Method and system for determining the absolute number of particles in a sample within a flow cytometer

Abstract

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

Description

Background Art

[0001] Optical detection is often used to characterize the components of a sample, for example, when the sample (e.g., a biological sample) is used for the diagnosis of a disease or condition. When the sample is irradiated, light is not only scattered by the sample and transmitted through the sample, but can also be emitted by the sample (e.g., by fluorescence).

[0002] Differences in sample components such as morphology, absorption rate, and the presence of fluorescent labels can cause variations in the light scattered, transmitted, or emitted by the sample. These variations can be used to characterize and identify the presence of components within the sample. To quantify these variations, light is collected and directed onto the surface of a detector.

[0003] One technique that utilizes optical detection to characterize components within a sample is flow cytometry. A flow cytometer includes an optical detection system consisting of an optical system, an optical detector, and electronics that enable efficient detection of optical signals and conversion of the optical signals into corresponding electrical signals. The electronic signals are processed to obtain parameters that can be utilized by a user to perform the desired analysis. A flow cytometer includes different types of optical detectors for detecting optical signals such as those from fluorescence, side scatter light, or forward scatter light. When an optical signal impinges on the optical detector, an electrical signal proportional to the incident optical signal is generated at the output of the optical detector. Flow cytometry provides a rapid method for quantifying cell characteristics.

[0004] However, most flow cytometers cannot directly provide the cell concentration or absolute number of cells in a sample. Absolute cell numbers are widely used in the quantification of cell populations and disease progression, including studies of the immune system or stem cells. Absolute cell numbers are generally obtained by using multiple platform assays to combine separate cell concentration measurements from a hematology analyzer with flow cytometric population data, or by using a single platform assay to add an internal microsphere counting standard to a flow cytometric sample. SUMMARY OF THE INVENTION

[0005] Aspects of the present disclosure include a method for determining the absolute number of particles in a sample within a flow cytometer. The method according to certain embodiments includes introducing air bubbles into a flow stream propagating a sample having particles, irradiating the flow stream with a light source, detecting light from the irradiated particles using a photodetector, detecting the presence of air bubbles in the flow stream using the photodetector, and determining the absolute number of particles in the sample based on data signals generated in response to light detected from the irradiated particles, data signals generated when air bubbles are introduced into the flow stream, and data signals generated in response to the detected air bubbles. Also described is a system (e.g., a flow cytometer) having a light source, a photodetection system, and a sample line configured to introduce air 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 the frost stream is irradiated with a light source and bubbles are introduced into the frost stream. In some embodiments, a data signal is generated when bubbles are introduced into the frost stream. In some cases, a data signal is generated when bubbles are introduced into the frost stream. In other cases, the data signal is generated at a predetermined time after bubbles are introduced into the frost stream, for example, 0.001 μs to 10 μs after bubbles are introduced into the frost stream. In some embodiments, bubbles are introduced into the frost stream by retracting the sample line from the sample source (source of the sample) to create an air gap within the sample in the frost stream. In certain embodiments, the sample line may be retracted from the sample source to create a gap within the sample in the frost stream, and the air intake replaces the intake of the sample into the frost stream for a duration of, for example, 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, for example, 0.001 μs to 10 μs after the sample line is retracted from the sample source. In some cases, the data signal generated when bubbles are introduced into the frost stream is an electronic data signal. In other cases, the data signal is an optical data signal.

[0007] In an embodiment, light is detected from the flow stream using a light detection system having a photodetector, 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 the bubbles in the flow stream, and detecting light from the flow stream when the bubbles pass through the inspection region. 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 the bubbles in the flow stream, and generating a data signal when the bubbles are irradiated within 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 cases, the data signal is generated from fluorescence detected from irradiated particles in the sample. In some cases, the data signal is generated from scattered light detected from irradiated particles in the sample. In a particular case, the data signal generated from detecting bubbles in the flow stream is from scattered light detected from irradiated bubbles.

[0008] The absolute number of particles in the sample is calculated in some cases using a data signal generated in response to light detected from irradiated particles and a predetermined volume between when bubbles are introduced into the flow stream and when the bubbles are detected. In some cases, the absolute number of particles is determined using the volume of a pre-calibrated sample line. In some cases, the method includes generating a data signal intensity plot of the data signal from the irradiated sample as a function of time. In a particular case, the data signal intensity plot includes at least the intensity of the data signal from irradiated particles collected from when bubbles are introduced into the flow stream until when the bubbles are detected. In a particular case, the time when bubbles are introduced into the flow stream is plotted on the data signal intensity plot.

[0009] Aspects of the present disclosure also include a flow cell configured to propagate a sample having particles in a flow stream, a sample line in fluid communication with a sample source and configured to introduce air bubbles into the flow stream, a light source for irradiating the flow stream, and a light detection system having a photodetector for detecting light from the irradiated flow stream (e.g., a flow cytometer). In some embodiments, the sample line is configured to create an air gap within the sample in the flow stream. In some cases, the system includes a sample line displacement component configured to retract the sample line from the sample source and incorporate air into the sample line while the sample line is being retracted from the sample source. Incorporation of air into the sample while the sample line is being retracted from the sample source creates an air gap within the flow stream. In certain embodiments, the system includes a gas source for introducing gaseous bubbles into the sample line.

[0010] In embodiments, the system includes a processor having a memory, the memory being operably coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to generate a data signal in response to air bubbles being introduced into the flow stream. In some cases, the memory includes instructions for generating an electronic signal when air bubbles are introduced into the flow stream. In some cases, the system includes a photodetector configured to generate an optical signal when air bubbles are introduced into the flow stream. In certain cases, the memory includes instructions for generating a data signal when air bubbles are introduced into the flow stream. In other cases, the memory includes instructions for generating a data signal at a predetermined time after air bubbles have been introduced into the flow stream, e.g., between 0.001 μs and 10 μs after air bubbles have been introduced into the flow stream.

[0011] In an embodiment, the system includes an optical detection system having a photodetector that detects light from a flow stream and generates a data signal in response to the detected light. In some embodiments, the optical detection system is configured to continuously detect light from irradiated particles before introducing bubbles into the flow stream, continuously detect light from upstream irradiated particles from bubbles within the flow stream, and detect light from the flow stream when the bubbles pass through the inspection region. 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 upstream irradiated particles from bubbles within the flow stream. In some embodiments, the system is configured to generate a data signal when the bubbles are irradiated within the flow stream. In some embodiments, the optical detection system includes one or more light absorption photodetectors, light scattering photodetectors, and light emission (e.g., fluorescence) photodetectors. In some cases, the optical detection system is configured to generate a data signal from fluorescence detected from irradiated particles in a sample. In some cases, the optical detection system is configured to generate a data signal from scattered light detected from irradiated particles in a sample. In a particular case, the optical detection system is configured to generate a data signal in a scattered light detection channel by detecting irradiated bubbles within the flow stream.

[0012] In an embodiment, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to calculate the absolute number of particles in a sample. In some embodiments, the memory includes instructions for calculating the absolute number of particles in a sample using a data signal generated in response to light detected from irradiated particles and a predetermined volume between when a bubble is introduced into the flow stream and when the bubble is detected. In some instances, the memory includes instructions for calculating the absolute number of particles using the volume of a pre-calibrated sample line. In some instances, the memory includes instructions for generating a data signal intensity plot of the data signal from the irradiated sample as a function of time. In a particular instance, the data signal intensity plot includes at least the intensity of the data signal from the irradiated particles collected from when a bubble is introduced into the flow stream until when the bubble is detected. In a particular instance, the time when a bubble is introduced into the flow stream is plotted on the data signal intensity plot.

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

[0014] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for introducing bubbles into the flow stream by retracting a sample line from a sample source to create an air gap within the flow stream. In some instances, the non-transitory computer-readable storage medium includes an algorithm for operating a sample line displacement component that retracts the sample line from the sample source. In certain instances, the non-transitory computer-readable storage medium includes an algorithm for introducing air or gas into the sample line while the sample line is being retracted to retract the sample line from the sample source. 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 has been retracted from the sample source, e.g., after bubbles have been introduced into the flow stream, for a period of 0.001 μs to 10 μs.

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

[0016] In an embodiment, the non - transitory computer - readable storage medium includes an algorithm for calculating the absolute number of particles in a sample. In some embodiments, the non - transitory computer - readable storage medium includes an algorithm for calculating the absolute number of particles in a sample using a data signal generated in response to light detected from irradiated particles and a predetermined volume between when a bubble is introduced into the flow stream and when the bubble is detected. In some cases, the non - transitory computer - readable storage medium includes an algorithm for calculating the absolute number of particles using the volume of a pre - calibrated sample line. In some cases, the non - transitory computer - readable storage medium includes an algorithm for generating a data - signal intensity plot of the data signal from an irradiated sample as a function of time. In a particular case, the data - signal intensity plot includes at least the intensity of the data signal from irradiated particles collected from when a bubble is introduced into the flow stream until when the bubble is detected. In a particular case, the time when a bubble is introduced into the flow stream is plotted on the data - signal intensity plot.

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. The drawings include the following figures.

[0018]

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[0019] Aspects of the present disclosure include a method for determining the absolute number of particles in a sample within a flow cytometer. The method according to a particular embodiment includes introducing air bubbles into a flow stream propagating a sample having particles, irradiating the flow stream with a light source, detecting light from the irradiated particles using a photodetector, detecting the presence of air bubbles in the flow stream using the photodetector, and determining the absolute number 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 air bubbles are introduced into the flow stream, and a data signal generated in response to the detected air bubbles. Also described is a system (e.g., a flow cytometer) having a light source, a light detection system, and a sample line configured to introduce air bubbles into the flow stream for practicing the subject method. A non-transitory computer-readable storage medium is also provided.

[0020] Before the present invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, and thus may, of course, vary. Also, it is to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0021] When ranges of values are provided, unless the context clearly dictates otherwise, each intervening value, to the tenth of the unit of the lower limit, is understood to be included between the upper and lower limits of the range and any other stated value or intervening value within the stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the invention, subject to any specific excluded limitations within the scope of the description. Ranges excluding any one or both of the included limitations, when the stated range includes one or both of the limitations, are also included within the invention.

[0022] A particular range is presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the exact number preceding the term and for numbers that are close to or approximately the number preceding the term. In determining whether a number is close to or approximately the specifically recited number, a number that is close to or approximately the unrecited number may be a number that provides substantial equivalence to the specifically recited number in the context presented.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, representative illustrative methods and materials are described herein.

[0024] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated as being incorporated by reference, and by being incorporated herein by reference, such publications disclose and describe the relevant methods and / or materials by which they are cited. The citation of any publication is for its disclosure prior to the filing date, and the present invention should not be construed as admitting that the invention has no right to antedate such publication by virtue of the features of the prior invention. Further, the dates of the publications provided may be different from the actual publication dates which may need to be independently confirmed.

[0025] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any optional element. Accordingly, this description is intended to serve as a precedent for the use of exclusive terms such as "solely", "only", etc. or the use of "negative" limitations in connection with the recitation of claim elements.

[0026] As will be apparent to those skilled in the art upon reading this disclosure, each of the distinct embodiments described and illustrated herein may be readily separated from, or combined with, the features of any of the various other embodiments without departing from the scope or spirit of the present invention. Any of the recited methods may be performed in the order of the recited events, or in any other order that is logically possible.

[0027] The apparatus and method are described, or are to be described, in functional terms for purposes of grammatical fluidity, but the claims are not to be construed as necessarily limited by a "means" or "step" limitation syntax unless expressly recited under 35 U.S.C. § 112, and are to be given the full scope of the definition provided by the claim and the equivalents thereof under the doctrine of equivalents, and it should be clearly understood that when the claims are expressly recited under 35 U.S.C. § 112, full statutory equivalents under 35 U.S.C. § 112 are to be given.

[0028] As summarized above, the present disclosure provides a method for determining the absolute number of particles in a sample within a flow cytometer. In a further description of embodiments of the present disclosure, methods for introducing bubbles into a flow stream carrying a sample having particles, methods for irradiating the flow stream with a light source, methods for detecting the presence of light from the irradiated particles and bubbles within the flow stream using a photodetector, and methods for determining the absolute number of particles in the sample are first described in more detail. Next, a system is described that includes a light source, a light detection system, and a sample line configured to introduce bubbles into the flow stream, and a non-transitory computer-readable storage medium for practicing the subject methods.

[0029] Method for determining the absolute number of particles in a sample within a flow cytometer Aspects of the present disclosure include a method for determining the absolute number of particles in a sample within a flow cytometer. The term "absolute number" 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 (as described in more detail below), and the subject method provides for determining the number of cells per volume in the biological sample. In some cases, the subject method provides an accurate count of the number of particles (or biological cells) per volume in the sample without using extraneous and often expensive components such as calibration bead particles, secondary sample loops, sample line flow meters, or syringe pumps. In certain cases, the method for determining the absolute number of particles in a flow cytometry sample described herein provides an improved accuracy of the count of the determined number of particles per volume, including, for example, 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, for example, 90% or more, and 99% or more, as compared to the absolute number determined using calibration beads, a sample line flow sensor, or a syringe pump. In certain embodiments, the subject method reduces the time required to determine the absolute number of particles in the sample, including, for example, 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, for example, 90% or more, and 99% or more.

[0030] In some cases, the sample described herein is a biological sample having cells. The term "biological sample" is used in its conventional meaning to refer to whole organisms, plants, fungi, or, in certain cases, a subset of animal tissues, cells, or components that can be found in, for example, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to both natural organisms or subsets of their tissues, as well as, without limitation, for example, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory, gastrointestinal, cardiovascular, and urinary organs, tears, saliva, milk, blood cells, tumors, homogenates, lysates, or extracts prepared from a biological or a subset of its tissues, including healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). A biological sample can be any type of biological tissue, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, 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., and in some cases, the sample is a blood sample including whole blood, such as blood obtained by venipuncture or finger stick (the blood may or may not be combined with any reagents such as preservatives, anticoagulants, etc. prior to the assay).

[0031] In certain embodiments, the sample source is a "mammal" or "mammalian animal", and these terms are widely used to describe organisms within the class of mammals, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The methods can be applied to samples obtained from human subjects of either sex at any stage of development (i.e., neonates, infants, juveniles, adolescents, adults), and in certain embodiments, the human subject is a juvenile, adolescent, or adult. It is understood that the present invention can be applied to samples from human subjects, but is not limited thereto, and can also be practiced on samples from other animal subjects (i.e., "non-human subjects") such as, without limitation, birds, mice, rats, dogs, cats, livestock, and horses.

