Method and system for monitoring flow rate of flow stream

Abstract

Aspects of the present disclosure include methods for monitoring the flow rate of a flow stream (e.g., in a flow cytometer). A method according to certain embodiments includes measuring a flow rate of a flow stream, comparing the measured flow rate of the flow stream to an absolute flow rate threshold and a moving window flow rate threshold, and generating an error alert when the measured flow rate of the flow stream exceeds the absolute flow rate threshold or the moving window flow rate threshold. Systems and non-transitory computer-readable storage media configured to perform the subject methods are also provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 705,166, filed October 9, 2024, pursuant to 35 USC § 119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for monitoring the flow rate of a flow stream, a system for configuring a flow cytometer, and a non-transient computer-readable storage medium for configuring a flow cytometer. Background Technology

[0004] Characterization of analytes in biofluids has become an important part of biological research, medical diagnostics, and the assessment of patients' overall health and well-being. Detection of analytes in biofluids (such as human blood or blood-derived products) can provide results that may play a role in determining treatment options for patients with various disease conditions.

[0005] Flow cytometry is a technique used to characterize and typically sort biological materials, such as cells in a blood sample or particles of interest in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample (e.g., a blood sample) and a sheath fluid reservoir containing sheath fluid. The flow cytometer delivers particles (including cells) from the fluid sample as a cell stream to a flow cell, while also guiding the sheath fluid into the flow cell. To characterize the components of the flow stream, the flow stream is illuminated. Changes in the material within the flow stream (e.g., the presence of morphological or fluorescent labels) can cause changes in the observed light, and these changes allow for characterization and separation. To characterize the components in the flow stream, light must be incident on the flow stream and collected. The light source in a flow cytometer can vary and may include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0006] Separation of biological particles has been achieved by adding sorting or collection capabilities to flow cytometers. Particles in a segregated stream that are detected as having one or more desired characteristics are individually separated from a sample stream by mechanical or electrical removal. One common flow sorting technique utilizes droplet sorting, in which a fluid stream containing linearly separated particles is broken into droplets. Droplets containing particles of interest are charged by an electric field and deflected into a collection tube. Typically, linearly separated particles in the stream are characterized as they pass through a viewing point just below the tip of the nozzle. Once a particle is identified as meeting one or more desired criteria, the time at which the particle reaches the droplet break point and is broken from the stream as a droplet can be predicted. Ideally, a brief charge is applied to the fluid stream just before the droplet containing the selected particle is about to break from the stream, and then grounded immediately after the droplet breaks. The droplet to be sorted retains its charge as it breaks from the fluid stream, while all other droplets are uncharged.

[0007] Flow cytometry data quality can be negatively impacted by sample quality or data acquisition issues, such as clogs or air bubbles in the sample core stream. This can lead to false conclusions from data analysis. Most flow cytometers do not have sample fluid error detection, or rely on air bubble detectors. These flow cytometers cannot provide real-time sample readings, and cannot detect sharp fluid changes in the flow stream. Changes in flow rate can indicate an error or in some cases a failure of the flow system to allow sample to flow through the system. SUMMARY

[0008] Aspects of the present disclosure include methods for monitoring flow rate of a flow stream, such as in a flow cytometer. The methods according to certain embodiments include measuring a flow rate of a flow stream, comparing the measured flow rate of the flow stream to an absolute flow rate threshold and a moving window flow rate threshold, and generating an error alert when the measured flow rate of the flow stream exceeds the absolute flow rate threshold or the moving window flow rate threshold. Systems and non-transitory computer-readable storage media configured to perform the subject methods are also provided.

[0009] In some embodiments, the flow rate of the flow stream is continuously measured. In some instances, the flow rate of the flow stream is measured at discrete intervals, such as where the flow rate is measured at intermittent intervals of a predetermined duration. In some instances, the flow rate of the flow stream is measured based on a change in temperature of the flow stream using a flow sensor. In some instances, the flow rate of the flow stream is measured based on a change in viscosity of the flow stream using a flow sensor.

[0010] In some instances, the absolute flow rate includes an upper threshold and a lower threshold. In some instances, the method includes continuously comparing the measured flow rate to both the upper and lower absolute flow rate thresholds. In some instances, an erroneous alarm is generated when the measured flow rate is determined to be less than the lower absolute flow rate threshold. In some instances, an erroneous alarm is generated when the measured flow rate is determined to be greater than the upper absolute flow rate threshold. In some instances, an erroneous alarm is generated when the measured flow rate is determined to be 1% or more (e.g., 3% or more) greater than the upper absolute flow rate threshold or 1% or more (e.g., 3% or more) less than the lower absolute flow rate threshold.

[0011] In some embodiments, the moving window flow rate thresholds include an upper threshold and a lower threshold. In some instances, the method includes comparing the measured flow rate with each moving window flow rate threshold at discrete intervals. In some instances, comparing the measured flow rate with each moving window flow rate threshold includes determining whether the flow rate measured during a predetermined time interval is greater than the upper moving window flow rate threshold. In some instances, comparing the measured flow rate with each moving window flow rate threshold includes determining whether the flow rate measured during a predetermined time interval is less than the lower moving window flow rate threshold. In some instances, the time interval of the moving window used to compare the measured flow rate with the flow rate threshold is 0.1 seconds to 10 seconds, for example, 1 second to 5 seconds. In some instances, the moving window time interval is 1 second.

[0012] In some embodiments, the generated error alarm indicates a malfunction in the flow cytometer. In some instances, the malfunction is a blockage in the flow stream. In some instances, the malfunction is the introduction of gas (e.g., air) into the flow stream or sample line. In some instances, the error alarm is generated in real time. In some instances, the error alarm is generated when the measured flow rate consistently exceeds an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration (e.g., 0.001 seconds or more, 0.01 seconds or more, 0.1 seconds or more, and including 5 seconds or more).

[0013] In some embodiments, the method includes changing one or more parameters of a flow cytometer in response to a generated error alarm. In some instances, the flow rate of the flow stream is adjusted in response to a generated error alarm. In some instances, the flow rate is increased in response to a generated error alarm. In some instances, the flow rate is decreased in response to a generated error alarm. In some instances, the flow rate is stopped in response to a generated error alarm. In some instances, the light source used to illuminate the flow stream is adjusted in response to a generated error alarm. In some instances, the light source is turned off in response to a generated error alarm. In some instances, the light configured to illuminate the flow stream is blocked in response to a generated error alarm.

[0014] In some embodiments, the flow includes a sheath fluid flow and a sample core fluid flow. In some instances, the method includes measuring the flow rate of the sheath fluid flow. In some instances, the method includes measuring the flow rate of the sample core fluid flow. In some instances, the method includes measuring the flow rates of the sheath fluid flow and the sample core fluid flow. In some embodiments, a sample containing particles is conveyed in the sample core fluid flow. In some instances, the method includes illuminating the sample with a light source and detecting light from the illuminating particles in the flow. In some instances, the light source includes a laser, such as multiple lasers. In some embodiments, light is detected in multiple photodetector channels.

[0015] This disclosure also includes systems for practicing methods of the subject matter (e.g., monitoring the flow rate of a flow stream (e.g., in a flow cytometer)). A system according to some embodiments includes a processor having memory operatively coupled to the processor, wherein the memory includes instructions stored thereon for measuring the flow rate, for comparing the measured flow rate with an absolute flow rate threshold and a moving window flow rate threshold, and for generating an error alarm when the measured flow rate exceeds either the absolute flow rate threshold or the moving window flow rate threshold.

[0016] In some embodiments, the system includes a flow velocity sensor configured to measure the velocity of a flowing stream. In some instances, the flow sensor is configured to measure the velocity of the flowing stream based on temperature changes in the flowing stream. In some instances, the flow sensor is configured to measure the velocity of the flowing stream based on viscosity changes in the flowing stream. In some instances, the system is configured to continuously measure the velocity of the flowing stream. In some instances, the system is configured to measure the velocity of the flowing stream at discrete intervals, such as intermittent intervals of predetermined duration.

[0017] In some instances, the absolute flow rate includes an upper threshold and a lower threshold. In some instances, the memory includes instructions for comparing the measured flow rate with the upper and lower absolute flow rate thresholds. In some instances, the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is less than the lower absolute flow rate threshold. In some instances, the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is greater than the upper absolute flow rate threshold. In some instances, the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is greater than the upper absolute flow rate threshold by % or more (e.g., 3% or more) or less than the lower absolute flow rate threshold by 1% or more (e.g., 3% or more).

[0018] In some embodiments, the moving window flow rate thresholds include an upper threshold and a lower threshold. In some instances, the memory includes instructions for comparing the measured flow rate with each moving window flow rate threshold at discrete intervals. In some instances, the memory includes instructions for comparing the measured flow rate with each moving window flow rate threshold by determining whether the flow rate measured during a predetermined time interval is greater than the upper moving window flow rate threshold. In some instances, the memory includes instructions for comparing the measured flow rate with each moving window flow rate threshold by determining whether the flow rate measured during a predetermined time interval is less than the lower moving window flow rate threshold. In some instances, the time interval of the moving window for comparing the measured flow rate with the flow rate thresholds is 0.1 seconds to 10 seconds, for example, 1 second to 5 seconds. In some instances, the moving window time interval is 1 second.

[0019] In some embodiments, the memory includes instructions for generating an error alarm indicating a fault in the flow cytometer. In some instances, the fault is a blockage in the flow stream. In some instances, the fault is the introduction of gas (e.g., air) into the flow stream or sample line. In some instances, the memory includes instructions for generating an error alarm in real time. In some instances, the memory includes instructions for generating an error alarm when the measured flow rate continuously exceeds an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration (e.g., 0.001 seconds or more, such as 0.01 seconds or more, such as 0.1 seconds or more, and including 5 seconds or more).

[0020] In some embodiments, the memory includes instructions for changing one or more parameters of the flow cytometer in response to a generated error alarm. In some instances, the memory includes instructions for adjusting the flow rate of the flow stream in response to a generated error alarm. In some instances, the memory includes instructions for increasing the flow rate in response to a generated error alarm. In some instances, the memory includes instructions for decreasing the flow rate in response to a generated error alarm. In some instances, the memory includes instructions for stopping the flow rate in response to a generated error alarm. In some instances, the memory includes instructions for adjusting the light source used to illuminate the flow stream in response to a generated error alarm. In some instances, the memory includes instructions for turning off the light source in response to a generated error alarm. In some instances, the memory includes instructions for blocking light configured to illuminate the flow stream in response to a generated error alarm.

[0021] In some embodiments, the system includes a flow stream formed by a sheath fluid flow stream and a sample core fluid flow stream. In some instances, the memory includes instructions for measuring the flow rate of the sheath fluid flow stream. In some instances, the memory includes instructions for measuring the flow rate of the sample core fluid flow stream. In some instances, the memory includes instructions for measuring the flow rates of the sheath fluid flow stream and the sample core fluid flow stream. In some embodiments, a sample containing particles is conveyed in the sample core fluid flow stream. In some instances, the memory includes instructions for illuminating the sample with a light source and instructions for detecting light from the illuminating particles in the flow stream. In some instances, the light source includes a laser, such as multiple lasers. In some embodiments, the system includes a light detection system configured to detect light in multiple photodetector channels.

[0022] This disclosure also includes non-transient computer-readable storage media, for example, for practicing one or more computer-implemented methods described herein. In some embodiments, the non-transient computer-readable storage medium includes algorithms for measuring the flow rate of a flow stream in a flow cytometer, algorithms for comparing the measured flow rate of the flow stream with an absolute flow rate threshold and a moving window flow rate threshold, and algorithms for generating an error alarm when the measured flow rate of the flow stream exceeds the absolute flow rate threshold or the moving window flow rate threshold.

[0023] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for continuously measuring the flow rate of a flowing stream. In some instances, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of a flowing stream at discrete intervals, such as wherein the flow rate is measured at intermittent intervals of predetermined duration.

[0024] In some instances, the absolute flow rate includes an upper threshold and a lower threshold. In some instances, the non-transient computer-readable storage medium includes an algorithm for continuously comparing the measured flow rate with both the upper and lower absolute flow rate thresholds. In some instances, the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when it is determined that the measured flow rate is less than the lower absolute flow rate threshold. In some instances, the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when it is determined that the measured flow rate is greater than the upper absolute flow rate threshold. In some instances, the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when it is determined that the measured flow rate is 1% or more greater than the upper absolute flow rate threshold (e.g., 3% or more) or less than the lower absolute flow rate threshold (e.g., 1% or more, e.g., 3% or more).

[0025] In some embodiments, the moving window flow rate threshold includes an upper threshold and a lower threshold. In some instances, the non-transient computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow rate threshold at discrete intervals. In some instances, the non-transient computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow rate threshold by determining whether the flow rate measured during a predetermined time interval is greater than the upper moving window flow rate threshold. In some instances, the non-transient computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow rate threshold by determining whether the flow rate measured during a predetermined time interval is less than the lower moving window flow rate threshold. In some instances, the time interval of the moving window for comparing the measured flow rate with the flow rate threshold is from 0.1 seconds to 10 seconds, for example, from 1 second to 5 seconds. In some instances, the moving window time interval is 1 second.

[0026] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm indicating a fault in the flow cytometer. In some instances, the fault is a blockage in the flow stream. In some instances, the fault is the introduction of gas (e.g., air) into the flow stream or sample line. In some instances, the non-transient computer-readable storage medium includes an algorithm for generating the erroneous alarm in real time. In some instances, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm when the measured flow rate continuously exceeds an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration (e.g., 0.001 seconds or more, 0.01 seconds or more, 0.1 seconds or more, and including 5 seconds or more).

[0027] In some embodiments, the non-transient computer-readable storage medium includes algorithms for changing one or more parameters of a flow cytometer in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for adjusting the flow rate of a flow stream in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for increasing the flow rate in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for decreasing the flow rate in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for stopping the flow rate in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for adjusting a light source used to illuminate the flow stream in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for turning off a light source in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for blocking light configured to illuminate the flow stream in response to a generated error alarm.

[0028] In some embodiments, the flow includes a sheath fluid flow and a sample core fluid flow. In some instances, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of the sheath fluid flow. In some instances, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of the sample core fluid flow. In some instances, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rates of the sheath fluid flow and the sample core fluid flow. Wherein a sample containing particles is conveyed in the sample core fluid flow, in some instances, the non-transient computer-readable storage medium includes an algorithm for illuminating the sample with a light source and an algorithm for detecting light from the illuminating particles in the flow. Attached Figure Description

[0029] This disclosure can be best understood by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures:

[0030] Figure 1A A flowchart illustrating the flow rate of a flow stream in a flow cytometer, according to certain embodiments, is described. Figure 1B The flow rate of a flow cytometer in which the flow stream is monitored is described according to certain embodiments.

[0031] Figure 2 A flow cytometry system according to certain embodiments is shown.

[0032] Figure 3 A particle sorter with image enabled according to certain embodiments is described.

[0033] Figure 4 A functional block diagram of a particle analysis system according to certain embodiments is depicted.

[0034] Figure 5 A functional block diagram of an example control system according to certain embodiments is depicted.

[0035] Figures 6A-6B A schematic diagram of a particle sorter system according to certain embodiments is depicted.

[0036] Figure 7 Aspects of a computer control system according to certain embodiments are described. Detailed Implementation

[0037] This disclosure includes methods for monitoring the flow rate of a flow stream (e.g., in a flow cytometer). A method according to some embodiments includes: measuring the flow rate of the flow stream, comparing the measured flow rate of the flow stream with an absolute flow rate threshold and a moving window flow rate threshold, and generating an error alarm when the measured flow rate of the flow stream exceeds the absolute flow rate threshold or the moving window flow rate threshold. A system and a non-transient computer-readable storage medium configured to perform the methods of this subject matter are also provided.

[0038] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure will be limited only by the appended claims.

[0039] Where a numerical range is provided, it should be understood that, unless the context explicitly states otherwise, every intermediate value between the upper and lower limits of the range (accurate to one-tenth of the lower limit unit), as well as any other value or intermediate value within the range, is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this disclosure, but are subject to any specific exclusions within the range. Where the range includes one or two limits, the range excluding any one or both of those included limits is also included in this disclosure.

[0040] Certain ranges presented in this article are preceded by the term "approximately". The term "approximately" is used here to provide textual support for the precise number that follows it, and to provide textual support for numbers that are close to or approximate to the number that follows the term. In determining whether a number is close to or approximate to a specifically cited number, a close to or approximate uncited number may be a number that provides a substantial equivalent to the specifically cited number in its presented context.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, representative illustrative methods and materials are now described.

[0042] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relating to the referenced publication. Any reference to a publication is for its publication prior to the filing date and should not be construed as an admission that this disclosure is not entitled to any prior publication due to a previous disclosure. Furthermore, the publication dates provided may differ from the actual publication dates, which require independent verification.

[0043] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a precondition for the use of exclusive terms such as “merely,” “only,” and / or “negative” limitations related to references to elements of the claim.

[0044] Those skilled in the art will understand upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any other several embodiments without departing from the scope or spirit of this disclosure. Any cited methods may be performed in the order in which the events are cited or in any other logically possible order.

[0045] While systems and methods have been or will be described by functional interpretation for the sake of grammatical fluency, it should be clearly understood that, unless expressly stated in accordance with 35 USC §112, claims should not be construed in any way as necessarily limited to the construction of “apparatus” or “step,” but should be given their meaning and the full scope of equivalents under the judicial principle of the doctrine of equivalents provided by the claims, and where claims are expressly stated in accordance with 35 USC §112, they should be given all legal equivalents in accordance with 35 USC §112.

[0046] Methods for monitoring the flow rate of a flow stream in a flow cytometer

[0047] This disclosure includes methods for monitoring the flow rate of a flow stream (e.g., in a flow cytometer). In embodiments, the flow rate is monitored to determine whether a change in flow rate has occurred, and in some instances, to determine whether such a change is caused by a sudden failure of the sample fluid in the system. As described in more detail below, the subject matter method for monitoring flow rate includes checking whether the flow rate remains within a stable range (e.g., between an upper and lower absolute flow rate threshold) and checking whether the flow rate exhibits any relative change within a moving window. In embodiments, monitoring the flow rate provides accurate and early detection of sample propagation errors in the system. In some instances, monitoring flow rate according to the subject matter method provides detection of sample fluid errors caused by sudden changes in flow rate, such as those caused by blockages or air introduction in the fluid system of a flow cytometer.

[0048] In some embodiments, the methods and systems of this subject provide for identifying patterns of flow rate and determining the cause of errors, such as distinguishing between blockages in the fluid, leaks in the sample or sheath fluid line, completion of sample propagation through the flow stream, and the introduction of gas into the flow stream (e.g., due to an error or malfunction or due to separation between samples). In some instances, an alarm is generated in response to a detected change in flow rate, which stops the sample flow and reduces sample loss upon error detection, for example, where sample loss is reduced by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including where sample loss is reduced by 99% or more. In some instances, monitoring the flow rate of the flow stream as described herein prevents any sample loss.

