Method for determining absolute count of particles in sample in flow cytometer and system therefor

Abstract

Aspects of the present disclosure include methods for determining an absolute count of particles in a sample in a flow cytometer. A method according to certain embodiments includes introducing a bubble into a flow stream propagating a sample having particles, irradiating the flow stream with a light source, detecting light from the irradiated particles with a photodetector, detecting the presence of the bubble in the flow stream with the photodetector, and detecting the presence of the bubble in the flow stream with the photodetector. And determining the absolute count of the particles in the sample based on a data signal generated in response to light detected from the irradiated particles, a data signal generated when the bubbles are introduced into the flow stream, and a data signal generated in response to detected bubbles. Also described are systems (e.g., flow cytometers) having a light source, a light detection system, and a sample line configured to introduce bubbles into a flow stream for practicing the subject methods. A non-transitory computer-readable storage medium is also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 USC § 119(e) to the filing date of U.S. Provisional Patent Application Serial No. 63 / 352,472, filed on June 15, 2022; the disclosure of which is incorporated herein by reference in its entirety. introduction

[0002] Light detection is often used to characterize the composition of a sample (e.g., a biological sample), such as when the sample is used for diagnosis of a disease or medical condition. When a sample is illuminated, light can be scattered by the sample, transmitted through the sample, and emitted by the sample (e.g., by fluorescence). Changes in the composition of the sample (such as morphology, absorbance, and the presence of fluorescent markers) may cause changes in the light scattered, transmitted, or emitted by the sample. These changes can be used to characterize and identify the presence of components in the sample. To quantify these changes, light is collected and directed to the surface of a detector.

[0003] One technique that uses light detection to characterize components in a sample is flow cytometry. A flow cytometer includes a photodetection system consisting of optics, photodetectors, and electronics that can effectively detect optical signals and convert them into corresponding electrical signals. The electronic signals are processed to obtain parameters that the user can use to perform the desired analysis. A flow cytometer includes different types of photodetectors to detect light signals, such as light signals from fluorescence, side scattered light, or forward scattered light. When an optical signal is incident on a photodetector, an electrical signal proportional to the incident optical signal is generated at its output. Flow cytometry provides a method for rapidly quantifying cell characteristics.

[0004] However, most flow cytometers cannot directly provide the cell concentration or absolute count of cells in a sample. Absolute cell counts have been used extensively to quantify cell populations and disease progression, including in the immune system or in the study of stem cells. Absolute cell counts are typically obtained by combining a separate cell concentration determination from a hematology analyzer with flow cytometric population data using multiplatform testing, or by adding an in-house microsphere counting standard to the flow cytometric sample using single-platform testing. Summary of the invention

[0005] Aspects of the present disclosure include methods for determining an absolute count of particles in a sample in a flow cytometer. Methods according to certain embodiments include introducing bubbles into a flow stream that propagates a sample having particles, irradiating the flow stream with a light source, detecting light from the irradiated particles with a photodetector, detecting the presence of bubbles in the flow stream with a photodetector, and determining an absolute count of particles in the sample based on the following: a data signal generated in response to light detected from the irradiated particles, a data signal generated when bubbles are introduced into the flow stream, and a data signal generated in response to detected bubbles. A system (e.g., a flow cytometer) having a light source, a light detection system, and a sample line is also described, the sample line being configured to introduce bubbles into the flow stream for practicing the subject method. A non-transitory computer-readable storage medium is also provided.

[0006] When practicing the subject method, a sample having particles in a flowing stream is irradiated with a light source, and bubbles are introduced into the flowing stream. In some embodiments, a data signal is generated when the bubble is introduced into the flowing stream. In some instances, a data signal is generated when the bubble is introduced into the flowing stream. In other instances, a data signal is generated at a predetermined time after the bubble is introduced into the flowing stream, such as 0.001 μs to 10 μs after the bubble is introduced into the flowing stream. In some embodiments, bubbles are introduced into the flowing stream by retracting the sample line from the source of the sample to produce an air gap in the sample in the flowing stream. In certain embodiments, in order to produce a gap in the sample in the flowing stream, the sample line can be retracted from the sample source, wherein air inhalation replaces the sample inhalation into the flowing stream, such as lasting 0.0001 seconds to 0.1 seconds. In some embodiments, a data signal is generated when the sample line is retracted from the sample source. In other embodiments, the data signal is generated at a predetermined time after the sample line is retracted from the sample source, such as 0.001 μs to 10 μs after the sample line is retracted from the sample source. In some examples, the data signal generated when the bubble is introduced into the flow stream is an electronic data signal. In other examples, the data signal is an optical data signal.

[0007] In an embodiment, a light detection system with a photodetector is used to detect light from a flow stream, and a data signal is generated in response to the detected light. In some embodiments, the method includes continuously detecting light from irradiated particles before introducing bubbles into the flow stream, continuously detecting light from irradiated particles upstream of bubbles in the flow stream, and detecting light from the flow stream when the bubble passes through an interrogation field. In certain embodiments, the method includes generating a data signal in response to light detected from irradiated particles before introducing bubbles into the flow stream, generating a data signal in response to light detected from irradiated particles upstream of bubbles in the flow stream, and generating a data signal when the bubble is irradiated in the flow stream. In some embodiments, the method includes detecting one or more of light absorption, light scattering, and light emission (e.g., fluorescence) from the flow stream. In some instances, the data signal is generated by fluorescence detected from particles irradiated in the sample. In some instances, the data signal is generated by scattered light detected from particles irradiated in the sample. In some instances, the data signal generated by detecting bubbles in the flow stream comes from scattered light detected from irradiated bubbles.

[0008] In some instances, an absolute count of particles in a sample is calculated using a data signal generated in response to light detected from an irradiated particle between when a bubble is introduced into the flow stream and when the bubble is detected and a predetermined volume. In some instances, the absolute count of particles is determined using a previously calibrated volume of a sample line. In some instances, the method includes generating a data signal intensity graph of the data signal from the irradiated sample as a function of time. In certain instances, the data signal intensity graph includes at least the intensity of the data signal from the irradiated particle collected from the time when the bubble is introduced into the flow stream until the time when the bubble is detected. In certain instances, the time when the bubble is introduced into the flow stream is plotted on the data signal intensity graph.

[0009] Aspects of the present disclosure also include systems (e.g., flow cytometers) having a flow cell configured to propagate a sample having particles in a flow stream; a sample line in fluid communication with a source of the sample, the sample line configured to introduce bubbles into the flow stream; a light source for illuminating the flow stream; and a light detection system having a photodetector for detecting light from the irradiated flow stream. In some embodiments, the sample line is configured to create an air gap in the sample in the flow stream. In some instances, the system includes a sample line displacement component configured to retract the sample line from the sample source and to draw air into the sample line when the sample line is retracted from the sample source. Air is drawn into the sample to create an air gap in the flow stream when the sample line is retracted from the sample source. In certain embodiments, the system includes a gas source for introducing gaseous bubbles into the sample line.

[0010] In an embodiment, the system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate a data signal in response to a bubble being introduced into a flow stream. In some instances, the memory includes instructions for generating an electronic signal when a bubble is introduced into the flow stream. In some instances, the system includes a photodetector configured to generate an optical signal when a bubble is introduced into the flow stream. In certain instances, the memory includes instructions for generating a data signal when a bubble is introduced into the flow stream. In other instances, the memory includes instructions for generating a data signal at a predetermined time after the bubble is introduced into the sample, such as 0.001 µs to 10 µs after the bubble is introduced into the flow stream.

[0011] In an embodiment, the system includes a light detection system having a photodetector for detecting light from a flow stream and generating a data signal in response to the detected light. In some embodiments, the light detection system is configured to continuously detect light from irradiated particles before introducing bubbles into the flow stream, continuously detect light from irradiated particles upstream of bubbles in the flow stream, and detect light from the flow stream when the bubbles pass through the query field. In some embodiments, the system is configured to generate a data signal in response to light detected from irradiated particles before introducing bubbles into the flow stream. In some embodiments, the system is configured to generate a data signal in response to light detected from irradiated particles upstream of bubbles in the flow stream. In some embodiments, the system is configured to generate a data signal when irradiating bubbles in the flow stream. In some embodiments, the light detection system includes one or more light absorption photodetectors, light scattering photodetectors, and light emission (e.g., fluorescence) photodetectors. In some instances, the light detection system is configured to generate a data signal from fluorescence detected from particles irradiated in the sample. In some examples, the light detection system is configured to generate a data signal from scattered light detected from illuminated particles in the sample. In certain examples, the light detection system is configured to generate a data signal in a scattered light detection channel by detecting illuminated bubbles in the flow stream.

[0012] In an embodiment, the system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to calculate an absolute count of particles in a sample. In some embodiments, the memory includes instructions for calculating an absolute count of particles in a sample using a data signal generated in response to light detected from an irradiated particle between when a bubble is introduced into the flow stream and when the bubble is detected and a predetermined volume. In some instances, the memory includes instructions for calculating an absolute count of particles using the volume of a previously calibrated sample line. In some instances, the memory includes instructions for generating a data signal intensity graph of a data signal from an irradiated sample varying over time. In some instances, the data signal intensity graph includes at least the intensity of the data signal from the irradiated particle collected from the time when the bubble is introduced into the flow stream until the time when the bubble is detected. In some instances, the time when the bubble is introduced into the flow stream is plotted on the data signal intensity graph.

[0013] Aspects of the present disclosure also include a non-transitory computer-readable storage medium for calculating an absolute count of particles in a sample in a flow cytometer. In an embodiment, the non-transitory computer-readable storage medium includes an algorithm for introducing bubbles into a flow stream that propagates a sample containing particles, an algorithm for illuminating the flow stream with a light source, an algorithm for detecting light from the irradiated particles with a photodetector, and an algorithm for detecting the presence of bubbles in the flow stream with a photodetector. In an embodiment, the non-transitory computer-readable storage medium further includes an algorithm for determining an absolute count of particles in a sample based on a data signal generated in response to light detected from the irradiated particles, a data signal generated when bubbles are introduced into the flow stream, and a data signal generated in response to detected bubbles.

[0014] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for introducing bubbles into the flow stream by retracting the sample line from the source of the sample to create an air gap in the flow stream. In some instances, the non-transitory computer-readable storage medium includes an algorithm for operating a sample line shifting component that retracts the sample line from the sample source. In certain instances, the non-transitory computer-readable storage medium includes an algorithm for retracting the sample line from the sample source and sucking air or gas into the sample line while retracting the sample line. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal when the sample line is retracted from the sample source. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal at a predetermined time after the sample line is retracted from the sample source, such as 0.001 µs to 10 µs after the bubble is introduced into the flow stream.

[0015] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for continuously detecting light from the irradiated particles before introducing the bubble into the flow stream, an algorithm for continuously detecting light from the irradiated particles upstream of the bubble in the flow stream, and an algorithm for detecting light from the flow stream as the bubble passes through the query field. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal in response to light detected from the irradiated particles before introducing the bubble into the flow stream, an algorithm for generating a data signal in response to light detected from the irradiated particles upstream of the bubble in the flow stream, and an algorithm for generating a data signal when the bubble is irradiated in the flow stream. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal in response to light detected from the irradiated particles downstream of the bubble in the flow stream.

[0016] In an embodiment, a non-transitory computer-readable storage medium includes an algorithm for calculating an absolute count of particles in a sample. In some embodiments, a non-transitory computer-readable storage medium includes an algorithm for calculating an absolute count of particles in a sample using a data signal generated in response to light detected from an irradiated particle between when a bubble is introduced into the flow stream and when a bubble is detected and a predetermined volume. In some instances, a non-transitory computer-readable storage medium includes an algorithm for calculating an absolute count of particles using the volume of a previously calibrated sample line. In some instances, a non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity graph of a data signal from an irradiated sample that varies over time. In some instances, the data signal intensity graph includes at least the intensity of the data signal from the irradiated particle collected from the time when the bubble is introduced into the flow stream until the time when the bubble is detected. In some instances, the time when the bubble is introduced into the flow stream is plotted on the data signal intensity graph. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which the following figures are included:

[0018] Figure 1 Depicted is introducing bubbles into a sample line and detecting the bubbles in the flow stream according to certain embodiments.

[0019] Figure 2 Depicted is a flow chart for determining absolute counts of a sample in a flow stream, according to certain embodiments.

[0020] Figure 3 Depicted are graphs of signal intensities of data generated from light detected from illuminated particles and bubbles introduced into a flow stream, according to certain embodiments.

[0021] Figure 4A Depicted is a functional block diagram of a particle analysis system according to certain embodiments. Figure 4B Depicted are flow cytometers according to certain embodiments.

[0022] Figure 5 Depicted is a functional block diagram of one example of a particle analyzer control system according to certain embodiments.

[0023] Fig. 6A Depicted is a schematic diagram of a particle sorting system according to certain embodiments.

[0024] Figure 6B Depicted is a schematic diagram of a particle sorting system according to certain embodiments.

[0025] Figure 7A block diagram of a computing system is depicted in accordance with certain embodiments. DETAILED DESCRIPTION

[0026] Aspects of the present disclosure include methods for determining an absolute count of particles in a sample in a flow cytometer. Methods according to certain embodiments include introducing bubbles into a flow stream that propagates a sample having particles, irradiating the flow stream with a light source, detecting light from the irradiated particles with a photodetector, detecting the presence of bubbles in the flow stream with a photodetector, and determining the absolute count of particles in the sample based on the following: a data signal generated in response to light detected from the irradiated particles, a data signal generated when the bubble is introduced into the flow stream, and a data signal generated in response to the detected bubble. A system (e.g., a flow cytometer) having a light source, a light detection system, and a sample line is also described, the sample line being configured to introduce bubbles into the flow stream for practicing the subject method. A non-transitory computer-readable storage medium is also provided.

[0027] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described, as such can certainly vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0028] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range (based on one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated or intervening value within the stated range are encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any explicitly excluded limits within the stated range. Where the stated range includes one or both limits, ranges excluding one or both of these included limits are also encompassed within the invention.

[0029] Certain ranges appearing herein are presented by preceding the numerical value with the term "about". The term "about" is used herein to provide literal support for the exact number that follows it as well as a number that is close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically cited number, the close or approximate unquoted number may be a number that provides a substantial equivalent of the specifically cited number in the context in which it appears.

[0030] 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 the 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 illustrative methods and materials are now described.

[0031] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and discloses and describes the methods and / or materials related thereto. 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.

[0032] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "only," "only," and the like, in connection with the recitation of claim elements or as a "negative" limitation.

[0033] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0034] Although the apparatus and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be clearly understood that unless expressly recited pursuant to 35 USC §112, the claims should not be construed as necessarily limited in any way to the "means" or "step" limitations, but should be given the full scope of meaning and equivalents provided by the claims pursuant to the judicial doctrine of equivalents, and to the extent a claim is expressly recited pursuant to 35 USC §112, it will be given full legal equivalents pursuant to 35 USC §112.

[0035] As described above, the present disclosure provides a method for determining an absolute count of particles in a sample in a flow cytometer. In further describing an embodiment of the present disclosure, a method for introducing bubbles into a flow stream that propagates a sample having particles, irradiating the flow stream with a light source, detecting light from the irradiated particles and the presence of bubbles in the flow stream with a photodetector, and determining an absolute count of particles in the sample is first described in more detail. Next, a system including a light source, a light detection system, and a sample pipeline configured to introduce bubbles into the flow stream is described, as well as a non-transitory computer-readable storage medium for practicing the subject method.

