Patent Publication Number: US-8524489-B2

Title: Particle or cell analyzer and method

Description:
RELATED APPLICATIONS 
     This application is a division of U.S. application Ser. No. 12/183,301, filed Jul. 31, 2008 issued as U.S. Pat. No. 7,972,559, which is a continuation of U.S. application Ser. No. 10/410,230, filed Apr. 8, 2003, issued as U.S. Pat. No. 7,410,809 on Aug. 12, 2008, which is a division of U.S. application Ser. No. 09/844,080, filed Apr. 26, 2001, abandoned, which claims priority to U.S. Provisional Application No. 60/230,380, filed Sep. 6, 2000. 
    
    
     BRIEF DESCRIPTION OF THE INVENTION 
     This invention relates generally to a particle or cell analyzer and method, and more particularly to a particle or cell analyzer and method in which the sample solution containing the particles or cells is drawn through a capillary for presentation to a shaped light beam. 
     BACKGROUND OF THE INVENTION 
     The detection and analysis of individual particles or cells is important in medical and biological research. It is particularly important to be able to measure characteristics of particles such as concentration, number, viability, identification and size. Individual particles or cells as herein defined include, for example, bacteria, viruses, DNA fragments, cells, molecules and constituents of whole blood. 
     Typically, such characteristics of particles are measured using flow cytometers. In flow cytometers, particles which are either intrinsically fluorescent or are labeled with a fluorescent marker or label, are hydrodynamically focused within a sheath fluid and caused to flow past a beam of radiant energy which excites the particles or labels to cause generation of fluorescent light. One or more photodetectors detect the fluorescent light emitted by the particles or labels at selected wavelengths as they flow through the light beam, and generates output signals representative of the particles. In most cytometers, a photodetector is also used to measure forward scatter of the light to generate signals indicative of the presence and size of all of the particles. 
     U.S. Pat. No. 5,547,849 describes a scanning imaging cytometer wherein an unprocessed biological fluid sample is reacted with a fluorescently labeled binding agent. The reacted sample undergoes minimal processing before it is enclosed in a capillary tube of predetermined size. The capillary tube with the enclosed sample is optically scanned and the fluorescent excitation is recorded from a plurality of columnar regions along the capillary tube. Each columnar region is generally defined by the spot size of the excitation beam and the depth dimension of the capillary tube. A spacial filter of sufficient pinhole diameter is selected to allow simultaneous volumetric detection of all fluorescent targets in each columnar region. The cellular components or particles are identified as is their concentration. 
     OBJECTS AND SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a particle analyzer and method having high particle selectivity. 
     It is another object of the present invention to provide a compact, high-sensitivity particle analyzer. 
     It is still another object of the present invention to provide a portable particle analyzer and method for use in immunology, microbiology, cell biology, hematology and cell analysis. 
     It is a further object of the present invention to provide a simple-to-use, less expensive, particle analyzing apparatus for counting particles in small volumes of sample fluids and determining their characteristics. 
     It is still another object of the present invention to provide a particle analyzer and method for analyzing low volumes of low-density sample fluids. 
     The foregoing and other objects of the invention are achieved by a particle analyzing apparatus which analyzes particles in a sample fluid flowing through a capillary tube which has a suspended sampling end for insertion into a sample fluid, and a pump coupled to the other end for drawing the sample fluid and particles through the capillary. An illumination source is provided for projecting a beam of light through a predetermined volume of the capillary to impinge upon the particles that flow through that volume. At least one detector is disposed to receive fluorescent light emitted by excited fluorescing particles and provide an output pulse for each fluorescing particle, and another detector senses the passage of all particles which flow through the volume and provides an output signal, whereby the output signals from the detectors can be used to characterize the particles. 
     A method of analyzing samples containing particles, which includes drawing the sample through a capillary volume where the particles are illuminated by a light source, and scattered light and fluorescent light from labeled particles excited by the light source is detected to provide output signals which are processed to provide an analysis of the sample. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing and other objects of the invention will be more clearly understood from the following detailed description when read in conjunction with the accompanying drawings in which: 
         FIG. 1  schematically shows a particle analyzer in accordance with the present invention. 
         FIG. 2  is a top plan view showing the optical components shown in  FIG. 1  mounted on a support shelf. 
         FIG. 3  is a front elevational view partly in section of  FIG. 2 . 
         FIG. 4  is a side elevational view of the beam-forming optical system. 
         FIG. 5  is a top plan view of the beam-forming optical system of  FIG. 4 . 
         FIG. 6  shows the sample fluid flow and pumping system. 