[0049] In some embodiments, monitoring the flow rate provides an optimized flow rate for sample detection, for example, by determining an optimized laser delay for irradiating sample particles in the flow. In some instances, the accuracy of the laser delay timing is improved by 10% or more, for example, by 25% or more, for example, by 50% or more, for example, by 75% or more, and includes improvements of 100% or more.

[0050] In some instances, error alarms are generated that can stop the sample flow upon error detection, thereby improving the reliability of flow cytometry data. In some instances, the methods in this subject matter improve the reliability of data generated from irradiation of a sample in a monitored flow stream, for example, by 10% or more, 25% or more, 50% or more, 75% or more, 100% or more, 150% or more, 200% or more, 250% or more, and including instances where data reliability is improved by 300% or more.

[0051] In practicing the methods of this subject, the flow rate of the flow is measured (e.g., in a flow cytometer). In some instances, the flow rate is measured continuously, for example, by continuously measuring the flow rate in real time. In some instances, the flow rate is measured at discrete intervals, such as at regular intervals of 0.00001 seconds or greater, such as at regular intervals of 0.00005 seconds or greater, such as at regular intervals of 0.0001 seconds or greater, such as at regular intervals of 0.0005 seconds or greater, such as at regular intervals of 0.001 seconds or greater, such as at regular intervals of 0.005 seconds or greater, such as at regular intervals of 0.01 seconds or greater, such as at regular intervals of 0.05 seconds or greater, such as at regular intervals of 0.1 seconds or greater, such as at regular intervals of 0.5 seconds or greater. For example, at regular intervals of 1 second or more, for example, at regular intervals of 2 seconds or more, for example, at regular intervals of 3 seconds or more, for example, at regular intervals of 4 seconds or more, for example, at regular intervals of 5 seconds or more, for example, at regular intervals of 6 seconds or more, for example, at regular intervals of 7 seconds or more, for example, at regular intervals of 8 seconds or more, for example, at regular intervals of 9 seconds or more, for example, at regular intervals of 10 seconds or more, for example, at regular intervals of 15 seconds or more, for example, at regular intervals of 30 seconds or more, and including regular intervals of 60 seconds or more to measure flow rate.

[0052] In some embodiments, the flow rate may be 1 μL / min or higher, such as 2 μL / min or higher, such as 3 μL / min or higher, such as 5 μL / min or higher, such as 10 μL / min or higher, such as 15 μL / min or higher, such as 25 μL / min or higher, such as 50 μL / min or higher, and includes 100 μL / min or higher, wherein in some instances the flow rate is 1 μL / sec or higher, such as 2 μL / sec or higher, such as 3 μL / sec or higher, such as 5 μL / sec or higher, such as 10 μL / sec or higher, such as 15 μL / sec or higher, such as 25 μL / sec or higher, such as 50 μL / sec or higher, and includes 100 μL / sec or higher. In some embodiments, the flow rate can be 25 μL / sec or higher, such as 50 μL / sec or higher, such as 75 μL / sec or higher, such as 100 μL / sec or higher, such as 250 μL / sec or higher, such as 500 μL / sec or higher, such as 750 μL / sec or higher, such as 1000 μL / sec or higher, and includes 2500 μL / sec or higher.

[0053] The flow velocity can be measured using any convenient method, where in some instances, the flow velocity is measured based on the temperature of the measured flow. In some instances, the flow velocity is measured based on the viscosity of the measured flow. In some instances, the flow velocity is measured at one or more detection locations along the flow, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more detection locations, and includes measuring the flow velocity at ten or more detection locations along the flow. In some instances, the detection locations for measuring the flow velocity span 0.001 mm or more of the flow, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 5 mm or more, and includes 10 mm or more of the flow. In some instances, the flow rate is measured based on temperature changes of the flow at two or more detection locations, such as three or more, for example four or more, five or more, six or more, seven or more, eight or more, nine or more detection locations, and includes temperature changes of the flow at ten or more different detection locations along the flow. In some instances, the flow rate is measured based on viscosity changes of the flow at two or more different detection locations, such as three or more, for example four or more, five or more, six or more, seven or more, eight or more, nine or more different detection locations, and includes viscosity changes of the flow at ten or more detection locations along the flow.

[0054] In some instances, temperature sensor flow meters are used to measure flow velocity. In some instances, the flow velocity sensor includes a controllable heating element located at the center of a pressure-stabilized membrane, and temperature sensors mounted upstream and downstream in the direction of fluid flow (e.g., symmetrically). In some instances, the flow causes heat transfer to the downstream temperature sensor, resulting in a measurable signal due to the temperature difference. In some instances, a microthermal flow sensor is integrated into a silicon-on-a-chip flow meter type sensor. In some instances, the flow sensor includes a temperature sensor that generates a signal that compensates for temperature effects. In some instances, the flow sensor includes a fluid flow sensor, such as those from the fluid flow sensor family commercially available from Sensiron (Switzerland). In some instances, flow sensors are used to measure flow velocity, such as those described in U.S. Patent Publication No. 2023 / 0324270, U.S. Patent No. 10,942,139, and European Patent Nos. EP3187881 and EP3404373, the disclosures of which are incorporated herein by reference.

[0055] In some embodiments, the flow stream includes a sheath fluid flow stream and a sample core fluid flow stream. In some embodiments, the sheath fluid injection system is configured to provide a sheath fluid flow into the flow cell chamber, for example, in combination with a sample fluid to generate a sheath fluid laminar flow around the sample core fluid flow stream. In some instances, the sheath fluid flow stream forms a laminar flow around the sample core fluid flow stream. In some instances, the method includes measuring the flow rate of the sheath fluid flow stream. In some instances, the flow rate of the sheath fluid flow stream is 25 μL / sec or higher, such as 50 μL / sec or higher, such as 75 μL / sec or higher, such as 100 μL / sec or higher, such as 250 μL / sec or higher, such as 500 μL / sec or higher, such as 750 μL / sec or higher, such as 1000 μL / sec or higher, and includes 2500 μL / sec or higher. In some instances, the method includes measuring the flow rate of the sample core fluid flow stream. In some instances, the flow rate of the sample core fluid flow is 1 μL / min or higher, for example, 2 μL / min or higher, for example, 3 μL / min or higher, for example, 5 μL / min or higher, for example, 10 μL / min or higher, for example, 15 μL / min or higher, for example, 25 μL / min or higher, for example, 50 μL / min or higher, and including 100 μL / min or higher. In some instances, the sample rate delivered to the flow cell chamber via the sample injection port is 1 μL / sec or higher, for example, 2 μL / sec or higher, for example, 3 μL / sec or higher, for example, 5 μL / sec or higher, for example, 10 μL / sec or higher, for example, 15 μL / sec or higher, for example, 25 μL / sec or higher, for example, 50 μL / sec or higher, and including 100 μL / sec or higher. In some instances, the method includes measuring the flow rate of the sheath fluid flow and the flow rate of the sample core fluid flow.

[0056] In some instances, the flow rate of the sheath fluid flow is compared to the flow rate of the sample core fluid flow. In some instances, the flow rate of the sheath fluid flow is determined to be the same as the flow rate of the sample core fluid flow. In some instances, the flow rate of the sheath fluid flow is determined to be less than the flow rate of the sample core fluid flow, for example, less than the flow rate of the sample core fluid flow by 0.01% or more, for example, less than 0.05% or more, for example, less than 0.1% or more, for example, less than 0.5% or more, for example, less than 1% or more, for example, less than 2% or more, for example, less than 3% or more, for example, less than 4% or more, for example, less than 5% or more, for example, less than 10% or more, for example, less than 15% or more, for example, less than 20% or more, for example, less than 25% or more, and including less than 50% or more. In some instances, the flow rate of the sheath fluid flow is determined to be greater than the flow rate of the sample core fluid flow, for example, by 0.01% or more, 0.05% or more, 0.1% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, and including 50% or more. When it is determined that the relative flow rates of the sheath fluid flow and the sample core fluid flow exceed a predetermined threshold, the method may include adjusting one or more of the flow rates of the sheath fluid flow and the sample core fluid flow. In some instances, when the relative flow rates of the sheath fluid flow and the sample core fluid flow exceed a threshold of 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, and including exceeding a threshold of 50% or more, the method includes adjusting the flow rates of one or more of the sheath fluid flow and the sample core fluid flow.

[0057] In some embodiments, flow rate measurement is initiated when a stable flow rate is detected. In some instances, flow rate is measured when the flow rate exhibits fluctuations (e.g., showing instantaneous changes) of 5% or less, such as 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.001% or less, and includes when the flow rate exhibits changes of 0.0001% or less. When the flow rate exhibits fluctuations exceeding a stability threshold, the system can be set to standby mode until the fluctuations are below the stability threshold for a predetermined duration, such as 1 second or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, and including 60 seconds or longer. Once the flow is determined to be sufficiently stable for monitoring, flow rate measurement can proceed to the monitoring phase. In some embodiments, when the flow rate exhibits instability or fluctuations exceeding a stability threshold during the monitoring phase, the system can re-enter standby mode and the monitoring phase is paused until the flow rate exhibits sufficient stability.

[0058] In some embodiments, absolute flow rate includes an upper threshold and a lower threshold. The term "absolute flow rate" refers to the flow rate of the flow at any time during the delivery of the flow. In some instances, absolute flow rate may be a real-time flow rate measured by a flow sensor. In other instances, absolute flow rate is the flow rate noticed at a specific time during a recording experiment, where the flow rate is recorded during the experiment. In some instances, absolute flow rate is continuously measured (and in some instances, recorded). In some instances, the method includes continuously comparing the measured flow rate of the flow with an upper absolute flow rate threshold. In some instances, an error alarm is generated when it is determined that the measured flow rate is greater than an upper limit absolute flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, and including 50% or more. In some instances, an error alarm is generated when it is determined that the measured flow rate is less than a lower absolute flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, and including 50% or more. When the flow rate is continuously measured, an error alarm may be generated when the flow rate exceeds one or more of the upper or lower threshold of the absolute flow rate threshold for a duration of 0.0001 seconds or longer (e.g., 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer).In some instances, an error alarm is generated when the flow rate exceeds the upper limit of the absolute flow rate threshold for 0.0001 seconds or longer (e.g., 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer). In some instances, an error alarm is generated when the flow rate is less than the lower limit of the absolute flow rate threshold for 0.0001 seconds or longer (e.g., 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer).

[0059] In some instances, the measured flow velocity is compared to an absolute velocity threshold at discrete intervals, such as every 0.00001 seconds or longer, for example, every 0.00005 seconds or longer, for example, every 0.0001 seconds or longer, for example, every 0.0005 seconds or longer, for example, every 0.001 seconds or longer, for example, every 0.005 seconds or longer, for example, every 0.01 seconds or longer, for example, every 0.05 seconds or longer, for example, every 0.1 seconds or longer, for example, every The measured flow rate is compared to an absolute flow rate threshold at regular intervals of 0.5 seconds or longer, such as every 1 second or longer, every 2 seconds or longer, every 3 seconds or longer, every 4 seconds or longer, every 5 seconds or longer, every 6 seconds or longer, every 7 seconds or longer, every 8 seconds or longer, every 9 seconds or longer, every 10 seconds or longer, every 15 seconds or longer, every 30 seconds or longer, and including regular intervals of every 60 seconds or longer. In some instances, an false alarm is generated if the measured flow rate exceeds the absolute flow rate threshold at each discrete comparison interval. In other instances, an false alarm is generated if the measured flow rate exceeds the threshold at more than one discrete comparison interval (e.g., in 2 or more, 3 or more, 4 or more, 5 or more, and including 10 or more discrete comparison intervals).

[0060] In an embodiment, the method includes comparing the measured flow rate of the flow with a moving window flow rate threshold. The term moving window flow rate refers to a predetermined time interval (i.e., duration) of the flow rate. For example, the moving window flow rate can be a moving average flow rate over a specific time interval, such as a moving average flow rate over a time interval of 0.1 seconds or longer, such as 0.5 seconds or longer, such as 1 second or longer, such as 2 seconds or longer, such as 3 seconds or longer, such as 4 seconds or longer, such as 5 seconds or longer, and including a moving average flow rate over a time interval of 10 seconds or longer. In some instances, the moving window flow rate is a snapshot of the measured flow rate of the flow over a specific duration, such as a snapshot of the measured flow rate over 0.1 seconds or longer, such as 0.5 seconds or longer, such as 1 second or longer, such as 2 seconds or longer, such as 3 seconds or longer, such as 4 seconds or longer, such as 5 seconds or longer, and including a snapshot of the measured flow rate over a time interval of 10 seconds or longer. In embodiments, the moving window flow rate can be the flow rate during a time interval of 0.1 seconds to 10 seconds, such as from 0.5 seconds to 9.5 seconds, such as from 1 second to 9 seconds, such as from 1.5 seconds to 8.5 seconds, such as from 2 seconds to 8 seconds, such as from 2.5 seconds to 7.5 seconds, and includes the flow rate during a time interval of 3 seconds to 7 seconds. In some instances, the method includes comparing the measured flow rate with a moving window flow rate threshold one or more times, such as two or more times, such as three or more times, such as four or more times, such as five or more times, such as ten or more times, such as 25 or more times, such as 50 or more times, such as 100 or more times, and including 250 or more times.

[0061] In some embodiments, each moving window flow rate threshold includes an upper threshold and a lower threshold. In some instances, the method includes comparing the measured flow rate to the upper threshold of the moving window flow rate threshold. In some instances, an error alarm is generated when it is determined that the measured flow rate is greater than the upper threshold of the moving window flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, for example, 0.05% or more, for example, 0.1% or more, for example, 0.5% or more, for example, 1% or more, for example, 2% or more, for example, 3% or more, for example, 4% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 20% or more, for example, 25% or more, and including 50% or more. In some instances, an error alarm is generated when it is determined that the measured flow rate is less than the lower limit of the moving window flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, and including 50% or more.

[0062] An error alarm may be generated when the flow rate exceeds one or more of the upper or lower thresholds of the moving window flow rate threshold for a duration of 0.0001 seconds or longer, for example, 0.0005 seconds or longer, for example, 0.001 seconds or longer, for example, 0.005 seconds or longer, for example, 0.01 seconds or longer, for example, 0.05 seconds or longer, for example, 0.1 seconds or longer, for example, 0.5 seconds or longer, for example, 1 second or longer, for example, 2 seconds or longer, for example, 3 seconds or longer, for example, 4 seconds or longer, for example, 5 seconds or longer, for example, 10 seconds or longer, for example, 15 seconds or longer, for example, 30 seconds or longer, for example, 45 seconds or longer, and including 60 seconds or longer. In some instances, an error alarm is generated when the flow rate exceeds the upper limit of the moving window flow rate threshold for 0.0001 seconds or longer, for example, 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer. In some instances, an error alarm is generated when the flow rate is less than the lower limit of the moving window flow rate threshold for 0.0001 seconds or longer, for example, 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer.

[0063] In some embodiments, the generated error alarm indicates a malfunction in the flow cytometer. In some instances, the malfunction is a blockage in the flow stream. In some instances, the malfunction is the introduction of gas (e.g., air) into the flow stream or sample line. In some instances, the error alarm is generated in real time. In some instances, the method includes determining the cause of the malfunction based on the generated error alarm. In some instances, flow rate patterns are analyzed based on measured flow rates to determine the cause of the malfunction. In some instances, the flow rate exhibits an increase followed by a decrease to below one or more of an absolute flow rate threshold and a moving window flow rate threshold, and it is determined that gas (e.g., bubbles) has been introduced into the flow stream. For example, the flow rate may exhibit an increase of 0.1% or more (e.g., 0.5% or more), 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, and including 10% or more, followed by a decrease to below one or more of an absolute flow rate threshold and a moving window flow rate threshold. In some instances, a flow rate increase above one or more of the upper limits of the absolute flow rate threshold and the moving window flow rate threshold, followed by a decrease below the lower limit of either the absolute flow rate threshold or the moving window flow rate threshold, indicates that gas has been introduced into the flow. In some instances, a blockage in the flow is determined when the flow rate decreases below one or more of the absolute flow rate threshold and the moving window flow rate threshold.

[0064] In some embodiments, the method includes changing one or more parameters of a flow cytometer in response to a generated error alarm. In some instances, the flow rate is adjusted in response to the generated error alarm. In some instances, the flow rate is increased in response to the generated error alarm, for example, by 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 25% or more, and including an increase of 50% or more in the flow rate. In some instances, the flow rate is decreased in response to the generated error alarm, such as by 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 25% or more, and including a decrease of 50% or more in the flow rate. In some instances, the flow rate is stopped in response to the generated error alarm. In some instances, in response to a generated error alarm, the flow rate is stopped for a predetermined time interval, such as 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer. In some instances, an experiment or calibration protocol is aborted in response to a generated error alarm. In some instances, the flow is flushed in response to a generated error alarm, for example, by flushing the flow with a predetermined volume of fluid buffer. In some instances, the flow is flushed for a predetermined time interval in response to a generated error alarm (e.g., when a blockage or air bubble is determined in the flow), such as for 5 seconds or longer, for example, for 15 seconds or longer, for example, for 30 seconds or longer, for example, for 60 seconds or longer, for example, for 300 seconds or longer, and including 600 seconds or longer.

[0065] As described in more detail below, in some instances, a light source illuminates a sample in a flowing stream. In some instances, the light source used to illuminate the flowing stream is adjusted in response to a generated error alarm. In some instances, the light source is turned off in response to a generated error alarm. In some instances, light configured to illuminate the flowing stream is blocked in response to a generated error alarm. In some instances, the light source is adjusted (e.g., blocked or turned off) when the flow rate exhibits a generated error, such as when the flow rate exhibits instability or exceeds one or more of an absolute flow rate threshold and a moving window flow rate threshold. In some instances, the flow rate is not measured in response to a generated error alarm, and the flow cytometer is put into standby mode. In some instances, the method includes adjusting one or more parameters of the light detection system in response to a generated error alarm. In some instances, one or more photodetector channels are turned off in response to a generated error alarm. In some instances, light to one or more photodetectors of the light detection system is blocked.