[0032] When practicing the subject method, bubbles are introduced into the flow stream of a flow cytometer that propagates a sample having particles. The term "bubble" is used herein to refer to a gap within the flow stream in which there are few or no particles (and in certain cases, no fluid) present within the flow stream. For example, a bubble introduced into the flow stream may contain, per volume of the sample, 1% or less of the particles, 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 0.00001% or less of the particles per volume of the sample. In certain cases, the bubbles introduced into the flow stream are free of the sample particles.

[0033] Any convenient protocol may be employed to introduce bubbles into the flow stream. In some embodiments, the gas is introduced into the flow stream during sample uptake into the flow stream. For example, an inert gas such as nitrogen, helium, or argon may be input into the flow stream during sample uptake into the flow stream. The volume of gas introduced as bubbles into the flow stream may vary depending on the volume of the sample and the sample line and may be, for example, 0.001 μL or more, including, for example, 0.005 μL or more, for example, 0.01 μL or more, for example, 0.05 μL or more, for example, 0.1 μL or more, for example, 0.5 μL or more, for example, 1 μL or more, for example, 2 μL or more, for example, 3 μL or more, for example, 4 μL or more, for example, 5 μL or more, and 10 μL or more.

[0034] In certain embodiments, bubbles are generated by creating an air gap within the flow stream. In some instances, the air gap is created by retracting a sample line that is in fluid communication with the sample source such that there is a time gap in the uptake of the sample composition from the sample source. During this time gap, ambient air is drawn into the sample line, providing separation of sample particles in the flow stream. As described in more detail below, the sample line can be retracted from the sample source by any convenient protocol such as using a displacement device (e.g., a well of a test tube or well plate) that lifts the sample line from the sample source. The sample line can be retracted from the sample source by, for example, 0.0001 seconds or more, including, for example, 0.0005 seconds or more, for example, 0.01 seconds or more, for example, 0.05 seconds or more, for example, 0.1 seconds or more, for example, 0.5 seconds or more, for example, 1 second or more, for example, 2 seconds or more, for example, 3 seconds or more, for example, 4 seconds or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 15 seconds or more, and 30 seconds or more.

[0035] As described in more detail below, the bubbles introduced into the flow stream provide a time gap in the data signal generated in response to light detected from irradiated particles in the flow stream. The time gap in the data signal can be a period of 0.0001 seconds or more, including, for example, 0.0005 seconds or more, for example, 0.01 seconds or more, for example, 0.05 seconds or more, for example, 0.1 seconds or more, for example, 0.5 seconds or more, for example, 1 second or more, for example, 2 seconds or more, for example, 3 seconds or more, for example, 4 seconds or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 15 seconds or more, and 30 seconds or more.

[0036] In some embodiments, the data signal is generated when bubbles are introduced into the flow stream. For example, the data signal can be generated when a volume of gas is input or taken into the sample line. In other embodiments, the data signal is generated at a predetermined time after bubbles are introduced into the flow stream, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2 μs or more, for example, 3 μs or more, for example, 5 μs or more, for example, 10 μs or more, for example, 100 μs or more, for example, 500 μs or more, and after bubbles are introduced into the flow stream, 1000 μs or more, for example, 0.001 μs or more after bubbles are introduced into the flow stream. In certain embodiments, when bubbles are introduced into the flow stream by retracting the sample line from the sample source, the data signal can be 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, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2 μs or more, for example, 3 μs or more, for example, 5 μs or more, for example, 10 μs or more, for example, 100 μs or more, for example, 500 μs or more, and after the sample line is retracted from the sample source, 1000 μs or more, for example, 0.001 μs or more after the sample line is retracted from the sample source. In other embodiments, the data signal is generated when the sample line is reinserted into the sample source (i.e., when sample uptake into the sample line is re-established).In still other embodiments, the data signal is generated, for example, after the sample line is reinserted into the sample source, at a predetermined time after the sample line is reinserted into the sample source, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2 μs or more, for example, 3 μs or more, for example, 5 μs or more, for example, 10 μs or more, for example, 100 μs or more, for example, 500 μs or more, and after the sample line is reinserted into the sample source, for example, 1000 μs or more, for example, 0.001 μs or more.

[0037] In some cases, the data signal generated when bubbles are introduced into the frost stream is an electronic data signal. The data signal generated when bubbles are introduced into the frost stream can be transmitted to a processor for use in calculating the number of particles in a sample (as described below). In other cases, the data signal generated when bubbles are introduced into the frost stream is an optical data signal. This optical data signal can be generated using a photodetector positioned to detect light from the sample line into which the bubbles are introduced. For example, a method according to a particular embodiment includes irradiating a frost stream into which bubbles are introduced with a light source such as an LED or a laser, detecting light from the bubbles introduced into the frost stream, and generating an optical data signal. When bubbles are introduced by retracting the sample line from a sample source, the photodetector and light source for detecting when bubbles are introduced into the frost stream may be adjacent to the end of the sample line or downstream from the end of the sample line. In some embodiments, the photodetector and light source for detecting the introduction of bubbles into the frost stream may be positioned downstream from the end of the sample line by 0.001 mm or more, including, for example, 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2 mm or more, for example, 3 mm or more, for example, 4 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and 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 use in calculating the number of particles in a sample.

[0038] In an embodiment, the sample is irradiated with a light source within the flow stream. The sample may be irradiated within the flow stream for a predetermined period before introducing bubbles into the flow stream. In some cases, the sample in the flow stream is irradiated for 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 5 seconds or more, such as 10 seconds or more, such as 15 seconds or more, such as 30 seconds or more, such as 60 seconds or more, and 90 seconds or more before introducing bubbles into the flow stream.

[0039] In some embodiments, the method includes irradiating a sample propagating through the flow stream across an inspection region of the flow stream of 5 μm or more, such as across an inspection region of the flow stream of 10 μm or more, such as 15 μm or more, such as 20 μm or more, such as 25 μm or more, such as 50 μm or more, such as 75 μm or more, such as 100 μm or more, such as 250 μm or more, such as 500 μm or more, such as 750 μm or more, and including 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 6 mm or more, such as 7 mm or more, such as 8 mm or more, such as 9 mm or more, and 10 mm or more.

[0040] In some embodiments, the sample in the flow stream is irradiated with a continuous light source, e.g., the light source provides a light beam that is not interrupted and maintains the irradiation of the particles of the sample in the flow stream with little or no undesirable change in light intensity. In some embodiments, this continuous light source emits non-pulsed or non-stroboscopic irradiation. In certain embodiments, the continuous light source provides a substantially constant emitted light intensity. For example, the method may include irradiating the sample in the flow stream with a continuous light source, and the continuous light source provides an emitted light intensity that varies by 10% or less, e.g., 9% or less, e.g., 8% or less, e.g., 7% or less, e.g., 6% or less, e.g., 5% or less, e.g., 4% or less, e.g., 3% or less, e.g., 2% or less, e.g., 1% or less, e.g., 0.5% or less, e.g., 0.1% or less, e.g., 0.01% or less, e.g., 0.001% or less, e.g., 0.0001% or less, e.g., 0.00001% or less during the irradiation time interval, including the case where the emitted light intensity varies by 0.000001% or less during the irradiation time interval. The intensity of the light output can be measured by any convenient protocol, including but not limited to a scanning slit profiler, a charge-coupled device (CCD such as an intensified charge-coupled device ICCD), a position sensor, a power sensor (e.g., a thermopile array power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector among other types of photodetectors.

[0041] In other embodiments, the method includes irradiating a sample in the flow stream with a pulsed light source, such as when 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 the sample with a pulsed light source in the inspection region of each flow stream with periodic light blinking. For example, the frequency of each light pulse can be 0.0001 kHz or more, including, for example, 0.0005 kHz or more, for example, 0.001 kHz or more, for example, 0.005 kHz or more, for example, 0.01 kHz or more, for example, 0.05 kHz or more, for example, 0.1 kHz or more, for example, 0.5 kHz or more, for example, 1 kHz or more, for example, 2.5 kHz or more, for example, 5 kHz or more, for example, 10 kHz or more, for example, 25 kHz or more, for example, 50 kHz or more, and 100 kHz or more. In certain cases, the frequency of pulsed irradiation by the light source is in the range of 0.00001 kHz to 1000 kHz, including, for example, 0.00005 kHz to 900 kHz, for example, 0.0001 kHz to 800 kHz, for example, 0.0005 kHz to 700 kHz, for example, 0.001 kHz to 600 kHz, for example, 0.005 kHz to 500 kHz, for example, 0.01 kHz to 400 kHz, for example, 0.05 kHz to 300 kHz, for example, 0.1 kHz to 200 kHz, and 1 kHz to 100 kHz. The duration of light irradiation for each light pulse (i.e., pulse width) can vary and can be 0.000001 millisecond or more, including, for example, 0.000005 millisecond or more, for example, 0.00001 millisecond or more, for example, 0.00005 millisecond or more, for example, 0.0001 millisecond or more, for example, 0.0005 millisecond or more, for example, 0.001 millisecond or more, for example, 0.005 millisecond or more, for example, 0.01 millisecond or more, for example, 0.05 millisecond or more, for example, 0.1 millisecond or more, for example, 0.5 millisecond or more, for example, 1 millisecond or more, for example, 2 millisecond or more, for example, 3 millisecond or more, for example, 4 millisecond or more, for example, 5 millisecond or more, for example, 10 millisecond or more, for example, 25 millisecond or more, for example, 50 millisecond or more, for example, 100 millisecond or more, and 500 millisecond or more.For example, the duration of light irradiation can be in the range of 0.000001 milliseconds to 1000 milliseconds, including, for example, 0.000005 milliseconds to 950 milliseconds, for example, 0.00001 milliseconds to 900 milliseconds, for example, 0.00005 milliseconds to 850 milliseconds, for example, 0.0001 milliseconds to 800 milliseconds, for example, 0.0005 milliseconds to 750 milliseconds, for example, 0.001 milliseconds to 700 milliseconds, for example, 0.005 milliseconds to 650 milliseconds, for example, 0.01 milliseconds to 600 milliseconds, for example, 0.05 milliseconds to 550 milliseconds, for example, 0.1 milliseconds to 500 milliseconds, for example, 0.5 milliseconds to 450 milliseconds, for example, 1 millisecond to 400 milliseconds, for example, 5 milliseconds to 350 milliseconds, and 10 milliseconds to 300 milliseconds.

[0042] The frost stream can be irradiated with any convenient light source, which may include a laser light source and a non-laser light source (e.g., a light-emitting diode). In certain embodiments, the method includes irradiating the particles with a laser such as a pulsed-wave laser or a continuous-wave laser. For example, the laser may be a diode laser such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluoride (ArF) excimer laser, a krypton-fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluoride (XeF) excimer laser, or a combination thereof. In other cases, the subject system includes a dye laser such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, the laser of interest includes a metal vapor laser such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and a combination thereof. In yet other cases, the subject system includes a solid laser such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a slim YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, and a combination thereof.

[0043] In some embodiments, the light source outputs specific wavelengths such as 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the continuous wave light source emits light having a wavelength of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm.

[0044] The frost stream can be irradiated by the light source from any suitable distance, such as a distance of 0.001 mm or more, including, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and 100 mm or more. In addition, the irradiation of the frost stream can be at any suitable angle, such as an angle in the range of 10° to 90°, including, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and 30° to 60°, for example, 90°.

[0045] In some embodiments, the method includes further 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, mirrors, pinholes, slits, gratings, photorefractive devices, and any combination thereof. In some cases, the collected light passes through one or more focusing lenses, such as to reduce the profile of the light. In other cases, the light emitted from the sample passes through one or more collimators to reduce the divergence of the light beam.

[0046] In certain embodiments, the method includes irradiating a sample with two or more beams of frequency-shifted light. As described above, an optical beam generator component having a laser and an acousto-optic device for frequency-shifting the laser light can be employed. In these embodiments, the method includes irradiating the acousto-optic device with the laser. Depending on the desired wavelength of the light generated by the output laser beam (e.g., for use in irradiating a sample in a flow stream), the laser can have a specific wavelength that varies between 200 nm and 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. The acousto-optic device can be irradiated with one or more lasers, including, for example, two or more lasers, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, and ten or more lasers. The laser can comprise any combination of several types of lasers. For example, in some embodiments, the method includes irradiating the acousto-optic device with a laser array, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0047] When two or more lasers are employed, the acousto-optic device can be irradiated with the lasers simultaneously, sequentially, or in a combination thereof. For example, the acousto-optic device can be irradiated simultaneously with each of the lasers. In other embodiments, the acousto-optic device is irradiated sequentially with each of the lasers. When two or more lasers are employed to irradiate the acousto-optic device sequentially, the time for each laser to irradiate the acousto-optic device can independently be, for example, 0.001 microseconds or more, including, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, and 60 microseconds or more. For example, the method can include irradiating the acousto-optic device with a laser for a duration in the range of 0.001 microseconds to 100 microseconds, including, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In embodiments where the acousto-optic device is irradiated sequentially with two or more lasers, the duration for which the acousto-optic device is irradiated by each laser can be the same or different.

[0048] In an embodiment, the method includes applying a high-frequency drive signal to the acousto-optic device to generate an angularly deflected laser beam. Two or more high-frequency drive signals can be applied to the acousto-optic device to generate an output laser beam having a desired number of angularly deflected laser beams, including, for example, three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals, and one hundred or more high-frequency drive signals.

[0049] The angle-deflected laser beams generated by the high-frequency drive signals each have an intensity based on the amplitude of the applied high-frequency drive signal. In some embodiments, the method includes applying a high-frequency drive signal having an amplitude sufficient to generate an angle-deflected laser beam at a desired intensity. In some cases, each applied high-frequency drive signal independently has an amplitude of from about 0.001 V to about 500 V, including, for example, from about 0.005 V to about 400 V, for example, from about 0.01 V to about 300 V, for example, from about 0.05 V to about 200 V, for example, from about 0.1 V to about 100 V, for example, from about 0.5 V to about 75 V, for example, from about 1 V to about 50 V, for example, from about 2 V to 40 V, for example, from 3 V to about 30 V, and from about 5 V to about 25 V. In some embodiments, each applied high-frequency drive signal has a frequency of from about 0.001 MHz to about 500 MHz, including, for example, from about 0.005 MHz to about 400 MHz, for example, from about 0.01 MHz to about 300 MHz, for example, from about 0.05 MHz to about 200 MHz, for example, from about 0.1 MHz to about 100 MHz, for example, from about 0.5 MHz to about 90 MHz, for example, from about 1 MHz to about 75 MHz, for example, from about 2 MHz to about 70 MHz, for example, from about 3 MHz to about 65 MHz, for example, from about 4 MHz to about 60 MHz, and from about 5 MHz to about 50 MHz.