[0036] Method for determining absolute counts of particles in a sample in a flow cytometer Aspects of the present disclosure include methods for determining the absolute count of particles in a sample in a flow cytometer. The term "absolute count" is used herein in its conventional sense to refer to the number of particles per volume in a sample. In some embodiments, the sample is a biological sample (described in more detail below), and the subject method provides a method for determining the number of cells per volume in a biological sample. In some instances, the subject method provides an accurate count of the number of particles (or biological cells) per volume in a sample without the use of additional and usually expensive components, such as calibration beads, secondary sample loops, sample line flowmeters or syringe pumps. In some instances, compared to the absolute count determined using calibration beads, sample line flow sensors or syringe pumps, the method for determining the absolute count of particles in a flow cytometry sample described herein provides an improved accuracy of the determined count of the number of particles per volume, such as an improvement of 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including 99% or more. In certain embodiments, the subject methods reduce the time required to determine the absolute count of particles in a sample, such as by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including 99% or more.

[0037] In some instances, the sample described herein is a biological sample with cells. The term "biological sample" is used to refer to a subset of a complete organism, plant, fungus or animal tissue, cell or component part in its conventional sense, which can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen in some instances. Therefore, "biological sample" refers to a subset of a natural organism or its tissue and refers to a homogenate, lysate or extract prepared from a subset of an organism or its tissue, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin, respiratory tract, gastrointestinal tract, cardiovascular and urogenital tract sections, tears, saliva, milk, blood cells, tumors, organs. Biological samples can be any type of organic tissue, including both healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, etc., wherein in some instances, the sample is a blood sample, including whole blood, such as blood obtained from a venipuncture or finger prick (wherein the blood may or may not be mixed with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0038] In certain embodiments, the source of the sample is "mammal" or "mammalian", wherein these terms are widely used to describe organisms in the class Mammalia, including Carnivora (e.g., dogs and cats), Rodents (e.g., mice, guinea pigs and rats) and Primates (e.g., humans, chimpanzees and monkeys). In some instances, the subject is a human. The method can be applied to samples obtained from both males and females and human subjects at any stage of development (i.e., newborns, infants, teenagers, adolescents, adults), wherein in certain embodiments, the human subject is a teenager, adolescent or adult. Although the present invention can be applied to samples from human subjects, it should be understood that the method can also be implemented on samples from other animal subjects (i.e., in "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

[0039] When practicing the subject method, bubbles are introduced into the flow stream of the flow cytometer that propagates the sample with particles. The term "bubble" is used herein to refer to the gap in the flow stream, in which there are almost no particles (and in some instances no fluid) in the flow stream. For example, the bubbles introduced into the flow stream can contain 1% or less particles per volume sample, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, and include 0.00001% or less particles per volume sample. In some instances, the bubbles introduced into the flow stream do not contain particles of the sample.

[0040] Any convenient scheme can be adopted to introduce bubbles into the flowing stream. In some embodiments, during the period when the sample is sucked into the flowing stream, gas is introduced into the flowing stream. For example, during the period when the sample is sucked into the flowing stream, an inert gas such as nitrogen, helium or argon can be input into the flowing stream. The volume of the gas introduced into the flowing stream as bubbles can vary according to the volume of the sample and the sample line, and can be 0.001 μL or more, such as 0.005 μL or more, such as 0.01 μL or more, such as 0.05 μL or more, such as 0.1 μL or more, such as 0.5 μL or more, such as 1 μL or more, such as 2 μL or more, such as 3 μL or more, such as 4 μL or more, such as 5 μL or more and include 10 μL or more.

[0041] In certain embodiments, bubbles are generated by creating an air gap in the flow stream. In some instances, an air gap is created by retracting a sample line that is in communication with the source fluid of the sample so that there is a time gap when the sample composition is sucked from the sample source. During this time gap, ambient air is sucked into the sample line for separating particles of the sample in the flow stream. As described in more detail below, the sample line can be retracted from the sample source by any convenient scheme, such as with a displacement device that lifts the sample line from the sample source (e.g., the hole of a test tube or orifice plate). The sample line may be retracted from the sample source for 0.0001 seconds or longer, such as for 0.0005 seconds or longer, such as for 0.01 seconds or longer, such as for 0.05 seconds or longer, such as for 0.1 seconds or longer, such as for 0.5 seconds or longer, such as for 1 second or longer, such as for 2 seconds or longer, such as for 3 seconds or longer, such as for 4 seconds or longer, such as for 5 seconds or longer, such as for 10 seconds or longer, such as for 15 seconds or longer and including for 30 seconds or longer.

[0042] As described in more detail below, the bubbles introduced into the flow stream provide time gaps in the data signal generated in response to light detected from particles irradiated in the flow stream. The time gaps in the data signal may last for a period of 0.0001 seconds or more, such as 0.0005 seconds or more, such as 0.01 seconds or more, such as 0.05 seconds or more, such as 0.1 seconds or more, such as 0.5 seconds or more, such as 1 second or more, such as 2 seconds or more, such as 3 seconds or more, such as 4 seconds or more, such as 5 seconds or more, such as 10 seconds or more, such as 15 seconds or more and including 30 seconds or more.

[0043] In some embodiments, a data signal is generated when a bubble is introduced into the flow stream. For example, a data signal can be generated when a certain volume of gas is input or sucked into the sample line. In other embodiments, a data signal is generated at a predetermined time after the bubble is introduced into the flow stream, such as 0.001 μs or longer, such as 0.005 μs or longer, such as 0.01 μs or longer, such as 0.05 μs or longer, such as 0.1 μs or longer, such as 0.5 μs or longer, such as 1 μs or longer, such as 2 μs or longer, such as 3 μs or longer, such as 5 μs or longer, such as 10 μs or longer, such as 100 μs or longer, such as 500 μs or longer, and including 1000 μs or longer after the bubble is introduced into the flow stream. In certain embodiments, in the case where bubbles are introduced into the flow stream by retracting the sample line from the sample source, a data signal may be generated when the sample line is retracted from the sample source. In other embodiments, a data signal is generated at a predetermined time period after the sample line is retracted from the sample source, such as 0.001 µs or more, such as 0.005 µs or more, such as 0.01 µs or more, such as 0.05 µs or more, such as 0.1 µs or more, such as 0.5 µs or more, such as 1 µs or more, such as 2 µs or more, such as 3 µs or more, such as 5 µs or more, such as 10 µs or more, such as 100 µs or more, such as 500 µs or more and including 1000 µs or more after the sample line is retracted from the sample source. In other embodiments, a data signal is generated when the sample line is reinserted into the sample source (i.e., when sample aspiration into the sample line is reestablished). In still other embodiments, a data signal is generated at a predetermined time after the sample line is reinserted into the sample source, such as 0.001 µs or longer, such as 0.005 µs or longer, such as 0.01 µs or longer, such as 0.05 µs or longer, such as 0.1 µs or longer, such as 0.5 µs or longer, such as 1 µs or longer, such as 2 µs or longer, such as 3 µs or longer, such as 5 µs or longer, such as 10 µs or longer, such as 100 µs or longer, such as 500 µs or longer, and including 1000 µs or longer after the sample line is reinserted into the sample source.

[0044] In some instances, the data signal generated when the bubble is introduced into the flow stream is an electronic data signal. The data signal generated when the bubble is introduced into the flow stream can be transmitted to a processor (described below) for calculating the particle count of the sample. In other instances, the data signal generated when the bubble is introduced into the flow stream is an optical data signal. The optical data signal can be generated using a photodetector positioned to detect light from a sample line into which the bubble is introduced. For example, a method according to certain embodiments includes irradiating a flow stream into which the bubble is introduced with a light source such as an LED or a laser, detecting light from the bubble introduced into the flow stream, and generating an optical data signal. In the case of introducing bubbles by retracting the sample line from a sample source, the photodetector and light source for detecting when the bubble is introduced into the flow stream can be adjacent to the end of the sample line or downstream of the end of the sample line. In some embodiments, the photodetector and light source for detecting the introduction of bubbles into the flow stream can be positioned 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 4 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and including 50 mm or more downstream from the end of the sample line. In some embodiments, the optical data signal can be transmitted to a processor for calculating the particle count of the sample.

[0045] In embodiments, a sample is irradiated in a flowing stream with a light source. Before bubbles are introduced into the flowing stream, the sample can be irradiated in the flowing stream for a predetermined time period. In some instances, before bubbles are introduced into the flowing stream, the sample irradiation in the flowing stream lasts for 0.01 seconds or longer, such as 0.05 seconds or longer, such as 0.1 seconds or longer, such as 0.5 seconds or longer, such as 1 second or longer, such as 5 seconds or longer, such as 10 seconds or longer, such as 15 seconds or longer, such as 30 seconds or longer, such as 60 seconds, and includes irradiating the sample in the flowing stream for 90 seconds or longer before bubbles are introduced into the flowing stream.

[0046] In some embodiments, the method includes irradiating the sample propagating through a flow stream, across an interrogation area of ​​the flow stream of 5 µm or greater, such as 10 µm or greater, such as 15 µm or greater, such as 20 µm or greater, such as 25 µm or greater, such as 50 µm or greater, such as 75 µm or greater, such as 100 µm or greater, such as 250 µm or greater, such as 500 µm or greater, such as 750 µm or greater, such as for example across an interrogation area of ​​1 mm or greater, such as 2 mm or greater, such as 3 mm or greater, such as 4 mm or greater, such as 5 mm or greater, such as 6 mm or greater, such as 7 mm or greater, such as 8 mm or greater, such as 9 mm or greater and including 10 mm or greater.

[0047] In some embodiments, a sample in a flowing stream is irradiated with a continuous wave light source, such as where the light source provides an uninterrupted light flux and maintains irradiation of particles of the sample in the flowing stream, and there is almost no undesirable change in light intensity. In some embodiments, a continuous light source emits non-pulsed irradiation or non-stroboscopic irradiation. In certain embodiments, a continuous light source provides a substantially constant emitted light intensity. For example, a method may include irradiating a sample in a flowing stream with a continuous light source, wherein the continuous light source provides a change of 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less of the emitted light intensity during the irradiated time interval, and including a case where the emitted light intensity changes 0.000001% or less during the irradiated time interval. The intensity of the light output can be measured using any convenient scheme, including but not limited to a scanning slit profilometer, a charge-coupled device (CCD, such as an intensified charge-coupled device (ICCD), a positioning sensor, a power sensor (e.g., a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, and other types of photodetectors.

[0048] In other embodiments, the method includes irradiating the sample in the flow stream with a pulsed light source, such as where light is emitted at predetermined time intervals, each time interval having a predetermined irradiation duration (i.e., pulse width). In certain embodiments, the method includes irradiating particles with periodic flashes in each interrogation region of the flow stream using a pulsed light source. For example, the frequency of each light pulse can be 0.0001 kHz or greater, such as 0.0005 kHz or greater, such as 0.001 kHz or greater, such as 0.005 kHz or greater, such as 0.01 kHz or greater, such as 0.05 kHz or greater, such as 0.1 kHz or greater, such as 0.5 kHz or greater, such as 1 kHz or greater, such as 2.5 kHz or greater, such as 5 kHz or greater, such as 10 kHz or greater, such as 25 kHz or greater, such as 50 kHz or greater and including 100 kHz or greater. In some instances, the frequency range of pulsed irradiation by the light source is 0.00001 kHz to 1000 kHz, such as 0.00005 kHz to 900 kHz, such as 0.0001 kHz to 800 kHz, such as 0.0005 kHz to 700 kHz, such as 0.001 kHz to 600 kHz, such as 0.005 kHz to 500 kHz, such as 0.01 kHz to 400 kHz, such as 0.05 kHz to 300 kHz, such as 0.1 kHz to 200 kHz and including 1 kHz to 100 kHz. The duration of light irradiation of each light pulse (i.e., pulse width) may vary and may be 0.000001 ms or longer, such as 0.000005 ms or longer, such as 0.00001 ms or longer, such as 0.00005 ms or longer, such as 0.0001 ms or longer, such as 0.0005 ms or longer, such as 0.001 ms or longer, such as 0.005 ms or longer, such as 0.01 ms or longer, such as 0.05 ms or longer, such as 0.1 ms or longer, such as 0.5 ms or longer, such as 1 ms or longer, such as 2 ms or longer, such as 3 ms or longer, such as 4 ms or longer, such as 5 ms or longer, such as 10 ms or longer, such as 25 ms or longer, such as 50 ms or longer, such as 100 ms or longer and including 500 ms or longer. ms or more.For example, the duration of light irradiation may be in the range of 0.000001 ms to 1000 ms, such as 0.000005 ms to 950 ms, such as 0.00001 ms to 900 ms, such as 0.00005 ms to 850 ms, such as 0.0001 ms to 800 ms, such as 0.0005 ms to 750 ms, such as 0.001 ms to 700 ms, such as 0.005 ms to 650 ms, such as 0.01 ms to 600 ms, such as 0.05 ms to 550 ms, such as 0.1 ms to 500 ms, such as 0.5 ms to 450 ms, such as 1 ms to 400 ms, such as 5 ms to 350 ms and including 10 ms to 300 ms.

[0049] The flow stream can be irradiated with any convenient light source, and can include lasers and non-laser light sources (e.g., light emitting diodes). In certain embodiments, the method includes irradiating the particles with a laser (such as a pulsed or continuous wave laser). For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible light diode laser, and a near infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some instances, 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 instances, the subject system includes a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In yet other examples, the lasers of interest include metal vapor lasers such as helium cadmium (HeCd) lasers, helium mercury (HeHg) lasers, helium selenium (HeSe) lasers, helium silver (HeAg) lasers, strontium lasers, neon copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof. In still other examples, the subject system includes a solid-state laser such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:YCa4O(BO3)3 laser, an Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, an ytterbium trioxide laser, or a cerium-doped laser, and combinations thereof.

[0050] In some embodiments, the light source outputs a specific wavelength, such as 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm and including 400 nm to 800 nm. In certain embodiments, the continuous wave light source emits light with a wavelength of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm or 850 nm.

[0051] The flow stream can be irradiated by the light source from any suitable distance, such as at a distance of 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more and including at a distance of 100 mm or more. In addition, the irradiation of the flow stream can be at any suitable angle, such as at an angle in the range of 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75° and including 30° to 60°, such as at an angle of 90°.

[0052] In some embodiments, the method further includes adjusting the light from the sample before detecting the light. For example, the light from the sample source can pass through one or more lenses, reflectors, pinholes, slits, gratings, light refractors and any combination thereof. In some instances, the collected light passes through one or more focusing lenses, such as to reduce the profile of the light. In other instances, the emitted light from the sample passes through one or more collimators to reduce beam divergence.

[0053] In some embodiments, the method includes irradiating the sample with two or more frequency shifted light. As described above, a beam generator component having a laser and an acousto-optic device can be used to frequency shift the laser light. In these embodiments, the method includes irradiating the acousto-optic device with a laser. Depending on the desired wavelength of the light generated in the output laser beam (for example, for irradiating a sample in a flowing stream), the laser can have a specific wavelength from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm and including changes from 400 nm to 800 nm. The acousto-optic device can be irradiated with one or more lasers, such as 2 or more lasers, such as 3 or more lasers, such as 4 or more lasers, such as 5 or more lasers and including 10 or more lasers. The laser can include a combination of any type of laser. For example, in some embodiments, the method includes illuminating the acousto-optic device with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0054] In the case of using more than one laser, the acousto-optic device can be irradiated by the laser simultaneously or sequentially or a combination thereof. For example, the acousto-optic device can be irradiated with each laser simultaneously. In other embodiments, the acousto-optic device is irradiated sequentially with each laser. In the case of using more than one laser to sequentially irradiate the acousto-optic device, the time for each laser to irradiate the acousto-optic device can be independently 0.001 microseconds or longer, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microsecond or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 30 microseconds or longer and including 60 microseconds or longer. For example, the method may include irradiating the acousto-optic device with a laser for a duration in the range of 0.001 microseconds to 100 microseconds, such as 0.01 microseconds to 75 microseconds, such as 0.1 microseconds to 50 microseconds, such as 1 microsecond to 25 microseconds and including 5 microseconds to 10 microseconds. In embodiments where the acousto-optic device is illuminated sequentially with two or more lasers, the duration of illumination of the acousto-optic device by each laser may be the same or different.