[0066] Figure 1A A flowchart illustrating the monitoring of flow rate in a flow cytometer according to certain embodiments is described. In step 101, the flow rate is measured. In some instances, the flow rate is measured continuously. In other instances, the flow rate is measured at discrete intervals. The flow rate can be measured using a flow sensor, such as one that determines the flow rate based on the temperature of the flow on the sensor. In step 102a, the measured flow rate is compared to an absolute flow rate threshold. The absolute flow rate threshold may include an upper threshold and a lower threshold, wherein the measured flow rate is compared to determine whether the flow rate at any given time is greater than the upper threshold or less than the lower threshold of the absolute flow rate threshold. The measured flow rate is also compared to a moving window flow rate threshold (step 102b), wherein the flow rate is compared to a threshold within a moving window time interval. For example, the moving window flow rate threshold may be an upper and lower rate over the past second. The moving window flow rate threshold may include an upper threshold and a lower threshold, wherein the measured flow rate during a time interval (e.g., the past 1 second) is compared to determine whether the flow rate during this time window is greater than the upper threshold of the moving window flow rate threshold or less than the lower threshold of the moving window flow rate threshold.

[0067] An error alarm is generated in step 103 when the measured flow rate exceeds one or more upper limits of an absolute flow rate threshold or a moving window flow rate threshold, or falls below one or more lower limits of an absolute flow rate threshold or a moving window flow rate threshold. The error alarm may indicate a malfunction in the flow cytometer, such as a blockage in the flow stream or the introduction of air into the flow stream (e.g., air bubbles). In some instances, the flow rate is analyzed to identify the source of the error alarm, for example, by analyzing the flow rate using a flow rate pattern. In some instances, one or more parameters of the flow cytometer are adjusted (step 104) in response to the generated error alarm. In some instances, the flow rate is adjusted in step 105a, which may include flushing the flow stream in step 106a (e.g., flushing with a fluid buffer solution), stopping the flow stream completely in step 106b, or increasing or decreasing the flow rate in step 106c. In some embodiments, one or more parameters of the optical detection system may be adjusted in step 105b in response to the generated error alarm. In some instances, adjusting the light detection system includes adjusting the photodetector gain, the photodetector signal amplitude, blocking one or more photodetectors of the light detection system, or shutting down one or more photodetector channels. In some embodiments, the light source of the flow cytometer can be adjusted, for example by turning off or blocking the light source as shown in step 105c.

[0068] Figure 1B The diagram depicts the monitoring of flow velocity in a flow cytometer according to certain embodiments. Screenshots illustrate, for example, a sample line where air enters the flow velocity when the sample source is depleted. The code currently divides the monitoring phase into four distinct steps or states. The code defines the acquisition-related fluid patterns monitored by the algorithm. 1) When the algorithm, executed in the firmware, begins, it exits the standby step and enters the fluid initiation step (time 0 seconds). The algorithm then enters the waiting step and waits for 10 seconds. In some instances, the nominal acquisition step may be a further waiting step (e.g., a dummy step) with an additional 3-second wait. During this phase, additional checks on the flow velocity (e.g., flow velocity stability) can be performed before entering the monitoring step. In some instances where immediate implementation is required, the further waiting step is bypassed. A recording mode can be implemented before the monitoring or standby step. Recording is a mode that records the flow velocity from the start of acquisition to a defined time. It ignores any fluid errors or changes and simply records the flow velocity data for observation.

[0069] During the monitoring of the flow velocity ( Figure 1BThe timeout is 13 seconds, during which two different checks are performed—absolute flow rate and relative flow rate. If the average flow rate buffer exceeds (is greater than or less than) the absolute flow rate threshold, a general fluid error can be reported. For low mode, due to the long wait time for mode development, a general fluid error is reported if the relative flow rate check fails. If the relative flow rate check fails in non-low mode, the algorithm then generates an alarm, recording the timestamp of the relative check failure, the maximum and minimum flow rates (…). Figure 1B The time interval is 22 seconds. Using this generated alarm, the algorithm then runs a pattern check function for an additional time interval (e.g., 15 seconds): 1) If the flow rate is above the flow rate before the relative check failure (the maximum value of the relative check error buffer), it indicates that air has been introduced into the sample line. An error alarm for air introduction (e.g., air bubbles) can be communicated to the user through the user interface. Figure 1B (1) The time interval is 32 seconds); 2) If the flow rate drops to a very low value or is far below the moving window flow rate threshold, it indicates that there is a blockage in the flow. A blockage error alarm can be communicated to the user through the user interface; 3) If neither 1 nor 2 is detected as a pattern within the pattern check interval, the algorithm can report a general error alarm and enter the error detection function. At this time, a fluid error is detected and the defined error is notified to the user. The next function (error detection function) can handle the internal state and variable reset and call the fluid mode to stop the sample and flush the sample line. In some instances, the algorithm is reset back to standby mode due to the state reset.

[0070] In some embodiments, the method includes conveying a sample containing particles through a flow stream. In some instances, the sample is conveyed in the flow stream after one or more errors in the flow rate have been corrected. In some instances, the sample is conveyed while the flow rate is being measured as described above. In some embodiments, the sample in the flow stream is illuminated with light from a light source. In some embodiments, the light source is a broadband light source that emits light with a wide wavelength range, such as spanning 50 nm or more, for example 100 nm or more, for example 150 nm or more, for example 200 nm or more, for example 250 nm or more, for example 300 nm or more, for example 350 nm or more, for example 400 nm or more, and including spanning 500 nm or more. For example, a suitable broadband light source emits light with wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with wavelengths from 400 nm to 1000 nm. When the method involves illumination with a broadband light source, the broadband light source schemes of interest may include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with continuous spectra, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof.

[0071] In other embodiments, the method includes illumination with a narrowband light source emitting a specific wavelength or a narrow wavelength range, such as illumination with a light source emitting light in a narrow wavelength range (e.g., 50 nm or less, 40 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, 2 nm or less), and illumination with a light source emitting light of a specific wavelength (i.e., monochromatic light). When the method includes illumination with a narrowband light source, the narrowband light source scheme of interest may include, but is not limited to, narrow-wavelength LEDs, laser diodes, or broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0072] In some embodiments, the method includes irradiating the sample with one or more lasers. As described above, the type and number of lasers will vary depending on the sample and the light to be collected, and can be gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other instances, the method includes irradiating the flow with a dye laser, such as a stilbene, coumarin, or rhodamine laser. In other instances, the method includes irradiating the flow with 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, or combinations thereof. In other instances, the method involves irradiating the flow with a solid-state laser, such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:yCa4O(BO3)3 laser, an Nd:YCOB laser, a titanite sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium₂O₃ laser, or a cerium-doped laser, or combinations thereof.

[0073] The sample can be irradiated with one or more of the aforementioned light sources, such as two or more, three or more, four or more, five or more, and including ten or more light sources. The light sources can include combinations of any type of light source. For example, in some embodiments, the method includes irradiating the sample in the flowing stream 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.

[0074] The sample can be illuminated with wavelengths ranging from 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm, and including 400 nm to 800 nm. For example, when the light source is a broadband light source, the sample can be illuminated with wavelengths from 200 nm to 900 nm. In other instances, when the light source comprises multiple narrowband light sources, the sample can be illuminated with specific wavelengths in the range of 200 nm to 900 nm. For example, the light source can be multiple narrowband LEDs (1 nm to 25 nm), each emitting light independently with wavelengths from 200 nm to 900 nm. In other embodiments, the narrowband light source comprises one or more lasers (e.g., a laser array) and illuminates the sample with specific wavelengths from 200 nm to 700 nm, for example, with a laser array having gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers as described above.

[0075] When using more than one light source, the sample can be illuminated simultaneously or sequentially, or in combination thereof. For example, the sample can be illuminated simultaneously with each light source. In other embodiments, the flow is illuminated sequentially with each light source. When illuminating the sample sequentially with more than one light source, the duration of illumination for each light source can be independently 0.001 microseconds or longer, for example, 0.01 microseconds or longer, for example, 0.1 microseconds or longer, for example, 1 microsecond or longer, for example, 5 microseconds or longer, for example, 10 microseconds or longer, for example, 30 microseconds or longer, and includes 60 microseconds or longer. For example, the method may include illuminating the sample with a light source (e.g., a laser) for a duration ranging from 0.001 microseconds to 100 microseconds, for example, from 0.01 microseconds to 75 microseconds, for example, from 0.1 microseconds to 50 microseconds, for example, from 1 microsecond to 25 microseconds, and includes 5 microseconds to 10 microseconds. In embodiments where the sample is illuminated sequentially with two or more light sources, the duration of illumination for each light source can be the same or different.

[0076] The time interval between each light source illumination can also vary as needed, independently spaced by delays of 0.001 microseconds or longer, such as 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 15 microseconds or longer, 30 microseconds or longer, and including extensions of 60 microseconds or longer. For example, the time interval between each light source illumination can range from 0.001 microseconds to 60 microseconds, such as from 0.01 microseconds to 50 microseconds, such as from 0.1 microseconds to 35 microseconds, such as from 1 microsecond to 25 microseconds, and includes extensions from 5 microseconds to 10 microseconds. In some embodiments, the time interval between each light source illumination is 10 microseconds. In embodiments where the sample is sequentially illuminated by more than two (i.e., three or more) light sources, the delays between each light source illumination can be the same or different.

[0077] The sample can be irradiated continuously or at discrete intervals. In some instances, the method includes irradiating the sample with a light source continuously. In other instances, the sample is irradiated with a light source 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 including every 1000 milliseconds, or at some other interval.

[0078] Depending on the light source, the sample can be illuminated from different distances, such as 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. Furthermore, the illumination angle can also vary from 10° to 90°, such as from 15° to 85°, from 20° to 80°, from 25° to 75°, and including angles from 30° to 60°, such as 90°.

[0079] In some embodiments, the method includes irradiating a sample with two or more frequency-shifted beams. As described above, a beam generator component having a laser and an acousto-optic device for frequency-shifting the laser can be employed. In these embodiments, the method includes irradiating the acousto-optic device with a laser. Depending on the desired wavelength of the light generated in the output laser beam (e.g., for irradiating a sample in a flowing stream), the laser can have a specific wavelength ranging from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including variations from 400 nm to 800 nm. The acousto-optic device can be irradiated with one or more lasers, such as two or more lasers, for example three or more lasers, for example four or more lasers, for example five or more lasers, and including ten or more lasers. The lasers can include any combination of laser types. For example, in some embodiments, the method includes irradiating an 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.

[0080] When using more than one laser, the lasers can be used to simultaneously or sequentially irradiate, or in combination, the acousto-optic device. For example, each laser can be used to simultaneously irradiate the acousto-optic device. In other embodiments, each laser is used to sequentially irradiate the acousto-optic device. When using more than one laser to sequentially irradiate the acousto-optic device, the duration for which each laser irradiates the acousto-optic device can be independently 0.001 microseconds or longer, for example, 0.01 microseconds or longer, for example, 0.1 microseconds or longer, for example, 1 microsecond or longer, for example, 5 microseconds or longer, for example, 10 microseconds or longer, for example, 30 microseconds or longer, and includes 60 microseconds or longer. For example, the method may include irradiating the acousto-optic device with a laser for a duration ranging from 0.001 microseconds to 100 microseconds, for example, from 0.01 microseconds to 75 microseconds, for example, from 0.1 microseconds to 50 microseconds, for example, from 1 microsecond to 25 microseconds, and includes 5 microseconds to 10 microseconds. In embodiments where two or more lasers are used to sequentially irradiate the acousto-optic device, the duration for which each laser irradiates the acousto-optic device may be the same or different.

[0081] The time interval between each laser irradiation can also vary as needed, independently spaced by a delay of 0.001 microseconds or longer, such as 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 15 microseconds or longer, 30 microseconds or longer, and including delays of 60 microseconds or longer. For example, the time interval between each light source irradiation can range from 0.001 microseconds to 60 microseconds, such as from 0.01 microseconds to 50 microseconds, such as from 0.1 microseconds to 35 microseconds, such as from 1 microsecond to 25 microseconds, and including delays from 5 microseconds to 10 microseconds. In some embodiments, the time interval between each laser irradiation is 10 microseconds. In embodiments where more than two (i.e., three or more) lasers sequentially irradiate the acousto-optic device, the delay between each laser irradiation can be the same or different.

[0082] The acousto-optic device can be illuminated continuously or at discrete intervals. In some instances, the method includes illuminating the acousto-optic device continuously with a laser. In other instances, the acousto-optic device is illuminated with a laser 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 including illuminating the acousto-optic device every 1000 milliseconds, or at other intervals.

[0083] Depending on the laser, the acousto-optic device can be illuminated from various distances, such as 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. Furthermore, the illumination angle can also vary from 10° to 90°, such as from 15° to 85°, from 20° to 80°, from 25° to 75°, and including angles from 30° to 60°, such as 90°.

[0084] In some embodiments, the method includes applying radio frequency (RF) drive signals to an acousto-optic device to generate an angle-deflected laser beam. Two or more RF drive signals may be applied to the acousto-optic device to generate an output laser beam having a desired number of angle-deflected laser beams. These two or more RF drive signals may be, for example, three or more RF drive signals, four or more RF drive signals, five or more RF drive signals, six or more RF drive signals, seven or more RF drive signals, eight or more RF drive signals, nine or more RF drive signals, ten or more RF drive signals, fifteen or more RF drive signals, twenty-five or more RF drive signals, fifty or more RF drive signals, and may include one hundred or more RF drive signals.

[0085] Each angle-deflecting laser beam generated by a radio frequency (RF) drive signal has an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal having a sufficient amplitude to generate an angle-deflecting laser beam with a desired intensity. In some instances, each applied RF drive signal independently has an amplitude from about 0.001 V to about 500 V, 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 50 V, for example from about 2 V to 40 V, for example from 3 V to about 30 V, and includes amplitudes from about 5 V to about 25 V. In some instances, each applied radio frequency drive signal independently has an amplitude from about 0.001 V to 100 V, such as from about 0.001 V to 200 V, such as from 0.001 V to 300 V, such as from 0.001 V to 400 V, and includes amplitudes from 0.001 V to 500 V. Each applied radio frequency drive signal has a frequency ranging from about 0.001 MHz to about 500 MHz in some embodiments, such as from about 0.005 MHz to about 400 MHz, such as from about 0.01 MHz to about 300 MHz, such as from about 0.05 MHz to about 200 MHz, such as from about 0.1 MHz to about 100 MHz, such as from about 0.5 MHz to about 90 MHz, such as from about 1 MHz to about 75 MHz, such as from about 2 MHz to about 70 MHz, such as from about 3 MHz to about 65 MHz, such as from about 4 MHz to about 60 MHz, and includes frequencies ranging from about 5 MHz to about 50 MHz. Each applied radio frequency drive signal has a frequency ranging from about 0.001 MHz to about 100 MHz in some embodiments, such as from 0.001 MHz to 200 MHz, such as from 0.001 MHz to 300 MHz, such as from 0.001 MHz to 400 MHz, such as from 0.001 MHz to 500 MHz.

[0086] In these embodiments, the angle-deflecting laser beams in the output laser beam are spatially separated. Depending on the applied RF drive signal and the desired illumination profile of the output laser beam, the angle-deflecting laser beams may be spaced 0.001 μm or more, for example 0.005 μm or more, for example 0.01 μm or more, for example 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm 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 including 5000 μm or more. In some embodiments, the angle-deflecting laser beams overlap, for example, with adjacent angle-deflecting laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., spot overlap) can be 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, and includes 100 μm or more.

[0087] In some instances, a flow is illuminated with multiple frequency-shifted beams, and particles in the flow are imaged, as 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, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 1 The disclosures described in 0,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369 and 11,946,851 are incorporated herein by reference.

[0088] In practicing the methods of this subject, light from each particle is detected by a light detection system. In embodiments, the light detection system includes one or more photodetectors, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, and including ten or more photodetectors. The photodetectors used to practice the methods of this subject can be any convenient light detection scheme, including but not limited to light sensors or photodetectors, such as avalanche photodetectors (APDs), active pixel sensors (APSs), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-mode charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or combinations thereof, and other photodetectors. In some embodiments, the photodetector is a photomultiplier tube, for example, with an effective detection surface area of ​​0.01 cm² per region. 2 Up to 10 cm 2 For example, 0.05 cm 2 up to 9 cm 2 For example, 0.1 cm 2 up to 8 cm 2 For example, 0.5 cm 2 up to 7 cm 2 And including 1 cm 2 up to 5 cm 2 A photomultiplier tube. It detects light from an irradiated sample in two or more photodetector channels, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 24 or more, 32 or more, 64 or more, 128 or more, 256 or more, and including 512 or more photodetector channels.

[0089] Light can be measured at one or more wavelengths using a photodetector, such as at two or more wavelengths, for example at five or more different wavelengths, for example at ten or more different wavelengths, for example at 25 or more different wavelengths, for example at 50 or more different wavelengths, for example at 100 or more different wavelengths, for example at 200 or more different wavelengths, for example at 300 or more different wavelengths, and including measuring light from particles in a flowing stream at 400 or more different wavelengths. Light can be measured continuously or at discrete intervals. In some instances, the detector of interest is configured to perform continuous light measurements. In other instances, the detector of interest is configured to perform measurements at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, and including measuring light every 1000 milliseconds, or at some other interval.

[0090] In some embodiments, the light detected from the sample is scattered light. As used herein, the term "scattered light" is used in its conventional sense to refer to the propagation of light energy from particles in the sample (e.g., flowing in a flow stream) that is deflected from the path of the incident beam, for example, by reflection, refraction, or deflection of the beam. In some instances, the scattered light detected from particles in the flow stream is forward scattered light (FSC). In other instances, the scattered light detected from particles in the flow stream is side scattered light (SSC). In still other instances, the scattered light detected from particles in the flow stream is backscattered light (BSC).

