Abstract:
An imaging flow cytometry system and method which includes a flow chamber, tracking mirror, microscope and imaging optics, image capturing system, device to regulate fluid flow through the chamber, and backlighting generator. The tracking mirror moves at a rate matched to the particle velocity in the flow chamber so as to enhance the sample flow rates possible with the system while maintaining clear and accurate imaging. The backlighting generator passes through the flow chamber and the objective before being focused on the image capturing system. Detected scatter events initiate tracking by the mirror, resulting in imaging with reduced motion blur even at high rates of flow.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates generally to an optical flow imaging and analysis configuration used in particle analysis instrumentation, and more particularly to an optical flow imaging system and method incorporating a flow chamber and a tracking mirror which sweeps at a rate which is matched to the fluid flow rate, enabling accurate imaging at flow rates much faster than previously enabled. 
       BACKGROUND OF THE INVENTION 
       [0002]    Various optical/flow systems employed for transporting a fluid within an analytical instrument to an imaging and optical analysis area exist in the art. A liquid sample is typically delivered into the bore of a flow chamber and the sample is interrogated in some way so as to generate analytical information concerning the nature or properties of the sample. For example, a laser beam may excite the sample present in the bore of the flow cell, and the emitted fluorescence energy provides signal information about the nature of the sample. 
         [0003]    If the system incorporates particle imaging, the imaging is generally accomplished by generating an extremely short flash to image the passing particle with a CCD or CMOS camera. A flash on the order of 100 microseconds is used, and it is necessary to keep the flow of the sample to less than one-tenth of a milliliter per minute to prevent motion blurring in the resulting images. 
         [0004]    The inefficiencies of standard methods of optically imaging with a very short flash, an objective lens and a CCD camera include using a very slow sample flow to prevent image blur, low image illumination energy from the sample, and accidental imaging of contamination on the walls of the flow cell. Therefore, there is a need in the art for an effective way to prevent image blur and allow longer exposures when imaging a rapidly-moving sample. 
       SUMMARY OF THE INVENTION 
       [0005]    It is an object of the present invention to provide an imaging flow cytometry system and method with improved sample fluid flow rate. It is also an object of the present invention to provide such an improved flow rate imaging flow cytometer system and method that may be incorporated into, or used with, existing imaging flow cytometers and provide better images with reduced blurring. These and other objects are achieved with the present invention, which enables imaging of higher than conventional sample flow rates through introduction of a tracking mirror, wherein the movement of the mirror is associated with the fluid flow rate. In other words, the tracking mirror is configured to track particles passing in the fluid at a known flow rate. In one embodiment, the imaging flow cytometry system and method of the present invention includes a scanning galvanometer mirror, a galvanometer driver circuit, one or more high current power supplies, a modification of an imaging flow cytometer&#39;s digital signal processor, and ramp generator electronic circuitry. The tracking minor allows the imaging system to track particles as they flow through the flow chamber, enabling clear imaging of the particles even when the sample is moving quickly. Specifically, when properly controlled, the tracking mirror reflects the magnified image of the particle obtained at particular moments by the backlighting generator to the same points on the face of the camera, correcting for motion associated with the sample flow. As such, the camera is able to image passing particles for a longer time without motion blur and obtain a clearer image of the particle than is otherwise possible. This configuration allows a dramatically improved sample flow rate suitable for analyzing large samples in a short span of time while obtaining clear and accurate images of particles in the sample. Use of the system and method of the present invention also prevent samples under examination from spoiling or deteriorating due to long processing times required when sample flow rates are low. 