[0050] In these embodiments, the angularly deflected laser beams within the output laser beam are spatially separated. Depending on the applied high-frequency drive signal of the output laser beam and the desired irradiation profile, the angularly deflected laser beams can be separated by 0.001 μm or more, including, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, and 5000 μm or more. In some embodiments, the angularly deflected laser beams overlap, for example, 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 more, including, for example, an overlap of 0.005 μm or more, for example, an overlap of 0.01 μm or more, for example, an overlap of 0.05 μm or more, for example, an overlap of 0.1 μm or more, for example, an overlap of 0.5 μm or more, for example, an overlap of 1 μm or more, for example, an overlap of 5 μm or more, for example, an overlap of 10 μm or more, and 100 μm or more.

[0051] In certain instances, the flow stream is irradiated with a plurality of frequency-shifted optical beams and the cells in the flow stream are imaged by fluorescence imaging using frequency-tagged luminescence (FIRE) as described in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013), as well as U.S. Pat. Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, and U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894 to generate frequency-encoded images such as those described therein, the disclosures of which are incorporated herein by reference.

[0052] The light from the irradiated flow stream is detected by a light detection system having a photodetector. The photodetector may be any convenient light detection protocol including, but not limited to, 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 solar cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor or photodiode, and combinations thereof. In certain embodiments, the photodetector is, for example, 0.05 cm 2 ~9 cm 2 For example, 0.1 cm 2 ~8 cm 2 For example, 0.5 cm 2 ~7 cm 2 And 1 cm 2 ~5 cm 2 including 0.01 cm2 ~10 cm 2 is an electron multiplier having an active detection surface area for each region in the range of.

[0053] Light can be measured by a photodetector at one or more wavelengths, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, including measuring light from particles in a frost stream at four hundred or more different wavelengths. In some embodiments, fluorescence from the sample is detected by a light detection system over one or more of the wavelength ranges from 200 nm to 1200 nm. In some cases, the method includes detecting fluorescence from the sample over a range of wavelengths, including, for example, 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 also 600 nm to 800 nm. In other cases, the method includes detecting fluorescence at one or more specific wavelengths. For example, fluorescence may be detected at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof, depending on the number of different fluorescence detectors in the light detection system of the subject. In certain embodiments, the method includes detecting the wavelength of light corresponding to the fluorescence peak wavelength of a specific fluorescent dye present in the sample.

[0054] Light can be measured continuously or at discrete intervals. In some cases, the detector of interest is configured to measure light continuously. In other cases, the detector of interest is configured to measure light at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at any other interval. The measurement of light from across the frost stream can be made one or more times during each discrete time interval, such as two or more times, such as three or more times, such as five or more times, and such as ten or more times. In certain embodiments, the light from the frost stream is measured two or more times by a light detector, and in certain cases, the data is averaged.

[0055] Each light detector may be positioned at any suitable distance from the frost stream as long as the available light signal is detectable. For example, the detector within the subject system may be positioned at 1 mm or more from the frost stream, including 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, 50 mm or more, 100 mm or more, 150 mm or more, 250 mm or more, and 500 mm or more from the frost stream. The detector may also be positioned at any angle from the frost stream. For example, the detector may be angled with respect to the vertical axis of the frost stream at 10° to 90°, including 15° to 85°, 20° to 80°, 25° to 75°, and 30° to 60°. In some cases, one or more detectors are positioned at 30° to 60° with respect to the vertical axis of the frost stream.

[0056] 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 optical signal from the irradiated flow stream may be detected by one or more detectors configured as forward scatter detectors. In these embodiments, the forward scatter detectors are positioned on the opposite side of the flow stream from the light source and are positioned to collect and detect forward propagating (e.g., scattered) light. In some embodiments, the light from the irradiated sample in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, the light from the irradiated sample in the flow stream is detected in one or more scattered light detector channels. In some embodiments, the light from the irradiated bubbles in the flow stream is detected in a side scatter detector channel. In some embodiments, the light from the irradiated bubbles in the flow stream is detected in a forward scatter detector channel.

[0057] In an embodiment, the data signal is generated in response to light detected from irradiated particles in the flow stream. In an embodiment, the light from the particles in the sample is detected in two or more light detector channels, including, for example, four or more, for example, eight or more, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty-four or more, for example, twenty-eight or more, for example, thirty-two or more, for example, thirty-six or more, for example, forty or more, for example, forty-four or more, for example, forty-eight or more, for example, fifty-two or more, for example, fifty-six or more, for example, sixty or more, and sixty-four or more light detector channels.

[0058] FIG. 1 depicts introducing bubbles into a sample line and detecting the bubbles within a flow stream according to a particular embodiment. 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 taken in by the sample line and propagated through the flow stream toward an inspection region 101c within a flow cell 101d and irradiated by a light source 101e (e.g., a laser). In a priming state 102, the sample composition is propagated through the flow stream to the inspection region and irradiated by the light source, generating a data signal in response to light detected from the irradiated particles such as a fluorescence detector channel and a light scattering detector channel. A bubble 101f is introduced in a state 103 where the sample line 101a is retracted from the sample source 101b, resulting in the intake of air into the sample line 101a. At least one data signal (e.g., an electronic data signal) is generated and communicated to a processor to indicate that a bubble such as a temporal air gap has been introduced into the flow stream. During a bubble flow state 104, the sample line 101a is reinserted into the sample source 101b after a predetermined period, and the bubble 101f propagates toward the inspection region 101c. After the bubble 101f reaches the inspection region 101c, it is irradiated by the laser 101e, and the light from the irradiated bubble 101f is detected in a bubble detection state 105. The bubble may be detected by light scattering or may be determined to be present in the flow stream based on a temporal gap in the data signal generated in the fluorescence detector channel.

[0059] In some embodiments, the data signal is generated in response to light detected from the irradiated flow stream before introducing bubbles into the flow stream. In some cases, light is detected from the irradiated flow stream, and the data signal is generated in one or more photodetector channels for at least 0.0001 seconds, such as at least 0.0005 seconds, such as at least 0.001 seconds, such as at least 0.005 seconds, such as at least 0.01 seconds, such as at least 0.05 seconds, at least 0.1 seconds, at least 0.5 seconds, such as at least 1 second, such as at least 2 seconds, such as at least 3 seconds, such as at least 4 seconds, such as at least 10 seconds, such as at least 15 seconds, such as at least 30 seconds, and up to 60 seconds before introducing bubbles into the flow stream, and includes generating the data signal from the detected light from the irradiated flow stream.

[0060] In some embodiments, the data signal is generated in response to light detected from irradiated particles of a sample upstream from the bubbles in the flow stream. In some cases, the data signal is generated in response to light from particles upstream from the bubbles for at least 0.0001 seconds, such as at least 0.0005 seconds, such as at least 0.001 seconds, such as at least 0.005 seconds, such as at least 0.01 seconds, such as at least 0.05 seconds, at least 0.1 seconds, at least 0.5 seconds, such as at least 1 second, such as at least 2 seconds, such as at least 3 seconds, such as at least 4 seconds, such as at least 10 seconds, such as at least 15 seconds, such as at least 30 seconds, such as at least 60 seconds, such as at least 5 minutes, such as at least 10 minutes, and includes generating the data signal in response to light from particles upstream from the bubbles for at least 15 minutes.

[0061] Light can be continuously detected from the irradiated frost stream before introducing bubbles into the frost stream, for example, for 0.0001 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, 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, before introducing bubbles into the frost stream, and includes detecting light from the irradiated frost stream for 60 seconds or more before introducing bubbles into the frost stream.

[0062] Also, light can be continuously detected, for example, for 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, such as 60 seconds or more, such as 5 minutes or more, such as 10 minutes or more, for 0.0001 seconds or more, from irradiated particles upstream of the introduced bubbles, such as continuously detecting light from irradiated particles upstream of the introduced bubbles, and includes continuously detecting light from irradiated particles upstream of the introduced bubbles for 15 minutes or more.

[0063] In some embodiments, light can also be continuously detected, for example, for 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, such as 60 seconds or more, such as 5 minutes or more, such as 10 minutes or more, for 0.0001 seconds or more, from irradiated particles downstream of the introduced bubbles, such as continuously detecting light from irradiated particles downstream of the introduced bubbles, and includes continuously detecting light from irradiated particles downstream of the introduced bubbles for 15 minutes or more.

[0064] In an embodiment, the bubble is irradiated by a light source as the bubble passes through the inspection area. The light from the bubble is detected by a photodetector of the light detection system. In some embodiments, scattered light is detected from the passing bubble, such as side scattered light. When light from the irradiated bubble is detected, for example, one or more data signals indicating the presence of bubbles in the flow stream may be generated, such as when generated within one or more light scattering photodetector channels, including within two or more light scattering photodetector channels, such as four or more, such as eight or more, such as twelve or more, such as sixteen or more, such as thirty-two or more, and sixty-four or more. In certain embodiments, the method includes determining that a bubble is present in the flow stream and that no light is detected in one or more of the fluorescence detector channels. In these embodiments, a plot of the fluorescence intensity data signal versus time includes a temporal gap in the generated data signal where the bubble passes through the inspection area and no fluorescence is detected by the light detection system. In certain embodiments, the absence of a data signal generated within the fluorescence detector channel correlates with the presence of a data signal generated within the light scattering photodetector channel for the irradiated bubble within the flow stream.

[0065] In some embodiments, the data signal is generated in response to light detected from irradiated particles of a sample downstream of the bubble in the flow stream. In some instances, the data signal is generated in response to light from particles downstream of the bubble 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, such as 60 seconds or more, such as 5 minutes or more, such as 10 minutes or more, and includes generating a data signal in response to light from particles downstream of the bubble for 15 minutes or more.

[0066] In some embodiments, the absolute number of particles in a sample is determined using a data signal generated when a bubble is introduced into the flow stream, a data signal generated when the bubble is detected in the flow stream, and a data signal generated in response to light detected from an irradiated particle between when the bubble is introduced into the flow stream and when the bubble is detected. In some embodiments, the method includes counting the number of particles in the sample between when a bubble is introduced into the flow stream and when the bubble is detected. The number of particles can be counted based on the number of fluorescence data signals generated within the time from when the bubble is introduced into the flow stream and when the bubble is detected. In some embodiments, the number of particles counted during this period is divided by a predetermined volume such as a pre-calibrated volume for the sample line. In other embodiments, the volume used in calculating the absolute number of particles is the determined volume of the sample line containing the particles irradiated between the time when the bubble is introduced into the flow stream and the time when the bubble is detected.

[0067] In certain embodiments, the method includes generating a data signal intensity plot of the generated data signals as a function of time. In some cases, the data signal intensity plot includes the data signals generated in the fluorescence detector channel, and each marker (e.g., dot) on the plot corresponds to the fluorescence detected from the irradiated particle. In some cases, the data signal intensity plot includes a visualization (e.g., a line boundary) indicating the time of the data signal generated when a bubble is introduced into the flow stream. In some cases, the data signal intensity plot includes the absence of the data signal when the bubble is detected. For example, the data signal intensity plot may include only the data signals generated within the fluorescence detector channel, and no fluorescence is detected when the bubble passes through the inspection region. In these embodiments, there is a time gap in the data signal intensity plot, indicating that the bubble has reached the inspection region.

[0068] Figure 2 depicts a flowchart for determining the absolute number of particles in a sample within a frost stream according to a particular embodiment. At step 201, particles propagating through the frost stream are irradiated with a light source such as a laser. At step 202, bubbles are introduced into the frost stream. In certain cases, the bubbles are introduced by retracting the sample line from the source of the sample composition, creating an air gap within the frost stream. Data signal 202a is generated in response to introducing bubbles into the frost stream. The light from the frost stream irradiated by the light source is detected at step 203 using a light detection system having a photodetector (e.g., fluorescence from particles in the sample). Data signal 203a within one or more photodetector channels is generated in response to the light detected from the irradiated particles in the frost stream. When a bubble reaches the inspection region, at step 204, the bubble is irradiated by the light source and detected by the light detection system. A data signal 204a indicating that the bubble has been detected is generated. In certain cases, the bubble is determined to be present based on the absence of the data signal generated in the fluorescence detector channel. In other cases, the data signal generated in the light scattering photodetector channel is generated in response to the light scattering detected from the irradiation of the bubble within the frost stream. The data signal generated from irradiating the particles in the flow, as well as the data signal generated when bubbles are introduced into the frost stream and detected by the light detection system, can be plotted on a data signal intensity plot at step 205. In other cases, the absolute number of particles in the sample is directly calculated at step 206 using data signals 202a, 203a, and 204a, for example, by using the count of data signal 203a generated from the time of the data signal (202a) when bubbles are introduced into the frost stream and the time of the data signal (204a) when the bubbles are detected. In a particular embodiment, the absolute number is calculated using the generated data signal intensity plot (step 205a).

[0069] Figure 3 depicts a data signal intensity plot 300 generated from light detected from irradiated particles and bubbles introduced into a flow stream, according to a particular embodiment. Each dot 301 on plot 300 corresponds to a data signal generated for the light detected from each particle. The intensity of the data signal is plotted on the y-axis and the time at which the data signal was generated is plotted on the x-axis. The time 302 at which a bubble is introduced into the flow stream is shown on the plot. For example, time 302 may be based on an electronic data signal generated when a sample line is retracted from a sample source. The time at which a bubble is detected is indicated by a temporal gap 303 in the data signals within the data signal intensity plot. The absolute number of particles in a sample can be calculated based on the volume of the sample line between time 302 and time 303 (i.e., the time between when a bubble is introduced into the flow stream and when the bubble is detected). In some instances, the volume is a pre-calibrated volume of the sample line.

[0070] A system configured to determine the absolute number of particles in a sample Aspects of the present disclosure also include a system (e.g., a flow cytometer) 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 a particular embodiment includes a flow cell configured to propagate a sample having particles in a flow stream, a sample line in fluid communication with a sample source configured to introduce bubbles into the flow stream, a light source for irradiating the flow stream, and a light detection system having a photodetector for detecting light from the irradiated flow stream.

[0071] In an embodiment, the system is configured to introduce bubbles into the frost stream. In certain cases, the system is configured to create an air gap within the frost stream. In some embodiments, the system is comprised of a gas source for introducing gas-containing bubbles into the frost stream. In some cases, the frost stream includes a gas input so that gas can be introduced into the frost stream. For example, the bubbles can be introduced into the frost stream via a gas input from an inert gas source, such as when nitrogen, helium, or argon is input into the frost stream during sample intake into the frost stream. The gas source can be configured to introduce any volume of gas into the frost stream, including, for example, 0.001 μL or more, for example, 0.005 μL or more, for example, 0.01 μL or more, for example, 0.05 μL or more, for example, 0.1 μL or more, for example, 0.5 μL or more, for example, 1 μL or more, for example, 2 μL or more, for example, 3 μL or more, for example, 4 μL or more, for example, 5 μL or more, and 10 μL or more, depending on the volume of the sample and the sample line.