[0091] In some embodiments, the light detected from each particle in the sample is transmitted light, such as light detected using a bright-field detector. In other embodiments, the light detected from each particle in the sample is emitted light, such as particle luminescence (i.e., fluorescence or phosphorescence). In these embodiments, each particle may include one or more fluorophores that emit fluorescence in response to illumination from two or more light sources. For example, each particle may include two or more fluorophores, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, and ten or more fluorophores. In some instances, each particle includes a first fluorophore and a second fluorophore, the first fluorophore emitting fluorescence in response to illumination from a first laser, and the second fluorophore emitting fluorescence in response to illumination from a second laser. In some embodiments, the fluorophore of interest may include, but is not limited to, dyes suitable for analytical applications (e.g., flow cytometry, imaging, etc.), such as acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone imine dyes, acridine dyes, eurhodin dyes, saffron dyes, indamins, indophenol dyes, fluorane dyes, oxazine dyes, oxazolone dyes, thiazine dyes, thiazolium dyes, xanthan dyes, fluorene dyes, pyronin dyes, fluorane dyes, rhodamine dyes, phenanthridine dyes, as well as dyes combining two or more of the above dyes (e.g., in tandem), polymer dyes having one or more monomer dye units, and mixtures of two or more of the above dyes. A wide range of dyes are commercially available from various sources, such as Molecular Probes (Eugene, Oregon), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, Missouri), Sirigen (Santa Barbara, California), and Exciton (Dayton, Ohio). For example, fluorophores may include 4-acetamido-4'-isothiocyanate stilbene-2,2'-disulfonic acid; acridine and its derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, polydinocyanin-chlorophyll, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalenedicarboximide-3,5-disulfonic acid (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; aminoanisinolamide; Brilliant Yellow;Coumarins and their derivatives, such as coumarins, 7-amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151), are also mentioned. 151)); cyanine and its derivatives, such as cyanin, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-diamidinyl-2-phenylindole (DAPI); 5',5''-dibromopyrogallol-sulfonylphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriaminepentaacetic acid ester; 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, Dansyl chloride; 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); Eosin and its derivatives, such as eosin and eosin isothiocyanate; Erythrosine and its derivatives, such as erythrosine B and erythrosine isothiocyanate; Ethidium bromide; Fluorescein and its derivatives, such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein and QFITC ​​(XRITC); Fluorescein; IR144; IR1446; Green fluorescent protein (GFP); Reef coral fluorescent protein (RCFP); Lissamine™; Lissamine rhodamine, Lucifer Yellow); Malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; paracinon red; Nile red; Oregon green; phenol red; β-phycoerythrin; phthalaldehyde; pyrene and its derivatives, such as pyrene, pyrene butyric acid and succinimide-1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); Rhodamine and its derivatives, such as 6-carboxy-X-rhodamine (ROX), 6-carboxy-rhodamine (R6G), 4,7-dichlororhodamine lissylamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulfonylrhodamine B, sulfonylrhodamine 101, sulfonylrhodamine 101 sulfonyl chloride derivatives (Texas) Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosinic acid and terbium chelate derivatives; xanthan;Dye-conjugated polymers (i.e., polymer-attached dyes), such as fluorescein isothiocyanate-dextran, and dyes combining two or more dyes (e.g., in tandem), polymer dyes having one or more monomeric dye units, and mixtures or combinations of two or more of the above dyes.

[0092] In some instances, the fluorophore (i.e., the dye) is a fluorescent polymer dye. The fluorescent polymer dyes used in the methods and systems of this subject matter are diverse. In some instances of the methods, the polymer dyes include conjugated polymers. Conjugated polymers (CPs) are characterized by a delocalized electronic structure comprising a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated bonds (e.g., single bonds), where π electrons can move from one bond to another. Thus, the conjugated backbone can endow polymer dyes with extended linear structures, where the bond angles between repeating units of the polymer are finite. For example, while proteins and nucleic acids are also polymers, they do not form extended-rod structures in certain cases, but rather fold into more advanced three-dimensional shapes. Furthermore, CPs may form “rigid-rod” polymer backbones and undergo finite twist (e.g., torsion) angles between monomer repeating units along the polymer backbone chain. In some instances, the polymer dyes include CPs with rigid-rod structures. As mentioned above, the structural features of polymer dyes can influence the fluorescence properties of the molecule.

[0093] Any readily available polymer dye can be used in the methods and systems of this subject. In some instances, the polymer dye is a multichromophore with a structure capable of collecting light to amplify the fluorescence output of a fluorophore. In some instances, the polymer dye is capable of collecting light and efficiently converting it into emitted light of longer wavelengths. In some cases, the polymer dye has a light-collecting multichromophore system that can efficiently transfer energy to a nearby luminescent species (e.g., a “signaling chromophore”). Energy transfer mechanisms include, for example, resonant energy transfer (e.g., Foster (or fluorescence) resonant energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some instances, these energy transfer mechanisms are relatively short-range; that is, the light-collecting multichromophore system provides efficient energy transfer by being close to the signaling chromophore. Under conditions of effective energy transfer, when the number of chromophores in the light-collecting multi-chromophore system is large, the emission from the signal chromophore will be amplified; that is, when the incident light ("excitation light") is at a wavelength that is absorbed by the light-collecting multi-chromophore system, the emission from the signal chromophore is stronger than when the signal chromophore is directly excited by the pump light.

[0094] Multichromophores can be conjugated polymers. Conjugated polymers (CPs) are characterized by their delocalized electronic structure and can be used as highly responsive optical reporter molecules targeting chemical and biological objectives. Because the effective conjugation length is much shorter than the polymer chain length, the backbone contains a large number of closely spaced conjugated segments. Therefore, conjugated polymers are efficient for light harvesting and achieve optical amplification through energy transfer.

[0095] In some instances, polymers can be used as direct fluorescent reporter molecules, such as fluorescent polymers with high extinction coefficients, high brightness, etc. In other instances, polymers can be used as strong chromophores, where color or optical density serves as an indicator.

[0096] Polymer dyes of interest include, but are not limited to, those described in Gaylord et al. in U.S. Publications 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986, 20130190193, and 20160025735, the disclosures of which are incorporated herein by reference in their entirety; and Gaylord et al., J. Am. Chem. Soc., 2001, 123 (26), pp. 6417–6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411–2419; and Traina et al., J. Am. Chem. Soc., 2011, 133 (32). The dyes described in sections 12600–12607 are incorporated herein by reference in their entirety.

[0097] In some instances, the sample is a biological sample. The term "biological sample," used in its conventional sense, refers to a subset of tissues, cells, or components of a whole organism, plant, fungus, or animal, which in some cases may be present in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, cord blood, urine, vaginal fluid, and semen. Therefore, "biological sample" refers both to a natural organism or a subset of its tissues and to homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, slices of skin, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. Biological samples can be any type of biological tissue, including healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic tissue, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some instances the sample is a blood sample, including whole blood, such as blood obtained by venipuncture or finger prick (wherein the blood may be bound or unbound to any reagents such as preservatives, anticoagulants, etc. before testing).

[0098] In some embodiments, the sample source is "mammal" or "mammal," terms widely used to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subject is a human. The method can be applied to samples obtained from male and female human subjects at any developmental stage (i.e., newborns, infants, toddlers, adolescents, and adults), where in some embodiments the human subject is a toddler, adolescent, or adult. While this disclosure can be applied to samples from human subjects, it should be understood that the method can also be implemented on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0099] Cells of interest can be targeted and characterized based on various parameters, such as phenotypic features identified by attaching specific fluorescent tags to the cells of interest. In some embodiments, the system is configured to deflect analytical droplets identified as containing target cells. Various cell types can be characterized using the methods described in this subject matter. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest also include cells with readily available cell surface markers or antigens that can be captured or labeled by readily available affinity agents or conjugates thereof. For example, target cells may include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialotetrahexosylganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) derived from whole blood, bone marrow, or umbilical cord blood.

[0100] When practicing the methods of this subject matter according to certain embodiments, a certain amount of initial fluid sample is injected into the flow cytometer. The amount of sample injected into the particle sorting module may vary, for example, from 0.001 mL to 1000 mL, from 0.005 mL to 900 mL, from 0.01 mL to 800 mL, from 0.05 mL to 700 mL, from 0.1 mL to 600 mL, from 0.5 mL to 500 mL, from 1 mL to 400 mL, from 2 mL to 300 mL, and includes samples from 5 mL to 100 mL.

[0101] In some embodiments, the method includes counting and optionally sorting labeled particles (e.g., target cells) in the sample. In practicing the method of this subject, a fluid sample containing particles is first introduced into a flow nozzle of the system. After exiting the flow nozzle, the particles pass through a sample interrogation region substantially one at a time, where each particle is illuminated by a light source, and measurements of light scattering parameters are recorded for each particle, and in some cases, fluorescence emission (e.g., measurements of two or more light scattering parameters and one or more fluorescence emission) is recorded as needed. Depending on the characteristics of the interrogated flow, the flow can be illuminated by light for 0.001 mm or more, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, and including 1 mm or more. In some embodiments, the method includes illuminating a planar cross-section of the flow in the sample interrogation region, for example, illuminating this planar cross-section with a laser (as described above). In other embodiments, the method includes illuminating a predetermined length of the flow in the sample interrogation region, for example, a predetermined length corresponding to the illumination profile of a diffuse laser beam or lamp.

[0102] In some embodiments, the method includes atomizing the flow at or near the flow cell nozzle orifice. For example, the method may include atomizing the flow at a location approximately 0.001 mm or more (e.g., 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, and including 1 mm or more). In some embodiments, the method includes atomizing the flow immediately adjacent to the flow cell nozzle orifice.

[0103] In embodiments of this method, detectors such as photomultiplier tubes (PMTs) are used to record light passing through each particle (in some cases referred to as forward scattering), light reflected orthogonally to the direction of particle flow through the sensing region (in some cases referred to as orthogonal or lateral scattering), and fluorescence emitted from the particle when it passes through the sensing region and is illuminated by an energy source, if the particle is labeled with one or more fluorescent markers. Each of forward scattering (FSC), lateral scattering (SSC), and fluorescence emission includes a separate parameter for each particle (or each "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle is analyzed in real time or stored in a data storage and analysis device such as a computer, as needed.

[0104] In some embodiments, particles are detected and uniquely identified, as needed, by exposing them to excitation light and measuring the fluorescence of each particle in one or more detection channels. The fluorescence emitted in the detection channels used to identify the particles and their associated binding complexes can be measured after excitation with a single light source, or individually after excitation with different light sources. If separate excitation sources are used to excite the particle tags, the tags can be selected such that all tags can be excited by each excitation source used.

[0105] The method in some embodiments further includes data acquisition, analysis, and recording, for example, using a computer for data acquisition, analysis, and recording, wherein multiple data channels record fluorescence and light scattering data emitted from each detector as each particle passes through the sample interrogation region of the particle sorting module. In these embodiments, the analysis includes classifying and counting particles such that each particle exists as a set of digitized parameter values. The system can be configured to be triggered based on selected parameters to distinguish particles of interest from background and noise. “Trigger” refers to a preset threshold for the detection parameter and can be used as a means of detecting particles passing through the light source. Detecting an event exceeding the threshold of the selected parameter triggers the acquisition of light scattering and fluorescence data of the particles. For particles or other components that elicit a response below the threshold in the medium being measured, no data is acquired. The trigger parameter can be the detection of forward scattered light caused by particles passing through the light beam. Flow cytometry then detects and collects the light scattering and fluorescence data of the particles.

[0106] Then, specific subpopulations of interest are further analyzed by “gating” based on data collected for the entire population. To select the appropriate gate, the data is plotted to obtain the best possible subpopulation separation. This process can be performed by plotting forward light scattering (FSC) and lateral (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. Subpopulations of particles (i.e., those cells within the gate) are then selected, and particles not within the gate are excluded. If needed, gates can be selected by drawing lines around the desired subpopulation using a cursor on the computer screen. These particles within the gate are then further analyzed by plotting other parameters (e.g., fluorescence) for them. If necessary, the above analysis can be configured to generate a count of particles of interest in the sample.

[0107] Methods of interest may also include the use of particles in research, laboratory testing, or therapeutics. In some embodiments, the methods of this subject matter include obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the methods of this subject matter include obtaining cells from a fluid or tissue sample for use as a research or diagnostic specimen for a disease, such as cancer. Similarly, the methods of this subject matter include obtaining cells from a fluid or tissue sample for therapeutic purposes. A cell therapy protocol is a protocol in which feasible cellular materials, including, for example, cells and tissues, can be prepared and introduced into a subject as a therapeutic treatment. Conditions that can be treated by administering samples sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, organ damage, etc.

[0108] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, genetic modification, in vitro culture and expansion, cell collection, sample volume reduction and washing, biopreservation, storage, and introduction of cells into a subject. The protocol may begin with the collection of live cells and tissues from the subject's source tissue to produce a sample of cells and / or tissues. Samples can be collected using any suitable procedure, including, for example, administration of cell mobilizing agents to the subject, aspiration of blood from the subject, removal of bone marrow from the subject, etc. After sample collection, cells can be enriched using several methods, including, for example, centrifugation-based methods, filter-based methods, washing, magnetic separation methods, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells can be genetically modified using any convenient method, such as nuclease-mediated gene editing. Genetically modified cells can then be cultured, activated, and expanded in vitro. In some cases, cells are preserved, for example, frozen, and stored for future use, where the cells are thawed and then administered to the patient; for example, cells can be injected into a patient.

[0109] system

[0110] This disclosure also includes systems for practicing methods of the subject matter, such as monitoring the flow rate of a flowing stream (e.g., in a flow cytometer). A system according to some embodiments includes a processor having memory operatively coupled to the processor, wherein the memory includes instructions stored thereon for measuring the flow rate of the flowing stream, for comparing the measured flow rate of the flowing stream with an absolute flow rate threshold and a moving window flow rate threshold, and for generating an error alarm when the measured flow rate of the flowing stream exceeds the absolute flow rate threshold or the moving window flow rate threshold.

[0111] A flow sensor used to measure the velocity of a flowing flow can be any convenient embodiment, wherein in some instances, the flow sensor is configured to measure the velocity based on the temperature of the measured flowing flow. In some instances, the flow sensor is configured to measure the velocity based on the viscosity of the measured flowing flow. In some instances, the flow sensor is configured to measure the velocity at one or more detection locations along the flowing flow, such as at two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more detection locations, and includes measuring the velocity at ten or more detection locations along the flowing flow. In some instances, the detection locations for measuring the velocity of the flowing flow span 0.001 mm or more, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2 mm or more, for example, 5 mm or more, and includes 10 mm or more of the flowing flow. In some instances, the flow sensor is configured to measure the flow velocity based on temperature changes in the flow at two or more detection locations, such as three or more, for example four or more, for example five or more, for example six or more, for example seven or more, for example eight or more, for example nine or more detection locations, and including temperature changes in the flow at ten or more different detection locations along the flow. In some instances, the flow sensor is configured to measure the flow velocity based on viscosity changes in the flow at two or more different detection locations, such as three or more, for example four or more, for example five or more, for example six or more, for example seven or more, for example eight or more, for example nine or more detection locations, and including viscosity changes in the flow at ten or more detection locations along the flow.

[0112] In some instances, the flow sensor is a temperature sensor flow meter. In some instances, the flow velocity sensor includes a controllable heating element located at the center of a pressure-stabilized membrane, and temperature sensors mounted upstream and downstream in the direction of fluid flow (e.g., symmetrically). In some instances, the flow causes heat transfer to the downstream temperature sensor, resulting in a measurable signal due to the temperature difference. In some instances, a micro-thermal flow sensor is integrated into a silicon chip flow meter-type sensor. In some instances, the flow sensor includes a temperature sensor that generates a signal that compensates for temperature effects. In some instances, the flow sensor includes a fluid flow sensor, such as those from the fluid flow sensor series commercially available from Sensiron (Switzerland). In some instances, a flow sensor is used to measure flow velocity, such as those described in U.S. Patent Publication No. 2023 / 0324270, U.S. Patent No. 10,942,139, and European Patent Nos. EP3187881 and EP3404373, the disclosures of which are incorporated herein by reference.

[0113] In one embodiment, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon for measuring the flow velocity. In some instances, the memory includes instructions for continuously measuring the flow velocity, for example, by continuously measuring the flow velocity through real-time monitoring. In some instances, the memory includes instructions for measuring the flow velocity at discrete intervals, such as at regular intervals of 0.00001 seconds or greater, for example, at regular intervals of 0.00005 seconds or greater, for example, at regular intervals of 0.0001 seconds or greater, for example, at regular intervals of 0.0005 seconds or greater, for example, at regular intervals of 0.001 seconds or greater, for example, at regular intervals of 0.005 seconds or greater, for example, at regular intervals of 0.01 seconds or greater, for example, at regular intervals of 0.05 seconds or greater, for example, at regular intervals of 0.1 seconds or greater, for example, at regular intervals of 0.5 seconds. Or larger rule intervals, such as every 1 second or larger rule intervals, such as every 2 seconds or larger rule intervals, such as every 3 seconds or larger rule intervals, such as every 4 seconds or larger rule intervals, such as every 5 seconds or larger rule intervals, such as every 6 seconds or larger rule intervals, such as every 7 seconds or larger rule intervals, such as every 8 seconds or larger rule intervals, such as every 9 seconds or larger rule intervals, such as every 10 seconds or larger rule intervals, such as every 15 seconds or larger rule intervals, such as every 30 seconds or larger rule intervals, and including every 60 seconds or larger rule intervals to measure flow rate.

[0114] In some embodiments, the flow rate can be 1 μL / min or higher, such as 2 μL / min or higher, such as 3 μL / min or higher, such as 5 μL / min or higher, such as 10 μL / min or higher, such as 15 μL / min or higher, such as 25 μL / min or higher, such as 50 μL / min or higher, and includes 100 μL / min or higher, wherein in some instances the flow rate is 1 μL / sec or higher, such as 2 μL / sec or higher, such as 3 μL / sec or higher, such as 5 μL / sec or higher, such as 10 μL / sec or higher, such as 15 μL / sec or higher, such as 25 μL / sec or higher, such as 50 μL / sec or higher, and includes 100 μL / sec or higher. In some embodiments, the flow rate can be 25 μL / sec or higher, such as 50 μL / sec or higher, such as 75 μL / sec or higher, such as 100 μL / sec or higher, such as 250 μL / sec or higher, such as 500 μL / sec or higher, such as 750 μL / sec or more, such as 1000 μL / sec or more, and includes 2500 μL / sec or more.

[0115] In some embodiments, the flow stream includes a sheath fluid flow stream and a sample core fluid flow stream. In some embodiments, the sheath fluid injection system is configured to provide a sheath fluid flow into the flow cell chamber, for example, in combination with the sample fluid to generate a sheath fluid laminar flow around the sample core fluid flow stream. In some instances, the sheath fluid flow stream forms a laminar flow around the sample core fluid flow stream. In some instances, the memory includes instructions for measuring the flow rate of the sheath fluid flow stream. In some instances, the flow rate of the sheath fluid flow stream is 25 μL / sec or higher, such as 50 μL / sec or higher, such as 75 μL / sec or higher, such as 100 μL / sec or higher, such as 250 μL / sec or higher, such as 500 μL / sec or higher, such as 750 μL / sec or higher, such as 1000 μL / sec or higher, and includes 2500 μL / sec or higher. In some instances, the memory includes instructions for measuring the flow rate of the sample core fluid flow stream. In some instances, the flow rate of the sample core fluid flow is 1 μL / min or higher, for example, 2 μL / min or higher, for example, 3 μL / min or higher, for example, 5 μL / min or higher, for example, 10 μL / min or higher, for example, 15 μL / min or higher, for example, 25 μL / min or higher, for example, 50 μL / min or higher, and including 100 μL / min or higher. In some instances, the sample rate delivered to the flow cell chamber via the sample injection port is 1 μL / sec or higher, for example, 2 μL / sec or higher, for example, 3 μL / sec or higher, for example, 5 μL / sec or higher, for example, 10 μL / sec or higher, for example, 15 μL / sec or higher, for example, 25 μL / sec or higher, for example, 50 μL / sec or higher, and including 100 μL / sec or higher. In some instances, the memory includes instructions for measuring the flow rate of the sheath fluid flow and the flow rate of the sample core fluid flow.