[0055] In an embodiment, the method includes applying a radio frequency drive signal to the acousto-optic device to generate an angularly deflected laser beam. Two or more radio frequency drive signals may be applied to the acousto-optic device to generate an output laser beam having a desired number of angularly deflected laser beams, such as 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals and including 100 or more radio frequency drive signals.

[0056] The angle-deflected laser beams generated by the RF drive signal each have an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal with an amplitude sufficient to generate an angle-deflected laser beam with a desired intensity. In some instances, each applied RF drive signal independently has an amplitude of about 0.001 V to about 500 V, such as about 0.005 V to about 400 V, such as about 0.01 V to about 300 V, such as about 0.05 V to about 200 V, such as about 0.1 V to about 100 V, such as about 0.5 V to about 75 V, such as about 1 V to 50 V, such as about 2 V to 40 V, such as 3 V to about 30 V and including about 5 V to about 25 V. In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz and including about 5 MHz to about 50 MHz.

[0057] In these embodiments, the angle-deflected laser beams in the output laser beam are spatially separated. Depending on the applied RF drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams can be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more and including 5000 μm or more. In some embodiments, the angle-deflected laser beams overlap, such as overlapping with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (such as the overlap of beam spots) can be an overlap of 0.001 μm or greater, such as an overlap of 0.005 μm or greater, such as an overlap of 0.01 μm or greater, such as an overlap of 0.05 μm or greater, such as an overlap of 0.1 μm or greater, such as an overlap of 0.5 μm or greater, such as an overlap of 1 μm or greater, such as an overlap of 5 μm or greater, such as an overlap of 10 μm or greater and including an overlap of 100 μm or greater.

[0058] In some instances, the flow stream is illuminated with multiple beams of frequency-shifted light, and cells in the flow stream are imaged by fluorescence imaging using radiofrequency tagged emission (FIRE) to generate frequency-encoded images, such as in Diebold et al., Nature Photonics, vol. 7(10); 806-810. (2013) and those described in U.S. Patent 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; and U.S. Patent Publication Nos. 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894 (the disclosures of which are incorporated herein by reference).

[0059] Light from the illuminated flow stream is detected using a light detection system having a photodetector. The photodetector can be any convenient light detection protocol, including but not limited to a photoelectric sensor or photodetector, such as an active pixel sensor (APS), an avalanche photodiode (APD), a quadrant photodiode, an image sensor, a charge coupled device (CCD), an intensified charge coupled device (ICCD), a light emitting diode, a photon counter, a calorimeter, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor or photodiode, and combinations thereof, and other photodetectors. In certain embodiments, the photodetector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area of ​​0.01 cm per region. 2 Up to 10 cm 2 , such as 0.05 cm 2 Up to 9 cm 2 , such as 0.1 cm 2 Up to 8 cm 2 , such as 0.5 cm 2 Up to 7 cm 2 And includes 1 cm 2 Up to 5 cm 2 Photomultiplier tube.

[0060] Light can be measured by photodetector at one or more wavelengths, such as at 2 or more wavelengths, such as at 5 or more different wavelengths, such as at 10 or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and including measuring the light of particles from the flow stream at 400 or more different wavelengths. In some embodiments, fluorescence from sample is detected in one or more wavelength ranges of 200 nm-1200 nm by light detection system. In some instances, method includes detecting fluorescence from sample in a certain wavelength range, such as 200 nm to 1200 nm, such as 300 nm to 1100 nm, such as 400 nm to 1000 nm, such as 500 nm to 900 nm and including 600 nm to 800 nm. In other instances, method includes detecting fluorescence at one or more specific wavelengths. For example, fluorescence can 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, and any combination thereof, depending on the number of different fluorescence detectors in the subject light detection system. In certain embodiments, the method includes detecting a wavelength of light corresponding to a peak wavelength of fluorescence of certain fluorochromes present in the sample.

[0061] Light can be measured continuously or at discrete intervals. In some instances, the detector of interest is configured to measure light continuously. In other instances, the detector of interest is configured to measure at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and include every 1000 milliseconds or some other intervals to measure light. During each discrete time interval, one or more measurements from the light across the flow stream can be carried out, such as 2 times or more, such as 3 times or more, such as 5 times or more and include 10 times or more. In certain embodiments, the light from the flow stream is measured 2 times or more by a photodetector, wherein data are averaged in certain instances.

[0062] Each photodetector can be positioned at any suitable distance from the flow stream, as long as the available light signal is detectable. For example, the detector in the subject system can be positioned 1 mm or more from the flow stream, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, such as 150 mm or more, such as 250 mm or more, and including 500 mm or more from the flow stream. The detector can also be positioned at any angle to the flow stream. For example, the detector can be at an angle of 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including 30° to 60° relative to the vertical axis of the flow stream. In some instances, one or more detectors are positioned at 30° to 60° relative to the vertical axis of the flow stream.

[0063] In an embodiment, the light detection system is configured to detect forward scattered light, side scattered light, emitted light, transmitted light, or a combination thereof. In certain embodiments, the light signal from the irradiated flow stream can be detected by one or more detectors configured as forward scattering detectors. In these embodiments, the forward scattering detector is positioned on the opposite side of the flow stream from the light source, and is positioned to collect and detect forward propagating (e.g., scattered) light. In some embodiments, light from irradiated samples in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, light from irradiated samples in the flow stream is detected in one or more scattered light detector channels. In some embodiments, light from irradiated bubbles in the flow stream is detected in the side scattering detector channel. In some embodiments, light from irradiated bubbles in the flow stream is detected in the forward scattering detector channel.

[0064] In an embodiment, a data signal is generated in response to light detected from particles irradiated in a flow stream. In some embodiments, light from particles in a sample is detected in two or more photodetector channels, such as 4 or more, such as 8 or more, such as 12 or more, such as 16 or more, such as 20 or more, such as 24 or more, such as 28 or more, such as 32 or more, such as 36 or more, such as 40 or more, such as 44 or more, such as 48 or more, such as 52 or more, such as 56 or more, such as 60 or more and including in 64 or more photodetector channels.

[0065] Figure 1Depicted is the introduction of bubbles into a sample line and detection of bubbles in a flow stream according to certain embodiments. In an initial state 101, a calibrated sample line 101a having a predetermined volume is inserted into a sample source 101b (e.g., a test tube) containing a sample composition having particles. The sample is absorbed by the sample line and propagates through the flow stream toward an interrogation region 101c in a flow cell 101d and is illuminated with a light source 101e (e.g., a laser). In a primed state 102, the sample composition propagates through the flow stream to the interrogation region and is illuminated by the light source, generating a data signal in response to light detected from the illuminated particles in channels such as a fluorescence photodetector channel and a light scattering photodetector channel. In a state 103, a bubble 101f is introduced, wherein the sample line 101a is retracted from the sample source 101b, causing air to be sucked into the sample line 101a. At least one data signal (e.g., an electronic data signal) can be generated and transmitted to a processor to indicate that a bubble such as a temporary air gap has been introduced into the flow stream. During the bubble flow state 104, after a predetermined period of time, the sample line 101a is reinserted into the sample source 101b and the bubble 101f propagates toward the interrogation region 101c. The bubble 101f is illuminated by the laser 101e after reaching the interrogation region 101c, and light from the illuminated bubble 101f is detected in the bubble detection state 105. The bubble may be detected by light scattering, or the presence of the bubble in the flow stream may be determined based on a time gap in the data signal generated in the fluorescence detector channel.

[0066] In some embodiments, a data signal is generated in response to light detected from the illuminated flow stream before the bubble is introduced into the flow stream. In some instances, light is detected from the illuminated flow stream and a data signal is generated in one or more photodetector channels for 0.0001 seconds or more, such as 0.0005 seconds or more, such as 0.001 seconds or more, such as 0.005 seconds or more, such as 0.01 seconds or more, such as 0.05 seconds or more, such as 0.1 seconds or more, such as 0.5 seconds or more, such as 1 second or more, such as 2 seconds or more, such as 3 seconds or more, such as 4 seconds or more, such as 10 seconds or more, such as 15 seconds or more, such as 30 seconds or more, and includes generating a data signal from light detected from the illuminated flow stream for 60 seconds or more before the bubble is introduced into the flow stream.

[0067] In some embodiments, a data signal is generated in response to light detected from an illuminated particle of a sample upstream of a bubble in a flow stream. In some instances, a data signal is generated in response to light from a particle upstream of a bubble within 0.0001 seconds or more, such as within 0.0005 seconds or more, such as within 0.001 seconds or more, such as within 0.005 seconds or more, such as within 0.01 seconds or more, such as within 0.05 seconds or more, such as within 0.1 seconds or more, such as within 0.5 seconds or more, such as within 1 second or more, such as within 2 seconds or more, such as within 3 seconds or more, such as within 4 seconds or more, such as within 10 seconds or more, such as within 15 seconds or more, such as within 30 seconds or more, such as within 60 seconds or more, such as within 5 minutes or more, such as within 10 minutes or more, and includes generating a data signal in response to light from a particle upstream of a bubble for 15 minutes or more.

[0068] Before the bubble is introduced into the flow stream, light may be continuously detected from the irradiated flow stream, such as, for example, for 0.0001 second or more, such as for 0.0005 seconds or more, such as for 0.001 second or more, such as for 0.005 seconds or more, such as for 0.01 second or more, such as for 0.05 seconds or more, such as for 0.1 second or more, such as for 0.5 seconds or more, such as for 1 second or more, such as for 2 seconds or more, such as for 3 seconds or more, such as for 4 seconds or more, such as for 10 seconds or more, such as for 15 seconds or more, such as for 30 seconds or more, and including detecting light from the irradiated flow stream for 60 seconds or more before the bubble is introduced into the flow stream.

[0069] Light can also be continuously detected from irradiated particles upstream of the introduced bubbles, such as within 0.0001 seconds or longer, such as within 0.0005 seconds or longer, such as within 0.001 seconds or longer, such as within 0.005 seconds or longer, such as within 0.01 seconds or longer, such as within 0.05 seconds or longer, such as within 0.1 seconds or longer, such as within 0.5 seconds or longer, such as within 1 second or longer, such as within 2 seconds or longer, such as within 3 seconds or longer, such as within 4 seconds or longer, such as within 10 seconds or longer, such as within 15 seconds or longer, such as within 30 seconds or longer, such as within 60 seconds or longer, such as within 5 minutes or longer, such as within 10 minutes or longer, and including continuously detecting light from irradiated particles upstream of the introduced bubbles for 15 minutes or longer.

[0070] In some embodiments, light is also continuously detected from irradiated particles downstream of the introduced bubbles, such as within 0.0001 seconds or longer, such as within 0.0005 seconds or longer, such as within 0.001 seconds or longer, such as within 0.005 seconds or longer, such as within 0.01 seconds or longer, such as within 0.05 seconds or longer, such as within 0.1 seconds or longer, such as within 0.5 seconds or longer, such as within 1 second or longer, such as within 2 seconds or longer, such as within 3 seconds or longer, such as within 4 seconds or longer, such as within 10 seconds or longer, such as within 15 seconds or longer, such as within 30 seconds or longer, such as within 60 seconds or longer, such as within 5 minutes or longer, such as within 10 minutes or longer, and including continuously detecting light from irradiated particles downstream of the introduced bubbles for 15 minutes or longer.

[0071] In an embodiment, when a bubble passes through the query field, the bubble is illuminated by a light source. The light from the bubble is detected by a photodetector of a light detection system. In some embodiments, scattered light, such as side scattered light, is detected from a passing bubble. When light from an irradiated bubble is detected, one or more data signals can be generated, indicating that there are bubbles in the flow stream, such as where data signals are generated in one or more light scattering photodetector channels, such as 2 or more light scattering photodetector channels, such as 4 or more, such as 8 or more, such as 12 or more, such as 16 or more, such as 32 or more and included in 64 or more light scattering photodetector channels. In certain embodiments, the method includes determining that there are bubbles in the flow stream in which light is not detected in one or more fluorescence photodetector channels. In these embodiments, the curve of the fluorescence intensity data signal relative to time includes a time gap in the generated data signal, where the bubble passes through the query area and no fluorescence is detected by the light detection system. In certain embodiments, the absence of the data signal generated in the fluorescence photodetector channel is related to the presence of the data signal generated in the light scattering photodetector channel for the irradiated bubble in the flow stream.

[0072] In some embodiments, a data signal is generated in response to light detected from an illuminated particle of a sample downstream of a bubble in a flow stream. In some instances, a data signal is generated in response to light from a particle downstream of a bubble within 0.0001 seconds or more, such as within 0.0005 seconds or more, such as within 0.001 seconds or more, such as within 0.005 seconds or more, such as within 0.01 seconds or more, such as within 0.05 seconds or more, such as within 0.1 seconds or more, such as within 0.5 seconds or more, such as within 1 second or more, such as within 2 seconds or more, such as within 3 seconds or more, such as within 4 seconds or more, such as within 10 seconds or more, such as within 15 seconds or more, such as within 30 seconds or more, such as within 60 seconds or more, such as within 5 minutes or more, such as within 10 minutes or more, and includes generating a data signal in response to light from a particle downstream of a bubble for 15 minutes or more.

[0073] In some embodiments, an absolute count of particles in a sample is determined using data signals generated when a bubble is introduced into a flow stream, data signals generated when a bubble is detected in the flow stream, and data signals generated in response to light detected from particles irradiated between the time when the bubble is introduced into the flow stream and the time when the bubble is detected. In some embodiments, the method includes counting the number of particles in the sample between the time when the bubble is introduced into the flow stream and the time when the bubble is detected. The number of particles can be counted based on the number of fluorescent data signals generated between the time when the bubble is introduced into the flow stream and the time when the bubble is detected. In some embodiments, the number of particles counted during the time period is divided by a predetermined volume, such as the volume of a previously calibrated sample line. In other embodiments, the volume used to calculate the absolute count of particles is a determined volume of the sample line containing particles irradiated between the time when the bubble is introduced into the flow stream and the time when the bubble is detected.

[0074] In certain embodiments, the method includes generating a data signal intensity graph of the generated data signal as a function of time. In some instances, the data signal intensity graph includes a data signal generated in a fluorescence detector channel, wherein each marker (e.g., a dot) on the graph corresponds to fluorescence detected from an irradiated particle. In some instances, the data signal intensity graph includes a visualization (e.g., a line demarcation) indicating the time of the data signal generated when a bubble is introduced into the flow stream. In some instances, the data signal intensity graph includes a lack of data signal when a bubble is detected. For example, the data signal intensity graph may include only data signals generated in a fluorescence detector channel, and no fluorescence is detected when the bubble passes through a query area. In these embodiments, there are time gaps in the data signal intensity graph to indicate that the bubble has reached the query area.