         [0006]    On the image capturing side, the present invention is an optical system and method including a light source and an image capturing system. In one embodiment, the present invention includes a backlighting generator, an image capturing system, a microscope objective, a rectangular flow chamber of known dimensions, a device which draws the sample through the flow chamber at a well regulated rate, an imaging objective, as well as an electronic ramp generator circuit, a galvanometer and mirror which can be controlled by the ramp generator, and a galvanometer driver circuit which can control the galvanometer with the ramp waveform. In this embodiment, high current power supplies are also needed for proper operation of the various elements. In a preferred embodiment, the image capturing system includes a camera. In a more preferred embodiment, the camera is a CCD or CMOS camera. 
         [0007]    If the tracking mirror involves a galvanometer, the galvanometer and mirror are controlled by a ramp generator to allow the camera to track particles in the flow of sample as they are passing in front of the objective within the flow chamber by matching the sweep of the mirror to the well-controlled sample flow rate. The flashing imaging light source generates light which passes through the flow chamber and then the objective before being focused onto the imaging camera. If fluorescence emissions are monitored by the system, they are deflected by another mirror to appropriate detectors. This combination enables high clarity images in the flow cytometry imaging system and method of the present invention. Specifically, the present system and method allow higher sample flow and higher quality images than available with existing imaging cytometry. Further, the invention allows the use of longer exposure times for imaging, resulting in brighter, less noisy images. In addition, the invention prevents imaging flow cytometers from imaging blemishes on the flow chamber walls since they are smeared or blurred beyond recognition. In contrast, state of the art imaging flow cytometers image the flow cell channel with a flash and consequently, will image any particles or blemishes on the channel walls clearly in addition to imaging desired particles in the sample. In the present invention, moving the mirror during the flash results in a motion smeared image of these particles or blemishes and will blend them in with the background, making such particles easier to avoid with image capturing. 
         [0008]    These and other advantages of the present invention will become more readily apparent upon review of the following detailed description, the accompanying drawings, and the appended claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]      FIG. 1  schematically illustrates one embodiment of the system of the present invention for imaging particles in a fluid. 
           [0010]      FIG. 2  is a block diagram of the signal processor designed for use in one embodiment of the invention. 
           [0011]      FIG. 3  illustrates the timing of the sweep and imaging signals in relation to the triggering light signal. 
           [0012]      FIG. 4  illustrates the details of the sweep and imaging signals. 
           [0013]      FIG. 5  illustrates a schematic of one embodiment of the programmable electronic ramp generator for use in one embodiment of the invention 
           [0014]      FIG. 6  is a schematic representation of the relationship between a particle in the fluid flow, the objective, the tracking mirror and the imaging device. 
           [0015]      FIG. 7  is a collection of images of marine plankton taken with a current state of the art imaging flow cytometer with a high speed sample flow rate. 
           [0016]      FIG. 8  is a collection of images of marine plankton taken with a sweeping mirror enhanced imaging flow cytometry system of the present invention operating at a high speed sample flow rate. 
           [0017]      FIG. 9  is another collection of images of marine plankton taken with a sweeping mirror enhanced imaging flow cytometry system of the present invention operating at a high speed sample flow rate. 
           [0018]      FIG. 10  is a flow diagram representing steps to be carried out using the sweeping mirror enhanced imaging flow cytometry system of the method of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0019]    One embodiment of a system  10  of the present invention suitable for high speed automated counting and/or imaging of particles in a fluid is shown in  FIG. 1 . The system  10  includes a flow chamber  12 , a backlighting generator  14 , particle scatter and fluorescence detectors  16 ,  18 , a signal processor  20 , an image capturing system  22 , a computing device  24 , a scan generator circuit  26  including high current power supplies and galvanometer driver electronics to control programmable ramp generator  56 , a scanning galvanometer and mirror combination  28 , and a pump  30  capable of delivering a controllable fluid flow rate. The embodiment of the system  10  depicted in  FIG. 1  also includes imaging and analysis optics such as the microscope objective  32 , dichroic mirror  34 , partial mirrors  36 ,  36 ′, and lenses  38  and  38 ′, although other configurations are possible. The combination of the components of the system  10  arranged and configured as described herein enable a user to detect and image particles without blurring in a fluid sample at flow rates not possible with existing imaging flow cytometers. 