[0116] In some instances, the memory includes instructions for comparing the flow rate of the sheath fluid flow with the flow rate of the sample core fluid flow. In some instances, the memory includes instructions for determining that the flow rate of the sheath fluid flow is the same as the flow rate of the sample core fluid flow. In some instances, the memory includes instructions for determining that the flow rate of the sheath fluid flow is less than the flow rate of the sample core fluid flow, for example, the flow rate of the sheath fluid flow is 0.01% or more less than the flow rate of the sample core fluid flow, such as 0.05% or more less, 0.1% or more less, 0.5% or more less, 1% or more less, 2% or more less, 3% or more less, 4% or more less, 5% or more less, 10% or more less, 15% or more less, 20% or more less, 25% or more less, and including less than 50% or more. In some instances, the memory includes instructions for determining that the flow rate of the sheath fluid flow is greater than the flow rate of the sample core fluid flow, for example, the flow rate of the sheath fluid flow is 0.01% or more greater than the flow rate of the sample core fluid flow, such as 0.05% or more, 0.1% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, and including 50% or more. When it is determined that the relative flow rates of the sheath fluid flow and the sample core fluid flow exceed a predetermined threshold, the memory includes instructions for adjusting one or more of the flow rates of the sheath fluid flow and the sample core fluid flow. In some instances, the memory includes instructions for adjusting the flow rate of one or more of the sheath fluid flow and the sample core fluid flow when the relative flow rate of the sheath fluid flow and the sample core fluid flow exceeds a threshold of 1% or more, for example 2% or more, for example 3% or more, for example 4% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 20% or more, for example 25% or more, and including 50% or more.

[0117] In some embodiments, the memory includes instructions for initiating flow rate measurement of the flow when a stable flow rate is detected. In some instances, the memory includes instructions for measuring the flow rate when the flow rate exhibits fluctuations (e.g., showing instantaneous measured changes) of 5% or less, such as 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.001% or less, including when the flow rate exhibits changes of 0.0001% or less. When the flow rate exhibits fluctuations exceeding a stability threshold, the system may be set to a standby mode until the fluctuations are below the stability threshold for a predetermined duration, such as 1 second or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, and including 60 seconds or longer. Once the flow is determined to be sufficiently stable for monitoring, the memory includes instructions for entering a monitoring phase to measure the flow rate. In some embodiments, when the flow rate exhibits instability or fluctuations exceeding a stability threshold during the monitoring phase, the system can be configured to re-enter standby mode and the monitoring phase is paused until the flow rate exhibits sufficient stability.

[0118] In some embodiments, the absolute flow rate includes an upper threshold and a lower threshold. In some instances, the memory includes instructions for continuously measuring the absolute flow rate. In some instances, the memory includes instructions for continuously comparing the measured flow rate with the upper absolute flow rate threshold. In some instances, the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is greater than the upper absolute flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, for example, 0.05% or more, for example, 0.1% or more, for example, 0.5% or more, for example, 1% or more, for example, 2% or more, for example, 3% or more, for example, 4% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 20% or more, for example, 25% or more, and including 50% or more. In some instances, the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is less than a lower absolute flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, for example 0.05% or more, for example 0.1% or more, for example 0.5% or more, for example 1% or more, for example 2% or more, for example 3% or more, for example 4% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 20% or more, for example 25% or more, and including 50% or more. When continuously measuring the flow rate, the memory includes instructions for generating an error alarm when the flow rate exceeds one or more of the upper or lower thresholds of the absolute flow rate threshold for a duration of 0.0001 seconds or longer (e.g., 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer).In some instances, the memory includes instructions for generating an error alarm when the flow rate exceeds an upper limit threshold of the absolute flow rate threshold for 0.0001 seconds or longer (e.g., 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer). In some instances, the memory includes instructions for generating an error alarm when the flow rate is less than a lower limit threshold of the absolute flow rate threshold for 0.0001 seconds or longer (e.g., 0.0005 seconds or longer, 0.001 seconds or longer, 0.005 seconds or longer, 0.01 seconds or longer, 0.05 seconds or longer, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer).

[0119] In some instances, the memory includes instructions for comparing the measured flow rate of the flow with an absolute flow rate threshold at discrete intervals, such as every 0.00001 seconds or longer, for example, every 0.00005 seconds or longer, for example, every 0.0001 seconds or longer, for example, every 0.0005 seconds or longer, for example, every 0.001 seconds or longer, for example, every 0.005 seconds or longer, for example, every 0.01 seconds or longer, for example, every 0.05 seconds or longer, for example, every 0.1 seconds or longer. The memory compares the measured flow rate to an absolute flow rate threshold at regular intervals, such as every 0.5 seconds or longer, every 1 second or longer, every 2 seconds or longer, every 3 seconds or longer, every 4 seconds or longer, every 5 seconds or longer, every 6 seconds or longer, every 7 seconds or longer, every 8 seconds or longer, every 9 seconds or longer, every 10 seconds or longer, every 15 seconds or longer, every 30 seconds or longer, and including regular intervals of every 60 seconds or longer. In some instances, the memory includes instructions for generating an error alarm if the measured flow rate exceeds the absolute flow rate threshold at each discrete comparison interval. In other instances, the memory includes instructions for generating an error alarm if the measured flow rate exceeds the threshold at more than one discrete comparison interval (e.g., in two or more, such as three or more, such as four or more, such as five or more, and including ten or more discrete comparison intervals).

[0120] In an embodiment, the memory includes instructions for comparing the measured flow rate of the flow with a moving window flow rate threshold. The predetermined time interval (i.e., duration) of each moving window can be a moving average flow rate of the flow over a specific time interval, such as a moving average flow rate over a time interval of 0.1 seconds or longer, such as 0.5 seconds or longer, such as 1 second or longer, such as 2 seconds or longer, such as 3 seconds or longer, such as 4 seconds or longer, such as 5 seconds or longer, and including a moving average flow rate over a time interval of 10 seconds or longer. In some instances, the moving window flow rate is a snapshot of the measured flow rate of the flow over a specific duration, such as a snapshot of the measured flow rate over a time interval of 0.1 seconds or longer, such as 0.5 seconds or longer, such as 1 second or longer, such as 2 seconds or longer, such as 3 seconds or longer, such as 4 seconds or longer, such as 5 seconds or longer, and including a snapshot of the measured flow rate over a time interval of 10 seconds or longer. In embodiments, the moving window flow rate can be a flow rate during a time interval of 0.1 seconds to 10 seconds, such as from 0.5 seconds to 9.5 seconds, such as from 1 second to 9 seconds, such as from 1.5 seconds to 8.5 seconds, such as from 2 seconds to 8 seconds, such as from 2.5 seconds to 7.5 seconds, and including flow rates during time intervals of 3 seconds to 7 seconds. In some instances, the memory includes instructions for comparing the measured flow rate with a moving window flow rate threshold one or more times, such as two or more times, such as three or more times, such as four or more times, such as five or more times, such as ten or more times, such as 25 or more times, such as 50 or more times, such as 100 or more times, and including 250 or more times.

[0121] In some embodiments, each moving window flow rate threshold includes an upper threshold and a lower threshold. In some instances, the memory includes instructions for comparing the measured flow rate with the upper threshold of the moving window flow rate threshold. In some instances, the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is greater than the upper threshold of the moving window flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, for example, 0.05% or more, for example, 0.1% or more, for example, 0.5% or more, for example, 1% or more, for example, 2% or more, for example, 3% or more, for example, 4% or more, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 20% or more, for example, 25% or more, and including 50% or more. In some instances, the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is less than a lower threshold of a moving window flow rate threshold, for example, when it is determined that the measured flow rate is 0.01% or more, for example 0.05% or more, for example 0.1% or more, for example 0.5% or more, for example 1% or more, for example 2% or more, for example 3% or more, for example 4% or more, for example 5% or more, for example 10% or more, for example 15% or more, for example 20% or more, for example 25% or more, and including 50% or more.

[0122] In some embodiments, the memory includes instructions for generating an error alarm when the flow rate exceeds one or more of the upper or lower thresholds of the moving window flow rate threshold for a duration of 0.0001 seconds or longer, for example, 0.0005 seconds or longer, for example, 0.001 seconds or longer, for example, 0.005 seconds or longer, for example, 0.01 seconds or longer, for example, 0.05 seconds or longer, for example, 0.1 seconds or longer, for example, 0.5 seconds or longer, for example, 1 second or longer, for example, 2 seconds or longer, for example, 3 seconds or longer, for example, 4 seconds or longer, for example, 5 seconds or longer, for example, 10 seconds or longer, for example, 15 seconds or longer, for example, 30 seconds or longer, for example, 45 seconds or longer, and including 60 seconds or longer. In some instances, the memory includes the following instruction: This instruction is used to generate an error alarm when the flow rate is greater than the upper limit threshold of the moving window flow rate threshold for 0.0001 seconds or longer, for example, 0.0005 seconds or longer, for example, 0.001 seconds or longer, for example, 0.005 seconds or longer, for example, 0.01 seconds or longer, for example, 0.05 seconds or longer, for example, 0.1 seconds or longer, for example, 0.5 seconds or longer, for example, 1 second or longer, for example, 2 seconds or longer, for example, 3 seconds or longer, for example, 4 seconds or longer, for example, 5 seconds or longer, for example, 10 seconds or longer, for example, 15 seconds or longer, for example, 30 seconds or longer, for example, 45 seconds or longer, and including 60 seconds or longer. In some instances, the memory includes the following instruction: This instruction is used to generate an error alarm when the flow rate is less than the lower threshold of the moving window flow rate threshold for 0.0001 seconds or longer, for example, 0.0005 seconds or longer, for example, 0.001 seconds or longer, for example, 0.005 seconds or longer, for example, 0.01 seconds or longer, for example, 0.05 seconds or longer, for example, 0.1 seconds or longer, for example, 0.5 seconds or longer, for example, 1 second or longer, for example, 2 seconds or longer, for example, 3 seconds or longer, for example, 4 seconds or longer, for example, 5 seconds or longer, for example, 10 seconds or longer, for example, 15 seconds or longer, for example, 30 seconds or longer, for example, 45 seconds or longer, and including 60 seconds or longer.

[0123] In some embodiments, the generated error alarm indicates a malfunction in the flow cytometer. In some instances, the malfunction is a blockage in the flow stream. In some instances, the malfunction is the introduction of gas (e.g., air) into the flow stream or sample line. In some instances, the memory includes instructions for generating error alarms in real time. In some instances, the memory includes instructions for determining the cause of the malfunction based on the generated error alarm. In some instances, the memory includes instructions for determining the cause of the malfunction based on flow rate analysis of measured flow rates. In some instances, the flow rate exhibits an increase followed by a decrease to below one or more of an absolute flow rate threshold and a moving window flow rate threshold, and the memory includes instructions for determining that gas (e.g., bubbles) has been introduced into the flow stream. For example, the flow rate may exhibit an increase of 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, and including 10% or more, followed by a decrease to below one or more of an absolute flow rate threshold and a moving window flow rate threshold. In some instances, the flow rate exhibits an increase above one or more upper thresholds of the absolute flow rate threshold and the moving window flow rate threshold, followed by a decrease below the lower threshold of either the absolute flow rate threshold or the moving window flow rate threshold, and the memory includes instructions for determining that gas has been introduced into the flow. In some instances, when the flow rate decreases below one or more of the absolute flow rate threshold and the moving window flow rate threshold, the memory includes instructions for determining that a blockage exists in the flow.

[0124] In some embodiments, the memory includes instructions for changing one or more parameters of the flow cytometer in response to a generated error alarm. In some instances, the memory includes instructions for adjusting the flow rate of the flow stream in response to a generated error alarm. In some instances, the memory includes instructions for increasing the flow rate in response to a generated error alarm, for example, wherein the flow rate is increased by 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 25% or more, and including wherein the flow rate is increased by 50% or more. In some instances, the memory includes instructions for decreasing the flow rate in response to a generated error alarm, such as decreasing the flow rate by 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 4% or more, such as 5% or more, such as 10% or more, such as 25% or more, and including wherein the flow rate is decreased by 50% or more. In some instances, the memory includes instructions for stopping the flow rate in response to a generated error alarm. In some instances, the flow rate is stopped for a predetermined time interval in response to a generated error alarm, such as 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 2 seconds or longer, 3 seconds or longer, 4 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 30 seconds or longer, 45 seconds or longer, and including 60 seconds or longer. In some instances, the memory includes instructions for aborting an experiment or calibration protocol in response to a generated error alarm. In some instances, the memory includes instructions for flushing the flow stream in response to a generated error alarm, for example, by flushing the flow stream with a predetermined volume of fluid buffer. In some instances, the memory includes instructions for flushing the flow stream for a predetermined time interval in response to a generated error alarm (e.g., when a blockage or air bubble is determined in the flow stream), such as flushing the flow stream for 5 seconds or longer, for example, up to 15 seconds or longer, for example, up to 30 seconds or longer, for example, up to 60 seconds or longer, for example, up to 300 seconds or longer, and including up to 600 seconds or longer.

[0125] As described in more detail below, the system includes a light source for illuminating the flowing stream. In some instances, the memory includes instructions for adjusting one or more parameters of the light source in response to a generated error alarm. In some instances, the memory includes instructions for shutting down the light source in response to a generated error alarm. In some instances, the memory includes instructions for blocking the light source in response to a generated error alarm. In some instances, the memory includes instructions for adjusting the light source when the flow rate exhibits a generated error, such as when the flow rate exhibits instability or exceeds one or more of an absolute flow rate threshold and a moving window flow rate threshold. In some instances, the memory includes instructions for stopping flow rate measurement in response to a generated error alarm and putting the flow cytometer into standby mode. In some instances, the memory includes instructions for adjusting one or more parameters of the light detection system in response to a generated error alarm. In some instances, the memory includes instructions for shutting down one or more photodetector channels in response to a generated error alarm. In some instances, the memory includes instructions for blocking light to one or more photodetectors of the light detection system.

[0126] The system according to some embodiments includes a light source configured to illuminate a flowing stream. In embodiments, the light source can be any suitable broadband or narrowband light source. The light source can be configured to emit light wavelengths ranging from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including light wavelengths varying from 400 nm to 800 nm. For example, the light source can include a broadband light source that emits light with wavelengths from 200 nm to 900 nm. In other instances, the light source includes a narrowband light source that emits light with wavelengths from 200 nm to 900 nm. For example, the light source can be a narrowband LED (1 nm to 25 nm) that emits light with wavelengths from 200 nm to 900 nm. In some embodiments, the light source is a laser. In some instances, the subject matter system includes gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other instances, the subject matter system includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In still other instances, lasers of interest include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, or combinations thereof. In other instances, the subject matter system includes solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:yCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers and combinations thereof.

[0127] In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to halogen lamps, deuterium arc lamps, xenon arc lamps, and light-emitting diodes (LEDs), such as broadband LEDs with a continuous spectrum, superluminescent LEDs, semiconductor LEDs, broadband LED white light sources, and multi-LED integration. In some instances, the non-laser light source is a stable fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0128] The light source can be positioned at any suitable distance from the flow, such as 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, and including distances of 100 mm or more. Furthermore, the light source illuminates the flow at any suitable angle (e.g., relative to the vertical axis of the flow), such angles ranging from 10° to 90°, from 15° to 85°, from 20° to 80°, from 25° to 75°, and including angles from 30° to 60°, such as 90°.

[0129] The light source can be configured to illuminate the sample continuously or at discrete intervals. In some instances, the system includes a light source configured to continuously illuminate the sample, such as a continuous-wave laser used to continuously illuminate the flow at the interrogation point in a flow cytometer. In other instances, the system of interest includes a light source configured to illuminate the sample at discrete intervals, such as every 0.001 ms, every 0.01 ms, every 0.1 ms, every 1 ms, every 10 ms, every 100 ms, and including every 1000 ms or some other interval. When the light source is configured to illuminate the sample at discrete intervals, the system may include one or more additional components to provide intermittent illumination of the sample by the light source. For example, the subject system in these embodiments may include one or more laser beam choppers, manually or computer-controlled beam stoppers for shielding the sample and exposing the sample to the light source.

[0130] In some embodiments, the light source is a laser. Lasers of interest may include pulsed lasers or continuous-wave lasers. For example, the laser may be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or 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, or a neon-copper (N) laser. eCu ​​lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:yCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or frequency doubling or third harmonics of any of the above lasers.

[0131] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some instances, the beam generator includes a laser, an RF generator configured to apply an RF drive signal to an acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser in the beam generator of interest can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, or a helium-selenium (He) laser. Se lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:yCa4O(BO3)3 lasers, Nd:YCOB lasers, titania-sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium₂O₃ lasers or cerium-doped lasers and combinations thereof.

[0132] The acousto-optic device can be any convenient acousto-optic scheme configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in this subject matter system is configured to generate an angle-deflected laser beam based on light from a laser and an applied radio frequency (RF) drive signal. Any suitable RF drive signal source, such as a direct digital synthesizer (DDS), arbitrary waveform generator (AWG), or electrical pulse generator, can be used to apply the RF drive signal to the acousto-optic device.

[0133] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angle-deflected laser beams in the output laser beam, for example, to apply 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and includes being further configured to apply 100 or more radio frequency drive signals.

[0134] In some instances, in order to generate an intensity profile of an angle-deflected laser beam in the output laser beam, the controller is configured to apply an radio frequency drive signal, the amplitude of which is, for example, from about 0.001 V to about 500 V, 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 50 V, for example from about 2 V to 40 V, for example from 3 V to about 30 V, and includes variations from about 5 V to about 25 V. Each applied radio frequency drive signal has a frequency ranging from about 0.001 MHz to about 500 MHz in some embodiments, such as from about 0.005 MHz to about 400 MHz, such as from about 0.01 MHz to about 300 MHz, such as from about 0.05 MHz to about 200 MHz, such as from about 0.1 MHz to about 100 MHz, such as from about 0.5 MHz to about 90 MHz, such as from about 1 MHz to about 75 MHz, such as from about 2 MHz to about 70 MHz, such as from about 3 MHz to about 65 MHz, such as from about 4 MHz to about 60 MHz, and includes frequencies ranging from about 5 MHz to about 50 MHz.

[0135] In some embodiments, the controller has a processor operatively coupled to the processor, such that a memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having angle-deflected laser beams with desired intensity profiles. For example, the memory may include instructions for generating two or more angle-deflected laser beams of equal intensity, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more angle-deflected laser beams, and the memory may include instructions for generating 100 or more angle-deflected laser beams of equal intensity. In other embodiments, they may include instructions for generating two or more angle-deflected laser beams of different intensities, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more angle-deflected laser beams, and the memory may include instructions for generating 100 or more angle-deflected laser beams of different intensities.

