Particle sorting machine

Abstract

A particle sorter that sorts particles carried by a fluid flow. The particle sorter comprises a particle detection system for detecting particles in a fluid flow, a particle deflection system for selectively deflecting particles from the fluid flow to sort them, an optical imaging system positioned to acquire images of particles in the fluid flow to identify particles at different locations in the fluid flow, an illumination source for selectively illuminating the fluid flow when acquiring images of particles, and an electronic control system. The particle sorter is configured such that, when the particle detection system outputs a signal for a detection event, the electronic control system controls the imaging system and the illumination source to acquire one or more images of particles in the fluid flow within one or more time delays from the detection event, and the electronic control system processes one or more images and the time delays of one or more time delays to determine one or more parameters for the sorting process.

Description

Technical Field

[0001] The present invention relates to a particle sorter.

Background Art

[0002] Equipment for particle sorting is widely used in biological research. The main application of particle sorting technology in this field is to sort biological cells. Equipment for sorting cells based on the measurement of fluorescent labels within the cells is typically known as fluorescence-activated cell sorting (FACS). Other applications of particle sorting include the sorting of solid beads or droplets of one liquid phase in a carrier fluid. For example, aqueous droplets in a non-aqueous carrier fluid can be used to contain cells.

[0003] In conventional particle sorters, after detecting individual particles in a flowing stream, jets are generated through nozzles, which are ejected through air and separated into droplets, and electrostatically guided to one or more output containers. One of the main drawbacks of conventional particle sorters when used for sorting biological cells is that the cell suspension is exposed to air and non-sterile surfaces. Thus, the particle sorter may pose a biological hazard to the user due to aerosol leakage. Also, in a particle sorter, contamination of the cell suspension by microorganisms on non-sterile surfaces, or cross-contamination of cells due to carry-over of a previous sample in the same wet portion, may occur. To enable sterile cell sorting and to obtain many other potential advantages, there are various microfluidic particle sorters. Similar to conventional sorters, microfluidic sorters measure individual cells in a fluid stream. However, microfluidic sorters employ a number of mechanisms for deflecting target particles.

[0004] All of the above particle sorters typically include means for measuring individual particles in a fluid flow, an electronic control system capable of distinguishing ("gate") particles into target and non-target populations in real time based on these measurements, and a deflection mechanism precise enough to deflect individual particles when the mechanism is activated when the particles are at a certain position in the fluid flow (referred to as the "operating position" or "deflection position"). In any of the above cell sorters, an "operating delay time," defined as the interval between a detection event and the activation of the deflection mechanism, must be set in the electronic control system. This operating delay time must be exactly equal to the transit time required for the target particles to move between the detection position and the operating position.

[0005] To determine the passage time and therefore set the operating delay time required for all types of particle sorters described above, the user must pass fluorescent tracer beads through the system before the cell sample flows through. The downstream region of the deflection is then observed using bright lighting and an electronic video camera with an exposure time long enough to image the average path of the fluorescent particles. The operating delay time is then scanned until the fluorescent particles are deflected to the desired path.

[0006] The drawback of the above method for setting the operating delay time is, firstly, that the passage time can be affected by many physical variables, such as the velocity of particles in the fluid flow, the temperature and viscosity of the medium, and the detection and operating positions. Even if the operating delay time is set immediately before the biological sample is passed through, many of these effects can cause rapid fluctuations in the passage time, potentially degrading the performance of the sorter. If the operating delay time and passage time are not equal, the target particles will not be deflected, resulting in a loss of purity and yield in the sorting process.

[0007] As described above, it is necessary to improve the accuracy of the sorting process and, preferably, simplify the manufacturing and setup of the particle sorter. It is also preferable to avoid the need to use tracer beads. [Overview of the project] [Means for solving the problem]

[0008] The present invention provides a particle sorter for sorting particles carried in a fluid flow, the sorter comprising: a particle detection system for detecting particles in a fluid flow; a particle deflection system for selectively deflecting particles from the fluid flow to sort them; an optical imaging system arranged to acquire images of particles in the fluid flow to identify particles at different locations in the fluid flow; an illumination source for selectively illuminating the fluid flow when acquiring images of particles; and an electronic control system, the particle sorter is configured, during use, to control the imaging system and the illumination source so that when the particle detection system outputs a signal of a detection event, the electronic control system acquires one or more images of particles in the fluid flow within one or more time delays from the detection event, and the electronic control system processes one or more images and the time delays of one or more time delays to determine one or more parameters for the sorting process.

