Method and system for evaluating flow cytometer data for coincidence events - Patents.com

Abstract

A method is provided for evaluating flow cytometer data for coincident events. The method of interest includes receiving flow cytometer data associated with a first particle. The received flow cytometer data is obtained from a detector after illuminating a first particle and a second particle in a flow stream with a first light source and a second light source, respectively. The method further includes receiving a first trigger signal and a second trigger signal associated with the first particle and the second particle. Additionally, the subject method evaluates the flow cytometer data for coincident events by generating a time interval for the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal, and determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time interval. A system and a computer readable medium are further provided.

Description

[Background technology]

[0001] Characterization of analytes in biological fluids has become an important part of biological research, medical diagnosis, and the evaluation 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 may be involved in determining treatment protocols for patients with various medical conditions.

[0002] Flow cytometry is a technique used to characterize and often sort biological material, such as cells in a blood sample or particles of interest in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer carries particles (including cells) in the fluid sample as a cell stream to a flow cell, while further directing the sheath fluid to the flow cell. To characterize the components in the flow stream, light is illuminated into the flow stream. Changes in the biological material in the flow stream, such as morphology or the presence of fluorescent labels, may change the light observed, and these changes allow for characterization and separation. To characterize the components in the flow stream, light must be directed into the flow stream and collected. The light source in the flow cytometer can be a variety of light sources and may include one or more broad spectrum lamps, light emitting diodes, and single wavelength lasers. The light source is aligned with the flow stream, and optical responses from the illuminated particles are collected and quantified. Summary of the Invention [Problem to be solved by the invention]

[0003] In spatially separated multi-laser flow cytometry systems, apertures are positioned to collect fluorescent emissions from cells as they pass through each laser beam in the system. The fluorescent emissions collected through these apertures are typically directed to individual detector arrays, one for each laser beam. Because the laser beams are spatially separated from one another and the apertures nominally block the fluorescent light emitted from cells passing through the non-primary lasers, it is generally assumed that any crosstalk between the lasers and detectors will be within acceptable levels. Current systems do not address the problem of crosstalk, relying on the relatively low frequency with which it occurs and the generally small magnitude of crosstalk when it does occur.

[0004] The inventors recognize that while the possibility of crosstalk between lasers and detectors was previously a tolerable defect, the increasing trend to include more colors in flow cytometry protocols is stressing the system to the extent that noise caused by this crosstalk can reduce the fidelity of the resulting data and generate confusing artifacts. Furthermore, as flow cytometers evolve and speed requirements increase, the likelihood of coincidences occurring increases. It is therefore desirable to at least identify crosstalk situations where data may be deemed suspect. Embodiments of the present invention fulfill this need. [Means for solving the problem]

[0005] Aspects of the invention include a method of evaluating flow cytometer data for coincident events. The method of interest includes receiving flow cytometer data associated with a first particle from a detector after illuminating a first particle and a second particle in a flow stream with a first light source and a second light source, respectively. The method further includes receiving a first trigger signal and a second trigger signal associated with the first particle and the second particle, respectively. The method further includes evaluating the flow cytometer data for coincident events by generating a time interval for the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal, and determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time interval. The first trigger signal and the second trigger signal, in embodiments, indicate a time when the first particle and the second particle are detected in the flow stream, respectively, and may be used to estimate a relative position of the first particle and the second particle within the flow stream. In some embodiments, when evaluating the flow cytometer data, the generated time interval is compared to a measurement window associated with the second light source, e.g., the measurement window defines a time when a coincidence occurs due to irradiation of the second particle. When evaluating the flow cytometer data, in some such embodiments, it may be determined based on the time interval whether the second particle is included within the measurement window of the second light source while the flow cytometer data associated with the first particle is obtained from the detector. In other cases, when evaluating the flow cytometer data, the time interval is compared to a reference value, e.g., by determining whether the time interval is lower than the reference value.

[0006] The method may further modify the flow cytometer data resulting from the coincidence event after evaluating the flow cytometer data. For example, in some embodiments, the method assigns a coincidence identifier to the flow cytometer data that includes the coincidence event. In further cases, the method discards the flow cytometer data that includes the coincidence event. In some cases, the method repeats the method with a different pair of the first particle and the second particle. In other words, the method evaluates the flow cytometer data associated with a plurality of different pairs of the first particle and the second particle from within the particle sample. In some cases, the first particle and the second particle are consecutive particles in the flow stream. In other cases, the first particle and the second particle are non-consecutive particles in the flow stream.

[0007] The flow cytometer data evaluated in the method may be received from an experiment already performed (i.e., storage / memory) or may be received in real-time from a flow cytometry experiment. If the flow cytometer data is received in real-time, the method may further include illuminating the first and second particles in the flow stream, respectively, with a first light source and a second light source. If a coincidence event is detected with respect to the first and second particles, embodiments of the subject method include deactivating the second light source during the coincidence event. In some cases, the first and second light sources are selected from a plurality of light sources (e.g., 2-10 different light sources). The plurality of light sources may include, for example, one or more lasers. Similarly, in some cases, the detector is selected from a plurality of detectors (e.g., 2-10 different detectors). The plurality of detectors may include, for example, one or more detector arrays.

[0008] Aspects of the invention further include systems. Systems of interest include a flow cell configured to carry a particle sample in a flow stream, a first light source and a second light source configured to illuminate first and second particles, respectively, in the particle sample, a detector configured to generate flow cytometer data associated with the first particle, and a processor. The subject processor is configured to perform the subject method (e.g., as described above) and to receive from the detector the flow cytometer data associated with the illuminated first particle and a first trigger signal and a second trigger signal associated with the first and second particles, respectively. Additionally, the processor is configured to generate a time interval for the first and second particles based on a difference between the first and second trigger signals, and evaluate the resulting flow cytometer data for coincidence events by determining whether the first and second particles were illuminated by the first and second light sources simultaneously based on the generated time interval. In some cases, the subject systems include a plurality of light sources (e.g., 2-10 different light sources), where the first light source and the second light source are selected from the plurality of light sources. The plurality of light sources may include, for example, one or more lasers. In further cases, the systems include a plurality of detectors (e.g., 2-10 different detectors), where the detectors are selected from the plurality of detectors. In some cases, the system is a flow cytometer. The system may further include a display, in some embodiments, configured to output flow cytometer data evaluated by the processor.

[0009] Aspects of the invention further include a non-transitory computer readable storage medium having stored thereon instructions for evaluating flow cytometer data for coincident events using the subject methods. In particular, the subject non-transitory computer readable storage medium has instructions for receiving flow cytometer data associated with a first particle obtained from a detector after illuminating a first particle and a second particle in a flow stream with a first light source and a second light source, respectively, and a first trigger signal and a second trigger signal associated with the first particle and the second particle, respectively. The non-transitory computer readable storage medium of interest further has instructions for evaluating the flow cytometer data for coincident events by generating a time interval for the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal, and determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time interval. [Brief description of the drawings]

[0010] The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, in which:

[0011] [Figure 1A] FIG. 1 shows a flow cytometry scenario without crosstalk. [Figure 1B] FIG. 1 illustrates a flow cytometry scenario involving crosstalk. [Diagram 2] 1 is a flow chart for evaluating flow cytometer data for coincidence events according to one embodiment. [Diagram 3] FIG. 1 is a functional block diagram illustrating a flow cytometry system according to an embodiment. [Figure 4] FIG. 1 illustrates a control system according to an embodiment. [Figure 5A] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 5B] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 6] FIG. 1 is a block diagram illustrating a computing system according to an embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] A method is provided for evaluating flow cytometer data for coincident events. The method of interest includes receiving flow cytometer data associated with a first particle. After illuminating the first and second particles in the flow stream with a first and second light source, respectively, the received flow cytometer data is obtained from a detector. The method further includes receiving a first trigger signal and a second trigger signal associated with the first and second particles, respectively. Additionally, the subject method evaluates the flow cytometer data for coincident events by generating a time interval for the first and second particles based on a difference between the first and second trigger signals, and determining whether the first and second particles were illuminated by the first and second light sources simultaneously based on the generated time interval. A system and computer readable medium are further provided.

[0013] Before the present invention is described in more detail, it should be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. The scope of the present invention will be limited only by the appended claims, and it should be further understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0014] When a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are further encompassed within the invention, subject to any specifically excluded limit 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 encompassed within the invention.

[0015] In this specification, a range is provided with the term "about" before the numerical values. The term "about" is used herein to literally support the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. 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 the specifically stated number is presented, provides a substantial equivalent to the specifically stated number.

[0016] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative and illustrative methods and materials are described.

[0017] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. 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 invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0018] 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. As such, this statement is intended to serve as a prelude to the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in relation to the recitation of claim elements.

[0019] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein comprises separate components and features which may be readily separated from, or combined with, any of the features of the other multiple embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0020] Although the systems and methods have been described or will be described for grammatical fluidity with functional descriptions, the claims should not be construed as necessarily limited in any way by limitations of "means" or "step" construction unless expressly recited under 35 U.S.C. 112, but should be accorded the full scope of meaning and equivalents of the definitions given by the claims under the judicial theory of equivalents, with the express understanding that if a claim is expressly recited under 35 U.S.C. 112, then the full statutory equivalents under 35 U.S.C. 112 should be accorded.

[0021] How to evaluate flow cytometer data As described above, aspects of the invention include methods for evaluating flow cytometer data for the presence or absence of coincident events. As described herein, a "coincident event" refers to flow cytometry bio-event data obtained from a flow cytometry protocol in which a detector collects light from a first particle illuminated by a first light source while a second light source illuminates a second particle. In other words, a coincident event exists in the flow cytometer data when a first particle and a second particle are illuminated by a first light source and a second light source simultaneously. As described in the Background, light emitted from a cell traveling through a non-primary laser may be collected by a detector associated with the primary laser. This creates "crosstalk" that can corrupt the data. The designations "primary" and "non-primary" may refer to the correspondence between a given light source and the particle it is illuminating. In an example where a particle passes through a beam of light emitted by a first light source when the second light source is active, the first light source is designated as the primary light source for that particle, while the second light source is a non-primary light source for the same particle. In some cases, evaluating flow cytometer data for the presence or absence of coincident events can improve the quality of the flow cytometer data, for example, by enabling the identification and / or removal of flow cytometer data that may be suspect and may contain artifacts.

[0022] The method of interest includes receiving flow cytometer data associated with a first particle. After illuminating the first particle and the second particle in the flow stream with a first light source and a second light source, respectively, the flow cytometer data of interest is obtained from a detector. The detector of interest may be any convenient detector configured to collect light from the illuminated particles in the flow stream. Detectors of interest may include, but are not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes (APD), image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other detectors. In some 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 some embodiments, the detector is selected from a plurality of detectors. In other words, a given flow cytometer receiving flow cytometer data of interest may include a plurality of detectors, and the detector of interest for the subject method may be selected from any one of these detectors. The number of detectors in the plurality of detectors may be in the range of 2 to 20, such as 2 to 10. In some cases, the detector in the plurality of detectors is a fluorescent light detector. Each detector in the plurality of detectors may be any suitable type of detector, including but not limited to the detectors listed above.