[0136] In some embodiments, the controller has a processor with a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity increases along a horizontal axis from the edge to the center. In these cases, the intensity of the laser beam angled at the center of the output beam can be from 0.1% to about 99%, for example 0.5% to about 95%, for example 1% to about 90%, for example about 2% to about 85%, for example about 3% to about 80%, for example about 4% to about 75%, for example about 5% to about 70%, for example about 6% to about 65%, for example about 7% to about 60%, for example about 8% to about 55%, and includes about 10% to about 50% of the intensity of the laser beam angled at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor with a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity increases along a horizontal axis from the edge to the center. In these cases, the intensity of the laser beam angled at the edge of the output beam can be from 0.1% to about 99%, for example 0.5% to about 95%, for example 1% to about 90%, for example about 2% to about 85%, for example about 3% to about 80%, for example about 4% to about 75%, for example about 5% to about 70%, for example about 6% to about 65%, for example about 7% to about 60%, for example about 8% to about 55%, and includes about 10% to about 50% of the intensity of the laser beam angled at the center of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor with a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having a Gaussian intensity profile along a horizontal axis. In other embodiments, the controller has a processor with a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity distribution along a horizontal axis.

[0137] In embodiments, the beam generator of interest can be configured to generate spatially separated angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam, the angle-deflected laser beams can be spaced 0.001 μm or more, for example 0.005 μm or more, for example 0.01 μm or more, for example 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm 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 include spacings of 5000 μm or more. In some embodiments, the system is configured to generate overlapping angle-deflected laser beams within the output laser beam, for example, overlapping with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., spot overlap) can be 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, and includes 100 μm or more.

[0138] In some instances, beam generators configured to generate two or more frequency-shifted beams include laser excitation modules, such as those described by Diebold et al., Nature Photonics, Vol. 7(10); 806-810. As described in (2013), and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, The contents of 11,692,926, 11,630,053, 11,774,343, 11,940,369 and 11,946,851 are described herein; their public information is incorporated herein by reference.

[0139] In embodiments, the system includes a light detection system having a photodetector configured to detect light. In some embodiments, the light detection system is configured to detect scattered light. In some instances, the light detection system includes a side-scattering light detector. In some instances, the light detection system includes a forward-scattering light detector. In other embodiments, the light detection system includes a plurality of scattered light detectors, such as two or more, three or more, four or more, and including five or more scattered light detectors. In some embodiments, the light detection system of this subject matter also includes a fluorescent photodetector configured to detect light of one or more fluorescence wavelengths. In other embodiments, the light detection system includes a plurality of fluorescent photodetectors, such as two or more, three or more, four or more, five or more, ten or more, fifteen or more, and including twenty or more fluorescent photodetectors.

[0140] The detector of interest may include, but is not limited to, optical sensors or detectors such as avalanche photodiodes (APDs), active pixel sensors (APSs), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-mode charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors, or combinations thereof, as well as other detectors. In some embodiments, a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an avalanche photodiode (APD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor is used to measure the collected light. In some embodiments, the detector is a photomultiplier tube, for example, with an effective detection surface area of ​​0.01 cm² per region. 2 Up to 10 cm 2 For example, 0.05 cm 2 up to 9cm 2 For example, 0.1 cm 2 up to 8 cm 2 For example, 0.5 cm 2 up to 7 cm 2 And including 1 cm 2 up to 5 cm 2 Photomultiplier tubes.

[0141] In systems comprising multiple fluorescence detectors, each fluorescence detector may be identical, or the collection of fluorescence detectors may be a combination of detectors of different types. For example, in systems comprising two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-type device, and the second fluorescence detector (or imaging sensor) is a CMOS-type device. In other embodiments, both the first and second fluorescence detectors are CCD-type devices. In other embodiments, both the first and second fluorescence detectors are CMOS-type devices. In other embodiments, the first fluorescence detector is a CCD-type device, and the second fluorescence detector is a photomultiplier tube (PMT). In other embodiments, the first fluorescence detector is a CMOS-type device, and the second fluorescence detector is a photomultiplier tube. In other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0142] In embodiments of this disclosure, the fluorescence detector of interest is configured to measure light collected at one or more wavelengths, such as at two or more wavelengths, such as at five or more different wavelengths, such as at ten or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and includes measuring light emitted by a sample in a flowing stream at 400 or more different wavelengths. In some embodiments, two or more detectors in the module described herein are configured to measure collected light at the same or overlapping wavelengths.

[0143] In some embodiments, the fluorescence detector of interest is configured to measure light collected within a wavelength range (e.g., 200 nm to 1000 nm). In some embodiments, the detector of interest is configured to collect a spectrum within a wavelength range. For example, a flow cytometer may include one or more detectors configured to collect a spectrum within one or more wavelength ranges from 200 nm to 1000 nm. In other embodiments, the detector of interest is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, the module may include one or more detectors configured to measure light at one or more wavelengths and combinations thereof, including 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, and 617 nm. In some embodiments, one or more detectors may be configured to pair with a specific fluorophore, such as those fluorophores that are used with the sample in a fluorescence assay.

[0144] The flow cytometer described herein may include one or more suitable mechanisms for providing sheath fluid and sample fluid to a sample fluid input coupler and a sheath fluid input coupler. For example, the sample fluid input coupler may be fluidly connected to a sample fluid line (e.g., a conduit) that is fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidly connected to a sheath fluid line that is fluidly connected to a sheath fluid reservoir. Similarly, the flow cytometer may include one or more suitable mechanisms for managing waste fluid from the flow stream. A fluid output coupler may be fluidly connected to a waste fluid line that is fluidly connected to a waste fluid reservoir. A fluid management system suitable for the flow cytometer of this subject is provided in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0145] In some embodiments, a flow cytometer includes a flow cell. The flow cell of interest includes a cuvette configured to deliver particles in a flow stream. As described herein, a “flow cell” in its conventional sense refers to a component containing a flow channel for delivering particles in a sheath fluid. The cuvette of interest has a channel (i.e., a flow channel) extending through it. The flow stream configured for the flow channel may include a liquid sample injected from a sample tube. In some cases, the flow cell includes a light-accessible flow channel. The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed of silica, such as fused silica. In some cases, the flow cell is configured to be illuminated with light from a light source at one or more query points. The “query point” discussed herein refers to an area within the flow cell where particles are illuminated by light from a light source for, for example, analysis. The size of the query point can vary as needed. For example, where 0 μm represents the optical axis of the light emitted by the light source, the query point can be from -50 μm to 50 μm, for example from -25 μm to 40 μm, and includes -15 μm to 30 μm. Depending on certain considerations (e.g., the number and arrangement of lasers), there may be multiple irradiation points within the flow cell.

[0146] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell, or configured for use with a sample injection port configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable sample flow into the flow cell chamber (i.e., the flow channel). Depending on the desired characteristics of the flow, the sample rate delivered to the flow cell via the sample injection port can be 1 μL / min or higher, for example, 2 μL / min or higher, for example, 3 μL / min or higher, for example, 5 μL / min or higher, for example, 10 μL / min or higher, for example, 15 μL / min or higher, for example, 25 μL / min or higher, for example, 50 μL / min or higher, and includes 100 μL / min or higher. In some instances, the sample rate delivered to the flow cell via the sample injection port is 1 μL / sec or higher, for example, 2 μL / sec or higher, for example, 3 μL / sec or higher, for example, 5 μL / sec or higher, for example, 10 μL / sec or higher, for example, 15 μL / sec or higher, for example, 25 μL / sec or higher, for example, 50 μL / sec or higher, and includes 100 μL / sec or higher.

[0147] The sample injection port can be an orifice located in the wall of the chamber, or it can be a conduit located at the proximal end of the chamber. When the sample injection port is an orifice located in the wall of the chamber, the orifice can be of any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses, etc.; and irregular shapes such as parabolic bottom portions coupled to a flat top portion. In some embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in some cases, the opening range of the sample injection port orifice is from 0.1 mm to 5.0 mm, 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 includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0148] In some cases, the sample injection port is a conduit located proximal to the flow cell chamber. For example, the sample injection port can be a conduit positioned such that its orifice is aligned with the flow cell orifice. When the sample injection port is a conduit positioned aligned with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including but not limited to: straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses; and irregular shapes such as parabolic bottom portions coupled to a flat top portion. The orifice of the conduit may vary depending on its shape; in some cases, the orifice opening ranges from 0.1 mm to 5.0 mm, 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 includes 1.25 mm to 1.75 mm, for example 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip with an angle of 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, and including a 5° angle.

[0149] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid into the flow cell chamber, for example, in combination with a sample to generate a laminar flow of sheath fluid around a sample flow. Depending on the desired characteristics of the flow flow, the rate of sheath fluid delivered to the flow cell chamber can be 25 μL / sec or higher, for example 50 μL / sec or higher, for example 75 μL / sec or higher, for example 100 μL / sec or higher, for example 250 μL / sec or higher, for example 500 μL / sec or higher, for example 750 μL / sec or higher, for example 1000 μL / sec or higher, and includes 2500 μL / sec or higher.

[0150] In some embodiments, the sheath injection port is an orifice located in the wall of the chamber. The sheath injection port orifice can be of any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses; and irregular shapes such as parabolic bottom portions coupled to a flat top portion. The size of the sheath injection port orifice may vary depending on the shape, and in some cases, the opening range of the sheath injection port orifice is 0.1 mm to 5.0 mm, for example 0.2 mm 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 includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0151] In some embodiments, the system includes or is operatively coupled to a flow cytometer. Suitable flow cytometry systems may include, but are not limited to, those described in the following literature: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (ed.), FlowCytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd Edition, 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; its published content is incorporated herein by reference. In some cases, flow cytometry systems of interest include BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSL Fortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TMFlow cytometer, BDBiosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BDBiosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BDBiosciences Via TM Cell sorters, including BD Biosciences Influx™, BD Biosciences Jazz™, BD Biosciences Aria™, BD Biosciences FACSAria™ II, BD Biosciences FACSAria™ III, BD Biosciences FACSAria™ Fusion, BD Biosciences FACSMelody™, and BD Biosciences FACSymphony. TM S6 cell sorter, BD Biosciences FACSDiscover™ cell sorter, etc.

[0152] In some embodiments, the subject matter system is a flow cytometry system, such as 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, and 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 Those described in 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766; the public information thereof is incorporated herein by reference in its entirety.

[0153] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the system of this subject is a flow cytometry system configured for imaging particles in a flowing stream using fluorescence imaging with radiofrequency tagged emission (FIRE), such as those described in the following literature: 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, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 1 0,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369 and 11,946,851; the public information thereof is incorporated herein by reference.

[0154] Figure 2 A system 200 for flow cytometry according to an illustrative embodiment of this disclosure is shown. The system 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. Although Figure 2 The example shows a single laser, but it should be understood that multiple lasers can also be used. The laser beam from laser 201 is directed to focusing lens 202, which focuses the beam onto the portion of the fluid flow within flow cell 210 where sample particles 211 are located. Flow cell 210 is part of a fluid system that directs particles (typically one at a time) in the flow flow to the focused laser beam for interrogation. Alternatively, in the case of a stream-in-air cytometer, a nozzle top can be used.

[0155] like Figure 2As shown, the flow chamber 210 is fluidly connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. Sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a conduit (i.e., a sheath fluid line) 207. Furthermore, sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to the sample injection port 206 via a conduit (i.e., a sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce particles 211 into the interior of the flow chamber 210. The particles 211 are hydrodynamically focused by the sheath fluid entering from the sheath fluid injection port 208, such that a flow stream 214 is formed downstream of the conical portion 212 of the flow chamber 210. Particles emitted at the distal end of the flow chamber 210 can be processed and / or collected by any suitable method. For example, depending on the type of flow cytometry performed, particles can be collected at the distal end of the flow chamber 210, for example, through a waste liquid line. Alternatively, the particles can be sorted.

[0156] Light from one or more laser beams interacts with particles 211 in the sample through diffraction, refraction, reflection, scattering, and absorption to be re-emitted at various wavelengths depending on the characteristics of the particles, such as their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be directed to one or more detectors. Specifically, forward scattered light (FSC) is directed to forward scattered light detector 223. Forward scattered light detector 223 is positioned slightly off-axis from the direct beam passing through flow cell 210 and configured to detect diffracted light, i.e., excitation light that propagates primarily in the forward direction through or around the particles. The intensity of the light detected by forward scattered light detector 223 depends on the overall size of the particles. The forward scattered detector may include, for example, a photodiode. Between forward scattered light detectors 223 are an optical filter 221a and a scattering rod 222. Optical filter 221a can be configured to filter out non-FSC light of at least one wavelength, while scattering rod 222 can be configured to prevent the incident beam (i.e. non-scattered light) from laser 201 from being detected by forward scattering detector 223.

[0157] Furthermore, side-scattered light (SSC) is detected by side-scattered light detector 224. In other words, side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of particle 211, which tends to increase with increasing particle structure complexity. Figure 2In the example, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to a side-scatter light detector 224 while allowing non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by the side-scatter light detector 224. Fluorescence detectors 225a-225c are also shown in the figure, each configured to detect fluorescence of a different wavelength. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a while allowing light of other wavelengths to pass through. The optical filter 221c may be configured to prevent light of at least one wavelength not corresponding to the first wavelength (or wavelength range) from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect FL light corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b while allowing light of a third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. Optical filter 221d is configured to prevent light of at least one wavelength that does not correspond to a second wavelength (or wavelength range) from being detected by fluorescence detector 225b. Furthermore, optical filter 221e is configured to prevent light of at least one wavelength that does not correspond to a third wavelength (or wavelength range) from being detected by fluorescence detector 225c.

[0158] Those skilled in the art will recognize that the flow cytometer according to embodiments of this disclosure is not limited to... Figure 2 The flow cytometer described herein may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in various different configurations. For example, although Figure 2 The example shown has three fluorescence detectors for illustrative purposes, but it should be understood that any suitable number of fluorescence detectors can be used.

[0159] During operation, the cytometer is controlled by controller / processor 290, and measurement data from the detector can be stored in memory 295 and processed by controller / processor 290. Although not explicitly shown, controller / processor 290 is coupled to the detector to receive its output signals and can also be coupled to the electrical and electromechanical components of the flow cytometer to control laser 201, fluid flow parameters, etc. Input / output (I / O) capability 297 may also be provided in the system. Memory 295, controller / processor 290, and I / O 297 may be provided entirely as an integral part of the flow cytometer. In such an embodiment, a display may also be formed as part of I / O capability 297 for presenting experimental data to the user of cytometer 200. Alternatively, memory 295 and controller / processor 290, along with some or all of the I / O capability, may be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, memory 295 and controller / processor 290 may communicate wirelessly or wiredly with cytometer 210. The controller / processor 290, combined with memory 295 and I / O 297, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0160] Different fluorescent molecules in the fluorescent dye panel used for flow cytometry experiments will emit light in their respective characteristic wavelength bands. Specific fluorescent tags used in the experiment and their associated fluorescence emission bands can be selected to typically coincide with the detector's filter window. I / O 297 can be configured to receive data about a flow cytometry experiment having a set of fluorescent tags and multiple cell populations with multiple labels, each cell population having a subset of those labels. I / O 297 can also be configured to receive biological data assigning one or more labels to one or more cell populations, label density data, emission spectral data, data on tag assignment to one or more labels, and flow cytometry configuration data. Flow cytometry experiment data, such as tag spectral characteristics and flow cytometry configuration data, can also be stored in memory 295. Controller / processor 290 can be configured to evaluate one or more assignments of tags to labels.

[0161] In some embodiments, the subject matter system is a particle sorting system configured to sort particles using enclosed particle sorting modules, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as those described in U.S. Patent Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting sample components includes a particle sorting module having deflection plates, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0162] In some embodiments, the system is a particle sorter that uses an RFID tag-transmitted image-enabled particle sorter, such as... Figure 3As depicted in the diagram, the particle sorter 300 includes an illumination component 300a, which includes a light source 301 (e.g., a 488 nm laser) that generates an output beam 301a, which is split into beams 302a and 302b by a beam splitter 302. Beam 302a propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a with one or more angle deflections. In some cases, the output beam 303a generated by the acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Beam 302b propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a with one or more angle deflections. In some cases, the output beam 304a generated by the acousto-optic device 304 includes a local oscillator beam and multiple radio frequency comb beams. Output beams 303a and 304a, generated by acousto-optic devices 303 and 304 respectively, are combined by beam splitter 305 to produce output beam 305a, which is transmitted through optical component 306 (e.g., objective lens) to illuminate particles in flow chamber 307. In some embodiments, acousto-optic device 303 (AOD) splits a single laser beam into a beam array, each beam having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then overlapped with the beam array at beam combiner 305. In some embodiments, the light illumination system having a light source and acousto-optic devices may also include those described in Schraivogel et al. (“High-speedfluorescence image-enabled cell sorting” Science (2022), 375(6578): 315-320) and U.S. Patent Publication No. 2021 / 0404943, the disclosure of which is incorporated herein by reference.

[0163] Output beam 305a irradiates sample particles 308 propagating through flow chamber 307 (e.g., together with sheath fluid 309) at irradiation region 310. As shown in irradiation region 310, multiple beams (e.g., RF offset beams depicted as points of angular deflection in irradiation region 310) overlap with a reference local oscillator beam (depicted as shaded lines in irradiation region 310). Because they have different optical frequencies, the overlapping beams exhibit beat frequency behavior, resulting in each beam carrying a different frequency f. 1-n Sine modulation.

[0164] Light from the irradiated sample is transmitted to a light detection system 300b comprising multiple photodetectors. The light detection system 300b includes a forward-scattering photodetector 311 for generating a forward-scattering image 311a and a side-scattering photodetector 312 for generating a side-scattering image 312a. The light detection system 300b also includes a bright-field photodetector 313 for generating a light loss image 313a. In some embodiments, the forward-scattering detector 311 and the side-scattering detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the irradiated sample is also detected by fluorescence photodetectors 314-317. In some cases, photodetectors 314-317 are photomultiplier tubes. Light from the irradiated sample is directed by a beamsplitter 320 to the side-scattering detection channel 312 and the fluorescence detection channels 314-317. The optical detection system 300b includes bandpass optics 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optics 321 is a 534 nm / 40 nm bandpass optic. In some cases, optics 322 is a 586 nm / 42 nm bandpass optic. In some cases, optics 323 is a 700 nm / 54 nm bandpass optic. In some cases, optics 324 is a 783 nm / 56 nm bandpass optic. The first number indicates the center of the spectral band. The second number provides the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm.