[0009] In the particle sorter of the present invention, each detection event triggers a camera and illumination with a precise time lag (also called a time delay) starting from the event detection. The position of the particles within the camera frame can be automatically detected using a machine vision algorithm. Therefore, in each frame, the precise particle position is measured with a precise time lag.

[0010] The particles to be sorted may be, for example, cells, beads, or droplets containing further particles. In this specification, the terms “cell sorter” and “particle sorter” are used interchangeably, and the inventors mean a device that can be used to sort any or all of the above particles suspended in a carrier fluid. The particle sorter may be a microfluidic particle sorter.

[0011] Particle deflection within the sorter may be generated in several ways, for example, by employing hydrodynamic forces via high-speed transducers such as sonic waves, electrophoretic forces, optical forces, magnetic forces, microscopic valves, or piezoelectric or thermal steam bubble actuators.

[0012] The particle sorter of the present invention allows the operation delay time (defined as the interval between a detection event and the operation of the deflection mechanism) to be set as follows: The value of the time lag can be adjusted until the particle is at the operation position. At this point, the time lag is equal to the transit time. The operation delay time is then set to be equal to this time. For this first function, the field of view of the imaging system preferably includes at least the operation location. The transit time it takes for the target particle to move between the detection position and the operation position is an example of a measurement parameter for the sorting process. The operation delay time is an example of a control parameter that is adjusted for the optimal operation of the particle sorter.

[0013] The particle sorter of the present invention may trigger a single flash to illuminate the fluid flow after a time delay, or trigger a camera frame to be acquired after a camera time delay, both of which may be triggered in sync with the corresponding particle detection event. The camera time delay can be selected so that the camera shutter opens when illumination occurs. The time delay may be varied to acquire various information about the particles being sorted. The time delay can be set to be equal to the operating time delay so that the particle position can be measured in the image when operating. A typical flash duration is currently 0.8 microseconds, and generally Furthermore, it should be on the order of less than 1 microsecond.

[0014] A second function of the particle sorter may be to adjust the operating amplitude. Here, the value of the time lag is increased so that the particle is imaged at a position where its output path is visible in the image. The operating amplitude can then be adjusted until the particle deflection corresponds to the target flow path. With respect to this second function, the field of view of the imaging system preferably includes at least one position downstream of the operating location after the particle deflection has occurred.

[0015] A third function of the particle sorter of the present invention may be to calculate the percentage of detection events sampled so that particles are deflected within a predetermined amplitude range so that they flow to the correct output (along the target flow path). The inventors of this application use the term "fidelity" for this ratio. Automatically measuring and informing the user of the fidelity in a cell sorter is useful because fidelity is a fundamental check that the sorting process provides a high-purity and high-yield output.

[0016] A fourth function of the particle sorter of the present invention may be to calculate the percentage of detection events sampled so that particles are present at the precise actuation position. The inventors of this application use the term “actuation presence” for this percentage. Automatically measuring and notifying the user of actuation presence in a cell sorter is useful because it serves as a basic check that particle detection events correspond to the precise time and location, and that the velocity of particles between the detection position and the actuation position is well controlled.

[0017] A fifth function of the particle sorter of the present invention may be to calculate the frequency of imaged particles present in the deflection path that are not synchronized with the deflection event. The value obtained by dividing this frequency by the sampling time in the deflection path is equal to the "output error rate". Automatically measuring the output error rate and notifying the user of it is useful as it serves as a basic check that there are no particle focusing failures that would adversely affect yield and purity.

[0018] A sixth function of the particle separator of the present invention may be to measure the standard deviation of particle positions in a direction perpendicular to the flow direction at the operating position. The inventors of this application define this measurement as "particle focusing quality." Automatically measuring and reporting particle focusing quality to the user is useful as it provides a further basic check to ensure that there are no particle focusing failures that could adversely affect yield and purity.

[0019] A seventh function of the particle sorter of the present invention may be to measure the standard deviation of particle positions in a direction parallel to the flow direction at the operating position. The inventors of this application define this measurement as "particle position jitter." Automatically measuring and reporting particle position jitter to the user is useful because it serves as a basic check to ensure that there are no failures in particle detection timing that could adversely affect yield and purity.

[0020] An eighth function of the particle separator of the present invention may be to measure the arrival and accumulation of debris within the particle separator. The inventors of the present application define several measurements depending on where the debris arrives. Each measurement is the area of ​​the camera frame occupied by the debris divided by the area associated with the location. For example, debris at the operating position or debris at a downstream position where the flow path branches is called the "actuation location ratio". It is defined as "gunk proportion" and "junction gunk proportion." Automatically measuring and notifying the user of the gunk proportion is useful because it serves as a basic check that the sample preparation is sufficient to avoid blocking in the particle sorter. This is useful because it allows the sorting machine to be automated to unblock the flow path by changing the flow path when a gunk is detected.