         FIG. 7  is a perspective view of a portion of a capillary used in connection with one embodiment of the present invention. 
         FIG. 8  is a perspective view of a portion of a capillary tube used in connection with another embodiment of the present invention. 
         FIG. 9  schematically shows a control and data acquisition system associated with the particle analyzer. 
         FIG. 10  is a timing and data acquisition diagram illustrating operation of the particle analyzer. 
         FIG. 11  is a schematic view of a four-color particle analyzer. 
         FIG. 12  schematically illustrates an analyzer having multiple analyzing stations along the capillary tube. 
         FIG. 13  shows an impedance detector for detecting particles as they flow past a detection region. 
         FIG. 14  schematically shows a circuit suitable for correlating signals from an impedance cell sensor with photomultiplier output signals 
         FIG. 15  shows a particle analyzer in accordance with another embodiment of the invention. 
     
    
    
     DESCRIPTION OF PREFERRED EMBODIMENT(S) 
     Referring to  FIG. 1 , there is schematically illustrated a particle analyzer in accordance with one embodiment of the present invention. As used herein, “particles” means particles or cells, for example, bacteria, viruses, DNA fragments, blood cells, molecules and constituents of whole blood. A capillary tube  11  has a suspended end  12  adapted to be immersed into a sample solution  13  retained in a cuvet or vial  14 . It will be apparent that, although a square capillary is illustrated, the capillary may be cylindrical or of other shape, such as a microchannel. Sample fluid is drawn into the end of the capillary as shown by the arrow  16 . As will be presently described, the fluid or liquid sample is drawn through the capillary by a calibrated pump connected to the other end of the capillary. The size or bore of the capillary tube  11  is selected such that the particles  18  are singulated as they pass a viewing or analyzing volume  19 . A light source, preferably a laser,  21  emits light  22  of selected wavelength. The light is received by an optical focusing system  23  which focuses said light and forms and directs a beam  24  to the capillary where it passes through the analyzing volume  19 . The optical focusing system is configured to form a flat, thin rectangular beam which impinges on the capillary tube  11 . The thickness of the flat beam and the walls of the capillary define the analyzing volume. In order to count all particles which traverse the detection volume, that is particles which are tagged to fluoresce and untagged particles, scattered light is detected. In one embodiment, a beam blocker  26  is positioned to intercept the beam after it passes through the capillary tube  11 . Light scattered by a particle that flows through the beam is directed onto a detector  27  by lens  28 . The detector provides an output signal such as the one illustrated by the peak  29 , when a particle passes through the beam and scatters the light. The size of the peak is dependent upon the size of the particle, and the occurrence of the peak indicates that a particle in the volume  19  (fluorescent or non-fluorescent) has traversed the thin beam of light. Another approach is to employ an off-axis detector, such as illustrated in  FIG. 15 , to measure the scattered light. In such event, a beam blocker is not required. There is also described below an impedance method of detecting particles. 
     If the particles are intrinsically fluorescent, or if the particles have been tagged with a fluorescent dye, they will emit light  31  at a characteristic wavelength as they pass through the volume defined by thin beam of light  24  which excites fluorescence. The fluorescent light is detected at an angle with respect to the beam axis so that no direct beam light is detected. In the embodiment of  FIGS. 1-3 , a collector lens  32  receives the fluorescent light from the particles and focuses it at detectors  36  and  37 . We have found that initially we included slits  33  or  34  oriented in the direction of the thin beam to block any stray light. However, we have found that if the beam is properly focused into a thin flat beam, stray light is not a problem. This greatly simplifies assembly of the analyzer, since there is no need to carefully align the slits. The light impinges onto a dichroic beam splitter  38  which passes light of selected wavelengths through filter  39  to detector  36 , and deflects light of other selected wavelengths through filter  41  to photodetector  37 . For example, the dichroic beam splitter reflects light having wavelengths less than 620 nm, and transmits light having a greater wavelength. The filters  39  and  41  are selected to pass the wavelengths corresponding to the fluorescence wavelength expected from the fluorescing particles. In one example, the filters  39  and  41  were selected to pass light at 580 nm and 675 nm, respectively. This permitted identification and counting of particles which had been tagged with fluorescent material which emits at these wavelengths in response to the optical beam. The outputs of the photodetectors are pulses such as those schematically illustrated at  42  and  43 ,  FIG. 1 . 
