Method for determining positional velocity of a flow stream and system therefor

Abstract

To provide a method for determining the positional velocity of particles in a flow stream of a particle analyzer.SOLUTION: Illuminating a sample having particles in a flow stream of a particle analyzer with a first laser of a first interrogation area and a second laser of a second interrogation area, detecting light from the illuminated particles with a first photodetector channel of the first interrogation area and a second photodetector channel of the second interrogation area, calculating a velocity of the particles in the flow stream based on the light detected by the first photodetector and the second photodetector, and determining a parameter of the particles in the flow stream; Based on the calculated velocities and parameters of the particles, the positional velocities of the particles in the flow stream are calculated.SELECTED DRAWING: None

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 665,875, filed June 28, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Introduction Characterization of analytes in biological fluids has become an important part of biological research, medical diagnosis, and the assessment of a patient's overall health and wellness. Detecting analytes in biological fluids, such as human blood or blood-derived products, can provide results that can play a role in determining treatment protocols for patients with various disease states.

[0003] Flow cytometry is a technique used to characterize and frequently separate biological materials, such as cells in a blood sample or particles of interest in another type of biological or chemical sample. Flow cytometers typically include a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer directs the sheath fluid through a flow cell while transporting particles (including cells) in the fluid sample as a stream of cells to the flow cell. To characterize components of the flow stream, light is irradiated onto the flow stream. Variations in materials within the flow stream, such as the form or presence of fluorescent labels, can cause variations in the observed light, which enable characterization and separation. To characterize components of the flow stream, light must be incident on the flow stream and collected. The light source for a flow cytometer can take a variety of forms and can include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0004] Isolation of biological particles has been achieved by adding sorting or collection capabilities to flow cytometers. Particles in the separated stream are detected as possessing one or more desired properties and are individually isolated from the sample stream by mechanical or electrical removal. A common flow sorting technique utilizes droplet sorting, in which a fluid stream containing linearly separated particles is split into droplets. Droplets containing the desired particle are electrically charged and deflected into a collection tube by passing through an electric field. Typically, linearly separated particles in the stream are characterized as they pass an observation point positioned directly below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, the time at which it will reach its breakoff point and break away from the stream can be predicted. Ideally, a charge is applied to the stream for a short period just before the droplet containing the selected particle breaks off from the fluid stream, and then grounded immediately after the droplet breaks off. The droplets to be sorted retain their charge as they break off from the fluid stream, while all other droplets remain uncharged.

[0005] Many flow cytometers move cells through multiple lasers and measure the light resulting from cell-laser interactions. To minimize laser crosstalk and limit baseline noise, most modern cytometers only take electronic measurements when a cell is interacting or about to interact with a given laser. To enable this technology, the transit time of the cell between lasers is best known and controlled. For fluid flow within a channel, temperature fluctuations cause changes in the viscosity of the fluid. While fluid temperature can generally be measured to predict changes in particle flow rate, these measurements are, at best, indirect indicators. Summary of the Invention [Means for solving the problem]

[0006] The inventors have recognized that cell-laser interactions generate laser crosstalk and baseline noise when particles in a flow stream within a flow cytometer move past lasers. Additionally, flow cytometers can only perform electronic measurements when cells are interacting or about to interact with the laser beam. To accurately illuminate passing particles, the transit time of a particle in the flow stream between lasers must be accurately determined. Temperature fluctuations in the flow stream can cause velocity fluctuations due to changes in fluid viscosity. Embodiments of the present disclosure address these and other challenges. The inventors have discovered that by accurately illuminating passing particles, the light emitted from the particle in the flow stream can be accurately measured. The inventors have discovered that the position of a particle can be predicted based on the particle's positional velocity in the flow stream. In certain embodiments, the subject method accounts for positional fluctuations in the particle flow and does not rely on indirect indicators (e.g., the temperature of the flow stream) to accurately measure the particle's position in the flow stream. Embodiments of the present disclosure minimize laser crosstalk, baseline noise, and can provide information about laser drift (e.g., whether one laser is drifting relative to another laser). Additionally, using the position velocity to adaptively adjust the laser delay reduces the need for long window extensions, which further reduces baseline noise in the data signal. Having individual measurements of all lasers in the system allows for determining an average velocity of the flow stream that can be used for stabilization, such that velocity changes in the flow stream (e.g., due to temperature fluctuations) are minimized.

[0007] Aspects of the present disclosure include methods for determining the positional velocity of particles in a flow stream of a particle analyzer. The method, according to certain embodiments, includes illuminating a sample having particles in the flow stream of the particle analyzer with a first laser in a first interrogation region and a second laser in a second interrogation region, detecting light from the illuminated particles with a first photodetector channel in the first interrogation region and a second photodetector channel in the second interrogation region, calculating the velocity of the particles in the flow stream based on the light detected by the first and second photodetectors, determining parameters of the particles in the flow stream, and calculating the positional velocity of the particles in the flow stream based on the calculated velocity and parameters of the particles. Systems and non-transitory computer-readable storage media configured to perform the subject methods are also provided.

[0008] In some embodiments, the position velocity of the particle is calculated based on the particle's distance from the center of the flow stream. In some cases, the parameter is a center of mass (COM) parameter of the particle in the flow stream. In some cases, the center of mass parameter is calculated from a generated image of the particle in the flow stream. In some embodiments, the second search region is located downstream from the first search region, e.g., no more than 200 μm downstream from the first search region, e.g., no more than 150 μm, including no more than 100 μm downstream from the first search region.

[0009] In some embodiments, the method includes detecting light from the particles with a first photodetector and generating a first photodetector signal pulse in response to the light emitted by the particles in a first investigation region, and detecting light from the particles with a second photodetector and generating a second photodetector signal pulse in response to the light emitted by the particles in a second investigation region. In some embodiments, each of the signal pulses is a voltage pulse output in a respective photodetector channel. In some embodiments, the signal pulse in each photodetector channel is generated from scattered light from the illuminated particles. In certain embodiments, the signal pulse in the first photodetector channel is generated in response to scattered light from each particle illuminated by a first laser, and the signal pulse in the second photodetector channel is generated in response to scattered light from each particle illuminated by a second laser. In some cases, the particles are illuminated with a trigger laser, and a trigger signal is generated in response to the illumination of the particles by the trigger laser. In certain cases, the first laser is a trigger laser, and the trigger signal is generated by the first photodetector (e.g., in the first photodetector channel).

[0010] In some cases, the amount of time between photodetector signal pulses is determined to calculate the velocity of the particles in the flow stream. In other cases, the time to the peak between the photodetector signal pulses is determined. In some cases, the velocity of each particle in the flow stream is calculated based on the amount of time between the first photodetector signal pulse and the second photodetector signal pulse and the distance between the interrogation area of ​​the first laser and the interrogation area of ​​the second laser. In other cases, the velocity of each particle in the flow stream is calculated based on the time between the peak of the first photodetector signal pulse and the peak of the second photodetector signal pulse and the distance between the interrogation area of ​​the first laser and the interrogation area of ​​the second laser.

[0011] In embodiments, a positional velocity of a particle in a flow stream is determined. In some cases, a position of the particle in the flow stream is determined. In some cases, a position of the particle relative to the center of the flow stream is determined. In some cases, the velocity of the particle depends on the particle's distance from the center of the flow stream. In some cases, if a particle is closer to the center of the flow stream, the particle has a higher velocity. In some cases, if a particle is farther from the center of the flow stream, the particle has a lower velocity. In some cases, a particle center of mass parameter is calculated for each particle. In some cases, the particle's position relative to the center of the flow stream is determined based on the calculated center of mass parameter. In some cases, the particle's positional velocity is determined based on the particle's calculated velocity and center of mass parameter.

[0012] In some embodiments, a method includes illuminating particles in a flowstream with multiple lasers at multiple different interrogation regions, detecting light from the illuminated particles with multiple photodetector channels, generating photodetector signal pulses in response to the light detected by each of the photodetector channels, calculating a velocity of the particles in the flowstream based on the photodetector signal pulses in two or more of the photodetector channels, determining a parameter (e.g., a center of mass parameter) of the particles in the flowstream, and calculating a positional velocity of the particles in the flowstream based on the calculated velocity and parameter of the particles. In some cases, each of the lasers is configured to illuminate the flowstream at interrogation regions spaced apart by 200 μm or less, including 150 μm or less, and 100 μm or less. In some cases, the plurality of lasers includes a second laser configured to irradiate the flow stream at a location downstream from the first laser, a third laser configured to irradiate the flow stream at a location downstream from the second laser, a fourth laser configured to irradiate the flow stream at a location downstream from the third laser, and a fifth laser configured to irradiate the flow stream at a location downstream from the fourth laser.

[0013] In some embodiments, the average velocity of particles is calculated between two or more of the plurality of search regions. In some cases, the method includes calculating the average velocity between three or more, such as four or more, search regions, including five or more, search regions. In some cases, the method includes evaluating the velocity between each of the search regions to determine whether there is an errant velocity between one or more of the search regions. In some cases, the method includes comparing the average velocity between the search regions and evaluating whether the velocity between any two different search regions is an errant velocity.

[0014] In some embodiments, the timing of illumination by each of the lasers is calculated. In some cases, the method includes adjusting the timing of illumination by one or more of the lasers based on a calculated velocity of the particles in the flowstream. In some cases, the timing of illumination by one or more of the lasers is adjusted based on a calculated average velocity of the particles in the flowstream. In some cases, the timing of illumination by one or more of the lasers is adjusted based on a calculated positional velocity of the particles in the flowstream.

[0015] In some embodiments, the particle analyzer includes a flow cell having a flow cell nozzle for generating droplets containing each particle in the sample, e.g., for sorting particles of the sample. In some embodiments, the method includes determining a laser delay of one or more of the lasers based on a calculated position velocity of the particles. In some cases, the method includes adjusting a laser delay of one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and laser delay of each particle in the flow stream. In certain cases, the method includes adjusting a laser delay of one or more of the lasers based on the position velocity of the particles.

[0016] In some embodiments, a drop delay is determined for each particle based on the particle's calculated position velocity. In some cases, the method includes adjusting the drop delay for each particle based on one or more of the particle's calculated velocity in the flow stream, a center of mass parameter, the calculated position velocity, and the calculated drop delay.

[0017] In some embodiments, one or more parameters of the particle analyzer are adjusted based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some embodiments, the parameter of the particle analyzer is timing of one or more of the lasers (e.g., the second laser). In some cases, the method includes adjusting the timing of illumination by one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In certain cases, the method includes adjusting the timing of illumination by one or more of the lasers based on the position velocity. In some cases, the flow rate of the flow stream is increased. In some cases, the flow rate of the flow stream is decreased. In some cases, the flow stream includes a flow of sheath liquid and a flow of sample core stream, and one or more of the flow of sheath liquid and the flow of sample core stream are adjusted based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some embodiments, the method includes adjusting the timing of droplet charging based on one or more of a calculated velocity, a center of mass parameter, a calculated position velocity, and a calculated drop delay of each particle in the flow stream.

[0018] Aspects of the present disclosure also include systems (e.g., particle analyzers) for carrying out the subject methods. The system, according to certain embodiments, includes a light source having at least a first laser and a second laser configured to illuminate a sample having a plurality of particles in a flow stream with the first laser in a first interrogation region and the second laser in a second interrogation region, and a light detection system configured to detect light from each particle in the sample with a first photodetector and a second photodetector. 、 The optical detection system includes: a processor having a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to calculate a velocity of a particle in the flow stream based on light detected in the first optical detector channel and the second optical detector channel, determine parameters of the particle in the flow stream, and calculate a position velocity of the particle in the flow stream based on the calculated velocity and parameters of the particle.

[0019] In some embodiments, the light detection system includes a plurality of light detectors. In some cases, the light detector includes one or more photomultiplier tubes. In some cases, the light detection system includes a light detector array. In certain cases, one or more of the light detectors in the array are photodiodes. In certain cases, one or more of the light detectors in the array are charge-coupled devices. In some cases, the light detector is configured to detect fluorescence from the particles. In some cases, the light detector is configured to detect scattered light from the particles. In certain cases, the scattered light is forward scattered light. In certain cases, the scattered light is side scattered light. In some cases, the light detector is a bright-field light detector. In some cases, the light detector is a dark-field light detector.

[0020] In some cases, the light detection system is configured to generate at least a first light detector signal pulse in response to light detected in a first light detector channel and a second light detector signal pulse in response to light detected in a second light detector channel. In some embodiments, each of the signal pulses is a voltage pulse output in each light detector channel. In some embodiments, the signal pulse in each light detector channel is generated from scattered light from the illuminated particles. In certain embodiments, the signal pulse in the first light detector channel is generated in response to scattered light from each particle illuminated by the first laser, and the signal pulse in the second light detector channel is generated in response to scattered light from each particle illuminated by the second laser.

[0021] In some embodiments, the memory includes instructions for calculating the velocity of particles in the flowstream by determining the amount of time between photodetector signal pulses. In other cases, the memory includes instructions for determining the time to peak between photodetector signal pulses. In some cases, the memory includes instructions for calculating the velocity of each particle in the flowstream based on the amount of time between a first photodetector signal pulse and a second photodetector signal pulse and the distance between the interrogation areas of the first laser and the second laser. In other cases, the memory includes instructions for calculating the velocity of each particle in the flowstream based on the time between the peak of the first photodetector signal pulse and the peak of the second photodetector signal pulse and the distance between the interrogation areas of the first laser and the second laser.

[0022] In embodiments, the memory includes instructions for determining a positional velocity of a particle in a flowstream. In some cases, the memory includes instructions for determining a position of a particle in the flowstream. In some cases, the memory includes instructions for determining a position of a particle relative to a center of the flowstream. In some cases, the memory includes instructions for calculating a particle's velocity relative to the particle's distance from the center of the flowstream. In some cases, if a particle is closer to the center of the flowstream, the particle has a higher velocity. In some cases, if a particle is farther from the center of the flowstream, the particle has a lower velocity. In some cases, the memory includes instructions for calculating a particle's center of mass parameter (COM) for each particle. In some cases, the memory includes instructions for generating images of the particles in the flowstream. In some cases, the memory includes instructions for calculating the center of mass parameter from the generated images of the particles. In some cases, the memory includes instructions for determining a particle's position relative to the center of the flowstream based on the calculated center of mass parameter. In some cases, the memory includes instructions for determining a particle's positional velocity based on the particle's calculated velocity and center of mass parameter.

[0023] In some embodiments, the memory includes instructions for illuminating particles in the flowstream with multiple lasers at multiple different interrogation regions; detecting light from the illuminated particles with multiple photodetector channels; generating photodetector signal pulses in response to the light detected by each of the photodetector channels; calculating a velocity of the particle in the flowstream based on the photodetector signal pulses in two or more of the photodetector channels; determining a parameter (e.g., a center of mass parameter) of the particle in the flowstream; and calculating a positional velocity of the particle in the flowstream based on the calculated velocity and parameter of the particle. In some cases, each of the lasers is configured to illuminate the flowstream at interrogation regions spaced apart by 200 μm or less, including 150 μm or less and 100 μm or less. In some cases, the light source includes multiple lasers, e.g., three or more, e.g., four or more, including five or more lasers. In some cases, the light source includes a trigger laser configured to generate a trigger signal by the light detection system when a particle in the flowstream is illuminated by the trigger laser. In some cases, the plurality of lasers includes a second laser configured to irradiate the flow stream at a location downstream from the first laser, a third laser configured to irradiate the flow stream at a location downstream from the second laser, a fourth laser configured to irradiate the flow stream at a location downstream from the third laser, and a fifth laser configured to irradiate the flow stream at a location downstream from the fourth laser.

[0024] In some embodiments, the memory includes instructions for calculating an average velocity of particles between two or more of the plurality of search areas. In some cases, the memory includes instructions for calculating an average velocity between three or more search areas, such as four or more, and including five or more search areas. In some cases, the memory includes instructions for evaluating the velocity between each of the search areas to determine whether there is an errant velocity between one or more of the search areas. In some cases, the memory includes instructions for comparing the average velocity between the search areas and for evaluating whether the velocity between any two different search areas is an errant velocity.

[0025] In some embodiments, the memory includes instructions for determining a laser delay of one or more of the lasers based on the calculated position velocity of the particles. In some cases, the memory includes instructions for adjusting a laser delay of one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and laser delay of each particle in the flow stream. In certain cases, the memory includes instructions for adjusting a laser delay of one or more of the lasers based on the position velocity of the particles.

[0026] In some embodiments, the memory includes instructions for calculating the timing of illumination by each of the lasers. In some cases, the memory includes instructions for adjusting the timing of illumination by one or more of the lasers based on a calculated velocity of the particles in the flow stream. In some cases, the memory includes instructions for adjusting the timing of illumination by one or more of the lasers based on a calculated average velocity of the particles in the flow stream. In some cases, the memory includes instructions for adjusting the timing of illumination by one or more of the lasers based on a calculated positional velocity of the particles in the flow stream.

[0027] In some embodiments, the particle analyzer includes a flow cell having a flow cell nozzle for generating droplets containing each particle in the sample, e.g., for sorting particles of the sample. In some embodiments, the memory includes instructions for determining a drop delay for each particle based on the calculated position velocity of the particle. In some cases, the memory includes instructions for adjusting the drop delay for each particle based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0028] In some embodiments, the memory includes instructions for adjusting one or more parameters of the particle analyzer based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the parameter is timing of one or more of the lasers (e.g., the second laser). In some cases, the memory includes instructions for increasing a flow rate of the flow stream. In some cases, the memory includes instructions for decreasing a flow rate of the flow stream. In some cases, the flow stream includes a flow of sheath fluid and a flow of a sample core stream, and the memory includes instructions for adjusting one or more of the flow of sheath fluid and the flow of the sample core stream based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some embodiments, the memory includes instructions for adjusting timing of droplet charging based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the memory includes instructions for adjusting the timing of irradiation by one or more of the lasers based on one or more of the calculated velocity, center of mass parameters, and calculated position velocity of each particle in the flow stream.

[0029] Aspects of the present disclosure also include non-transitory computer-readable storage media for carrying out, for example, one or more computer-implemented methods described herein. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for illuminating a sample containing particles in a flow stream of a particle analyzer with a first laser in a first interrogation region and a second laser in a second interrogation region, an algorithm for detecting light from the illuminated particles with a first photodetector in the first interrogation region and a second photodetector in the second interrogation region, an algorithm for calculating the velocity of the particles in the flow stream based on the light detected in the first photodetector channel and the second photodetector channel, an algorithm for determining parameters of the particles in the flow stream, and an algorithm for calculating the position velocity of the particles in the flow stream based on the calculated velocity and parameters of the particles.

[0030] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the velocity of particles in the flowstream by determining the amount of time between photodetector signal pulses. In other cases, the non-transitory computer-readable storage medium includes an algorithm for determining the time to peak between photodetector signal pulses. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the velocity of each particle in the flowstream based on the amount of time between a first photodetector signal pulse and a second photodetector signal pulse and the distance between the interrogation areas of the first laser and the second laser. In other cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the velocity of each particle in the flowstream based on the time between the peak of the first photodetector signal pulse and the peak of the second photodetector signal pulse and the distance between the interrogation areas of the first laser and the second laser.

[0031] In embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining the positional velocity of particles in a flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining the position of a particle in a flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining the position of a particle relative to the center of the flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the velocity of a particle relative to the particle's distance from the center of the flowstream. In some cases, if a particle is closer to the center of the flowstream, the particle has a higher velocity. In some cases, if a particle is farther from the center of the flowstream, the particle has a lower velocity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating a particle center of mass parameter (COM) for each particle. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating images of particles in the flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the center of mass parameter from the generated images of the particles. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining a position of a particle relative to a center of a flow stream based on a calculated center of mass parameter. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining a position velocity of a particle based on a calculated velocity and center of mass parameter of the particle.

[0032] In some embodiments, a non-transitory computer-readable storage medium includes an algorithm for illuminating particles in a flow stream with multiple lasers at multiple different interrogation regions; an algorithm for detecting light from the illuminated particles with multiple photodetector channels; instructions for generating photodetector signal pulses in response to the light detected by each of the photodetector channels; an algorithm for calculating the velocity of the particles in the flow stream based on the photodetector signal pulses in two or more of the photodetector channels; instructions for determining parameters (e.g., center of mass parameters) of the particles in the flow stream; and an algorithm for calculating the position velocity of the particles in the flow stream based on the calculated velocity and parameters of the particles.

[0033] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating an average velocity of particles between two or more of a plurality of search regions. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating average velocities between three or more search regions, such as four or more, including between five or more search regions. In some cases, the non-transitory computer-readable storage medium includes an algorithm for evaluating the velocities between each of the search regions to determine whether there is an errant velocity between one or more of the search regions. In some cases, the non-transitory computer-readable storage medium includes an algorithm for comparing the average velocities between the search regions and instructions for evaluating whether the velocity between any two different search regions is an errant velocity.

[0034] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining a laser delay of one or more of the lasers based on a calculated position velocity of the particles. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting a laser delay of one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and laser delay of each particle in the flow stream. In certain cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting a laser delay of one or more of the lasers based on a position velocity of the particles.

[0035] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the timing of illumination by each of the lasers. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting the timing of illumination by one or more of the lasers based on a calculated velocity of the particles in the flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting the timing of illumination by one or more of the lasers based on a calculated average velocity of the particles in the flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting the timing of illumination by one or more of the lasers based on a calculated positional velocity of the particles in the flowstream.