[0023] In some embodiments, the plurality of detectors comprises one or more detector arrays, e.g., arrays of photodiodes. In these embodiments, each detector array may have 4 or more detectors, such as 10 or more detectors, for example 25 or more detectors, such as 50 or more detectors, for example 100 or more detectors, for example 250 or more detectors, such as 500 or more detectors, for example 750 or more detectors, for example 1000 or more detectors. For example, a detector may be a photodiode array having 4 or more photodiodes, such as 10 or more photodiodes, for example 25 or more photodiodes, such as 50 or more photodiodes, for example 100 or more photodiodes, such as 250 or more photodiodes, for example 500 or more photodiodes, for example 750 or more photodiodes, for example 1000 or more photodiodes.

[0024] The detectors may be arranged in any geometric configuration as desired, with configurations of interest including, but not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The detectors of the detector array may be oriented at angles (with respect to the XZ plane) in the range of 10° to 180°, such as 15° to 170°, such as 20° to 160°, such as 25° to 150°, such as 30° to 120°, such as 45° to 90°, relative to the other detectors. The detector array may have any suitable shape, including rectilinear shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, curvilinear shapes, such as circular, elliptical, and irregular shapes, such as a parabolic base joined to a planar top. In an embodiment, the detector array has a rectangular active surface.

[0025] In addition, the first and second light sources used to irradiate the first and second particles may be any convenient light source configured to independently irradiate particles in a flow stream. For example, in some embodiments, one or more of the first and second light sources are lasers. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and 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 CO2 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 flow cytometer of interest includes a dye laser, such as a stilbene laser, a coumarin laser, or a 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 flow cytometer 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 Yb2O3 laser, or a cerium-doped laser, and combinations thereof. In some cases, the first light source and the second light source are selected from a plurality of light sources.In other words, a given flow cytometer from which flow cytometer data of interest is received may include multiple light sources, and the first and second light sources of interest for the subject methods may be selected from any two of these light sources. The number of light sources in the multiple light sources may be in the range of 2 to 20, such as 2 to 10. The light sources (e.g., lasers) in the multiple light sources may be any convenient type of light source, including but not limited to those light sources described above.

[0026] Aspects of the invention further include receiving a first trigger signal and a second trigger signal associated with the first particle and the second particle, respectively. By "trigger signal" is meant the time when a given particle is first detected in the flow stream. Thus, in some cases, the first trigger signal and the second trigger signal indicate the time when the first particle and the second particle are detected in the flow stream, respectively. The trigger signal of interest may be received from any suitable detector in the flow cytometer. For example, in some cases, the trigger signal is received from a forward scatter detector. In further embodiments, the trigger signal is received from a side scatter detector. In further embodiments, the trigger signal is received from an axial light loss (ALL) detector. In further embodiments, the trigger signal is received from a fluorescent detector. In embodiments of the invention, a trigger signal is received for each particle in the flow stream. In some embodiments, each trigger signal may be used to provide an approximation of the position of the particle in the flow stream at any time during the particle's passage. In these embodiments, the position of the particle may be approximated using the trigger signal and the flow rate of the flow stream.

[0027] In an aspect of the subject method, after receiving the flow cytometer data and the first and second trigger signals, a time interval for the first and second particles is generated based on the difference between the first and second trigger signals. For example, t1 is a time point associated with the first trigger signal, t2 is a time point associated with the second trigger signal, and the time interval may be calculated by determining |t2-t1|. In some cases, the time interval indicates the amount of time that elapses between when the first particle is detected in the flow stream (i.e., the first trigger signal) and when the second particle is detected in the flow stream (i.e., the second trigger signal). In some cases, the generated time interval provides an approximation of the relative positions of the first and second particles in the flow stream. In other words, assuming a constant flow rate, the generated time interval corresponds to a particular (e.g., constant) distance measured relative to the flow axis separating the first and second particles as they are transported.

[0028] The method of interest further includes, after generating the time interval, evaluating the flow cytometer data for a coincidence event based on the generated time interval. In other words, the method determines whether the first particle and the second particle are illuminated by the first light source and the second light source at the same time. Any convenient method for determining the coincidence event may be applied. In some cases, the time interval is compared to a measurement window associated with the second light source when evaluating the flow cytometer data. By "measurement window" is meant a time during which a coincidence event occurs due to illumination of the second particle by the second light source. In other words, during illumination of the first particle by the first light source, a second particle that is estimated to be within the investigation zone of the second light source based on the generated time interval is within the measurement window associated with the second light source. As described herein, an "investigation zone" refers to an area in a flow stream where particles are illuminated by light from a light source, for example, for analysis. The size of the investigation zone may vary as desired. For example, where 0 μm represents the axis of the light emitted by the light-relevant light source, the interrogation zone may be in the range of −100 μm to 100 μm, such as −50 μm to 50 μm, such as −25 μm to 40 μm, such as −15 μm to 30 μm. Thus, according to these embodiments, when evaluating the flow cytometer data, a determination is made as to whether a second particle is included within the measurement window of the second light source during the time period during which flow cytometer data associated with a first particle is obtained from the detector, based on a time interval. Since the time interval is related to the distance that separates the first and second particles as they travel through the flow stream (as described above), it can be determined whether a first and second particle separated by such a distance are simultaneously present within the interrogation zones of the first and second light sources at a point during their passage through the flow stream, i.e., as opposed to particles associated with a larger or smaller time interval that may not be simultaneously present within the interrogation zones of the first and second light sources. In some embodiments, the method determines whether the generated time interval falls within a range of time values ​​associated with the measurement window.If the time interval falls within the range of time values ​​associated with the measurement window, then it may be determined that the resulting flow cytometer data contains a coincident event. If the time interval does not fall within the range of time values ​​associated with the measurement window (i.e., the time interval is higher or lower than the range), then it may be determined that the resulting flow cytometer data does not contain a coincident event.

[0029] In other cases, the time interval is compared to a reference value when evaluating the flow cytometer data. As described above, only particles that are a certain distance apart in the flow stream may be illuminated by the first and second light sources simultaneously. Thus, one method of identifying coincidence events is to consider particles that have a separation distance (i.e., as determined by the time interval) that is less than the reference value as possibly contributing to crosstalk. In other words, in these embodiments, when evaluating the flow cytometer data, it is determined whether the time interval is lower than the reference value. In some cases, the reference value is a length of time. If the time interval is less than the length of time associated with the reference value, it may be determined that the resulting flow cytometer data is associated with a coincidence event. In some embodiments, a reference value is used when it is desired to be more conservative in evaluating the flow cytometer data for coincidence events, i.e., when designating flow cytometer data that may contain coincidence as including coincidence events.

[0030] Additionally, embodiments of the method evaluate flow cytometer data associated with multiple different pairs of first and second particles from within the particle sample. In other words, the method is repeated such that different particles are designated as first and second particles. In some embodiments, a particle that was once a first particle in a previous iteration of the method may then be considered a second particle, and so on. In some cases, the method is repeated such that every particle in the particle sample being analyzed is designated as a first and / or second particle at least once. The detectors and the first and second light sources may be the same or different between iterations of the method. For example, different sets of light sources within the plurality of light sources may be designated as first and second light sources of interest for the method in different iterations. Similarly, different detectors within the plurality of detectors may be designated as detectors of interest for the method in different iterations. For a particular combination of light source and detector, if the flow cytometer data associated with a given set of a first particle and a second particle is determined to include a coincident event, then the flow cytometer data received by one or more additional detectors from this particular first particle (as this particular first particle travels through the flow stream) may also be presumed to include a coincident event. Assuming that the flow rate does not change, it may be assumed that the distance separating the first particle and the second particle remains the same as the particle travels through the flow cell. Thus, a first particle and a second particle associated with a time interval that is found to be within a measurement window or below a reference value, for example, may continue to generate flow cytometer data having a coincident event when measured by other light sources and detectors. Thus, the flow cytometer data measured from this particle at each detector may similarly include a coincident event. The first particle and the second particle may or may not be consecutive particles in the flow stream. In some cases, the first particle and the second particle are consecutive particles in the flow stream (i.e., there are no particles separating the first particle and the second particle). In other cases, the first particle and the second particle are non-contiguous particles in the flow stream (ie, there are one or more particles that separate the first particle and the second particle).

[0031] 1A and 1B show the contrast between a flow cytometry scenario without crosstalk (FIG. 1A) and one with crosstalk (FIG. 1B). A particle 102 is flowing through a flow cell 101 in a direction d. In the scenario of FIG. 1A, five different lasers 103a-103e irradiate the flow stream at interrogation zones 104a-104e, respectively. A first particle is irradiated by laser 103a at interrogation zone 104a. Particle-modulated light resulting from the irradiation of the particle is collected by detector 105. However, there is no second particle in the illumination zone of any of the other light sources (i.e., 103b-103e). Therefore, the particle being irradiated in FIG. 1A is not associated with flow cytometry data having crosstalk or coincidence events. In contrast, FIG. 1B shows a scenario that may result in coincidence events. As shown in FIG. 1B, a first particle is illuminated by laser 103a in interrogation zone 104a, while a second particle is illuminated by laser 103b in interrogation zone 104b. Particle-modulated light resulting from illumination of the particle in illumination zone 104a is collected by detector 105, while particle-modulated light resulting from illumination of the particle in illumination zone 104b is collected by detector 106. However, crosstalk between the lasers may be observed. As shown in area 107, there is a slight overlap in the light detected by each of detectors 105 and 106. In other words, a portion of the light from interrogation zone 104b is collected by detector 105, and a portion of the light from interrogation zone 104a is collected by detector 106. Thus, the flow cytometer data collected by detectors 105 and 106 in this scenario includes coincident events. The present invention anticipates this scenario by generating a time interval for the first particle and the second particle. It is also worth noting that the first particle and the second particle are spaced apart such that as they travel through the flow cell in direction d, they are simultaneously irradiated by laser 103b and laser 103c, simultaneously irradiated by laser 103c and laser 103d, and simultaneously irradiated by laser 103d and laser 103e.Thus, particle modulated light received by detectors associated with these light sources (not shown) may also result in coincidence events. As a result, embodiments of the method further predict that two particles having a particular time separation will result in coincidence events at multiple locations.

[0032] If the evaluated flow cytometer data includes a coincidence event (as determined above), embodiments of the method may further modify the evaluated flow cytometer data. In some cases, when modifying the evaluated flow cytometer data, the flow cytometer data including the coincidence event is discarded. By "discarding" the flow cytometer data, it is meant removing the affected flow cytometer data from the remaining data collected in the experiment. In other embodiments, the method assigns a coincidence identifier to the flow cytometer data including the coincidence event. In other words, the affected flow cytometer data is marked as potentially corrupted. Any convenient coincidence identifier may be used. For example, when the flow cytometer data is displayed to a user, the affected flow cytometer data may be displayed in a color that indicates to the user that the data includes a coincidence event. In other cases, the method provides a notification to the user regarding the presence and location of the affected data. An individual interpreting the flow cytometer data may then make an informed assessment of the impact of these data on the overall quality of the data.