[0165] Processors 350 and 351 process data signals generated in response to light detected in scattered light detection channels 311 and 312, bright field light detection channel 313, and fluorescence detection channels 314-317 via real-time digital processing. Based on the data signals generated in processors 350 and 351, images 311a-317a can be generated in each light detection channel. Image-enabled sorting is performed in response to a sorting signal generated in sorting trigger 352. Sorting component 300c includes a deflector plate 331 for deflecting particles into sample container 332 or waste stream 333. In some cases, sorting component 300c is configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0166] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4 It can be used to analyze and characterize particles, whether or not the particles are physically sorted into a collection container. Figure 4 A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 includes a fluid system 402. The fluid system 402 may include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, wherein particles 403 (e.g., cells) of the sample move along a common sample path 409 in the moving fluid column.

[0167] The particle analysis system 401 includes a detection system 404 configured to collect signals from each sample as each particle passes through one or more detection stations along a common sample path. Detection stations 408 typically refer to monitored areas 407 along the common sample path. In some embodiments, detection may include detecting light or one or more other properties of the particle as it passes through monitored areas 407. Figure 4 The image shows a detection station 408 with a monitored area 407. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations can monitor more than one area.

[0168] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be referred to as event data. The data point can be a multidimensional data point, which includes values ​​of the corresponding attributes measured for the particle. The detection system 404 is configured to collect a series of such data points within a first time interval.

[0169] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system shown may be operationally associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the number of data points collected by the detection system 404 during the first time interval and their Poisson distribution. The control system 406 may also be configured to generate an experimental signal frequency based on the number of data points in that portion of the first time interval. The control system 406 may also compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0170] Figure 5 A functional block diagram of an example particle analyzer control system (e.g., analysis controller (i.e., processor) 500) is shown for analyzing and displaying biological events. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0171] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 through this data communication channel. The analysis controller 500 can be a processor configured to perform the methods of the present invention, for example, by applying a distance-based classification model to determine a density discrimination threshold in a size-based analyte feature space, applying a density-based clustering algorithm to separate the analyte data into high-density clusters and low-density clusters based on the density threshold, and classifying the analyte data based on the high-density clusters and low-density clusters according to the size-based analyte feature space.

[0172] Analysis controller 500 may be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display of a first plot including the biological event data to display device 506. Analysis controller 500 may also be configured to render regions of interest as gates around the population of biological event data displayed on display device 506, for example, overlaying them onto the first plot. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest plotted on a single-parameter histogram or bivariate plot. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0173] The analysis controller 500 can also be configured to display the biological event data on the display device 506 inside the door in a manner different from other events outside the door in the biological event data. For example, the analysis controller 500 can be configured to distinguish the color of the biological event data inside the door from the color of the biological event data outside the door. The display device 506 can be a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0174] The analysis controller 500 can be configured to receive a door selection signal from a first input device to identify the door. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a door selection signal to the analysis controller 500 to identify a door to be displayed on or operated via the display device 506 (e.g., by clicking the desired door at the cursor location or clicking within the desired door). In some embodiments, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, optical detector, or voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function can be considered an input device. For example, such as... Figure 5 As shown, mouse 510 may include a right button and a left button, and each of the right and left buttons can generate a trigger event.

[0175] This triggering event can cause the analysis controller 500 to change the way the data is displayed, the portion of the data actually displayed on the display device 506, and / or provide input for further processing (such as selecting groups of interest for particle sorting).

[0176] In some embodiments, the analysis controller 500 may be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 may also be configured to automatically modify the plotting visualization to facilitate the gating process. This modification may be based on a specific distribution of the biological event data received by the analysis controller 500.

[0177] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can also be configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0178] Display device 506 can be configured to receive display data from analysis controller 500. The display data may include plots of biological event data and gates outlining portions of the plots. Display device 506 can also be configured to change the presented information based on input received from analysis controller 500 in conjunction with input received from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0179] In some implementations, the analysis controller 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include controls for receiving sample events or sample images. The sample events, images, or sample gates may be provided before collecting event data for the sample, or based on an initial set of events for a portion of the sample.

[0180] Figure 6A This is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Figure 6A As shown, a droplet formation sensor 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, 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 flow). Inside the moving fluid column 608, particles 609 (e.g., cells) align in a single file across a monitored area 611 (e.g., where a laser intersects with the flow) and are irradiated by an irradiation source 612 (e.g., a laser). Vibration of the droplet formation sensor 602 causes the moving fluid column 608 to break into multiple droplets 610, some of which contain particles 609.

[0181] In operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitored area 611. The detection station 614 is fed into a timing circuit 628, which in turn feeds into a flash-charging circuit 630. At the droplet breakpoint, notified by a timing drop delay (Δt), a flash charge can be applied to the moving fluid column 608, causing the droplet of interest to carry a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect it into a container (e.g., a collection tube or a porous or microporous sample plate), where pores or micropores can be associated with the droplet of particular interest. Figure 6A As shown, the droplets can be collected in the discharge receiver 638.

[0182] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a particle of interest passes through the monitored region 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 616 allows the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may be fed an amplitude signal 620 and / or a phase signal 618, which in turn (via amplifier 622) feeds an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624, in turn, control the droplet formation sensor 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0183] In some embodiments, sorting electronics (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory configured to store detected events and sorting decisions based on the detected events. The sorting decisions may be included in the event data of the particles. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or communication coupling, such that event measurements can be collected by one of detection system 616 or detection station 614 and provided to non-collecting elements.

[0184] Figure 6B This is a schematic diagram of a particle sorting system according to an embodiment of the present document. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via a current-charging wire in the barbs. This generates a droplet stream 610 containing particles 610 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The particle information is analyzed, for example, by sorting electronics or other detection systems. Figure 6B(Not shown in the image) Particle information is sorted. Deflecting plates 652 and 654 can be independently controlled to attract or repel charged droplets, thereby guiding the droplets toward a target collection receiver (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflection plates 652 and 654 can be controlled to guide particles toward receiver 674 along a first path 662, or toward receiver 678 along a second path 668. 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 along flow path 664. Such uncharged droplets can be drawn into waste liquid receiver via suction device 670.

[0185] It may include sorting electronics to initiate measurement collection, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to induce particle sorting. Figure 6B An example implementation of the illustrated embodiment includes the BD FACSAria™ series flow cytometer, commercially available from Becton, Dickinson and Company (Franklin Lake, New Jersey).

[0186] Computer control system

[0187] The system may include a display and operator input devices. Operator input devices may be, for example, a keyboard, mouse, etc. The processing module includes a processor that can access memory storing instructions thereon for performing steps of the methods of this subject. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or other existing or future processors. The processor executes the operating system, which interfaces with firmware and hardware in a well-known manner and assists the processor in coordinating and executing the functions of various computer programs written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control (e.g., negative feedback control).

[0188] System memory can be any of a variety of known or future memory storage devices. Examples include any common random access memory (RAM), magnetic media (such as resident hard disks or magnetic tapes), optical media (such as optical discs), flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, or floppy disk drives. Such memory storage devices typically read from and / or write to program storage media (not shown) (such as optical discs). Any of these program storage media, or other media currently in use or that may be developed in the future, can be considered computer program products. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs (also known as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0189] In some embodiments, a computer program product is described, comprising a computer-usable medium having control logic (computer software program, including program code) stored therein. When executed by a computer's processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. The implementation of a hardware state machine to perform the functions described herein will be apparent to those skilled in the art.

[0190] The memory can be any suitable device in which a processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including disks, optical discs, magnetic tapes, RAM, or any other suitable device, whether fixed or portable). The processor can include a general-purpose digital microprocessor that is appropriately programmed from a computer-readable medium carrying the necessary program code. Programming can be provided to the processor remotely via a communication channel or previously stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium, using any of these devices connected to memory. For example, a disk or optical disc can carry the programming and can be read by a disk writer / reader. The systems disclosed herein also include programming, such as programming in the form of a computer program product for practicing the methods described above, or an algorithm. Programming according to this disclosure can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROMs; electronic storage media such as RAM and ROM; portable flash drives; and mixtures of these categories, such as magnetic / optical storage media.

[0191] The processor can also access communication channels to communicate with users at remote locations. A remote location refers to a user who does not have direct contact with the system but instead relays input information to the input manager from an external device, such as a computer connected to a wide area network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0192] In some embodiments, the system according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or other devices. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., RFID, Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth). ® Communication protocols and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0193] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces like USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject system and other external devices, such as computer terminals configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0194] In one embodiment, the communication interface is configured for infrared communication and Bluetooth. ® Communication or any other suitable wireless communication protocol to enable the system of this subject to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user can use in conjunction with them.

[0195] In one embodiment, the communication interface is configured to provide data transmission connectivity via Internet Protocol (IP) through a cellular telephone network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0196] In one embodiment, the system is configured to communicate wirelessly with a server device via a communication interface, such as using common standards like 802.11 or Bluetooth. ®Wireless communication is achieved using RF protocols or IrDA infrared protocols. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices such as buttons, keyboard, mouse, or touchscreen.

[0197] In some embodiments, the communication interface is configured to automatically or semi-automatically transmit data stored in the subject system, such as an optional data storage unit, to a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0198] The output controller may include controllers for any variety of known display devices for presenting information to a user (whether human or machine, local or remote). If one of the display devices provides visual information, that information may typically be logically and / or physically organized as an array of picture elements. The graphical user interface (GUI) controller may include any 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. Functional elements of the computer may communicate with each other via a system bus. In alternative embodiments, some of these communications may be accomplished using a network or other types of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location, according to known technologies such as via the Internet, telephone, or satellite networks. The output manager may present data according to a variety of known technologies. As some examples, the data may include SQL, HTML, or XML documents, emails, or other files, or other forms of data. The data may include Internet URLs so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the system of this subject may be any type of known computer platform or type to be developed in the future, although they generally fall into the category of computers commonly referred to as servers. However, they may also be mainframes, workstations, or other types of computers. They can be connected via any known or future type of cabling or other communication system (including wireless systems), whether networked or otherwise. They can be co-located or physically separated. Various operating systems can be deployed on any computer platform, depending on the type and / or brand of the chosen platform. Suitable operating systems include Windows. ® NT ® Windows ® XP, Windows ® 7. Windows ® 8. Windows ® 10. iOS® macOS ® Linux ® Ubuntu ® Fedora ® OS / 400 ® i5 / OS ® IBM i ® Android™, SGI IRIX ® Oracle Solaris ® wait.

[0199] Figure 7 A general architecture of an example computing device 700 according to certain embodiments is described. Figure 7 The general architecture of the computing device 700 depicted includes the arrangement of computer hardware and software components. However, it is not necessary to show all these generally conventional components to provide a disclosure of what can be achieved. As shown, 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 communicate with each other via a communication bus. The network interface 720 can provide connectivity to one or more networks or computing systems. Therefore, the processing unit 710 can receive information and instructions from other computing systems or services via the network. The processing unit 710 can also communicate with memory 770 and further provide output information to the optional display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or programs such as FlowJoy) stored as executable instructions in the non-transient memory of the analysis system. ® It can display flow cytometry event data to the user. The input / output device interface 740 can also accept input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input device.

[0200] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments), which processing unit 710 executes to implement one or more embodiments. Memory 770 typically 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 general management and operation of computing device 700 by processing unit 710. Data may be stored in data storage device 790. Memory 770 may also include computer program instructions and other information for implementing various aspects of this disclosure.

[0201] Non-transient computer-readable storage media

[0202] This disclosure further includes a non-transitory computer-readable storage medium having instructions for practicing the methods of this subject matter, such as for practicing one or more computer-implemented methods described herein. The computer-readable storage medium can be deployed on one or more computers for fully or partially automating systems used to practice the methods described herein. In some embodiments, instructions according to the methods described herein can be encoded onto a computer-readable medium in a “programmed” form, wherein the term “computer-readable medium” as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network attached storage (NAS), whether such devices are internal or external to a computer. Files containing information can be “stored” on a computer-readable medium, where “stored” means recording the information so that it can be accessed and retrieved by a computer at a later date. The computer-implemented methods described herein can be executed using programming, which can be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Python, Java, JavaScript, C, C#, C++, Go, R, Swift, PHP, and many other languages.

[0203] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of a flow stream in a flow cytometer, an algorithm for comparing the measured flow rate of the flow stream with an absolute flow rate threshold and a moving window flow rate threshold, and an algorithm for generating an error alarm when the measured flow rate of the flow stream exceeds the absolute flow rate threshold or the moving window flow rate threshold.

[0204] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for continuously measuring the flow velocity of a flowing stream. In some cases, the non-transient computer-readable storage medium includes an algorithm for measuring the flow velocity of a flowing stream at discrete intervals, for example, wherein the flow velocity is measured at intervals of predetermined duration.

[0205] In some cases, the absolute flow rate includes an upper threshold and a lower threshold. In some cases, the non-transient computer-readable storage medium includes an algorithm for continuously comparing the measured flow rate with the upper and lower absolute flow rate thresholds. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm when it is determined that the measured flow rate is less than the lower absolute flow rate threshold. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm when it is determined that the measured flow rate is greater than the upper absolute flow rate threshold. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm when it is determined that the measured flow rate is 1% or more (e.g., 3% or more) greater than the upper absolute flow rate threshold or 1% or more (e.g., 3% or more) less than the lower absolute flow rate threshold.

[0206] In some embodiments, the moving window flow rate threshold includes an upper threshold and a lower threshold. In some cases, the non-transient computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow rate threshold at discrete intervals. In some cases, the non-transient computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow rate threshold by determining whether the measured flow rate is greater than the upper moving window flow rate threshold during a predetermined time interval. In some cases, the non-transient computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow rate threshold by determining whether the measured flow rate is less than the lower moving window flow rate threshold during a predetermined time interval. In some cases, the time interval of the moving window for comparing the measured flow rate with the flow rate threshold is from 0.1 seconds to 10 seconds, for example, from 1 second to 5 seconds. In some cases, the moving window time interval is 1 second.

[0207] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm indicating a fault in the flow cytometer. In some cases, the fault is a blockage in the flow stream. In some instances, the fault is the introduction of gas (e.g., air) into the flow stream or sample tubing. In some instances, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm in real time. In some instances, the non-transient computer-readable storage medium includes an algorithm for generating an erroneous alarm when the measured flow rate continuously exceeds an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration (e.g., 0.001 seconds or longer, 0.01 seconds or longer, 0.1 seconds or longer, and including 5 seconds or longer).

[0208] In some embodiments, the non-transient computer-readable storage medium includes algorithms for changing one or more parameters of a flow cytometer in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for adjusting the flow rate of a flow stream in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for increasing the flow rate in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for decreasing the flow rate in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for stopping the flow rate in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for adjusting a light source used to illuminate the flow stream in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for turning off a light source in response to a generated error alarm. In some instances, the non-transient computer-readable storage medium includes algorithms for blocking light configured to illuminate the flow stream in response to a generated error alarm.

[0209] In some embodiments, the flow includes a sheath fluid flow and a sample core fluid flow. In some instances, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of the sheath fluid flow. In some instances, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of the sample core fluid flow. In some instances, the non-transient computer-readable storage medium includes an algorithm for measuring the flow rates of the sheath fluid flow and the sample core fluid flow. Wherein a sample containing particles is conveyed in the sample core fluid flow, in some instances, the non-transient computer-readable storage medium includes an algorithm for illuminating the sample with a light source and an algorithm for detecting light from the irradiated particles in the flow.

[0210] Non-transient computer-readable storage media can be deployed on one or more computer systems having displays and operator input devices. Operator input devices can be, for example, keyboards, mice, etc. The processing module includes a processor that can access memory on which instructions for performing the steps of the methods of this subject are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup units, and many other devices. The processor can be a commercially available processor or other existing or future processors. The processor executes the operating system, which interfaces with firmware and hardware in well-known ways and assists the processor in coordinating and executing the functions of various computer programs written in various programming languages, such as those described above, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services according to known techniques.

[0211] kit

[0212] This disclosure also includes kits comprising storage media such as magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network attached storage (NAS). Any of these program storage media, or other media currently in use or that may be developed in the future, may be included in this subject matter kit. In embodiments, the program storage media includes instructions for monitoring the flow rate of a flow in a method and for use with the system. In embodiments, instructions contained on a computer-readable medium provided in this subject matter kit, or portions thereof, may be implemented as software components for data analysis software. In these embodiments, a computer control system according to this disclosure may be used as a software "plugin" for existing software packages such as FlowJo®.

[0213] In addition to the components described above, this subject matter kit may also include (in some embodiments) instructions. These instructions may exist in various forms within the subject matter kit; one or more types of instructions may be present in the kit. One form of these instructions may be printed information on a suitable medium or substrate (e.g., one or more sheets of paper on which information is printed), in the packaging of the kit, in packaging inserts, etc. Another form of these instructions is a computer-readable medium on which information is recorded, such as a floppy disk, optical disc (CD), portable flash drive, etc. Yet another form of these instructions may be a website address, which can be used via the Internet to access information at a remote site.

[0214] practicality

[0215] The methods, systems, and computer systems of this subject matter are useful in a variety of applications where the analysis of particles in a flow stream needs to be calibrated or optimized. In some instances, monitoring the flow rate helps maintain the consistency and accuracy of analytical data acquired by the systems of this subject matter, thereby reducing variability between samples and preventing errors in the system during analysis. In some instances, the methods of this subject matter provide the use of optical detection systems (e.g., with photodetectors) to optimize the analysis of particles in a flow stream, for example, in a particle analyzer. The methods and systems of this subject matter are also applicable to optical detection systems used for analyzing and sorting particulate components in samples in fluid media (e.g., biological samples). This disclosure is also applicable to flow cytometry where a flow cytometer is required to provide 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, this disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In some embodiments, the methods and systems of this subject matter provide a fully automated scheme so that adjustments to the flow cytometer during use require little, if any, manual input.

[0216] Notwithstanding the appended claims, this disclosure is also defined by the following provisions:

[0217] 1. A method comprising:

[0218] Measure the flow rate of the flow stream in a flow cytometer;

[0219] The measured flow velocity is compared with the following:

[0220] Absolute flow rate threshold; and

[0221] Moving window flow rate threshold; and

[0222] An error alarm is generated when the measured flow velocity exceeds the absolute velocity threshold or the moving window velocity threshold.

[0223] 2. The method according to Clause 1, wherein the method includes continuously measuring the flow velocity of the flow.

[0224] 3. The method according to Clause 1, wherein the method includes measuring the flow velocity of the flow at discrete intervals.

[0225] 4. The method according to any one of Clauses 1-3, wherein the absolute flow rate threshold includes an upper limit threshold and a lower limit threshold.

[0226] 5. The method according to any one of clauses 1-4, wherein the method comprises continuously comparing the measured flow velocity with each absolute flow velocity threshold.

[0227] 6. The method according to any one of Clauses 4-5, wherein an error alarm is generated when it is determined that the measured flow rate is less than the lower limit absolute flow rate threshold.

[0228] 7. The method according to any one of Clauses 4-5, wherein an error alarm is generated when it is determined that the measured flow rate is greater than the upper limit absolute flow rate threshold.