[0036] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining a drop delay for each particle based on the particle's calculated position velocity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting a drop delay for each particle based on one or more of the particle's calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay in the flow stream. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for adjusting one or more parameters of a particle analyzer based on one or more of the particle's calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay in the flow stream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for increasing a flow rate of the flow stream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for decreasing a flow rate of the flow stream. In some cases, the flow stream includes a flow of sheath liquid and a flow of sample core stream, and the non-transitory computer-readable storage medium includes an algorithm for adjusting one or more of the flow of sheath liquid and the flow of sample core stream based on one or more of a calculated velocity, a center of mass parameter, and a calculated position velocity of each particle in the flow stream. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for adjusting timing of droplet charging based on one or more of a calculated velocity, a center of mass parameter, a calculated position velocity, and a calculated drop delay of each particle in the flow stream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting timing of irradiation by one or more of the lasers based on one or more of a calculated velocity, a center of mass parameter, and a calculated position velocity of each particle in the flow stream. [Brief explanation of the drawings]

[0037] The present disclosure can be best understood from the following detailed description when read in conjunction with the accompanying drawings, which include the following figures:

[0038] [Figure 1A] 1 is a flowchart for calculating the position velocity of a particle in a flow stream, according to certain embodiments. [Figure 1B] 1 illustrates the generation of a signal pulse in response to irradiation of a particle using five spatially separated lasers, according to certain embodiments. [Figure 1C] 1 illustrates a measure of the position dependence of the velocity of particles in a flow stream, according to certain embodiments. [Figure 2] 1 illustrates a flow cytometry system, according to certain embodiments. [Figure 3-1] 1 illustrates an image-enabled particle sorter, according to certain embodiments. [Figure 3-2] 1 illustrates an image-enabled particle sorter, according to certain embodiments. [Figure 4] FIG. 1 illustrates a functional block diagram of a particle analysis system, according to certain embodiments. [Figure 5] FIG. 1 illustrates a functional block diagram of an example control system, in accordance with certain embodiments. [Figure 6A] 1 shows a schematic diagram of a particle sorter system, according to certain embodiments. [Figure 6B] 1 shows a schematic diagram of a particle sorter system, according to certain embodiments. [Figure 7] 1 illustrates aspects of a computer control system, according to certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0039] Aspects of the present disclosure include methods for determining the positional velocity of particles in a flow stream of a particle analyzer. The method, according to certain embodiments, includes illuminating a sample having particles in the flow stream of the particle analyzer with a first laser in a first interrogation region and a second laser in a second interrogation region, detecting light from the illuminated particles with a first photodetector channel in the first interrogation region and a second photodetector channel in the second interrogation region, calculating the velocity of the particles in the flow stream based on the light detected by the first and second photodetectors, determining parameters of the particles in the flow stream, and calculating the positional velocity of the particles in the flow stream based on the calculated velocity and parameters of the particles. Systems and non-transitory computer-readable storage media configured to perform the subject methods are also provided.

[0040] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0041] When a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0042] Certain ranges are presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the exact number preceded by the term, as well as a number that is close to or approximately the number preceded by the term. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately may be a number that, in the context in which it is presented, represents a substantial equivalent to the specifically stated number.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, representative illustrative methods and materials are now described.

[0044] All publications and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0045] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0046] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0047] Although the systems and methods have been or will be described for grammatical fluidity with functional descriptions, it is expressly understood that the claims should not be construed as necessarily limited by "means" or "step" limitation constructions unless expressly formulated under 35 U.S.C. 112, but should be given the full scope of meaning and equivalents of the definitions provided by the claims under the judicial doctrine of equivalents, and that if a claim is expressly formulated under 35 U.S.C. 112, it should be given full legal equivalents under 35 U.S.C. 112.

[0048] Method for determining the position velocity of particles in a flow stream - Patent Application 20070122947 Aspects of the present disclosure include methods for determining the positional velocity of particles in a flow stream of a particle analyzer (e.g., a flow cytometer). In some embodiments, the subject methods provide for minimizing crosstalk between lasers in the particle analyzer, such as when laser illumination by multiple sequential lasers exhibits little or no interference with each other (e.g., crosstalk) or electronic noise. In some cases, interference between the lasers is reduced by 90% or more, e.g., 95% or more, e.g., 97% or more, e.g., 99% or more, e.g., 99.5% or more, and including 99.9% or more. In some embodiments, the methods described herein provide improved accuracy of laser timing (i.e., timing of illumination of particles in the flow stream). In some embodiments, the methods provide dynamic particle velocity determination for each individual particle in the flow stream. In certain cases, the subject methods provide for individually determining the positional velocity of all particles of interest in an illuminated sample. In some cases, the improved accuracy of the laser timing and measurement of positional velocity in the flow stream provides a more accurate drop delay calculated for each particle. In some cases, particle sorting accuracy is improved, including when particle sorting according to the subject disclosure exhibits a greater overall particle sorting yield of 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more, and when the purity of the collected sample is increased by 99% or more. The methods described herein also provide for dynamic particle drop delay determination in real time, and for determining the unique drop delay of each particle of interest in a sample. In certain cases, the subject methods provide for individually determining the drop delay of all particles of interest in an irradiated sample. In some embodiments, particle sorting parameters can be determined and adjusted, e.g., in real time, without any further user intervention.

[0049] In carrying out a method according to certain embodiments, a sample having a plurality of particles in a flowstream is illuminated with two or more spatially separated lasers. In some cases, the particles in the flowstream are illuminated for one or more predetermined time intervals with a continuous wave light source. The term "continuous wave light source" is used herein in its conventional sense to refer to a light source that provides an uninterrupted light beam and maintains illumination of the particles in the flowstream with little or no undesirable changes in light intensity. In some embodiments, the continuous light source emits non-pulsed or non-stroboscopic illumination. In certain embodiments, the continuous light source provides a substantially constant emitted light intensity. For example, a continuous light source may provide a radiated light intensity that varies by 10% or less, such as 9% or less, for example 8% or less, such as 7% or less, for example 6% or less, such as 5% or less, for example 4% or less, for example 3% or less, such as 2% or less, for example 1% or less, such as 0.5% or less, for example 0.1% or less, for example 0.01% or less, such as 0.001% or less, for example 0.0001% or less, for example 0.00001% or less, during a time interval of illumination, including cases where the radiated light intensity varies by 0.000001% or less during a time interval of illumination. The intensity of the light output may be measured by any convenient protocol, including, but not limited to, a scanning slit profiler, a charge-coupled device (CCD, e.g., an intensified charge-coupled device, ICCD), a positioning sensor, a power sensor (e.g., a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, among other types of photodetectors.

[0050] In certain embodiments, the light source is a laser, such as a pulsed or continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCL) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the target system includes a dye laser, such as a stilbene, coumarin, or rhodamine laser. In still other cases, the laser of interest includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In still other cases, the system of interest includes a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium 2O3 laser, or a cerium-doped laser, and combinations thereof.

[0051] In some embodiments, the light source is a continuous wave laser. In some embodiments, particles in the flow stream are illuminated with a pulsed light source for one or more predetermined time intervals. The term "pulsed light source" is used herein in its conventional sense to refer to a light source that emits light in predetermined time intervals, each time interval having a predetermined illumination duration (i.e., pulse width). In certain embodiments, the pulsed light source is configured to illuminate the photodetector with periodic flashes of light. For example, the frequency of each light pulse can be 0.0001 kHz or greater, e.g., 0.0005 kHz or greater, e.g., 0.001 kHz or greater, e.g., 0.005 kHz or greater, e.g., 0.01 kHz or greater, e.g., 0.05 kHz or greater, e.g., 0.1 kHz or greater, e.g., 0.5 kHz or greater, e.g., 1 kHz or greater, e.g., 2.5 kHz or greater, e.g., 5 kHz or greater, e.g., 10 kHz or greater, e.g., 25 kHz or greater, e.g., 50 kHz or greater, including 100 kHz or greater. In certain cases, the frequency of the pulsed irradiation by the light source is in the range including 0.00001 kHz to 1000 kHz, such as 0.00005 kHz to 900 kHz, for example 0.0001 kHz to 800 kHz, for example 0.0005 kHz to 700 kHz, for example 0.001 kHz to 600 kHz, for example 0.005 kHz to 500 kHz, for example 0.01 kHz to 400 kHz, for example 0.05 kHz to 300 kHz, for example 0.1 kHz to 200 kHz, and 1 kHz to 100 kHz. The duration of light irradiation for each light pulse (i.e. pulse width) may vary and may be 0.000001 ms or more, such as 0.000005 ms or more, for example 0.00001 ms or more, such as 0.00005 ms or more, for example 0.0001 ms or more, such as 0.0005 ms or more, for example 0.001 ms or more, such as 0.005 ms or more, for example 0.01 ms or more, such as 0.05 ms or more, for example 0.1 ms or more, such as 0.5 ms or more, for example 1 ms or more, such as 2 ms or more, for example 3 ms or more, such as 4 ms or more, for example 5 ms or more, such as 10 ms or more, for example 25 ms or more, such as 50 ms or more, for example 100 ms or more, including 500 ms or more.For example, the duration of light irradiation may be in the range of 0.000001 ms to 1000 ms, such as 0.000005 ms to 950 ms, for example 0.00001 ms to 900 ms, for example 0.00005 ms to 850 ms, for example 0.0001 ms to 800 ms, such as 0.0005 ms to 750 ms, for example 0.001 ms to 700 ms, for example 0.005 ms to 650 ms, for example 0.01 ms to 600 ms, such as 0.05 ms to 550 ms, for example 0.1 ms to 500 ms, for example 0.5 ms to 450 ms, for example 1 ms to 400 ms, for example 5 ms to 350 ms, and 10 ms to 300 ms.

[0052] In embodiments, the light source may be any convenient light source, including laser and non-laser light sources. In certain embodiments, the light source is a non-laser light source, such as a narrow-band light source that emits a specific wavelength or a narrow range of wavelengths. In some cases, the narrow-band light source emits light having a narrow wavelength range, such as 50 nm or less, for example 40 nm or less, for example 30 nm or less, for example 25 nm or less, for example 20 nm or less, for example 15 nm or less, for example 10 nm or less, for example 5 nm or less, for example 2 nm or less, and includes a light source that emits light of a specific wavelength (i.e., monochromatic light). Any convenient narrow-band light source protocol, such as a narrow-wavelength LED, may be used.

[0053] In other embodiments, the light source is a broadband light source, such as a broadband light source coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof. In some cases, the broadband light source emits light having a wide range of wavelengths, including those ranging from 50 nm or more, e.g., 100 nm or more, e.g., 150 nm or more, e.g., 200 nm or more, e.g., 250 nm or more, e.g., 300 nm or more, e.g., 350 nm or more, e.g., 400 nm or more, and even 500 nm or more. For example, any suitable broadband light source emits light having a wavelength between 200 nm and 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having a wavelength between 400 nm and 1000 nm. Any convenient broadband light source protocol may use, among other broadband light sources, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, superluminescent light emitting diodes, semiconductor light emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, or any combination thereof. In certain embodiments, the light source includes an array of LEDs. In certain cases, the light source includes multiple monochromatic light emitting diodes, each monochromatic light emitting diode outputting light having a different wavelength. In some cases, the light source includes a plurality of multi-color light emitting diodes that output light having a predetermined spectral width, for example, the plurality of multi-color light emitting diodes collectively output light having a spectral width in a range including 200 nm to 1500 nm, for example 225 nm to 1475 nm, for example 250 nm to 1450 nm, for example 275 nm to 1425 nm, for example 300 nm to 1400 nm, for example 325 nm to 1375 nm, for example 350 nm to 1350 nm, for example 375 nm to 1325 nm, for example 400 nm to 1300 nm, for example 425 nm to 1275 nm, for example 450 nm to 1250 nm, for example 475 nm to 1225 nm and 500 nm to 1200 nm.

[0054] In some embodiments, the light source is a narrow bandwidth light source. In some cases, the light source is a light source that outputs a specific wavelength, including 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the continuous wave light source emits light having a wavelength of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm.

[0055] In some embodiments, the light source emits light having wavelengths where the output spectra of one or more components of the light source overlap by 1 nm or more, such as 2 nm or more, for example 3 nm or more, for example 4 nm or more, for example 5 nm or more, for example 6 nm or more, for example 7 nm or more, for example 8 nm or more, for example 9 nm or more, for example 10 nm or more, and including 20 nm or more. In some embodiments, the wavelengths of the light emitted by the light source do not overlap. For example, the output spectra of the light sources may be separated by 1 nm or more, such as 2 nm or more, for example 3 nm or more, for example 4 nm or more, for example 5 nm or more, for example 6 nm or more, for example 7 nm or more, for example 8 nm or more, for example 9 nm or more, for example 10 nm or more, and including 20 nm or more.

[0056] In embodiments, particles in the flowstream are illuminated with two or more, such as three or more, for example four or more, for example five or more, such as six or more spatially separated light sources, including seven or more spatially separated light sources. In some embodiments, one or more light sources are lasers, and particles in the flowstream are illuminated with one or more, such as two or more, for example three or more, for example four or more, for example five or more, such as six or more spatially separated lasers, including seven or more spatially separated lasers. As described in more detail below, each light source is configured to illuminate a different location on the flowstream. In some cases, each of the light sources is configured to independently illuminate locations on the flow stream that are spaced apart from each other by 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm or more, such as 1 μm or more, for example 2 μm or more, for example 3 μm or more, such as 4 μm or more, for example 5 μm or more, such as 10 μm or more, for example 15 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 150 μm or more, such as 200 μm or more, for example 250 μm or more, such as 500 μm or more, for example 750 μm or more, and including 1000 μm or more. For example, each of the light sources is configured to independently illuminate locations on the flow stream that are spaced apart from one another by a distance including 0.01 μm to 2500 μm, such as 0.05 μm to 2000 μm, for example 0.1 μm to 1500 μm, for example 0.5 μm to 1000 μm, for example 1 μm to 500 μm, for example 5 μm to 400 μm, and 10 μm to 100 μm. In certain cases, each of the light sources is configured to independently illuminate locations on the flow stream that are spaced apart from one another by 10 μm or less, such as 9 μm or less, for example 8 μm or less, for example 7 μm or less, for example 6 μm or less, and locations on the flow stream that are spaced apart by 5 μm or less.

[0057] In some embodiments, the method includes illuminating particles in the flow stream with a first laser in a first interrogation region and a second laser in a second interrogation region. The size of each interrogation region can vary depending on the characteristics of the flow nozzle used to generate the flow stream, such as the size of the nozzle orifice and the size of the sample injection port (as described in more detail below). In embodiments, the interrogation region can have a width of 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 5 mm or more, including 10 mm or more. The length of the interrogation region can also vary along a length in some cases of 0.01 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 1.5 mm or more, such as 2 mm or more, such as 3 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 20 mm or more, such as 25 mm or more, including 50 mm or more.

[0058] The flow stream can be illuminated by each light source from any suitable distance, including, for example, from a distance of 0.001 mm or more, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and from a distance of 100 mm or more. Additionally, illumination of the flow stream can be at any suitable angle, such as an angle ranging from 10° to 90°, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and including 30° to 60°, for example, an angle of 90°.

[0059] When performing the subject method, light from each particle is detected by a light detection system. In embodiments, the light detection system includes a light detector, including one or more, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, and ten or more light detectors. The light detector for performing the subject method can be any convenient light detection protocol, including, but not limited to, light sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or photodiodes, and combinations thereof, among other light detectors. In certain embodiments, the light detector has a resolution of 0.01 cm. 2 ~10cm 2 , e.g. 0.05cm 2 ~9cm 2 , e.g. 0.1cm 2 ~8cm 2 , e.g. 0.5cm 2 ~7cm 2 , and e.g. 1 cm 2 ~5cm 2 and a photomultiplier tube such as a photomultiplier tube having an active detection surface area in each region that is in the range of .mu.m.

[0060] The light can be measured by a photodetector at one or more wavelengths, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, and even four hundred or more different wavelengths. The light can be measured continuously or at discrete intervals. In some cases, the detector is configured to obtain continuous light measurements. In other cases, the detector is configured to perform measurements at discrete intervals, such as measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or some other interval.

[0061] Measurements of light from the light source may be taken one or more times during each discrete time interval, including two or more times, such as three or more times, such as five or more times, and ten or more times. In certain embodiments, the light from the light source is measured by the photodetector two or more times, and in certain cases, the data is averaged.

[0062] In certain embodiments, the light detected from the sample is scattered light. The term "scattered light" is used herein in its conventional sense to refer to the propagation of light energy from particles in the sample (e.g., flowing in a flow stream) that have been deflected from an incident beam path, such as by reflection, refraction, or deflection of the light beam. In certain cases, the scattered light detected from particles in the flow stream is forward scattered light (FSC). In other cases, the scattered light detected from particles in the flow stream is side scattered light. In still other cases, the scattered light detected from particles in the flow stream is backscattered light.

[0063] In some embodiments, the light detected from each particle in the sample is transmitted light, such as light detected by a bright-field photodetector. In other embodiments, the light detected from each particle in the sample is emitted light, such as particle luminescence (i.e., fluorescence or phosphorescence). In these embodiments, each particle may include one or more fluorophores that emit fluorescence in response to illumination by two or more light sources. For example, each particle may include two or more fluorophores, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, and including ten or more fluorophores. In some cases, each particle includes a first fluorophore that emits fluorescence in response to illumination by a first laser and a second fluorophore that emits fluorescence in response to illumination by a second laser. In some embodiments, fluorophores of interest may include, but are not limited to, dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.), such as acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinoneimine dyes, azine dyes, eurodine dyes, safranine dyes, indamines, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronine dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, as well as combinations (e.g., in tandem) of two or more of the foregoing dyes, polymeric dyes having one or more monomeric dye units, and mixtures of two or more of the foregoing dyes. Many dyes are commercially available from a variety of sources, including Molecular Probes (Eugene, OR), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, MO), Sirigen, Inc. (Santa Barbara, CA), and Exciton (Dayton, OH). For example, the fluorophore is 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acids; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, peridinin - chlorophyll proteins, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow V) S); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; brilliant yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumaran 151); cyanine and derivatives such as cyanosine, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-Dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; Diethylaminocoumarin; Diethylenetriaminepentaacetate; 4,4'-Diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[Dimethylamino]naphthalene-1-sulfonyl chloride ( DNS, dansyl chloride; 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosine and derivatives such as erythrosine B and erythrosine isothiocyanate; ethidium; fluorescein and 5-carboxyfluorescein (FAM), 5-(4,6-Dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and derivatives such as QFITC ​​(XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); coral fluorescent protein (RCFP); Lissamine (trademark); Lissamine rhodamine, Lucifer Yellow; Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon Green; phenol red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate, and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine, as well as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine Lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and derivatives such as tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthenes; dye-conjugated polymers (i.e., polymer-bound dyes) such as fluorescein isothiocyanate-dextran, as well as dyes combining two or more dyes (e.g., in tandem), polymeric dyes having one or more monomeric dye units, and mixtures of two or more of the foregoing dyes or combinations thereof.

[0064] In some cases, the fluorophore (i.e., dye) is a fluorescent polymer dye. The fluorescent polymer dyes found for use in the subject methods and systems are diverse. In some cases of the present methods, the polymer dye comprises a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure comprising a backbone of alternating unsaturated (e.g., double and / or triple) and saturated (e.g., single) bonds, with π electrons able to move from one bond to another. In this manner, the conjugated backbone can impart an elongated, linear structure to the polymer dye, with limited bond angles between repeat units of the polymer. For example, proteins and nucleic acids are also polymers, but in some instances do not form elongated rod structures but rather fold into highly ordered three-dimensional shapes. Furthermore, CPs can form "rigid rod" polymer backbones, experiencing limited twist angles (e.g., opposite twist angles) between monomer repeat units along the polymer backbone chain. In some cases, the polymer dye comprises a CP having a rigid rod structure. As summarized above, the structural features of the polymeric dye can affect the fluorescent properties of the molecule.

[0065] Any convenient polymer dye can be utilized in the subject methods and systems. In some cases, the polymer dye is a multichromophore having a structure capable of harvesting light to amplify the fluorescent output of the fluorophore. In some cases, the polymer dye can harvest light and efficiently convert it to longer wavelength emission. In some cases, the polymer dye has a light-harvesting multichromophore system that can efficiently transfer energy to a nearby luminescent species (e.g., a "signaling chromophore"). Mechanisms for energy transfer include, for example, resonance energy transfer (e.g., Förster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some cases, these energy transfer mechanisms are relatively short-range, i.e., the proximity of the light-harvesting multichromophore system to the signaling chromophore provides efficient energy transfer. Under conditions for efficient energy transfer, amplification of emission from the signaling chromophore occurs when the number of individual chromophores in the light-harvesting multichromophore system is large. That is, the emission from the signaling chromophore is stronger when the incident light (the "excitation light") is at a wavelength that is absorbed by the light-harvesting multichromophore system than when the signaling chromophore is directly excited by the pump light.

[0066] The multichromophore can be a conjugated polymer. Conjugated polymers (CPs) feature a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is significantly shorter than the length of the polymer chain, the backbone contains many closely spaced conjugated segments. Therefore, conjugated polymers are efficient at light harvesting, enabling light amplification via energy transfer.

[0067] In some cases, the polymers can be used as direct fluorescent reporters, e.g., fluorescent polymers with high extinction coefficients, high brightness, etc. In some cases, the polymers can be used as strong chromophores where color or optical density is used as an indicator.

[0068] Polymeric dyes of interest include those disclosed by Gaylord et al. in U.S. Patent Application Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986, 20130190193, and 20160025735, the disclosures of which are incorporated herein by reference in their entireties, as well as those disclosed in Gaylord et al., J. Am. Chem. Soc., 2001, 123(26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419, and Traina et al. al., J. Am. Chem. Soc., 2011, 133(32), pp 12600-12607, the disclosures of which are incorporated herein by reference in their entireties.

[0069] Light from the flowstream in each illuminated interrogation region is detected in two or more, such as three or more, for example four or more, for example five or more, for example six or more, for example seven or more, for example eight or more, for example nine or more, for example ten or more, for example twelve or more, for example sixteen or more, for example twenty-four or more, for example twenty-four or more, for example thirty-two or more, for example sixty-four or more, for example one hundred twenty-eight or more, for example twenty-five or more, and ... In certain embodiments, scattered light from each particle in the flow stream illuminated by the first laser is detected by a first photodetector channel, and scattered light from each particle in the flow stream illuminated by the second laser is detected by a second photodetector channel.