[0033] In some embodiments, the method stops operation of the second light source during the coincidence event. In other words, the method predicts when a coincidence event is likely to occur and stops operation of the second (i.e., non-primary) light source accordingly to prevent the flow cytometer data from being affected. The method may further resume operation of the second light source when the time when the coincidence is likely has passed.

[0034] FIG. 2 is a flow chart for evaluating flow cytometer data for coincidence events according to an embodiment. In step 201, flow cytometer data associated with a first particle and a first trigger signal and a second trigger signal associated with the first particle and a second particle, respectively, are received. In step 202, a time interval between the first particle and the second particle is generated based on a difference between the first trigger signal and the second trigger signal. In step 203, the flow cytometer data is evaluated for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time interval. In step 204, it is determined whether a coincidence event is present in the flow cytometer data evaluated in step 203. If the evaluated flow cytometer data includes a coincidence event, in step 205, the evaluated flow cytometer data is modified (e.g., by discarding the evaluated flow cytometer data or by assigning a coincidence identifier to the evaluated flow cytometer data). The method then repeats for a different pair of the first particle and the second particle in step 206. If the evaluated flow cytometer data does not include a coincidence event, the method proceeds directly to step 206 without modifying the evaluated flow cytometer data.

[0035] The flow cytometer data evaluated in the method may be received from an experiment already performed (e.g., storage / memory) or may be received in real time from a flow cytometry experiment. In other words, the time at which the flow cytometer data is obtained from the detector after the first and second light sources illuminate the first and second particles in the flow stream may be in the past or may be part of an ongoing experiment. If the flow cytometer data is received from an experiment already performed, the time at which the flow cytometer data was obtained may range from, for example, 1 second or more in the past to 25 years or more in the past. For example, the time at which the flow cytometer data was obtained may be 1 second or more in the past, 5 seconds or more in the past, 10 seconds or more in the past, 1 minute or more in the past, 5 minutes or more in the past, 10 minutes or more in the past, 30 minutes or more in the past, 1 hour or more in the past, 5 hours or more in the past, 10 hours or more in the past, 1 day or more in the past, 3 days or more in the past, 1 week or more in the past, 3 weeks or more in the past, 1 month or more in the past, 3 months or more in the past, 6 months or more in the past, 1 year or more in the past, 3 years or more in the past, 6 years or more in the past, 15 years or more in the past, 20 years or more in the past, 25 years or more in the past. In some embodiments, the flow cytometer data received from previously performed experiments is stored in a memory device. The flow cytometer data obtained from previously performed experiments may be stored in memory, which 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 resident hard disk or tape, optical media, such as a read-write compact disk, flash memory device, cloud storage device, or other memory storage device.

[0036] In embodiments of the method receiving real-time flow cytometer data from an experiment, aspects of the invention further include illuminating the first and second particles in the flow stream using a first light source and a second light source, respectively. In performing the subject method, a sample having at least the first and second particles (e.g., in a flow stream of a flow cytometer) is illuminated with light from at least the first and second light sources. In some embodiments, at least one light source is a broadband light source, emitting light having a wide range of wavelengths, e.g., 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, e.g., 500 nm or more. For example, one 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 emitting light having a wavelength between 400 nm and 1000 nm. When the method involves illumination with a broadband light source, broadband light source protocols of interest may include, but are not limited to, 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, broad spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof, among other broadband light sources.

[0037] In other embodiments, the method includes irradiating with at least one narrowband light source emitting at a specific wavelength or narrow range of wavelengths, e.g., irradiating with a light source emitting light in a narrow range of wavelengths, e.g., 50 nm or less, e.g., 40 nm or less, e.g., 30 nm or less, e.g., 25 nm or less, e.g., 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less, e.g., 5 nm or less, e.g., 2 nm or less, e.g., irradiating with a light source emitting light of a specific wavelength (i.e., monochromatic light). When the method includes irradiating with a narrowband light source, narrowband light source protocols of interest include, but are not limited to, narrow wavelength LEDs, laser diodes, or broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0038] In aspects of the invention, the fluorescence is collected using a fluorescence detector. The fluorescence detector may optionally be configured to detect fluorescence emission from fluorescent molecules associated with the particles in the flow cell, e.g., a labeled specific binding member (such as a labeled antibody that specifically binds to a marker of interest). In an embodiment, the method detects fluorescence from the sample using one or more fluorescence detectors, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 6 or more, e.g., 7 or more, e.g., 8 or more, e.g., 9 or more, e.g., 10 or more, e.g., 15 or more, e.g., 25 or more fluorescence detectors. In an embodiment, each of the fluorescence detectors is configured to generate a fluorescence data signal. The fluorescence from the sample may be detected independently by each fluorescence detector over one or more of a wavelength range of 200 nm to 1200 nm. In some cases, the method detects fluorescence from the sample over a wavelength range of, e.g., 200 nm to 1200 nm, e.g., 300 nm to 1100 nm, e.g., 400 nm to 1000 nm, e.g., 500 nm to 900 nm, e.g., 600 nm to 800 nm, etc. In other cases, the method detects fluorescence at one or more specific wavelengths using each fluorescence detector. For example, fluorescence may be detected at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, or any combination thereof, depending on the number of different fluorescence photodetectors in the subject light detection system. In an embodiment, the method detects wavelengths of light corresponding to the fluorescence peak wavelengths of certain fluorescent dyes present in the sample. In an embodiment, the fluorescence flow cytometer data is received from one or more fluorescence photodetectors (e.g., one or more detection channels), such as 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 8 or more fluorescence photodetectors (e.g., 8 or more detection channels).

[0039] In some cases, the sample to be analyzed is a particle sample. In other words, the sample contains a plurality of particles (e.g., cells, beads, polynucleotides, proteins, etc.). In some cases, the sample to be analyzed in the present method is a biological sample. The term "biological sample" is used in the conventional sense to refer to a whole organism, a plant, a fungus, or a subset of tissues, cells, or components of an animal, where the subset of tissues, cells, or components of an animal may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, or semen, as the case may be. Thus, "biological sample" refers to both a naturally occurring organism or a subset of its tissues, and homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. The biological sample may be tissue from any type of organism, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, such as plasma, tears, urine, semen, etc., and in some cases, the sample is a blood sample, including whole blood, such as blood obtained from a venipuncture or finger prick (which may or may not be bound with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0040] In some embodiments, the sample source is a "mammal" or "mammalian," which terms are used broadly to describe organisms belonging to the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult), and in some embodiments, the human subject is a juvenile, adolescent, or adult. While the present invention may be applied to samples from human subjects, it should be understood that the method may also be performed on samples from other animal subjects (i.e., "non-human subjects"), including, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0041] The cells of interest may be subject to characterization according to various parameters, such as phenotype, identified by attaching a specific fluorescent label to the cells of interest. In some embodiments, the system is configured to deflect analysis droplets determined to contain target cells. A variety of cells may be characterized using the subject methods. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and red blood cells. Target cells of interest include cells bearing convenient cell surface markers or antigens that may be taken up or labeled by convenient affinity agents or conjugates thereof. For example, the target cell may comprise cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0042] The featured methods may further use the particles in research, clinical trials, or therapy. In some embodiments, the subject methods obtain individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods obtain cells from a fluid or tissue sample used as a research or diagnostic specimen for diseases such as cancer. Similarly, the subject methods obtain cells from a fluid or tissue sample used for therapy. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissue, may be prepared and introduced into a subject as a therapeutic treatment. Conditions that may be treated by administration of flow cytometrically sorted samples include, but are not limited to, blood disorders, immune system disorders, organ damage, and the like.

[0043] A typical cell therapy protocol may include the steps of sample collection, cell isolation, genetic modification, culture, in vitro expansion, cell harvesting, sample volume reduction, sample washing, biopreservation, storage, and introduction of cells into a subject. The protocol may begin by harvesting viable cells and tissue from a tissue source of the subject to generate a cell and / or tissue sample. The sample may be harvested by any suitable procedure, including, for example, administering a cell mobilizing agent to the subject, drawing blood from the subject, removing bone marrow from the subject, and the like. After harvesting the sample, cell enrichment may be performed by a number of methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, fluorescence-activated cell sorting (FACS), and the like. In some cases, the enriched cells may be genetically modified by any convenient method, for example, nuclease-mediated gene editing. The genetically modified cells may be cultured, activated, and expanded in vitro. In some cases, the cells are preserved, for example, cryopreserved, and stored for future use. When in use, the cells may be thawed and then administered to the patient, for example, the cells may be infused into the patient.

[0044] System for evaluating flow cytometer data - Patents.com Aspects of the invention further include a system (e.g., a flow cytometer) for evaluating a flow cytometer. Systems of interest include a flow cell configured to carry a particle sample in a flow stream, a first light source and a second light source configured to irradiate first and second particles, respectively, in the particle sample, a detector configured to generate flow cytometer data associated with the first particle, and a processor configured to evaluate the flow cytometer data. The processor of interest is configured to receive the flow cytometer data associated with the illuminated first particle from the detector and a first trigger signal and a second trigger signal associated with the first and second particles, respectively. Additionally, the processor of interest generates a time interval for the first and second particles based on a difference between the first and second trigger signals, and evaluates the resulting flow cytometer data for coincidence events by determining whether the first and second particles were illuminated by the first and second light sources simultaneously based on the generated time interval.

[0045] As described herein, a "flow cell" is used in the conventional sense to refer to a component, such as a cuvette, that includes a flow path having a liquid flow stream for carrying particles within a sheath fluid. A cuvette of interest includes a container with a passageway therethrough. The flow stream may include a liquid sample injected from a sample tube. A flow cell of interest includes a flow path accessible to light. In some cases, the flow cell includes a transparent material (e.g., quartz) that allows the passage of light. In some embodiments, the flow cell is a stream-in-air flow cell in which optical interrogation of particles occurs in free space. In some cases, the flow stream is configured to be illuminated with light from a first light source and a second light source in an interrogation zone (e.g., as described above). The flow stream, in which the flow path is configured, may include a liquid sample injected from a sample tube. In some embodiments, the flow stream may include a narrow, rapidly flowing stream of liquid that is arranged such that linearly separated particles carried therein are in-line and separated from one another. After illumination of the particles in the flow cell, particle modulated light may be observed. By "particle modulated light" is meant light received from particles in the flow stream after illumination of the particles with light from a light source. In some cases, the particle-modulated light is side-scattered light. As described herein, side-scattered light refers to light that refracts and reflects off the surface and internal structures of a particle. In further embodiments, the particle-modulated light includes forward-scattered light (i.e., light that travels primarily through or around the particle in a forward direction). In still other cases, the particle-modulated light includes fluorescent light (i.e., light emitted from a fluorescent dye after illumination with an excitation wavelength light).