[0072] In certain embodiments, bubbles are generated by creating an air gap within the flow stream. In some cases, the system includes a sample line displacement component configured to retract the sample line from the sample source to create an air gap within the flow stream. In some embodiments, while the sample line is being retracted from the sample source, the sample line displacement component retracts the sample from the sample source and the sample line entraps air. The sample line displacement component is configured to reinsert the sample line into the sample source when an air gap is created within the flow stream. The sample line displacement component can be any convenient protocol for retracting the sample line from the sample source, such as when the displacement is manual, mechanical, or uses a motor-driven displacement device. In certain embodiments, the sample line is retracted using a motor-driven actuator such as a motor-operated geared actuation device employing a motor-driven lead screw assembly, a stepper motor, a servo motor, a brushless electric motor, a brushed DC motor, a microstep drive motor, a high-resolution stepper motor, among other types of motors.

[0073] 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 sample source by at least 0.00001 mm, including, for example, at least 0.00005 mm, for example, at least 0.0001 mm, for example, at least 0.0005 mm, for example, at least 0.001 mm, for example, at least 0.005 mm, for example, at least 0.01 mm, for example, at least 0.05 mm, for example, at least 0.1 mm, for example, at least 0.5 mm, for example, at least 1 mm, for example, at least 2 mm, for example, at least 3 mm, for example, at least 5 mm, for example, at least 10 mm, for example, at least 15 mm, for example, at least 25 mm, and at least 50 mm. The sample line displacement component can be configured to retract the sample line from the sample source for at least 0.0001 seconds, including, for example, at least 0.0005 seconds, for example, at least 0.01 seconds, for example, at least 0.05 seconds, for example, at least 0.1 seconds, for example, at least 0.5 seconds, for example, at least 1 second, for example, at least 2 seconds, for example, at least 3 seconds, for example, at least 4 seconds, for example, at least 5 seconds, for example, at least 10 seconds, for example, at least 15 seconds, and at least 30 seconds.

[0074] In embodiments, the system includes a light source for irradiating the flow stream. In embodiments, the light source can be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, beads, non-cellular particles, etc.), the light source can be configured to emit light at wavelengths that vary in the range of 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and for example, 400 nm to 800 nm. For example, the light source can include a broadband light source that emits light having wavelengths in the range of 200 nm to 900 nm. In other cases, the light source includes a narrowband light source that emits wavelengths in the range of 200 nm to 900 nm. For example, the light source can be a narrowband LED (1 nm to 25 nm) that emits light having wavelengths in the range of 200 nm to 900 nm.

[0075] 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-fluoride (ArF) excimer laser, a krypton-fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser or a xenon-fluoride (XeF) excimer laser, or a combination thereof, a dye laser such as a stilbene, coumarin, or rhodamine laser, a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and a combination thereof, a metal-vapor laser, a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, and a combination thereof, a solid-state laser, a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or an embodiment of frequency doubling or frequency tripling of any of the above lasers.

[0076] In other embodiments, the light source is a non-laser light source including, but not limited to, a halogen lamp, a deuterium arc lamp, a lamp including a xenon arc lamp, a broadband LED having a continuous spectrum, a superluminescent light emitting diode, a semiconductor light emitting diode, a wide area spectrum LED white light source, a multi-LED integration, etc. In some cases, the non-laser light source is, among other light sources, a stabilized fiber-coupled broadband light source, a white light source, or any combination thereof.

[0077] In certain embodiments, the light source is an optical beam generator configured to generate two or more beams of frequency-shifted light. In some cases, this optical beam generator includes a laser and a high-frequency generator configured to apply a high-frequency drive signal to an 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 optical 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-fluoride (ArF) excimer laser, a krypton-fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser or a xenon-fluoride (XeF) excimer laser, or a combination thereof, a dye laser such as a stilbene, coumarin, or 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 (NeCu) laser, a copper laser, or a gold laser, and a combination thereof, a solid-state laser such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, and a combination thereof.

[0078] An acousto-optic device can be any convenient acousto-optic protocol configured to frequency shift a laser beam using an applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device within the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. This high-frequency drive signal can be applied to the acousto-optic device from any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0079] In an embodiment, the controller is configured to apply a high-frequency drive signal to the acousto-optic device, such as three or more high-frequency drive signals, such as four or more high-frequency drive signals, such as five or more high-frequency drive signals, such as six or more high-frequency drive signals, such as seven or more high-frequency drive signals, such as eight or more high-frequency drive signals, such as nine or more high-frequency drive signals, such as ten or more high-frequency drive signals, such as fifteen or more high-frequency drive signals, such as twenty-five or more high-frequency drive signals, such as fifty or more high-frequency drive signals, such as one hundred or more high-frequency drive signals, to generate a desired number of angularly deflected laser beams within the output laser beam.

[0080] In some cases, to generate an intensity profile of an angularly deflected laser beam within an output laser beam, the controller is configured to apply a high-frequency drive signal having an amplitude that varies, for example, from about 0.001V to about 500V, including, for example, from about 0.005V to about 400V, for example, from about 0.01V to about 300V, for example, from about 0.05V to about 200V, for example, from about 0.1V to about 100V, for example, from about 0.5V to about 75V, for example, from about 1V to 50V, for example, from about 2V to 40V, for example, from 3V to about 30V, and from about 5V to about 25V. In some embodiments, each applied high-frequency drive signal has a frequency of, for example, from about 0.001MHz to about 500MHz, including, for example, from about 0.005MHz to about 400MHz, for example, from about 0.01MHz to about 300MHz, for example, from about 0.05MHz to about 200MHz, for example, from about 0.1MHz to about 100MHz, for example, from about 0.5MHz to about 90MHz, for example, from about 1MHz to about 75MHz, for example, from about 2MHz to about 70MHz, for example, from about 3MHz to about 65MHz, for example, from about 4MHz to about 60MHz, and from about 5MHz to about 50MHz.

[0081] In certain embodiments, the controller has a processor operably coupled to a memory such that the memory contains instructions stored thereon, and the instructions, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam with a desired intensity profile. For example, the memory may contain instructions to generate two or more angularly deflected laser beams having the same intensity, such as three or more, four or more, five or more, ten or more, twenty-five or more, fifty or more, and the memory may contain instructions to generate one hundred or more angularly deflected laser beams having the same intensity. In other embodiments, the memory may contain instructions to generate two or more angularly deflected laser beams having different intensities, such as three or more, four or more, five or more, ten or more, twenty-five or more, fifty or more, and including that the memory may contain instructions to generate one hundred or more angularly deflected laser beams having different intensities.

[0082] In certain embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory contains instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center along the horizontal axis. In these instances, the intensity of the angularly deflected laser beam at the center of the output beam can range from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, including, for example, from 0.5% to about 95%, from 1% to about 90%, from about 2% to about 85%, from about 3% to about 80%, from about 4% to about 75%, from about 5% to about 70%, from about 6% to about 65%, from about 7% to about 60%, from about 8% to about 55%, and 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 contains instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center along the horizontal axis. In these instances, the intensity of the angularly deflected laser beam at the edge of the output beam can range from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, including, for example, from 0.5% to about 95%, from 1% to about 90%, from about 2% to about 85%, from about 3% to about 80%, from about 4% to about 75%, from about 5% to about 70%, from about 6% to about 65%, from about 7% to about 60%, from about 8% to about 55%, and 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 yet other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory contains instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile with a Gaussian distribution 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 contains instructions 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.

[0083] In an embodiment, the light beam generator of interest may be configured to generate angularly deflected laser beams within the output laser beam that are spatially separated. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, such as 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, 100 μm or more, 500 μm or more, 1000 μm or more, and 5000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams within the output laser beam that overlap, such as adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (such as overlap of beam spots) can be an overlap of 0.001 μm or more, such as an overlap of 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, and 100 μm or more.

[0084] In certain instances, a light beam generator configured to generate two or more beams of frequency-shifted 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 Publications 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0085] The light source can be positioned at any suitable distance from the flow stream, such as a distance of 0.001 mm or more from a frost stream that includes distances of, for example, 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 100 mm or more. Additionally, the light source irradiates the flow stream at an angle in the range of 10° to 90°, including, for example, any suitable angle (e.g., with respect to the vertical axis of the flow stream), such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and 30° to 60°, such as 90°.

[0086] The light source can be configured to irradiate the sample continuously or in discrete periods. In some cases, the system includes a light source configured to continuously irradiate the sample, such as a continuous wave laser that continuously irradiates the flow stream at the interrogation point of a flow cytometer. In other cases, the system of interest includes a light source configured to irradiate the sample in discrete periods that include, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or some other period. When the light source is configured to irradiate the sample in discrete periods, the system can include one or more additional components for providing intermittent irradiation of the sample using the light source. For example, the system of the subject matter in these embodiments can include one or more laser beam choppers that are manual or computer-controlled beam stops for blocking and exposing the sample to the light source.

[0087] The system includes an optical detection system having an optical detector for detecting light from an irradiated frost stream. The optical detector may be any convenient optical detection protocol including, but not limited to, among other optical detectors, active pixel sensors (APS), avalanche photodiodes (APD), quadrant photodiodes, image sensors, charge coupled devices (CCD), intensified charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the optical detector is, for example, 0.05 cm 2 ~9 cm 2 、for example, 0.1 cm 2 ~8 cm 2 、for example, 0.5 cm 2 ~7 cm 2 、and 1 cm 2 ~5 cm 2 and includes a photomultiplier tube having an active detection surface area in the range of 0.01 cm 2 ~10 cm 2 for each region.

[0088] In some embodiments, the light detection system of interest includes a plurality of photodetectors. In some cases, the light detection system includes a plurality of solid-state detectors such as photodiodes. In certain cases, the light detection system includes an array of photodetectors such as a photodiode array. In these embodiments, the photodetector array can include four or more photodetectors, for example, 10 or more photodetectors, for example, 25 or more photodetectors, for example, 50 or more photodetectors, for example, 100 or more photodetectors, for example, 250 or more photodetectors, for example, 500 or more photodetectors, for example, 750 or more photodetectors, and 1000 or more photodetectors. For example, the detector can be a photodiode array having four or more photodiodes, for example, 10 or more photodiodes, for example, 25 or more photodiodes, for example, 50 or more photodiodes, for example, 100 or more photodiodes, for example, 250 or more photodiodes, for example, 500 or more photodiodes, for example, 750 or more photodiodes, and 1000 or more photodiodes.

[0089] The photodetectors can be arranged in any geometric configuration as needed, in which case the arrangement of interest includes, but is not limited to, a square configuration, a rectangular configuration, a trapezoidal configuration, a triangular configuration, a hexagonal configuration, a heptagonal configuration, an octagonal configuration, a nonagonal configuration, a decagonal configuration, a dodecagonal configuration, a circular configuration, an oval configuration, and an irregularly patterned configuration. The photodetectors in the photodetector array can be oriented at an angle ranging from 10° to 180°, for example, 15° to 170°, for example, 20° to 160°, for example, 25° to 150°, for example, 30° to 120°, and 45° to 90° with respect to the other plane (as referenced in the X-Z plane). The photodetector array can be of any suitable shape, a shape consisting of straight lines, for example, square, rectangular, trapezoidal, triangular, hexagonal, etc., a shape consisting of curves, for example, circular, oval, and an irregular shape, for example, a parabolic base coupled to the upper part of the plane. In certain embodiments, the photodetector array has a rectangular-shaped effective surface.

[0090] Each photodetector (e.g., a photodiode) in the array may have an effective surface having a width in the range of 5 μm to 250 μm, including, for example, 10 μm to 225 μm, for example, 15 μm to 200 μm, for example, 20 μm to 175 μm, for example, 25 μm to 150 μm, for example, 30 μm to 125 μm, and 50 μm to 100 μm, and a length in the range of 5 μm to 250 μm, including, for example, 10 μm to 225 μm, for example, 15 μm to 200 μm, for example, 20 μm to 175 μm, for example, 25 μm to 150 μm, for example, 30 μm to 125 μm, and 50 μm to 100 μm. In this case, the surface area of each photodetector (e.g., a photodiode) in the array is, for example, 50 μm 2 ~9000 μm 2 、for example, 75 μm 2 ~8000 μm 2 、for example, 100 μm 2 ~7000 μm 2 、for example, 150 μm 2 ~6000 μm 2 、and 200 μm 2 ~5000 μm 2 including, 25 μm 2 ~10000 μm 2 ranging over.

[0091] The size of the photodetector array can vary according to the amount and intensity of light, the number of photodetectors, and the desired sensitivity, and can have a length in the range of 0.01 mm to 100 mm, including, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, and 5 mm to 25 mm. The width of the photodetector array can also vary in the range of 0.01 mm to 100 mm, including, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, and 5 mm to 25 mm. Therefore, the effective surface of the photodetector array is, for example, 0.5 mm 2 ~5000 mm 2 、for example, 1 mm 2~1000 mm 2 For example, 5 mm 2 ~500 mm 2 and 10 mm 2 ~100 mm 2 including 0.1 mm 2 ~10000 mm 2 can be in the range of

[0092] The light can be measured by a photodetector at one or more wavelengths, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, and includes measuring the light from particles in a frost stream at four hundred or more different wavelengths. In some embodiments, the fluorescence from the sample is detected by a light detection system over one or more of the wavelength ranges from 200 nm to 1200 nm. In some cases, the method includes detecting fluorescence from the sample over a range of wavelengths including, for example, 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 600 nm to 800 nm. In other cases, the method includes detecting fluorescence at one or more specific wavelengths. For example, the fluorescence may be detected at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof, depending on the number of different fluorescence detectors in the light detection system of the subject matter.

[0093] Light can be measured continuously or at discrete intervals. In some cases, the detector of interest is configured to measure light continuously. In other cases, the detector of interest is configured to measure light at discrete intervals, such as, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at any other interval. The measurement of light across the flow stream can be made one or more times during each discrete time interval, including, for example, two or more times, three or more times, five or more times, and ten or more times. In certain embodiments, the light from the flow stream is measured two or more times by a light detector, and in certain cases, the data is averaged.

[0094] Each light detector may be positioned at any suitable distance from the flow stream as long as the available light signal is detectable. For example, the detector within the subject system may be positioned at 1 mm or more from the flow stream, including, for example, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, 50 mm or more, 100 mm or more, 150 mm or more, 250 mm or more, and 500 mm or more from the flow stream. The detector may also be positioned at any angle from the flow stream. For example, the detector may be angled with respect to the vertical axis of the flow stream at 10° to 90°, including, for example, 15° to 85°, 20° to 80°, 25° to 75°, and 30° to 60°. In some cases, one or more detectors are positioned at 30° to 60° with respect to the vertical axis of the flow stream.