[0070] In embodiments, one or more signal pulses are generated in each photodetector channel in response to the detected light. In some embodiments, a plurality of signal pulses are generated in each photodetector channel in response to the detected light, e.g., two or more signal pulses, e.g., three or more signal pulses, e.g., four or more signal pulses, e.g., five or more signal pulses, e.g., six or more signal pulses, e.g., seven or more signal pulses, e.g., eight or more signal pulses, e.g., nine or more signal pulses, including generating ten or more signal pulses in each photodetector channel in response to the detected light. In some embodiments, one or more signal pulses are generated in different photodetector channels in response to light detected from particles illuminated by each light source (e.g., each laser). For example, if particles in a flow stream are illuminated by a first laser and a second laser, a signal pulse can be generated in a first photodetector channel in response to light detected from each particle illuminated by the first laser, and a signal pulse can be generated in a second photodetector channel in response to light detected from each particle illuminated by the second laser.

[0071] In some embodiments, the signal pulse is a voltage pulse.The voltage pulse can comprise 0.001mV or more, for example 0.005mV or more, for example 0.01mV or more, for example 0.05mV or more, for example 0.1mV or more, for example 0.5mV or more, for example 1mV or more, for example 5mV or more, for example 10mV or more, for example 25mV or more, for example 50mV or more, for example 100mV or more, for example 250mV or more, for example 500mV or more, for example 1000mV or more, for example 2500mV or more and 5000mV or more. In some embodiments a signal pulse is generated in each photodetector channel when the detected light produces a voltage above a predetermined threshold of 0.00001 mV or more, such as 0.00005 mV or more, for example 0.0001 mV or more, such as 0.0005 mV or more, for example 0.001 mV or more, such as 0.005 mV or more, for example 0.01 mV or more, such as 0.05 mV or more, for example 0.1 mV or more, such as 0.5 mV or more, for example 1 mV or more, such as 5 mV or more, for example 10 mV or more, such as 25 mV or more, for example 50 mV or more, such as 100 mV or more, for example 250 mV or more, such as 500 mV or more, for example 1000 mV or more, such as 2500 mV or more, and above a predetermined threshold of 5000 mV or more.

[0072] In some embodiments, the method includes determining an amount of time between photodetector signal pulses, in some cases the method includes determining an amount of time between signal pulses in 2 or more, such as 3 or more, for example 4 or more, such as 5 or more, for example 6 or more, such as 7 or more, for example 8 or more, such as 9 or more, for example 10 or more, such as 12 or more, for example 16 or more, such as 24 or more, for example 24 or more, such as 32 or more, for example 64 or more, such as 128 or more, for example 256 or more different photodetector channels, including determining an amount of time between signal pulses in 512 or more different photodetector channels. In some cases, the time between signal pulses in two or more different photodetector channels may be 0.00001 μs or more, such as 0.00005 μs or more, for example 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs, for example 0.01 μs or more, for example 0.05 μs or more, such as 0.1 μs or more, for example 0.5 μs or more, such as 1 μs or more, for example 5 μs or more, such as 10 μs or more, for example 25 μs or more, such as 50 μs or more, for example 100 μs or more, such as 500 μs or more, and may include 1000 μs or more. In certain cases, the time between signal pulses in two or more different photodetector channels is in the range of 0.00001 μs to 5000 μs, such as 0.0001 μs to 4000 μs, such as 0.001 μs to 3000 μs, such as 0.01 μs to 2000 μs, such as 0.1 μs to 1000 μs, including 1 μs to 500 μs.

[0073] In some embodiments, the method includes determining the duration to peak between photodetector signal pulses. The term "time to peak" is used herein in its conventional sense to refer to the duration between the peaks (e.g., the highest detected voltage value in a photodetector channel during a signal pulse) of the generated signal pulses in each photodetector channel. For example, the duration to peak can be determined between signal pulses in 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 12 or more, such as 16 or more, such as 24 or more, such as 24 or more, such as 32 or more, such as 64 or more, such as 128 or more, such as 256 or more different photodetector channels, including determining the duration to peak between signal pulses in 512 or more different photodetector channels. In some cases, the time to peak between signal pulses in two or more different photodetector channels may be 0.00001 μs or more, such as 0.00005 μs or more, for example 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs, for example 0.01 μs or more, such as 0.05 μs or more, for example 0.1 μs or more, such as 0.5 μs or more, for example 1 μs or more, such as 5 μs or more, for example 10 μs or more, such as 25 μs or more, for example 50 μs or more, for example 100 μs or more, such as 500 μs or more, and may include 1000 μs or more. In certain cases, the peak-to-peak duration between signal pulses in two or more different photodetector channels is in the range of 0.00001 μs to 5000 μs, such as 0.0001 μs to 4000 μs, such as 0.001 μs to 3000 μs, such as 0.01 μs to 2000 μs, such as 0.1 μs to 1000 μs, including 1 μs to 500 μs.

[0074] In some embodiments, the method includes calculating the velocity of each particle of interest in the flow stream. In some cases, the velocities of one or more particles in the sample, e.g., the velocities of two or more particles in the sample, e.g., the velocities of five or more particles in the sample, e.g., the velocities of ten or more particles in the sample, e.g., the velocities of twenty-five or more particles in the sample, e.g., the velocities of fifty or more particles in the sample, e.g., the velocities of one ... For example, the velocity of 0.0001% or more, such as 0.0005% or more, for example 0.001% or more, such as 0.005% or more, for example 0.01% or more, such as 0.05% or more, for example 0.1% or more, such as 0.5% or more, for example 1% or more, such as 5% or more, for example 10% or more, such as 25% or more, for example 50% or more, such as 75% or more of the particles in the sample can be independently calculated, including independently calculating the velocity of 90% or more of the particles in the sample.

[0075] In some embodiments, the velocity of each particle is calculated based on the amount of time between signal pulses in two or more photodetector channels and the distance between two or more interrogation areas. In one embodiment, the velocity of each particle is calculated based on the amount of time between a signal pulse from a first photodetector channel and a signal pulse from a second photodetector channel and the distance between the interrogation area of ​​the first laser and the interrogation area of ​​the second laser. In some cases, the distance between the first interrogation area and the second interrogation area is determined based on the distance between the location on the flowstream illuminated by the first laser and the location on the flowstream illuminated by the second laser. For example, depending on the particle analyzer in a particular case, the distance between the first interrogation area and the second interrogation area may be 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 2 μm or more, for example 3 μm or more, such as 4 μm or more, for example 5 μm or more, such as 10 μm or more, for example 15 μm or more, such as 25 μm or more, for example 50 μm or more, including 100 μm or more. In other cases, the distance between the first interrogation area and the second interrogation area may be in the range of 0.01 μm to 500 μm, such as 0.05 μm to 450 μm, for example 0.1 μm to 400 μm, such as 0.5 μm to 350 μm, for example 1 μm to 200 μm, such as 5 μm to 150 μm, including 10 μm to 100 μm.

[0076] In some embodiments, particles in the flowstream are characterized by non-uniform velocity, where particles in the flowstream have independently different velocities. In some cases, particle velocity varies between one or more interrogation regions of the flowstream, such as when particles exhibit a greater flow rate between two or more of the interrogation regions. In some cases, particles exhibit a slower flow rate between two of the interrogation regions of the flowstream. In some cases, the method includes determining flow rates between two or more of the interrogation regions, e.g., between three or more, e.g., between four or more, and including between five or more interrogation regions. In some cases, the method includes determining whether there are diverging velocities between the interrogation regions. In some embodiments, the method includes determining whether there is laser pointing instability based on one or more diverging velocities, e.g., determining that the laser has pointing instability if there are diverging velocities between the interrogation regions.

[0077] In some cases, the non-uniformity of particle velocity depends on the particle's position in the flow stream. In some cases, the flow stream includes a sheath fluid flow and a sample core stream flow. In a flow stream having a core stream and a surrounding sheath flow, in certain cases, the velocity of particles flowing near the center of the core stream is faster than the velocity of particles flowing along the periphery of the core stream.

[0078] In some embodiments, the positional velocity of a particle in a flowstream is correlated (proportional or inversely proportional) to its distance from the center of the flowstream (e.g., radius r from the center of the flowstream). In certain cases, the positional velocity is proportional (e.g., linearly or exponentially) to the distance to the center of the flowstream. In some cases, the closer a particle is to the center of the flowstream, the higher the velocity the particle will have in the flowstream.

[0079] In embodiments, the method includes calculating a positional velocity of the particle in the flowstream. In some cases, the velocity of the particle is determined at different positions from the center of the flowstream, such as where the velocity is determined at predetermined increments of distance from the center of the flowstream. In some cases, the velocity of the particle in the flowstream is determined at increments of 0.0001 μm or more from the center of the flowstream (e.g., 0.0001 μm from the center of the flowstream, 0.0002 μm from the center of the flowstream, 0.0003 μm from the center of the flowstream, etc.), such as 0.0005 μm or more, such as 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm increments, and at increments of 1 μm or more from the center of the flowstream.

[0080] In some embodiments, the position of a particle in a flow stream is determined by plotting the flow stream on a Cartesian plot and determining the Cartesian coordinates of the particle. For example, the Cartesian plot can be a plot of a cross-section of the flow stream, such that a cross-sectional position on the Cartesian plot (e.g., a position on the x-axis and a position on the y-axis) is determined for the particle. In some cases, the position of a particle in a flow stream is determined based on the polar coordinates of the flow stream. In one particular case, a cross-section of the flow stream is plotted, and the polar coordinates of the particle in the flow stream are determined as the position of the particle in the flow stream.

[0081] In some embodiments the position of the particle is measured as a distance from the center of the flow stream, for example the position of the particle is 0.0001 μm or more from the center of the flow stream, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the center of the flow stream. For example, the particle location may be between 0.0001 μm and 10000 μm from the center of the flow stream, such as between 0.0005 μm and 5000 μm, such as between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, such as between 0.01 μm and 100 μm, such as between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the center of the flow stream.

[0082] In some embodiments, the position of the particle is measured as a distance from the wall of the flow channel (e.g., the periphery of the flow stream), for example the position of the particle is 0.0001 μm or more from the wall of the flow channel, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the wall of the flow channel. For example, the particle may be located between 0.0001 μm and 10000 μm from the wall of the flow channel, such as between 0.0005 μm and 5000 μm, for example between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, for example between 0.01 μm and 100 μm, for example between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the wall of the flow channel.

[0083] In calculating the positional velocity of a particle in a flow stream according to certain embodiments of the present disclosure, a parameter of the particle is determined. In some cases, the parameter is an image parameter of the particle in the flow stream. In some cases, the image parameter is generated from imaging data of the particle from measured light. In some cases, the image parameter is generated from an image of the particle from detected light. One or more images can be generated from the measured light. In some embodiments, a single image is generated for each particle from each form of detected light. In other embodiments, multiple images are generated for each particle, e.g., two or more, e.g., three or more, e.g., five or more, e.g., ten or more, including 25 or more images of the particle. For example, a first image of the particle is generated from detected light absorption, a second image of the cell is generated from detected light scattering, and a third image of the cell is generated from detected light emission. In other embodiments, two or more, e.g., three or more, e.g., four or more, e.g., five or more, images are generated from each form of detected light, including ten or more images or combinations thereof.

[0084] In some cases, the method includes generating one or more grayscale images of the particles. The term "grayscale" is used herein in its conventional sense to refer to an image of cells in a flow stream composed of various shades of gray based on the intensity of light at each pixel. In some embodiments, a pixel intensity threshold is determined from the grayscale image, and the pixel intensity threshold is used to convert each pixel to a binary value that is used to generate the image of the particle. In certain cases, the image of the particle is a binary pixel image of the particle, e.g., each pixel is assigned a binary pixel value of 1 (e.g., if the intensity of the pixel exceeds a predetermined threshold) or a binary pixel value of 0 (e.g., if the intensity of the pixel is below a predetermined threshold).

[0085] In some embodiments, one or more image parameters are calculated from the generated images of the particles. In some cases, a center of mass image parameter is calculated from the generated images. In some cases, a delta center of mass image parameter is calculated from the generated images. In some cases, a diffuse image parameter is calculated from the generated images. In some cases, an eccentricity image parameter is calculated from the generated images. In some cases, a major axis moment image parameter is calculated from the generated images. In some cases, a maximum intensity image parameter is calculated from the generated images. In some cases, a radial moment image parameter is calculated from the generated images. In some cases, a minor axis moment image parameter is calculated from the generated images. In some cases, a particle size image parameter is calculated from the generated images. In some cases, a total intensity image parameter is calculated from the generated images. In some cases, a particle light loss image parameter is calculated from the generated images. In some cases, a forward scatter image parameter is calculated from the generated images. In some cases, a side scatter image parameter is calculated from the generated images. In some cases, image moments are calculated from the generated images. The term "image moment" is used herein in its conventional sense to refer to a weighted average of pixel intensities in an image. In some cases, the center of mass of an image may be calculated from the image moments of an image. In other cases, the orientation of a cell may be calculated from the image moments of an image. In still other cases, the eccentricity of a cell may be calculated from the image moments of an image.

[0086] In one particular embodiment, imaging parameters (as described below) calculated from the images generated for use in generating a gating strategy are summarized in Table 1.

[0087] [Table 1-1] [Table 1-2]

[0088] In some embodiments, the position velocity of a particle in a flow stream is calculated based on the particle's velocity and a center of mass (COM) parameter. In some cases, the center of mass parameter is calculated from the particle's image moments and the generated image. For example, the particle's center of mass can be determined from the calculated image moments and the generated image according to:

[0089]

number

[0090] In some embodiments, the center of mass parameter includes a position of the particle in the flowstream. In some cases, the position of the particle in the flowstream is based on Cartesian coordinates of the particle plotted on a Cartesian plot. For example, the Cartesian plot can be a plot of a cross-section of the flowstream such that a cross-sectional position on the Cartesian plot (e.g., a position on the x-axis and a position on the y-axis) is determined for the particle. In some cases, the position of the particle in the flowstream is based on polar coordinates of the particle in the flowstream.

[0091] In some embodiments, the particle position for determining the center of mass parameter is the distance of the particle from the center of the flow stream, for example the particle position is 0.0001 μm or more from the center of the flow stream, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the center of the flow stream. For example, the particle location may be between 0.0001 μm and 10000 μm from the center of the flow stream, such as between 0.0005 μm and 5000 μm, such as between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, such as between 0.01 μm and 100 μm, such as between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the center of the flow stream.

[0092] In some embodiments, the position of the particle for determining the center of mass parameter is the distance of the particle from the wall of the flow channel (e.g., the periphery of the flow stream), for example the position of the particle is 0.0001 μm or more from the wall of the flow channel, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the wall of the flow channel. For example, the particle may be located between 0.0001 μm and 10000 μm from the wall of the flow channel, such as between 0.0005 μm and 5000 μm, for example between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, for example between 0.01 μm and 100 μm, for example between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the wall of the flow channel.

[0093] In some embodiments, the particle analyzer includes a flow cell having a flow nozzle for generating droplets containing each particle in the flow stream (as described in more detail below). In some embodiments, the method includes determining a laser delay of one or more of the lasers based on a calculated position velocity of the particles. In some cases, the method includes adjusting a laser delay of one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and laser delay of each particle in the flow stream. In certain cases, the method includes adjusting a laser delay of one or more of the lasers based on the position velocity of the particle.

[0094] In some cases, the method includes determining a drop delay for each particle based on the particle's calculated position velocity and a distance between an interrogation region of one or more of the light sources and a flow cell nozzle orifice. In some embodiments, the drop delay for each particle is independently determined by multiplying the calculated position velocity of each particle of interest by the distance between the interrogation region of one or more of the light sources and the flow cell nozzle orifice. For example, depending on the particle analyzer in a particular case, the distance between each interrogation region and the flow cell nozzle orifice may independently be 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 2 μm or more, such as 3 μm or more, such as 4 μm or more, such as 5 μm or more, such as 10 μm or more, such as 15 μm or more, such as 25 μm or more, such as 50 μm or more, including 100 μm or more. In other cases, the distance between each interrogation area and the flow cell nozzle orifice is in the range of 0.01 μm to 500 μm, such as 0.05 μm to 450 μm, for example 0.1 μm to 400 μm, for example 0.5 μm to 350 μm, for example 1 μm to 200 μm, for example 5 μm to 150 μm, including 10 μm to 100 μm.

[0095] In some embodiments, the method includes adjusting one or more parameters of the particle analyzer based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the flow rate of the flow stream is increased. In certain cases, the flow rate can be increased by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including increasing the flow rate of the flow stream by 99.9% or more. In some embodiments, the flow rate of the flow stream is increased by 1 μL / sec or more, such as 2 μL / sec or more, for example 5 μL / sec or more, such as 10 μL / sec or more, for example 25 μL / sec or more, such as 50 μL / sec or more, for example 100 μL / sec or more, such as 250 μL / sec or more, for example 500 μL / sec or more, such as 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the flow rate of the flow stream is increased by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, such as 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, such as 5 mm / sec or more, including when the flow rate of the flow stream is increased by 10 mm / sec or more.

[0096] In other cases, the flow rate of the flow stream is reduced. In certain cases, the flow rate can be reduced by 1% or more, such as 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, for example 90% or more, for example 95% or more, for example 97% or more, for example 99% or more, including reducing the flow rate of the flow stream by 99.9% or more. In some embodiments, the flow rate of the flow stream is reduced by 1 μL / sec or more, for example 2 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, for example 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the flow rate of the flow stream is reduced by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, such as 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, such as 5 mm / sec or more, including where the flow rate of the flow stream is reduced by 10 mm / sec or more.

[0097] In some cases, the flow rate of the sheath fluid is increased based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the flow rate of the sheath fluid is increased by 1% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, including increasing the flow rate of the sheath fluid by 99.9% or more. In some embodiments, the flow rate of the sheath fluid is increased by 1 μL / sec or more, for example, 2 μL / sec or more, for example, 5 μL / sec or more, for example, 10 μL / sec or more, for example, 25 μL / sec or more, for example, 50 μL / sec or more, for example, 100 μL / sec or more, for example, 250 μL / sec or more, for example, 500 μL / sec or more, for example, 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the flow rate of the sheath fluid is increased by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, for example 0.05 mm / sec or more, for example 0.1 mm / sec or more, for example 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including when the flow rate of the sheath fluid is increased by 10 mm / sec or more.

[0098] In some cases, the flow rate of the sheath fluid is reduced based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the flow rate of the sheath fluid is reduced by 1% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, including reducing the flow rate of the sheath fluid by 99.9% or more. In some embodiments, the flow rate of the sheath fluid is reduced by 1 μL / sec or more, for example, 2 μL / sec or more, for example, 5 μL / sec or more, for example, 10 μL / sec or more, for example, 25 μL / sec or more, for example, 50 μL / sec or more, for example, 100 μL / sec or more, for example, 250 μL / sec or more, for example, 500 μL / sec or more, for example, 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the flow rate of the sheath fluid is reduced by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, for example 0.05 mm / sec or more, such as 0.1 mm / sec or more, for example 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including where the flow rate of the sheath fluid is reduced by 10 mm / sec or more.

[0099] In some cases, the flow of the sample core liquid is increased based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the flow of the sample core liquid is increased by 1% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, including increasing the flow rate of the sample core liquid stream by 99.9% or more. In some embodiments, the flow of the sample core liquid stream is increased by 1 μL / sec or more, for example, 2 μL / sec or more, for example, 5 μL / sec or more, for example, 10 μL / sec or more, for example, 25 μL / sec or more, for example, 50 μL / sec or more, for example, 100 μL / sec or more, for example, 250 μL / sec or more, for example, 500 μL / sec or more, for example, 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the flow of sample core liquid is increased by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, such as 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including where the flow rate of sample core liquid is increased by 10 mm / sec or more.

[0100] In some cases, the flow of sample core liquid is reduced based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the flow of sample core liquid is reduced by 1% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, including reducing the flow rate of the sample core liquid stream by 99.9% or more. In some embodiments, the flow of the sample core liquid stream is reduced by 1 μL / sec or more, for example, 2 μL / sec or more, for example, 5 μL / sec or more, for example, 10 μL / sec or more, for example, 25 μL / sec or more, for example, 50 μL / sec or more, for example, 100 μL / sec or more, for example, 250 μL / sec or more, for example, 500 μL / sec or more, for example, 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the flow of sample core liquid is reduced by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, for example 0.05 mm / sec or more, such as 0.1 mm / sec or more, for example 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including where the flow rate of sample core liquid is reduced by 10 mm / sec or more.