[0021] Examples of sorting process parameters include particle position related to the operating position, the strength of the measured particle deflection, the presence of particles in the target flow path, the time for a particle to pass from the detection position to the operating position, fidelity, and operating presence.

[0022] Examples of control parameters include operating time delay, operating amplitude, illumination (e.g., strobe) time delay, and camera time delay. Further, the control parameters can include one or more operating control parameters that control how the deflection system operates, for example, defining a specific voltage waveform to achieve the desired deflection of the particles, defining the simple magnitude and / or duration of the voltage signal, or defining the amplitude and / or duration of the acoustic wave in the case of acoustic wave operation.

Brief Description of the Drawings

[0023] [Figure 1] FIG. 1 shows a schematic configuration diagram of a prior art particle sorter, FIG. 1A shows a general-purpose particle sorter that explains both a conventional particle sorter and a microfluidic particle sorter, FIG. 1B shows a conventional particle sorter, and FIG. 1C shows a vortex-driven particle sorter. [Figure 2] FIG. 2 shows a schematic diagram of the optical measurement system of the particle sorter used in the present invention. [Figure 3] FIG. 3 shows a configuration diagram of the optical measurement system of the particle sorter according to the first embodiment of the present invention. [Figure 4] FIG. 4 shows a schematic diagram of the control system of an exemplary particle sorter according to the present invention. [Figure 5] FIG. 5 shows the control system of the particle sorter according to the first embodiment of the present invention. [Figure 6] FIG. 6 shows a schematic diagram of the machine vision algorithm of the particle sorter capable of identifying particles within the camera frame used in the present invention. [Figure 7] FIG. 7 shows the machine vision algorithm of the particle sorter capable of identifying particles within the camera frame according to the first example of the present invention. [Figure 8]FIG. 8 shows an example of the results of using the particle sorter according to the present invention to set the actuation delay time and the actuation amplitude. [Figure 9] FIG. 9 shows the machine vision detection mask of the cell sorter within the camera frame according to the first embodiment. [Figure 10] FIG. 10 shows exemplary results of notifying a user of functional measurement values via a graphical user interface. BEST MODE FOR CARRYING OUT THE INVENTION

[0024] Referring to the drawings, FIG. 1 shows the configuration in prior art sorting. A schematic diagram of a general particle sorter is shown in FIG. 1A. In this apparatus, the particle stream is focused into a flow path 101 that passes through a detection region 102 including one or more detection positions 103, 104, 105 where the particles are measured. As each particle passes through a detection position, a time-resolved signal is provided to the particle detection system. When there are multiple detection positions, the signals from the upstream detection positions 103, 104 are aligned with the signal from the downstream detection position 105 to give an "event". An event is the passage of a particle that occurs at a certain time at a certain detection position, at which time a set of measurements including fluorescence measurement, light scattering measurement, and time measurement at one or more detection positions are performed on the particle. The event is processed by an electronic control system, which determines based on the set of particle measurements whether the particle belongs to one or more target gates, and thus whether to sort the particle into the output streams corresponding to each target gate. For example, the general particle sorter of FIG. 1A has two output streams 108 ("two-way sorter") corresponding to two target gates, as well as a waste stream 109 for non-target particles. When a decision is made to deflect a particular particle, the deflection is performed by a deflection mechanism 106, and the deflection mechanism 106 acts on the particle downstream of the detection position at the operating position 107, which is a specific location through which the particle passes. The electronic control system is programmed to activate the deflection mechanism with a precise "operating amplitude" and a precise "operating delay time" after the event, so that the particle enters the required output stream. The operating delay time and operating amplitude are parameters required by the electronic control system and both must be precisely determined. The operating delay time must be set to be equal to the time it takes for the target particle to pass between the detection position and the operating position; otherwise, the target particle will not be sorted. The operating amplitude must be set so that the particle at a particular target gate is deflected and sent to the corresponding output stream. As described above, a decrease in the accuracy of these control parameters can very quickly lead to inefficient sorting and malfunction.

