A method for monitoring the flow rate of a flowstream, and a system for doing so.

Abstract

Aspects of the present disclosure include methods for monitoring the flow rate of a flow stream (for example, in a flow cytometer). In one embodiment, the method measures the flow rate of the flow stream, compares the measured flow rate of the flow stream to an absolute flow threshold and a moving window flow threshold, and generates an error alert if the measured flow rate of the flow stream exceeds the absolute flow threshold or the moving window flow threshold. A system and a non-temporary computer-readable storage medium configured to carry out the method of the subject are further provided.

Description

[Background technology]

[0001] The characterization of analytes in biological fluids is a crucial part of biological research, medical diagnosis, and the assessment of a patient's overall health and well-being. By detecting analytes in biological fluids such as human blood or blood-derived products, results can be obtained that may be relevant in determining treatment protocols for patients with various medical conditions.

[0002] Flow cytometry is a technique used to characterize and often sort biomaterials, such as cells in blood samples or particles of interest in other types of biological or chemical samples. A flow cytometer typically comprises a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer transports particles (including cells) from the fluid sample as a cellular flow into a flow cell, while further guiding the sheath fluid into the flow cell. Light is shone into the flow stream to characterize its components. Changes in biomaterials within the flow stream, such as morphology or the presence of fluorescent labels, can alter the observed light, and these changes enable characterization and separation. Light must be shone into the flow stream and collected to characterize its components. The light source of a flow cytometer can be a variety of light sources, including 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 irradiated particles is collected and quantified.

[0003] The separation of biological particles is achieved by adding a sorting or collection function to a flow cytometer. Particles present in the separated flow and identified as possessing one or more desired characteristics are individually separated from the sample flow by mechanical or electrical removal. Common flow sorting techniques utilize droplet sorting, which divides a fluid stream containing linearly separated particles into droplets. Droplets containing the particles of interest are electrically charged and deflected into a collection tube by passing through an electric field. Typically, linearly separated particles in the flow are characterized as they pass through an observation point directly below the nozzle tip. Once particles are identified as meeting one or more desired criteria, the time it takes for those particles to reach the droplet separation point and separate from the flow into droplets can be predicted. Ideally, the fluid stream is briefly charged just before the droplet containing the selected particles separates from the fluid stream, and then grounded immediately after the droplet separates. The sorted droplet retains its charge when separating from the fluid stream, while all other droplets remain uncharged. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The quality of flow cytometry data can be negatively affected by sample quality or problems during data acquisition, such as blockages or bubbles in the sample core stream. This can lead to erroneous conclusions from data analysis. Most flow cytometers lack error detection capabilities in the sample fluid control system or rely on bubble detectors. These flow cytometers cannot provide measurements for living samples and cannot detect rapid fluid changes in the flow stream. Changes in flow rate can indicate errors and, in some cases, indicate a flow system malfunction that obstructs the flow of sample through the flow system. [Means for solving the problem]

[0005] Aspects of this disclosure include methods for monitoring the flow rate of a flow stream (e.g., in a flow cytometer). In one embodiment, the method measures the flow rate of the flow stream, compares the measured flow rate of the flow stream to an absolute flow threshold and a moving window flow threshold, and generates an error alert if the measured flow rate of the flow stream exceeds the absolute flow threshold or the moving window flow threshold. A system and a non-temporary computer-readable storage medium configured to carry out the method of the subject are further provided.

[0006] In some embodiments, the flow rate of the flowstream is measured continuously. In some cases, the flow rate of the flowstream is measured at discrete intervals, for example, at intermittent intervals of a predetermined duration. In some cases, the flow rate of the flowstream is measured using a flow sensor based on the temperature change of the flowstream. In some cases, the flow rate of the flowstream is measured using a flow sensor based on the viscosity change of the flowstream.

[0007] In some cases, the absolute flow rate has upper and lower thresholds. In some cases, the method continuously compares the measured flow rate of the flow stream with the upper and lower absolute flow rate thresholds. In some cases, an error alert is generated if the measured flow rate is determined to be less than the lower absolute flow rate threshold. In some cases, an error alert is generated if the measured flow rate is determined to be greater than the upper absolute flow rate threshold. In some cases, an error alert is generated if the measured flow rate is determined to be 1% or more greater than the upper absolute flow rate threshold, e.g., 3% or more, or 1% or more less than the lower absolute flow rate threshold, e.g., 3% or more.