[0095] 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 optical signal from the irradiated flow stream may be detected by one or more detectors configured as forward scatter detectors. In these embodiments, the forward scatter detectors are positioned on the opposite side of the flow stream from the light source and are positioned to collect and detect forward propagating (e.g., scattered) light. In some embodiments, the light from the irradiated sample in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, the light from the irradiated sample in the flow stream is detected in one or more scattered light detector channels. In some embodiments, the light from the irradiated bubbles in the flow stream is detected in a side scatter detector channel. In some embodiments, the light from the irradiated bubbles in the flow stream is detected in a forward scatter detector channel.

[0096] 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 may be physically coupled to the light detection system by, for example, an adhesive, co-molded with the light detection system, or integrated with 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 light collection system is coupled to the light detection system by a connector such as a hook and loop fastener, a magnet, a latch, a notch, a socket, a counterbore, a groove, a pin, a tether, a hinge, Velcro®, a non-permanent adhesive, or a combination thereof.

[0097] 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 may be positioned at least 0.001 mm, such as at least 0.005 mm, at least 0.01 mm, at least 0.05 mm, at least 0.1 mm, at least 0.5 mm, at least 1 mm, at least 10 mm, at least 25 mm, at least 50 mm, and up to 100 mm or more from the light detection system.

[0098] In certain embodiments, the optical collection system includes an optical fiber. For example, the optical collection system may be an optical fiber light relay bundle, and light from the plume stream is transmitted through the optical fiber light relay bundle to a first set of linear variable optical filters of the light detection system. Any optical fiber light relay system may be employed to transmit light, and in certain embodiments, suitable optical fiber light relay systems include, but are not limited to, those described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0099] 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 an optical propagation that employs a configuration of one or more optical components to direct light from a sample through free space to a light detection system. 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 lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof. For example, in some embodiments, the free-space optical relay system of interest includes one or more focusing lenses. In other embodiments, the free-space optical relay system of the subject includes one or more mirrors. In still other embodiments, the free-space optical relay system includes a collimating lens. In certain embodiments, a suitable free-space optical relay system for propagating light from a sample to a first set of linear variable optical filters of a light detection system includes, but is not limited to, optical relay systems such as those described in U.S. Patent Nos. 7,643,142, 7,728,974, and 8,223,445, the disclosures of which are incorporated herein by reference.

[0100] In some embodiments, the system includes a processor having a memory, the memory being operably coupled to the processor, the memory including instructions stored thereon, the instructions causing the processor to generate a data signal to be introduced into the flow stream when executed by the processor. In some cases, the memory includes instructions for generating a data signal when the volume of gas is input into or taken into the sample line. In other examples, the memory includes instructions for generating a data signal at a predetermined time after a bubble has been introduced into the flow stream, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2 μs or more, for example, 3 μs or more, for example, 5 μs or more, for example, 10 μs or more, for example, 100 μs or more, for example, 500 μs or more, and 1000 μs or more after a bubble has been introduced into the flow stream, for example, 0.001 μs or more.

[0101] In certain embodiments, the memory includes instructions for generating a data signal when a bubble is introduced into the flow stream by retracting the sample line from the sample source. In some cases, the memory includes instructions for generating a data signal when the sample line is retracted from the sample source. In other embodiments, the memory includes instructions for generating a data signal at a predetermined time after the sample line has been retracted from the sample source, for example, at least 0.005 μs, for example, at least 0.01 μs, for example, at least 0.05 μs, for example, at least 0.1 μs, for example, at least 0.5 μs, for example, at least 1 μs, for example, at least 2 μs, for example, at least 3 μs, for example, at least 5 μs, for example, at least 10 μs, for example, at least 100 μs, for example, at least 500 μs, and after the sample line has been retracted from the sample source, at least 1000 μs, for example, after the sample line has been retracted from the sample source, for example, at least 0.001 μs. 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 uptake into the sample line is re-established). In yet another embodiment, the memory includes instructions for generating a data signal at a predetermined time after the sample line has been reinserted into the sample source, for example, at least 0.005 μs, for example, at least 0.01 μs, for example, at least 0.05 μs, for example, at least 0.1 μs, for example, at least 0.5 μs, for example, at least 1 μs, for example, at least 2 μs, for example, at least 3 μs, for example, at least 5 μs, for example, at least 10 μs, for example, at least 100 μs, for example, at least 500 μs, and after the sample line has been reinserted into the sample source, at least 1000 μs, after the sample line has been retracted from the sample source, for example, at least 0.001 μs.

[0102] In some cases, the memory includes instructions for generating an electronic data signal when bubbles are introduced into the flow stream. In other cases, the memory includes instructions for generating an optical data signal when bubbles are introduced into the flow stream. In some cases, the system includes one or more photodetectors positioned to detect light from a sample line into which bubbles are introduced. For example, the system may include a light source, such as an LED or a laser, for irradiating the flow stream into which bubbles are introduced, and one or more photodetectors (e.g., a scattered light detector) for detecting light from the introduced bubbles within the flow stream. In certain embodiments, the above-described light detection system may include one or more photodetector channels for detecting light from the introduced bubbles within the flow stream.

[0103] If the system is configured to introduce bubbles by retracting the sample line from the sample source, the photodetector and the light source for detecting when bubbles are introduced into the flow stream may be adjacent to the end of the sample line or downstream from the end of the sample line. In some embodiments, the photodetector and the light source for detecting the introduction of bubbles into the flow stream may be positioned downstream from the end of the sample line by 0.001 mm or more, including, for example, 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2 mm or more, for example, 3 mm or more, for example, 4 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and 50 mm or more downstream from the end of the sample line. In some embodiments, the memory includes instructions for transmitting the optical data signal to a processor for use in calculating the particle count of the sample.

[0104] In some embodiments, the system includes a memory having instructions for generating a data signal in response to light detected from irradiated particles in a flow stream. In certain embodiments, the memory includes instructions for generating a data signal in response to light from particles in a sample within two or more photodetector channels, including within, for example, four or more, for example, eight or more, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty-four or more, for example, twenty-eight or more, for example, thirty-two or more, for example, thirty-six or more, for example, forty or more, for example, forty-four or more, for example, forty-eight or more, for example, fifty-two or more, for example, fifty-six or more, for example, sixty or more, and sixty-four or more photodetector channels.

[0105] In some embodiments, the memory includes instructions for generating a data signal in response to light detected from an irradiated flow stream before introducing bubbles into the flow stream. In some cases, the memory includes instructions for generating a data signal from detected light within one or more photodetector channels from the irradiated flow stream for, for example, 0.0001 seconds or more, for example, 0.0005 seconds or more, for example, 0.001 seconds or more, for example, 0.005 seconds or more, for example, 0.01 seconds or more, for example, 0.05 seconds or more, for example, 0.1 seconds or more, for example, 0.5 seconds or more, for example, 1 second or more, for example, 2 seconds or more, for example, 3 seconds or more, for example, 4 seconds or more, for example, 10 seconds or more, for example, 15 seconds or more, for example, 30 seconds or more before bubbles are introduced into the flow stream, and includes instructions for generating a data signal from detected light from the irradiated flow stream for 60 seconds or more before bubbles are introduced into the flow stream.

[0106] In some embodiments, the memory includes instructions for generating a data signal in response to light detected from irradiated particles of a sample upstream from bubbles in a flow stream. In some cases, the memory includes instructions for generating a data signal in response to light from particles upstream from bubbles for, for example, 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, such as 60 seconds or more, such as 5 minutes or more, such as 10 minutes or more. The case where the memory includes instructions for generating a data signal in response to light from particles upstream from bubbles for 15 minutes or more is included.

[0107] In some cases, the memory includes instructions for continuously detecting light from a flow stream irradiated before introducing bubbles into the flow stream, such as for 0.0001 seconds or more before introducing bubbles into the flow stream, 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. The case where the memory includes instructions for detecting light from a flow stream irradiated before introducing bubbles into the flow stream for 60 seconds or more is included.

[0108] In some cases, the memory includes instructions for continuously detecting light from irradiated particles upstream of the introduced bubbles, for example, for 0.0001 seconds or more, such as 0.0005 seconds or more, for example 0.001 seconds or more, for example 0.005 seconds or more, for example 0.01 seconds or more, for example 0.05 seconds or more, for example 0.1 seconds or more, for example 0.5 seconds or more, for example 1 second or more, for example 2 seconds or more, for example 3 seconds or more, for example 4 seconds or more, for example 10 seconds or more, for example 15 seconds or more, for example 30 seconds or more, for example 60 seconds or more, for example 5 minutes or more, for example 10 minutes or more, and includes cases where the memory includes instructions for continuously detecting light from irradiated particles upstream of the introduced bubbles for 15 minutes or more.

[0109] In some cases, the memory includes instructions for continuously detecting light from irradiated particles downstream of the introduced bubbles, for example, for 0.0001 seconds or more, such as 0.0005 seconds or more, for example 0.001 seconds or more, for example 0.005 seconds or more, for example 0.01 seconds or more, for example 0.05 seconds or more, for example 0.1 seconds or more, for example 0.5 seconds or more, for example 1 second or more, for example 2 seconds or more, for example 3 seconds or more, for example 4 seconds or more, for example 10 seconds or more, for example 15 seconds or more, for example 30 seconds or more, for example 60 seconds or more, for example 5 minutes or more, for example 10 minutes or more, and includes cases where the memory includes instructions for continuously detecting light from irradiated particles downstream of the introduced bubbles for 15 minutes or more.

[0110] In some embodiments, the memory includes instructions for generating a data signal in response to light detected from irradiated particles of a sample downstream from bubbles in the flow stream. In some cases, the memory includes instructions for generating a data signal in response to light from particles downstream from bubbles for a time of 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, such as 60 seconds or more, such as 5 minutes or more, such as 10 minutes or more, including cases where the memory includes instructions for generating a data signal in response to light from particles downstream from bubbles for 15 minutes or more.

[0111] In some embodiments, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to use data signals generated when bubbles are introduced into the flow stream, data signals generated when bubbles are detected in the flow stream, and data signals generated in response to light detected from particles irradiated between the time bubbles are introduced into the flow stream and the time bubbles are detected, to determine the absolute number of particles in a sample. In some embodiments, the memory includes instructions for counting the number of particles in the sample between the time bubbles are introduced into the flow stream and the time bubbles are detected. The number of particles can be counted based on the number of fluorescence data signals generated within the time from when bubbles are introduced into the flow stream and when bubbles are detected. In some embodiments, the memory includes instructions for counting the number of particles during this period and dividing the count by a predetermined volume such as a volume pre-calibrated for the sample line. In other embodiments, the memory includes instructions for determining the volume of the sample line containing particles irradiated between the time bubbles are introduced into the flow stream and the time bubbles are detected. In some cases, the memory includes instructions for calculating the absolute number using the determined volume of the sample line containing particles irradiated between the time bubbles are introduced into the flow stream and the time bubbles are detected.

[0112] In certain embodiments, the memory includes instructions for generating a data signal intensity plot of the generated data signal as a function of time. In some cases, the memory includes instructions for generating a data signal intensity plot that includes the data signals generated in the fluorescence detector channels. In some cases, the memory includes instructions for generating a data signal intensity plot where each marker (e.g., dot) on the plot corresponds to the fluorescence detected from the irradiated particles. In some cases, the memory includes instructions for generating a data signal intensity plot that includes a visualization (e.g., line boundary) of the data signals generated when bubbles are introduced into the flow stream. In some cases, the data signal intensity plot includes the absence of data signals when bubbles are detected. For example, the data signal intensity plot may include only the data signals generated within the fluorescence detector channels, and no fluorescence is detected when the bubbles pass through the inspection area. In these embodiments, there is a time gap in the data signal intensity plot, indicating that the bubbles have reached the inspection area.

[0113] In certain embodiments, the system further includes a flow cell configured to propagate a sample in a flow stream. Any convenient flow cell for propagating a fluid sample to a sample inspection area may be employed. In some embodiments, the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal conical portion terminating in a flat surface having an orifice that is transverse to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) can vary in the range of 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, for example, 2 mm to 10 mm, for example, 3 mm to 9 mm, and 4 mm to 8 mm. The length of the distal frustoconical portion (measured along the longitudinal axis) can also vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, and 4 mm to 7 mm. The diameter of the flow cell nozzle chamber can vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, and 4 mm to 7 mm in some embodiments.

[0114] In certain cases, the flow cell does not include a cylindrical portion, and the entire internal chamber of the flow cell is formed in a frustum shape. In these embodiments, the length of the frustum-shaped internal chamber (measured along the longitudinal axis transverse to the nozzle orifice) can be in the range of 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, for example, 2 mm to 10 mm, for example, 3 mm to 9 mm, and 4 mm to 8 mm. The diameter of the proximal portion of the frustum-shaped nozzle chamber can be in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, and 4 mm to 7 mm.

[0115] In some embodiments, the sample flow stream is emitted from the orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the flow cell orifice can be of any suitable shape, and the cross-sectional shapes of interest include, but are not limited to, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., for example, curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as, for example, a parabolic bottom portion coupled to a planar upper portion. In certain embodiments, the flow cell of interest has a circular orifice. The size of the nozzle orifice can vary in some embodiments in the range of 1 μm to 20,000 μm, including, for example, 2 μm to 17,500 μm, for example, 5 μm to 15,000 μm, for example, 10 μm to 12,500 μm, for example, 15 μm to 10,000 μm, for example, 25 μm to 7,500 μm, for example, 50 μm to 5,000 μm, for example, 75 μm to 1,000 μm, for example, 100 μm to 750 μm, and 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0116] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell. In an embodiment, the sample injection system is configured to provide a suitable flow of the sample into the flow cell interior chamber. Depending on the desired characteristics of the flow stream, the rate of the sample transmitted to the flow cell chamber by the sample injection port can be, for example, 1 μL / min or more, including, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 15 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, and 100 μL / min or more. In some cases, the rate of the sample transmitted to the flow cell chamber by the sample injection port can be, for example, 1 μL / sec or more, including, for example, 2 μL / sec or more, for example, 3 μL / sec or more, for example, 5 μL / sec or more, for example, 10 μL / sec or more, for example, 15 μL / sec or more, for example, 25 μL / sec or more, for example, 50 μL / sec or more, and 100 μL / sec or more.