[0101] In some embodiments, the method includes adjusting timing of laser firing of one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the timing of laser firing by one or more of the lasers is adjusted to be delayed by 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, and including 50% or more. For example, the timing of the laser firing can be delayed by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs or more, for example 0.01 μs or more, such as 0.05 μs or more, for example 0.1 μs or more, such as 0.5 μs or more, for example 1 μs or more, such as 5 μs or more, for example 10 μs or more, such as 25 μs or more, for example 50 μs or more, for example 100 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream, including delaying laser firing by one or more of the lasers by 500 μs. In other cases, the timing of laser irradiation by one or more of the lasers is adjusted to be earlier by 0.01% or more, such as 0.05% or more, for example 0.1% or more, for example 0.5% or more, such as 1% or more, for example 2% or more, such as 5% or more, for example 10% or more, such as 15% or more, for example 25% or more, and including 50% or more. For example, the timing of laser firing by one or more of the lasers can be adjusted to be earlier by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs or more, for example 0.01 μs or more, such as 0.05 μs or more, for example 0.1 μs or more, such as 0.5 μs or more, for example 1 μs or more, such as 5 μs or more, for example 10 μs or more, such as 25 μs or more, for example 50 μs or more, such as 100 μs or more, including 500 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0102] In some embodiments, the method includes adjusting the timing of droplet charging based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the timing of droplet charging is adjusted to be delayed by 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, and such as 50% or more. For example, the timing of droplet charging can be delayed by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs or more, for example 0.001 μs or more, for example 0.005 μs or more, for example 0.01 μs or more, such as 0.05 μs or more, for example 0.1 μs or more, such as 0.5 μs or more, for example 1 μs or more, such as 5 μs or more, for example 10 μs or more, such as 25 μs or more, for example 50 μs or more, for example 100 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream, including delaying droplet charging by 500 μs or more in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In other cases, the timing of droplet charging is adjusted to be earlier by 0.01% or more, such as 0.05% or more, for example 0.1% or more, for example 0.5% or more, such as 1% or more, for example 2% or more, such as 5% or more, for example 10% or more, such as 15% or more, for example 25% or more, and 50% or more. For example, the timing of droplet charging can be adjusted to be earlier by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs or more, for example 0.01 μs or more, such as 0.05 μs or more, for example 0.1 μs or more, such as 0.5 μs or more, for example 1 μs or more, such as 5 μs or more, for example 10 μs or more, such as 25 μs or more, for example 50 μs or more, such as 100 μs or more, including 500 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0103] In other embodiments, the method includes adjusting the drop drive frequency based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the drop drive frequency is increased by 0.01 Hz or more, for example, 0.05 Hz or more, for example, 0.1 Hz or more, for example, 0.25 Hz or more, for example, 0.5 Hz or more, for example, 1 Hz or more, for example, 2.5 Hz or more, for example, 5 Hz or more, for example, 10 Hz or more, including 25 Hz or more. For example, the drop drive frequency can be increased by 1% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, including increasing the drop drive frequency by 90% or more. In other cases, the drop drive frequency is reduced by 0.01 Hz or more, such as 0.05 Hz or more, for example 0.1 Hz or more, for example 0.25 Hz or more, for example 0.5 Hz or more, for example 1 Hz or more, for example 2.5 Hz or more, for example 5 Hz or more, for example 10 Hz or more, including 25 Hz or more. For example, the drop drive frequency can be reduced by 1% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, including reducing the drop frequency by 90% or more.

[0104] In yet another embodiment, the method includes adjusting the drop delay based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the drop delay is increased by, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.3 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2.5 μs or more, for example, 5 μs or more, for example, 7.5 μs or more, including increasing the drop delay by 10 μs or more. For example, the drop delay can be increased by 1% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, including increasing the drop delay by 90% or more. In other cases, the drop frequency is reduced by, for example, 0.01 μs or more, for example 0.05 μs or more, for example 0.1 μs or more, for example 0.3 μs or more, for example 0.5 μs or more, for example 1 μs or more, for example 2.5 μs or more, for example 5 μs or more, for example 7.5 μs or more, including reducing the drop delay by 10 μs or more. For example, the drop delay can be reduced by 1% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, including reducing the drop delay by 90% or more.

[0105] FIG. 1A shows a flowchart for calculating the positional velocity of a particle in a flowstream according to one specific embodiment. In step 101, a particle in the flowstream is illuminated with a first laser in a first investigation region, generating a signal pulse in a first photodetector channel. In step 102, the particle is illuminated with a second laser in a second investigation region, generating a signal pulse in a second photodetector channel. The particle's velocity is calculated in step 103a, where the velocity is calculated based on the time of a pulse peak generated by the first laser's illumination in the first investigation region and the time of a pulse peak generated by the second laser's illumination in the second investigation region, as well as the distance between the location on the flowstream where the first laser was illuminated and the location on the flowstream where the second laser was illuminated. In step 103b, an image of the particle in the flowstream can be generated. Particle center of mass parameters are determined for the particle in step 104, such as using the imaging data or image generated in step 103b. In step 105, the positional velocity of the particle in the flowstream is calculated based on the image parameters and the particle's velocity. One or more parameters of the particle analyzer, such as the drop delay, the timing of illumination by each laser in the system, the timing of droplet charging, and the droplet drive frequency, can be determined using the calculated position velocity of the particle (step 106). In some cases, one or more parameters of the particle analyzer are adjusted based on the calculated position velocity in step 107.

[0106] FIG. 1B illustrates the generation of signal pulses in response to irradiation of particles using five spatially separated lasers, according to a specific embodiment. Particles (cells) in a flow stream are illuminated by five spatially separated lasers having different laser wavelengths (ultraviolet, red, green, yellow-green, and blue lasers). A signal pulse is generated in a first photodetector channel in response to light detected from irradiation of the particles by the ultraviolet laser. A second signal pulse is generated in a second photodetector channel in response to light detected from irradiation by the red laser. A third signal pulse is generated in a third photodetector channel in response to light detected from irradiation by the green laser. A fourth signal pulse is generated in a fourth photodetector channel in response to light detected from irradiation by the yellow-green laser. A fifth signal pulse is generated in a fifth photodetector channel in response to light detected from irradiation by the blue laser.

[0107] The time to peak (ttp) is the duration measured between the peaks of the signal pulses generated in response to illumination by the different lasers. The red time to peak (ttp) is a measure of the duration between the peak of the signal pulse generated in the first photodetector channel in response to illumination by the ultraviolet laser and the peak of the signal pulse generated in the second photodetector channel in response to illumination by the red laser. The green time to peak (ttp) is a measure of the duration between the peak of the signal pulse generated in the second photodetector channel in response to illumination by the red laser and the peak of the signal pulse generated in the third photodetector channel in response to illumination by the green laser. The yellow-green time to peak (ttp) is a measure of the duration between the peak of the signal pulse generated in the third photodetector channel in response to illumination by the green laser and the peak of the signal pulse generated in the fourth photodetector channel in response to illumination by the yellow-green laser. The time to blue peak (TTP) is a measure of the duration between the peak of the signal pulse generated in the fourth photodetector channel in response to illumination by the yellow-green laser and the peak of the signal pulse generated in the fifth photodetector channel in response to illumination by the blue laser. The particle velocity is estimated by dividing the distance between each illumination location by the duration of the time to peak.

[0108] FIG. 1C shows a measure of the positional dependence of particle velocity in a flow stream, according to certain embodiments. A blue laser is used to illuminate particles in the flow stream, with each particle flowing through a different position in the flow stream. Particle velocity is determined as described above using the time to peak of the blue laser pulse. Particle velocity is plotted as a function of the center of mass (COM) parameter calculated for each particle. A positive correlation in the plot indicates that as the COM increases, there is a corresponding increase in the time to peak. This correlation indicates a positional change in velocity, demonstrating that cells farther from the central axis move slower and take longer to reach the subsequent laser.

[0109] In certain embodiments, the method also includes sorting particles of the sample. The term "sorting" is used herein in its conventional sense to refer to separating components of a sample (e.g., droplets containing cells, droplets containing non-cellular particles such as biological macromolecules) and, in some cases, delivering the separated components to one or more sample collection vessels. For example, the method may include sorting two or more components of the sample, e.g., three or more components, e.g., four or more components, e.g., five or more components, e.g., ten or more components, e.g., fifteen or more components, including sorting twenty-five or more components of the sample. In embodiments, the method includes sorting cells based on a generated image mask of the cells.

[0110] In sorting cells, the method includes data acquisition (e.g., by determining one or more of the velocity, center of mass parameter, position velocity, and drop delay of each particle in the flow stream), analysis, and recording, such as by a computer, where multiple data channels record data from each detector (e.g., scatter detector, bright field detector, or fluorescence detector) used to generate the cell image, image mask, or mask image. In these embodiments, the analysis includes classifying and counting particles such that each particle is represented as a set of digitized parameter values. The subject system (described below) may be configured to trigger on selected parameters to distinguish particles of interest from background and noise.

[0111] Specific subpopulations of interest can then be further analyzed by "gating" based on the data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible subpopulation separation. This procedure may be performed by plotting forward light scatter (FSC) versus side (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot plot. A subpopulation of particles is then selected (i.e., those cells within the gate) and particles not within the gate are excluded. Optionally, a gate may be selected by drawing a line around the desired subpopulation using a cursor on the computer screen. Only those particles within the gate are then further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis can be configured to result in counting the particles of interest in the sample.

[0112] In some embodiments, the method is carried out using a method described in, for example, U.S. Patent Nos. 10,006,852, 9,952,076, 9,933,341, 9,784,661, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 8,544,475, and 8,544,475.

[0010] In some embodiments, the method of separating components of a sample includes separating particles (e.g., cells in a biological sample) in an enclosed particle separation module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, the disclosure of which is incorporated herein by reference in its entirety. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having multiple sorting determination units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781 (the disclosure of which is incorporated herein by reference). In some embodiments, a method of sorting components of a sample includes sorting particles (e.g., cells in a biological sample) in a particle sorting module having a deflector plate, such as that described in U.S. Patent Application Publication No. 2017 / 0299493 (filed March 28, 2017, the disclosure of which is incorporated herein by reference).

[0113] System for determining the position velocity of particles in a flow stream - Patent Application 20070122967 Aspects of the present disclosure also include systems (e.g., particle analyzers) for implementing the subject methods. The system, according to certain embodiments, includes: a light source having at least a first laser and a second laser configured to illuminate a sample having a plurality of particles in a flowstream with the first laser in a first interrogation region and the second laser in a second interrogation region; a light detection system comprising photodetectors configured to detect light from each particle in the sample in a first photodetector channel and a second photodetector channel; and a processor having a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to calculate velocities of the particles in the flowstream based on the light detected by the first and second photodetectors, determine parameters of the particles in the flowstream, and calculate positional velocities of the particles in the flowstream based on the calculated velocities and parameters of the particles.

[0114] In embodiments, the system includes two or more spatially separated light sources configured to illuminate a plurality of particles in the flowstream. In some embodiments, the light source is a continuous wave light source, e.g., the light source provides an uninterrupted light beam, maintaining illumination of the particles in the flowstream with little or no undesired changes in light intensity. In some embodiments, the continuous light source emits non-pulsed or non-stroboscopic illumination. In certain embodiments, the continuous light source provides a substantially constant emitted light intensity. For example, the continuous light source can provide an emitted light intensity that varies by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less, during a time interval of illumination, including cases where the emitted light intensity varies by 0.000001% or less during a time interval of illumination. The intensity of the light output can be measured with any convenient protocol, including but not limited to, a scanning slit profiler, a charge-coupled device (CCD, e.g., an intensified charge-coupled device, ICCD), a positioning sensor, a power sensor (e.g., a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, or other types of light detectors.

[0115] In certain embodiments, the light source is a laser, such as a pulsed or continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCL) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the target system includes a dye laser, such as a stilbene, coumarin, or rhodamine laser. In still other cases, the laser of interest includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In still other cases, the system of interest includes a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium 2O3 laser, or a cerium-doped laser, and combinations thereof.

[0116] In some embodiments, the light source includes one or more pulsed light sources, e.g., light is emitted at predetermined time intervals, each time interval having a predetermined illumination duration (i.e., pulse width). In certain embodiments, the pulsed light source is configured to illuminate the photodetector with periodic flashes of light. For example, the frequency of each light pulse may be 0.0001 kHz or greater, e.g., 0.0005 kHz or greater, e.g., 0.001 kHz or greater, e.g., 0.005 kHz or greater, e.g., 0.01 kHz or greater, e.g., 0.05 kHz or greater, e.g., 0.1 kHz or greater, e.g., 0.5 kHz or greater, e.g., 1 kHz or greater, e.g., 2.5 kHz or greater, e.g., 5 kHz or greater, e.g., 10 kHz or greater, e.g., 25 kHz or greater, e.g., 50 kHz or greater, including 100 kHz or greater. In certain cases, the frequency of the pulsed irradiation by the light source is in the range including 0.00001 kHz to 1000 kHz, such as 0.00005 kHz to 900 kHz, for example 0.0001 kHz to 800 kHz, for example 0.0005 kHz to 700 kHz, for example 0.001 kHz to 600 kHz, for example 0.005 kHz to 500 kHz, for example 0.01 kHz to 400 kHz, for example 0.05 kHz to 300 kHz, for example 0.1 kHz to 200 kHz, and 1 kHz to 100 kHz. The duration of light irradiation for each light pulse (i.e. pulse width) may vary and may be 0.000001 ms or more, such as 0.000005 ms or more, for example 0.00001 ms or more, such as 0.00005 ms or more, for example 0.0001 ms or more, such as 0.0005 ms or more, for example 0.001 ms or more, such as 0.005 ms or more, for example 0.01 ms or more, such as 0.05 ms or more, for example 0.1 ms or more, such as 0.5 ms or more, for example 1 ms or more, such as 2 ms or more, for example 3 ms or more, such as 4 ms or more, for example 5 ms or more, such as 10 ms or more, for example 25 ms or more, such as 50 ms or more, for example 100 ms or more, including 500 ms or more.For example, the duration of light irradiation may be in the range of 0.000001 ms to 1000 ms, such as 0.000005 ms to 950 ms, for example 0.00001 ms to 900 ms, for example 0.00005 ms to 850 ms, for example 0.0001 ms to 800 ms, such as 0.0005 ms to 750 ms, for example 0.001 ms to 700 ms, for example 0.005 ms to 650 ms, for example 0.01 ms to 600 ms, such as 0.05 ms to 550 ms, for example 0.1 ms to 500 ms, for example 0.5 ms to 450 ms, for example 1 ms to 400 ms, for example 5 ms to 350 ms, and 10 ms to 300 ms.

[0117] In embodiments, the system may include any convenient light source, including laser and non-laser light sources. In certain embodiments, the light source is a non-laser light source, such as a narrow-band light source that emits a specific wavelength or a narrow range of wavelengths. In some cases, the narrow-band light source emits light having a narrow wavelength range, such as 50 nm or less, for example 40 nm or less, for example 30 nm or less, for example 25 nm or less, for example 20 nm or less, for example 15 nm or less, for example 10 nm or less, for example 5 nm or less, for example 2 nm or less, and includes a light source that emits light of a specific wavelength (i.e., monochromatic light). Any convenient narrow-band light source protocol, such as a narrow-wavelength LED, may be used.

[0118] In other embodiments, the light source is a broadband light source, such as a broadband light source coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof. In some cases, the broadband light source emits light having a wide range of wavelengths, including those ranging from 50 nm or more, e.g., 100 nm or more, e.g., 150 nm or more, e.g., 200 nm or more, e.g., 250 nm or more, e.g., 300 nm or more, e.g., 350 nm or more, e.g., 400 nm or more, and even 500 nm or more. For example, any suitable broadband light source emits light having a wavelength between 200 nm and 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having a wavelength between 400 nm and 1000 nm. Any convenient broadband light source protocol may use, among other broadband light sources, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, superluminescent light emitting diodes, semiconductor light emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, or any combination thereof. In certain embodiments, the light source includes an array of LEDs. In certain cases, the light source includes multiple monochromatic light emitting diodes, each monochromatic light emitting diode outputting light having a different wavelength. In some cases, the light source includes a plurality of multi-color light emitting diodes that output light having a predetermined spectral width, for example, the plurality of multi-color light emitting diodes collectively output light having a spectral width in a range including 200 nm to 1500 nm, for example 225 nm to 1475 nm, for example 250 nm to 1450 nm, for example 275 nm to 1425 nm, for example 300 nm to 1400 nm, for example 325 nm to 1375 nm, for example 350 nm to 1350 nm, for example 375 nm to 1325 nm, for example 400 nm to 1300 nm, for example 425 nm to 1275 nm, for example 450 nm to 1250 nm, for example 475 nm to 1225 nm and 500 nm to 1200 nm.

[0119] In some embodiments, the light source is a narrow bandwidth light source. In some cases, the light source is a light source that outputs a specific wavelength, including 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the continuous wave light source emits light having a wavelength of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm.

[0120] In some embodiments, the light source emits light having wavelengths where the output spectra of one or more components of the light source overlap by 1 nm or more, such as 2 nm or more, for example 3 nm or more, for example 4 nm or more, for example 5 nm or more, for example 6 nm or more, for example 7 nm or more, for example 8 nm or more, for example 9 nm or more, for example 10 nm or more, and including 20 nm or more. In some embodiments, the wavelengths of the light emitted by the light source do not overlap. For example, the output spectra of the light sources may be separated by 1 nm or more, such as 2 nm or more, for example 3 nm or more, for example 4 nm or more, for example 5 nm or more, for example 6 nm or more, for example 7 nm or more, for example 8 nm or more, for example 9 nm or more, for example 10 nm or more, and including 20 nm or more.

[0121] In embodiments, the light sources of the subject systems are spatially separated. In some embodiments, the system includes two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more spatially separated light sources, and includes seven or more spatially separated light sources. In some embodiments, one or more light sources are lasers, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more spatially separated lasers, and includes seven or more spatially separated lasers. Each light source is configured to illuminate a separate position on the flow stream. In some cases, each of the light sources is configured to independently illuminate locations on the flow stream that are spaced apart from each other by 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm or more, such as 1 μm or more, for example 2 μm or more, for example 3 μm or more, such as 4 μm or more, for example 5 μm or more, such as 10 μm or more, for example 15 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 150 μm or more, such as 200 μm or more, for example 250 μm or more, such as 500 μm or more, for example 750 μm or more, and including 1000 μm or more. For example, each of the light sources is configured to independently illuminate locations on the flow stream that are spaced apart from one another by a distance including 0.01 μm to 2500 μm, such as 0.05 μm to 2000 μm, for example 0.1 μm to 1500 μm, for example 0.5 μm to 1000 μm, for example 1 μm to 500 μm, for example 5 μm to 400 μm, and 10 μm to 100 μm. In certain cases, each of the light sources is configured to independently illuminate locations on the flow stream that are spaced apart by 10 μm or less, such as 9 μm or less, for example 8 μm or less, for example 7 μm or less, for example 6 μm or less, including locations on the flow stream that are spaced apart by 5 μm or less.

[0122] In some embodiments, the system includes a first laser configured to illuminate the flow stream in a first interrogation region and a second laser in a second interrogation region. In some cases, the system includes multiple lasers, and the lasers are configured to illuminate multiple interrogation regions along the flow stream. The size of each interrogation region can vary depending on the characteristics of the flow nozzle used to generate the flow stream, such as the size of the nozzle orifice and the size of the sample injection port. In some embodiments, the interrogation region can have a width of 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 5 mm or more, including 10 mm or more. The length of the interrogation region can also vary along the flow stream in some cases by 0.01 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 1.5 mm or more, such as 2 mm or more, such as 3 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 20 mm or more, such as 25 mm or more, including 50 mm or more.

[0123] Each interrogation region in certain cases may be configured to facilitate illumination of a planar cross-section of the diverging flow stream, or may be configured to facilitate illumination of a diffuse field (e.g., using a diffuse laser or lamp) of a predetermined length. In some embodiments, the interrogation region of a flow stream in a system of interest includes a transparent window that facilitates illumination of the diverging flow stream of a predetermined length, e.g., 1 mm or more, e.g., 2 mm or more, e.g., 3 mm or more, e.g., 4 mm or more, e.g., 5 mm or more, including 10 mm or more. Depending on the light source used to illuminate the diverging flow stream (as described below), the interrogation region may be configured to pass light in the ranges of 100 nm to 1500 nm, e.g., 150 nm to 1400 nm, e.g., 200 nm to 1300 nm, e.g., 250 nm to 1200 nm, e.g., 300 nm to 1100 nm, e.g., 350 nm to 1000 nm, e.g., 400 nm to 900 nm, and e.g., 500 nm to 800 nm.

[0124] Each light source may be positioned at any suitable distance from the flow stream, including, for example, at a distance of 0.001 mm or more, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and 100 mm or more. Additionally, each light source may be positioned at any suitable angle relative to the flow stream, for example, at an angle in the range of 10° to 90°, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and 30° to 60°, for example, 90°.

[0125] A light source according to certain embodiments may also include one or more optical conditioning components. The term "optical conditioning" is used herein in its conventional sense and applies to any device that can change the spatial width of the illumination or some other characteristic of the illumination from the light source, such as the illumination direction, wavelength, beam width, beam intensity, and focus. An optical conditioning protocol may be any convenient device that adjusts one or more characteristics of the light source, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In certain embodiments, the objective system includes one or more focusing lenses. The focusing lens may, in one example, be a reduction lens. In another example, the focusing lens is a magnification lens. In other embodiments, the objective system includes one or more mirrors. In yet other embodiments, the objective system includes an optical fiber.

[0126] When the optical conditioning component is configured to move, the optical conditioning component may be configured to move continuously or in discrete intervals. In some embodiments, the movement of the optical conditioning component is continuous. In other embodiments, the optical conditioning component can be moved in discrete intervals, including increments of, for example, 0.01 microns or more, for example, 0.05 microns or more, for example, 0.1 microns or more, for example, 0.5 microns or more, for example, 1 micron or more, for example, 10 microns or more, for example, 100 microns or more, for example, 500 microns or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, and 25 mm or more.

[0127] Any displacement protocol may be employed to move the optical adjustment component structure, such as those coupled to a movable support stage or employing a motorized translation stage, a lead screw translation assembly, a geared translation device, e.g., a stepper motor, a servo motor, a brushless electric motor, a brushed DC motor, a microstep drive motor, a high resolution stepper motor, among other types of motors.

[0128] The light detector of the subject system can be any convenient light detection protocol, including, but not limited to, optical sensors or photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, other detectors, and combinations thereof. In certain embodiments, the collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector has a resolution of 0.01 cm. 2 ~10cm 2 , e.g. 0.05cm 2 ~9cm 2, e.g. 0.1cm 2 ~8cm 2 , e.g. 0.5cm 2 ~7cm 2 Range (1cm 2 ~5cm 2 and a photomultiplier tube such as a photomultiplier tube having an active detection surface area of ​​each region that is 100 nm to 150 nm.

[0129] In embodiments of the present disclosure, the photodetector may be configured to detect light at one or more wavelengths, such as two or more wavelengths, for example five or more different wavelengths, such as ten or more different wavelengths, for example twenty-five or more different wavelengths, such as fifty or more different wavelengths, for example one hundred or more different wavelengths, such as two hundred or more different wavelengths, for example three hundred or more different wavelengths, and including measuring light at four hundred or more different wavelengths.

[0130] In embodiments, the light detector may be configured to measure light continuously or at discrete intervals. In some cases, the detector is configured to obtain light measurements continuously. In other cases, the detector is configured to obtain measurements at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or some other interval.