[0025] In conventional particle sorters, the operating position is the point where droplets separate from the flow. The solution to the timing problem is, firstly, to have a stable particle velocity (requiring stable pressure and temperature of the equipment and fluid medium), and secondly, to stabilize the droplet separation point (requiring a stable flow stimulus). Droplet separation is typically monitored using stroboscopic video imaging. The flow is imaged by a video camera at the point where droplets separate. Illumination is provided by regular pulses at a frequency equal to the droplet stimulus frequency. The phase of the illumination pulses is then scanned to determine the precise location of droplet formation and to ensure that this location does not move. In a typical setup, the camera exposure period is much longer than the stimulus period. Therefore, many illumination pulses are captured in a single camera frame to provide an averaged image of droplet formation over many stimulus cycles. In this way, the stroboscopic imaging system determines the operating position as defined above. However, this stroboscopic system cannot determine the operating delay timing for two reasons. Firstly, since particles cannot be observed within the droplet, the velocity of particles in the flow is unknown and therefore cannot be measured by this method. Secondly, stroboscopic illumination is synchronized with the vibration of the droplet stimulus, not with the detection event. The detection event does not actually correlate with the phase of the vibration of the droplet stimulus.

[0026] Another prior art particle separator is shown in Figure 1B, which has a similar configuration to the particle separator in Figure 1A. The flow enters the cuvette 111 under high pressure. The particles are focused into the channel 112 by hydrodynamic focusing (upstream in the figure (not shown)), and the channel passes the particles through a series of laser foci 113, which are the detection positions described above. A set of fluorescence, light scattering, and time measurements is performed for each particle. The particle suspension exits through the nozzle 114 in a jet 115 that separates the particles into droplets. The fluid is acoustically stimulated so that each droplet separates in a precise phase of the stimulation period. The position 116 where the droplets separate is the operating position described above. With a precise operating time delay after the detection event, the control system applies a precise voltage to an electrode 117 that is in contact with the particle suspension upstream of the nozzle. In this example, this voltage is the operating amplitude. The voltage generates electric polarization of the jet, thereby electrostatically charging the droplets in proportion to the voltage at the time of separation. The droplet 120 then flies toward the set of output receptacles 121, but the electrostatic field between the two charged plates 118 causes the droplet to deflect in proportion to the charge trapped in the droplet. One flow ("waste flow") is set to receive non-target particles 119. The operating amplitude needs to be further adjusted to compensate for the electrostatic interaction between the current droplet and the preceding droplet. It is important that the jet velocity and droplet separation position are stable, because fluctuations in either the jet velocity or the droplet separation position will cause the operating delay time to not equal the transit time, reducing the accuracy of sorting.

[0027] Another microfluidic particle sorter is shown in Figure 1C, which has a similar configuration to the particle sorters in Figures 1A and 1B. The flow is generated within the microfluidic channel on the tip 131. The particles enter a focused flow 132 that passes through one or more laser foci (133, 134) at precise points within the microfluidic channel 135. These laser foci are the detection points described above. The particle measurements from all laser focal points are combined within the electronically controlled system such that each event in which a particle passes through a downstream laser focal point 133 is accompanied by a set of particle measurements (fluorescence, scattering, and time measurements from all laser focal points). Then, during the activation delay time after the event, the electronically controlled system applies a voltage pulse across the microresistor 136. The voltage pulse is defined by its amplitude (activation amplitude) and time interval. This voltage pulse heats the resistor, causing a film of superheated liquid in contact with the resistor to form a thermal vapor bubble. The thermal vapor bubble expands rapidly within a few microseconds, causing a transient flow across the vortex-generating tip 137 in the channel wall, thereby forming a transient vortex in the flow downstream of this tip. If, when the activation occurs, the particle is at the activation position 138 close to the tip 137, the particle flows downstream simultaneously with the vortex, causing its deflection from the non-deflection channel 139 to the deflection channel 140. These two paths enter two corresponding output sections 142 and 143 downstream of a junction 141 defined by the walls of the microfluidic channel. The two output channels are referred to as “Sort0” (un-deflected) and “Sort1” (deflected). Figure 1C shows a device with a single output 143 for deflected particles (a “unidirectional sorter”), although this type of sorter can accommodate several output channels branching off from the input channel. The voltage of the operating pulse determines the strength of the transient vortex that changes the amplitude of the deflection perpendicular to the streamlines of the undeflected particles. Thus, this deflection mechanism can direct particles toward a selected output channel from among multiple output channels (in embodiments including multiple output channels). This provides accurate and reliable sorting, although high control parameter precision is still required to maintain it.

[0028] Figure 2A shows a schematic diagram of the optical instrumentation of the particle sorter used in the present invention. The channel 250 carries the fluid flow through which the particles are transported. The channel 250 is illuminated by one or more lasers, and at least one of forward scattered light, occultation, side scattered light, and fluorescence is measured. (Occultation is the loss of light from a non-bent beam due to scattering and absorption. Occultation is a nearly constant signal obtained by subtracting forward scattered light from the direct beam, and therefore has nearly the same information as true forward scattered light, and is therefore commonly referred to as "forward scattering" in the art of this application.) The measurement of at least one of forward scattered light, occultation, side scattered light, and fluorescence is performed by a particle detection system to detect particles in the fluid flow of the channel.