         [0020]    The flow chamber  12  includes an inlet  40  for receiving the particle containing fluid to be observed, and an outlet  42  through which the fluid passes out of the flow chamber  12  after imaging and particle optical measurement functions have been performed. The flow chamber  12  is a low fluorescence structure of known dimensions. That is, it must be fabricated of a material that does not readily fluoresce, for example, but not limited to, microscope glass or rectangular glass extrusions. The flow chamber  12  is of rectangular shape and defines a channel  44  through which the fluid flows at a predetermined controllable rate. In some embodiments, the channel  44  within the flow chamber  12  is of rectangular configuration with a known cross sectional depth (D) and width (W). An example of a suitable form of the flow chamber  12  is a W1050 Vitxotube from Vitrocom, Inc. (River Lakes, N.J., US). The inlet  40  of the flow chamber  12  is connectable to a fluid source such as sample  46  and the outlet  42  is connectable to a downstream device for transferring the fluid away from the flow chamber  12  at a well-controlled, steady and adjustable rate. A suitable example of such a fluid transfer device is the pump  30 , which may be a model 210 programmable syringe pump from KD Scientific, Inc. (Holliston, Mass., US). 
         [0021]    A light source  48  is used to generate fluorescence and scatter light directed to the flow chamber  12 , resulting in particle fluorescence and/or light scatter. The light source  48  may be a laser with, an excitation filter  50 . The light source  48  may be, but is not limited to, a 473 nanometer (nm), 488 nm or 532 nm solid state model laser available from an array of manufacturers known to those of skill in the art. The excitation filter  50  should at least have the characteristic of being able to transmit light at wavelengths longer than the wavelengths of light generated by the light source  48 . An example of a suitable form of the excitation filter  50  is a 505DCLP longpass filter available from Chroma Technologies (Rockingham, Vt., US), which can be used with a 488 nm laser. Those of skill in the art will recognize that other suitable filters may be employed for the excitation filter  50 . 
         [0022]    Any particle fluorescence emissions from the flow chamber  12  that have a wavelength of 535 to 900 nm are detected by the detection system, which includes at least one or more emission filters  52  and one or more high sensitivity photomultiplier tubes (PMTs)  54  within the fluorescence detector  18 . The emission filters  52  should at least have the characteristic of being transparent to the fluorescence emissions of a desired fluorophore. An example of a suitable form of an emission filter  52  is a 570/40 phycoerithryn emission filter available from Chroma Technologies (Rockingham, Vt., US); those of skill in the art will recognize that other suitable filters may be employed for the emission filter  52 . The PMTs  54  should at least have the characteristic of being sensitive to the fluorescence emissions desired. An example of a suitable PMT is the H9656-20 model available from Hamamatsu (Bridgewater, N.J., US); those of skill in the art will recognize that other equivalent PMTs may be employed for the PMT  54 . 
         [0023]    Preferably, the signal processor  20  includes a user adjusted threshold setting which determines the amount of fluorescence or scatter required for the system  10  to acknowledge a passing particle. For example, and in no means limiting the scope of the invention, the user may set the threshold to be 200 (dimensionless cytometer fluorescence or scatter units). One embodiment of a signal processor  20  that can be used in the system  10  or method of the present invention is shown in  FIG. 2 . Scatter and fluorescence inputs are processed by conditioning amplifiers where they may be amplified and/or converted to their logarithm for better dynamic range as is commonly done in flow cytometers. These signals are then converted to digital signals which are analyzed by the signal processor  20 . Programming of the signal processor  20  determines how it analyzes and reacts to these inputs. In this invention, the signal processor  20  is programmed to monitor the scatter and fluorescence inputs and, if any of these inputs are greater than a predetermined threshold, initiate the signal sequence, also called the particle tracking interval, seen in  FIGS. 3 and 4 . 