[0131] The photodetector may be configured to obtain measurements of light from the light source one or more times during each discrete time interval, including two or more times, for example, three or more times, for example, five or more times, and ten or more times. In certain embodiments, the light from the light source is measured by the photodetector two or more times, and in certain cases, the data is averaged.

[0132] In some embodiments, the optical detection system is configured to detect emitted light from particles in the sample, such as particle luminescence (i.e., fluorescence or phosphorescence). In other embodiments, the optical detection system is configured to detect transmitted light, for example, the optical detection system includes a bright field photodetector.

[0133] In certain embodiments, the light detection system is configured to detect scattered light. In embodiments, the scattered light according to the present disclosure is not fluorescence or phosphorescence. In some embodiments, the scattered light detected by the scattered light detector of the system of interest comprises Mie scattering by particles in the flow stream. In other embodiments, the scattered light detected by the scattered light detector of the system of interest comprises Rayleigh scattering by particles in the flow stream. In still other embodiments, the scattered light detected by the scattered light detector of the system of interest comprises Mie scattering and Rayleigh scattering by particles in the flow stream. The scattered light detector may be a side scattered light detector, a forward scattered light detector, a back scattered light detector, or combinations thereof.

[0134] A light detection system according to certain embodiments includes an unfiltered light scattering detector. The term "unfiltered" is used herein to refer to a light scattering detector that receives light from a sample that has not been transmitted through optical components configured to restrict, reduce, or otherwise limit the propagation of one or more wavelengths of light (e.g., the wavelengths of light of a laser used to irradiate the sample) from the sample to the active surface of the light scattering detector. For example, in some embodiments, the intended unfiltered light scattering detector is not in optical communication with the sample through a bandpass filter. In other embodiments, the intended unfiltered light scattering detector is not in optical communication with the sample through a dichroic mirror. In certain cases, scattered light from the sample is transmitted directly to the active surface of the unfiltered light scattering detector. In other cases, scattered light from the sample is transmitted to the active surface of the unfiltered light scattering detector through one or more light-transmitting optical components, such as optical components that change the direction or focus of the light beam without reducing, restricting, or limiting the propagation of one or more wavelengths of light. In certain embodiments, scattered light from the sample is transmitted to the active surface of the unfiltered light scattering detector using one or more beam splitters, mirrors, lenses, or collimators.

[0135] In some embodiments, the light detection system includes one or more filtered light scattering detectors. The term "filtered" is used herein to refer to a light scattering detector that receives light from a sample that has been conveyed through optical components configured to restrict, reduce, or limit the propagation of at least one or more wavelengths of light (e.g., one or more of the wavelengths of light of a laser used to irradiate the sample) from the sample to the active surface of the light scattering detector. The light conveyed to the light scattering detector can include optical components that restrict the propagation of one or more, e.g., 5 or more, e.g., 10 or more, e.g., 25 or more, e.g., 50 or more, e.g., 100 or more, e.g., 200 or more, e.g., 300 or more, different wavelengths of light, including limiting the propagation of 500 or more different wavelengths of light. For example, in some embodiments, scattered light from the sample is conveyed to the active surface of the filtered light scattering detector through a bandpass filter. In other embodiments, scattered light from the sample is conveyed to the active surface of the filtered light scattering detector through a dichroic mirror.

[0136] The scattered light may be detected by each photodetector at an angle of, for example, 1° or more, for example, 10° or more, for example, 15° or more, for example, 20° or more, for example, 25° or more, for example, 30° or more, for example, 45° or more, for example, 60° or more, for example, 75° or more, for example, 90° or more, for example, 135° or more, or for example, 150° or more, relative to the incident beam of light illumination, including when the scattered light detector is configured to detect light from particles in the sample at an angle of 180° or more relative to the incident beam of light illumination. In certain cases, one or more of the filtered light scattering detector and the unfiltered light scattering detector is a side scattered light detector, for example, where the photodetector is positioned to detect scattered light propagating at an angle between 30° and 120°, including between 60° and 90°, relative to the incident beam of light illumination, such as between 45° and 105°. In certain cases, one or more of the filtered light scattering detector and the unfiltered light scattering detector is a side scattered light detector positioned at an angle of 90° relative to the incident beam of light illumination. In other cases, one or more of the filtered light scatter detector and the unfiltered light scatter detector are forward scatter detectors, e.g., where the detectors are positioned to detect scattered light propagated at angles between 120° and 240°, e.g., between 100° and 220°, e.g., between 120° and 200°, including between 140° and 180°, relative to the incident beam of light illumination. In certain cases, one or more of the filtered light scatter detector and the unfiltered light scatter detector are forward scatter detectors positioned to detect scattered light propagated at angles between 180° and 180°, relative to the incident beam of light illumination. In still other cases, one or more of the filtered light scatter detector and the unfiltered light scatter detector are backscatter detectors positioned to detect scattered light propagated at angles between 1° and 30°, e.g., between 5° and 25°, including between 10° and 20°, relative to the incident beam of light illumination.In certain cases, one or more of the filtered light scattering detector and the unfiltered light scattering detector is a backscattered light detector positioned to detect scattered light propagated at an angle of 30° relative to the incident beam of light illumination.

[0137] Light from each interrogation region on the flowstream is detected by two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, and even ten or more photodetector channels. In some embodiments, light is detected by a different photodetector for each light source (e.g., each laser) used to illuminate the flowstream. For example, if particles in the flowstream are illuminated by a first laser and a second laser, light from each particle in the flowstream illuminated by the first laser can be detected by a first photodetector channel, and light from each particle in the flowstream illuminated by the second laser can be detected by a second photodetector channel. In certain embodiments, scattered light from each particle in the flowstream illuminated by the first laser is detected by a first photodetector channel, and scattered light from each particle in the flowstream illuminated by the second laser is detected by a second photodetector channel.

[0138] In embodiments, the memory includes instructions that cause the processor to generate one or more signal pulses in each photodetector channel in response to the detected light. In some embodiments, the memory includes instructions that cause the processor to generate a plurality of signal pulses in each photodetector channel in response to the detected light, e.g., two or more signal pulses, e.g., three or more signal pulses, e.g., four or more signal pulses, e.g., five or more signal pulses, e.g., six or more signal pulses, e.g., seven or more signal pulses, e.g., eight or more signal pulses, e.g., nine or more signal pulses, including generating ten or more signal pulses in each photodetector channel in response to the detected light. In some embodiments, the memory includes instructions that generate one or more signal pulses in different photodetector channels in response to light detected from particles illuminated by each light source (e.g., each laser). For example, if particles in a flow stream are illuminated by a first laser and a second laser, a signal pulse can be generated in a first photodetector channel in response to light detected from each particle illuminated by the first laser, and a signal pulse can be generated in a second photodetector channel in response to light detected from each particle illuminated by the second laser.

[0139] In some embodiments, the signal pulse is a voltage pulse.The voltage pulse can comprise 0.001mV or more, for example 0.005mV or more, for example 0.01mV or more, for example 0.05mV or more, for example 0.1mV or more, for example 0.5mV or more, for example 1mV or more, for example 5mV or more, for example 10mV or more, for example 25mV or more, for example 50mV or more, for example 100mV or more, for example 250mV or more, for example 500mV or more, for example 1000mV or more, for example 2500mV or more and 5000mV or more. In some embodiments a signal pulse is generated in each photodetector channel when the detected light produces a voltage above a predetermined threshold of 0.00001 mV or more, such as 0.00005 mV or more, for example 0.0001 mV or more, such as 0.0005 mV or more, for example 0.001 mV or more, such as 0.005 mV or more, for example 0.01 mV or more, such as 0.05 mV or more, for example 0.1 mV or more, such as 0.5 mV or more, for example 1 mV or more, such as 5 mV or more, for example 10 mV or more, such as 25 mV or more, for example 50 mV or more, such as 100 mV or more, for example 250 mV or more, such as 500 mV or more, for example 1000 mV or more, such as 2500 mV or more, and including a predetermined threshold of 5000 mV or more.

[0140] In some embodiments, the memory comprises instructions for determining the amount of time between photodetector signal pulses, in some cases the memory comprises instructions for determining the amount of time between signal pulses on two or more, for example three or more, for example four or more, for example five or more, for example six or more, for example seven or more, for example eight or more, for example nine or more, for example ten or more, for example twelve or more, for example sixteen or more, for example twenty-four or more, for example twenty-four or more, for example thirty-two or more, for example sixty-four or more, for example 128 or more, for example 256 or more different photodetector channels, and including determining the amount of time between signal pulses on 512 or more different photodetector channels. In some cases, the time between signal pulses in two or more different photodetector channels may be 0.00001 μs or more, such as 0.00005 μs or more, for example 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs, for example 0.01 μs or more, for example 0.05 μs or more, such as 0.1 μs or more, for example 0.5 μs or more, such as 1 μs or more, for example 5 μs or more, such as 10 μs or more, for example 25 μs or more, such as 50 μs or more, for example 100 μs or more, such as 500 μs or more, and may include 1000 μs or more. In certain cases, the time between signal pulses in two or more different photodetector channels is in the range of 0.00001 μs to 5000 μs, such as 0.0001 μs to 4000 μs, such as 0.001 μs to 3000 μs, such as 0.01 μs to 2000 μs, such as 0.1 μs to 1000 μs, including 1 μs to 500 μs.

[0141] In some embodiments, the memory includes instructions for determining the duration to peak between photodetector signal pulses. The term "time to peak" is used herein in its conventional sense to refer to the duration between the peaks (e.g., the highest detected voltage values ​​in a photodetector channel during a signal pulse) of the generated signal pulses in each photodetector channel. For example, the duration to peak can be determined between signal pulses in 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 12 or more, such as 16 or more, such as 24 or more, such as 24 or more, such as 32 or more, such as 64 or more, such as 128 or more, such as 256 or more different photodetector channels, including determining the duration to peak between signal pulses in 512 or more different photodetector channels. In some cases, the time to peak between signal pulses in two or more different photodetector channels may be 0.00001 μs or more, such as 0.00005 μs or more, for example 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs, for example 0.01 μs or more, such as 0.05 μs or more, for example 0.1 μs or more, such as 0.5 μs or more, for example 1 μs or more, such as 5 μs or more, for example 10 μs or more, such as 25 μs or more, for example 50 μs or more, for example 100 μs or more, such as 500 μs or more, and may include 1000 μs or more. In certain cases, the peak-to-peak duration between signal pulses in two or more different photodetector channels is in the range of 0.00001 μs to 5000 μs, such as 0.0001 μs to 4000 μs, such as 0.001 μs to 3000 μs, such as 0.01 μs to 2000 μs, such as 0.1 μs to 1000 μs, including 1 μs to 500 μs.

[0142] In some embodiments, the memory includes instructions for calculating the velocity of each particle of interest in the flow stream. In some cases, the velocities of one or more particles in the sample are independently determined, such as the velocities of two or more particles in the sample, such as the velocities of five or more particles in the sample, such as the velocities of ten or more particles in the sample, such as the velocities of twenty-five or more particles in the sample, such as the velocities of fifty or more particles in the sample, such as the velocities of one hundred or more particles in the sample, such as the velocities of two or more particles in the sample, such as the velocities of five or more particles in the sample, including independently calculating the velocities of five hundred or more particles in the sample. For example, the velocity of 0.0001% or more, such as 0.0005% or more, for example 0.001% or more, such as 0.005% or more, for example 0.01% or more, such as 0.05% or more, for example 0.1% or more, such as 0.5% or more, for example 1% or more, such as 5% or more, for example 10% or more, such as 25% or more, for example 50% or more, such as 75% or more of the particles in the sample can be independently calculated, including independently calculating the velocity of 90% or more of the particles in the sample.

[0143] In some embodiments, the memory includes instructions for calculating a velocity of each particle based on the amount of time between signal pulses in two or more photodetector channels and the distance between two or more interrogation areas. In one embodiment, the memory includes instructions for calculating a velocity of each particle based on the amount of time between a signal pulse from a first photodetector channel and a signal pulse from a second photodetector channel and the distance between the interrogation area of ​​the first laser and the interrogation area of ​​the second laser. In some cases, the memory includes instructions for determining a distance between a first interrogation area and a second interrogation area based on the distance between a location on the flowstream illuminated by the first laser and a location on the flowstream illuminated by the second laser. For example, depending on the particle analyzer in a particular case, the distance between the first interrogation area and the second interrogation area may be 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 2 μm or more, for example 3 μm or more, such as 4 μm or more, for example 5 μm or more, such as 10 μm or more, for example 15 μm or more, such as 25 μm or more, for example 50 μm or more, including 100 μm or more. In other cases, the distance between the first interrogation area and the second interrogation area may be in the range of 0.01 μm to 500 μm, such as 0.05 μm to 450 μm, for example 0.1 μm to 400 μm, such as 0.5 μm to 350 μm, for example 1 μm to 200 μm, such as 5 μm to 150 μm, including 10 μm to 100 μm.

[0144] In some embodiments, particles in the flowstream are characterized by non-uniform velocities, where particles in the flowstream have independently different velocities. In some cases, the velocities of particles vary between one or more of the investigation regions of the flowstream, such as when particles exhibit a greater flow rate between two or more of the investigation regions. In some cases, particles exhibit a slower flow rate between two of the investigation regions of the flowstream. In some cases, the memory includes instructions for determining flow rates between two or more of the investigation regions, including between three or more, such as between four or more, and including between five or more investigation regions. In some cases, the memory includes instructions for determining whether there are diverging velocities between the investigation regions. In some embodiments, the memory includes instructions for determining whether there is laser pointing instability based on one or more diverging velocities, e.g., the laser is determined to have pointing instability if there is a diverging velocity between the investigation regions.

[0145] In some cases, the non-uniformity of particle velocity depends on the particle's position in the flow stream. In some cases, the flow stream includes a sheath fluid flow and a sample core stream flow. In a flow stream having a core stream and a surrounding sheath flow, in certain cases, the velocity of particles flowing near the center of the core stream is faster than the velocity of particles flowing along the periphery of the core stream.

[0146] In embodiments, the memory includes instructions for calculating the positional velocity of a particle in the flowstream. In some cases, the memory includes instructions for determining the velocity of the particle at different positions from the center of the flowstream, for example, the velocity is determined at a predetermined increment of distance from the center of the flowstream. In some cases, the memory includes instructions for determining the velocity of the particle in the flowstream at increments of 0.0001 μm or more from the center of the flowstream (e.g., 0.0001 μm from the center of the flowstream, 0.0002 μm from the center of the flowstream, 0.0003 μm from the center of the flowstream, etc.), for example, 0.0005 μm or more, for example, 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm increments, and including increments of 1 μm or more from the center of the flowstream.

[0147] In some embodiments, the memory includes instructions for determining the position of a particle in a flowstream by plotting the flowstream on a Cartesian plot and determining the Cartesian coordinates of the particle. For example, the Cartesian plot can be a plot of a cross-section of the flowstream, such that a cross-sectional position on the Cartesian plot (e.g., a position on the x-axis and a position on the y-axis) is determined for the particle. In some cases, the position of the particle in the flowstream is determined based on polar coordinates of the flowstream. In one particular case, a cross-section of the flowstream is plotted, and the polar coordinates of the particle in the flowstream are determined as the position of the particle in the flowstream.

[0148] In some embodiments the position of the particle is measured as a distance from the center of the flow stream, for example the position of the particle is 0.0001 μm or more from the center of the flow stream, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the center of the flow stream. For example, the particle location may be between 0.0001 μm and 10000 μm from the center of the flow stream, such as between 0.0005 μm and 5000 μm, such as between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, such as between 0.01 μm and 100 μm, such as between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the center of the flow stream.

[0149] In some embodiments, the position of the particle is measured as a distance from the wall of the flow channel (e.g., the periphery of the flow stream), for example the position of the particle is 0.0001 μm or more from the wall of the flow channel, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the wall of the flow channel. For example, the particle may be located between 0.0001 μm and 10000 μm from the wall of the flow channel, such as between 0.0005 μm and 5000 μm, for example between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, for example between 0.01 μm and 100 μm, for example between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the wall of the flow channel.

[0150] In some cases, the memory includes instructions for using the determined parameters of the particle to calculate the positional velocity of the particle in the flow stream accordingly. In some cases, the memory includes instructions for determining image parameters of the particle in the flow stream. In some cases, the memory includes instructions for generating image parameters from imaging data of the particle from the measured light. In some cases, the memory includes instructions for generating image parameters from images of the particle from the detected light. One or more images can be generated from the measured light. In some embodiments, the memory includes instructions for generating a single image for each particle from each form of detected light. In other embodiments, the memory includes instructions for generating an image for each particle, including multiple images of the particle, e.g., two or more, e.g., three or more, e.g., five or more, e.g., ten or more, and twenty-five or more. For example, the memory includes instructions for generating a first image of the particle from detected light absorption; instructions for generating a second image of the cell from detected light scattering; and instructions for generating a third image of the cell from detected light emission. In other embodiments, the memory includes instructions for generating two or more images, such as three or more, such as four or more, such as five or more, and ten or more images, or combinations thereof, from each form of detected light.

[0151] In some cases, the memory includes instructions for generating one or more grayscale images of the particles. In some embodiments, the memory includes instructions for determining a pixel intensity threshold from the grayscale image, the pixel intensity threshold being used to convert each pixel to a binary value that is used to generate the image of the particle. In certain cases, the image of the particle is a binary pixel image of the particle, e.g., each pixel is assigned a binary pixel value of 1 (e.g., if the intensity of the pixel exceeds a predetermined threshold) or a binary pixel value of 0 (e.g., if the intensity of the pixel is below a predetermined threshold).

[0152] In some embodiments, the memory includes instructions for calculating one or more image parameters from the generated images of the particles. In some cases, the memory includes instructions for calculating a center of mass image parameter from the generated images. In some cases, the memory includes instructions for calculating a delta center of mass image parameter from the generated images. In some cases, the memory includes instructions for calculating a diffuse image parameter from the generated images. In some cases, the memory includes instructions for calculating an eccentricity image parameter from the generated images. In some cases, the memory includes instructions for calculating a major axis moment image parameter from the generated images. In some cases, the memory includes instructions for calculating a maximum intensity image parameter from the generated images. In some cases, the memory includes instructions for calculating a radial moment image parameter from the generated images. In some cases, the memory includes instructions for calculating a minor axis moment image parameter from the generated images. In some cases, the memory includes instructions for calculating a particle size image parameter from the generated images. In some cases, the memory includes instructions for calculating a total intensity image parameter from the generated images. In some cases, the memory includes instructions for calculating a particle light loss image parameter from the generated images. In some cases, the memory includes instructions for calculating forward scatter image parameters from the generated images. In some cases, the memory includes instructions for calculating side scatter image parameters from the generated images. In some cases, the memory includes instructions for calculating image moments from the generated images. In some cases, the memory includes instructions for calculating center of mass parameters from the image moments of the images. In other cases, the memory includes instructions for calculating cell orientation from the image moments of the images. In still other cases, the memory includes instructions for calculating cell eccentricity from the image moments of the images.

[0153] In some embodiments, the memory includes instructions for calculating a position velocity of a particle in a flow stream based on the particle's velocity and a center of mass (COM) parameter. In some cases, the memory includes instructions for calculating the center of mass parameter from image moments and generated images of the particle. For example, the center of mass of a particle can be determined from the calculated image moments and generated images according to:

[0154]

number

[0155] In some embodiments, the center of mass parameter includes a position of the particle in the flowstream. In some cases, the position of the particle in the flowstream is based on Cartesian coordinates of the particle plotted on a Cartesian plot. For example, the Cartesian plot can be a plot of a cross-section of the flowstream such that a cross-sectional position on the Cartesian plot (e.g., a position on the x-axis and a position on the y-axis) is determined for the particle. In some cases, the position of the particle in the flowstream is based on polar coordinates of the particle in the flowstream.

[0156] In some embodiments, the particle position for determining the center of mass parameter is the distance of the particle from the center of the flow stream, for example the particle position is 0.0001 μm or more from the center of the flow stream, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the center of the flow stream. For example, the particle location may be between 0.0001 μm and 10000 μm from the center of the flow stream, such as between 0.0005 μm and 5000 μm, such as between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, such as between 0.01 μm and 100 μm, such as between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the center of the flow stream.

[0157] In some embodiments, the position of the particle for determining the center of mass parameter is the distance of the particle from the wall of the flow channel (e.g., the periphery of the flow stream), for example the position of the particle is 0.0001 μm or more from the wall of the flow channel, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 5 μm or more, for example 10 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, for example 1000 μm or more, including 5000 μm or more from the wall of the flow channel. For example, the particle may be located between 0.0001 μm and 10000 μm from the wall of the flow channel, such as between 0.0005 μm and 5000 μm, for example between 0.001 μm and 1000 μm, such as between 0.005 μm and 500 μm, for example between 0.01 μm and 100 μm, for example between 0.05 μm and 50 μm, including between 0.1 μm and 25 μm from the wall of the flow channel.

[0158] In some embodiments, the memory includes instructions for determining a laser delay of one or more of the lasers based on the calculated position velocity of the particles. In some cases, the memory includes instructions for adjusting a laser delay of one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and laser delay of each particle in the flow stream. In certain cases, the memory includes instructions for adjusting a laser delay of one or more of the lasers based on the position velocity of the particles.

[0159] In some embodiments, the memory includes instructions for determining a drop delay for each particle based on the particle's calculated position velocity and the distance between the interrogation region of one or more of the light sources and the flow cell nozzle orifice. In some embodiments, the memory includes instructions for independently determining a drop delay for each particle of interest by multiplying the calculated position velocity of each particle of interest by the distance between the interrogation region of one or more of the light sources and the flow cell nozzle orifice. For example, depending on the particle analyzer in a particular instance, the distance between each interrogation region and the flow cell nozzle orifice may independently be 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 2 μm or more, such as 3 μm or more, such as 4 μm or more, such as 5 μm or more, such as 10 μm or more, such as 15 μm or more, such as 25 μm or more, such as 50 μm or more, including 100 μm or more. In other cases, the distance between each interrogation area and the flow cell nozzle orifice is in the range of 0.01 μm to 500 μm, such as 0.05 μm to 450 μm, for example 0.1 μm to 400 μm, for example 0.5 μm to 350 μm, for example 1 μm to 200 μm, for example 5 μm to 150 μm, including 10 μm to 100 μm.