[0029] An illumination source is provided, and the camera is configured to image at least a portion of the flow path 250. Illumination and measurement equipment may use lenses, filters, and dichroic mirrors in various combinations. For example, the illumination source and the camera may share an objective lens (epitaxial illumination), which may be further combined with laser illumination, side-scattered light, fluorescence detection, or forward-scattered light.

[0030] The lighting and camera may be of the strobe type, but are not required. Advantageously, as will be further described below, the lighting source and camera can be controlled to acquire camera frames in which the camera shutter is open when the lighting (e.g., strobe flash) occurs. The camera shutter is preferably open only for the duration required to capture a single illumination so that the target particle is imaged. In some cases, multiple particles, including the target particle, can be imaged.

[0031] Figure 2B shows a more detailed schematic diagram of the optical instrumentation of the particle sorter according to the present invention. One or more lasers 201 are each focused and shaped by one or more lenses 202, coupled by a series of dichroic mirrors 203, and then coupled by a projection lens 204, which focuses the laser light into a channel 205 that may be contained in a cuvette or tip. If the channel contains a reflective surface, a forward focusing lens 206 is positioned to focus specularly reflected light. If the channel is transparent, the forward focusing lens is positioned to focus transmitted light. The beams are positioned as shown (not shown). In either case, the collimated light is split by a series of dichroic mirrors 207. Each beam is then focused onto the detector 209 by a lens 208. Optionally, a beam stop 240 is placed to stop the unscattered light of each beam. If a beam stop is present, the detector measures forward scattered light. If a beam stop is not present, the detector measures occlusion. The objective lens 210 projects illumination light onto the channel and focuses the side scattered light, fluorescence emission, and illumination reflection from the channel. The dichroic 211 separates the illumination light from the laser excitation light. The illumination wavelength is selected to suitably separate the fluorescence emission set for detection by the instrument. The side scattered light and fluorescence pass through the dichroic and are split into side scattered light and one or more fluorescence emission bands by a further series of dichroic mirrors 212. Each light-emitting band is focused onto the photodetector 214 213, and the side-scattered light is similarly focused onto the detector 216 215. Illumination light travels from the light source 218 (which may be a strobe light source) and is collimated by the lens 219. The partial silver mirror 217 sends the illumination light to the chip via the dichroic 211 and objective lens 210, and returns the reflected light along the same path. Some of the reflected light passes through 217 and is focused onto the camera 221 by the lens 220.

[0032] Figure 3 shows the optical instrumentation layout of the particle sorter according to the present invention. All components are mounted on a base plate 300. A microfluidic chip 301 is positioned so that the incident laser beam 302 is focused onto the flow path at the detection position described above (chip mount is not shown). Fluorescence, side-scattered light, and imaging 303 from the chip are collected separately from specular reflection 304, as described below. A laser projection block 305 receives two collimated and shaped laser beams (a 488 nm "blue" beam and a 640 nm "red" beam) as input (laser module and beam shaping stage are not shown). The beams are guided and coupled by a movable mirror 306 and a fixed dichroic mirror 307 before being projected onto the chip by a lens 308. A forward scatter collection block is shown in 309. The specularly reflected laser light exits the chip and enters the forward scattering focusing lens 310, where it is separated into red and blue components by the dichroic mirror 312, reflected by mirror 311, and focused by the photodiode detector 313. Block 314 houses fluorescence focusing, side scattering, illumination, and imaging. The objective lens 315 projects illumination onto the chip and focuses and collimates the side-scattered light, fluorescence emission, and illumination reflection from the chip. The collimated light is split into fluorescence bands by a series of dichroic mirrors 316 and knife-edge pick-off mirrors 322, which are measured by a set of silicon photomultiplier tube detectors 324. The side-scattered light is measured by the photodiode 325. The illumination component in this example uses an LED 318 with a wavelength (850 nm) selected to be away from the laser wavelength and fluorescence emission. Light is projected onto the chip, and the reflected light returns via the same path through the dichroic mirror because the wavelength of the dichroic mirror does not change. This light is split by the partial silver mirror 317 and then focused onto the video camera 321 by the tube lens 320.