[0046] In some cases, the subject processor operates with programmable logic, which may be implemented in hardware, software, firmware, or any combination thereof, for example, to evaluate the flow cytometer data for the presence or absence of a coincidence event. For example, if the programmable logic is implemented in software, the evaluation of the flow cytometer data for the coincidence event may be at least partially achieved by a computer readable data storage medium including a program code having instructions configured, when executed, to receive flow cytometer data associated with an illuminated first particle from a detector, and a first trigger signal and a second trigger signal associated with the first particle and the second particle, respectively. The program code may further include instructions for generating a time interval of the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal. For example, if t1 is a time point associated with the first trigger signal and t2 is a time point associated with the second trigger signal, the time interval may be calculated by determining |t2-t1|. In some cases, the time interval indicates an amount of time that elapses between when a first particle is detected in the flow stream (i.e., the first trigger signal) and when a second particle is detected in the flow stream (i.e., the second trigger signal). The program code may further include instructions for evaluating the acquired flow cytometer data for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time interval. In some cases, when evaluating the flow cytometer data, the time interval is compared to a measurement window associated with the second light source, e.g., by determining whether the second particle falls within the measurement window associated with the second light source while acquiring flow cytometer data associated with the first particle from the detector based on the time interval. In other cases, when evaluating the flow cytometer data, the time interval is compared to a reference value, e.g., by determining whether the time interval is lower than the reference value.

[0047] If a coincidence event is identified by the programmable logic, the subject processor may be further configured to modify the affected flow cytometer data. In some cases, modifying the evaluated flow cytometer data involves discarding the flow cytometer data that includes the coincidence event (i.e., removing the affected flow cytometer data from the remaining data collected in the experiment). In other embodiments, the method assigns a coincidence identifier to the flow cytometer data that includes the coincidence event. In other words, the affected flow cytometer data is marked as potentially corrupted. An individual interpreting the flow cytometer data may then make an informed assessment of the impact of these data on the quality of the data.

[0048] The programmable logic of interest may be embodied in any of a variety of devices, such as a specially 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 combination of a DSP and a microprocessor, multiple microprocessors, one or more microprocessors in combination with a DSP core, or any other such configuration in at least partial data connectivity, may implement one or more of the features described herein.

[0049] In some cases, the system further includes a display configured to output the flow cytometer data evaluated by the processor, so that a user may then view the flow cytometer data evaluated by the processor. Targeted displays may include, but are not limited to, monitors, tablet computers, smartphones, or other electronic devices configured to provide a graphical interface.

[0050] As described above, aspects of the invention further include a first light source and a second light source configured to illuminate particles passing through the flow cell at an interrogation point. Any convenient light source may be used as the first light source and the second light source described herein. In some embodiments, the light source or light sources are lasers. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and 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 CO2 laser, a CO2 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 flow cytometer of interest includes a dye laser, such as a stilbene laser, a coumarin laser, or a 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 flow cytometer 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 Yb2O3 laser, or a cerium-doped laser, and combinations thereof.

[0051] The laser light source according to an embodiment may further include one or more optical adjustment components. In an embodiment, the optical adjustment components are disposed between the light source and the flow cell and may include any device capable of changing the spatial width of the illumination from the light source or other characteristics of the illumination, such as the illumination direction, wavelength, beam width, beam intensity, and focus. The optical adjustment protocol may include 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 an embodiment, the flow cytometer of interest includes one or more focusing lenses. The focusing lens may be, in one example, a reduction lens. In yet another embodiment, the flow cytometer of interest includes optical fibers.

[0052] If the optical adjustment component is configured to move, it may be configured to move continuously or at discrete intervals, for example in increments of 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 10 μm or more, for example 100 μm or more, such as 500 μm or more, for example 1 mm or more, such as 5 mm or more, for example 10 mm or more, such as 25 mm or more.

[0053] Any movement protocol may be used to move the optical adjustment component structure, for example coupled to a movable support stage or directly coupled to a motor-driven translation stage, a lead screw translation assembly, a geared translation device, for example using stepper motors, servo motors, brushless electric motors, brushed DC motors, microstep drive motors, and high resolution stepper motors, among other types of motors.

[0054] The light source may be positioned at any suitable distance from the flow cell, for example the light source and the flow cell are separated by 0.005 mm or more, such as 0.01 mm or more, for example 0.05 mm or more, such as 0.1 mm or more, for example 0.5 mm or more, such as 1 mm or more, for example 5 mm or more, such as 10 mm or more, for example 25 mm or more, for example 100 mm or more. In addition, the light source may be positioned at any suitable angle relative to the flow cell, for example at an angle between 10 degrees and 90 degrees, for example 15 degrees and 85 degrees, for example 20 degrees and 80 degrees, for example 25 degrees and 75 degrees, for example 30 degrees and 60 degrees, for example at an angle of 90 degrees.

[0055] In some embodiments, the light source of interest may include multiple lasers, e.g., 2 or more lasers, e.g., 3 or more lasers, e.g., 4 or more lasers, e.g., 5 or more lasers, e.g., 10 or more lasers, e.g., 15 or more lasers, configured to provide laser light for separate application to the flow stream. Depending on the desired wavelength of light for irradiating the flow stream, each laser may have a particular wavelength within the range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. In some embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0056] As discussed above, particle analyzers of interest may further include one or more particle modulated light detectors for detecting intensity data of the particle modulated light. In some embodiments, the one or more particle modulated light detectors include one or more forward scattered light detectors configured to detect forward scattered light. For example, particle analyzers of interest may include one forward scattered light detector or multiple forward scattered light detectors, such as two or more, such as three or more, such as four or more, such as five or more forward scattered light detectors. In some embodiments, the particle analyzer includes one forward scattered light detector. In other embodiments, the particle analyzer includes two forward scattered light detectors.

[0057] Any convenient detector for detecting collected light may be used with the forward scattered light detector described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof, among other detectors. In some 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 some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10cm 2 , e.g. 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2The photomultiplier tube has an active detection surface area of ​​each region within the range of .

[0058] In embodiments, the forward scattered light detector is configured to measure light continuously or at discrete intervals. In some cases, the detector of interest is configured to measure the collected light continuously. In other cases, the detector of interest is configured to measure 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, such as every 1000 milliseconds, or other intervals.

[0059] In further embodiments, the one or more detectors may comprise one or more side scatter light detectors for detecting side scattered wavelengths of light (i.e., light that is refracted and reflected off the surface and internal structure of the particle). In some embodiments, the particle analyzer comprises one side scatter light detector. In other embodiments, the particle analyzer comprises multiple side scatter light detectors, for example 2 or more, for example 3 or more, for example 4 or more, for example 5 or more side scatter light detectors.

[0060] Any convenient detector for detecting collected light may be used in the side scatter light detector described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof, among other detectors. In some 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 some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10cm 2 , e.g. 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .

[0061] In embodiments, a subject particle analyzer further comprises a fluorescence detector configured to detect light at one or more fluorescence wavelengths, hi other embodiments, the particle analyzer comprises a plurality of fluorescence detectors, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, 5 or more, 10 or more, 15 or more, e.g., 20 or more fluorescence detectors.

[0062] Any convenient detector for detecting collected light may be used with the fluorescence photodetectors described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof, among other detectors. In some 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 some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10cm 2 , e.g. 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .

[0063] When a particle analyzer of interest has multiple fluorescence detectors, each of the fluorescence detectors may be identical, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, when a particle analyzer of interest has two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector (or image sensor) is a CMOS type device. In other embodiments, both the first and second fluorescence detectors are CCD type devices. In yet other embodiments, both the first and second fluorescence detectors are CMOS type devices. In yet other embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector is a photomultiplier tube (PMT). In yet other embodiments, the first fluorescence detector is a CMOS type device and the second fluorescence detector is a photomultiplier tube. In yet other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0064] In embodiments of the present disclosure, the fluorescent light detector of interest is configured to measure collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., twenty-five or more different wavelengths, e.g., fifty or more different wavelengths, e.g., one or more than one hundred different wavelengths, e.g., two or more than one hundred different wavelengths, e.g., two or more than one hundred different wavelengths, e.g., three or more than one hundred different wavelengths, e.g., four ...

[0065] In some embodiments, the fluorescent light detector of interest is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect a spectrum of light over a range of wavelengths. For example, a particle analyzer may have one or more detectors configured to collect a spectrum of light over one or more of the wavelength ranges of 200 nm to 1000 nm. In yet other embodiments, the detector of interest is configured to measure light emitted from a sample in the flow stream at one or more particular wavelengths. For example, the particle analyzer may have one or more detectors configured to measure light at one or more of: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In some embodiments, the one or more detectors may be configured to pair with a particular fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0066] In some embodiments, one or more of the particle modulation light detectors include one or more detector arrays, e.g., an array of photodiodes. In these embodiments, each detector array may have 4 or more detectors, such as 10 or more detectors, for example 25 or more detectors, for example 50 or more detectors, for example 100 or more detectors, for example 250 or more detectors, for example 500 or more detectors, for example 750 or more detectors, for example 1000 or more detectors. For example, a detector may be a photodiode array having 4 or more photodiodes, such as 10 or more photodiodes, for example 25 or more photodiodes, for example 50 or more photodiodes, for example 100 or more photodiodes, for example 250 or more photodiodes, for example 500 or more photodiodes, for example 750 or more photodiodes, for example 1000 or more photodiodes.

[0067] The detectors may be arranged in any geometric configuration as desired, with configurations of interest including, but not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The detectors of the detector array may be oriented at angles (with respect to the XZ plane) in the range of 10° to 180°, such as 15° to 170°, such as 20° to 160°, such as 25° to 150°, such as 30° to 120°, such as 45° to 90°, relative to the other detectors. The detector array may have any suitable shape, including rectilinear shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, curvilinear shapes, such as circular, elliptical, and irregular shapes, such as a parabolic base joined to a planar top. In an embodiment, the detector array has a rectangular active surface.

[0068] In some embodiments, the particle analyzer comprises one or more wavelength separators disposed between the flow cell and the one or more particle modulation photodetectors. The term "wavelength separator" is used herein in its conventional sense to refer to an optical component configured to separate light collected from a sample into predetermined spectral regions. In some embodiments, the particle analyzer comprises one wavelength separator. In other embodiments, the particle analyzer comprises multiple wavelength separators, e.g., 2 or more wavelength separators, e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 6 or more, e.g., 7 or more, e.g., 8 or more, e.g., 9 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, e.g., 100 or more wavelength separators. In some embodiments, the wavelength separator is configured to separate light collected from a sample into predetermined spectral regions by passing light having a predetermined spectral region and reflecting light in one or more remaining spectral regions. In other embodiments, the wavelength separator is configured to separate light collected from a sample into predetermined spectral regions by passing light having a predetermined spectral region and absorbing light in one or more remaining spectral regions. In yet other embodiments, the wavelength separator is configured to spatially diffract light collected from the sample into a predetermined spectral region. Each wavelength separator may be any convenient light separation protocol, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In certain embodiments, the wavelength separator of the subject light detection system is a dichroic mirror.

[0069] As discussed above, aspects of flow cytometers of interest include a flow cell configured to transport particles within a flow stream. Any convenient flow cell that transports a fluid sample to a sample interrogation region may be used, and in some embodiments the flow cell is a cylindrical flow cell, a frusto-conical flow cell, or a flow cell that includes a proximal cylindrical portion defining a longitudinal axis and a distal frusto-conical portion terminating in a flat surface having an orifice perpendicular to the longitudinal axis.