[0117] The sample injection port can be an orifice positioned in the wall of the interior chamber or a conduit positioned at the proximal end of the interior chamber. When the sample injection port is an orifice positioned in the wall of the interior chamber, the sample injection port orifice can be of any suitable shape, and examples of cross-sectional shapes of interest include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In a particular embodiment, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on the shape, and in a particular case, it has an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0118] In certain cases, the sample injection port is a conduit positioned at the proximal end of the flow cell internal chamber. For example, the sample injection port can be a conduit positioned to have an orifice of the sample injection port along the flow cell orifice. When the sample injection port is a conduit positioned along the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, and examples of cross-sectional shapes of interest include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. The orifice of the conduit can vary depending on the shape, and in certain cases, for example, it has an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, for example, 1.5 mm. The shape of the tip of the sample injection port can be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port can include a tapered tip having a bevel angle in the range of 1° to 10°, including, for example, 2° to 9°, for example, 3° to 8°, for example, 4° to 7°, and 5°.

[0119] In some embodiments, the flow cell also includes a sheath fluid injection port configured to provide a sheath fluid to the flow cell. In an embodiment, the sheath fluid injection system is configured to provide a flow of sheath fluid to the flow cell internal chamber, for example, in combination with the sample, to generate a laminar flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid transmitted to the flow cell chamber can be, for example, 25 μL / second or more, including, for example, 50 μL / second or more, for example, 75 μL / second or more, for example, 100 μL / second or more, for example, 250 μL / second or more, for example, 500 μL / second or more, for example, 750 μL / second or more, for example, 1000 μL / second or more, and 2500 μL / second or more.

[0120] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the inner chamber. The sheath fluid injection port orifice can be of any suitable shape, and the cross-sectional shape of interest includes, but is not limited to, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., for example, curved cross-sectional shapes such as circular, elliptical, etc., and irregular shapes such as, for example, a parabolic bottom coupled to a planar top. The size of the sample injection port orifice can vary depending on the shape, and in certain cases, for example, has an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0121] In some embodiments, the system further includes a pump that is in fluid communication with the flow cell to propagate a flow stream through the flow cell. Any convenient fluid pump protocol can be employed to control the flow of the flow stream through the flow cell. In certain instances, the system includes a peristaltic pump having a pulse damper. The pump within the system of the present subject matter is configured to transfer 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 the sample flow within the flow cell is, for example, 1 μL / min (microliters per minute) or more, including, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, for example, 75 μL / min or more, for example, 100 μL / min or more, for example, 250 μL / min or more, for example, 500 μL / min or more, for example, 750 μL / min or more, and 1000 μL / min or more. For example, the system can include a pump configured to flow a sample through the flow cell at a rate in the range of 1 μL / min to 500 μL / min, including, for example, 1 μL / min to 250 μL / min, for example, 1 μL / min to 100 μL / min, for example, 2 μL / min to 90 μL / min, for example, 3 μL / min to 80 μL / min, for example, 4 μL / min to 70 μL / min, for example, 5 μL / min to 60 μL / min, and 10 μL / min to 50 μL / min. In certain embodiments, the flow rate of the flow stream is 5 μL / min to 6 μL / min.

[0122] In certain embodiments, a light detection system having a plurality of photodetectors as described above is part of, or is positioned within, a particle analyzer such as a particle sorter. In certain embodiments, the subject system is a flow cytometric system that includes photodiode and amplifier components as part of a light detection system for detecting light emitted by a sample in a flow stream. Suitable flow cytometry systems 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 No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1):17-28; Linden, et.al., Semin Throm Hemost. 2004 Oct; 30(5):502-11, Alison, et al. J Pathol, 2010 Dec; 222(4):335-344, and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference.In certain cases, the flow cytometry systems of interest include the BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and the BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, and BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, and the like.

[0123] In some embodiments, the subject system is a flow cytometric system such as those described in U.S. Patent 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; 9,095,494; 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 entirety.

[0124] 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, sample particles (e.g., cells) are sorted using a sorting determination module having a plurality of sorting determination 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 having a deflector plate such as those described in U.S. Patent Publication No. 2017 / 0299493, filed Mar. 28, 2017, the disclosure of which is incorporated herein by reference.

[0125] In certain instances, the flow cytometry system of the present invention is configured to image particles in a flow stream by fluorescence imaging using high-frequency tagged emission (FIRE), such as those described in Diebold, et al. Nature Photonics Vol.7(10);806-810(2013), and 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 Publications 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894. These disclosures are incorporated herein by reference.

[0126] In some embodiments, the system is a particle analyzer that can analyze and characterize particles, with or without physically sorting the particles into a collection container, using a particle analysis system 401 (FIG. 4A). FIG. 4A shows a functional block diagram of a particle analysis system for computer-based sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 shown in FIG. 4A can be configured to perform the methods described herein, in whole or in part. 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 through which sample particles 403 (e.g., cells) move along a common sample path 409.

[0127] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as the particles pass through one or more detection stations along a common sample path. Detection station 408 generally refers to the monitoring area 407 of the common sample path. In some embodiments, detection may include detecting the light of those particles, or one or more other characteristics, as the particles 403 pass through the monitoring area 407. In FIG. 4A, one detection station 408 having one monitoring area 407 is shown. Some embodiments of the particle analysis system 401 can include multiple detection stations. Further, some detection stations can monitor two or more areas.

[0128] Each signal is assigned a signal value for forming a data point for each particle. As described above, this data can be referred to as event data. The data point can be a multi-dimensional data point that includes the value of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0129] The particle analysis system 401 can also include a control system 306. The control system 406 can include one or more processors, an amplitude control circuit, and / or a frequency control circuit. The control system shown can be operably associated with the fluidics system 402. The control system can be configured to generate a signal frequency calculated for at least a portion of the first period based on a Poisson distribution and the number of data points collected by the detection system 404 during the first period. The control system 406 can be further configured to generate an experimental signal frequency based on the number of data points in a portion of the first period. Additionally, the control system 406 can compare the experimental signal frequency to that of the calculated signal frequency or a predetermined signal frequency.

[0130] Figure 4B shows a system 400 for flow cytometry according to an exemplary embodiment of the present invention. This 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.

[0131] The excitation lasers 115a - 115c emit light in the form of laser beams. The wavelengths of the laser beams emitted from the excitation lasers 415a - 415c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system of Figure 4B. The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. The beam splitter 445b transmits UV light (light having wavelengths in the range of 10 - 400 nm) and reflects light at 488 nm and 633 nm.

[0132] The laser beams are then directed to the focusing lens 420, which focuses the beams onto the portion of the fluid stream in the flow chamber 425 where the particles of the sample are located. The flow chamber is part of a fluidics system that directs the particles in the stream, typically one at a time, into the focused laser beam for investigation. The flow chamber can comprise a flow cell within a benchtop flow cytometer or a nozzle tip within a stream - in - air cytometer.

[0133] The light from the laser beam interacts with the particles in the sample through diffraction, refraction, reflection, scattering, and absorption with re - emission at various different wavelengths, depending on particle characteristics such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally occurring on or in the particle. Fluorescent emission, as well as diffracted light, refracted light, reflected light, and scattered light can be routed through one or more of beam splitters 445a - 445g, band - pass filters 450a - 450e, long - pass filters 455a - 455b, and fluorescence focusing lens 440 to one or more of forward scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a - 460f.

[0134] The fluorescence collection lens 440 collects the light emitted from the interaction between the particle and the laser beam and routes that light towards one or more beam splitters and filters. Band - pass filters such as band - pass filters 450a - 450e allow a narrow wavelength range to pass through the filter. For example, band - pass 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. Thus, the 510 / 20 filter spreads 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short - pass filters transmit wavelengths of light below a specified wavelength. Long - pass filters such as long - pass filters 455a - 455b transmit wavelengths of light above a specified wavelength. For example, long - pass filter 455a, which is a 670 nm long - pass filter, transmits light of 670 nm and above. Filters are often selected to optimize the detector's specificity for a particular fluorescent dye. Those filters can be configured so that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0135] The beam splitter guides light of different wavelengths in different directions. The beam splitter can be characterized by filter characteristics such as short pass and long pass. For example, beam splitter 445g is a 620SP beam splitter, which means that beam splitter 445g transmits light with a wavelength of 620 nm or less and reflects light with a wavelength longer than 620 nm in a different direction. In one embodiment, beam splitters 445a - 445g can include an optical mirror such as a dichroic mirror.

[0136] The forward scatter detector 430 is positioned slightly off - axis from the direct beam passing through the flow cell and is configured to detect diffracted light, excitation light that moves mostly in the forward direction through or around the particles. The intensity of the light detected by the forward scatter detector depends on the overall size of the particles. The forward scatter detector can include a photodiode. The side scatter detector 435 is configured to detect diffracted and reflected light from the surface and internal structure of the particles and tends to increase as the particle structure becomes more complex. Fluorescent emission from fluorescent molecules associated with the particles can be detected by one or more fluorescence detectors 460a - 460f. The side scatter detector 435 and the fluorescence detectors can include photomultiplier tubes. Signals detected by the forward scatter detector 430, the side scatter detector 435, and the fluorescence detectors can be converted by the detectors into electronic signals (voltages). This data can provide information about the sample.

[0137] Those skilled in the art will recognize that a flow cytometer according to an embodiment of the present invention is not limited to the flow cytometer depicted in FIG. 4B and can include any flow cytometer known in the art. For example, the flow cytometer can have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in various different configurations.

[0138] During operation, the operation of the flow cytometer is controlled by the controller / processor 490, and the measurement data from the detector is stored in the memory 495 and can be processed by the controller / processor 490. Although not explicitly shown, the controller / processor 190 is coupled to the detector to receive the output signal therefrom, and is also coupled to the electrical components and electromechanical parts of the flow cytometer 400 to control the laser, fluid flow parameters, etc. An input / output (I / O) functional unit 497 may also be provided within the system. The memory 495, the controller / processor 490, and the I / O 497 may be provided as an integral part of the flow cytometer 410. In such an embodiment, the display may also form part of the I / O functional unit 497 for presenting experimental data to the user of the flow cytometer 400. Alternatively, some or all of the memory 495, the controller / processor 490, and the I / O functional unit may 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 wirelessly or wired with the flow cytometer 410. Together with the memory 495 and the I / O 497, the controller / processor 490 may be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0139] The system illustrated in FIG. 4B includes six different detectors that detect fluorescence within six different wavelength bands (which may be referred to herein as "filter windows" for any detector), as defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in flow cytometry experiments emit light in their own characteristic wavelength bands. The specific fluorescent labels used in the experiment, and their associated fluorescence emission bands, can be selected to generally coincide with the filter windows of the detectors. However, due to the provision of more detectors and the utilization of more labels, a perfect correspondence between the filter windows and the fluorescence emission spectra is not possible. The peak of the emission spectrum of a particular fluorescent molecule may be within the filter window of one particular detector, but it is generally true that a portion of the emission spectrum of that label also overlaps with the filter windows of one or more other detectors. This may be referred to as spillover signal. I / O 497 can be configured to receive data related to flow cytometry experiments having a panel of fluorescent labels and multiple cell parent populations having a plurality of markers, each cell parent population having a subset of the plurality of markers. I / O 497 can also be configured to receive biological data that assigns one or more markers to one or more cell parent populations, marker concentration data, emission spectrum data, data that assigns labels to one or more markers, and cytometer configuration data. Flow cytometry experiment data such as label spectral characteristics and flow cytometer configuration data can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more assignments of labels to markers.

[0140] FIG. 5 shows a functional block diagram of an example of a particle analyzer control system, such as analysis controller 500, for analyzing and displaying biological events. Analysis controller 500 can be configured to implement various processes for controlling the graphic display of biological events.

[0141] The particle analyzer or particle sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometric event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or particle 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.

[0142] The analysis controller 500 can be configured to receive biological event data from the particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 can include flow cytometric event data. The analysis controller 500 can be configured to provide a graphical display including a first plot of the biological event data to the display device 506. The analysis controller 500 can be further configured to render a region of interest as a gate around the population of biological event data indicated by the display device 506, for example, superimposed on the first plot. In some embodiments, the gate can be a logical combination of one or more graphical regions of interest drawn on a histogram of a single parameter or a bivariate plot. In some embodiments, the display can be used to display particle parameters or saturated detector data.

[0143] The analysis controller 500 can be further configured to display the biological event data on the display device 506 within the gate differently from other events within the biological event data outside the gate. For example, the analysis controller 500 can be configured to render the color of the biological event data included within the gate to be distinguishable from the color of the biological event data outside the gate. The display device 506 can be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0144] The analysis controller 500 can be configured to receive a gate selection signal that identifies a gate from a first input device. For example, the first input device can be implemented as a mouse 510. This mouse 510 can initiate a gate selection signal to the analysis controller 500 that identifies a gate to be displayed or manipulated via the display device 506 (e.g., by clicking on the desired gate when positioning the cursor there). In some embodiments, the first device can be implemented as a keyboard 508, or as other means for providing an input signal to the analysis controller 500, such as a touch screen, input pen, light detector, or voice recognition system. Some input devices can include multiple input functions. In such embodiments, each input function can be considered an input device. For example, as shown in FIG. 5, the mouse 510 can include a right mouse button and a left mouse button, each of which can generate an activation event.

[0145] The activation event can cause the analysis controller 500 to change how data is displayed, which portions of the data are actually displayed on the display device 506, and / or provide an input to further processing, such as the selection of a population of interest for particle sorting.

[0146] In some embodiments, the analysis controller 500 can be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the visualization of the plot to facilitate the gate control process. This modification can be based on a particular distribution of the biological event data received by the analysis controller 500.

[0147] The analysis controller 500 may be connected to the memory device 504. This memory device 504 may be configured to receive and store biometric event data from the analysis controller 500. The memory device 504 may also be configured to receive and store flow cytometric event data from the analysis controller 500. The memory device 504 may be further configured by the analysis controller 500 to enable the search of biometric event data such as flow cytometric event data.

[0148] The display device 506 may be configured to receive display data from the analysis controller 500. The display data may include a plot of biometric event data and a gate depicting the contour of the plot's compartments. The display device 506 may be further configured to change the information presented according to the input received from the analysis controller 500 in conjunction with the input from the particle analyzer 502, the memory device 504, the keyboard 508, and / or the mouse 510.

[0149] In some embodiments, the analysis controller 500 can generate a user interface and receive exemplary events for sorting. For example, this user interface can include controls for receiving exemplary events or exemplary images. The exemplary events or images, or exemplary gates, can be provided before the collection of event data for the sample or based on an initial set of events for a portion of the sample.