[0033] Figure 4 shows a schematic diagram of the control system of the particle sorter according to the present invention. One or more measurement channels ("n channels" may include forward scattered light, occlusion, side scattered light, and one or more fluorescence bands) are input to a signal processing system. The signal processing system detects events in the measurement channels. Each event is characterized by the detection time and the set of measurements by the n measurement channels. Each event may include information from particles passing through multiple lasers at different times. This information then needs to be spliced ​​to identify the same particle in separate signals. The events are fed to a sorting logic system, which applies a set of threshold criteria ("gates") to distinguish target events from non-target events and determine which target events to sort to which output. For each event determined to be sorted ("sorted event"), a trigger signal is sent to an actuator (e.g., a particle deflection system) after an activation time delay ("activation trigger"). ). For at least some of these sorting events, a decision is also made to acquire a camera frame ("strobe event"). For each strobe event, a strobe trigger is sent after a strobe time delay and a camera trigger is sent after a camera time delay, both synchronized with the corresponding sorting event. The camera time delay is selected so that the camera shutter opens when illumination occurs. The strobe time delay can be modified to capture various information about the sorting event. In the first function, the strobe time delay is set to be equal to the operation time delay, so that particle positions can be measured in the image at operation. Further functions are described below.

[0034] Figure 5 shows an exemplary control system for a particle sorter according to the present invention. Two measurement channels are provided, each from separate lasers (Laser 1 and Laser 2), both sent to an analog-to-digital converter (ADC) and then to a field-programmable gate array (FPGA). The Laser 2 channel is delayed in a buffering block by a laser delay parameter, and both signals are fed to signal processing blocks, each including a finite impulse response (FIR) filter, baseline correction (BLC correction), and peak detection. Each signal processing block sends a stream of event records to a peak matching block, which splices the records from the two laser channels. Each spliced ​​event record represents a single particle, and the time difference ("delta T") between the signals in the two lasers is calculated. The spliced ​​event records enter further processing blocks that perform cytometry compensation, gating, and sorting logic, which makes the decision whether or not to sort each event. The event records are sent to further blocks that generate an actuation pulse trigger with an actuation delay time and select a subset of events for imaging. Several parameters of the selection process are measured from the images, as described below, and are used to update control parameters such as the operating delay time and illumination delay time within the FPGA for various functions, as described below. This block also sends a stream of events to the microcontroller unit (MCU) for determining the laser delay parameters. For each event captured, the block generates a camera trigger and a strobe trigger. The strobe trigger activates an LED flash of approximately 1 microsecond. The camera trigger is timed a few microseconds before the strobe trigger so that the camera shutter opens when the LED flash occurs. The MCU automatically analyzes the event data and updates the laser delay in the following way: The median delta T is calculated over a given period. This median is a measure of error in the laser delay used to calculate the new laser delay.The new laser delay is fed back to the FPGA buffering block. The camera image is supplied to an embedded PC for automated image analysis. Image analysis in the embedded PC determines the position of the particle during the operating delay time. This position is supplied to a control system block that determines several adjustments. In the first function, the control system calculates what adjustments should be made to the operating delay time in order to move the particle to the operating position.

[0035] Figure 6 shows a schematic diagram of the image analysis and control system for a particle sorter according to the present invention. Particles are first identified in each image by a particle detection algorithm. Next, the particle positions are correlated with event times. Various correlation methods can be employed here. In a direct method using indexed images (each image is associated with an indexed event), events are acquired that are sufficiently far from their neighbors so that only one particle exists within the relevant region of each image. Thus, in a single image, there is a clear association between particle position and illumination time delay. The particle positions are then fed to a controller that determines an increment of the operating time delay based on the operating position setpoint. Following this determination, the illumination delay is set to a new operating time delay. A new sorting event is triggered, a new image is acquired, and particles are sorted. The control loop is completed when the results are fed back to the child detection algorithm.

[0036] Figure 7 shows a particle sorter image analysis capable of identifying particles in a camera image according to the present invention, and the associated control loop. The particle detection algorithm includes several steps: (1) background subtraction: the absolute difference between the new image and a background image without particles is obtained. Then, (2) this image is thresholded, and (3) noise reduction is performed by morphological aperture (erosion followed by dilation). The processed image is (4) fed to a clustering algorithm, for which the known algorithm DBScan is used to generate a set of particles in the image. Next, (5) these particles are filtered by size, weight, density, and circularity to remove image artifacts. The filtered particle locations are correlated by an accumulator algorithm using particle locations from several frames to generate clear particle locations corresponding to illumination time delays. The accumulator algorithm is employed as a countermeasure for various cases where many particles are present in each frame, leading to ambiguity as to which event corresponds to which particle image. Various accumulator algorithms are available. The approach employed in this example involves assigning each particle position as a weight to one position in a one-dimensional position array, where this position corresponds to the distance along the contour line ("stretch line") through which the particle passes in the image in the sorter. Particles from several ("n") frames are accumulated on this array, accumulating specific weights. This array is then convolved using a smoothing function that calculates the sum of a positive Gaussian (width equal to the expected uncertainty of particle position caused by a single illumination time delay) and a negative Gaussian (width is a larger width corresponding to the possible positions of the nearest neighbor particle). Thus, this function, having the same illumination time delay, is more likely to find a position corresponding to the target event and less likely to find a neighboring particle. Next, a proportional controller calculates the difference between the particle position and the operating position setpoint and determines the operating position increment as a percentage of this difference. More generally, a PID (proportional-integral-derivative) controller may be used, but the present inventors have found that a proportional controller also yields sufficient results.The operating position increment is fed to the velocity compensation function, which calculates the operating time delay increment based on the measured velocity of particles in the flow due to their passage between the two lasers. The illumination delay is then set to the new operating time delay, and a new sorting event triggers the acquisition of a new image, which is fed back to the particle detection algorithm to complete the loop.