[0160] In some embodiments, the memory includes instructions for adjusting one or more parameters of the particle analyzer based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the memory includes instructions for increasing the flow rate of the flow stream. In certain cases, the flow rate can be increased by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including increasing the flow rate of the flow stream by 99.9% or more. In some embodiments, the memory comprises instructions for increasing the flow rate of the flow stream by 1 μL / sec or more, such as 2 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, such as 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more, such as 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the memory comprises instructions for increasing the flow rate of the flow stream by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, such as 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including instructions for increasing the flow rate of the flow stream by 10 mm / sec or more.

[0161] In other cases, the memory includes instructions for decreasing the flow rate of the flow stream. In certain cases, the memory includes instructions for decreasing the flow rate by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including decreasing the flow rate of the flow stream by 99.9% or more. In some embodiments, the memory includes instructions for decreasing the flow rate of the flow stream by 1 μL / sec or more, such as 2 μL / sec or more, such as 5 μL / sec or more, such as 10 μL / sec or more, such as 25 μL / sec or more, such as 50 μL / sec or more, such as 100 μL / sec or more, such as 250 μL / sec or more, such as 500 μL / sec or more, such as 750 μL / sec or more, and including 1000 μL / sec or more. In other embodiments, the memory includes instructions for reducing the flow rate of the flow stream by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, such as 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, such as 5 mm / sec or more, including where the memory includes instructions for reducing the flow rate of the flow stream by 10 mm / sec or more.

[0162] In some cases, the memory includes instructions for increasing the flow of sheath liquid based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the memory includes instructions for increasing the flow of sheath liquid by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including increasing the flow rate of sheath liquid by 99.9% or more. In some embodiments, the memory includes instructions for increasing the flow rate of the sheath fluid by 1 μL / sec or more, such as 2 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, such as 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the memory includes instructions for increasing the flow rate of the sheath fluid by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, for example 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including where the memory includes instructions for increasing the flow rate of the sheath fluid by 10 mm / sec or more.

[0163] In some cases, the memory includes instructions for reducing the flow of sheath liquid based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the memory includes instructions for reducing the flow of sheath liquid by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including reducing the flow rate of sheath liquid by 99.9% or more. In some embodiments, the memory includes instructions for decreasing the flow rate of the sheath fluid by 1 μL / sec or more, such as 2 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, such as 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, including 1000 μL / sec or more. In other embodiments, the memory includes instructions for decreasing the flow rate of the sheath fluid by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, for example 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including when the memory includes instructions for decreasing the flow rate of the sheath fluid by 10 mm / sec or more.

[0164] In some cases, the memory includes instructions for increasing the flow of the sample core liquid based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the flow of the sample core liquid is increased by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including increasing the flow rate of the sample core liquid by 99.9% or more. In some embodiments, the memory comprises instructions for increasing the flow of the sample core fluid stream by 1 μL / sec or more, such as 2 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, such as 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more, such as 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, and including 1000 μL / sec or more. In other embodiments, the flow of the sample core fluid is increased by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, such as 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, such as 5 mm / sec or more, including when the flow rate of the sample core fluid stream is increased by 10 mm / sec or more.

[0165] In some cases, the memory includes instructions for reducing the flow of sample core liquid based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream. In some cases, the memory includes instructions for reducing the flow of sample core liquid by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including reducing the flow rate of the sample core liquid by 99.9% or more. In some embodiments, the memory comprises instructions for decreasing the flow of the sample core fluid stream by 1 μL / sec or more, such as 2 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, such as 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more, such as 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, and including 1000 μL / sec or more. In other embodiments, the memory comprises instructions for decreasing the flow of the sample core fluid by 0.001 mm / sec or more, such as 0.005 mm / sec or more, for example 0.01 mm / sec or more, such as 0.05 mm / sec or more, for example 0.1 mm / sec or more, such as 0.5 mm / sec or more, for example 1 mm / sec or more, for example 5 mm / sec or more, including where the memory comprises instructions for decreasing the flow rate of the sample core fluid stream by 10 mm / sec or more.

[0166] In some embodiments, the memory includes instructions for adjusting the timing of laser firing by one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the memory includes instructions for adjusting the timing of laser firing by one or more of the lasers to delay by 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, and including 50% or more. For example, the memory may include instructions for delaying the timing of laser firing by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, for example 0.005 μs or more, for example 0.01 μs or more, for example 0.05 μs or more, for example 0.1 μs or more, for example 0.5 μs or more, such as 1 μs or more, for example 5 μs or more, for example 10 μs or more, for example 25 μs or more, for example 50 μs or more, for example 100 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream, and may include instructions for delaying laser firing by one or more of the lasers by 500 μs. In other cases, the memory includes instructions for adjusting the timing of laser irradiation by one or more of the lasers to be earlier, including by 0.01% or more, for example 0.05% or more, for example 0.1% or more, for example 0.5% or more, for example 1% or more, for example 2% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, and 50% or more.For example, the memory includes instructions for adjusting the timing of laser firing by one or more of the lasers to be earlier, including by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, such as 0.005 μs or more, for example 0.001 μs or more, such as 0.005 μs or more, for example 0.01 μs or more, such as 0.05 μs or more, for example 0.1 μs or more, such as 0.5 μs or more, for example 1 μs or more, such as 5 μs or more, for example 10 μs or more, such as 25 μs or more, for example 50 μs or more, for example 100 μs or more, and 500 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0167] In some embodiments, the memory includes instructions for adjusting the timing of droplet charging based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the memory includes instructions for adjusting the timing of droplet charging to delay by 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, and including 50% or more. For example, the memory may include instructions for delaying the timing of droplet charging by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, for example 0.005 μs or more, for example 0.001 μs or more, for example 0.005 μs or more, for example 0.01 μs or more, for example 0.05 μs or more, for example 0.1 μs or more, for example 0.5 μs or more, for example 1 μs or more, for example 5 μs or more, for example 10 μs or more, for example 25 μs or more, for example 50 μs or more, for example 100 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream, including delaying droplet charging by 500 μs or more in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In other cases, the memory includes instructions to adjust the timing of droplet charging to be earlier by 0.01% or more, such as 0.05% or more, for example 0.1% or more, for example 0.5% or more, such as 1% or more, for example 2% or more, such as 5% or more, for example 10% or more, such as 15% or more, for example 25% or more, and including 50% or more.For example, the memory includes instructions for adjusting the timing of droplet charging to be earlier, including by 0.0001 μs or more, such as 0.0005 μs or more, for example 0.001 μs or more, for example 0.005 μs or more, such as 0.01 μs or more, for example 0.05 μs or more, such as 0.1 μs or more, for example 0.5 μs or more, such as 1 μs or more, for example 5 μs or more, such as 10 μs or more, for example 25 μs or more, for example 50 μs or more, such as 100 μs or more, and 500 μs or more, in response to one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0168] In other embodiments, the memory includes instructions for adjusting the drop drive frequency based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the memory includes instructions for increasing the drop drive frequency by 0.01 Hz or more, e.g., 0.05 Hz or more, e.g., 0.1 Hz or more, e.g., 0.25 Hz or more, e.g., 0.5 Hz or more, e.g., 1 Hz or more, e.g., 2.5 Hz or more, e.g., 5 Hz or more, e.g., 10 Hz or more, and including 25 Hz or more. For example, the drop drive frequency can be increased by 1% or more, e.g., 5% or more, e.g., 10% or more, e.g., 15% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, including increasing the drop drive frequency by 90% or more. In other cases, the memory includes instructions to reduce the drop drive frequency by 0.01 Hz or more, for example 0.05 Hz or more, for example 0.1 Hz or more, for example 0.25 Hz or more, for example 0.5 Hz or more, for example 1 Hz or more, for example 2.5 Hz or more, for example 5 Hz or more, for example 10 Hz or more, and including 25 Hz or more. For example, the drop drive frequency can be reduced by 1% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, including reducing the drop frequency by 90% or more.

[0169] In yet other embodiments, the memory includes instructions for adjusting the drop delay based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream. In some cases, the memory includes instructions for increasing the drop delay by, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.3 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2.5 μs or more, for example, 5 μs or more, for example, 7.5 μs or more, including increasing the drop delay by 10 μs or more. For example, the memory includes instructions for increasing the drop delay by 1% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, including instructions for increasing the drop delay by 90% or more. In other cases, the memory includes instructions to reduce the drop frequency by, for example, 0.01 μs or more, for example 0.05 μs or more, for example 0.1 μs or more, for example 0.3 μs or more, for example 0.5 μs or more, for example 1 μs or more, for example 2.5 μs or more, for example 5 μs or more, for example 7.5 μs or more, including instructions to reduce the drop delay by 10 μs or more. For example, the memory includes instructions to reduce the drop delay by 1% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, including reducing the drop delay by 90% or more.

[0170] In some embodiments, the system is a flow cytometer. In some cases, the subject flow cytometer includes a flow cell. The subject flow cell includes a cuvette configured to transport particles in a flow stream. As discussed herein, "flow cell" is described in its conventional sense, referring to a component including a flow path with a liquid flow stream for transporting particles of sheath fluid. The subject cuvette has a passageway (i.e., a flow channel) therethrough. The flow stream comprising the flow channel may include a liquid sample injected from a sample tube. In certain cases, the flow cell includes an optically accessible flow channel. The cuvette may be constructed of, for example, quartz, glass, clear plastic, etc. In some embodiments, the cuvette is formed from silica, such as fused silica. In some cases, the flow cell is configured to be illuminated with light from a light source at one or more interrogation points. As discussed herein, "interrogation point" refers to an area within the flow cell where particles are illuminated by light from a light source, for example, for analysis. The size of the interrogation point may vary as needed. For example, if 0 μm represents the optical axis of the light emitted by the light source, the interrogation point may be in the range of -50 μm to 50 μm, e.g., -25 μm to 40 μm (including -15 μm to 30 μm). Depending on specific considerations (e.g., the number and placement of lasers), multiple illumination points may be present within the flow cell.

[0171] In some embodiments, the flow cell includes or is configured for use with a sample injection port configured to provide a sample to the flow cell, hi embodiments, the sample injection system is configured to provide a suitable flow of sample into the flow cell internal chamber (e.g., flow channel). Depending on the desired characteristics of the flow stream, the rate at which sample is delivered by the sample injection port to the flow cell chamber may be 1 μL / min or more, such as 2 μL / min or more, for example 3 μL / min or more, such as 5 μL / min or more, for example 10 μL / min or more, for example 15 μL / min or more, such as 25 μL / min or more, for example 50 μL / min or more (including 100 μL / min or more), and in some cases the rate at which sample is delivered by the sample injection port to the flow cell chamber is 1 μL / sec or more, such as 2 μL / sec or more, for example 3 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, for example 15 μL / sec or more, for example 25 μL / sec or more, for example 50 μL / sec or more (including 100 μL / sec or more).

[0172] The sample injection port can be an orifice disposed in the wall of the internal chamber or a conduit disposed at the proximal end of the internal chamber. When the sample injection port is an orifice disposed in the wall of the internal chamber, the sample injection port orifice can have any suitable cross-sectional shape, including, but not limited to, rectilinear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal, curvilinear cross-sectional shapes such as circular and elliptical, as well as irregular shapes such as a parabolic bottom joined to a flat top. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on the shape, with openings ranging from 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm (including 1.25 mm to 1.75 mm, e.g., 1.5 mm).

[0173] In certain cases, the sample injection port is a conduit located at the proximal end of the flow cell internal chamber. For example, the sample injection port may be a conduit positioned so that the orifice of the sample injection port is aligned with the flow cell orifice. When the sample injection port is a conduit aligned with the flow cell orifice, the cross-sectional shape of the sample injection tube may be any suitable shape, including, but not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal, curved cross-sectional shapes such as circular and elliptical, as well as irregular shapes such as a parabolic bottom joined to a flat top. In certain cases, the orifice of the conduit may have an opening ranging from 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm (including 1.25 mm to 1.75 mm, e.g., 1.5 mm), and may vary depending on the shape. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip having a bevel angle in the range of 1° to 10° (e.g., 2° to 9°, e.g., 3° ​​to 8°, e.g., 4° to 7°), including a bevel angle of 5°.

[0174] In some embodiments, the flow cell also includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid to the flow cell interior chamber, e.g., in conjunction with the sample, to generate a stacked flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of sheath fluid delivered to the flow cell by the sheath fluid injection port can be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more (including 2500 μL / sec or more).

[0175] In some embodiments, the sheath fluid injection port is an orifice disposed in the wall of the internal chamber. The sheath fluid injection port orifice may be of any suitable shape, including, but not limited to, rectilinear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal, curvilinear cross-sectional shapes such as circular and elliptical, as well as irregular shapes such as a parabolic bottom joined to a flat top. The size of the sheath fluid injection port orifice may vary depending on the shape, with certain cases ranging from 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm (including 1.25 mm to 1.75 mm), e.g., having an opening of 1.5 mm.

[0176] The flow cytometer may include any suitable mechanism for supplying sheath fluid and sample fluid to the sample fluid input coupler and sheath fluid input coupler. For example, the sample fluid input coupler may be fluidly connected to a sample fluid line (e.g., tubing) that is fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidly connected to a sheath fluid line that is fluidly connected to a sheath fluid reservoir. Similarly, the flow cytometer may include any suitable mechanism for managing waste from the flow stream. The fluid output coupler may be fluidly connected to a waste line that is fluidly connected to a waste reservoir. A fluid management system that may be adapted for use with the subject flow cytometers is provided in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0177] Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1):17-28; Linden, et al., Semin Thromb Hemost. 2004 Oct; 30(5):502-11; Alison, et al. J Pathol. 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference.In certain instances, the flow cytometry system of interest is a BD Biosciences FACSCanto™ flow cytometer, a BD Biosciences FACSCanto™ II flow cytometer, a BD Accuri™ flow cytometer, a BD Accuri™ C6 Plus flow cytometer, a BD Biosciences FACSCelesta™ flow cytometer, a BD Biosciences FACSLyric™ flow cytometer, a BD Biosciences FACSVerse™ flow cytometer, a BD Biosciences FACSymphony™ flow cytometer, a BD Biosciences LSRFortessa™ flow cytometer, a BD Biosciences LSRFortessa™ X-20 flow cytometer, a BD Biosciences FACSPresto™ flow cytometer, a BD Biosciences FACSVia™ flow cytometer, and a BD Biosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cell sorter, a BD Biosciences These include the FACSLyric™ cell sorter, BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, BD Biosciences FACSDiscover™ cell sorter, and others.

[0178] In some embodiments, the subject systems may be implemented using the same or similar technology as disclosed in U.S. Patent Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, and 10,578,469, the disclosures of which are incorporated herein by reference in their entireties. No. 10,481,074, No. 10,302,545, No. 10,145,793, No. 10,113,967, No. 10,006,852, No. No. 9,952,076, No. 9,933,341, No. 9,726,527, No. 9,453,789, No. 9,200,334, No. 9,097,6 No. 40, No. 9,095,494, No. 9,092,034, No. 8,975,595, No. 8,753,573, No. 8,233,146, No. 8 ,140,300, No. 7,544,326, No. 7,201,875, No. 7,129,505, No. 6,821,740, No. 6,813,017 Nos. 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766.

[0179] In some embodiments, the flow cytometer is configured as an imaging flow cytometer.For example, in certain cases, the target system is a flow cytometry system configured to image particles in a flow stream by fluorescence imaging using radiofrequency tagged emission (FIRE), as described in, for example, Diebold, et al. Nature Photonics Vol.7(10), 806-810(2013) and U.S. Patent Nos. 9,423,353, 9,784,661 and 10,006,852, and U.S. Patent Application Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.In some embodiments where the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter. Image-enabled particle sorters are described in US Provisional Patent Applications Nos. 63 / 431,803 and 63 / 465,057, the disclosures of which are incorporated herein by reference in their entireties.

[0180] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses a memory in which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are or become available. The processor executes an operating system, which interfaces with firmware and hardware in well-known ways and facilitates the processor's coordination and execution of the functions of various computer programs, which may be written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0181] System memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a permanent hard disk or tape, optical media such as a read-and-write compact disc, flash memory devices, or other memory storage devices. The memory storage device may also be any of a variety of known or future devices, including a compact disc drive, tape drive, or diskette drive. Such types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disc. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with the memory storage devices.

[0182] In some embodiments, a computer program product is described that includes a computer-usable medium having stored thereon control logic (a computer software program including program code). The control logic, when executed by a processor of a computer, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementing a hardware state machine to perform the functions described herein will be apparent to one skilled in the art.

[0183] The subject programmable logic may be implemented in any of a variety of devices, such as a specifically programmed event processing computer, a wireless communication device, an integrated circuit device, etc. In some embodiments, the programmable logic may be executed by a specially programmed processor, which may include one or more processors, such as one or more digital signal processors (DSPs), configurable microprocessors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or other equivalent integrated or discrete logic circuits. Combinations of computing devices, such as a DSP with a microprocessor, multiple microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration in at least partial data connection, may implement one or more of the features described herein.

[0184] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, or tape, or RAM, or any other suitable device, fixed or portable). The processor may include a general-purpose digital microprocessor that is appropriately programmed from a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communications channel or may be pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium using any of these devices in conjunction with the memory. For example, a magnetic or optical disk may carry the program and be read by a disk writer / reader. The system of the present disclosure also includes programming in the form of a computer program product, e.g., algorithms for use in implementing the above-described methods. Programming according to the present disclosure may be recorded on a computer-readable medium, e.g., any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape, optical storage media such as CD-ROMs, electrical storage media such as RAM, ROM, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0185] The processor may also have access to a communication channel for communicating with a user at a remote location, meaning that the user is not in direct contact with the system but relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0186] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications (e.g., radio frequency identification (RFID), Zigbee communications protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0187] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., a physical port or interface such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port that enables data communication between the subject system and other external devices, such as a computer terminal (e.g., in a doctor's office or hospital environment) configured for similar complementary data communication.

[0188] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the target system to communicate with computer terminals and / or other devices such as networks, communication-enabled mobile phones, personal digital assistants, or any other communication device that a user can integrate and use.

[0189] In one embodiment, the communication interface is configured to provide connectivity for data transfer using Internet Protocol (IP) over a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) in a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0190] In one embodiment, the target system is configured to wirelessly communicate with a server device via a communications interface using common standards such as, for example, 802.11 or Bluetooth® RF protocols, or the IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0191] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the target system, e.g., any data storage unit, with a network or server device using one or more of the above communication protocols and / or mechanisms.

[0192] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of graphical elements. A graphical user interface (GUI) controller provides a graphical input / output interface between the system and the user and may include any of a variety of known or future software programs for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing modules to users at remote locations, for example, via the Internet, telephone, or satellite networks, according to known techniques. Presentation of data by the output manager may be performed according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or data in other formats. The data may also include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the subject system can be any type of known or future-developed computer platform, but they are typically computers of a class commonly referred to as servers. However, they may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cabling or other communication system, including wireless systems, and may or may not be networked. They may be co-located or physically separated.A variety of operating systems may be employed on any computer platform depending on the type and / or manufacturer of the computer platform selected. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, Windows 10, iOS, macOS, Linux, Ubuntu, Fedora, OS / 400, i5 / OS, IBM i, Android, SGI IRIX, Oracle Solaris, and the like.

[0193] FIG. 2 illustrates a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 includes a laser 201 configured to illuminate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. While the example of FIG. 2 shows a single laser, it is understood that multiple lasers can also be used. The laser beam from the laser 201 is directed toward a focusing lens 202, which focuses the beam onto a portion of the fluid stream where the sample particles 211 are located within the flow cell 210. The flow cell 210 is part of a fluid system that directs particles, typically one at a time, within the stream toward the focused laser beam for interrogation (interrogation). Alternatively, if the flow cytometer is a stream-in air cytometer, a nozzle top can be used.

[0194] As shown in FIG. 2 , the flow cell 210 is fluidly connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. The sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a conduit (i.e., sheath fluid line) 207. Additionally, the sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a conduit (i.e., sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce the particles 211 into the interior of the flow cell 210. The particles 211 are hydrodynamically focused via the sheath fluid entering the sheath fluid injection port 208 such that a flow stream 214 is formed downstream of the tapered portion 212 of the flow cell 210. Particles emitting at the distal end of the flow cell 210 can be disposed of and / or collected via any suitable protocol. For example, depending on the type of flow cytometry being performed, particles may be collected at the distal end of flow cell 210, for example, via a waste line. Alternatively, particles may be sorted.

[0195] Light from the laser beam interacts with sample particles through diffraction, refraction, reflection, scattering, and absorption by re-emission at various different wavelengths, depending on particle characteristics such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The fluorescent light and the diffracted, refracted, reflected, and scattered light may be sent to one or more detectors. In particular, forward-scattered light (FSC) is sent to a forward-scattered light detector 223. The forward-scattered light detector 223 is positioned slightly off-axis from the direct beam passing through the flow cell 210 and is configured to detect diffracted light, excitation light traveling primarily in the forward direction through or around the particle. The intensity of the light detected by the forward-scattered light detector 223 depends on the overall size of the particle. The forward-scattered light detector may include, for example, a photodiode. An optical filter 221a and a scattering bar 222 are positioned between the forward-scattered light detectors 223. The optical filter 221a may be configured to remove at least one wavelength of non-FSC light, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scattered light detector 223.

[0196] Additionally, side-scattered light (SSC) is detected by a side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of the particle 211, which tends to increase as the particle's structure becomes more complex. In the example of FIG. 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered light detector 224 while passing non-SSC (e.g., fluorescent) light. An optical filter 221b is configured to prevent at least one wavelength of non-SSC light from being detected by the side-scattered light detector 224. Also shown are fluorescence detectors 225a-225c, each configured to detect fluorescence of a different wavelength. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a while passing light of other wavelengths. Optical filter 221c may be configured to prevent at least one wavelength of light that does not correspond to the first wavelength (or wavelength range) from being detected by fluorescence detector 225a. Similarly, dichroic mirror 220c is configured to reflect FL light corresponding to the second wavelength (or wavelength range) to fluorescence detector 225b, while passing light of a third wavelength (or wavelength range) for detection by fluorescence detector 225c. Optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to the second wavelength (or wavelength range) from being detected by fluorescence detector 225b. Furthermore, optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to the third wavelength (or wavelength range) from being detected by fluorescence detector 225c.