[0070] In some embodiments, the sample flow stream is emitted from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the orifice of the flow cell may have any suitable shape, including but not limited to rectilinear cross-sectional shapes, e.g., square, rectangular, trapezoidal, triangular, hexagonal, curvilinear cross-sectional shapes, e.g., circular, oval, and irregular shapes, e.g., a parabolic bottom joined to a planar top. In some embodiments, the flow cell of interest has a circular orifice. The size of the nozzle orifice may vary and in some embodiments may be in the range of 1 μm to 10,000 μm, e.g., 25 μm to 7,500 μm, e.g., 50 μm to 5,000 μm, e.g., 75 μm to 1,000 μm, e.g., 100 μm to 750 μm, e.g., 150 μm to 500 μm. In some embodiments, the nozzle orifice is 100 μm.

[0071] In some embodiments, the flow cell has a sample injection port configured to deliver a sample to the flow cell. The sample injection port may be an orifice in the wall of the internal chamber or a tube in the proximal end of the internal chamber. When the sample injection port is an orifice in the wall of the internal chamber, the orifice of the sample injection port may have any suitable shape, and cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, e.g., square, rectangular, trapezoidal, triangular, hexagonal, curvilinear cross-sectional shapes, e.g., circular, oval, and irregular shapes, e.g., a parabolic bottom joined to a planar top. In some embodiments, the sample injection port has a circular orifice. The size of the orifice of the sample injection port may vary depending on the shape, and in some cases may have an opening within the range of 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, such as 1.25 mm to 1.75 mm, for example an opening of 1.5 mm.

[0072] In some cases, the sample injection port is a tube provided at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a tube that is arranged such that the orifice of the sample injection port is aligned with the orifice of the flow cell. When the sample injection port is a tube that is arranged such that it is aligned with the orifice of the flow cell, the cross-sectional shape of the sample injection tube may be any suitable shape, cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, e.g., square, rectangular, trapezoidal, triangular, hexagonal, curvilinear cross-sectional shapes, e.g., circular, oval, and irregular shapes, e.g., a parabolic bottom joined to a planar top. The orifice of the tube may vary depending on the shape, and in some cases may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 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, e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm. 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 have a bevel angle in the range of 1° to 10°, for example, 2° to 9°, for example, 3° to 8°, for example, 4° to 7°, for example, a bevel angle of 5°.

[0073] In some embodiments, the flow cell further comprises a sheath fluid injection port configured to supply a sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid, e.g., together with the sample, to the internal chamber of the flow cell to generate a laminated flow stream of sheath fluid surrounding the sample flow stream. The velocity of the sheath fluid delivered to the chamber of the flow cell may be between 25 μL / sec and 2500 μL / sec, e.g., between 50 μL / sec and 1000 μL / sec, e.g., between 75 μL / sec and 750 μL / sec, depending on the desired characteristics of the flow stream.

[0074] In some embodiments, the sheath fluid injection port is an orifice in the wall of the internal chamber. The orifice of the sheath fluid injection port may be of any suitable shape, with cross-sectional shapes of interest including, but not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, curvilinear cross-sectional shapes, such as circular, oval, and irregular shapes, such as a parabolic bottom joined to a planar top. The size of the orifice of the sample injection port may vary depending on the shape, and may in some cases have an opening in the range of 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, such as 1.25 mm to 1.75 mm, such as an opening of 1.5 mm.

[0075] In some embodiments, the system further comprises a pump in fluid communication with the flow cell for propagating the flow stream through the flow cell. Any convenient fluid pumping protocol may be used to control the flow of the flow stream through the flow cell. In some cases, the system comprises a peristaltic pump, e.g., a peristaltic pump having a pulse damper. The pump of the subject system is configured to convey fluid through the flow cell at a rate suitable for multiphoton counting of light from the sample in the flow stream. For example, the system may comprise a pump configured to flow the sample through the flow cell at a rate in the range of 1 nL / min to 500 nL / min, e.g., 1 nL / min to 250 nL / min, e.g., 1 nL / min to 100 nL / min, e.g., 2 nL / min to 90 nL / min, e.g., 3 nL / min to 80 nL / min, e.g., 4 nL / min to 70 nL / min, e.g., 5 nL / min to 60 nL / min, e.g., 10 nL / min to 50 nL / min. In one embodiment, the flow rate of the flow stream is between 5 nL / min and 6 nL / min.

[0076] Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. 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 Throm 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.In some cases, the flow cytometry systems of interest are BD Biosciences FACSCanto™ flow cytometers, BD Biosciences FACSCanto™ II flow cytometers, BD Accuri™ flow cytometers, BD Accuri™ C6 Plus flow cytometers, BD Biosciences FACSCelesta™ flow cytometers, BD Biosciences FACSLyric™ flow cytometers, BD Biosciences FACSVerse™ flow cytometers, BD Biosciences FACSymphony™ flow cytometers, BD Biosciences LSRFortessa™ flow cytometers, BD Biosciences LSRFortessa™ X-20 flow cytometers, BD Biosciences FACSPresto™ flow cytometers, BD Biosciences FACSVia™ flow cytometers, as well as BD Biosciences FACSCalibur™ cell sorters, BD Biosciences FACSCount™ cell sorters, BD Biosciences FACSLyric™ cell sorters, BD These include 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, etc.

[0077] In some embodiments, the subject system may be configured as described in U.S. Pat. Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, 10,302,545, 10,145,793, 10,113,967, 10,006,852, 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,097,641, 9,100,972, 9,200,334, 9,300,972 ...0, 9,100,972, 9,100,972, 9,200,972, 9,300,972, 9,453,789, 9,200,334, 9,097,640, 9,100,972, 9,100,972, 9,200,972, 9,3 Nos. 5494, 9092034, 8975595, 8753573, 8233146, 8140300, 7544326, 7201875, 7129505, 6821740, 6813017, 6809804, 6372506, 5700692, 5643796, 5627040, 5620842, 5602039, 4987086, and 4498766.

[0078] In some cases, the flow cytometry system of the present invention may be implemented using the techniques disclosed in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013), the disclosures of which are incorporated herein by reference, as well as U.S. Pat. Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,820,121, 10,840,132, 10,820,141, 10,840,152, 10,840,162, 10,840,171, 10,840,185, 10,840,191, 10,840,192, 10,840,162, 10,840,173, 10,840,191, 10,840,162, 10,840,173, 10,840,185, 10,840,192, 10,840,16 ... The present invention has been adapted to image particles in a flow stream by Fluorescence Imaging using Radio Frequency Tag Emission (FIRE), as described in U.S. Patent Application Publication No. 2019 / 0376895, U.S. Patent Application Publication No. 2019 / 0376894, U.S. Patent Application Publication No. 2019 / 0376895, U.S. Patent Application Publication No. 2019 / 0376894, U.S. Patent Application Publication No. 2019 / 0376895, U.S. Patent Application Publication No. 2019 / 0376894, and U.S. Patent Application Publication No. 2019 / 0376894.

[0079] 3 illustrates a system 300 for flow cytometry according to an exemplary embodiment of the invention. System 300 includes a flow cytometer 310, a controller / processor 390, and a memory 395. Flow cytometer 310 includes one or more excitation lasers 315a-315c, a focusing lens 320, a flow chamber 325, a forward scatter detector 330, a side scatter detector 335, a fluorescence collection lens 340, one or more beam splitters 345a-345g, one or more bandpass filters 350a-350e, one or more longpass (LP) filters 355a-355b, and one or more fluorescence detectors 360a-360f.

[0080] The pump lasers 315a-315c emit light in the form of laser beams. The wavelengths of the laser beams emitted from the pump lasers 315a-315c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system of FIG. 3. The laser beams are first directed through one or more of beam splitters 345a and 345b. Beam splitter 345a transmits 488 nm light and reflects 633 nm light. Beam splitter 345b transmits UV light (light having a wavelength in the range of 10-400 nm) and reflects 488 nm and 633 nm light.

[0081] The laser beam is then directed to a focusing lens 320, which focuses the laser beam onto the portion of the fluid stream in which the sample particles reside in a flow chamber 325. The flow chamber is the part of the fluid system that directs particles in the stream, usually one at a time, into the focused laser beam for investigation. The flow chamber can comprise a flow cell in a benchtop flow cytometer or a nozzle tip in a stream-in-air cytometer.

[0082] Light from the laser beam(s) interacts with particles in the sample by diffraction, refraction, reflection, scattering and absorption, and is re-emitted at a variety of different wavelengths depending on the particle's properties, such as the particle's size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The fluorescent radiation, as well as the diffracted, refracted, reflected and scattered light, may be sent through one or more of beam splitters 345c-g, bandpass filters 350a-e, longpass filters 355a-b and fluorescence collection lens 340 to one or more of forward scatter detector 330, side scatter detector 335, and one or more fluorescence detectors 360a-f.

[0083] Fluorescence collection lens 340 collects light emitted by particle-laser beam interactions and directs the light toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 350a-350e, allow a narrow range of wavelengths to pass through the bandpass filter. For example, bandpass filter 350a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number represents the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, from 500 nm to 520 nm. Shortpass filters transmit light with wavelengths equal to or less than a specified wavelength. Longpass filters, such as longpass filters 355a-355b, transmit light with wavelengths equal to or greater than a specified wavelength of light. For example, longpass filter 355b, a 670 nm longpass filter, transmits light above 670 nm. Filters are often selected to optimize the specificity of the detector for a particular fluorochrome. The filters may be configured so that the spectral band of light transmitted to the detector approximates the emission peak of the fluorescent dye.

[0084] The forward scatter detector 330 is positioned slightly off-axis from the direct beam through the flow cell and is configured to detect diffracted light, i.e., excitation light traveling primarily in a forward direction through or around the particle. The intensity of light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 335 is configured to detect light that is refracted and reflected off the surface and internal structure of the particle, which tends to increase as the structural complexity of the particle increases. Fluorescent emission from fluorescent molecules bound to the particle may be detected by one or more of the fluorescence detectors 360a-360f. The side scatter detector 335 and the fluorescence detector may include photomultiplier tubes. The signals detected by the forward scatter detector 330, the side scatter detector 335 and the fluorescence detector may be converted to electronic signals (voltage) by the detectors. This data may provide information about the sample.

[0085] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to the flow cytometer shown in Figure 3, 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.

[0086] During operation, the operation of the flow cytometer is controlled by the controller / processor 390, and measurement data from the detectors may be stored in memory 395 and processed by the controller / processor 390. Although not explicitly shown, the controller / processor 390 is coupled to the detectors to receive output signals from the detectors, and may further be coupled to electrical and electromechanical components of the flow cytometer 310 to control lasers, fluid flow parameters, and the like. Input / output (I / O) functionality 397 may also be provided in the system. The memory 395, controller / processor 390, and I / O functionality 397 may be provided entirely as an integral part of the flow cytometer 310. In such an embodiment, a display may also form part of the I / O functionality 397 to present experimental data to a user of the flow cytometer 310. Alternatively, some or all of the memory 395 and the controller / processor 390 and 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 the memory 395 and controller / processor 390 may be in wireless or wired communication with the flow cytometer 310. The controller / processor 390 in conjunction with the memory 395 and I / O functionality 397 may be configured to perform a variety of functions associated with the preparation and analysis of flow cytometer experiments.