[0150] FIG. 6A is a schematic diagram of a particle sorter system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. As shown in FIG. 6A, a droplet 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 mobility fluid column 608, the particles 609 (e.g., cells) line up in a single file and cross a monitoring area 611 (e.g., where a laser stream intersects) and are irradiated by an irradiation source 612 (e.g., a laser). Due to the vibration of the droplet formation transducer 602, the mobility fluid column 608 is split into a plurality of droplets 610, some of which contain the particles 609.

[0151] During operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring area 611. The detection station 614 supplies an input to a timing circuit 628, which then supplies an input to a flash charge circuit 630. At the droplet splitting point notified by the timed droplet delay (Δt), flash charge can be applied to the moving fluid column 608, and thus the droplet of interest carries a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplet is then sorted by activating a deflection plate (not shown) and deflected into a container such as a collection tube, or a multi-well or microwell sample plate where wells or microwells can be associated with specific droplets of interest. As shown in FIG. 6A, the droplets can be collected in a drain container 638.

[0152] The detection system 616 (e.g., droplet boundary detector) serves to automatically determine the phase of the droplet drive signal when a particle of interest passes through the area 611 being monitored. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is hereby incorporated by reference in its entirety. The detection system 616 enables the device to accurately calculate the position of each detected particle in the droplet. The detection system 616 can supply an input to the amplitude signal 620 and / or the phase 618 signal, which are then supplied as inputs (via the amplifier 622) to the amplitude control circuit 626 and / or the frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then controls the droplet formation transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included within the control system.

[0153] In some embodiments, the sorting electronics (e.g., the detection system 616, the detection station 614, and the processor 640) can be coupled to a memory configured to store the detected events and the sorting decisions based thereon. The sorting decisions may be included in the particle event data. In some embodiments, the detection system 616 and the detection station 614 can be implemented as a single detection unit, or communicatively coupled such that event measurements can be collected by one of the detection system 616 or the detection station 614 and provided to the non-collecting element.

[0154] FIG. 6B is a schematic diagram of a particle sorter system according to one embodiment presented herein. The particle sorter system 600 shown in FIG. 6B includes deflection plates 652 and 654. Charge can be applied via a stream charging wire within the barb. This creates a stream of droplets 610 that encompasses particles 610 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed, such as by sorting electronics or other detection systems (not shown in FIG. 6B). The deflection plates 652 and 654 are independently controlled to attract or repel charged droplets and direct the droplets towards a desired collection container (e.g., one of 672, 674, 676, or 678). As shown in FIG. 6B, the deflection plates 652 and 654 are controlled to direct the particles along a first path 662 towards container 674 or along a second path 668 towards container 678. If the particles are not of interest (e.g., do not exhibit scattering or illumination information within a specified sorting range), the deflection plates can allow the particles to continue to travel along flow path 664. Such uncharged droplets can be transferred into a waste container, such as via aspirator 670.

[0155] Sorting electronics can include starting the collection of measurements, receiving fluorescence signals regarding the particles, and determining how to adjust the deflection plates to cause sorting of the particles. An exemplary implementation of the embodiment shown in FIG. 6B includes the BD FACSAria® flow cytometer commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0156] Computer control system Aspects of the present disclosure further include a computer control system, which 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 storing a computer program, which, when loaded into the computer, includes instructions for calculating the absolute number of particles in a sample within a flow cytometer. In some embodiments, the computer program includes instructions for introducing air bubbles into a flow stream propagating a sample containing particles, instructions for irradiating the flow stream with a light source, an algorithm for detecting light from the particles irradiated by a photodetector, and instructions for detecting the presence of air bubbles in the flow stream with a photodetector. In an embodiment, the computer program further includes instructions for determining the absolute number of particles in the sample based on data signals generated in response to light detected from the irradiated particles, data signals generated when air bubbles are introduced into the flow stream, and data signals generated in response to the detected air bubbles.

[0157] In some embodiments, the computer program includes instructions for introducing air bubbles into the flow stream by retracting the sample line from the sample source to create an air gap within the flow stream. In some cases, the computer program includes instructions for operating a sample line displacement component that retracts the sample line from the sample source. In a particular case, the computer program includes instructions for retracting the sample line from the sample source while the sample line is being retracted to draw air or gas into the sample line. In some cases, the computer program includes instructions for generating a data signal when the sample line is retracted from the sample source. In some cases, the computer program includes instructions for generating a data signal at a predetermined time after the sample line has been retracted from the sample source, for example, after air bubbles have been introduced into the flow stream, between 0.001 μs and 10 μs.

[0158] In some embodiments, the computer program includes instructions for continuously detecting light from irradiated particles before introducing bubbles into the flow stream, instructions for continuously detecting light from irradiated particles upstream of the bubbles in the flow stream, and instructions for detecting light from the flow stream when the bubbles pass through the inspection region. In some embodiments, the computer program includes instructions for generating a data signal in response to light detected from irradiated particles before introducing bubbles into the flow stream, instructions for generating a data signal in response to light detected from irradiated particles upstream of the bubbles in the flow stream, and instructions for generating a data signal when the bubbles are irradiated within the flow stream. In some embodiments, the computer program includes instructions for generating a data signal in response to light detected from irradiated particles downstream of the bubbles in the flow stream.

[0159] In embodiments, the computer program includes instructions for calculating the absolute number of particles in the sample. In some embodiments, the computer program includes instructions for calculating the absolute number of particles in the sample using the data signal generated in response to light detected from irradiated particles between when a bubble is introduced into the flow stream and when the bubble is detected, and a predetermined volume. In some cases, the computer program includes instructions for calculating the absolute number of particles using the volume of a pre-calibrated sample line. In some cases, the computer program includes instructions for generating a data signal intensity plot of the data signal from the irradiated sample as a function of time. In a particular case, the computer program includes instructions for generating a data signal intensity plot that includes at least the intensity of the data signal from irradiated particles collected from when a bubble is introduced into the flow stream until the bubble is detected. In a particular case, the computer program includes instructions for generating a data signal intensity plot on which the time when a bubble is introduced into the flow stream is plotted.

[0160] In an embodiment, the system includes an input module, a processing module, and an output module. The system of the subject matter may include both hardware components and software components. The hardware components may take the form of, for example, one or more platforms in the form of a server. As a result, the functional elements of the system, i.e., those elements of the system that perform specific tasks (such as management of input / output of information, processing of information, etc.), may be executed by the execution of software applications on and across one or more computer platforms represented by the system.

[0161] The system may include 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 accesses a memory having instructions stored therein to execute the steps of the subject method. 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, as well as many other devices. The processor can be a commercially available processor, or one of other processors that are available or will become available. The processor executes an operating system, and the operating system interfaces with firmware and hardware in a well-known manner and facilitates the processor to cooperate and execute the functions of various computer programs that can be described 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, all in accordance with well-known techniques. The processor can be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that enable a user to manually align a light source with a frost stream based on a first optical signal and a second optical signal. In some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0162] The system memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read / write compact disks, flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including a compact disk drive, tape drive, removable hard disk drive, or disk drive. Such types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk, magnetic tape, removable hard disk, or magnetic disk. Any of these program storage media, or others currently in use or that may be developed later, can be considered a computer program product. As is understood, these program storage media typically store computer software programs and / or data. A computer software program, also referred to as computer control logic, is typically stored in the system memory and / or a program storage device used in conjunction with the memory storage device.

[0163] In some embodiments, a computer program product is described that comprises a computer-usable medium having control logic (a computer software program including program code) stored therein. When the control logic is executed by a processor, the computer causes the processor to perform the functions described herein. In other embodiments, some of the functions are implemented primarily in hardware, using, for example, a hardware state machine. Embodiments of the hardware state machine for performing the functions described herein will be apparent to those skilled in the relevant art.

[0164] The memory can be any suitable device such as a magnetic, optical, or solid-state storage device (including magnetic or optical disks, or tapes, or RAM, or any other suitable device, either fixed or portable). The processor can include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming can be provided remotely to the processor via a communication channel or can be pre-stored using any of those devices together with the memory in a computer program product such as a memory or any other portable or fixed computer-readable storage medium. For example, a magnetic or optical disk can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product and algorithms for use in practicing the above method. The programming according to the present invention can be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as magnetic disks, hard disk storage media, and magnetic tapes, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0165] The processor can also have access to a communication channel for communicating with a user located at a remote location. A remote location means that the user is not in direct contact with the system and that input information is relayed from an external device such as a computer connected to any other suitable communication channel including a wide area network (“WAN”), a telephone network, a satellite network, or a cellular phone (i.e., a smartphone) to the input manager.

[0166] In some embodiments, the system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface may include 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) for mobile communication).

[0167] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces such as USB ports, RS-232 ports, or any other suitable electrical connection ports, to enable data communication between the subject system and other external devices, such as computer terminals configured for similar complementary data communication (e.g., in a clinic or hospital environment).

[0168] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the subject system to communicate with other devices, such as computer terminals and / or networks, communicable mobile phones, personal digital assistants, or any other communication device that the user may use in combination.

[0169] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet protocol (IP) via a cellular phone network, short message service (SMS), wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or WiFi connection to the Internet at a WiFi hotspot.

[0170] In one embodiment, the subject system is configured to wirelessly communicate with a server device via a communication interface using a common standard such as, for example, the 802.11 or Bluetooth® RF protocol, or the IrDA infrared protocol. The server device may be another portable device such as a smartphone, a personal digital assistant (PDA), or a notebook computer, or a larger device such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display such as a liquid crystal display (LCD), and an input device such as buttons, a keyboard, a mouse, or a touch screen.

[0171] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored within the subject system, e.g., within an optional data storage unit, with a network or a server device using one or more of the communication protocols and / or mechanisms described above.

[0172] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The 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 achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to the user at a remote location, for example, via the Internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include an Internet URL address so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present within the subject system are typically of a class of computers commonly referred to as servers, but can be of any type of known computer platform or a type to be developed in the future. However, they can be mainframe computers, workstations, or other computer types. They can be connected via other communication systems including any known or future type of cable wiring or either a wired or wireless system, whether networked or not. They can be located in the same place or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems may be adopted on any of the computer platforms. Suitable operating systems include Windows, iOS, Oracle Solaris, Linux (registered trademark), IBM i, Unix, and the like.

[0173] FIG. 7 depicts a general architecture of an exemplary computing device 700 according to a particular embodiment. The general architecture of the computing device 700 depicted in FIG. 7 includes the arrangement of computer hardware and software components. The computing device 700 may include more (or fewer) elements than those shown in FIG. 7. However, not all of these generally traditional elements necessarily need to be shown in order to provide an effective disclosure. As illustrated, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which may communicate with each other via a communication bus. The network interface 720 may provide a connection to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via the network. The processing unit 710 may also communicate with a memory 770 and may further provide output information for an optional display 750 via the input / output device interface 740. The input / output device interface 740 may also receive input from an optional input device 760 such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device.

[0174] Memory 770 may contain computer program instructions (grouped as modules or components in some embodiments) that the processing unit 710 executes in sequence to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. 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 computing device 700. Memory 770 may further contain computer program instructions and other information for implementing aspects of the present disclosure.

[0175] Non-transitory computer-readable storage medium Aspects of the present disclosure further include a non-transitory computer-readable storage medium having instructions for practicing the methods of the subject matter. The computer-readable storage medium can be employed on one or more computers for the complete or partial automation of a system for practicing the methods described herein. In certain embodiments, the instructions according to the methods described herein can be encoded in a computer-readable medium in the form of a "programming", in which case the term "computer-readable storage medium", as used herein, refers to any non-transitory storage medium involved in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include magnetic disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid state disks, and network-connected storage devices (NAS), whether such devices are internal or external to the computer. A file containing information can be "stored" on a computer-readable medium, where "storing" means recording the information such that it is accessible and searchable by a computer at a later date. The computer-implemented methods described herein can be executed using programming 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 number of many other languages.

[0176] 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 irradiating the flow stream with a light source, an algorithm for detecting light from the irradiated particles using a photodetector, and an algorithm for detecting the presence of bubbles in the flow stream using a photodetector. In an embodiment, the non-transitory computer-readable storage medium further includes an algorithm for determining the absolute number of particles in the sample based on a data signal generated in response to the 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 the detected bubbles.

[0177] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for introducing bubbles into the flow stream by retracting a sample line from a sample source to create an air gap in the flow stream. In some cases, the non-transitory computer-readable storage medium includes an algorithm for operating a sample line displacement component that retracts the sample line from the sample source. In a particular case, the non-transitory computer-readable storage medium includes an algorithm for introducing air or gas into the sample line while the sample line is being retracted to retract the sample line from the sample source. In some cases, 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 cases, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal at a predetermined time after the sample line has been retracted from the sample source, for example, after bubbles have been introduced into the flow stream, in the range of 0.001 μs to 10 μs.

[0178] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for continuously detecting light from irradiated particles before introducing bubbles into the flow stream, an algorithm for continuously detecting light from irradiated particles upstream from bubbles in the flow stream, and an algorithm for detecting light from the flow stream when the bubbles pass through the inspection region. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal in response to light detected from irradiated particles before introducing bubbles into the flow stream, an algorithm for generating a data signal in response to light detected from irradiated particles upstream from bubbles in the flow stream, and an algorithm for generating a data signal when the bubbles are irradiated within 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 irradiated particles downstream from bubbles in the flow stream.

[0179] In an embodiment, the non-transitory computer-readable storage medium includes an algorithm for calculating the absolute number of particles in a sample. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the absolute number of particles in a sample using a data signal generated in response to light detected from irradiated particles and a predetermined volume during the interval between when a bubble is introduced into the flow stream and when the bubble is detected. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the absolute number of particles using the volume of a pre-calibrated sample line. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity plot of the data signal from the irradiated sample as a function of time. In a particular case, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity plot that includes at least the intensity of the data signal from irradiated particles collected between when a bubble is introduced into the flow stream and when the bubble is detected. In a particular case, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity plot on which the time when an air bubble is introduced into the flow stream is plotted.

[0180] A non-transitory computer-readable storage medium can be employed on one or more computer systems having 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 accesses a memory having instructions stored therein for performing the steps of the subject method. 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 one of other processors that are available or will become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs written in various programming languages such as other high-level or low-level languages and combinations thereof, as known in the art. The operating system typically coordinates and executes the functions of other components of the computer in cooperation with the processor. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0181] Kit Aspects of the present disclosure further include a kit, the kit including one or more of the components of the systems described herein. In some embodiments, the kit includes a computer-readable medium (e.g., flash drive, USB storage, compact disc, DVD, Blue-ray disc, etc.), or instructions in the form for downloading programming from an Internet web protocol or cloud server, etc., a plurality of photodetectors and programming for the subject system. The kit may also include optical adjustment components such as lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating protocols, and combinations thereof.