[0197] Those skilled in the art will recognize that flow cytometers according to embodiments of the present disclosure are not limited to the flow cytometer shown in FIG. 2 , but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and in a variety of different configurations. For example, while the embodiment of FIG. 2 shows three fluorescence detectors for illustrative purposes, it will be understood that any suitable number of fluorescence detectors may be used.

[0198] During operation, the operation of the cytometer is controlled by controller / processor 290, and measurement data from the detectors may be stored in memory 295 and processed by controller / processor 290. While not explicitly shown, controller / processor 290 is coupled to the detectors to receive output signals from the detectors, and may also be coupled to electrical and electromechanical components of the flow cytometer to control laser 201, fluid flow parameters, etc. Input / output (I / O) functionality 297 may also be provided within the system. Memory 295, controller / processor 290, and I / O 297 may be provided entirely as an integral part of the flow cytometer. In such embodiments, a display may form part of I / O functionality 297 for presenting experimental data to a user of cytometer 200. Alternatively, memory 295 and controller / processor 290 and some or all of the I / O functionality may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of memory 295 and controller / processor 290 may be in wireless or wired communication with cytometer 210. In conjunction with memory 295 and I / O 297, controller / processor 290 can be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0199] Different fluorescent molecules in a panel of fluorescent dyes used in a flow cytometer experiment emit light in their own characteristic wavelength bands. The particular fluorescent labels used in the experiment and their associated fluorescence emission bands can be selected to generally match the filter window of the detector. I / O 297 can be configured to receive data regarding a flow cytometer experiment having a panel of fluorescent labels and multiple cell populations having multiple markers, each cell population having a subset of the multiple markers. I / O 297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker concentration data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 295. Controller / processor 290 can be configured to evaluate one or more assignments of labels to markers.

[0200] In some embodiments, the subject system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having multiple sorting determination units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module with deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0201] In certain embodiments, the system is a fluorescence imaging using a radio frequency tagged luminescence imaging enabled particle sorter as shown in FIG. 3. Particle sorter 300 includes a light illumination component 300a including a light source 301 (e.g., a 488 nm laser) that generates an output beam of light 301a that is split into beams 302a and 302b by a beam splitter 302. Light beam 302a propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a having one or more angularly deflected light beams. In some cases, output beam 303a generated from acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Light beam 302b propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a having one or more angularly deflected light beams. In some cases, output beam 304a generated from acousto-optic device 304 includes a local oscillator beam and multiple radio frequency comb beams. Output beams 303a and 304a generated from acousto-optical devices 303 and 304, respectively, are combined with beam splitter 305 to generate output beam 305a, which is conveyed through optical component 306 (e.g., an objective lens) to illuminate particles in flow cell 307. In certain embodiments, acousto-optical device 303 (AOD) splits a single laser beam into an array of beamlets, each having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then overlapped with the array of beamlets at beam combiner 305. In certain embodiments, the light illumination system having a light source and acousto-optical device can also include those described in Schraivogel, et al. (“High-speed fluorescence image-enabled cell sorting,” Science (2022), 375(6578):315-320) and U.S. Patent Application Publication No. 2021 / 0404943, the disclosures of which are incorporated herein by reference.

[0202] Output beam 305a illuminates sample particles 308 propagating through flow cell 307 (e.g., with sheath fluid 309) in illumination region 310. As shown in illumination region 310, multiple beams (e.g., angularly deflected, high-frequency shifted optical beams shown as dots across illumination region 310) overlap with a reference local oscillator beam (shown as hatched across illumination region 310). Due to their different optical frequencies, the overlapping beams exhibit beating behavior, whereby each beamlet emits a distinct frequency f 1~n carries a sinusoidal modulation.

[0203] Light from the illuminated sample is conveyed to a light detection system 300b, which includes multiple photodetectors. The light detection system 300b includes a forward-scattered light photodetector 311 for generating a forward-scattered image 311a and a side-scattered light photodetector 312 for generating a side-scattered image 312a. The light detection system 300b also includes a bright-field photodetector 313 for generating a light loss image 313a. In some embodiments, the forward-scattered light detector 311 and the side-scattered light detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the illuminated sample is also detected by fluorescence photodetectors 314-317. In some cases, the photodetectors 314-317 are photomultiplier tubes. The light from the illuminated sample is directed through a beam splitter 320 to the side-scattered light detection channel 312 and the fluorescence detection channels 314-317. Light detection system 300b includes bandpass optical components 321, 322, 323, and 324 (e.g., dichroic mirrors) for transmitting light of predetermined wavelengths to light detectors 314-317. In some cases, optical component 321 is a 534 nm / 40 nm bandpass. In some cases, optical component 322 is a 586 nm / 42 nm bandpass. In some cases, optical component 323 is a 700 nm / 54 nm bandpass. In some cases, optical component 324 is a 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number indicates the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, i.e., from 500 nm to 520 nm.

[0204] Data signals generated in response to light detected in scattered light detection channels 311 and 312, bright-field light detection channel 313, and fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated by processors 350 and 351. Image-enabled sorting is performed in response to a sorting signal generated by sorting trigger 352. Sorting component 300c includes deflection plates 331 for deflecting particles into a sample container 332 or to a waste stream 333. In some cases, sorting component 300c is configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In one particular embodiment, the sorting component 300c includes a sorting determination module having multiple sorting determination units, such as those described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0205] In some embodiments, the system is a particle analyzer, and particle analysis system 401 (FIG. 4) can be used to analyze and characterize particles, with or without physically sorting the particles into a collection vessel. FIG. 4 shows a functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, particle analysis system 401 is a flow system. Particle analysis system 401 includes a fluidic system 402. Fluidic system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube through which particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0206] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. A detection station 408 generally refers to a monitoring area 407 of the common sample path. Detection, in some implementations, may include detecting light or one or more other characteristics of the particle 403 as it passes through the monitoring area 407. FIG. 4 shows one detection station 408 with one monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Additionally, some detection stations may monitor more than one area.

[0207] Each signal is assigned a signal value to form a data point for each particle. This data may be referred to as event data, as described above. The data points may be multidimensional data points that include values ​​for each property measured for the particle. The detection system 404 is configured to collect such data points continuously over a first time interval.

[0208] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system 406 may further compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0209] 5 shows a functional block diagram of an example particle analyzer control system for analyzing and displaying biological events, such as an analysis controller (e.g., processor) 500. Analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0210] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel. The analysis controller 500 can be a processor configured to perform the methods of the present invention, for example, by applying a distance-based classification model to determine a density distinction threshold in a size-based analyte feature space, applying a density-based clustering algorithm to separate the analyte data into high-density clusters and low-density clusters based on the density threshold, and classifying the analyte data based on the high-density clusters and low-density clusters based on the size-based analyte feature space.

[0211] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 can include flow cytometry event data. The analysis controller 500 can be configured to provide a graphical display on a display device 506 that includes a first plot of the biological event data. The analysis controller 500 can be further configured to render a region of interest, for example, as a gate around a population of the biological event data shown by the display device 506, overlaid on the first plot. In some embodiments, the gate can be a logical combination of one or more graphical regions of interest depicted on a histogram or bivariate plot of a single parameter. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0212] Analysis controller 500 can be further configured to display biological event data within the gate on display device 506 differently from other events in the biological event data outside the gate. For example, analysis controller 500 can be configured to render the color of the biological event data contained within the gate differently from the color of the biological event data outside the gate. Display device 506 can be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0213] The analysis controller 500 can be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a gate selection signal to the analysis controller 500 identifying a gate to be displayed on or operated via the display device 506 (e.g., by clicking the desired gate when a cursor is positioned there). In some implementations, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, a stylus, a photodetector, or a voice recognition system. Some input devices may include multiple input functions. In such implementations, each input function can be considered an input device. For example, as shown in FIG. 5, the mouse 510 can include a right mouse button and a left mouse button, each capable of generating a trigger event.

[0214] The trigger event can cause the analysis controller 500 to change how the data is displayed, what portions of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting a population for particle sorting purposes.

[0215] In some embodiments, the analysis controller 500 can be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the visualization of the plot to facilitate the gating process. The modification can be based on a particular distribution of the biological event data received by the analysis controller 500.

[0216] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can be further configured to enable retrieval of biological event data, such as flow cytometry event data, by the analysis controller 500.

[0217] The display device 506 can be configured to receive display data from the analysis controller 500. The display data can include a plot of the biological event data and a gate that delineates a section of the plot. The display device 506 can be further configured to modify the information presented according to input received from the analysis controller 500, along with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0218] In some implementations, the analysis controller 500 can generate a user interface for receiving exemplary events for sorting. For example, the user interface can include controls for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates can be provided prior to collection of event data for the sample or based on an initial set of events for a subset of the sample.

[0219] FIG. 6A is a schematic diagram of a particle sorter system 600 (e.g., particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. As shown in FIG. 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which can be coupled to, include, or be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned in single file across a monitoring area 611 (e.g., where laser streams intersect) illuminated by an illumination source 612 (e.g., a laser). Vibration of droplet-forming transducer 602 causes moving fluid column 608 to break up into multiple droplets 610 , some of which contain particles 609 .

[0220] During operation, the detection station 614 (e.g., an event detector) identifies when a particle of interest (or cell of interest) crosses the monitoring area 611. The detection station 614 inputs a timing circuit 628, which in turn inputs a flash charge circuit 630. At a droplet break-off point, signaled by a timed drop delay (Δt), a flash charge can be applied to the moving fluid column 608 so that the droplet of interest carries a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the droplet into a container, such as a collection tube or a multi-well or microwell sample plate, and a well or microwell can be associated with the particular droplet of interest. As shown in FIG. 6A, the droplets can be collected in a waste receptacle 638.

[0221] Detection system 616 (e.g., a droplet boundary detector) helps automatically determine the phase of the drop drive signal as a particle of interest passes through monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. Detection system 616 allows the instrument to accurately calculate the location of each detected particle in the droplet. Detection system 616 can provide amplitude signal 620 and / or phase 618 signals, which are then provided (via amplifier 622) to amplitude control circuit 626 and / or frequency control circuit 624. Amplitude control circuit 626 and / or frequency control circuit 624 then control droplet forming transducer 602. Amplitude control circuit 626 and / or frequency control circuit 624 can be included in a control system.

[0222] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the particle's event data. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit or can be communicatively coupled such that event measurements can be collected by either the detection system 616 or the detection station 614 and provided to a non-collecting element.

[0223] FIG. 6B is a schematic diagram of a particle sorter system according to one embodiment presented herein. The particle sorter system 600 shown in FIG. 6B includes deflection plates 652 and 654. An electric charge can be applied via stream charging wires within the barbs. This creates a stream of droplets 610 containing particles 610 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The information about the particles is analyzed, such as by sorting electronics or another detection system (not shown in FIG. 6B). Deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, directing them toward a destination collection receptacle (e.g., any of 672, 674, 676, or 678). 6B, deflector plates 652 and 654 can be controlled to direct particles along a first path 662 toward a receptacle 674 or along a second path 668 toward a receptacle 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector plates can allow the particle to continue along flow path 664. Such uncharged droplets can enter a waste receptacle, such as via an aspirator 670.

[0224] Sorting electronics may be included to initiate the collection of measurements, receive fluorescent signals about the particles, and determine how to adjust the deflection plates to cause particle sorting. An exemplary implementation of the embodiment shown in Figure 6B includes the BD FACSAria™ line of flow cytometers commercially offered by Becton, Dickinson and Company (Franklin Lakes, NJ).

[0225] FIG. 7 illustrates the general architecture of an exemplary computing device 700 according to certain embodiments. The general architecture of computing device 700 illustrated in FIG. 7 includes an arrangement of computer hardware and software components. However, not all of these generally conventional elements need be shown to form an enabling disclosure. As illustrated, computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and input devices 760, all of which can communicate with each other via a communication bus. Network interface 720 can connect to one or more networks or computing systems. Thus, processing unit 710 can receive information and instructions from other computing systems or services over a network. Processing unit 710 also communicates with memory 770 and can further communicate output information to optional display 750 via input / output device interface 740. For example, analysis software (e.g., data analysis software or a program such as FlowJo®) stored as executable instructions in the analysis system's non-transitory memory can display flow cytometry event data to a user. The input / output device interface 740 can also accept input from optional input devices 760 such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0226] Memory 770 may include computer program instructions (grouped in some embodiments as modules or components) that processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that communicates computer program instructions used by processing unit 710 in the general management and operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0227] Non-transitory computer-readable storage medium Aspects of the present disclosure further include non-transitory computer-readable storage media having instructions for performing the subject methods, such as performing one or more computer-implemented methods described herein. The computer-readable storage medium may be used by one or more computers to fully or partially automate a system for performing the methods described herein. In certain embodiments, instructions according to the methods described herein may be encoded on a computer-readable medium in the form of "programming," and the term "computer-readable medium" as used herein refers to any non-transitory storage medium involved in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state disks, and network-attached storage (NAS), whether such devices are internal or external to the computer. Files containing information may be "stored" on a computer-readable medium, where "storing" means recording information so that it can be accessed and retrieved at a later date by a computer. The computer-implemented methods described herein may be performed using programming that may be written in one or more of any number of computer programming languages, including, for example, Python, Java, JavaScript, C, C#, C++, Go, R, Swift, PHP, and many others.

[0228] In some embodiments, a non-transitory computer-readable storage medium includes an algorithm for illuminating a sample containing particles in a flow stream of a particle analyzer with a first laser in a first interrogation region and a second laser in a second interrogation region; detecting light from the illuminated particles with a first photodetector in the first interrogation region and a second photodetector in the second interrogation region; calculating the velocity of the particles in the flow stream based on the light detected in the first and second photodetector channels; determining parameters of the particles in the flow stream; and calculating the positional velocity of the particles in the flow stream based on the calculated velocity and parameters of the particles.

[0229] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the velocity of particles in the flowstream by determining the amount of time between photodetector signal pulses. In other cases, the non-transitory computer-readable storage medium includes an algorithm for determining the time to peak between photodetector signal pulses. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the velocity of each particle in the flowstream based on the amount of time between a first photodetector signal pulse and a second photodetector signal pulse and the distance between the interrogation areas of the first laser and the second laser. In other cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the velocity of each particle in the flowstream based on the time between the peak of the first photodetector signal pulse and the peak of the second photodetector signal pulse and the distance between the interrogation areas of the first laser and the second laser.

[0230] In embodiments, a non-transitory computer-readable storage medium includes an algorithm for determining a positional velocity of a particle in a flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining a position of a particle in a flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining a position of a particle relative to a center of the flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating a particle's velocity relative to the particle's distance from the center of the flowstream. In some cases, if a particle is closer to the center of the flowstream, the particle has a higher velocity. In some cases, if a particle is farther from the center of the flowstream, the particle has a lower velocity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating a particle's center of mass parameter (COM) for each particle. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining the particle's position relative to the center of the flowstream based on the calculated center of mass parameter. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining a particle's positional velocity based on the particle's calculated velocity and center of mass parameter.

[0231] In some embodiments, a non-transitory computer-readable storage medium includes an algorithm for illuminating particles in a flow stream with multiple lasers at multiple different interrogation regions; an algorithm for detecting light from the illuminated particles with multiple photodetector channels; generating photodetector signal pulses in response to the light detected by each of the photodetector channels; an algorithm for calculating the velocity of the particles in the flow stream based on the photodetector signal pulses in two or more of the photodetector channels; instructions for determining parameters (e.g., center of mass parameters) of the particles in the flow stream; and an algorithm for calculating the position velocity of the particles in the flow stream based on the calculated velocity and parameters of the particles.

[0232] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating an average velocity of particles between two or more of a plurality of search regions. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating average velocities between three or more search regions, such as four or more, including between five or more search regions. In some cases, the non-transitory computer-readable storage medium includes an algorithm for evaluating the velocities between each of the search regions to determine whether there is an errant velocity between one or more of the search regions. In some cases, the non-transitory computer-readable storage medium includes an algorithm for comparing the average velocities between the search regions and instructions for evaluating whether the velocity between any two different search regions is an errant velocity.

[0233] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining a laser delay of one or more of the lasers based on a calculated position velocity of the particles. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting a laser delay of one or more of the lasers based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and laser delay of each particle in the flow stream. In certain cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting a laser delay of one or more of the lasers based on a position velocity of the particles.

[0234] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the timing of illumination by each of the lasers. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting the timing of illumination by one or more of the lasers based on a calculated velocity of the particles in the flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting the timing of illumination by one or more of the lasers based on a calculated average velocity of the particles in the flowstream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting the timing of illumination by one or more of the lasers based on a calculated positional velocity of the particles in the flowstream.

[0235] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining a drop delay for each particle based on the particle's calculated position velocity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting a drop delay for each particle based on one or more of the particle's calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay in the flow stream. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for adjusting one or more parameters of a particle analyzer based on one or more of the particle's calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay in the flow stream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for increasing a flow rate of the flow stream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for decreasing a flow rate of the flow stream. In some cases, the flow stream includes a flow of sheath liquid and a flow of sample core stream, and the non-transitory computer-readable storage medium includes an algorithm for adjusting one or more of the flow of sheath liquid and the flow of sample core stream based on one or more of a calculated velocity, a center of mass parameter, and a calculated position velocity of each particle in the flow stream. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for adjusting timing of droplet charging based on one or more of a calculated velocity, a center of mass parameter, a calculated position velocity, and a calculated drop delay of each particle in the flow stream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting timing of irradiation by one or more of the lasers based on one or more of a calculated velocity, a center of mass parameter, and a calculated position velocity of each particle in the flow stream.

[0236] The non-transitory computer-readable storage medium may be used in one or more computer systems having a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses a memory in which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are or become available. The processor executes an operating system, which interfaces with firmware and hardware in well-known manners and facilitates the processor's coordination and execution of the functions of various computer programs, which may be written in various programming languages, such as those mentioned above, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0237] kit Kits containing one or more components of the subject system are also provided. In certain embodiments, the kit includes two or more light sources, such as two continuous wave lasers and a photodetector (e.g., a photomultiplier tube). The kit may also include a flow cell nozzle and a cuvette for illuminating the flow stream. The kit may also include optical adjustment components, such as lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof.

[0238] In some embodiments, the kits include storage media such as optical magnetic disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state disks, and network-attached storage (NAS). Some of these program storage media, or others now in use or that may later be developed, may be included in the subject kits. In embodiments, the program storage media include instructions for analyzing flow cytometer data for use with the methods and systems described herein. In embodiments, the instructions included on computer-readable media provided in the subject kits, or portions thereof, may be implemented as software components of software for analyzing data. In these embodiments, a computer-controlled system according to the present disclosure may function as a software "plug-in" for an existing software package (e.g., FlowJo®).

[0239] In addition to the above components, the subject kits may further include (in some embodiments) instructions for carrying out the subject methods. These instructions may be present in the subject kits in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, package inserts, etc. Another form in which these instructions may be present is a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Another form in which these instructions may be present is a website address that can be used via the Internet to access the information at a remote site.

[0240] Utilities The subject methods, systems, and computer systems find use in a variety of applications where it is desirable to optimize particle identification, characterization, and sorting. The subject methods and systems provide for dynamic particle velocity and drop delay determination in real time. The present disclosure also finds use in flow cytometry, where it is desirable to provide a flow cytometer with improved cell sorting accuracy, increased particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting. In embodiments, the present disclosure reduces the need for user input or manual adjustments during sample analysis by a flow cytometer. In certain embodiments, the subject methods and systems provide fully automated protocols so that adjustments to the flow cytometer during use require little, if any, human input.

[0241] Notwithstanding the appended claims, the present disclosure is also defined by the following paragraphs.

[0242] 1. A method comprising: irradiating a sample containing particles in a flow stream of a particle analyzer with a first laser in a first interrogation region and a second laser in a second interrogation region; detecting light from the illuminated particle with a first photodetector channel in a first interrogation region and a second photodetector channel in a second interrogation region; calculating a velocity of the particle in the flow stream based on the light detected by the first photodetector channel and the second photodetector channel; determining parameters of particles in the flow stream; calculating a position velocity of the particle in the flow stream based on the calculated velocity and the parameter of the particle; A method comprising:

[0243] 2. The method of clause 1, wherein the method includes calculating a position velocity of the particle based on the distance of the particle from the center of the flow stream.

[0244] 3. The method of clause 1 or 2, wherein the method includes calculating a center of mass (COM) parameter of particles in the flow stream.

[0245] 4. The method of any one of clauses 1-3, wherein the second laser is configured to irradiate the flow stream at a location downstream from the first laser.

[0246] 5. The method of clause 4, wherein the second laser is configured to irradiate the flow stream at a location no more than 200 μm downstream from the first laser.

[0247] 6. How to generating a first photodetector signal pulse in response to light detected by the first photodetector channel; generating a second photodetector signal pulse in response to light detected by a second photodetector channel; 6. The method of any one of clauses 1 to 5, comprising:

[0248] 7. The method of clause 6, wherein the method includes determining an amount of time between a first photodetector signal pulse and a second photodetector signal pulse.

[0249] 8. The method of clause 6, wherein the method further includes determining a time between a peak of the first photodetector signal pulse and a peak of the second photodetector signal pulse.

[0250] 9. The method of any one of clauses 6 to 8, wherein each photodetector signal pulse is a voltage pulse.

[0251] 10. The method of any one of clauses 1-9, wherein the light detected from the particles comprises scattered light.

[0252] 11. The method of clause 10, wherein the light detected by the first photodetector channel comprises scattered light from particles illuminated by the first laser.

[0253] 12. The method of clause 10 or 11, wherein the light detected by the second photodetector channel comprises scattered light from particles illuminated by the second laser.

