows another sample output from digital processing unit  318 . When an microparticle  104  which has been tagged by fluorescent substance  106  passes through test volume  204 , the it generates a burst of fluorescence light with a time signature  406  and spectral signature  408  The time signature  406  and spectral signature  408  is then processed by the digital signal processing unit  318  and compared with the expected time and spectral signatures of microparticle  104  and fluorescent substance  106  to be detected. 
     Because a wide range of particles and organisms naturally fluoresce at a wide range of wavelengths, it is crucial to spectrally differentiate an microparticle to reliably detect it. Multiple laser sources and detectors may be used in close wavelength proximity to spectrally pinpoint the fluorescence pattern of the dye attached to the microparticle targeted for detection. The electronic signal analysis techniques can be tailored to the understanding of the pre-defined shape and spectral properties of the target microparticle prior to detection. 
     The use of multiple signals provides normalization and improved selectivity. Measurements at more than one fluorescence emission wavelength and/or at more than one excitation wavelength give spectral selectivity which can distinguish different dye sources. Because the dyes used for immunofluorescence commonly have relatively narrow emission peaks compared to background fluorescence sources, ratios of on-peak to off-peak signals may reliably distinguish dye-labeled particles from background events of similar absolute fluorescence. 
     Background particle signals are rejected through the use of electronic filtering, thereby allowing a sustained and very high sampling data rate. Electronic filtering involves the use of several detectors and is made possible by the uniqueness of a particle&#39;s light scattering signature and the presence of at least two fluorescent wavelength signatures. Based on the differential signal analysis of each of the detected log amplified signals, the capability of singling out the tagged microparticle at a data frequency rate of up to 50 kHz can be achieved. 
     FIG. 5A shows emission spectra of various fluorescent cyanide dyes which may be used to tag microparticles  104 : Cy5, Cy5.5, and Cy7, with maximum absorption peaks of 650 nm, 675 nm, and 743 nm, respectively. Multiple detector array  318  may be used to first record the fluorescent spectrum of the particular dye. The fluorescent spectra emitted by the tagged microparticles  104  in the sample is then compared to the recorded fluorescent spectrum of the dye. In this fashion, tagged microparticles  104  can be identified and distinguished from other fluorescently tagged microparticles  104 . In addition, because the flow rate is controlled, the width of the trace signal can be considered proportional to the diameter of microparticle  104  crossing test volume  204 . 
     FIG. 5B shows a flowchart of a signal processing strategy to detect a Cy5 tagged microparticle  104  in the presence of other fluorescently labeled microparticles. Four detectors are used. Detector D 1  is centered on the resonant laser excitation, in this case 635 nm. Detectors D 2 , D 3 , and D 4  are centered at 650 nm, 670 nm, and 690 nm, corresponding to features of the Cy5 fluorescence emission. 
     When a microparticle  104  is detected in test volume  204 , first, the time trace of the signal detected by detector D 1  is analyzed (block  502 ) and the signal intensity S1 and the pulse width W1 are compared with the expected time trace (Sc, Wc) generated by the passage of the particular microparticle in the test volume (block  504 ). If the detected signal does not meet this criteria, then the data is rejected (block  506 ). If the detected signal passes this first test, then the fluorescence intensity ratio of the detectors D 2 , D 3 , and D 4  are analyzed (block  508 ). The fluorescent ratio of S2/S3 and S2/S4 are compared with the expected fluorescence ratios corresponding to the Cy5 fluorescence spectra Sca and Scb, respectively (block  510 ). If these two tests are positive, a microparticle is counted (block  512 ); if the tests are negative the data is rejected (block  514 ). 
     EXAMPLE 1 
     Flow carrier system  100  was calibrated using water samples with known fluosphere concentrations. A reference solution of 3×10 9  beads/ml was diluted 1000 times. Then water samples containing 0, 3, 500, 7000, 14000 and 28000 beads/ml were prepared with a 10% confidence interval using a 20 μl micropipet. These water samples were passed through the device. FIG. 6A shows detected fluorescence peak counts versus expected counts for the calibration samples. An excellent correlation was consistently obtained. 
     A reference sample of 10 7    E. coli  SYTO™ 60 DNA-stained was prepared by first killing the bacteria using a 70% isopropanol exposure for one hour and then following with three sterile washes. The  E. coli  bacteria population was then stained with a 5 μmol concentration SYTO™ 60 dye. The spectral characteristics of the SYTO™ 60 dye (Abs=650 nm, Em=678 nm) are very well suited for the laser-based system of the present invention. 