[0008] In some embodiments, the moving window flow threshold has an upper and lower threshold. In some cases, the method compares the measured flow rate with each moving window flow threshold at discrete intervals. In some cases, when comparing the measured flow rate with each moving window flow threshold, it is determined whether the measured flow rate is greater than the upper moving window flow threshold within a given time interval. In some cases, when comparing the measured flow rate with each moving window flow threshold, it is determined whether the measured flow rate is less than the lower moving window flow threshold within a given time interval. In some cases, the time interval of the moving window for comparing the measured flow rate with the flow threshold is in the range of 0.1 seconds to 10 seconds, for example, 1 second to 5 seconds. In some cases, the time interval of the moving window is 1 second.

[0009] In some embodiments, the generated error alert indicates a malfunction in the flow cytometer. In some cases, the malfunction is a blockage in the flow stream. In some cases, the malfunction is the ingress of gas (e.g., air) into the flow stream or sample line. In some cases, the error alert is generated in real time. In some cases, an error alert is generated if the measured flow rate continues to exceed an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration, e.g., 0.001 seconds or longer, e.g., 0.01 seconds or longer, e.g., 0.1 seconds or longer, e.g., 5 seconds or longer.

[0010] In some embodiments, the method modifies one or more parameters of the flow cytometer in response to a generated error alert. In some cases, the flow rate of the flow stream is adjusted in response to a generated error alert. In some cases, the flow rate is increased in response to a generated error alert. In some cases, the flow rate is decreased in response to a generated error alert. In some cases, the flow rate is stopped in response to a generated error alert. In some cases, the light source for illuminating the flow stream is adjusted in response to a generated error alert. In some cases, the light source is turned off in response to a generated error alert. In some cases, the light configured to illuminate the flow stream is blocked in response to a generated error alert.

[0011] In some embodiments, the flowstream includes a sheath fluid flowstream and a sample core fluid flowstream. In some cases, the method measures the flow rate of the sheath fluid flowstream. In some cases, the method measures the flow rate of the sample core fluid flowstream. In some cases, the method measures the flow rates of both the sheath fluid flowstream and the sample core fluid flowstream. In some embodiments, a sample containing particles is carried in the sample core fluid flowstream. In some cases, the method irradiates the sample using a light source and detects light from the irradiated particles in the flowstream. In some cases, the light source has lasers, for example, multiple lasers. In some embodiments, light is detected by multiple photodetector channels.

[0012] Aspects of the present disclosure further include a system for carrying out the subject method for monitoring the flow rate of a flow stream, for example (such as in a flow cytometer). One embodiment of the system includes a processor operably coupled to a memory storing instructions, the memory including instructions for measuring the flow rate of a flow stream, instructions for comparing the measured flow rate of the flow stream with an absolute flow threshold and a moving window flow threshold, and instructions for generating an error alert if the measured flow rate of the flow stream exceeds the absolute flow threshold or the moving window flow threshold.

[0013] In some embodiments, the system includes a flow sensor configured to measure the flow rate of a flowstream. In some cases, the flow sensor is configured to measure the flow rate of a flowstream based on a change in the temperature of the flowstream. In some cases, the flow sensor is configured to measure the flow rate of a flowstream based on a change in the viscosity of the flowstream. In some cases, the system is configured to measure the flow rate of a flowstream continuously. In some cases, the system is configured to measure the flow rate of a flowstream at discrete intervals, such as measuring the flow rate of a flowstream at intermittent intervals of a predetermined duration.

[0014] In some cases, the absolute flow rate has upper and lower thresholds. In some cases, the memory includes instructions for continuously comparing the measured flow rate of the flow stream with the upper and lower absolute flow rate thresholds. In some cases, the memory includes instructions for generating an error alert if the measured flow rate is determined to be less than the lower absolute flow rate threshold. In some cases, the memory includes instructions for generating an error alert if the measured flow rate is determined to be greater than the upper absolute flow rate threshold. In some cases, the memory includes instructions for generating an error alert if the measured flow rate is determined to be 1% or more greater than the upper absolute flow rate threshold, e.g., 3% or more, or 1% or more less than the lower absolute flow rate threshold, e.g., 3% or more.

[0015] In some embodiments, the moving window flow threshold has an upper and lower threshold. In some cases, the memory includes instructions for comparing the measured flow rate with each moving window flow threshold at discrete intervals. In some cases, the memory includes instructions for comparing the measured flow rate with each moving window flow threshold by determining whether the measured flow rate is greater than the upper moving window flow threshold during a given time interval. In some cases, the memory includes instructions for comparing the measured flow rate with each moving window flow threshold by determining whether the measured flow rate is less than the lower moving window flow threshold during a given time interval. In some cases, the time interval of the moving window for comparing the measured flow rate with the flow threshold is in the range of 0.1 seconds to 10 seconds, for example, 1 second to 5 seconds. In some cases, the time interval of the moving window is 1 second.