[0075] Figure 2A flow chart for determining an absolute count of a sample in a flow stream according to certain embodiments is depicted. In step 201, particles propagating through the flow stream are illuminated with a light source (such as a laser). In step 202, bubbles are introduced into the flow stream. In some instances, the bubbles are introduced by retracting a sample line from a source of the sample composition to create an air gap in the flow stream. A data signal 202a is generated in response to the introduction of the bubble into the flow stream. In step 203, a light detection system with a photodetector is used to detect light from the flow stream illuminated by the light source (e.g., fluorescence from particles in the sample). Data signals 203a in one or more photodetector channels are generated in response to light detected from the illuminated particles in the flow stream. When the bubble reaches the query area, in step 204, the bubble is illuminated by the light source and detected by the light detection system. A data signal 204a indicating that a bubble has been detected is generated. In some instances, the presence of a bubble is determined based on the absence of a data signal generated in a fluorescence photodetector channel. In other examples, the data signal generated in the light scattering photodetector channel is generated in response to light scattering detected from illumination of a bubble in the flow stream. In step 205, the data signal generated from illuminating particles in the flow stream and the data signal generated when the bubble is introduced into the flow stream and detected by the light detection system can be plotted on a data signal intensity graph. In other examples, in step 206, the absolute count of particles in the sample is directly calculated using data signals 202a, 203a, and 204a, such as by using the count of data signal 203a generated from the time of the data signal when the bubble was introduced (202a) into the flow stream and the time of the data signal when the bubble was detected (204a). In some embodiments, the absolute count is calculated using the generated data signal intensity graph (step 205a).

[0076] Figure 3 A data signal intensity graph 300 is depicted according to certain embodiments, which is generated by light detected from irradiated particles and bubbles introduced into the flow stream. Each point 301 on the graph 300 corresponds to a data signal generated for light detected from each particle. The intensity of the data signal is plotted on the y-axis, and the time when the data signal is generated is plotted on the x-axis. The time 302 when the bubble is introduced into the flow stream is shown in the figure. For example, time 302 can be based on an electronic data signal generated when the sample line is retracted from the sample source. The time when the bubble is detected is shown by a time gap 303 in the data signal in the data signal intensity graph. The absolute count of particles in the sample can be calculated based on the volume of the sample line between time 302 and time 303 (i.e., the time between the time when the bubble is introduced into the flow stream and the time when the bubble is detected). In some instances, the volume is the volume of the sample line previously calibrated.

[0077] A system configured to determine an absolute count of particles in a sample Aspects of the present disclosure also include systems (e.g., flow cytometers) having a light source, a light detection system, and a sample line configured to introduce bubbles into a flow stream for practicing the methods described herein. A system according to certain embodiments includes: a flow cell configured to propagate a sample having particles in a flow stream; a sample line in fluid communication with a source of the sample, the sample line configured to introduce bubbles into the flow stream; a light source for illuminating the flow stream; and a light detection system having a photodetector for detecting light from the illuminated flow stream.

[0078] In embodiments, the system is configured to introduce bubbles into the flow stream. In certain instances, the system is configured to produce air gaps in the flow stream. In some embodiments, the system is configured with a gas source for introducing gas-containing bubbles into the flow stream. In some instances, the flow stream includes a gas input so that gas can be introduced into the flow stream. For example, bubbles can be introduced into the flow stream by gas input from an inert gas source, such as wherein nitrogen, helium or argon can be input into the flow stream during sample suction into the flow stream. The gas source can be configured to introduce any volume of gas into the flow stream according to the volume of the sample and the sample line, such as 0.001 μL or more, such as 0.005 μL or more, such as 0.01 μL or more, such as 0.05 μL or more, such as 0.1 μL or more, such as 0.5 μL or more, such as 1 μL or more, such as 2 μL or more, such as 3 μL or more, such as 4 μL or more, such as 5 μL or more and including 10 μL or more.

[0079] In certain embodiments, bubble is generated by producing air gap in flowing stream.In some instances, system comprises sample line displacement component, and described sample line displacement component is configured to retract sample line from sample source and produce air gap in flowing stream.In some embodiments, sample line displacement component makes sample retract from sample source, and when retracting sample line from sample source, sample line inhales air.Once air gap has been produced in flowing stream, sample line displacement component is configured to reinsert sample line back into sample source.Sample line displacement component can be any convenient protocol for retracting sample line from sample source, such as wherein displacing with manual, mechanical or motor-driven displacement device.In certain embodiments, sample line retracts with motor-driven actuator, such as motor-driven lead screw assembly, gear actuation device, servomotor, brushless electric motor, brushed DC motor, micro-stepping drive motor, high resolution stepping motor and other types of motors.

[0080] In some embodiments, the sample line displacement component is configured to retract the sample line from the sample source by a distance sufficient to draw air into the sample line and create an air gap in the flow stream. In some embodiments, the sample line displacement component is configured to retract the sample line from the source of the sample by 0.00001 mm or more, such as 0.00005 mm or more, such as 0.0001 mm or more, such as 0.0005 mm or more, such as 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more and including 50 mm or more. The sample line shifting component can be configured to retract the sample line from the sample source for 0.0001 seconds or longer, such as for 0.0005 seconds or longer, such as for 0.01 seconds or longer, such as for 0.05 seconds or longer, such as for 0.1 seconds or longer, such as for 0.5 seconds or longer, such as for 1 second or longer, such as for 2 seconds or longer, such as for 3 seconds or longer, such as for 4 seconds or longer, such as for 5 seconds or longer, such as for 10 seconds or longer, such as 15 seconds or longer and including for 30 seconds or longer.

[0081] In embodiments, system includes a light source for illuminating a flow stream. In embodiments, the light source can be any suitable broadband or narrowband light source. Depending on the components (e.g., cells, beads, non-cellular particles, etc.) in the sample, the light source can be configured to emit a wavelength of 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm and including a wavelength that changes in the range of 400 nm to 800 nm. For example, the light source can include a broadband light source that emits light with a wavelength of 200 nm to 900 nm. In other examples, the light source includes a narrowband light source that emits a wavelength in the range of 200 nm to 900 nm. For example, the light source can be a narrowband LED (1 nm–25 nm) that emits light with a wavelength in the range between 200 nm to 900 nm.

[0082] In some embodiments, the light source is a laser. The laser of interest may include a pulsed laser or a continuous wave laser. For example, the laser may be 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; a dye laser such as a stilbene laser, a coumarin laser, or a rhodamine laser; 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 ( solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium trioxide lasers or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL) or frequency doping or tripling of any of the above mentioned lasers.

[0083] In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a light emitting diode, such as a broadband LED with a continuous spectrum, a superluminescent diode, a semiconductor light emitting diode, a wide spectrum LED white light source, an integrated multi-LED. In some examples, the non-laser light source is a stabilized fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0084] In some embodiments, the light source is a beam generator configured to generate two or more beams of frequency-shifted light. In some instances, the beam generator includes a laser, a radio frequency generator configured to apply a radio frequency drive signal to the acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous wave laser. For example, the laser in the beam generator of interest can be 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; a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser; a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium ( solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium trioxide lasers or cerium-doped lasers and combinations thereof.

[0085] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied RF drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0086] In an embodiment, the controller is configured to apply an RF drive signal to the acousto-optic device to produce a desired number of angularly deflected laser beams in the output laser beam, such as being configured to apply 3 or more RF drive signals, such as 4 or more RF drive signals, such as 5 or more RF drive signals, such as 6 or more RF drive signals, such as 7 or more RF drive signals, such as 8 or more RF drive signals, such as 9 or more RF drive signals, such as 10 or more RF drive signals, such as 15 or more RF drive signals, such as 25 or more RF drive signals, such as 50 or more RF drive signals, and including being configured to apply 100 or more RF drive signals.

[0087] In some examples, to produce an intensity profile of an angularly deflected laser beam in the output laser beam, the controller is configured to apply a radio frequency drive signal having an amplitude varying, such as from about 0.001 V to about 500 V, such as from about 0.005 V to about 400 V, such as from about 0.01 V to about 300 V, such as from about 0.05 V to about 200 V, such as from about 0.1 V to about 100 V, such as from about 0.5 V to about 75 V, such as from about 1 V to 50 V, such as from about 2 V to 40 V, such as from 3 V to about 30 V and including from about 5 V to about 25 V. In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz and including about 5 MHz to about 50 MHz.

[0088] In certain embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam having a desired intensity profile. For example, the memory may include instructions for generating two or more angularly deflected laser beams having the same intensity, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, and including the memory may include instructions for generating 100 or more angularly deflected laser beams having the same intensity. In other embodiments, instructions for generating two or more angularly deflected laser beams having different intensities may be included, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, and including the memory may include instructions for generating 100 or more angularly deflected laser beams having different intensities.

[0089] In certain embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an increasing intensity from the edge to the center of the output laser beam along the horizontal axis. In these examples, the intensity of the angularly deflected laser beam at the center of the output beam may be in the range of 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and includes about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an increasing intensity from the edge to the center of the output laser beam along the horizontal axis. In these examples, the intensity of the angularly deflected laser beam at the edge of the output beam may be in the range of 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and includes about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In still other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile having a Gaussian distribution along a horizontal axis. In still other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0090] In an embodiment, the beam generator of interest can be configured to generate spatially separated angularly deflected laser beams in the output laser beam. Depending on the applied RF drive signal and the desired irradiation profile of the output laser beam, the angularly deflected laser beams can be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more and including 5000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams in the output laser beam, and the angularly deflected laser beams overlap, such as overlapping with adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (such as the overlap of beam spots) can be an overlap of 0.001 μm or greater, such as an overlap of 0.005 μm or greater, such as an overlap of 0.01 μm or greater, such as an overlap of 0.05 μm or greater, such as an overlap of 0.1 μm or greater, such as an overlap of 0.5 μm or greater, such as an overlap of 1 μm or greater, such as an overlap of 5 μm or greater, such as an overlap of 10 μm or greater and including an overlap of 100 μm or greater.

[0091] In some instances, a beam generator configured to generate two or more frequency-shifted beams of light includes a laser excitation module as described in U.S. Patent Nos. 9,423,353; 9,784,661 and 10,006,852 and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803 (the disclosures of which are incorporated herein by reference).

[0092] The light source may be positioned at any suitable distance from the flow stream, such as at a distance of 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and including at a distance of 100 mm or more. In addition, the light source may illuminate the flow stream at any suitable angle (e.g., relative to the vertical axis of the flow stream), for example, at an angle in the range of 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including 30° to 60°, for example, at an angle of 90°.

[0093] The light source can be configured to irradiate the sample continuously or at discrete intervals. In some instances, the system includes a light source configured to irradiate the sample continuously, such as a continuous wave laser with a flow stream continuously irradiating the query point in the flow cytometer. In other instances, the system of interest includes a light source, and the light source is configured to irradiate the sample at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or some other intervals. When the light source is configured to irradiate the sample at discrete intervals, the system can include one or more other parts to provide the intermittent irradiation of the sample with the light source. For example, the subject system in these embodiments can include one or more laser beam choppers, manually or computer-controlled beam blockers, for blocking the sample and exposing the sample to the light source.

[0094] The system includes a light detection system having a photodetector for detecting light from the irradiated flow stream. The photodetector can be any convenient light detection protocol, including but not limited to a photoelectric sensor or photodetector, such as an active pixel sensor (APS), an avalanche photodiode (APD), a quadrant photodiode, an image sensor, a charge coupled device (CCD), an intensified charge coupled device (ICCD), a light emitting diode, a photon counter, a bolometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor or photodiode, and combinations thereof, and other photodetectors. In certain embodiments, the photodetector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area in the range of 0.01 cm per region. 2 Up to 10 cm 2 , such as 0.05 cm 2 Up to 9 cm 2 , such as 0.1 cm 2 Up to 8 cm 2 , such as 0.5 cm 2 Up to 7 cm 2 And includes 1 cm 2 Up to 5 cm 2 Photomultiplier tube.

[0095] In some embodiments, the light detection system of interest includes multiple photodetectors. In some instances, the light detection system includes multiple solid-state detectors, such as photodiodes. In some instances, the light detection system includes a photodetector array, such as an array of photodiodes. In these embodiments, the photodetector array may include 4 or more photodetectors, such as 10 or more photodetectors, such as 25 or more photodetectors, such as 50 or more photodetectors, such as 100 or more photodetectors, such as 250 or more photodetectors, such as 500 or more photodetectors, such as 750 or more photodetectors, and include 1000 or more photodetectors. For example, the detector may be a photodiode array with 4 or more photodiodes, such as 10 or more photodiodes, such as 25 or more photodiodes, such as 50 or more photodiodes, such as 100 or more photodiodes, such as 250 or more photodiodes, such as 500 or more photodiodes, such as 750 or more photodiodes, and include 1000 or more photodiodes.

[0096] The photodetectors can be arranged in any geometric configuration as desired, where arrangements of interest include, but are not limited to, square configurations, rectangular configurations, trapezoidal configurations, triangular configurations, hexagonal configurations, heptagonal configurations, octagonal configurations, nonagonal configurations, decagonal configurations, dodecagonal configurations, circular configurations, elliptical configurations, and irregular patterned configurations. The photodetectors in the photodetector array can be oriented relative to one another (as referenced in the XZ plane) at an angle 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°, and including 45° to 90°. The photodetector array can be of any suitable shape, and can be a rectilinear shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). In some embodiments, the photodetector array has an active surface in a rectangular shape.

[0097] Each photodetector (e.g., photodiode) in the array may have a width ranging from 5 μm to 250 μm, such as 10 μm to 225 μm, such as 15 μm to 200 μm, such as 20 μm to 175 μm, such as 25 μm to 150 μm, such as 30 μm to 125 μm and including 50 μm to 100 μm, and a length ranging from 5 μm to 250 μm, such as 10 μm to 225 μm, such as 15 μm to 200 μm, such as 20 μm to 175 μm, such as 25 μm to 150 μm, such as 30 μm to 125 μm and including 50 μm to 100 μm, wherein the surface area of ​​each photodetector (e.g., photodiode) in the array is within 25 μm 2 Up to 10000 μm 2 , such as 50 μm 2 Up to 9000 μm 2 , such as 75 μm 2 Up to 8000 μm 2 , such as 100 μm 2 Up to 7000 μm 2 , such as 150 μm 2 Up to 6000 μm 2 And including 200 μm 2 Up to 5000 μm 2 within the range.

[0098] The size of the photodetector array may vary depending on the amount and intensity of light, the number of photodetectors, and the desired sensitivity, and may have a length in the range of 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm, and including 5 mm to 25 mm. The width of the photodetector array may also vary, ranging from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm, and including 5 mm to 25 mm. Thus, the active surface of the photodetector array may be within 0.1 mm 2 Up to 10000 mm 2 , such as 0.5 mm 2 Up to 5000 mm 2 , such as 1 mm2 Up to 1000 mm 2 , such as 5 mm 2 Up to 500 mm 2 And includes 10 mm 2 Up to 100 mm 2 within the range.

[0099] Light can be measured by photodetector at one or more wavelengths, such as at 2 or more wavelengths, such as at 5 or more different wavelengths, such as at 10 or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and including measuring light from particles in the flow stream at 400 or more different wavelengths. In some embodiments, fluorescence from sample is detected in one or more wavelength ranges of 200 nm-1200 nm by light detection system. In some instances, method includes detecting fluorescence from sample in a certain wavelength range, such as 200 nm to 1200 nm, such as 300 nm to 1100 nm, such as 400 nm to 1000 nm, such as 500 nm to 900 nm and including 600 nm to 800 nm. In other instances, method includes detecting fluorescence at one or more specific wavelengths. For example, fluorescence can 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, and any combination thereof, depending on the number of different fluorescence detectors in the subject light detection system.