     Five graded concentration samples from 10 7  to 0  E. coli  per ml stained were prepared using a 20 μl micropipette and 2 μm filtered de-ionized water. A 100 μl solution of each sample was drawn using a 1 ml syringe. The syringe was placed onto a syringe pump, and a 10 μl/min flow rate of the solution was injected into a 70 μm single capillary towards the test volume. The fluorescent test volume was defined by a 20 μm focused laser beam using a 635 nm, 5 mW laser diode and a single 10 mm focal lens. The test volume was imaged onto a 3 mm×3 mm slit using a 40× objective microscope. The photodetector signal was digitized at 3000 Hz and 8 byte dynamic range. The digitized signal was transmitted through a single serial cable onto a laptop computer. The signal was displayed on-line on a window screen using proprietary software. A time series corresponding to an injection of each sample at 10 μl/min flow rate during 30 sec was recorded. A triplicate experiment was performed for each sample, which corresponds to a 90 sec injection. By controlling the flow rate, the injection time, and the expected concentration, an expected fluorescent peak count was calculated and compared with the actual measured count. For each sample, the average peak residence time, peak intensity, and peak power (peak integral) were also computed. 
     The arrival process of the particle across the test volume was assumed to follow a random arrival process and therefore follow a Poisson process. The expected count number was corrected accordingly, to take into account the probability of having more than one particle arriving into the test volume during a time window equivalent to the particle transit time across the laser beam, taking into account the fact that a single detected count could be attributed to more than one particle. 
     FIG. 6B represents the particle arrival rate distribution at the test volume for the organism concentration investigated. For concentrations greater than 10 5  p/ml at a 10 μl/min flow rate, a Poisson statistical correction is necessary. In particular, at a concentration of 10 7    E. coli  per ml, there is more than one  E. coli  crossing the test volume 80% of the time. 
     FIG. 6C represents a correlation between the expected concentration and the integrated detected fluorescence signal corresponding to the passage of individual stained  E. coli  through the laser test volume. The integrated signal was computed as the product of the number of detected peaks corrected by Poisson statistics, and the average peak power (V/ms). The correlation is excellent, with a 98% slope. However, when there was no  E. coli  present in the sample, an integrated background noise of 4 peaks every 30 seconds was detected. These background peaks where attributed to bubbles deflecting the beam reflection into the photo-detector slit or to naturally fluorescent particles. The use of multiple wavelength detection arrays and a light scattering detector may eliminate these false positive counts. 
     EXAMPLE 2 
     Water quality monitoring using rotifers was performed using a capillary tube with an internal diameter of 70 μm, a narrow band, 635 nm, 3 mW diode laser with a beam diameter of 40 μm was used. Interference filters were selected to transmit 12% at 670 nm (20 nm FWM), and 10 −6  at all other wavelengths. 
     Method 
     1. A live  B. plicatilis  rotifer culture was obtained from Aqua-Farms, Fla. These rotifers were chosen because they are easy to raise, and the influence of toxic samples on their feeding, reproduction, and death rates have been studied extensively. The average concentration of rotifers in a 100 ml vial was counted using five 20 μl samples examined under a 50× microscope. An average count of 10 rotifers per 20 μl sample was measured, or about 500 rotifers/ml. 
     2. Two samples of 8 ml each were used to make the feeding rate measurements, a reference sample and test sample. These two vials were filled with the 500 rotifers/ml reference solution. 
     3. A 2000 ppm diazanon solution was prepared using the rotifer medium solution, so as to maintain water quality parameters such as pH, O 2 , alkalinity, salinity, and temperature as constant as possible. A 20 μl amount of the diazanon solution was added to test sample. 
     4. After a 5 minute incubation, 20 μl of a 40×10 6  beads/ml solution of crimson fluospheres was added to both the reference and test samples. The time was noted as t=0. 
     5. Using two identical syringes connected with a luer union to a 100 μm piece of nylon tubing terminated with a 20 μm plankton filter, a 50 μl sample was extracted from the reference and test samples. In both cases, the organisms were filtered out from the bead solution. 
     6. The reference and test samples were successively passed through the test chamber using the syringe pump at a flow rate of 15 μl/min. The data from data acquisition were stored for later analysis. 
     7. Steps 5 and 6 were repeated at t=5, 10, 15, and 20 minutes. 
     Data Analysis 
     Each data file was retrieved, using TOXANA™, a time series data analysis software program. This program allows visualization of the digitized photo-multiplier trace signal on a 0-5 V scale for each data file. An assessment of the signal baseline mean and variance values (where no peak is detected) was made for each file. From this measurement, a peak detection threshold, T d , was computed as follows: 
     