[0229] 8. The method according to any one of Clauses 6-7, wherein an error alarm is generated when it is determined that the measured flow rate is 1% or more greater or less than the absolute flow rate threshold.

[0230] 9. The method according to any one of Clauses 6-7, wherein an error alarm is generated when the measured flow rate is determined to be 3% or more greater or less than the absolute flow rate threshold.

[0231] 10. The method according to any one of Clauses 1-9, wherein the moving window flow rate threshold includes an upper limit threshold and a lower limit threshold.

[0232] 11. The method according to any one of clauses 1-9, wherein the method comprises comparing the measured flow rate with a flow rate threshold for each moving window at discrete intervals.

[0233] 12. The method according to any one of Clauses 10-11, wherein comparing the measured flow rate with a flow rate threshold for each moving window comprises: determining whether the measured flow rate is greater than an upper moving window flow rate threshold during a predetermined time interval.

[0234] 13. The method according to any one of Clauses 10-11, wherein comparing the measured flow rate with a moving window flow rate threshold comprises: determining whether the measured flow rate is less than a lower moving window flow rate threshold during a predetermined time interval.

[0235] 14. The method according to any one of Clauses 12-13, wherein the predetermined time interval is from 0.1 seconds to 10 seconds.

[0236] 15. The method according to Clause 14, wherein the predetermined time interval of the moving window is 1 to 5 seconds.

[0237] 16. The method according to any one of Clauses 14-15, wherein the predetermined time interval is 1 second.

[0238] 17. The method according to any one of clauses 1-16, wherein the generated error alarm indicates a malfunction in the flow cytometer.

[0239] 18. The method according to Clause 17, wherein the fault includes a blockage in the flow.

[0240] 19. The method according to Clause 17, wherein the fault includes the introduction of gas into the flow stream.

[0241] 20. The method according to any one of Clauses 1-19, wherein the error alarm is generated in real time.

[0242] 21. The method according to Clause 20, wherein an error alarm is generated when the measured flow rate continuously exceeds an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration.

[0243] 22. The method according to any one of clauses 1-21, wherein the method further comprises adjusting the flow rate of the flow in response to a generated error alarm.

[0244] 23. The method according to Clause 22, wherein the method includes increasing the flow rate of the flow in response to a generated error alarm.

[0245] 24. The method according to Clause 22, wherein the method includes reducing the flow rate of the flow in response to a generated error alarm.

[0246] 25. The method according to Clause 22, wherein the method includes stopping the flow of the flow in response to a generated error alarm.

[0247] 26. The method according to Clause 22, wherein the method includes flushing the flow in response to a generated error alarm.

[0248] 27. The method according to any one of clauses 1-26, wherein the flow rate of the flow is measured using a flow sensor based on one or more of the temperature and viscosity of the flow.

[0249] 28. The method according to any one of clauses 1-26, wherein the flow stream comprises a sheath fluid flow stream and a sample core fluid flow stream.

[0250] 29. The method according to Clause 28, wherein the method includes measuring the flow rate of the sheath fluid flow.

[0251] 30. The method according to Clause 28, wherein the method includes measuring the flow rate of the fluid flow in the core of the sample.

[0252] 31. The method according to any one of clauses 28-30, wherein the method comprises measuring the relative flow rates of the sheath fluid flow and the sample core fluid flow.

[0253] 32. The method according to any one of clauses 1-31, wherein the method further comprises conveying a sample containing particles in a flowing stream.

[0254] 33. The method according to Clause 32, wherein the method includes illuminating a sample with a light source and detecting light from the irradiated particles in the flowing stream.

[0255] 34. A system for configuring a flow cytometer, the system comprising:

[0256] A processor, including memory operatively coupled to the processor, wherein the memory includes instructions stored thereon, the memory comprising:

[0257] Instructions used to measure the flow rate of a flow stream in a flow cytometer;

[0258] Instructions for comparing the measured flow velocity with the following:

[0259] Absolute flow rate threshold; and

[0260] Moving window flow rate threshold; and

[0261] Instructions used to generate an error alarm when the measured flow velocity exceeds an absolute velocity threshold or a moving window velocity threshold.

[0262] 35. The system according to Clause 34, wherein the memory includes instructions for continuously measuring the flow rate of the flow.

[0263] 36. The system according to Clause 34, wherein the memory includes instructions for measuring the flow rate of the flow at discrete intervals.

[0264] 37. The system according to any one of clauses 34-36, wherein the absolute flow rate threshold includes an upper limit threshold and a lower limit threshold.

[0265] 38. The system according to any one of clauses 34-37, wherein the memory includes instructions for continuously comparing the measured flow rate of the flow with each absolute flow rate threshold.

[0266] 39. The system according to any one of clauses 37-38, wherein the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is less than a lower limit absolute flow rate threshold.

[0267] 40. The system according to any one of clauses 37-38, wherein the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is greater than an upper limit absolute flow rate threshold.

[0268] 41. The system according to any one of clauses 39-40, wherein the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is 1% or more greater or less than an absolute flow rate threshold.

[0269] 42. The system according to any one of clauses 39-40, wherein the memory includes instructions for generating an error alarm when it is determined that the measured flow rate is 3% or more greater or less than an absolute flow rate threshold.

[0270] 43. The system according to any one of clauses 34-42, wherein the moving window flow rate threshold includes an upper limit threshold and a lower limit threshold.

[0271] 44. The system according to any one of clauses 34-42, wherein the memory includes instructions for comparing the measured flow rate with a flow rate threshold for each moving window at discrete intervals.

[0272] 45. The system according to any one of clauses 43-44, wherein the memory includes instructions for comparing the measured flow rate with each moving window flow rate threshold by determining whether the measured flow rate is greater than an upper limit moving window flow rate threshold during a predetermined time interval.

[0273] 46. ​​The system according to any one of clauses 43-44, wherein the memory includes instructions for comparing the measured flow rate with the moving window flow rate threshold by determining whether the measured flow rate is less than the lower limit moving window flow rate threshold during a predetermined time interval.

[0274] 47. The system according to any one of clauses 45-46, wherein the predetermined time interval is from 0.1 seconds to 10 seconds.

[0275] 48. The system according to Clause 47, wherein the predetermined time interval of the moving window is 1 to 5 seconds.

[0276] 49. The system according to any one of Clauses 47-48, wherein the predetermined time interval is 1 second.

[0277] 50. A system according to any one of clauses 34-49, wherein the generated error alarm indicates a malfunction in the flow cytometer.

[0278] 51. The system according to Clause 50, wherein the fault includes a blockage in the flow.

[0279] 52. The system according to Clause 50, wherein the fault includes gas being introduced into the flow stream.

[0280] 53. The system according to any one of clauses 34-52, wherein the memory includes instructions for generating error alerts in real time.

[0281] 54. The system according to Clause 53, wherein the memory includes instructions for generating an error alarm when the measured flow rate continuously exceeds an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration.

[0282] 55. The system according to any one of clauses 34-54, wherein the memory includes instructions for adjusting the flow rate of the flow in response to a generated error alarm.

[0283] 56. The system according to Clause 55, wherein the memory includes instructions for increasing the flow rate of the flow in response to a generated error alarm.

[0284] 57. The system according to Clause 55, wherein the memory includes instructions for reducing the flow rate of the flow in response to a generated error alarm.

[0285] 58. The system according to Clause 55, wherein the memory includes instructions for stopping the flow of the flow stream in response to a generated error alarm.

[0286] 59. The system according to Clause 55, wherein the memory includes instructions for flushing the flow in response to a generated error alarm.

[0287] 60. The system according to any one of clauses 34-59, wherein the system includes a flow velocity sensor configured to measure the flow velocity of the flow based on one or more of the temperature and viscosity of the flow.

[0288] 61. The system according to any one of clauses 34-60, wherein the flow includes a sheath fluid flow and a sample core fluid flow.

[0289] 62. The system according to Clause 61, wherein the memory includes instructions for measuring the flow rate of the sheath fluid flow.

[0290] 63. The system according to Clause 61, wherein the memory includes instructions for measuring the flow rate of the sample core fluid flow.

[0291] 64. The system according to any one of clauses 61-63, wherein the memory includes instructions for measuring the relative flow rates of the sheath fluid flow and the sample core fluid flow.

[0292] 65. The system according to any one of clauses 34-64, wherein the system is configured to deliver a sample containing particles in a flow stream.

[0293] 66. The system according to Clause 65, wherein the system comprises:

[0294] A light source for illuminating a sample in a flowing stream; and

[0295] A light detection system, including a photodetector, is used to detect light from irradiated particles.

[0296] 67. A non-transient computer-readable storage medium for configuring a flow cytometer, comprising instructions stored thereon, the instructions comprising:

[0297] An algorithm for measuring the flow rate of a flow stream in a flow cytometer;

[0298] An algorithm used to compare the measured flow velocity with the following:

[0299] Absolute flow rate threshold; and

[0300] Moving window flow rate threshold; and

[0301] An algorithm for generating error alarms when the measured flow velocity exceeds an absolute velocity threshold or a moving window velocity threshold.

[0302] 68. The non-transient computer-readable storage medium according to Clause 67, wherein the non-transient computer-readable storage medium includes an algorithm for continuously measuring the flow rate of the flow.

[0303] 69. The non-transient computer-readable storage medium according to Clause 67, wherein the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of the flow at discrete intervals.

[0304] 70. A non-transient computer-readable storage medium according to any one of clauses 67-69, wherein the absolute flow rate threshold includes an upper limit threshold and a lower limit threshold.

[0305] 71. A non-transient computer-readable storage medium according to any one of clauses 67-70, wherein the non-transient computer-readable storage medium includes an algorithm for continuously comparing the measured flow velocity of a flow with each absolute flow velocity threshold.

[0306] 72. A non-transient computer-readable storage medium according to any one of Clauses 70-71, wherein the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when it is determined that the measured flow rate is less than a lower limit absolute flow rate threshold.

[0307] 73. A non-transient computer-readable storage medium according to any one of Clauses 70-71, wherein the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when it is determined that the measured flow rate is greater than an upper limit absolute flow rate threshold.

[0308] 74. A non-transient computer-readable storage medium according to any one of clauses 72-73, wherein the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when it is determined that the measured flow rate is 1% or more greater or less than an absolute flow rate threshold.

[0309] 75. A non-transient computer-readable storage medium according to any one of clauses 72-73, wherein the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when it is determined that the measured flow rate is 3% or more greater or less than an absolute flow rate threshold.

[0310] 76. A non-transient computer-readable storage medium according to any one of clauses 67-75, wherein the moving window flow rate threshold includes an upper limit threshold and a lower limit threshold.

[0311] 77. A non-transient computer-readable storage medium according to any one of clauses 67-75, wherein the non-transient computer-readable storage medium includes an algorithm for comparing a measured flow rate with a flow rate threshold for each moving window at discrete intervals.

[0312] 78. A non-transient computer-readable storage medium according to any one of clauses 76-77, wherein the non-transient computer-readable storage medium includes an algorithm for comparing a measured flow rate with each moving window flow rate threshold by determining whether the measured flow rate is greater than an upper limit moving window flow rate threshold during a predetermined time interval.

[0313] 79. A non-transient computer-readable storage medium according to any one of clauses 76-78, wherein the non-transient computer-readable storage medium includes an algorithm for comparing a measured flow rate with a moving window flow rate threshold by determining whether the measured flow rate is less than a lower moving window flow rate threshold during a predetermined time interval.

[0314] 80. A non-transient computer-readable storage medium according to any one of clauses 78-79, wherein the predetermined time interval is from 0.1 seconds to 10 seconds.

[0315] 81. The non-transient computer-readable storage medium as described in Clause 80, wherein the predetermined time interval for the moving window is 1 to 5 seconds.

[0316] 82. A non-transient computer-readable storage medium according to any one of clauses 80-81, wherein the predetermined time interval is 1 second.

[0317] 83. A non-transient computer-readable storage medium according to any one of clauses 67-82, wherein the generated error alarm indicates a fault in the flow cytometer.

[0318] 84. The non-transient computer-readable storage medium as described in Clause 83, wherein the fault includes a blockage in the flow.

[0319] 85. The non-transient computer-readable storage medium as described in Clause 83, wherein the fault includes the introduction of gas into the flow stream.

[0320] 86. A non-transient computer-readable storage medium according to any one of clauses 67-85, wherein the non-transient computer-readable storage medium includes an algorithm for generating error alerts in real time.

[0321] 87. The non-transient computer-readable storage medium as described in Clause 86, wherein the non-transient computer-readable storage medium includes an algorithm for generating an error alarm when a measured flow rate continuously exceeds an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration.

[0322] 88. A non-transient computer-readable storage medium according to any one of clauses 67-87, wherein the non-transient computer-readable storage medium includes an algorithm for adjusting the flow rate of a flow in response to a generated error alarm.

[0323] 89. The non-transient computer-readable storage medium as described in Clause 88, wherein the non-transient computer-readable storage medium includes an algorithm for increasing the flow rate of the flow in response to a generated error alarm.

[0324] 90. A non-transient computer-readable storage medium as described in Clause 88, wherein the non-transient computer-readable storage medium includes an algorithm for reducing the flow rate of a flow in response to a generated error alarm.

[0325] 91. A non-transient computer-readable storage medium as described in Clause 88, wherein the non-transient computer-readable storage medium includes an algorithm for stopping the flow of a flow in response to a generated error alarm.

[0326] 92. The non-transient computer-readable storage medium as described in Clause 88, wherein the non-transient computer-readable storage medium includes an algorithm for flushing the flow in response to a generated error alarm.

[0327] 93. A non-transient computer-readable storage medium according to any one of Clauses 34-60, wherein the flow stream includes a sheath fluid flow stream and a sample core fluid flow stream.

[0328] 94. The non-transient computer-readable storage medium according to Clause 93, wherein the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of a sheath fluid flow.

[0329] 95. The non-transient computer-readable storage medium according to Clause 93, wherein the non-transient computer-readable storage medium includes an algorithm for measuring the flow rate of a fluid flow in the core of a sample.

[0330] 96. A non-transient computer-readable storage medium according to any one of clauses 93-95, wherein the non-transient computer-readable storage medium includes an algorithm for measuring the relative flow rates of a sheath fluid flow and a sample core fluid flow.

[0331] Although the foregoing disclosure has been described in detail by way of illustration and example for purposes of clarity, those skilled in the art will readily recognize, based on the teachings of this disclosure, that some changes and modifications may be made to this disclosure without departing from the spirit or scope of the appended claims.

[0332] Therefore, the foregoing only illustrates the principles of this disclosure. It should be understood that those skilled in the art will be able to design various arrangements that, while not explicitly described or shown herein, embody the principles of this disclosure and are included within its spirit and scope. Furthermore, all examples and conditional language cited herein are primarily intended to assist the reader in understanding the principles of this disclosure and the concepts it contributes to advancing the art, and should be construed as not being limited to these specifically cited examples and conditions. Moreover, all statements and specific examples of the principles, aspects, and embodiments of this disclosure herein are intended to cover their structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and future development equivalents, i.e., any developed elements performing the same function, regardless of structure. Furthermore, nothing disclosed herein is intended for public use only, whether or not such disclosure is expressly stated in the claims.

[0333] Therefore, the scope of this disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied in the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is invoked only if the exact phrase “means for %” or the exact phrase “step for ” is expressly recited at the beginning of the claim; if such an exact phrase is not used in the limitation of the claim, 35 USC §112(f) or 35 USC §112(6) is not invoked.

Claims

1. A method comprising: Measure the flow rate of the flow stream in a flow cytometer; The measured flow velocity is compared with the following: Absolute flow rate threshold; and Moving window flow rate threshold; and An error alarm is generated when the measured flow velocity exceeds the absolute flow velocity threshold or the moving window flow velocity threshold.

2. The method of claim 1, wherein the method includes continuously measuring the flow velocity of the flow.

3. The method of claim 1, wherein the method includes measuring the flow velocity of the flow at discrete intervals.

4. The method according to any one of claims 1-3, wherein the absolute flow rate threshold includes an upper limit threshold and a lower limit threshold.

5. The method according to any one of claims 1-4, wherein the method comprises continuously comparing the measured flow velocity with each absolute flow velocity threshold.

6. The method according to any one of claims 4-5, wherein an error alarm is generated when it is determined that the measured flow rate is less than a lower absolute flow rate threshold or greater than an upper absolute flow rate threshold.

7. The method of claim 6, wherein an error alarm is generated when it is determined that the measured flow rate is 1% or more greater or less than the absolute flow rate threshold.

8. The method according to any one of claims 1-7, wherein the moving window flow rate threshold includes an upper limit threshold and a lower limit threshold.

9. The method according to any one of claims 1-7, wherein the method comprises comparing the measured flow rate with a flow rate threshold for each moving window at discrete intervals.

10. The method according to any one of claims 1-9, wherein the error alarm is generated in real time.

11. The method of claim 10, wherein an error alarm is generated when the measured flow rate continuously exceeds the absolute flow rate threshold or the moving window flow rate threshold for a predetermined duration.

12. The method according to any one of claims 1-10, wherein the method further comprises adjusting the flow rate of the flow in response to a generated error alarm.

13. The method according to any one of claims 1-12, wherein the flow rate of the flow is measured using a flow sensor based on one or more of the temperature and viscosity of the flow.

14. The method according to any one of claims 1-13, wherein the flow stream comprises a sheath fluid flow stream and a sample core fluid flow stream.

15. The method according to any one of claims 1-14, wherein the method further comprises conveying a sample containing particles in a flow stream.

16. The method of claim 15, wherein the method comprises illuminating a sample with a light source and detecting light from the illuminating particles in the flow stream.

17. A system for configuring a flow cytometer, the system comprising: A processor, including memory operatively coupled to the processor, wherein the memory includes instructions stored thereon, the memory comprising: Instructions used to measure the flow rate of a flow stream in a flow cytometer; Instructions for comparing the measured flow velocity with the following: Absolute flow rate threshold; and Moving window flow rate threshold; and Instructions for generating an error alarm when the measured flow velocity exceeds the absolute flow velocity threshold or the moving window flow velocity threshold.

18. The system of claim 17, wherein the memory includes instructions for continuously measuring the flow rate of the flow or measuring the flow rate of the flow at discrete intervals.

19. A non-transient computer-readable storage medium for configuring a flow cytometer, comprising instructions stored thereon, the instructions comprising: An algorithm for measuring the flow rate of a flow stream in a flow cytometer; An algorithm used to compare the measured flow velocity with the following: Absolute flow rate threshold; and Moving window flow rate threshold; and An algorithm for generating an error alarm when the measured flow velocity exceeds the absolute flow velocity threshold or the moving window flow velocity threshold.

20. The non-transient computer-readable storage medium of claim 19, wherein the non-transient computer-readable storage medium includes an algorithm for continuously measuring the flow velocity of the flow or measuring the flow velocity of the flow at discrete intervals.

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