[0037] The second function of the automatic particle sorter, which adjusts the operating amplitude, is the same as the description of the particle detection and control loop above, with a few modifications. In this second function, the particle position is determined in the downstream region after deflection occurs. The strobe delay time is set to the "verification delay time" instead of the start delay time. The correlation algorithm measures the particle deflection from the undefended particle path. The controller obtains the setpoint deflection and calculates the increment of the operating amplitude required to reach this setpoint.

[0038] Figure 8 shows exemplary results using the particle sorter of the present invention to set the operating delay time and operating amplitude. 801 shows an image triggered by the operating time delay, details of which are shown in 802. The particle detection region ("operating mask") 804 around the operating position and the graphical output of the accumulator algorithm 803, showing both a weight histogram (bars) and convolution function (line graph) along the streak line, are superimposed on the image. Particles are detected in image 805, and the operating position setting point is shown in 806. A further image frame 807, triggered by the validation time delay, is shown. Superimposed on this image is the particle detection region downstream of the particle deflection ("validation mask") 808, showing the deflected particles 809.

[0039] Using the control system and image analysis algorithm of the particle sorter described above, several Further functionality becomes possible. A third function of the particle sorter is to measure the sorter's fidelity, i.e., the percentage of images triggered at a verification time delay where particles are deflected within a range of amplitude so that they flow into a specific deflected output channel instead of an undefended output channel. In the first example, these output channels are the Sort1 and Sort0 channels described above. A fourth function of the particle sorter is to measure the presence of actions, i.e., the percentage of images, that are triggered at an action time delay where particles are present at the precise action location. A fifth function of the particle sorter is to measure the output error rate, i.e., the frequency of particles present in a sampled image that are triggered at an action time delay, where the particles are present in the deflected output channel and therefore not synchronized with the deflection event. In the first embodiment, the output error rate may be expressed as the expected rate of false positives in the Sort1 output, indicating the expected rate of asynchronous co-occurring events. In other words, the output error rate is equal to the percentage of frames in which deflected particles are detected in the Sort1 output, divided by the time it is expected that the flowing particles are within the imaging area of ​​the Sort1 output, minus the sorting rate, asynchronously with respect to the operating time delay. The sixth and seventh functions of the particle sorter are to measure particle focusing quality and particle position jitter, respectively. These are defined as the standard deviation of the particle position at the operating point in the "y" (vertical axis, perpendicular to the streak line) and "x" (horizontal axis, parallel to the streak line) of the image triggered by the operating delay time, using axes defined according to the image shown in Figure 8. The eighth function of the particle sorter is to measure gunks in several regions of interest ("gunk masks") within the image field of view.

[0040] Gunk is measured as the percentage of each gunk mask that has a significant difference from a reference image without gunk. Gunk measurement is used in a particle sorter to trigger a separate gunk removal mechanism. In the first embodiment, the gunk removal mechanism consists of transient shutoff of the Sort1 output by a solenoid valve outside the sorter tip and, optionally, the application of more electrical energy to the actuation than is required for the deflection to actuate.

[0041] Figure 9 shows the machine vision detection masks for a particle sorter in a camera frame according to the present invention. Each mask is a region of interest for the image analysis algorithm described herein. 901 shows the schematic configuration of the microfluidic channels of the particle sorter. 902 shows the working mask. 903 shows the verification mask. Three gunk detection masks are shown in the detection region 904, the working region 905, and the junction 906 between the Sort1 output channel and the Sort0 output channel.

[0042] Figure 10 shows exemplary results of reporting functional measurements to the user via a graphical user interface according to the present invention. Particle position jitter, particle focusing quality, gunk ratio (in the junction region), presence of operation, deflection, fidelity, and output error rate are shown.