       
           T   d =mean+2{square root over (variance)} 
       
     
     The number of peaks with an intensity above T d  were calculated for each file, as well as the average peak intensity, the average peak width, and the average peak area. 
     Analytical Model 
     The clearance volume V Cl  for an organism with an average motility Ω and clearance diameter d can be assumed to be:                V   Cl     =     Ω   ·   π   ·       d   2     4               (     Eq   .              1     )                                
     The number of beads present in the clearance volume per unit of time is equal to:                       N   b            t       =         C   b          (   t   )       ·     V   Cl               (     Eq   .              2     )                                
     Assume that a volume V contains organisms with an average motility Ω. In this volume the concentration of food particles or beads is C b (t). 
     The change in bead concentration varies per unit of time:                C   b     =              (       C   b          (   t   )       )            t       =       -              N   b          (   t   )              t         ·       N   0     V                 (     Eq   .              3     )                                
     By substituting (1) and (2) into (3), the rate at which the bead concentration varies with time is governed by the differential equation:                       (       C   b          (   t   )       )            t       =       -       C   b          (   t   )         ·   π   ·       d   2     4     ·   Ω   ·       N   0     4               (     Eq   .              4     )                                
     Define the constant K as:              K   =     π   ·       d   2     4     ·   Ω   ·       N   0     V               (     Eq   .              5     )                                
     Then 1/K is a time constant which reflects the rate at which the bead concentration decreases. Then (5) becomes:                       (       C   b          (   t   )       )            t       =       -   K     ·       C   b          (   t   )                 (     Eq   .              6     )                                
     Integrating (6) gives: 
     
       
           C   b ( t )=λ· e   −Kt   (Eq. 7) 
       
     
     When t=0 and C N (t=0)=C B0 , (7) becomes: 
     
       
           C   b ( t )= C   B0   ·e   −Kt   (Eq. 8) 
       
     
     The feeding rate is defined as the number of beads ingested per organism and per unit of time. It can be expressed by:              FR   =                N   b            t       ·     1     N   0         =              (     C   b     )            t       ·     V     N   0                   (     Eq   .              9     )                                
     Combining (8) and (9), F can be expressed as follows:              FR   =     K   ·       C   b          (   t   )       ·     V     N   0                 (     Eq   .              10     )                                
     Assuming that d(C b )/dt is small compared with C b (t), F becomes a constant which can be expressed as:              FR   =     K   ·     C   b     ·     V     N   0                 (     Eq   .              11     )                                
     FIG. 7A shows normalized bead concentrations versus feeding time according to the model given in Eq. 8 for three different organism concentrations: 100 rotifers/ml, 250 rotifers/ml, and 1000 rotifers/ml. The analytical value used to model the feeding rate (FR) in this case is 2.7 beads/min. 
     This value was computed based on the following assumptions: the organism is a rotifer and its clearance rate is proportional to a 150 μm diameter section with a motility of 15 cm/s. It is important to note that the sensitivity of the technique strongly depends strongly on the organism concentration. 
     Results 
     FIG. 7B shows normalized fluosphere concentrations versus feeding time for a test sample containing 1 ppm of diazanon and a control sample. To compare the experimental results to the model, a concentration of 500 rotifers/ml was used. 
     For the control sample the correlation between model and experiment is excellent for the first 600 seconds. In this region, the average feeding rate is 29×10 −3  beads/sec. The departure of experiment from the model after 900 seconds can be attributed to two factors. First, the model assumes that the variation of C b  remains small compared to C b  and therefore the feeding rate is a constant. In fact, the feeding rate depends on the food concentration, which after 900 seconds has dropped by 50%. This variation cannot be neglected. Second, the rotifers have an average digestion transit time of 1200 seconds. Since the fluospheres are not metabolized by the rotifers, they are ejected back into the sample by the rotifer after 1000 seconds, which may contribute to an increase in bead concentration. 
     For the test sample the concentration of fluospheres decreases slightly with a slope corresponding to an ingestion rate per organism of 10 −3  beads/sec. Here, the feeding rate was reduced by a factor of 30 from exposure to 1 ppm of diazanon. 
     FIG. 7C shows normalized fluosphere concentrations versus feeding time for a test sample containing 1 ppm of diazanon and a control sample. The concentration of organisms is now 600 rotifers/ml. 
     Again, for the control sample, the agreement between model and experiment is excellent for the first 600 seconds. The feeding rate is now 3 beads/min per organism, compared well to the 1.7 beads/min per organism value obtained earlier. For the test sample, the concentration of beads remains almost unchanged with time and indicates a feeding rate of less than 0.05 beads/min. This measurement is consistent with previous experiments. 
     FIG. 7D shows normalized fluosphere concentrations versus feeding time for a 600 rotifers/ml sample and an 1000 rotifers/ml sample. The two samples are exposed to concentrated 2 μm fluospheres for 1700 seconds. The fluosphere concentrations are monitored continuously and the normalized concentrations are reported and compared to the clearance rate model described earlier. The agreement between model and experiment is optimum for an average feeding rate per organism equal to 4.8 beads/min. 
     To express feeding rate in terms of mass, the following equation may be used: 
     
       
           M   ·   =FR·ρ·V   fluo   (Eq. 12) 
       
     
     where FR, ρ, and V fluo  are the average feeding rate per individual rotifer, the fluosphere density, and the individual fluosphere volume, respectively. Here, for a feeding rate of 4.8 beads/min and spherical fluospheres with a density of 1.055 g/ml and a diameter of 2 μm, M-dot=21×10 −12  g/min per organism. 
     The foregoing description of the 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. Obviously, many modifications and variations will be apparent to practitioners skilled in this art. It is intended that the scope of the invention be defined by the following claims and their equivalents.