[0100] Light can be measured continuously or at discrete intervals. In some instances, the detector of interest is configured to measure light continuously. In other instances, the detector of interest is configured to measure at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and include every 1000 milliseconds or some other intervals to measure light. During each discrete time interval, one or more measurements from the light across the flow stream can be carried out, such as 2 times or more, such as 3 times or more, such as 5 times or more and include 10 times or more. In certain embodiments, the light from the flow stream is measured 2 times or more by a photodetector, wherein data are averaged in certain instances.

[0101] Each photodetector can be positioned at any suitable distance from the flow stream, as long as the available light signal is detectable. For example, the detector in the subject system can be positioned 1 mm or more from the flow stream, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, such as 150 mm or more, such as 250 mm or more, and including 500 mm or more from the flow stream. The detector can also be positioned at any angle to the flow stream. For example, the detector can be at an angle of 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including 30° to 60° relative to the vertical axis of the flow stream. In some instances, one or more detectors are positioned at 30° to 60° relative to the vertical axis of the flow stream.

[0102] In an embodiment, the light detection system is configured to detect forward scattered light, side scattered light, emitted light, transmitted light, or a combination thereof. In certain embodiments, the light signal from the irradiated flow stream can be detected by one or more detectors configured as forward scattering detectors. In these embodiments, the forward scattering detector is positioned on the opposite side of the flow stream from the light source, and is positioned to collect and detect forward propagating (e.g., scattered) light. In some embodiments, light from irradiated samples in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, light from irradiated samples in the flow stream is detected in one or more scattered light detector channels. In some embodiments, light from irradiated bubbles in the flow stream is detected in the side scattering detector channel. In some embodiments, light from irradiated bubbles in the flow stream is detected in the forward scattering detector channel.

[0103] In some embodiments, the system includes an optical collection system for collecting light from the flow stream and directing it to the light detection system. The optical collection system can be physically coupled to the light detection system, such as with an adhesive, co-molded together, or integrated into the light detection system. In certain embodiments, the optical collection system and the light detection system are integrated into a single unit. In other embodiments, the optical collection system is coupled to the light detection system through a connector, such as by hook and loop fasteners, magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, Velcro, non-permanent adhesives, or combinations thereof.

[0104] In other embodiments, the light detection system and the optical collection system are in optical communication, but not in physical contact. For example, the optical collection system can be positioned 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 10 mm or more, such as 25 mm or more, such as 50 mm or more, and including 100 mm or more from the light detection system.

[0105] In some embodiments, the optical collection system includes an optical fiber. For example, the optical collection system can be a fiber optic optical relay bundle, and the light from the flow stream is transmitted to the first set of linear variable filters of the optical detection system through the fiber optic optical relay bundle. Any fiber optic optical relay system can be used to transmit light, wherein in some embodiments, suitable fiber optic optical relay systems include, but are not limited to, fiber optic optical relay systems such as those described in U.S. Patent No. 6,809,804 (the disclosure of which is incorporated herein by reference).

[0106] In other embodiments, the optical collection system is a free space optical relay system. The phrase "free space optical relay" is used herein in its conventional sense to refer to the light propagation of light from a sample through free space to a light detection system using a configuration of one or more optical components. In certain embodiments, the free space optical relay system includes a housing having a proximal end and a distal end, the proximal end being coupled to a light detection module. The free space relay system can include any combination of different optical components, such as one or more of a lens, a reflector, a slit, a pinhole, a wavelength separator, or a combination thereof. For example, in some embodiments, the free space optical relay system of interest includes one or more focusing lenses. In other embodiments, the subject free space optical relay system includes one or more reflectors. In yet other embodiments, the free space optical relay system includes a collimating lens. In certain embodiments, suitable free-space optical relay systems for propagating light from the sample to the first set of linear variable filters of the optical detection system include, but are not limited to, optical relay systems such as those described in U.S. Pat. Nos. 7,643,142; 7,728,974; and 8,223,445 (the disclosures of which are incorporated herein by reference).

[0107] In some embodiments, the system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate a data signal when a gas bubble is introduced into the flow stream. In some examples, the memory includes instructions for generating a data signal when a volume of gas is input or drawn into the sample line. In other instances, the memory includes instructions for generating a data signal at a predetermined time after the bubble is introduced into the flow stream, such as 0.001 µs or longer, such as 0.005 µs or longer, such as 0.01 µs or longer, such as 0.05 µs or longer, such as 0.1 µs or longer, such as 0.5 µs or longer, such as 1 µs or longer, such as 2 µs or longer, such as 3 µs or longer, such as 5 µs or longer, such as 10 µs or longer, such as 100 µs or longer, such as 500 µs or longer, and including 1000 µs or longer after the bubble is introduced into the flow stream.

[0108] In certain embodiments, the memory includes instructions for generating data signals when bubbles are introduced into the flow stream by retracting the sample line from the sample source. In some instances, the memory includes instructions for generating data signals when the sample line is retracted from the sample source. In other embodiments, the memory includes instructions for generating data signals in a predetermined time period after the sample line is retracted from the sample source, such as 0.001 µs seconds or longer after the sample line is retracted from the sample source, such as 0.005 µs or longer, such as 0.01 µs or longer, such as 0.05 µs or longer, such as 0.1 µs or longer, such as 0.5 µs or longer, such as 1 µs or longer, such as 2 µs or longer, such as 3 µs or longer, such as 5 µs or longer, such as 10 µs or longer, such as 100 µs or longer, such as 500 µs or longer, and included in 1000 µs or longer after the sample line is retracted from the sample source. In other embodiments, the memory includes instructions for generating a data signal when the sample line is reinserted into the sample source (i.e., when sample aspiration into the sample line is reestablished). In still other embodiments, the memory includes instructions for generating a data signal at a predetermined time after the sample line is reinserted into the sample source, such as 0.001 µs or more, such as 0.005 µs or more, such as 0.01 µs or more, such as 0.05 µs or more, such as 0.1 µs or more, such as 0.5 µs or more, such as 1 µs or more, such as 2 µs or more, such as 3 µs or more, such as 5 µs or more, such as 10 µs or more, such as 100 µs or more, such as 500 µs or more, and including 1000 µs or more after the sample line is reinserted into the sample source.

[0109] In some instances, the memory includes instructions for generating an electronic data signal when a bubble is introduced into the flow stream. In other instances, the memory includes instructions for generating an optical data signal when a bubble is introduced into the flow stream. In some instances, the system includes one or more photodetectors positioned to detect light from a sample line into which the bubble is introduced. For example, the system may include a light source such as an LED or a laser for illuminating the flow stream into which the bubble is introduced, and one or more photodetectors (e.g., a scattered light detector) for detecting light from the bubble introduced into the flow stream. In certain embodiments, the light detection system described above may include one or more photodetector channels for detecting light from bubbles introduced into the flow stream.

[0110] In the case where the system is configured to introduce bubbles by retracting the sample line from the sample source, the photodetector and light source for detecting when the bubble is introduced into the flow stream can be adjacent to the end of the sample line or downstream of the end of the sample line. In some embodiments, the photodetector and light source for detecting the introduction of bubbles into the flow stream can be positioned 0.001 mm or more downstream from the end of the sample line, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 4 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and including 50 mm or more downstream from the end of the sample line. In some embodiments, the memory includes instructions for transmitting an optical data signal to a processor for calculating a particle count of the sample.

[0111] In some embodiments, the system includes a memory having instructions for generating a data signal in response to light detected from particles illuminated in a flow stream. In some embodiments, the memory includes instructions for generating a data signal in response to light from particles in a sample in two or more photodetector channels, such as 4 or more, such as 8 or more, such as 12 or more, such as 16 or more, such as 20 or more, such as 24 or more, such as 28 or more, such as 32 or more, such as 36 or more, such as 40 or more, such as 44 or more, such as 48 or more, such as 52 or more, such as 56 or more, such as 60 or more and included in 64 or more photodetector channels.

[0112] In some embodiments, the memory includes instructions for generating a data signal in response to light detected from the illuminated flow stream prior to introducing the gas bubble into the flow stream. In some instances, the memory includes instructions for generating a data signal from light detected from an irradiated flow stream in one or more photodetector channels for 0.0001 seconds or longer, such as for 0.0005 seconds or longer, such as for 0.001 seconds or longer, such as for 0.005 seconds or longer, such as for 0.01 seconds or longer, such as for 0.05 seconds or longer, such as for 0.1 seconds or longer, such as for 0.5 seconds or longer, such as for 1 second or longer, such as for 2 seconds or longer, such as for 3 seconds or longer, such as for 4 seconds or longer, such as for 10 seconds or longer, such as for 15 seconds or longer, such as for 30 seconds or longer, and includes instructions for generating a data signal from light detected from an irradiated flow stream for 60 seconds or longer before a bubble is introduced into the flow stream.

[0113] In some embodiments, the memory includes instructions for generating a data signal in response to light detected from an illuminated particle of the sample upstream of the bubble in the flow stream. In some instances, the memory includes generating a data signal in response to light from particles upstream of the bubble within 0.0001 seconds or more, such as within 0.0005 seconds or more, such as within 0.001 seconds or more, such as within 0.005 seconds or more, such as within 0.01 seconds or more, such as within 0.05 seconds or more, such as within 0.1 seconds or more, such as within 0.5 seconds or more, such as within 1 second or more, such as within 2 seconds or more, such as within 3 seconds or more, such as within 4 seconds or more, such as within 10 seconds or more, such as within 15 seconds or more, such as within 30 seconds or more, such as within 60 seconds or more, such as within 5 minutes or more, such as within 10 minutes or more and includes instructions for generating a data signal in response to light from particles upstream of the bubble within 15 minutes or more.

[0114] In some instances, the memory includes instructions for continuously detecting light from the irradiated flow stream before introducing a bubble into the flow stream, such as for 0.0001 seconds or more, such as for 0.0005 seconds or more, such as for 0.001 seconds or more, such as for 0.005 seconds or more, such as for 0.01 seconds or more, such as for 0.05 seconds or more, such as for 0.1 seconds or more, such as for 0.5 seconds or more, such as for 1 second or more, such as for 2 seconds or more, such as for 3 seconds or more, such as for 4 seconds or more, such as for 10 seconds or more, such as for 15 seconds or more, such as for 30 seconds or more, and including where the memory includes instructions for detecting light from the irradiated flow stream for 60 seconds or more before introducing a bubble into the flow stream.

[0115] In some examples, the memory includes instructions for continuously detecting light from the irradiated particles upstream of the introduced bubble, such as for 0.0001 seconds or more, such as 0.0005 seconds or more, such as 0.001 seconds or more, such as 0.005 seconds or more, such as 0.01 seconds or more, such as 0.05 seconds or more, such as 0.1 seconds or more, such as 0.5 seconds or more, such as 1 second or more, such as 2 seconds or more. The memory includes instructions for continuously detecting light from irradiated particles upstream of introduced bubbles for a longer period of time, such as 3 seconds or longer, such as 4 seconds or longer, such as 10 seconds or longer, such as 15 seconds or longer, such as 30 seconds or longer, such as 60 seconds or longer, such as 5 minutes or longer, such as 10 minutes or longer, and includes instructions for continuously detecting light from irradiated particles upstream of introduced bubbles for 15 minutes or longer.

[0116] In some embodiments, the memory includes instructions for continuously detecting light from the irradiated particles downstream of the introduced bubble, such as for 0.0001 seconds or more, such as 0.0005 seconds or more, such as 0.001 seconds or more, such as 0.005 seconds or more, such as 0.01 seconds or more, such as 0.05 seconds or more, such as 0.1 seconds or more, such as 0.5 seconds or more, such as 1 second or more, such as 2 seconds. or longer, such as in 3 seconds or more, such as in 4 seconds or more, such as in 10 seconds or more, such as in 15 seconds or more, such as in 30 seconds or more, such as in 60 seconds or more, such as in 5 minutes or more, such as in 10 minutes or more, and includes instructions for continuously detecting light from irradiated particles downstream of the introduced bubbles for 15 minutes or more.

[0117] In some embodiments, the memory includes instructions for generating a data signal in response to light detected from an illuminated particle of the sample downstream of the bubble in the flow stream. In some instances, the memory includes instructions for generating a data signal in response to light from particles downstream of the bubble within 0.0001 seconds or more, such as within 0.0005 seconds or more, such as within 0.001 seconds or more, such as within 0.005 seconds or more, such as within 0.01 seconds or more, such as within 0.05 seconds or more, such as within 0.1 seconds or more, such as within 0.5 seconds or more, such as within 1 second or more, such as within 2 seconds or more, such as within 3 seconds or more, such as within 4 seconds or more, such as within 10 seconds or more, such as within 15 seconds or more, such as within 30 seconds or more, such as within 60 seconds or more, such as within 5 minutes or more, such as within 10 minutes or more, and includes instructions wherein the memory includes instructions for generating a data signal in response to light from particles downstream of the bubble within 15 minutes or more.

[0118] In some embodiments, the system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine an absolute count of particles in a sample using: a data signal generated when a bubble is introduced into a flow stream, a data signal generated when a bubble is detected in the flow stream, and a data signal generated in response to light detected from particles illuminated between a time when the bubble is introduced into the flow stream and a time when the bubble is detected. In some embodiments, the memory includes instructions for counting the number of particles in the sample between a time when the bubble is introduced into the flow stream and a time when the bubble is detected. The number of particles can be counted based on the number of fluorescent data signals generated between a time when the bubble is introduced into the flow stream and a time when the bubble is detected. In some embodiments, the memory includes instructions for counting the number of particles during the time period and dividing the count by a predetermined volume, such as a previously calibrated volume of a sample line. In other embodiments, the memory includes instructions for determining the volume of the sample line containing irradiated particles between the time when the bubble was introduced into the flow stream and the time when the bubble was detected. In some examples, the memory includes instructions for calculating an absolute count using the determined volume of the sample line containing irradiated particles between the time when the bubble was introduced into the flow stream and the time when the bubble was detected.

[0119] In some embodiments, the memory includes instructions for generating a data signal intensity map of the generated data signal over time. In some instances, the memory includes instructions for generating a data signal intensity map, the data signal intensity map including the data signal generated in the fluorescence detector channel. In some instances, the memory includes instructions for generating a data signal intensity map, wherein each marker (e.g., point) on the map corresponds to the fluorescence detected from the irradiated particles. In some instances, the memory includes instructions for generating a data signal intensity map, the data signal intensity map including a visualization (e.g., line demarcation) of the data signal generated when a bubble is introduced into the flow stream. In some instances, the data signal intensity map includes the absence of the data signal when the bubble is detected. For example, the data signal intensity map may include only the data signal generated in the fluorescence detector channel, and when the bubble passes through the query area, no fluorescence is detected. In these embodiments, there is a time gap in the data signal intensity map to indicate that the bubble has reached the query area.

[0120] In certain embodiments, the system further includes a flow cell configured to propagate the sample in the flow stream. Any convenient flow cell that propagates the fluid sample to the sample interrogation area can be used, wherein in some embodiments, the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminating in a flat surface having an orifice transverse to the longitudinal axis. The length of the proximal cylindrical portion (as measured along the longitudinal axis) can vary in the range of 1 mm to 15 mm, such as 1.5 mm to 12.5 mm, such as 2 mm to 10 mm, such as 3 mm to 9 mm and including 4 mm to 8 mm. The length of the distal truncated conical portion (as measured along the longitudinal axis) can also vary in the range of 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm and including 4 mm to 7 mm. In some embodiments, the diameter of the flow cell nozzle chamber can vary in the range of 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm and including 4 mm to 7 mm.