[0087] The system illustrated in FIG. 3 includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as "filter windows" for a given detector) as defined by the configuration of filters and / or splitters in the beam path from the flow cell 325 to each detector. Different fluorescent molecules used in a flow cytometer experiment emit light in their own characteristic wavelength bands. The particular fluorescent labels used in the experiment and the associated fluorescent emission bands of the fluorescent labels may be selected to approximately match the filter windows of the detectors. The I / O function 397 may 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 multiple markers. The I / O function 397 may be further 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, may be further stored in the memory 395. The controller / processor 390 may be configured to evaluate one or more assignments of labels to markers.

[0088] In some embodiments, the subject system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In an embodiment, particles (e.g., cells) of a sample are sorted using a sorting determination module having a plurality of sorting determination units, such as 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 having deflection plates, such as described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0089] 4 is a functional block diagram of an example of a system for analyzing and displaying biological events, such as a processor 400. The processor 400 may be configured to perform various processes for controlling the graphical display of biological events.

[0090] A flow cytometer or sorting system 402 may be configured to acquire bio-event data. For example, a flow cytometer may generate flow cytometry event data (e.g., particle-modulated light data). The flow cytometer 402 may be configured to provide the bio-event data to the processor 400. A data communication channel may be provided between the flow cytometer 402 and the processor 400. The bio-event data may be provided to the processor 400 via the data communication channel.

[0091] The processor 400 may be configured to receive bio-event data from the flow cytometer 402. The bio-event data received from the flow cytometer 402 may include flow cytometry event data. The processor 400 may be configured to evaluate the data received from the flow cytometer 402, for example, as described above. For example, the processor 400 may be configured to receive flow cytometer data from the detector related to the illuminated first particle and a first trigger signal and a second trigger signal related to the first particle and the second particle, respectively. In addition, the processor 400 may be configured to evaluate the obtained flow cytometer data for the presence or absence of a coincident event by generating a time interval for the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal, and determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time interval.

[0092] The processor 400 may be configured to provide a graphical display including the bio-event data to the display device 406. In some cases, the processor 400 prevents the flow cytometer data including the coincident event from being displayed in the graphical display (i.e., discards the affected flow cytometer data). In other cases, the processor 400 assigns a coincidence identifier to the affected flow cytometer data. For example, the affected flow cytometer data may be marked in a particular color in the graphical display, or a notice is displayed drawing the user's attention to the affected data. The processor 400 may further be configured to render a region of interest as a gate around the population of bio-event data displayed by the display device 406, e.g., overlaid on the first plot. In some embodiments, the gate may be a logical combination of one or more illustrated regions of interest plotted on a histogram or bivariate plot of one parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0093] The processor 400 may further be configured to display the bioevent data on the display device 406 within the gate differently than other events within the bioevent data outside the gate. For example, the processor 400 may be configured to render the color of the bioevent data included within the gate differently than the color of the bioevent data outside the gate. The display device 406 may be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to provide a graphical interface.

[0094] The processor 400 may be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device may be implemented as a mouse 410. The mouse 410 may transmit a gate selection signal identifying a gate to be displayed on or manipulated via the display device 406 (e.g., by clicking on or within the desired gate when the cursor is on the desired gate) to the processor 400. In some implementations, the first input device may be implemented as a keyboard 408 or other means for providing input signals to the processor 400, such as a touch screen, a pen, a photodetector, or a voice recognition system. Some input devices may include multiple input functions. In such implementations, each input function may be considered an input device. For example, as shown in FIG. 4, the mouse 410 may include a right mouse button and a left mouse button, and the right mouse button and the left mouse button may each generate a trigger event.

[0095] A trigger event can cause the processor 400 to change how the data is displayed, what portions of the data are actually displayed on the display device 406, and / or provide input for further processing, such as selection of a population of interest for particle sorting.

[0096] In some embodiments, the processor 400 may be configured to detect when a gate selection is initiated by the mouse 410. The processor 400 may further be configured to automatically modify the visualization of the plot to facilitate gating. This modification may be made based on a particular distribution of the bio-event data received by the processor 400. In some embodiments, the processor 400 extends the first gate such that a second gate is generated (e.g., as described above).

[0097] The processor 400 may be connected to a storage device 404. The storage device 404 may be configured to receive and store bio-event data from the processor 400. The storage device 404 may be further configured to receive and store flow cytometry event data from the processor 400. The storage device 404 may be further configured to enable retrieval of bio-event data, such as flow cytometry event data, by the processor 400.

[0098] The display device 406 may be configured to receive display data from the processor 400. The display data may include plots of the biological event data and gates outlining sections of the plots. The display device 406 may be further configured to modify the information displayed in response to input received from the processor 400 in conjunction with input from the flow cytometer 402, the storage device 404, the keyboard 408, and / or the mouse 410.

[0099] In some embodiments, the processor 400 can generate a user interface for receiving exemplary events for screening. For example, the user interface can include a mechanism 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 a sample, or based on an initial set of events for a portion of a sample.

[0100] FIG. 5A is a schematic diagram illustrating a particle sorting system 500 (e.g., flow cytometer 402) according to one embodiment presented herein. In some embodiments, the particle sorting system 500 is a cell sorting system. As shown in FIG. 5A, a droplet forming transducer 502 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 501, which may be coupled to, include, or be a nozzle 503. Within the fluid conduit 501, a sheath fluid 504 hydrodynamically focuses a sample fluid 506 containing particles 509 into a moving fluid column 508 (e.g., a stream). Within the moving fluid column 508, the particles 509 (e.g., cells) are aligned in a single file across a monitoring region 511 (e.g., where a laser and a stream intersect) and are illuminated by an illumination source 512 (e.g., a laser). Vibration of the droplet forming transducer 502 causes the moving fluid column 508 to break up into a number of droplets 510 , some of which contain particles 509 .

[0101] In operation, a detection station 514 (e.g., an event detector) identifies when a particle of interest (or cell of interest) crosses the monitoring region 511. The detection station 514 feeds a timing circuit 528, which in turn feeds a flash charge circuit 530. At a droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 508 such that the droplet of interest carries a charge. The droplet of interest can 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 charged droplets into a receptacle, such as a collection tube or a multi-well or microwell sample plate, where a well or microwell can be specifically associated with the droplet of interest. As shown in FIG. 5A, the droplets can be collected in a drain receptacle 538.

[0102] The detection system 516 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal when a particle of interest passes through the monitoring region 511. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 516 enables the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 516 may provide an amplitude signal 520 and / or a phase signal 518, which are then provided (via an amplifier 522) to an amplitude control circuit 526 and / or a frequency control circuit 524. The amplitude control circuit 526 and / or the frequency control circuit 524 then control the droplet forming transducer 502. The amplitude control circuit 526 and / or the frequency control circuit 524 may be included in the control system.

[0103] In some embodiments, the sorting electronics (e.g., detection system 516, detection station 514, and processor 540) may be coupled to a memory configured to store detected events and sorting decisions based on the detected events. The sorting decisions may be included in the event data for the particles. In some embodiments, detection system 516 and detection station 514 may be implemented as a single detection unit or may be communicatively coupled such that event measurements may be collected by either detection system 516 or detection station 514 and provided to a non-collection element.

[0104] FIG. 5B is a schematic diagram showing a particle sorting system according to one embodiment presented herein. The particle sorting system 500 shown in FIG. 5B includes deflection plates 552 and 554. An electric charge can be applied via a stream charging wire in the barb, thus generating a stream of droplets 510 containing the particles 509 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 (not shown in FIG. 5B) or other detection systems. Deflection plates 552 and 554 can be independently controlled to attract or repel the charged droplets and direct them toward a desired collection vessel (e.g., one of 572, 574, 576, or 578). 5B, deflector 552 and deflector 554 can be controlled to direct particles along a first path 562 toward a container 574 or along a second path 568 toward a container 578. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector may allow the particle to continue along flow path 564. Such uncharged droplets may be channeled into a waste container, such as via an aspirator 570.

[0105] Sorting electronics can be included to initiate measurement collection, receive fluorescent signals for the particles, and determine how to adjust the deflection plates to sort the particles. Exemplary implementations of the embodiment shown in Figure 5B include the BD FACSAria™ line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0106] Computer Control System Aspects of the present disclosure further include a computer control system, the computer control system comprising one or more computers for full or partial automation. In some embodiments, the system comprises a computer having a non-transitory computer readable storage medium having a computer program stored thereon, the computer program having instructions for receiving flow cytometer data associated with a first particle and a first trigger signal and a second trigger signal associated with the first particle and a second particle, respectively, when loaded into the computer. The computer program of interest further comprises instructions for evaluating the flow cytometer data for coincidence events by generating a time interval for the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal, and determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time interval.

[0107] 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 has a processor that can access a memory in which instructions for performing 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, and 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 available or become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor coordinating and executing 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 is known in the art. The operating system generally cooperates with the processor to coordinate and execute the functions of the other elements of the computer. The operating system further 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.

[0108] The 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 resident hard disks or tapes, optical media such as read-write compact disks, flash memory devices, or other memory storage devices. The memory storage devices may be any of a variety of known or future devices including compact disk drives, tape drives, or diskette drives. Such types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk. Any of these program storage media, or other program storage media now in use or that may be developed in the future, 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 referred to as computer control logic, are typically stored in the system memory and / or in program storage devices used in conjunction with the memory storage devices.

[0109] 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, 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. The implementation of hardware state machines to perform the functions described herein will be apparent to one of ordinary skill in the relevant art.

[0110] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic device, an optical device, or a solid-state storage device (including a magnetic or optical disk or a tape or a RAM or any other suitable device, fixed or portable). The processor may include a general-purpose digital microprocessor suitably programmed from a computer-readable medium storing the necessary program code. The program may be provided remotely to the processor via a communication channel, or may be pre-recorded in a computer program product such as a memory or in other portable or fixed computer-readable storage media using any of those devices connected to the memory. For example, a magnetic or optical disk may store the program and may be read by a disk writer / reader. The system of the present invention further comprises a program, for example in the form of a computer program product, an algorithm for use in implementing the method as described above. The program of the present invention may be recorded in a computer-readable medium, for example 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 magnetic disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROM; electrical storage media, such as RAM and ROM; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0111] The processor may also access a communication channel to communicate with a user at a remote location, meaning that the user does not have 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, such as a mobile phone (i.e., smartphone).

[0112] In some embodiments, a system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication, such as radio frequency identification (RFID), ZigBee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).

[0113] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communication.

[0114] In one embodiment, the communications interface is configured for infrared communications, Bluetooth® communications, or any other suitable wireless communications protocol, allowing the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communications devices that a user may use in conjunction therewith.

[0115] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing Internet Protocol (IP) via a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0116] In one embodiment, the subject system is configured to communicate wirelessly with the server device via a communication interface using a common standard such as 802.11, Bluetooth RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smart phone, a personal digital assistant (PDA), or a notebook computer; or a larger device such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as a button, a keyboard, a mouse, or a touch screen.