         [0024]    When an input is greater than a predetermined threshold, indicating presence of a particle to be imaged, for example, the signal processor  20  initiates a particle tracking interval, as shown in  FIGS. 3 and 4 . The first step of the particle tracking interval is initiation of a mirror pulse, which is converted to a mirror ramp signal by the programmable ramp generator  56 . After initiation of the mirror pulse and ramp, a camera trigger and then a flash signal to the backlighting generator  14  are initiated. The exposure of the camera and resultant imaging overlap the period where the sample is illuminated by the flash. Representative samples of the time periods for each element of the particle tracking interval are shown in  FIG. 4 . Input from a scatter and/or fluorescence detector initiates the particle tracking interval, which starts with initiation of the mirror pulse after a brief delay. The mirror pulse is converted to the ramp signal, and the pulse and ramp may run for approximately 1000 μseconds. After approximately 200 μseconds the mirror is moving sufficiently to start tracking and imaging particles and a brief camera trigger signal is initiated. The trigger initiates a flash and the camera exposure, which is of controlled duration. In  FIG. 4  the flash and associated imaging are shown as occurring over approximately 100 μseconds. The time periods described herein are examples only, and it is to be understood that other time periods or timing conditions may be established without deviating from the invention. 
         [0025]    Programmable ramp generator  56  may be configured to sweep its output voltage at different rates, depending on its setting. The functions of the ramp generator  56  are achieved by the structure shown in the schematic of one specific embodiment shown in  FIG. 5 . The ramp generator  56  receives a ramp parameter control signal from the computing device  24  which sets the internal resistance R of the digital potentiometer U 1 . This resistance determines the rate at which the ramp voltage rises. Together, components R, R 5  and C 1  determine the change rate of this ramp voltage with time when transistor Q 3  is turned off. The voltage change rate is determined from the charge rate of capacitor C 1 , which generates a voltage of 0.632 times the voltage +5V in a time of (R+R 5 )*C 1  in this example. When the mirror pulse signal from the signal processor  20  makes a high to low transition, the bipolar transistor Q 3  turns off and the capacitor C 1  begins charging at this charge rate. 
         [0026]    It is to be understood that  FIG. 5  depicts only one type of ramp generator  56  suitable for use in the present invention. Those skilled in the art can readily envisage alternative computer interfaces that could be used with different ramp generators  56  to achieve the same results. Provided that one skilled in the art knows the flow rate of the pump and the voltage to angle galvanometer constant (that is, the change in the angle of the galvanometer corresponding to a particular voltage increase), the digital potentiometer of the ramp generator can be set so that the ramp generator will match the mirror sweep rate to the predicted particle speeds. 
         [0027]    If a sufficiently fluorescent or light scattering particle passes through the flow chamber  12 , a signal from the scatter detector  16 , fluorescence detector  18 , or PMT  54  is sent to the signal processor  20 . The signal processor  20  then generates a trigger signal which is transmitted to the imaging camera  22  through the computing device  24 , and a pulse is also sent to the ramp generator  56 . An example of a suitable computing device  24  is a desktop or laptop Pentium class processor based personal computer. The primary functions of the computing device  24  are to control the signal processor  20  and ramp generator  56  and to read in and analyze the images from the image capturing system  22  and the measurements from the signal processor  20  and to collate the measurements and images. 