[0121] In some examples, the flow cell does not include a cylindrical portion, and the entire flow cell chamber is truncated conical. In these embodiments, the length of the truncated conical chamber (as measured along the longitudinal axis transverse to the nozzle orifice) can be in the range of 1 mm to 15 mm, such as 1.5 mm to 12.5 mm, such as 2 mm to 10 mm, such as 3 mm to 9 mm and including 4 mm to 8 mm. The diameter of the proximal portion of the truncated conical chamber can be in the range of 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm and including 4 mm to 7 mm.

[0122] In some embodiments, the sample flow stream emanates from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the flow cell orifice can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curved cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., a parabolic bottom portion coupled to a planar top portion). In certain embodiments, the flow cell of interest has a circular orifice. The size of the nozzle orifice can vary, ranging in some embodiments from 1 μm to 20000 μm, such as 2 μm to 17500 μm, such as 5 μm to 15000 μm, such as 10 μm to 12500 μm, such as 15 μm to 10000 μm, such as 25 μm to 7500 μm, such as 50 μm to 5000 μm, such as 75 μm to 1000 μm, such as 100 μm to 750 μm and including 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0123] In some embodiments, the flow cell comprises a sample injection port configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable sample flow to the flow cell chamber. Depending on the desired characteristics of the flow stream, the rate of sample delivered to the flow cell chamber through the sample injection port can be 1 μL / min or greater, such as 2 μL / min or greater, such as 3 μL / min or greater, such as 5 μL / min or greater, such as 10 μL / min or greater, such as 15 μL / min or greater, such as 25 μL / min or greater, such as 50 μL / min or greater and including 100 μL / min or greater, wherein in some instances, the rate of sample delivered to the flow cell chamber through the sample injection port is 1 μL / sec or greater, such as 2 μL / sec or greater, such as 3 μL / sec or greater, such as 5 μL / sec or greater, such as 10 μL / sec or greater, such as 15 μL / sec or greater, such as 25 μL / sec or greater, such as 50 μL / sec or greater and including 100 μL / sec or greater.

[0124] The sample injection port can be an orifice positioned in the wall of the inner chamber, or can be a conduit positioned at the proximal end of the inner chamber. In the case where the sample injection port is an orifice positioned in the wall of the inner chamber, the sample injection port orifice can be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical, etc.), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary according to the shape, and in certain instances, has a 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 and including 1.25 mm to 1.75 mm, such as an opening of 1.5 mm.

[0125] In some instances, the sample injection port is a conduit positioned at the proximal end of the flow cell chamber. For example, the sample injection port can be a conduit positioned so that the orifice of the sample injection port is consistent with the orifice of the flow cell. In the case where the sample injection port is a conduit positioned to be consistent with the orifice of the flow cell, the cross-sectional shape of the sample injection tube can be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). The orifice of the conduit can vary according to the shape, and in some instances, has a 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 and including 1.25 mm to 1.75 mm, such as an opening of 1.5 mm. The shape of the tip of the sample injection port can be the same or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a bevel tip with an angle ranging from 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7° and including a bevel angle of 5°.

[0126] In some embodiments, the flow cell also includes a sheath liquid injection port configured to provide a sheath liquid to the flow cell. In an embodiment, the sheath liquid injection system is configured to provide a sheath liquid flow to the flow cell inner chamber, for example, in combination with the sample to produce a laminar flow of the sheath liquid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the rate of the sheath liquid delivered to the flow cell chamber can be 25 μL / sec or greater, such as 50 μL / sec or greater, such as 75 μL / sec or greater, such as 100 μL / sec or greater, such as 250 μL / sec or greater, such as 500 μL / sec or greater, such as 750 μL / sec or greater, such as 1000 μL / sec or greater and including 2500 μL / sec or greater.

[0127] In some embodiments, the sheath injection port is an orifice positioned in the wall of the inner chamber. The sheath injection port orifice can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to, straight cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curved cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., a parabolic bottom portion coupled to a planar top portion). The size of the sample injection port orifice can vary depending on the shape, and in some instances, has an opening ranging from 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 and including 1.25 mm to 1.75 mm, such as 1.5 mm.

[0128] In some embodiments, the system further includes a pump in fluid communication with the flow cell to propagate the flow stream through the flow cell. Any convenient fluid pump scheme can be used to control the flow of the flow stream through the flow cell. In some instances, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the subject system is configured to transport the fluid through the flow cell at a rate suitable for detecting light from a sample in the flow stream. In some instances, the rate of sample flow in the flow cell is 1 μL / min (microliter / minute) or greater, such as 2 μL / min or greater, such as 3 μL / min or greater, such as 5 μL / min or greater, such as 10 μL / min or greater, such as 25 μL / min or greater, such as 50 μL / min or greater, such as 75 μL / min or greater, such as 100 μL / min or greater, such as 250 μL / min or greater, such as 500 μL / min or greater, such as 750 μL / min or greater and including 1000 μL / min or greater. For example, the system may include a pump configured to flow the sample through the flow cell at a rate of 1 μL / min to 500 μL / min, such as 1 μL / min to 250 μL / min, such as 1 μL / min to 100 μL / min, such as 2 μL / min to 90 μL / min, such as 3 μL / min to 80 μL / min, such as 4 μL / min to 70 μL / min, such as 5 μL / min to 60 μL / min and including 10 μL / min to 50 μL / min. In certain embodiments, the flow rate of the flowing stream is 5 μL / min to 6 μL / min.

[0129] In some embodiments, a light detection system having a plurality of photodetectors as described above is part of or positioned in a particle analyzer, such as a particle sorter. In some embodiments, the subject system is a flow cytometry system that includes a photodiode and amplifier component as part of a light detection system for detecting light emitted by a sample in a flowing stream. Suitable flow cytometry systems may include, but are not limited to, 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 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. Oct 2004;30(5):502-11; Alison et al. J Pathol. Pathol, 2010 Dec;222(4):335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference. In certain instances, the flow cytometry system of interest includes the BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TMFlow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell Sorters, 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 TM S6 cell sorter, etc.

[0130] In some embodiments, the subject system is a flow cytometry system, such 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, those described in 095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766; the disclosures of which are incorporated herein by reference in their entireties.

[0131] In some embodiments, the subject system is a particle sorting system configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493 (the disclosure of which is incorporated herein by reference). In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781 (the disclosure of which is incorporated herein by reference). In some embodiments, the subject system includes a particle sorting module with a deflection plate, such as described in U.S. Patent Publication No. 2017 / 0299493 filed on March 28, 2017 (the disclosure of which is incorporated herein by reference).

[0132] In certain embodiments, the flow cytometry system of the present invention is configured to image particles in a flow stream by fluorescence imaging using radio frequency labeled emission (FIRE), such as described in Diebold et al., Nature Photonics, Vol. 7 (10); 806-810. (2013) and those described in U.S. Patent 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; and U.S. Patent Publication Nos. 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894, the disclosures of which are incorporated herein by reference.

[0133] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4A ) can be used to analyze and characterize particles with or without physical sorting of the particles into collection vessels. Figure 4A A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, particle analysis system 401 is a flow system. Figure 4A The particle analysis system 401 shown in the figure can be configured to perform, in whole or in part, methods such as those described herein. The particle analysis system 401 includes a fluidics system 402. The fluidics system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, wherein particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0134] Particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as each particle passes through one or more detection stations along a common sample path. Detection station 408 generally refers to monitoring area 407 of the common sample path. In some embodiments, detection can include detecting light or one or more other properties of particles 403 as they pass through monitoring area 407. Figure 4A , one detection station 408 is shown having one monitoring area 407. Some embodiments of particle analysis system 401 may include multiple detection stations. In addition, some detection stations may monitor more than one area.

[0135] Each signal is assigned a signal value to form a data point for each particle. As described above, this data can be referred to as event data. The data point can be a multidimensional data point, including the value of the corresponding property measured for the particle. The detection system 404 is configured to collect a series of such data points in a first time interval.

[0136] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, an amplitude control circuit, and / or a frequency control circuit. The control system shown may be operatively associated with the fluidics system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system 406 may additionally compare the experimental signal frequency with the calculated signal frequency or a predetermined signal frequency.

[0137] Figure 4B A system 400 for flow cytometry is shown according to an illustrative embodiment of the invention. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long pass ("LP") filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0138] The excitation lasers 115a-c emit light in the form of laser beams. Figure 4B In an exemplary system of , the wavelengths of the laser beams emitted from the excitation lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits the 488 nm light and reflects the 633 nm light. The beam splitter 445b transmits UV light (light with a wavelength in the range of 10 to 400 nm) and reflects the 488 nm and 633 nm light.

[0139] The laser beam is then directed to a focusing lens 420 which focuses the beam onto the portion of the fluid stream within a flow cell 425 where the particles of the sample are located. The flow cell is the portion of the fluidics system that directs the particles in the stream (usually one particle at a time) to the focused laser beam for interrogation. The flow cell may include a flow cell in a benchtop cytometer or a nozzle tip in an air flow cytometer.

[0140] Light from the laser beam interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various wavelengths depending on the characteristics of the particles, such as the size of the particles, the internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or in the particles. The fluorescence emission and the diffracted, refracted, reflected, and scattered light can be routed to one or more of the forward scatter detector 430, the side scatter detector 435, and one or more fluorescence detectors 460a-460f through one or more of the beam splitters 445a-445g, the bandpass filters 450a-450e, the longpass filters 455a-455b, and the fluorescence collection lens 440.

[0141] Fluorescence collection lens 440 collects light emitted from the particle-laser beam interaction and routes the light toward one or more beam splitters and filters. Bandpass filters (such as bandpass filters 450a-450e) allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Therefore, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit light with a wavelength equal to or shorter than a specified wavelength. Long-pass filters (such as long-pass filters 455a-455b) transmit light with a wavelength equal to or longer than a specified wavelength. For example, long-pass filter 455a (which is a 670 nm long-pass filter) transmits light equal to or longer than 670 nm. Filters are typically selected to optimize the specificity of the detector for a particular fluorescent dye. The filter can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0142] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be characterized by filter properties such as short pass and long pass. For example, beam splitter 445g is a 620 SP beam splitter, which means that beam splitter 445g transmits light with a wavelength of 620 nm or shorter and reflects light with a wavelength greater than 620 nm in different directions. In one embodiment, beam splitters 445a-445g can include optical mirrors, such as dichroic mirrors.

[0143] The forward scatter detector 430 is positioned slightly off the axis of the direct beam passing through the flow cell and is configured to detect diffracted light, i.e., excitation light that travels through or around particles primarily in the forward direction. The intensity of the 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 435 is configured to detect refracted and reflected light from the surface and internal structure of the particle, and tends to increase with the increase in the complexity of the particle structure. Fluorescence emission from fluorescent molecules associated with the particle may be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detector may include a photomultiplier tube. The signals detected at the forward scatter detector 430, the side scatter detector 435, and the fluorescence detector may be converted into electronic signals (voltages) by the detectors. The data may provide information about the sample.

[0144] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to Figure 4B The flow cytometer depicted in the embodiment of the present invention may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in various different configurations.

[0145] In operation, the operation of the cytometer is controlled by the controller / processor 490, and the measurement data from the detector can be stored in the memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 190 is coupled to the detector to receive output signals therefrom, and can also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, etc. Input / output (I / O) capabilities 497 can also be provided in the system. The memory 495, the controller / processor 490, and the I / O 497 can be provided entirely as an integral part of the flow cytometer 410. In such embodiments, the display can also form part of the I / O capabilities 497 for presenting experimental data to the user of the cytometer 400. Alternatively, the memory 495 and the controller / processor 490 and some or all of the I / O capabilities can be part of one or more external devices such as a general purpose computer. In some embodiments, some or all of the memory 495 and the controller / processor 490 can communicate with the cytometer 410 wirelessly or by wire. Controller / processor 490 in conjunction with memory 495 and I / O 497 may be configured to perform various functions associated with the preparation and analysis of flow cytometric experiments.

[0146] Figure 4BThe system illustrated in 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 beam splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in a flow cytometer experiment will emit light in their own characteristic wavelength bands. The specific fluorescent markers used in the experiment and their associated fluorescence emission bands may be selected to generally coincide with the filter windows of the detectors. However, as more detectors are provided and more markers are utilized, a perfect correspondence between the filter windows and the fluorescence emission spectra is not possible. It is generally true that, although the peak of the emission spectrum of a particular fluorescent molecule may be within the filter window of a particular detector, some of the emission spectra of the marker will also overlap with the filter windows of one or more other detectors. This may be referred to as spillover. I / O 497 may be configured to receive data about a flow cytometer experiment having a set of fluorescent markers and multiple cell populations having multiple markers, each cell population having a subset of multiple markers. The I / O 497 may also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning markers to one or more markers, and cytometer configuration data. Flow cytometer experimental data such as marker spectral characteristics and flow cytometer configuration data may also be stored in the memory 495. The controller / processor 490 may be configured to evaluate one or more assignments of markers to markers.

[0147] Figure 5 A functional block diagram of one example of a particle analyzer control system, such as analysis controller 500, for analyzing and displaying biological events is shown. Analysis controller 500 may be configured to implement various processes for controlling the graphical display of biological events.

[0148] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0149] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 can include flow cytometry event data. The analysis controller 500 can be configured to provide a graphical display of a first plot including the biological event data to a display device 506. For example, the analysis controller 500 can be further configured to present an area of ​​interest as a gate around a population of biological event data displayed by the display device 506, overlaid on the first plot. In some embodiments, the gate can be a logical combination of one or more graphical areas of interest plotted on a single parameter histogram or a bivariate plot. In some embodiments, the display can be used to display particle parameters or saturated detector data.

[0150] Analysis controller 500 can be further configured to display biological event data on display device 506 inside the door, different from other events in the biological event data outside the door. For example, analysis controller 500 can be configured to display the biological event data contained inside the door in a different color than the biological event data outside the door. Display device 506 can be implemented as a monitor, tablet computer, smart phone, or other electronic device configured to present a graphical interface.

[0151] The analysis controller 500 can be configured to receive a door selection signal identifying a door from a first input device. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a door selection signal to the analysis controller 500, identifying a door to be displayed on the display device 506 or operated via the display device 506 (for example, by clicking on the desired door or in the desired door when the cursor is located on or in the desired door). In some embodiments, the first device can be implemented as a keyboard 508 or other device for providing input signals to the analysis controller 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, the input functions can each be considered an input device. For example, as Figure 5 As shown in , the mouse 510 may include a right mouse button and a left mouse button, each of which may generate a trigger event.

[0152] A triggering event may cause analysis controller 500 to change the manner in which data is displayed, which portions of the data are actually displayed on display device 506, and / or provide input for further processing, such as selecting a population of interest for particle sorting.

[0153] In some embodiments, the analysis controller 500 can be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a particular distribution of the bio-event data received by the analysis controller 500.

[0154] Analysis controller 500 may be connected to storage device 504. Storage device 504 may be configured to receive and store biological event data from analysis controller 500. Storage device 504 may also be configured to receive and store flow cytometry event data from analysis controller 500. Storage device 504 may further be configured to allow retrieval of biological event data, such as flow cytometry event data, by analysis controller 500.

[0155] The display device 506 can be configured to receive display data from the analysis controller 500. The display data can include plots of the biological event data and gates outlining portions of the plot. The display device 506 can be further configured to change the information presented based on input received from the analysis controller 500 and in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0156] In some embodiments, the analysis controller 500 can generate a user interface to receive exemplary events for sorting. For example, the user interface can include a control for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates can be provided before collecting event data for the sample, or based on an initial set of events for a portion of the sample.