[0117] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored in the subject system, e.g., any data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0118] 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. Where one of the display devices provides visual information, this information may generally be logically and / or physically organized as an array of pixels. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input / output interface between the system and the user, and 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 accomplished using a network or other type of remote communication in alternative embodiments. The output manager may further provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, according to known techniques. Presentation of data by the output manager may be performed according to a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, emails, or other files, or other forms of data. The data may include Internet URL addresses so that the user may retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The platform or platforms present in the subject system are typically a class of computers commonly referred to as servers, but may be any type of computer platform now known or later developed. However, the platforms may also be mainframe computers, workstations, or other computer types. The platforms may be connected via any type of cabling, now known or later, or other communication systems, including wireless systems, whether networked or not. The platforms may be co-located or physically separated.Various operating systems may be used on any of the computer platforms, depending in some cases on the type and / or configuration 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.

[0119] FIG. 6 illustrates a general configuration of an exemplary computing device 600 according to an embodiment. The general configuration of the computing device 600 illustrated in FIG. 6 includes an arrangement of computer hardware and software components. However, not all of these general conventional elements need to be illustrated to provide a useful disclosure. As illustrated, the computing device 600 includes a processing unit 610, a network interface 620, a computer-readable medium drive 630, an input / output device interface 640, a display 650, and an input device 660, all of which may communicate with each other via a communication bus. The network interface 620 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 610 may receive information and instructions from other computing systems or services via the network. The processing unit 610 may further communicate with a memory 670 and may further provide output information for an optional display 650 via the input / output device interface 640. For example, analysis software (e.g., data analysis software or programs, such as FlowJo®), stored as executable instructions in non-transitory memory of the analysis system, can display flow cytometry event data to a user. Input / output device interface 640 may further accept input from any input device 660, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device.

[0120] Memory 670 may include computer program instructions (grouped in some embodiments as modules or components) that processing unit 610 executes to implement one or more embodiments. Memory 670 typically includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 670 may store an operating system 672 that provides computer program instructions for use by processing unit 610 in the general management and operation of computing device 600. Data may be stored in data storage device 690. Memory 670 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0121] usefulness The subject methods, systems and computer systems are used in a variety of applications where it is desirable to analyze and optionally sort particulate components in a sample in a fluid medium, such as a biological sample, and then store the sorted product for subsequent use, such as for example therapeutic applications. The invention is particularly used when it is desirable to evaluate flow cytometer data for the presence or absence of coincidence events. As flow cytometer data collection speeds increase, coincidence events become more likely to occur. Thus, the invention is used in situations where it is desirable to increase the speed of analysis while maintaining data integrity. In addition, the subject methods, systems and computer systems may be used when it is desirable to include more colors in a flow cytometry protocol. Because the trend to include more colors stresses existing systems to the extent that noise caused by this crosstalk can reduce the fidelity of the resulting data and generate confusing artifacts, the invention may be used to increase the feasibility of protocols that include an increased number of colors by identifying and mitigating the effects of crosstalk.

[0122] The embodiments of the present invention are further used in applications where cells prepared from biological samples may be desired for use in research, clinical trials, or therapy. In some embodiments, the subject methods and devices may facilitate obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods and systems may facilitate obtaining cells from a fluid or tissue sample used as a research or diagnostic specimen for a disease such as cancer. Similarly, the subject methods and systems may facilitate obtaining cells from a fluid or tissue sample used for therapy. The disclosed methods and devices allow for the separation and collection of cells from biological samples (e.g., organs, tissues, tissue slices, fluids) with high efficiency and low cost compared to conventional flow cytometry systems.

[0123] kit Aspects of the present disclosure further include kits, the kits having storage media, such as 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). Any of these program storage media, or other program storage media now in use or that may be developed in the future, may be included in the subject kits. In embodiments, the program storage media have instructions for evaluating flow cytometer data for the presence or absence of coincidence events. In embodiments, the instructions included in the 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, the computer control system of the present disclosure may function as a software "plug-in" to an existing software package.

[0124] In addition to the above components, the subject kits may further comprise (in some embodiments) instructions for use, for example, for installing a plug-in to an existing software package. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be provided is information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, the kit packaging, a package insert, etc. Yet 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. Yet another form in which these instructions may be present is a website address that may be used via the internet to access the information at a remote location.

[0125] Regardless of the scope of the appended claims, the present disclosure is further defined by the following caveats.

[0126] Appendix 1. A method for evaluating flow cytometer data for the presence or absence of coincidence events, comprising: receiving flow cytometer data associated with the first particle from the detector and a first trigger signal and a second trigger signal associated with the first particle and the second particle, respectively, after illuminating the first particle and the second particle in the flow stream with a first light source and a second light source, respectively; generating a time interval between the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal; The method evaluates the flow cytometer data for coincidence events by determining whether a first particle and a second particle were illuminated by a first light source and a second light source simultaneously based on the generated time intervals.

[0127] Appendix 2. The method of appendix 1, wherein when evaluating the flow cytometer data, the time interval is compared to a measurement window associated with a second light source.

[0128] Clause 3. The method of clause 2, wherein the measurement window defines a time during which a coincidence event occurs upon irradiation of a second particle.

[0129] Appendix 4. The method of appendix 2 or 3, wherein when evaluating the flow cytometer data, determining whether a second particle is included within a measurement window of a second light source while flow cytometer data related to a first particle is obtained from a detector based on a time interval.

[0130] Appendix 5. The method of appendix 1, wherein time intervals are compared to a reference value when evaluating flow cytometer data.

[0131] Appendix 6. The method of Appendix 5, wherein when evaluating the flow cytometer data, it is determined whether the time interval is lower than a reference value.

[0132] Appendix 7. The method of any one of appendices 1-6, wherein flow cytometer data including coincidence events are assigned a coincidence identifier.

[0133] Appendix 8. The method of any one of appendices 1-6, discarding flow cytometer data that includes coincident events.

[0134] Clause 9. The method of any one of clauses 1-8, wherein the first trigger signal and the second trigger signal indicate when a first particle and a second particle are detected in the flow stream, respectively.

[0135] Clause 10. The method of any one of clauses 1-9, further comprising estimating a relative position of the first particle and the second particle in the flow stream based on the generated time intervals.

[0136] Addendum 11. The method of any one of Addendums 1-10, comprising evaluating flow cytometer data associated with a plurality of different pairs of a first particle and a second particle from within a particle sample.

[0137] Clause 12. The method of any one of clauses 1 to 11, wherein the first particle and the second particle are consecutive particles in the flow stream.

[0138] Clause 13. The method of any one of clauses 1-11, wherein the first particle and the second particle are non-contiguous particles within the flow stream.

[0139] Clause 14. The method of any one of clauses 1 to 13, wherein a first light source and a second light source are used to illuminate the first particle and the second particle, respectively, in the flow stream.

[0140] Clause 15. The method of clause 14, further comprising deactivating the second light source during the coincidence event.

[0141] Addendum 16. The method of any one of Addendums 1 to 15, wherein the first light source and the second light source are selected from a plurality of light sources.

[0142] Addendum 17. The method of Addendum 16, wherein the number of light sources in the plurality of light sources is in the range of 2 to 10.

[0143] Addendum 18. The method of any one of Addendums 16 to 17, wherein the multiple light sources include one or more lasers.

[0144] Addendum 19. The method of any one of Addendums 1 to 18, wherein the detector is selected from a plurality of detectors.

[0145] Addendum 20. The method of Addendum 19, wherein the number of detectors in the plurality of detectors is in the range of 2 to 10.

[0146] Clause 21. The method of clause 19 or 20, wherein the plurality of detectors comprises one or more detector arrays.

[0147] Clause 22. A flow cell configured to carry a particle sample in a flow stream; a first light source and a second light source configured to irradiate first particles and second particles, respectively, in a particle sample; a detector configured to generate flow cytometer data related to the first particle; receiving flow cytometer data from the detector related to the illuminated first particle and a first trigger signal and a second trigger signal related to the first particle and the second particle, respectively; generating a time interval between the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal; Evaluating the resulting flow cytometer data for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time intervals. and a processor configured as The system comprises:

[0148] Addendum 23. The system of Addendum 22, wherein when evaluating the flow cytometer data, the time interval is compared to a measurement window associated with the second light source.

[0149] Addendum 24. The system of Addendum 23, wherein the measurement window defines a time during which a coincidence event occurs upon irradiation of a second particle.

[0150] Addendum 25. The system of Addendum 23 or 24, wherein when evaluating the flow cytometer data, a determination is made based on a time interval as to whether a second particle is included within a measurement window associated with a second light source while flow cytometer data associated with a first particle is obtained from a detector.

[0151] Addendum 26. The system of Addendum 22, wherein the time intervals are compared to a reference value when evaluating the flow cytometer data.

[0152] Addendum 27. The system of Addendum 26, wherein when evaluating the flow cytometer data, it determines whether the time interval is below a reference value.

[0153] Addendum 28. A system according to any one of Addendums 22 to 27, wherein the processor is configured to assign a coincidence identifier to flow cytometer data including a coincidence event.

[0154] Addendum 29. The system of any one of Addendums 22-27, wherein the processor is configured to discard flow cytometer data that includes coincident events.

[0155] Addendum 30. The system of any one of Addendums 22-29, wherein the first trigger signal and the second trigger signal indicate when a first particle and a second particle are detected in the flow stream, respectively.

[0156] Addendum 31. The system of any one of Addendums 22-30, wherein the processor is configured to estimate a relative position of the first particle and the second particle in the flow stream based on the generated time intervals.

[0157] Addendum 32. A system according to any one of Addendums 22 to 31, wherein the processor is configured to evaluate flow cytometer data relating to a plurality of different pairs of a first particle and a second particle from within the particle sample.

[0158] Addendum 33. The system of any one of Addendums 22-32, wherein the processor is configured to stop operation of the second light source during the coincidence event.

[0159] Addendum 34. The system of any one of Addendums 22-33, wherein the first light source and the second light source are selected from a plurality of light sources.

[0160] Addendum 35. The system of Addendum 34, wherein the number of light sources in the plurality of light sources is in the range of 2 to 10.

[0161] Addendum 36. The system of any one of Addendums 34 to 35, wherein the multiple light sources include one or more lasers.

[0162] Addendum 37. The system of any one of Addendums 22-36, wherein the detector is selected from a plurality of detectors.

[0163] Addendum 38. The system of Addendum 37, wherein the number of detectors in the plurality of detectors is in the range of 2 to 10.

[0164] Addendum 39. The system of any one of Addendums 37 to 38, wherein the multiple detectors include one or more detector arrays.

[0165] Addendum 40. The system of any one of Addendums 22 to 39, which is a flow cytometer.

[0166] Addendum 41. The system of any one of Addendums 22 to 40, further comprising a display configured to output flow cytometer data evaluated by the processor.

[0167] Clause 42. Illuminating a first particle and a second particle in a flow stream with a first light source and a second light source, respectively, and then receiving flow cytometer data related to the first particle from a detector and a first trigger signal and a second trigger signal related to the first particle and the second particle, respectively; generating a time interval between the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal; Evaluating the flow cytometer data for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time intervals. A non-transitory computer readable storage medium having stored thereon instructions for evaluating flow cytometer data for the presence or absence of coincidence events by a method.