[0182] The kit may further include instructions for implementing the subject method. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece of paper on which information is printed, within the package of the kit, within an accompanying document, etc. Yet another form of these instructions is a computer-readable medium on which information is recorded, e.g., a diskette, a compact disc (CD), a portable flash drive, etc. Yet another form of these instructions that may be present is a website address that may be used via the Internet to access the information at a removed site.

[0183] Significance The subject methods, systems, and computer systems find use in a variety of applications where it is desirable to optimize the photodetectors of a photodetection system. The subject methods and systems also find use for a photodetection system having a plurality of photodetectors that is used to analyze and sort particle components in a sample in a fluid medium, such as a biological sample. The present disclosure also finds use in flow cytometry, where it is desirable to provide a flow cytometer having 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 embodiments, the present disclosure reduces the need for user input or manual adjustment during sample analysis by a flow cytometer. In certain embodiments, the subject methods and systems provide a fully automated protocol such that adjustment to the flow cytometer in use requires little, if any, human input.

[0184] Notwithstanding the appended claims, the present disclosure is also defined by the following appendices. 1. A method for determining the absolute number of particles in a sample within a flow cytometer, comprising: introducing bubbles into a flow stream propagating a sample containing particles; irradiating the flow stream with a light source; detecting light from the irradiated particles using a photodetector; detecting the presence of bubbles in the flow stream using a photodetector; determining the absolute number 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 bubbles are introduced into the flow stream, and a data signal generated in response to the detected bubbles. The method of claim 1, wherein introducing bubbles into the flow stream comprises retracting the sample line from the sample source to create an air gap in the flow stream. 2. The method of appendix 1, wherein introducing bubbles into the flow stream comprises retracting the sample line from the sample source to create an air gap in the flow stream. 3. The method according to appendix 2, comprising generating a data signal when the sample line is retracted from the sample source. 4. The method according to appendix 2, comprising generating a data signal at a predetermined time after the sample line has been retracted from the sample source. 5. The method according to appendix 4, wherein the data signal is generated in the range of 0.001 μs to 10 μs after the sample line has been retracted from the sample source.

[0185] 6. The method according to any one of appendices 1 to 5, wherein the data signal generated when introducing bubbles into the flow stream includes an electronic data signal. 7. The method according to any one of appendices 1 to 5, wherein the data signal generated when introducing bubbles into the flow stream includes an optical data signal. 8. The method according to any one of appendices 1 to 7, wherein the flow stream is continuously irradiated by a light source. 9. The method according to appendix 8, wherein the light source includes a laser. 10. The method according to appendix 8 or 9, wherein the flow stream is continuously irradiated by a light source having a wavelength in the range of 200 nm to 800 nm.

[0186] 11. The method comprises generating a data signal in response to the light detected from the irradiated particles before introducing bubbles into the flow stream, generating a data signal in response to the light detected from the irradiated particles upstream from the bubbles in the flow stream, and generating a data signal when the bubbles are irradiated in the flow stream and is the method according to any one of appendices 1 to 10. 12. The method according to appendix 11, further comprising generating a data signal in response to the light detected from the irradiated particles downstream from the bubbles in the flow stream. 13. The method comprises continuously detecting the light from the irradiated particles before introducing bubbles into the flow stream, Continuously detecting light from upstream irradiated particles from bubbles in the frost stream; Detecting light from the frost stream when a bubble passes through the inspection area; The method according to appendix 11, comprising: 14. The method according to any one of appendices 1 to 13, wherein the detected light includes fluorescence. 15. The method according to any one of appendices 1 to 13, wherein the detected light includes scattered light.

[0187] 16. The method according to any one of appendices 1 to 15, wherein the presence of bubbles in the frost stream is determined based on detection of scattered light from the irradiated bubbles. 17. The method, wherein the method further includes calculating the absolute number of particles in the sample using: A data signal generated in response to light detected from the irradiated particles between when a bubble is introduced into the frost stream and when the bubble is detected; A predetermined volume; The method according to any one of appendices 1 to 16. 18. The method according to any one of appendices 1 to 17, further comprising generating a data signal intensity plot of the generated data signal as a function of time. 19. The method according to appendix 18, further comprising plotting the time when a bubble is introduced into the frost stream on the data signal intensity plot.

[0188] 20. A system, comprising: A flow cell configured to propagate a sample containing particles in a frost stream; A sample line in fluid communication with a sample source and the flow cell, the sample line being configured to introduce bubbles into the frost stream; A light source configured to irradiate the sample in the frost stream; An optical detection system including a photodetector for detecting light from the irradiated frost stream; A processor, including a memory operably coupled to the processor, the memory including instructions stored in the memory, the instructions causing the processor, when executed by the processor, to determine the absolute number of particles in a sample from a data signal generated in response to light detected from irradiated particles, a data signal generated when bubbles are introduced into the flow stream, and calculate based on a data signal generated in response to detected bubbles. A system comprising the processor. 21. The system according to appendix 20, comprising a sample line displacement component configured to retract the sample line from the sample source and introduce an air gap into the flow stream. 22. The system according to appendix 20 or 21, wherein the memory includes instructions for generating a data signal when bubbles are introduced into the flow stream. 23. The system according to appendix 22, wherein the memory includes instructions for generating an electronic data signal when the sample line is retracted from the sample source. 24. The system according to appendix 22, wherein the memory includes instructions for generating an electronic data signal at a predetermined time after the sample line has been retracted from the sample source.

[0189] 25. The system according to appendix 24, wherein the data signal is generated from 0.001 μs to 10 μs after the sample line has been retracted from the sample source. 26. The system according to any one of appendices 20 to 25, wherein the light source includes a laser. 27. The system according to appendix 26, wherein the laser is a continuous wave laser. 28. The system according to appendix 26 or 27, wherein the light source is configured to continuously irradiate the flow stream with light having a wavelength of 200 nm to 800 nm. 29. The light detection system generates a data signal in response to light detected from irradiated particles before bubbles are introduced into the flow stream, In response to light detected from particles irradiated upstream from bubbles in the frost stream, generate a data signal, The system according to any one of appendices 20 to 28, configured to generate a data signal when a bubble is irradiated in the frost stream.

[0190] 30. The system according to appendix 29, further configured such that the light detection system generates a data signal in response to light detected from particles irradiated downstream from bubbles in the frost stream. 31. The light detection system is continuously detect light from irradiated particles before introducing bubbles into the frost stream, continuously detect light from particles irradiated upstream from bubbles in the frost stream, The system according to appendix 30, configured to detect light from the frost stream when a bubble passes through the inspection region. 32. The system according to any one of appendices 20 to 31, wherein the light detection system comprises a detector configured to detect fluorescence from a sample. 33. The system according to any one of appendices 20 to 31, wherein the light detection system comprises a detector configured to detect scattered light. 34. The system according to any one of appendices 20 to 33, wherein the memory includes instructions for determining the presence of bubbles in the frost stream based on the scattered light detector signal.

[0191] 35. The memory is configured to determine the absolute number of particles in the sample between when a bubble is introduced into the frost stream and when the bubble is detected, using a data signal generated in response to light detected from irradiated particles and a predetermined volume. The system according to any one of appendices 20 to 34 includes instructions for calculating. 36. The system according to any one of appendices 20 to 35, wherein the memory includes instructions for generating a data signal intensity plot of the generated data signal as a function of time. 37. The system according to appendix 36, wherein the memory includes instructions for plotting on a data signal strength plot the time at which bubbles are introduced into the flow stream.

[0192] 38. A non - transitory computer - readable storage medium, comprising instructions stored on the non - transitory computer - readable storage medium for calculating the absolute number of particles in a sample within a flow cytometer, the instructions including an algorithm for introducing bubbles into a flow stream propagating a sample containing particles, an algorithm for irradiating the flow stream with a light source, an algorithm for detecting light from the irradiated particles using a photodetector, an algorithm for detecting the presence of bubbles within the flow stream, and an algorithm for determining the absolute number 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 bubbles are introduced into the flow stream, and a data signal generated in response to the detected bubbles. A non - transitory computer - readable storage medium comprising the same. 39. The non - transitory computer - readable storage medium according to appendix 38, wherein the non - transitory computer - readable storage medium includes an algorithm for creating an air gap within the flow stream by introducing bubbles into the flow stream by retracting a sample line from a sample source. 40. The non - transitory computer - readable storage medium according to appendix 39, wherein 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. 41. The non - transitory computer - readable storage medium according to appendix 39, wherein the non - transitory computer - readable storage medium includes an algorithm for generating a data signal at a predetermined time after the sample line has been retracted from the sample source. 42. The non-transitory computer-readable storage medium is the non-transitory computer-readable storage medium according to Appendix 41, including an algorithm for generating a data signal in the range of 0.001 μs to 10 μs after the sample is retracted from the sample source.

[0193] 43. The non-transitory computer-readable storage medium is the non-transitory computer-readable storage medium according to any one of Appendices 38 to 42, including an algorithm for generating an electronic data signal when bubbles are introduced into the frost stream. 44. The non-transitory computer-readable storage medium is the non-transitory computer-readable storage medium according to any one of Appendices 38 to 42, including an algorithm for generating an optical data signal when bubbles are introduced into the frost stream. 45. The non-transitory computer-readable storage medium includes an algorithm for generating a data signal in response to the light detected from the irradiated particles before introducing bubbles into the frost stream, and an algorithm for generating a data signal in response to the light detected from the irradiated particles upstream from the bubbles in the frost stream, and an algorithm for generating a data signal when the bubbles are irradiated in the frost stream, and is the non-transitory computer-readable storage medium according to any one of Appendices 38 to 44. 46. The non-transitory computer-readable storage medium includes an algorithm for generating a data signal in response to the light detected from the irradiated particles downstream from the bubbles in the frost stream, and is the non-transitory computer-readable storage medium according to Appendix 45. 47. The non-transitory computer-readable storage medium includes an algorithm for continuously detecting the light from the irradiated particles before introducing bubbles into the frost stream, and an algorithm for continuously detecting the light from the irradiated particles upstream from the bubbles in the frost stream, and an algorithm for detecting the light from the frost stream when the bubbles pass through the inspection area, and is the non-transitory computer-readable storage medium according to Appendix 45.

[0194] 48. A non-transitory computer-readable storage medium includes an algorithm for calculating the absolute number of particles in a sample using a data signal generated in response to light detected from irradiated particles between when a bubble is introduced into a flow stream and when the bubble is detected, and a predetermined volume, the non-transitory computer-readable storage medium according to any one of Appendices 37 to 47. 48. A non-transitory computer-readable storage medium includes an algorithm for calculating the absolute number of particles in a sample using a data signal generated in response to light detected from irradiated particles between when a bubble is introduced into a flow stream and when the bubble is detected, and a predetermined volume, the non-transitory computer-readable storage medium according to any one of Appendices 37 to 47. 48. A non-transitory computer-readable storage medium includes an algorithm for calculating the absolute number of particles in a sample using a data signal generated in response to light detected from irradiated particles between when a bubble is introduced into a flow stream and when the bubble is detected, and a predetermined volume, the non-transitory computer-readable storage medium according to any one of Appendices 37 to 47. 49. A non-transitory computer-readable storage medium includes an algorithm for generating a data signal intensity plot of a generated data signal as a function of time, the non-transitory computer-readable storage medium according to any one of Appendices 37 to 48. 50. The non-transitory computer-readable storage medium according to Appendix 49 includes an algorithm for plotting on the data signal intensity plot the time when a bubble is introduced into the flow stream.

[0195] The above invention has been described in some detail by way of illustration and example for the purpose of clear understanding. However, it will be readily apparent to those skilled in the art that certain changes and modifications can be made to those inventions without departing from the spirit or scope of the appended claims in light of the teachings of the present invention.

[0196] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be appreciated by those skilled in the art that, although not explicitly described or illustrated herein, various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised. Further, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Additionally, all descriptions in this specification of the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function regardless of structure. Further, what is disclosed herein is not intended to be dedicated to the public whether or not such disclosure is expressly recited in the claims.

[0197] Accordingly, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined to be invoked for a claim limitation only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such a limitation in the claim, and 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked when such exact phrase is not used in the claim limitation.

[0198] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to the filing date of U.S. Provisional Patent Application No. 63 / 352,472, filed on June 15, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

Claims

1. A method for determining the absolute number of particles in a sample within a flow cytometer, comprising: introducing air bubbles into a flow stream propagating a sample containing particles; irradiating the flow stream with a light source; detecting light from the irradiated particles using a photodetector; detecting the presence of the air bubbles in the flow stream using the photodetector; determining the absolute number 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 air bubbles are introduced into the flow stream, and a data signal generated in response to the detected air bubbles. A method comprising the above steps.

2. The method according to claim 1, wherein introducing the air bubbles into the flow stream includes retracting a sample line from a sample source to create an air gap within the flow stream.

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

4. The method according to claim 2, wherein the method includes generating a data signal at a predetermined time after the sample line is retracted from the sample source.

5. The method according to any one of claims 1 to 4, wherein the data signal generated when introducing the air bubbles into the flow stream includes 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 continuously irradiated by the light source.

7. The method includes: generating a data signal in response to light detected from the irradiated particles before introducing the air bubbles into the flow stream; generating a data signal in response to light detected from the irradiated particles upstream of the air bubbles in the flow stream; and generating a data signal when the air bubbles are irradiated within the flow stream. The method according to any one of claims 1 to 6.

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

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

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

11. The method calculates the absolute number of the particles in the sample by 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, a predetermined volume, and The method according to any one of claims 1 to 10.

12. The method according to any one of claims 1 to 11, including generating a data signal intensity plot of the generated data signal as a function of time.

13. A system comprising: a flow cell configured to propagate a sample containing particles within a flow stream; a sample line in fluid communication with a sample source and the flow cell, the sample line being configured to introduce bubbles into the flow stream; a light source configured to irradiate the sample in the flow stream; an optical detection system including a photodetector for detecting light from the irradiated flow stream; a processor including a memory operably coupled to the processor, the memory including instructions stored thereon, the instructions causing the processor, when executed by the processor, to calculate the absolute number of the particles in the sample by using 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, The system.

14. A non-transitory computer-readable storage medium comprising: instructions stored on the non-transitory computer-readable storage medium for calculating the absolute number of particles in a sample within a flow cytometer, the instructions including an algorithm for introducing bubbles into a flow stream propagating a sample containing particles; an algorithm for irradiating the flow stream with a light source; an algorithm for detecting light from the irradiated particles using a photodetector; an algorithm for detecting the presence of the bubbles within the flow stream; the absolute number of the particles in the sample by using 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 An algorithm for making a determination based on and A non-transitory computer-readable storage medium including.

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