       FIGS. 2 and 3  show the components of a particle analyzer in accordance with the above-described embodiment mounted on a support plate  51 . The support plate  51  carries an optical block  52  adapted to receive and support the suspended capillary tube  11 . Capillary tube  11  includes a hub  53 ,  FIGS. 3 and 6 , which is received in a well  54  to retain and position the capillary in the optical block. The capillary  11  is positioned in the optical block  52  by threading it through a narrow slot (not shown) and held in position by nylon-tipped set screws inserted in threaded holes  56  and  57 . As it is inserted through the block, the capillary tube can be viewed through the viewing port  58 . The end of the capillary tube is suspended and extends downwardly for insertion into a vial or cuvet  14  which contains the sample fluid or specimen. It is apparent that the capillary can be positioned and suspended by other supporting arrangements. 
     In one embodiment, a rotatable vial support member having two arms  59  is rotatably and slidably received by a guide post  60  secured to the base. A vial holder  61  is disposed at the end of each arm. In operation, the support is moved downwardly along the post  60 , rotated to bring a vial under the capillary, and moved upwardly whereby the end of the capillary is immersed in the sample fluid. As the sample is being analyzed, another vial with another sample can be placed in the other holder whereby it can be brought into cooperative relationship with the capillary tip as soon as the analysis of the prior sample has been completed. 
     The housing  23  for the laser and optical focusing system which forms the beam  24  is carried on mounting block  62 . The optical system is shown in  FIGS. 4 and 5 . It receives collimated light  22  from the laser  21 , and generates the light beam  24 , which impinges upon the capillary tube  11 . The optical system may include, for example, a first plano-convex lens  63 , a second plano-convex lens  64  and a cylindrical lens  66 . The action of the lens assembly is to form a sheet-like thin rectangular beam which in one example was 20 μm in thickness along the longitudinal direction of the capillary, and 400 μm broad in the perpendicular direction, whereby a rectangular volume of sample was illuminated. The arrows  67  and  68 ,  FIGS. 4 and 5 , show the thin and broad configuration of the beam, respectively. 
     The photodetector  27  is mounted on the block  52  and supported axially with respect to the axis of the beam  24 . The beam blocker bar  26  is mounted in the block  52  and intercepts and blocks out the direct beam after it passes through the capillary  11 . The scattered light which passes around beam-blocking bar  26  is focused onto the detector  27  by a lens  28 . Thus, the scattered light will provide an output signal for any tagged or untagged particle flowing past the observation volume  19 , thus providing a total particle count. The output of the detector is then representative of the passage of a particle or cluster of particles and the size of the particle or cluster of particles. As will be explained below, this, taken together with the fluorescent signal, enables analysis of the sample. If the detector  27  is located off-axis, it will only receive scattered light and there is no need for a beam-blocking bar. Furthermore, this would be less sensitive to stray light in the forward direction which carries broadband laser noise which can mask out low level particle signals. 
     As described above, light emitted by fluorescence from intrinsically fluorescent particles, or particles which have been tagged with a fluorescent dye or material, is detected at an angle with respect to the beam axis. Referring to  FIG. 2 , the condenser lens  31  is carried by the block  52 . The lens  31  receives the fluorescent light and focuses it at the detectors  36  and  37 , which may be photomultipliers, charge-coupled diodes (CCDs), or other photodetectors. More particularly, the fluorescent light from the lens  31  impinges upon a dichroic beam splitter  38  which splits the beam into two wavelengths, one which passes through the beam splitter and one which is deflected by the dichroic beam splitter  38 . Filters  39  and  41  filter the transmitted and reflected light to pass only light at the wavelength of the fluorescence of the particles to reject light at other wavelengths. If slits  33  and  34  are present, they reject any stray light from regions outside of the volume  19  defined by the thin rectangular beam  24 . However, as discussed above, slits may not be required because the effect of stray light is minimized. The photo-multipliers or other photodetectors each provide an output signal representing the intensity of light at the filtered wavelength. As described above, the dichroic beam splitter reflects light having wavelengths less than 620 nm, and transmits light having greater wavelengths. The filter  39  passes light at 580 nm, while the filter  41  passes light at 675 nm. This permits analysis of particles which have been tagged with fluorescent substances which emit light at 580 nm and 675 nm to be individually counted. The output of the photo-multipliers are pulses  42  and  43 , one for each particle emitting light at the particular wavelength, such as those schematically illustrated in  FIG. 1 . It is apparent that the wavelengths selected for the filters depends upon the fluorescent wavelength of the marker or label affixed to the particles. 