[0157] Fig. 6A is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Fig. 6A As shown in FIG. 6 , a droplet formation transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, may include, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are arranged in a single file to span a monitoring area 611 (e.g., where the laser-stream intersects) illuminated by an illumination source 612 (e.g., a laser). The vibration of the droplet formation transducer 602 causes the moving fluid column 608 to break into a plurality of droplets 610, some of which contain particles 609.

[0158] In operation, a detection station 614 (e.g., an event detector) identifies when a particle of interest (or cell of interest) crosses the monitoring region 611. The detection station 614 feeds into a timing circuit 628, which in turn feeds into a flash charging circuit 630. At a droplet break point informed by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608 so that the droplet of interest carries a charge. The droplet of interest may include one or more particles or cells to be sorted. The charged droplets may then be sorted by activating a deflection plate (not shown) to deflect the charged droplets into a container such as a collection tube or a multi-well or micro-well sample plate, where the wells or micro-wells may be associated with droplets of particular interest. As shown in FIG. Fig. 6A As shown in , the droplets can be collected in a discharge container 638.

[0159] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as particles of interest pass through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039 (which is incorporated herein by reference in its entirety). The detection system 616 allows the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 can feed into an amplitude signal 620 and / or a phase signal 618, which in turn feeds (via an amplifier 622) into an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 in turn control the droplet formation transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 can be included in the control system.

[0160] In some embodiments, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store detected events and sorting decisions based on the detected events. The sorting decision can be included in the event data of the particle. In some embodiments, the detection system 616 and the detection station 614 can be implemented as a single detection unit or communicatively coupled so that event measurements can be collected by one of the detection system 616 or the detection station 614 and provided to a non-collecting element.

[0161] Figure 6B is a schematic diagram of a particle sorting system according to one embodiment presented herein. Figure 6B The particle sorting system 600 shown in FIG. 6 includes deflection plates 652 and 654. A charge can be applied via a stream of charge wires in the barbs. This creates a stream of droplets 610 containing particles 610 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The particles can be detected by a sorting electronics or other detection system (e.g., in FIG. 6 ). Figure 6BDeflector plates 652 and 654 can be independently controlled to attract or repel charged droplets, thereby directing the droplets toward a target collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown in FIG, deflector plates 652 and 654 can be controlled to direct particles along a first path 662 toward a container 674 or along a second path 668 toward a container 678. If the particle is not of interest (e.g., does not show scattering or illumination information within a specified sorting range), the deflector plates can allow the particle to continue along flow path 664. Such uncharged droplets can enter a waste container, such as via an aspirator 670.

[0162] Sorting electronics may be included to initiate the collection of measurements, receive the fluorescence signal of the particles, and determine how to adjust the deflection plates to cause sorting of the particles. Figure 6B An exemplary implementation of the embodiment shown in includes a BD FACSAria™ series flow cytometer commercially provided by Becton, Dickinson and Company (Franklin Lakes, NJ).

[0163] Computer controlled systems Aspects of the present disclosure further include a computer-controlled system, wherein the system further includes one or more computers for full or partial automation of the methods described herein. In some embodiments, the system includes a computer having a computer-readable storage medium, on which a computer program is stored, wherein when loaded onto the computer, the computer program includes instructions for calculating the absolute count of particles in a sample in a flow cytometer. In some embodiments, the computer program includes instructions for introducing bubbles into a flow stream that propagates a sample containing particles, instructions for irradiating the flow stream with a light source, algorithms for detecting light from irradiated particles with a photodetector, and instructions for detecting the presence of bubbles in the flow stream with a photodetector. In an embodiment, the computer program further includes instructions for determining the absolute count of particles in the sample based on a data signal generated in response to light detected from the irradiated particles, a data signal generated when the bubble is introduced into the flow stream, and a data signal generated in response to a detected bubble.

[0164] In some embodiments, the computer program includes instructions for introducing bubbles into the flow stream by retracting the sample line from the source of the sample to create an air gap in the flow stream. In some instances, the computer program includes instructions for operating a sample line displacement component that retracts the sample line from the sample source. In certain instances, the computer program includes instructions for retracting the sample line from the sample source and causing air or gas to be sucked into the sample line while the sample line is retracted. In some instances, the computer program includes instructions for generating a data signal when the sample line is retracted from the sample source. In some instances, the computer program includes instructions for generating a data signal at a predetermined time after the sample line is retracted from the sample source, such as 0.001 µs to 10 µs after the bubble is introduced into the flow stream.

[0165] In some embodiments, the computer program includes instructions for continuously detecting light from the irradiated particles before introducing the bubble into the flow stream, instructions for continuously detecting light from the irradiated particles upstream of the bubble in the flow stream, and instructions for detecting light from the flow stream as the bubble passes through the interrogation field. In some embodiments, the computer program includes instructions for generating a data signal in response to light detected from the irradiated particles before introducing the bubble into the flow stream, instructions for generating a data signal in response to light detected from the irradiated particles upstream of the bubble in the flow stream, and instructions for generating a data signal while the bubble is irradiated in the flow stream. In some embodiments, the computer program includes instructions for generating a data signal in response to light detected from the irradiated particles downstream of the bubble in the flow stream.

[0166] In an embodiment, the computer program includes instructions for calculating the absolute count of particles in the sample. In some embodiments, the computer program includes instructions for using a data signal generated in response to light detected from the irradiated particles between when the bubble is introduced into the flow stream and when the bubble is detected and a predetermined volume to calculate the absolute count of particles in the sample. In some instances, the computer program includes instructions for using the volume of a previously calibrated sample line to calculate the absolute count of particles. In some instances, the computer program includes instructions for generating a data signal intensity graph of the data signal from the irradiated sample that varies over time. In certain instances, the computer program includes instructions for generating a data signal intensity graph, the data signal intensity graph including at least the intensity of the data signal from the irradiated particles collected from the time when the bubble is introduced into the flow stream until the time when the bubble is detected. In certain instances, the computer program includes instructions for generating a data signal intensity graph, wherein the time when the bubble is introduced into the flow stream is plotted on the data signal intensity graph.

[0167] In an embodiment, the system includes an input module, a processing module, and an output module. The subject system may include both hardware components and software components, wherein the hardware components may take the form of one or more platforms, such as in the form of a server, so that functional elements, i.e., those elements of the system that perform specific tasks of the system (such as managing the input and output of information, processing information, etc.) can be implemented by executing software applications on and across one or more computer platforms represented by the system.

[0168] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that is capable of accessing a memory on which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device 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 it may be one of other processors that are available or will become available. The processor executes the operating system, and the operating system interfaces with firmware and hardware in a well-known manner, and helps the processor coordinate and execute the functions of various computer programs, which can be written in various programming languages ​​(such as Java, Perl, C++, other high-level or low-level languages ​​and combinations thereof), as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services according to known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align the light source with the flow stream based on the first light signal and the second light signal. In some embodiments, the processor includes analog electronics that provide feedback control (such as, for example, negative feedback control).

[0169] 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 a resident hard disk or tape), optical media (such as a read-write optical disk), flash memory devices, or other memory storage devices. The memory storage device may be any of a variety of known or future devices, including an optical drive, a tape drive, a removable hard drive, or a floppy disk drive. This type of memory storage device typically reads from and / or writes to a program storage medium (not shown), such as an optical disk, a tape, a removable hard disk, or a floppy disk, respectively. Any of these program storage media, or other media currently in use or that may be developed later, may be considered a computer program product. As will be understood, 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 system memory and / or program storage devices used in conjunction with memory storage devices.

[0170] In some embodiments, a computer program product is described that includes a computer usable medium having control logic (computer software program, including program code) stored therein. When executed by a processor of a computer, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementing a hardware state machine to perform the functions described herein will be apparent to those skilled in the relevant art.

[0171] The memory may be any suitable device in which the processor can store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including a magnetic disk or optical disk or tape or RAM, or any other suitable device, fixed or portable). The processor may include a general-purpose digital microprocessor that is appropriately programmed by a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely through a communication channel, or pre-saved in a computer program product (such as a memory or some other portable or fixed computer-readable storage medium) using any of those devices associated with the memory. For example, a magnetic disk or optical disk may carry the programming and may be read by a disk writer / reader. The system of the present invention also includes programming, for example in the form of a computer program product, an algorithm for practicing the method described above. The programming according to the present invention may be recorded on a computer-readable medium (e.g., any medium that can be directly read and accessed by a computer). Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROMs; portable flash drives; and hybrids of these categories, such as magnetic storage media / optical storage media.

[0172] The processor may also access a communication channel to communicate with a user at a remote location. A remote location means that the user does not interact directly with the system, but rather relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smart phone).

[0173] 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 (e.g., radio frequency identification (RFID), Zigbee communication protocol, WiFi, 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).

[0174] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to allow data communications between the subject system and other external devices, such as computer terminals configured for similar supplemental data communications (e.g., in a doctor's office or in a hospital environment).

[0175] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as a computer terminal and / or network, a communication-enabled mobile phone, a personal digital assistant, or any other communication device that a user may use in conjunction with it.

[0176] In one embodiment, the communication interface is configured to provide a connection for data transmission using Internet Protocol (IP) through a cellular telephone network, a short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0177] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, for example using a common standard such as 802.11 or Bluetooth® RF protocol or IrDA infrared protocol. The server device can be another portable device such as a smart phone, a personal digital assistant (PDA), or a laptop; 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.

[0178] In some embodiments, the communication interface is configured to automatically or semi-automatically transfer data stored in the subject system, such as data stored in the optional data storage unit, with a network or server device using one or more of the above-described communication protocols and / or mechanisms.

[0179] The output controller may include a controller for any of various known display devices for presenting information to a user, whether the user is a person or a machine, whether local or remote. If one of the display devices provides visual information, the information can generally be logically and / or physically organized as an array of image elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and 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 implemented using a network or other type of remote communication in an alternative embodiment. The output manager may also provide information generated by the processing module to a user at a remote location, such as through the Internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML or XML documents, emails or other files, or data in other forms. The data may include an Internet URL address so that a user can retrieve additional SQL, HTML, XML or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or a type to be developed in the future, although they will generally belong to a class of computers commonly referred to as servers. However, they may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cables or other communication systems (networked or otherwise) including wireless systems. They may be co-located, or they may be physically separate. Various operating systems may be employed on any computer platform, possibly depending on the type and / or configuration of the computer platform selected. Suitable operating systems include Windows, iOS, Oracle Solaris, Linux, IBM i, Unix, etc.

[0180] Figure 7 Depicted is the overall architecture of an exemplary computing device 700 in accordance with certain embodiments. Figure 7 The overall architecture of the computing device 700 depicted in FIG. 7 includes an arrangement of computer hardware and software components. The computing device 700 may include Figure 7700. The computing device 700 may be a computer system or a computing device ...

[0181] The memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) that the processing unit 710 executes to implement one or more embodiments. The memory 770 typically includes RAM, ROM, and / or other persistent computer-readable media, auxiliary computer-readable media, or non-transitory computer-readable media. The memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. The memory 770 may further include computer program instructions and other information for implementing various aspects of the present disclosure.

[0182] Non-transitory computer readable storage medium Aspects of the present disclosure further include a non-transitory computer-readable storage medium with instructions for practicing the subject method. A computer-readable storage medium can be used on one or more computers for full or partial automation of the system for practicing the method described herein. In certain embodiments, the instructions according to the method described herein can be encoded on a computer-readable medium in the form of "programming", wherein the term "computer-readable medium" as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state disks, and network attached storage (NAS), whether such devices are inside or outside the computer. Files containing information can be "stored" on a computer-readable medium, wherein "storage" refers to recording information so that the information can be accessed and retrieved by a computer later. The computer-implemented method described herein can be performed using programming, which can be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Python, Java, JavaScript, C, C#, C++, Go, R, Swift, PHP, and any of many others.

[0183] In an embodiment, a non-transitory computer-readable storage medium includes an algorithm for introducing bubbles into a flow stream propagating a sample containing particles, an algorithm for illuminating the flow stream with a light source, an algorithm for detecting light from the illuminated particles with a photodetector, and an algorithm for detecting the presence of bubbles in the flow stream with the photodetector. In an embodiment, the non-transitory computer-readable storage medium further includes an algorithm for determining an absolute count of particles in the sample based on a data signal generated in response to light detected from the illuminated particles, a data signal generated when bubbles are introduced into the flow stream, and a data signal generated in response to detected bubbles.

[0184] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for introducing bubbles into the flow stream by retracting the sample line from the source of the sample to create an air gap in the flow stream. In some instances, the non-transitory computer-readable storage medium includes an algorithm for operating a sample line shifting component that retracts the sample line from the sample source. In certain instances, the non-transitory computer-readable storage medium includes an algorithm for retracting the sample line from the sample source and sucking air or gas into the sample line while retracting the sample line. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal when the sample line is retracted from the sample source. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal at a predetermined time after the sample line is retracted from the sample source, such as 0.001 µs to 10 µs after the bubble is introduced into the flow stream.

[0185] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for continuously detecting light from the irradiated particles before introducing the bubble into the flow stream, an algorithm for continuously detecting light from the irradiated particles upstream of the bubble in the flow stream, and an algorithm for detecting light from the flow stream as the bubble passes through the query field. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal in response to light detected from the irradiated particles before introducing the bubble into the flow stream, an algorithm for generating a data signal in response to light detected from the irradiated particles upstream of the bubble in the flow stream, and an algorithm for generating a data signal when the bubble is irradiated in the flow stream. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal in response to light detected from the irradiated particles downstream of the bubble in the flow stream.

[0186] In an embodiment, a non-transitory computer-readable storage medium includes an algorithm for calculating an absolute count of particles in a sample. In some embodiments, a non-transitory computer-readable storage medium includes an algorithm for calculating an absolute count of particles in a sample using a data signal generated in response to light detected from an irradiated particle between when a bubble is introduced into the flow stream and when a bubble is detected and a predetermined volume. In some instances, a non-transitory computer-readable storage medium includes an algorithm for calculating an absolute count of particles using the volume of a previously calibrated sample line. In some instances, a non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity map of a data signal from an irradiated sample that varies over time. In some instances, a non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity map, the data signal intensity map including at least the intensity of the data signal from the irradiated particle collected from the time when the bubble is introduced into the flow stream until the time when the bubble is detected. In some instances, a non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity map, wherein the time when the bubble is introduced into the flow stream is plotted on the data signal intensity map.

[0187] The non-transitory computer-readable storage medium can be used on one or more computer systems with a display and an operator input device. The operator input device can be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access a memory on which instructions for executing the steps of the subject method are stored. The processing module can 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 can be a commercially available processor, or it can be one of other processors that are available or will become available. The processor executes the operating system, and the operating system interfaces with firmware and hardware in a well-known manner, and helps the processor coordinate and execute the functions of various computer programs, which can be written in various programming languages ​​(such as those mentioned above), other high-level or low-level languages, and combinations thereof, as known in the art. The operating system usually cooperates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services according to known technologies.

[0188] Reagent test kit Aspects of the present disclosure further include a kit, wherein the kit includes one or more components of the system described herein. In some embodiments, the kit includes multiple photodetectors and programming for the subject system, such as in the form of a computer readable medium (e.g., a flash drive, USB memory, optical disc, DVD, Blu-ray disc, etc.) or instructions for downloading programming from an Internet web protocol or cloud server. The kit can also include optical adjustment components, such as lenses, reflectors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof.