[0254] 13. The velocity of the particle in the flow stream is the amount of time between the first photodetector signal pulse and the second photodetector signal pulse; and The distance between the investigation area of ​​the first laser and the investigation area of ​​the second laser The method according to any one of clauses 6 to 12, wherein the calculation is based on

[0255] 14. 14. The method of any one of clauses 1-13, wherein the particle analyzer comprises a flow cell having a flow cell nozzle for generating droplets containing each particle.

[0256] 15. 15. The method of any one of clauses 1-14, wherein the first laser comprises a trigger laser and the trigger signal is generated in a first photodetector channel.

[0257] 16. 16. The method of any one of clauses 1-15, wherein the method further comprises determining a drop delay for each particle based on the calculated position velocity of the particle.

[0258] 17. 17. The method of claim 16, further comprising adjusting the drop delay for each particle based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0259] 18. 18. The method of any one of clauses 1-17, wherein the method further comprises adjusting one or more parameters of the particle analyzer based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0260] 19. 19. The method of claim 18, wherein the method includes increasing the flow rate of the flow stream.

[0261] 20. 19. The method of clause 18, wherein the method includes reducing the flow rate of the flow stream.

[0262] twenty one. 21. The method of any one of clauses 18-20, wherein the method includes adjusting the timing of droplet charging based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated droplet delay of each particle in the flow stream.

[0263] twenty two. 21. The method of any one of clauses 18-20, wherein the method includes adjusting timing of illumination by the second laser in the second interrogation region based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream.

[0264] twenty three. The method is: irradiating particles in the flowstream with a plurality of lasers at a plurality of different interrogation regions; detecting light from the illuminated particles in a plurality of photodetector channels; generating a photodetector signal pulse in response to light detected by each of the photodetector channels; calculating a velocity of the particle in the flow stream based on the photodetector signal pulses in two or more of the photodetector channels; determining parameters of particles in the flow stream; calculating a position velocity of the particle in the flow stream based on the calculated velocity and the parameter of the particle; 23. The method of any one of clauses 1 to 22, comprising:

[0265] twenty four. 24. The method of claim 23, wherein the plurality of lasers are configured to irradiate the flow stream at locations spaced apart from each other by no more than 200 μm.

[0266] twenty five. The plurality of lasers may be a second laser configured to illuminate the flow stream at a location downstream from the first laser; a third laser configured to illuminate the flow stream at a location downstream from the second laser; a fourth laser configured to illuminate the flow stream at a location downstream from the third laser; a fifth laser configured to illuminate the flow stream at a location downstream from the fourth laser; 25. The method according to clause 23 or 24, comprising:

[0267] 26. 26. The method of any one of clauses 23-25, further comprising calculating an average velocity of particles in the flow stream between each of the plurality of interrogation regions.

[0268] 27. 27. The method of any one of clauses 23-26, further comprising calculating the timing of illumination of the flow stream by each of the lasers.

[0269] 28. 28. The method of claim 26 or 27, further comprising assessing laser drift for one or more of the lasers based on the calculated average velocity of particles in the flow stream between each of the plurality of interrogation regions.

[0270] 29. 28. The method of claim 26 or 27, further comprising adjusting timing of illumination by one or more of the lasers based on the calculated average velocity of the particles between each of the plurality of interrogation regions.

[0271] 30. 30. The method of any one of clauses 23-29, wherein the velocity of each particle in the flow stream is calculated based on photodetector signal pulses in three or more of the photodetector channels.

[0272] 31. 30. The method of any one of clauses 1-29, wherein one or more of the lasers is a continuous wave laser.

[0273] 32. 1. A particle analyzer comprising: a light source comprising a first laser and a second laser configured to illuminate a sample including a plurality of particles in a flow stream with the first laser in a first interrogation region and the second laser in a second interrogation region; a light detection system comprising a light detector configured to detect light from each illuminated particle in the sample with a first light detector channel and a second light detector channel; 1. A processor comprising: a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to: calculating a velocity of the particle in the flow stream based on the light detected by the first photodetector channel and the second photodetector channel; determining parameters of particles in the flow stream; a processor that calculates a position velocity of the particle in the flow stream based on the calculated velocity and the parameters of the particle; A particle analyzer comprising:

[0274] 33. 33. The particle analyzer of claim 32, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine a position velocity of the particle based on a distance of the particle from a center of the flow stream.

[0275] 34. 34. The particle analyzer of clause 32 or 33, wherein the memory stores instructions that, when executed by the processor, cause the processor to calculate center of mass (COM) parameters of particles in the flow stream.

[0276] 35. 35. The particle analyzer of any one of clauses 32-34, wherein the second laser is configured to illuminate the flow stream at a location downstream from the first laser.

[0277] 36. 36. The particle analyzer of clause 35, wherein the second laser is configured to illuminate the flow stream at a location no more than 200 μm downstream from the first laser.

[0278] 37. The memory stores instructions that, when executed by the processor, cause the processor to: generating a first photodetector signal pulse in response to light detected in the first photodetector channel; 37. A particle analyzer according to any one of clauses 32 to 36, generating a second photodetector signal pulse in response to light detected in a second photodetector channel.

[0279] 38. 38. The particle analyzer of clause 37, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine an amount of time between a first photodetector signal pulse and a second photodetector signal pulse.

[0280] 39. 38. The particle analyzer of clause 37, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine a time between a peak of the first photodetector signal pulse and a peak of the second photodetector signal pulse.

[0281] 40. 40. A particle analyzer according to any one of clauses 37 to 39, wherein each photodetector signal pulse is a voltage pulse.

[0282] 41. 37. A particle analyzer according to any one of clauses 29 to 36, wherein the photodetector is configured to detect scattered light from the particles.

[0283] 42. 42. The particle analyzer of clause 41, wherein the light detected by the first photodetector channel comprises scattered light from particles illuminated by the first laser.

[0284] 43. 43. The particle analyzer of clause 41 or 42, wherein the light detected by the second photodetector channel comprises scattered light from particles illuminated by the second laser.

[0285] 44. The memory stores instructions that, when executed by the processor, cause the processor to: the amount of time between the first photodetector signal pulse and the second photodetector signal pulse; and The distance between the investigation area of ​​the first laser and the investigation area of ​​the second laser 44. A particle analyzer according to any one of clauses 32 to 43, wherein the particle analyzer calculates the velocity of each particle in the flow stream based on:

[0286] 45. 45. A particle analyzer as described in clauses 32-44, wherein the light detection system comprises a scattered light detector configured to detect scattered light by the first light detector channel and scattered light by the second light detector channel.

[0287] 46. The memory stores instructions that, when executed by the processor, cause the processor to: the amount of time between the first photodetector signal pulse and the second photodetector signal pulse; and The distance between the investigation area of ​​the first laser and the investigation area of ​​the second laser 46. ​​The particle analyzer of any one of clauses 37 to 45, wherein the particle analyzer calculates the velocity of each particle in the flow stream based on:

[0288] 47. 47. A particle analyzer according to any one of clauses 32 to 46, wherein the particle analyzer comprises a flow cell having a flow cell nozzle for producing droplets containing each particle.

[0289] 48. 48. A particle analyzer according to any one of clauses 32 to 47, wherein the first laser comprises a trigger laser and generates a trigger signal in the first photodetector channel.

[0290] 49. 49. A particle analyzer as described in any one of clauses 32 to 48, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine a drop delay for each particle based on the calculated position velocity of the particle.

[0291] 50. 49. The particle analyzer of claim 49, wherein the memory stores instructions that, when executed by the processor, cause the processor to adjust the drop delay of each particle based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0292] 51. 51. The particle analyzer of any one of clauses 32-50, wherein the memory stores instructions that, when executed by the processor, cause the processor to adjust one or more parameters of the particle analyzer based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0293] 52. 52. The particle analyzer of clause 51, wherein the memory stores instructions that, when executed by the processor, cause the processor to increase a flow rate of the flow stream.

[0294] 53. 52. The particle analyzer of clause 51, wherein the memory stores instructions that, when executed by the processor, cause the processor to decrease a flow rate of the flow stream.

[0295] 54. 54. A particle analyzer as described in any one of clauses 51 to 53, wherein the memory stores instructions that, when executed by the processor, cause the processor to adjust the timing of droplet charging based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0296] 55. 54. A particle analyzer as described in any one of clauses 51 to 53, wherein the memory stores instructions that, when executed by the processor, cause the processor to adjust the timing of irradiation by the second laser in the second interrogation region based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream.

[0297] 56. The system is a light source including a plurality of lasers; a light detection system comprising a photodetector configured to detect light from the illuminated particles with a plurality of photodetector channels; 1. A processor comprising a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to: detecting light from the particles with a plurality of photodetector channels; generating photodetector signal pulses in response to light detected by each of the photodetector channels; calculating a velocity of the particle in the flow stream based on the photodetector signal pulses in two or more of the photodetector channels; determining parameters of particles in the flow stream; a processor that calculates a position velocity of the particle in the flow stream based on the calculated velocity and the parameters of the particle; 56. A particle analyzer according to any one of clauses 32 to 55, comprising:

[0298] 57. 57. The particle analyzer of clause 56, wherein the plurality of lasers are configured to illuminate the flow stream at locations spaced apart from each other by no more than 200 μm.

[0299] 58. The plurality of lasers may be a second laser configured to illuminate the flow stream at a location downstream from the first laser; a third laser configured to illuminate the flow stream at a location downstream from the second laser; a fourth laser configured to illuminate the flow stream at a location downstream from the third laser; a fifth laser configured to illuminate the flow stream at a location downstream from the fourth laser; 58. A particle analyzer according to clause 56 or 57, comprising:

[0300] 59. 59. A particle analyzer as described in any one of clauses 56 to 58, wherein the memory stores instructions that, when executed by the processor, cause the processor to calculate an average velocity of particles in the flow stream between each of the plurality of interrogation regions.

[0301] 60. 60. A particle analyzer as described in any one of clauses 56 to 59, wherein the memory stores instructions that, when executed by the processor, cause the processor to calculate the timing of irradiation of the flow stream by each of the lasers.

[0302] 61. 60. The particle analyzer of any one of clauses 56-59, wherein the memory stores instructions that, when executed by the processor, cause the processor to assess laser drift for one or more of the lasers based on the calculated average velocity of particles in the flow stream between each of the plurality of interrogation regions.

[0303] 62. 60. A particle analyzer as described in any one of clauses 56 to 59, wherein the memory stores instructions that, when executed by the processor, cause the processor to adjust the timing of irradiation by one or more of the lasers based on the calculated average velocity of the particles between each of the plurality of interrogation regions.

[0304] 63. 63. A particle analyzer as described in any one of clauses 56-62, wherein the memory stores instructions that, when executed by the processor, cause the processor to calculate the velocity of each particle in the flow stream based on the photodetector signal pulses in three or more of the photodetector channels.

[0305] 64. 64. A particle analyzer according to any one of clauses 32 to 63, wherein one or more of the lasers is a continuous wave laser.

[0306] 65. 65. A particle analyzer according to any one of clauses 32 to 64, wherein the optical detector comprises a photodiode.

[0307] 66. 65. A particle analyzer according to any one of clauses 32 to 64, wherein the light detector comprises a photomultiplier tube.

[0308] 67. 67. A particle analyzer according to any one of clauses 32 to 66, wherein the particle analyzer is part of a flow cytometer.

[0309] 68. 68. A particle analyzer according to any one of clauses 32 to 67, wherein the system comprises a flow cell comprising a sheath fluid input and a sample input.

[0310] 69. 69. The particle analyzer of claim 68, wherein the memory stores instructions that, when executed by the processor, cause the processor to increase an input flow rate of sheath fluid based on the calculated positional velocity of each particle in the flow stream.

[0311] 70. 70. The particle analyzer of claim 69, wherein the memory stores instructions that, when executed by the processor, cause the processor to increase an input flow rate of the sample based on the calculated positional velocity of each particle in the flow stream.

[0312] 71. 1. A non-transitory computer-readable storage medium storing instructions for determining drop delays of a plurality of particles in a flow stream in a particle analyzer, the instructions comprising: an algorithm for irradiating a sample containing particles in a flow stream of a particle analyzer with a first laser in a first interrogation region and a second laser in a second interrogation region; an algorithm for detecting light from the illuminated particle with a first photodetector channel in a first interrogation region and a second photodetector channel in a second interrogation region; an algorithm for calculating a velocity of the particle in the flow stream based on the light detected by the first photodetector channel and the second photodetector channel; an algorithm for determining parameters of particles in the flow stream; an algorithm for calculating a position velocity of the particle in the flow stream based on the calculated velocity and parameters of the particle; a non-transitory computer-readable storage medium,

[0313] 72. 72. The non-transitory computer readable storage medium of clause 71, wherein the non-transitory computer readable storage medium includes an algorithm for calculating a position velocity of a particle based on a distance of the particle from a center of the flow stream.

[0314] 73. 73. The non-transitory computer readable storage medium of clause 71 or 72, wherein the non-transitory computer readable storage medium comprises an algorithm for calculating a center of mass (COM) parameter of a particle in a flow stream.

[0315] 74. 73. The non-transitory computer readable storage medium of any one of clauses 71 to 72, wherein the non-transitory computer readable storage medium includes an algorithm for irradiating the flow stream with a second laser at a location downstream from the first laser.

[0316] 75. The non-transitory computer-readable storage medium comprises: an algorithm for generating a first photodetector signal pulse in response to light detected by the first photodetector channel; an algorithm for generating a second photodetector signal pulse in response to light detected by the second photodetector channel; A non-transitory computer-readable storage medium according to any one of clauses 71 to 74, comprising:

[0317] 76. 76. The non-transitory computer readable storage medium of clause 75, wherein the non-transitory computer readable storage medium includes an algorithm for determining an amount of time between a first photodetector signal pulse and a second photodetector signal pulse.

[0318] 77. 77. The non-transitory computer readable storage medium of clause 76, wherein the non-transitory computer readable storage medium includes an algorithm for determining a time between a peak of a first photodetector signal pulse and a peak of a second photodetector signal pulse.

[0319] 78. The non-transitory computer-readable storage medium comprises: the amount of time between the first photodetector signal pulse and the second photodetector signal pulse; and Distance between the first and second lasers 78. The non-transitory computer readable storage medium of any one of clauses 75 to 77, comprising an algorithm for calculating the velocity of each particle in a flow stream based on:

[0320] 79. 79. The non-transitory computer readable storage medium of any one of clauses 71-78, wherein the particle analyzer comprises a flow cell having a flow cell nozzle for generating droplets containing particles, and the non-transitory computer readable storage medium includes an algorithm for calculating a drop delay of each particle in the flow stream based on the calculated positional velocity of the particle.

[0321] 80. 80. The non-transitory computer readable storage medium of clause 79, comprising an algorithm for adjusting the drop delay of each particle based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0322] 81. 81. The non-transitory computer readable storage medium of any one of clauses 71-80, wherein the non-transitory computer readable storage medium includes an algorithm for adjusting one or more parameters of the particle analyzer based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0323] 82. 82. The non-transitory computer readable storage medium of clause 81, wherein the non-transitory computer readable storage medium includes an algorithm for increasing a flow rate of the flow stream.

[0324] 83. 82. The non-transitory computer readable storage medium of clause 81, wherein the non-transitory computer readable storage medium includes an algorithm for reducing a flow rate of the flow stream.

[0325] 84. 84. The non-transitory computer readable storage medium of any one of clauses 81-83, wherein the non-transitory computer readable storage medium includes an algorithm for adjusting the timing of droplet charging based on one or more of the calculated velocity, center of mass parameter, calculated position velocity, and calculated drop delay of each particle in the flow stream.

[0326] 85. 84. The non-transitory computer readable storage medium of any one of clauses 81-83, wherein the non-transitory computer readable storage medium includes an algorithm for adjusting the timing of irradiation by the second laser in the second interrogation region based on one or more of the calculated velocity, center of mass parameter, and calculated position velocity of each particle in the flow stream.

[0327] 86. The command is, an algorithm for irradiating particles in the flow stream with multiple lasers at multiple different interrogation regions; an algorithm for detecting light from the illuminated particles by a plurality of photodetector channels; an algorithm for generating photodetector signal pulses in response to light detected by each of the photodetector channels; an algorithm for calculating the velocity of the particle in the flow stream based on the photodetector signal pulses in two or more of the photodetector channels; an algorithm for determining parameters of particles in the flow stream; an algorithm for calculating a position velocity of the particle in the flow stream based on the calculated velocity and parameters of the particle; A non-transitory computer-readable storage medium according to any one of clauses 71 to 85, comprising:

[0328] 87. 87. The non-transitory computer readable storage medium of clause 86, wherein the non-transitory computer readable storage medium includes an algorithm for calculating an average velocity of particles in the flow stream between each of the plurality of interrogation regions.

[0329] 88. 88. The non-transitory computer readable storage medium of clause 86 or 87, wherein the non-transitory computer readable storage medium includes an algorithm for calculating the timing of illumination of the flow stream by each of the lasers.

[0330] 89. 89. The non-transitory computer readable storage medium of any one of clauses 86-88, wherein the non-transitory computer readable storage medium includes an algorithm for assessing laser drift for one or more of the lasers based on a calculated average velocity of particles in the flow stream between each of the plurality of interrogation regions.

[0331] 90. 90. The non-transitory computer readable storage medium of any one of clauses 86-89, wherein the non-transitory computer readable storage medium includes an algorithm for adjusting the timing of irradiation by one or more of the lasers based on the calculated average velocity of the particles between each of the plurality of interrogation regions.

[0332] 91. 91. The non-transitory computer readable storage medium of any one of clauses 85-90, wherein the non-transitory computer readable storage medium includes an algorithm for calculating the velocity of each particle in the flow stream based on the photodetector signal pulses in three or more of the photodetector channels.

[0333] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0334] Thus, the foregoing merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, that embody the principles of the present disclosure and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present disclosure and the concepts that the present disclosure has contributed to advancing the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present disclosure, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended as a dedication to the public, regardless of whether such disclosure is expressly recited in the claims.

[0335] Accordingly, the scope of the present disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present disclosure are embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) are expressly defined as being invoked for a limitation in a claim only if the exact phrase "means for" or the exact phrase "step" appears at the beginning of such limitation in the claim. If such exact phrases are not used in a claim limitation, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is not invoked.

Claims

1. It is a method, The sample containing particles in the flow stream of the particle analyzer is irradiated with a first laser in the first investigation area and a second laser in the second investigation area, The light from the irradiated particles is detected by the first photodetector channel in the first investigation area and the second photodetector channel in the second investigation area, Based on the light detected by the first and second photodetectors, the velocity of the particles in the flow stream is calculated, Determining the parameters of the particles in the flow stream, Based on the calculated velocity of the particle and the parameters, the position velocity of the particle in the flow stream is calculated, Methods that include...

2. The method according to claim 1, wherein the position velocity of the particle is calculated based on the distance of the particle from the center of the flowstream.

3. The method according to claim 1 or 2, wherein the parameter includes the center of mass (COM) parameter of the particle in the flowstream.

4. The method according to claim 1 or 2, wherein the second investigation area is located downstream from the first investigation area.

5. Detecting light from the particles with the first photodetector includes generating a first photodetector signal pulse in response to the light emitted by the particles in the first investigation area, The method according to claim 1 or 2, wherein detecting light from the particles with the second photodetector includes generating a second photodetector signal pulse in response to the light emitted by the particles in the second investigation area.

6. The method according to claim 5, wherein the velocity of the particle is calculated by determining the amount of time between the first photodetector signal pulse and the second photodetector signal pulse.

7. The method according to claim 6, wherein determining the amount of time between the first photodetector signal pulse and the second photodetector signal pulse includes determining the time between the peak of the first photodetector signal pulse and the peak of the second photodetector signal pulse.

8. The velocity of the particles in the flow stream is The amount of time between the first photodetector signal pulse and the second photodetector signal pulse, and The distance between the first survey area and the second survey area. The method according to claim 6, calculated based on the method described in claim 6.

9. The method according to claim 1 or 2, wherein the first laser includes a trigger laser and a trigger signal is generated by the first photodetector.

10. The method according to claim 1 or 2, further comprising determining the laser delay of at least the second laser based on the calculated position velocity of the particle.

11. The method according to claim 1 or 2, further comprising adjusting one or more parameters of the particle analyzer based on the position velocity.

12. The method according to claim 11, wherein the parameter includes the timing of the second laser.

13. The method according to claim 11, further comprising measuring the temperature of the sample, wherein the one or more parameters of the particle analyzer are further adjusted based on the measured temperature.

14. A particle analyzer, A light source comprising a first laser and a second laser, configured to irradiate a sample containing a plurality of particles in a flow stream with the first laser in a first investigation area and the second laser in a second investigation area, A photodetection system comprising a photodetector configured to detect light from each irradiated particle in the sample by a first photodetector channel in the first investigation area and a second photodetector channel in the second investigation area, A processor comprising a memory operably coupled to the processor, wherein the memory stores instructions, and when an instruction is executed by the processor, the processor... Based on the light detected by the first and second photodetectors, the velocity of the particles in the flow stream is calculated. Determine the parameters of the particles in the flow stream. A processor that calculates the position velocity of the particle in the flow stream based on the calculated velocity of the particle and the parameters of the particle, A particle analyzer equipped with the following features.

15. A non-temporary computer-readable storage medium that stores instructions for determining the dropping delay of multiple particles in a flow stream in a particle analyzer, wherein the instructions An algorithm for irradiating a sample containing particles in the flow stream of a particle analyzer with a first laser in a first investigation area and a second laser in a second investigation area, An algorithm for detecting light from irradiated particles using a first photodetector channel in the first investigation area and a second photodetector channel in the second investigation area, An algorithm for calculating the velocity of the particles in the flow stream based on the light detected by the first photodetector and the second photodetector, An algorithm for determining the parameters of the particles in the flow stream, An algorithm for calculating the position velocity of the particle in the flow stream based on the calculated velocity of the particle and the parameters, Includes non-temporary computer-readable storage media.

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