[0016] In some embodiments, the memory includes instructions for generating an error alert indicating a malfunction in the flow cytometer. In some cases, the malfunction is a blockage in the flow stream. In some cases, the malfunction is the ingress of gas (e.g., air) into the flow stream or sample line. In some cases, the memory includes instructions for generating an error alert in real time. In some cases, the memory includes instructions for generating an error alert if the measured flow rate continues to exceed an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration, e.g., 0.001 seconds or longer, e.g., 0.01 seconds or longer, e.g., 0.1 seconds or longer, e.g., 5 seconds or longer.

[0017] In some embodiments, the memory includes commands to modify one or more parameters of the flow cytometer in response to generated error alerts. In some cases, the memory includes commands to adjust the flow rate of the flow stream in response to generated error alerts. In some cases, the memory includes commands to increase the flow rate in response to generated error alerts. In some cases, the memory includes commands to decrease the flow rate in response to generated error alerts. In some cases, the memory includes commands to stop the flow rate in response to generated error alerts. In some cases, the memory includes commands to adjust the light source for illuminating the flow stream in response to generated error alerts. In some cases, the memory includes commands to turn off the light source in response to generated error alerts. In some cases, the memory includes commands to block the light configured to illuminate the flow stream in response to generated error alerts.

[0018] In some embodiments, the system has a flow stream formed from a sheath fluid flow stream and a sample core fluid flow stream. In some cases, the memory includes instructions for measuring the flow rate of the sheath fluid flow stream. In some cases, the memory includes instructions for measuring the flow rate of the sample core fluid flow stream. In some cases, the memory includes instructions for measuring the flow rates of both the sheath fluid flow stream and the sample core fluid flow stream. In some embodiments, a sample containing particles is carried in the sample core fluid flow stream. In some cases, the memory includes instructions for irradiating the sample using a light source and for detecting light from the irradiated particles in the flow stream. In some cases, the light source has a laser, for example, multiple lasers. In some embodiments, the system includes a light detection system configured to detect light within a plurality of photodetector channels.

[0019] Aspects of the present disclosure further include, for example, a non - transient computer - readable storage medium for implementing one or more of the computer - implemented methods described herein. 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 to an absolute flow rate threshold and a moving window flow rate threshold, and an algorithm for generating an error alert if the measured flow rate of the flow stream exceeds the absolute flow rate threshold or the moving window flow rate threshold.

[0020] In some embodiments, the non - transient computer - readable storage medium includes an algorithm for continuously measuring the flow rate of the flow stream. In some cases, the non - transient computer - readable storage medium includes an algorithm for measuring the flow rate of the flow stream at discrete intervals, such as at intermittent intervals for a predetermined duration.

[0021] In some cases, the absolute flow rate has an upper threshold value and a lower threshold value. In some cases, the non-transitory computer-readable storage medium includes an algorithm for continuously comparing the measured flow rate of the flow stream with the upper absolute flow rate threshold value and the lower absolute flow rate threshold value. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating an error alert when it is determined that the measured flow rate is less than the lower absolute flow rate threshold value. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating an error alert when it is determined that the measured flow rate is greater than the upper absolute flow rate threshold value. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating an error alert when it is determined that the measured flow rate is 1% or more, for example 3% or more, greater than the upper absolute flow rate threshold value or 1% or more, for example 3% or more, less than the lower absolute flow rate threshold value.

[0022] In some embodiments, the moving window flow rate threshold has an upper threshold value and a lower threshold value. In some cases, the non-transitory 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-transitory 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-transitory 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 within the range of 0.1 second to 10 seconds, for example 1 second to 5 seconds. In some cases, the time interval of the moving window is 1 second.

[0023] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for generating error alerts indicating a malfunction in the flow cytometer. In some cases, the malfunction is a blockage in the flow stream. In some cases, the malfunction is the ingress of gas (e.g., air) into the flow stream or sample line. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating error alerts in real time. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating an error alert if the measured flow rate continues to exceed an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration, e.g., 0.001 seconds or longer, e.g., 0.01 seconds or longer, e.g., 0.1 seconds or longer, e.g., 5 seconds or longer.

[0024] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for modifying one or more parameters of the flow cytometer in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for adjusting the flow rate of the flow stream in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for increasing the flow rate in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for decreasing the flow rate in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for stopping the flow rate in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for adjusting the light source for illuminating the flow stream in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for turning off the light source in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for blocking light configured to illuminate the flow stream in response to generated error alerts.