         [0028]    Once the ramp pulse is sent to the ramp generator  56 , the ramp generator  56  generates a voltage ramp which is used to steer the scanning galvanometer and mirror combination  28  to track the passing particle. An example of a suitable galvanometer and mirror combination  28  is model 6210H galvanometer with a 6 mm diameter mirror available from Cambridge Technology, Inc., (Cambridge, Mass., USA). An example of suitable galvanometer driver electronics is a model 677 circuit board from Cambridge Technology, Inc. Prior to the beginning of a run of images and fluorescence and scatter measurements, the ramp generator  56  is programmed to sweep the galvanometer and mirror combination  28  at a rate which allows for the camera  22  to track the passing particles. As shown in  FIG. 6 , a particle which is passing at velocity v generates an image from the microscope objective  32  which moves across the mirror at a speed of Mv, where M is the system magnification. To compensate for this, the galvanometer and mirror combination  28  which is a distance r from the camera must turn at an angular rate of δθ/δτ=Mv/r in order to reflect the image of the particle to the same spot on the camera for as long as possible. Given the flow rate and flow chamber/cell  12  dimensions, the galvanometer and mirror combination  28  must move at an angular velocity of θ/δτ=Flow/(D×W) where D and W are the depth and width of the flow chamber  12 . 
         [0029]    In other embodiments, the tracking mirror scan rate may be adjusted manually or automatically without requiring knowledge of the dimensions of the flow chamber  12 . Manual adjustment of the galvanometer/mirror combination  28  embodiment is possible if the instrument is placed in an image acquisition mode with the value of the digital potentiometer adjustable via a computer “dialog box” or “computer controlled slider” and if the user is able to adjust the image clarity while looking at the acquired images. In an automatic adjustment mode, it is possible that the image acquisition software can adjust the image clarity by changing the value of the resistance R of the digital potentiometer. Since the image clarity is measured by the image “edge gradient,” in an automated adjustment scenario, the edge gradient may be maximized by the software while the software is adjusting the value of R. 
         [0030]    The backlighting generator  14  is configured to flash while the galvanometer/mirror combination  28  is sweeping, as shown in  FIGS. 3 and 4 . In the fluorescence and scatter mode of operation, when a fluorescent or light scattering particle passes through the area illuminated by the light source, the particle generates a signal which the signal processor  20  monitors. The signal processor  20  carries out an analysis interval to determine if the signal is strong enough to track, i.e., above the predetermined threshold. For example, particles of interest should emit signals significantly stronger than simply noise or small particles of debris in the sample. If the signal is strong enough as determined during the analysis interval, the signal processor  20  initiates a particle tracking interval with a mirror pulse. The mirror pulse is converted to a mirror ramp signal by the programmable ramp generator  56 . The mirror pulse/ramp is followed by a camera trigger pulse and then a flash signal to the backlighting generator  14 . The computing device  24  then reads in the resulting image and data regarding the scatter and/or fluorescence data. The computing device  24  is programmed to store the information received from the signal processor  20  and to make calculations associated with the particles detected. For example, but not limited thereto, the computing device  24  may be programmed to provide specific information regarding the fluorescence of the detected particles; the shape of the particles, dimensions of the particles, and specific features of the particles. The computing device  24  may be any sort of computing system suitable for receiving information, running software on its one or more processors, and producing output of information, including, but not limited to, images and data that may be observed on a user interface. 
         [0031]    The signal processor  20  is also connected to the backlighting generator  14 . The signal processor  20  may include an arrangement whereby a user of the system  10  may alternatively select a setting to automatically generate a particle tracking interval at a selectable time point or at particular time intervals. The particle tracking interval generated produces a signal to activate the operation of the galvanometer ramp generator  56  and the backlighting generator  14  so that a light flash is generated. Specifically, the backlighting generator  14  may be a light emitting diode (LED) or other suitable light generating means that produces a light of sufficient intensity to backlight the flow chamber  12  and image the passing particles. In one embodiment the backlighting generator  14  may be a very high intensity LED flash such as a 670 nm LED flash, or a flash of another suitable wavelength, which is flashed on one side of the flow chamber  12  for 200 μsec (or less). At the same time, the image capturing system  22  positioned on the opposing side of the flow chamber  12  is activated to capture an instantaneous image of the particles in the fluid as “frozen” when the high intensity flash occurs and the galvanometer/mirror combination  28  tracks the particle. The image capturing system  22  is arranged to either retain the captured image, transfer it to the computing device  24 , or a combination of the two. The image capturing system  22  includes characteristics of a digital camera or an analog camera with a framegrabber or other means for retaining images. For example, but in no way limiting what this particular component of the system may be, the image capturing system  22  may be a CCD firewire, a CCD USB-based camera, a CMOS camera, or other suitable device that can be used to capture images and that further preferably includes intrinsic computing means or that may be coupled to computing device  24  for the purpose of retaining images and to manipulate those images as desired. The computing device  24  may be programmed to measure the size and shape of the particle captured by the image capturing system  22  and/or to store the data for later analysis. 