[0168] Addendum 43. The non-transitory computer-readable storage medium of Addendum 42, wherein when evaluating the flow cytometer data, the time interval is compared to a measurement window associated with the second light source.

[0169] Clause 44. The non-transitory computer-readable storage medium of clause 43, wherein the measurement window defines a time during which a coincidence event occurs upon irradiation of the second particle.

[0170] Addendum 45. The non-transitory computer-readable storage medium of Addendum 43 or 44, wherein when evaluating the flow cytometer data, determining whether a second particle is included within a measurement window associated with a second light source while flow cytometer data associated with a first particle is obtained from a detector based on a time interval.

[0171] Clause 46. The non-transitory computer-readable storage medium of clause 42, wherein when evaluating the flow cytometer data, the time intervals are compared to a reference value.

[0172] Clause 47. The non-transitory computer-readable storage medium of clause 46, wherein when evaluating the flow cytometer data, it is determined whether the time interval is below a reference value.

[0173] Addendum 48. The non-transitory computer-readable storage medium of any one of Addendums 42 to 47, further comprising the step of assigning a coincidence identifier to flow cytometer data including coincidence events.

[0174] Addendum 49. The non-transitory computer-readable storage medium of any one of Addendums 42 to 47, wherein the method further comprises discarding flow cytometer data that includes coincidence events.

[0175] Addendum 50. A non-transitory computer-readable storage medium according to any one of Addendums 42-49, wherein the first trigger signal and the second trigger signal indicate a time when a first particle and a second particle are detected in the flow stream, respectively.

[0176] Addendum 51. The non-transitory computer-readable storage medium of any one of Addendums 42-50, wherein the method further comprises estimating a relative position of the first particle and the second particle in the flow stream based on the generated time interval.

[0177] Addendum 52. A non-transitory computer-readable storage medium according to any one of Addendums 42 to 51, wherein the method further comprises evaluating flow cytometer data associated with a plurality of different pairs of a first particle and a second particle from within the particle sample.

[0178] Addendum 53. The non-transitory computer-readable storage medium of any one of Addendums 42 to 52, wherein the method further comprises deactivating the second light source during the coincidence event.

[0179] Addendum 54. The non-transitory computer-readable storage medium of any one of Addendums 42 to 53, wherein the first light source and the second light source are selected from a plurality of light sources.

[0180] Addendum 55. The non-transitory computer-readable storage medium of Addendum 54, wherein the number of light sources in the plurality of light sources is in the range of 2 to 10.

[0181] Addendum 56. The non-transitory computer-readable storage medium of any one of Addendums 54 to 55, wherein the multiple light sources include one or more lasers.

[0182] Addendum 57. The non-transitory computer-readable storage medium of any one of Addendums 42 to 56, wherein the detector is selected from a plurality of detectors.

[0183] Addendum 58. The non-transitory computer-readable storage medium of Addendum 57, wherein the number of detectors in the plurality of detectors is in the range of 2 to 10.

[0184] Addendum 59. The non-transitory computer-readable storage medium of any one of Addendums 57 to 58, wherein the plurality of detectors includes one or more detector arrays.

[0185] Addendum 60. A method for evaluating flow cytometer data, comprising: (a) a flow cell configured to carry a particle sample in a flow stream; a first light source and a second light source configured to irradiate first particles and second particles, respectively, in a particle sample; a detector configured to generate flow cytometer data related to the first particle; receiving flow cytometer data from the detector related to the illuminated first particle and a first trigger signal and a second trigger signal related to the first particle and the second particle, respectively; generating a time interval between the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal; Evaluating the resulting flow cytometer data for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time intervals. and a processor configured as introducing a particle sample into a system comprising: (b) receiving the evaluated flow cytometer data from the processor.

[0186] Addendum 61. The method of Addendum 60, wherein when evaluating the flow cytometer data, the time interval is compared to a measurement window associated with a second light source.

[0187] Addendum 62. The method of Addendum 61, wherein the measurement window defines a time during which a coincidence event occurs upon irradiation of a second particle.

[0188] Addendum 63. The method of Addendum 61 or 62, wherein when evaluating the flow cytometer data, it is determined whether a second particle is included within a measurement window associated with a second light source based on a time interval while flow cytometer data associated with a first particle is obtained from a detector.

[0189] Addendum 64. The method of Addendum 60, wherein time intervals are compared to a reference value when evaluating flow cytometer data.

[0190] Addendum 65. The method of Addendum 64, wherein when evaluating the flow cytometer data, it is determined whether the time interval is lower than a reference value.

[0191] Addendum 66. The method of any one of Addendums 60 to 65, wherein the processor is configured to assign a coincidence identifier to flow cytometer data that includes a coincidence event.

[0192] Addendum 67. The method of any one of Addendums 60 to 65, wherein the processor is configured to discard flow cytometer data that includes coincidence events.

[0193] Addendum 68. The method of any one of Addendums 60-67, wherein the first trigger signal and the second trigger signal indicate when a first particle and a second particle are detected in the flow stream, respectively.

[0194] Addendum 69. The method of any one of Addendums 60-68, wherein the processor is configured to estimate a relative position of the first particle and the second particle within the flow stream based on the generated time intervals.

[0195] Addendum 70. The method of any one of Addendums 60-69, wherein the processor is configured to evaluate flow cytometer data associated with a plurality of different pairs of a first particle and a second particle from within the particle sample.

[0196] Addendum 71. The method of any one of Addendums 60-70, wherein the first particle and the second particle are consecutive particles in the flow stream.

[0197] Addendum 72. The method of any one of Addendums 60-70, wherein the first particle and the second particle are non-contiguous particles within the flow stream.

[0198] Addendum 73. The method of any one of Addendums 60-72, wherein the first light source and the second light source are selected from a plurality of light sources.

[0199] Addendum 74. The method of Addendum 73, wherein the number of light sources in the plurality of light sources is in the range of 2 to 10.

[0200] Addendum 75. The method of any one of Addendum 73 to 74, wherein the multiple light sources include one or more lasers.

[0201] Addendum 76. The method of any one of Addendums 60-75, wherein the detector is selected from a plurality of detectors.

[0202] Addendum 77. The method of Addendum 76, wherein the number of detectors in the plurality of detectors is in the range of 2 to 10.

[0203] Addendum 78. The method of any one of Addendum 76 to 77, wherein the multiple detectors include one or more detector arrays.

[0204] Addendum 79. The method of any one of Addendums 60-78, wherein the processor is configured to stop operation of the second light source during the coincidence event.

[0205] Addendum 80. The method of any one of Addendums 60 to 79, wherein the system is a flow cytometer.

[0206] Addendum 81. The method of any one of Addendums 60 to 80, wherein the system further comprises a display configured to output flow cytometer data evaluated by the processor.

[0207] Although the foregoing invention has been described in some detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art, in view of the teachings of the invention, that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

[0208] Thus, the foregoing merely illustrates the nature of the invention. It is clear that those skilled in the art may devise various configurations that embody the nature of the invention and are within the spirit and scope of the invention, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended essentially to aid the reader in understanding the nature of the invention and the concepts given by the inventor to advance the art, and should not be construed as being limited to the examples and conditions specifically described as such. Furthermore, all descriptions herein that describe the nature, aspects, and embodiments of the invention, as well as specific examples of the invention, are intended to include both structural and functional equivalents of the invention. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., all elements developed that perform the same function, regardless of structure. Furthermore, the descriptions disclosed herein are not intended to be public, regardless of whether such disclosure is expressly set forth in the claims.

[0209] Therefore, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied in the appended claims. With respect to claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly provided to be invoked with respect to a limitation in a claim only when the precise phrase "means for" or "step for" is recited at the beginning of such limitation in the claim, and if such precise phrase is not used in the limitation of the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0210] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Application No. 63 / 313,391, filed February 24, 2022, the entire disclosure of which is incorporated herein by reference.

Claims

1. 1. A method for evaluating flow cytometer data for the presence or absence of coincidence events, comprising: receiving flow cytometer data associated with the first particle from a detector after illuminating a first particle and a second particle in the flow stream with a first light source and a second light source, respectively, and a first trigger signal and a second trigger signal associated with the first particle and the second particle, respectively; generating a time interval between the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal; evaluating the flow cytometer data for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time intervals.

2. The method of claim 1 , further comprising comparing the time interval to a measurement window associated with the second light source when evaluating the flow cytometer data.

3. The method of claim 2 , wherein the measurement window defines a time during which irradiation of the second particle results in a coincidence event.

4. 4. The method of claim 2 or 3, wherein when evaluating the flow cytometer data, it is determined whether the second particle is included within a measurement window of the second light source while flow cytometer data related to the first particle is obtained from the detector based on the time interval.

5. The method of claim 1 , wherein said time intervals are compared to a reference value when evaluating said flow cytometer data.

6. The method of claim 5 , further comprising: determining whether the time interval is less than the reference value when evaluating the flow cytometer data.

7. The method of any one of claims 1 to 6, further comprising assigning a coincidence identifier to said flow cytometer data containing coincidence events.

8. The method of any one of claims 1 to 6, further comprising discarding the flow cytometer data that includes coincidence events.

9. The method of any one of claims 1 to 8, wherein the first trigger signal and the second trigger signal indicate when the first particle and the second particle are detected in the flow stream, respectively.

10. The method of any one of claims 1 to 9, further comprising estimating a relative position of a first particle and a second particle in the flow stream based on the generated time intervals.

11. A method according to any one of claims 1 to 10, comprising evaluating flow cytometer data relating to a plurality of different pairs of a first particle and a second particle from within a particle sample.

12. The method of any one of claims 1 to 11, wherein the first particle and the second particle are consecutive particles in the flow stream.

13. The method of any one of claims 1 to 11, wherein the first particle and the second particle are non-contiguous particles in the flow stream.

14. a flow cell configured to carry a particle sample in a flow stream; a first light source and a second light source configured to illuminate first particles and second particles, respectively, in the particle sample; a detector configured to generate flow cytometer data related to the first particles; receiving flow cytometer data from the detector related to the illuminated first particle and a first trigger signal and a second trigger signal related to the first particle and the second particle, respectively; generating a time interval between the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal; Evaluating the resulting flow cytometer data for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time intervals. and a processor configured as The system comprises:

15. 1. A method for evaluating flow cytometer data, comprising: (a) a flow cell configured to carry a particle sample in a flow stream; a first light source and a second light source configured to illuminate first particles and second particles, respectively, in the particle sample; a detector configured to generate flow cytometer data related to the first particles; receiving flow cytometer data from the detector related to the illuminated first particle and a first trigger signal and a second trigger signal related to the first particle and the second particle, respectively; generating a time interval between the first particle and the second particle based on a difference between the first trigger signal and the second trigger signal; Evaluating the resulting flow cytometer data for coincidence events by determining whether the first particle and the second particle were illuminated by the first light source and the second light source simultaneously based on the generated time intervals. and a processor configured as introducing a particle sample into a system comprising: (b) receiving evaluated flow cytometer data from said processor.

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