     In order to identify and count the particles in the fluid in a volumetric manner, the volume of fluid must be correlated with the number of particles detected in a given volume. In the present invention, the fluid sample is drawn through the capillary tube at a constant rate by an electrically operated calibrated pump or syringe  71 ,  FIG. 6 . The pump may be any other type of pump which can draw known volume samples through the capillary. The pump is connected to the capillary tube by a conduit or tube  72 . This permits changing capillaries  11  to substitute a clean capillary or a capillary having a different diameter which may be needed for various types and sizes of particles or cells. As illustrated, the pump comprises a syringe pump in which sample fluid is drawn into the capillary by moving the plunger  73 . The pump  71  is also connected to a waste or drain conduit  74  which includes a valve  76 . When the valve is closed, the pump draws sample from the vial or cuvet through the capillary tube  11  past the detection volume  19 . After an analysis has been completed, the valve  76  is opened, whereby reversal of direction of the plunger  73  causes fluid to flow through conduit  74  into the waste container  77 . In accordance with a feature of the present invention, the diameter of the waste tube  74  is selected to be many times, 10 or more than that of the capillary, whereby substantially all of the fluid from the syringe is discharged into the waste. For example, if there is a factor of ten ratio in diameter, only 1/10,000 of the fluid will travel back through the capillary, a negligible amount. 
     The pump is designed such that a predetermined movement of the plunger  73  will draw a known volume of sample through the capillary tube. The pump can be calibrated for each capillary by drawing a fluid into the pump by moving the plunger a known distance and then discharging the fluid and measuring the volume of the discharged fluid. Thereafter, for a given movement of the plunger, the volume of sample which flows through the analyzing volume is known. The volume can either be determined by measuring the movement of the plunger or measuring the time the plunger is moved if it is calibrated as a function of time. Although a syringe pump is described, other types of pumps which can draw known volumes of fluid through the capillary can be used. 
     Preferably, the capillary tubes are of rectangular configuration.  FIGS. 7 and 8  show a capillary tube that includes an opaque coating  81  which is removed over an area  82 ,  FIG. 7 , or  83 ,  FIG. 8 . In the embodiment of  FIG. 7 , the beam projects through the window  82  which has a rectangular configuration to accept of the beam  24 . In  FIG. 8 , the slit masks the walls of the capillary tube and prevents diffraction of light by the walls. A combination of the two masks would confine the detected light to that emitted by a particle traveling through the capillary to block out any stray light. 
     An example of the operation of the apparatus to analyze a sample containing particles which do not fluoresce, and particles which intrinsically fluoresce or are marked or tagged to fluoresce, at two different wavelengths, for example 580 nm and 675 nm, is now provided with reference to  FIGS. 9 and 10 . 
     With the syringe pump plunger  73  extended to empty the pump, the sample vial containing the particles is positioned to immerse the end of the capillary  11  in the sample. The sample is then drawn through the capillary by applying a control signal from the controller  121  to start the pump  71 . The controller receives the command from processor  122 . The pump  71  is driven at a constant rate whereby the volume of sample passing through the analyzing volume  19  can be measured by timing the counting period. After sample has been drawn from the vial for a predetermined time to assure that the new sample has reached the volume  19 , the processor begins to process the output  29  from the scatter photodetector and the outputs  42  and  43  from the photodetectors and does so for a predetermined time which will represent a known volume of sample passing through the analyzing volume. The processing time is schematically illustrated in  FIG. 10A  by the curve  123 .  FIG. 10B  illustrates the output pulses  28  from the scatter detector. It is seen that there are individual particles which provide a trace  124  and a cluster of particles which provides a trace  126 .  FIG. 10C  shows traces  126  for particles which fluoresce at a first wavelength, for example 675 nm. Of note is the fact that the cluster  126  includes three such particles.  FIG. 10D  shows traces  127  for particles which fluoresce at another wavelength, for example 750 nm. Of note is the fact that there is also one such particle  128  in the cluster  126 . The processor can call for a number of analyzing cycles. Finally, when an analysis is completed, the processor instructs the controller to open the valve  76  and reverse the pump to discharge the analyzed sample into the waste  77 . A new sample cuvette can then be installed and a new sample analyzed. The processor can be configured to average the counts over a number of cycles and to process the counts to provide outputs representing the concentration of the various particles, the number of particles, etc. Using suitable labels or markers one can conduct viability assays and antibody screening assays or monitor apoptosis. 