         [0032]    The advantages associated with the sweeping mirror enhanced imaging flow cytometer system  10  of the present invention may be readily observed by viewing the images represented in  FIGS. 7-9 .  FIG. 7  shows a plurality of images of individual marine phytoplankton contained in a fluid as captured using an imaging flow cytometry system without a tracking mirror with a sample flow rate of 2.5 ml per minute, which is 10 times the normal sample processing rate for a system of this configuration. A 100×2000 micrometer flow chamber cross section, a magnification of 10× and an imaging flash duration of 100 microseconds were used.  FIG. 8  shows a plurality of images of individual marine phytoplankton cells from the same fluid but as captured using the system  10  of the present invention with a sample flow rate of 2.5 ml per minute, a 100×2000 micrometer flow chamber cross section, a magnification of 10× and an imaging flash duration of 100 microseconds.  FIG. 9  shows a plurality of images from the same sample but as captured using the system  10  of the present invention with a sample flow rate of 4 ml per minute, a 100×2000 micrometer flow chamber cross section, a magnification of 10× and an imaging flash duration of 100 microseconds. It can be easily observed that the system  10  of the present invention generates substantially sharper, less blurry images than available with the prior system even when operating at much higher sample flow rates than would otherwise be possible. 
         [0033]    As represented in  FIG. 10 , a method  200  of the present invention includes steps associated with capturing images with the system  10  of the present invention. Several processes occur on a continuous basis during normal operation. For example, in one embodiment, the pump  30  draws the sample through the flow chamber  12  at a constant rate. The flow chamber  12  is illuminated with excitation light from the laser  48  continuously. The scatter and fluorescence detectors  16 ,  18  provide fluorescence and scatter analog waveforms to the inputs of the signal processor  20 . Finally, the signal processor  20  continuously reads these signals. 
         [0034]    In addition to these continuous processes, discrete steps are carried out. During step  201 , fluorescence signals from the PMTs  54 , and/or scatter detector  16 , are compared to a preset threshold. If the signals are not greater than the threshold, the waveforms are measured again in step  202 . If they are greater than the threshold, the digital signal processor  20  executes step  203 , where the signal processor  20  generates a particle tracking interval by initiating the timers that control the mirror pulse and ramp, camera trigger, and flash signals. Executing step  203  causes the programmable ramp generator  56  to generate a mirror pulse and ramp, generating a voltage ramp which is used to steer the scanning galvanometer and mirror combination  28 . This causes the galvanometer/mirror combination  28  to track the passing particle. Executing step  203  also activates the image capturing system and flash so that the system  10  can capture an image of the passing particle while the high intensity flash occurs. The tracking, triggering and the imaging flash all occur within the period that the mirror pulse and ramp are occurring, as shown in  FIGS. 3 and 4 . During step  204  of the method of the present invention the image capturing system  22  transfers the captured image to the computing device  24 . During the image analysis step  205 , the computing device analyzes the image for particles and if any particles with acceptable characteristics are found, the device stores their images and their fluorescence, scatter and other measurements. 
         [0035]    The present invention has been described with respect to various examples. Nevertheless, it is to be understood that various modifications may be made without departing from the spirit and scope of the invention. All equivalents are deemed to fall within the scope of this description of the invention.