[0189] Test kit may further include instructions for practicing the subject method. These instructions may be present in the subject test kit in various forms, wherein one or more forms may be present in the test kit. A form in which these instructions may exist is to be present on a suitable medium or substrate as printed information, such as one or more sheets of paper with information printed thereon, in the packaging of the test kit, in a package insert, or the like. Another form of these instructions is a computer-readable medium having information recorded thereon, such as a disk, a compact disk (CD), a portable flash drive, etc. Another form of these instructions that may exist is a website address, which may be used to access information at a site removed via the Internet.

[0190] utility The subject methods, systems, and computer systems find use in various applications where it is desired to optimize the photodetectors of a light detection system. The subject methods and systems also find use in light detection systems having multiple photodetectors that are used to analyze and sort particle components in a sample (such as a biological sample) in a fluid medium. The present disclosure also finds use in flow cytometry, where it is desired to provide a flow cytometer that has improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In an embodiment, the present disclosure reduces the need for user input or manual adjustments during sample analysis with a flow cytometer. In certain embodiments, the subject methods and systems provide a fully automated solution so that little, if any, manual input is required for adjustments to the flow cytometer during use.

[0191] Notwithstanding the appended claims, the present disclosure is defined by the following clauses: 1. A method for determining an absolute count of particles in a sample in a flow cytometer, the method comprising: introducing bubbles into a flow stream that propagates a sample containing particles; illuminating the flow stream with a light source; detecting light from the irradiated particles with a photodetector; detecting the presence of the bubbles in the flow stream with the photodetector; and The absolute count of the particles in the sample is determined based on: a data signal generated in response to light detected from the illuminated particle; a data signal generated when the bubble is introduced into the flow stream; and A data signal is generated in response to the detected bubble.

[0192] 2. The method of clause 1, wherein introducing the gas bubbles into the flow stream comprises retracting a sample line from a source of the sample to create an air gap in the flow stream.

[0193] 3. The method of clause 2, wherein the method includes generating a data signal when the sample line is retracted from the sample source.

[0194] 4. The method of clause 2, wherein the method includes generating a data signal a predetermined time after retracting the sample line from the sample source.

[0195] 5. The method of clause 4, wherein the data signal is generated 0.001 µs to 10 µs after the sample line is retracted from the sample source.

[0196] 6. A method according to any one of clauses 1 to 5, wherein the data signal generated when the gas bubble is introduced into the flow stream comprises an electronic data signal.

[0197] 7. A method according to any one of clauses 1 to 5, wherein the data signal generated when the gas bubble is introduced into the flow stream comprises an optical data signal.

[0198] 8. A method according to any one of clauses 1 to 7, wherein the flow stream is illuminated continuously by the light source.

[0199] 9. The method of clause 8, wherein the light source comprises a laser.

[0200] 10. A method according to any one of clauses 8 to 9, wherein the flow stream is illuminated continuously with the light source having a wavelength of 200 nm to 800 nm.

[0201] 11. A method according to any one of clauses 1 to 10, wherein the method comprises: generating a data signal in response to light detected from the illuminated particles prior to introducing the bubble into the flow stream; generating a data signal in response to light detected from an illuminated particle upstream of the bubble in the flow stream; and A data signal is generated when the bubble is illuminated in the flow stream.

[0202] 12. The method of clause 11, wherein the method further comprises generating a data signal in response to light detected from an illuminated particle downstream of the bubble in the flow stream.

[0203] 13. A method according to clause 11, wherein the method comprises: continuously detecting light from the illuminated particles prior to introducing the gas bubble into the flow stream; continuously detecting light from illuminated particles upstream of the bubble in the flow stream; and Light is detected from the flow stream as the bubble passes through an interrogation field.

[0204] 14. A method according to any one of clauses 1 to 13, wherein the detected light comprises fluorescence.

[0205] 15. A method according to any one of clauses 1 to 13, wherein the detected light comprises scattered light.

[0206] 16. A method according to any one of clauses 1 to 15, wherein the presence of the bubbles in the flow stream is determined based on detecting scattered light from the illuminated bubbles.

[0207] 17. A method according to any one of clauses 1 to 16, wherein the method comprises calculating the absolute count of particles in the sample using: a data signal generated in response to light detected from the illuminated particle between when the bubble is introduced into the flow stream and when the bubble is detected; and Predetermined volume.

[0208] 18. A method according to any one of clauses 1 to 17, wherein the method further comprises generating a data signal strength graph of the generated data signal over time.

[0209] 19. The method of clause 18, wherein the method further comprises plotting the time when the bubble is introduced into the flow stream onto the data signal strength graph.

[0210] 20. A system comprising: a flow cell configured to propagate a sample comprising particles in a flow stream; a sample line in fluid communication with a source of the sample and the flow cell, wherein the sample line is configured to introduce bubbles into the flow stream; a light source configured to illuminate the sample in the flow stream; a light detection system comprising a photodetector for detecting light from the illuminated flow stream; and a processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to calculate the absolute count of the particles in the sample based on: a data signal generated in response to light detected from the illuminated particle; a data signal generated when the bubble is introduced into the flow stream; and A data signal is generated in response to the detected bubble.

[0211] 21. The system of clause 20, wherein the system comprises a sample line displacement component configured to retract the sample line from the sample source and introduce an air gap into the flow stream.

[0212] 22. A system according to any of clauses 20 to 21, wherein the memory comprises instructions for generating a data signal when the bubble is introduced into the flow stream.

[0213] 23. The system of clause 22, wherein the memory includes instructions for generating an electronic data signal when the sample line is retracted from the sample source.

[0214] 24. The system of clause 22, wherein the memory includes instructions for generating an electronic data signal at a predetermined time after the sample line is retracted from the sample source.

[0215] 25. The system of clause 24, wherein the electronic data signal is generated 0.001 µs to 10 µs after the sample line is retracted from the sample source.

[0216] 26. A system according to any of clauses 20 to 25, wherein the light source comprises a laser.

[0217] 27. The system of clause 26, wherein the laser is a continuous wave laser.

[0218] 28. A system according to any of clauses 26 to 27, wherein the light source is configured to continuously illuminate the flow stream with light having a wavelength of 200 nm to 800 nm.

[0219] 29. A system according to any one of clauses 20 to 28, wherein the light detection system is configured to: generating a data signal in response to light detected from the illuminated particles prior to introducing the bubble into the flow stream; generating a data signal in response to light detected from an illuminated particle upstream of the bubble in the flow stream; A data signal is generated when the bubble is illuminated in the flow stream.

[0220] 30. The system of clause 29, wherein the light detection system is further configured to generate a data signal in response to light detected from an illuminated particle downstream of the bubble in the flow stream.

[0221] 31. A system according to clause 30, wherein the light detection system is configured to: continuously detecting light from the illuminated particles prior to introducing the gas bubble into the flow stream; continuously detecting light from illuminated particles upstream of the bubble in the flow stream; Light is detected from the flow stream as the bubble passes through an interrogation field.

[0222] 32. A system according to any of clauses 20 to 31, wherein the light detection system comprises a photodetector configured to detect fluorescence from the sample.

[0223] 33. A system according to any of clauses 20 to 31, wherein the light detection system comprises a photodetector configured to detect scattered light.

[0224] 34. A system according to any of clauses 20 to 33, wherein the memory comprises instructions for determining the presence of the bubbles in the flow stream based on a scattered light detector signal.

[0225] 35. A system according to any one of clauses 20 to 34, wherein the memory comprises instructions for calculating the absolute count of particles in the sample using: a data signal generated in response to light detected from the illuminated particle between when the bubble is introduced into the flow stream and when the bubble is detected; and Predetermined volume.

[0226] 36. A system according to any of clauses 20 to 35, wherein the memory comprises instructions for generating a data signal strength graph of the generated data signal over time.

[0227] 37. The system of clause 36, wherein the memory includes instructions for plotting the time when the bubble was introduced into the flow stream onto the data signal strength graph.

[0228] 38. A non-transitory computer readable storage medium comprising instructions stored thereon for calculating an absolute count of particles in a sample in a flow cytometer, the instructions comprising: an algorithm for introducing bubbles into a flow stream propagating a sample containing particles; an algorithm for illuminating the flow stream with a light source; an algorithm for detecting light from the illuminated particles with a photodetector; an algorithm for detecting the presence of said bubbles in said flow stream with said photodetector; and An algorithm for determining the absolute count of the particles in the sample based on: a data signal generated in response to light detected from the illuminated particle; a data signal generated when the bubble is introduced into the flow stream; and A data signal is generated in response to the detected bubble.

[0229] 39. The non-transitory computer readable storage medium of clause 38, wherein the non-transitory computer readable storage medium comprises an algorithm for introducing the gas bubble into the flow stream by retracting a sample line from a source of the sample to create an air gap in the flow stream.

[0230] 40. The non-transitory computer readable storage medium of clause 39, wherein the non-transitory computer readable storage medium comprises an algorithm for generating a data signal when the sample line is retracted from the sample source.

[0231] 41. The non-transitory computer readable storage medium of clause 39, wherein the non-transitory computer readable storage medium comprises an algorithm for generating a data signal at a predetermined time after the sample line is retracted from the sample source.

[0232] 42. The non-transitory computer readable storage medium of clause 41, wherein the non-transitory computer readable storage medium comprises an algorithm for generating a data signal 0.001 µs to 10 µs after retracting the sample line from the sample source.

[0233] 43. The non-transitory computer readable storage medium of any one of clauses 38 to 42, wherein the non-transitory computer readable storage medium comprises an algorithm for generating an electronic data signal when the gas bubble is introduced into the flow stream.

[0234] 44. The non-transitory computer readable storage medium of any one of clauses 38 to 42, wherein the non-transitory computer readable storage medium comprises an algorithm for generating an optical data signal when the gas bubble is introduced into the flow stream.

[0235] 45. The non-transitory computer-readable storage medium of any one of clauses 38 to 44, wherein the non-transitory computer-readable storage medium comprises: an algorithm for generating a data signal in response to light detected from the illuminated particles prior to introducing the bubble into the flow stream; an algorithm for generating a data signal in response to light detected from an illuminated particle upstream of the bubble in the flow stream; and An algorithm for generating a data signal when the bubble is illuminated in the flow stream.

[0236] 46. ​​The non-transitory computer readable storage medium of clause 45, wherein the non-transitory computer readable storage medium comprises an algorithm for generating a data signal in response to light detected from an illuminated particle downstream of the bubble in the flow stream.

[0237] 47. The non-transitory computer-readable storage medium of clause 45, wherein the non-transitory computer-readable storage medium comprises: an algorithm for continuously detecting light from the illuminated particles prior to introducing the bubble into the flow stream; an algorithm for continuously detecting light from illuminated particles upstream of the bubble in the flow stream; and An algorithm is provided for detecting light from the flow stream as the bubble passes through an interrogation field.

[0238] 48. The non-transitory computer-readable storage medium of any one of clauses 37 to 47, wherein the non-transitory computer-readable storage medium comprises an algorithm for calculating the absolute count of particles in the sample using: a data signal generated in response to light detected from the illuminated particle between when the bubble is introduced into the flow stream and when the bubble is detected; and Predetermined volume.

[0239] 49. The non-transitory computer readable storage medium of any of clauses 37 to 48, wherein the non-transitory computer readable storage medium comprises an algorithm for generating a data signal strength map of the generated data signal over time.

[0240] 50. The non-transitory computer readable storage medium of clause 49, wherein the non-transitory computer readable storage medium comprises an algorithm for plotting the time when the bubble was introduced into the flow stream onto the data signal strength graph.

[0241] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0242] Therefore, the above only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language described in this article are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventor to promote this area, and should be interpreted as not being limited to such specific examples and conditions.In addition, all statements of the principles, aspects and embodiments of the present invention and its specific examples described herein are intended to cover both its structural equivalents and functional equivalents.In addition, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any element that performs the same function developed, regardless of the structure.In addition, regardless of whether such disclosure is clearly described in the claims, any content disclosed herein is not intended to be dedicated to the public.

[0243] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 USC § 112 (f) or 35 USC § 112 (6) is expressly defined as being cited for the limitation in the claim only when the exact phrase "means for..." or the exact phrase "step for..." is cited at the beginning of such limitation in the claim; if such exact phrase is not used in the limitation in the claim, 35 USC § 112 (f) or 35 USC § 112 (6) is not cited.

Claims

1. A method for determining an absolute count of particles in a sample in a flow cytometer, the method comprising: introducing bubbles into a flow stream that propagates a sample containing particles; illuminating the flow stream with a light source; detecting light from the irradiated particles with a photodetector; detecting the presence of the bubbles in the flow stream with the photodetector; as well as The absolute count of the particles in the sample is determined based on: a data signal generated in response to light detected from the illuminated particle; a data signal generated when the bubble is introduced into the flow stream; and A data signal is generated in response to the detected bubble. 2 . The method of claim 1 , wherein introducing the gas bubbles into the flow stream comprises retracting a sample line from a source of the sample to create an air gap in the flow stream.

3. The method of claim 2, wherein the method comprises generating a data signal when the sample line is retracted from the sample source.

4. The method of claim 2, wherein the method includes generating a data signal a predetermined time after retracting the sample line from the sample source.

5. The method of any one of claims 1 to 4, wherein the data signal generated when the gas bubble is introduced into the flow stream comprises an electronic data signal or an optical data signal.

6. The method according to any one of claims 1 to 5, wherein the flow stream is illuminated continuously by the light source.

7. The method according to any one of claims 1 to 6, wherein the method comprises: generating a data signal in response to light detected from the illuminated particles prior to introducing the bubble into the flow stream; generating a data signal in response to light detected from an illuminated particle upstream of the bubble in the flow stream; as well as A data signal is generated when the bubble is illuminated in the flow stream.

8. The method of any one of claims 1 to 7, wherein the detected light comprises fluorescence.

9. The method of any one of claims 1 to 7, wherein the detected light comprises scattered light.

10. The method of any one of claims 1 to 9, wherein the presence of the bubbles in the flow stream is determined based on detecting scattered light from the illuminated bubbles.

11. The method according to any one of claims 1 to 10, wherein the method comprises calculating the absolute count of particles in the sample using: a data signal generated in response to light detected from the illuminated particle between when the bubble is introduced into the flow stream and when the bubble is detected; and Predetermined volume.

12. The method according to any one of claims 1 to 11, wherein the method further comprises generating a data signal strength graph of the generated data signal over time.

13. A system comprising: a flow cell configured to propagate a sample comprising particles in a flow stream; a sample line in fluid communication with a source of the sample and the flow cell, wherein the sample line is configured to introduce bubbles into the flow stream; a light source configured to illuminate the sample in the flow stream; a light detection system comprising a photodetector for detecting light from the illuminated flow stream; as well as a processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to calculate the absolute count of the particles in the sample based on: a data signal generated in response to light detected from the illuminated particle; a data signal generated when the bubble is introduced into the flow stream; and A data signal is generated in response to the detected bubble.

14. A non-transitory computer readable storage medium comprising instructions stored thereon for calculating an absolute count of particles in a sample in a flow cytometer, the instructions comprising: an algorithm for introducing bubbles into a flow stream propagating a sample containing particles; an algorithm for illuminating the flow stream with a light source; an algorithm for detecting light from the illuminated particles with a photodetector; an algorithm for detecting the presence of said bubbles in said flow stream with said photodetector; as well as An algorithm for determining the absolute count of the particles in the sample based on: a data signal generated in response to light detected from the illuminated particle; a data signal generated when the bubble is introduced into the flow stream; and A data signal is generated in response to the detected bubble.

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