     Although the apparatus has been described for a two-color analysis it can easily be modified for four-color analysis. This is schematically shown in  FIG. 11 . The input light beam  24  impinges upon the analyzing volume  19 . The photodetector  27  and associated lens  28  provide the scatter signal. The fluorescent light  31  is focused by lens  32  to pass through three dichroic beam splitters  81 ,  82  and  83  which reflect light at three different wavelengths through filters  86 ,  87  and  88  onto photodetectors  91 ,  92  and  93 . The light at the fourth wavelength, passed by the three dichroic beam splitters  81 ,  82  and  83  passes through filter  94  onto photodetector  96 . Thus, up to four different particles which intrinsically fluoresce or are labeled to fluoresce at four different wavelengths can be analyzed by choosing the proper reflecting wavelengths for the dichroic beam splitter and the filters. 
       FIG. 12  schematically shows a system using a plurality of light sources (not shown) projecting light beams  106 ,  107  and  108  to analyzing volumes  111 ,  112  and  113 , which are at a predetermined distance apart. The scattered light indicated by arrows  114 ,  116 ,  117  is detected by individual detecting systems of the type described. The fluorescent light represented by arrows  118 ,  119  and  121  is detected by individual analyzing systems of the type described above. This arrangement permits analyzing particles which have been tagged with different labels by selecting the wavelength of the light source to excite different fluorescent tags or markers. Alternatively, the plurality of light beams may project onto a single analyzing volume and the individual analyzing systems receive the different fluorescent wavelengths. 
     Rather than sensing particles by light scattered by the particles, a change in electrical current can detect the particles as they travel past spaced electrodes disposed on opposite sides of the flow path. Referring to  FIG. 13 , a capillary  11  is shown with spaced electrodes  123  and  124  which extend into the capillary  11 . The electrodes are spaced along the capillary from the analyzing volume  19 . As the cell or particle flows between the electrodes, the electrically conductive working fluid is displaced and the resulting change in current (impedance) can be detected. This method avoids any laser noise problem. It is usually most convenient mechanically to place the electrodes along the fluid flow path either before or after the point where the laser beam  24  impinges onto the capillary. This creates a timing problem in that the impedance detector will detect a cell at a different point in time than the fluorescence detector, and it is possible that a second cell near the first may create a signal in the fluorescence detector at the same time as the first cell creates a signal in the impedance channel. This necessitates the use of a delay element to shift one signal in time with respect to the other by an amount equal to the distance between the two detectors divided by the flow rate, so that the two signals from one cell become congruent. This delay element may be implemented in hardware with a delay line or circuit.  FIG. 14  shows the output signal  126  from the impedance cell sensor and the fluorescent signal  42  or  43  ( FIG. 1 ) with a delay  127  in the photomultiplier signal, whereby the signals are correlated. This can also be implemented in software by sampling the signal from each detector into its own data stream and then shifting one data stream with respect to the other. A further feature of this arrangement is that, if the physical distance between the two detectors is known, then the actual flow rate can be deduced by finding the delay that corresponds to the best correlation between the two channels; this might be helpful when trying to identify a clogged capillary. 
     As explained above, we have discovered that because the illumination traversing the capillary is in the form of a thin rectangular beam the detection volume is accurately defined by the thickness of the beam and the walls of the capillary  11 . With this in mind, we conducted experiments in which the slits  33  and  34  were eliminated. We found that the results obtained in tests of variously labeled particles were comparable to those obtained with slits. Referring to  FIG. 15 , an embodiment of the invention making use of this discovery is schematically illustrated. The particle detector includes a light source, for example a laser, whose output is optically focused by the optics  23  to form a thin, flat beam  24  as described above. The beam traverses the capillary  11  to define the detection volume  19 . The scatter detector includes an off-axis detector assembly including a collection lens  26   a  and a detector  27   a . As much as possible of the emitted fluorescent light from the tagged cells or particles is gathered or intercepted by an off-axis detector assembly. It can be gathered by a condenser lens as illustrated in  FIG. 1 . However, in the present embodiment, it is collected by a light guide  141  which receives the light  142  and conveys it to the beam splitter  143 . The light beam is directed to optical filters  144  and  146  and directly to detectors  147  and  148 . The output signals from the detectors and the scatter signals are processed to provide particle counts, cell viability, antibody screening, etc. 
     There has been provided a simple-to-use particle analyzing apparatus for characterizing particles such as determining their count, viability, concentration and identification. The analyzing apparatus detects particles in a sample fluid flowing through a capillary tube which has a sampling end for insertion into a sample fluid, and a pump coupled to the other end for drawing sample through the capillary. A light source is provided for projecting a beam of light through a predetermined analyzing volume of the capillary tube to excite fluorescence in particles that flow through the volume. At least one detector is disposed to receive the fluorescent light from excited particles and another detector is disposed to provide a signal representing all particles which flow through the analyzing volume. The output of said detectors provides signals which can be processed to provide the characteristics of the particles. 
     The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best use the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.