[0043] As can be understood from the above description, the present invention can improve the operation and accuracy of particle sorting by identifying process parameters using a relatively simple image acquisition device. The adoption of a delayed-trigger illumination and image capture configuration enables the use of relatively inexpensive and simple equipment.

Claims

1. A particle sorting machine that sorts particles carried by a fluid flow, A particle detection system for detecting particles in the aforementioned fluid flow, A particle deflection system that selectively deflects particles from the fluid flow to sort them, An optical imaging system arranged to acquire images of particles in the fluid flow in order to identify particles at different locations in the fluid flow, An illumination source that selectively illuminates the fluid flow when acquiring an image of particles, Electronic control system, It has, When the particle sorter is in use, the particle detection system outputs a signal indicating a detection event. The electronic control system controls the imaging system and the illumination light source so as to acquire one or more images of the particles in the fluid flow within one or more respective time delays from the detection event. The electronic control system processes the one or more images and the one or more time delays of each to determine one or more parameters for the sorting process. It is configured in such a way A particle sorting machine characterized by the following features.

2. The particle separator according to claim 1, further characterized in that the control system is configured to adjust at least one control parameter of the particle separator based on the determined one or more sorting process parameters.

3. The particle sorter according to claim 2, characterized in that the adjusted control parameters are one or more of the following: the time delay for acquiring the one or more images; the time delay for activating the illumination light source for acquiring the one or more images; the operation control parameters for controlling the operation of the particle detection system; and the timing and / or intensity of the deflection of the particles by the particle detection system.

4. The particle sorter according to any one of claims 1 to 3, characterized in that the one or more sorting parameters are determined by identifying the position of one or more particles in the one or more acquired images.

5. The particle sorter according to any one of claims 1 to 4, characterized in that the control system processes one or more acquired images and calculates the proportion of sampled detection events that are deflected within a predetermined amplitude range so that the particles flow to the correct output.

6. The particle sorter according to any one of claims 1 to 5, characterized in that the control system processes one or more acquired images to calculate the percentage of particle detection events sampled such that the particles are located at the operating position deflected by the particle deflection system.

7. The particle sorter according to any one of claims 1 to 6, characterized in that the control system processes one or more acquired images to determine the frequency of particles present in the deflection path that are not synchronized with the deflection event.

8. The control system processes the acquired one or more images and determines the direction of the particles perpendicular to the flow direction at the operating position where the particles are deflected by the particle deflection system. A particle sorting machine according to any one of claims 1 to 7, characterized by determining the standard deviation of position.

9. The particle sorting machine according to any one of claims 1 to 8, characterized in that the control system processes one or more acquired images to determine the standard deviation of the particle positions in a direction parallel to the flow direction at the operating position.

10. The particle separator according to any one of claims 1 to 9, characterized in that the control system processes one or more acquired images to determine the arrival and accumulation of debris in the particle separator.

11. The particle sorter according to any one of claims 1 to 10, characterized in that the optical imaging system and the illumination light source are arranged to acquire one or more images of the particles in the fluid flow when the particles are in an operating position where they are deflected by the particle deflection system.

12. The particle sorter according to any one of claims 1 to 11, characterized in that the control system controls the optical imaging system and the illumination light source so that a single flash of light is directed at the fluid flow when acquiring an image.

13. The particle separator according to any one of claims 1 to 12, characterized in that the particle separator includes a microfluidic particle separator.

14. The particle separator includes a single joint separator, The particle sorter has an input channel, one or more output sorting channels, and an output waste channel. Each of the one or more output sorting channels and the output discard channel are connected to the input channel that receives the fluid flow. The particle deflection system selectively deflects particles to one or more output sorting channels or output discard channels. A particle separator according to any one of claims 1 to 13.

15. The particle sorter further, when in use, when the particle detection system outputs a signal for an additional detection event, The control system controls the imaging system and the illumination light source so that it acquires one or more additional images of additional particles in the fluid flow at each of one or more additional time delays from the additional detection event. The control system processes each of the one or more additional images and the one or more additional time delays to adjust the one or more parameters of the sorting process. A particle separator according to any one of claims 1 to 14, characterized in that it is configured as follows.

16. A step of supplying a fluid flow to a particle separator according to any one of claims 1 to 15, The steps include detecting particles in the fluid flow, A step of deflecting the detected particles and A particle sorting method having [a specific characteristic].

17. The particles in the fluid flow include the particles to be sorted. The step of deflecting the detected particles includes sorting the particles. The particle sorting method according to claim 16.

18. The particle sorter according to claim 16 or 17, characterized in that the particles include at least one of cells, beads, and droplets containing further particles.

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