[0025] In some embodiments, the flowstream includes a sheath fluid flowstream and a sample core fluid flowstream. In some cases, a non-temporary computer-readable storage medium includes an algorithm for measuring the flow rate of the sheath fluid flowstream. In some cases, a non-temporary computer-readable storage medium includes an algorithm for measuring the flow rate of the sample core fluid flowstream. In some cases, a non-temporary computer-readable storage medium includes an algorithm for measuring the flow rates of the sheath fluid flowstream and the sample core fluid flowstream. When a sample containing particles is carried in the sample core fluid flowstream, the non-temporary computer-readable storage medium may include an algorithm for irradiating the sample with a light source and an algorithm for detecting light from the irradiated particles in the flowstream. [Brief explanation of the drawing]

[0026] This disclosure can be best understood from the following detailed description when read in conjunction with the attached drawings. The drawings include the following figures.

[0027] [Figure 1A] This flowchart shows the process of monitoring the flow rate of a flow stream in a flow cytometer according to a certain embodiment. [Figure 1B] This is a diagram showing the results of monitoring the flow rate of a flow stream in a flow cytometer according to a certain embodiment. [Figure 2] This is a diagram showing a flow cytometry system according to one embodiment. [Figure 3-1] This figure shows an image-compatible particle sorter according to one embodiment. [Figure 3-2] This figure shows an image-compatible particle sorter according to one embodiment. [Figure 4] This is a functional block diagram showing a particle analysis system according to a certain embodiment. [Figure 5] This is a functional block diagram showing an example of a control system according to a certain embodiment. [Figure 6A]This is a schematic diagram showing a particle sorting system according to one embodiment. [Figure 6B] This is a schematic diagram showing a particle sorting system according to one embodiment. [Figure 7] This is a diagram showing an embodiment of a computer control system according to one particular example. [Modes for carrying out the invention]

[0028] Aspects of this disclosure include methods for monitoring the flow rate of a flow stream (e.g., in a flow cytometer). In one embodiment, the method measures the flow rate of the flow stream, compares the measured flow rate of the flow stream to an absolute flow threshold and a moving window flow threshold, and generates an error alert if the measured flow rate of the flow stream exceeds the absolute flow threshold or the moving window flow threshold. A system and a non-temporary computer-readable storage medium configured to carry out the method of the subject are further provided.

[0029] Before this disclosure is described in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, as it is, needless to say, subject to change. Since the scope of this disclosure is limited only by the appended claims, it should be further understood that the terms used herein are intended to describe, and not to limit, specific embodiments.

[0030] If a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless the context otherwise expresses, and all other stated or intervening values ​​within that stated range are included in this disclosure. These smaller upper and lower limits may be independently included within the smaller range and are further included in this disclosure, subject to any specifically excluded limits within the stated range. If the stated range includes one or both limits, the range excluding one or both of those included limits is also included in this disclosure.

[0031] In this specification, a range is presented preceded by the term “approximately.” The term “approximately” is used here to literally support the exact number preceding it, and any number that is close to or approximates it. When determining whether a number is close to or approximates a specifically stated number, the close or approximate unstated number may be a number that, in the context in which the specifically stated number is presented, gives a substantial equivalent of the specifically stated number.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar to or equivalent to those described herein may be further used in the practice or testing of this disclosure, but representative and exemplary methods and materials are described herein.

[0033] All publications and patents referenced herein are incorporated herein by reference as if each individual publication or patent were specifically and individually incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relating to the references of the publications. Any reference to a publication is for its disclosure prior to the filing date, and this disclosure should not be construed as acknowledging that such publication has no prior rights on the grounds of prior invention. Furthermore, the dates of the publications provided may differ from the actual publication dates and may need to be verified individually.

[0034] It should be noted that, as used in this specification and the attached claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context otherwise indicates. It should also be noted that claims may be drafted to exclude any particular element. Therefore, this statement is intended to serve as a prior art for the use of exclusive terms such as "only," "only," or "negative" limitations relating to the description of elements of a claim.

[0035] As will be apparent to those skilled in the art upon reading this disclosure, each of the embodiments described and illustrated herein has separate components and features, which may be readily separated from or readily combined with any of the features of any of the other embodiments without departing from the scope or spirit of this disclosure. All described methods may be performed in the order of the described events or in any other logically possible order.

[0036] While systems and methods are described or stipulated for grammatical fluidity with respect to functional descriptions, claims should not necessarily be construed as being limited in any way by a limitation of the composition of “means” or “steps” unless expressly described under Section 112 of the United States Patent Act, and should be granted the meaning of the definitions and the full scope of equivalents given by claims under the judicial theory of equivalents, and should be clearly understood that if claims are expressly described under Section 112 of the United States Patent Act, they should be granted the full statutory equivalents under Section 112 of the United States Patent Act.

[0037] A method for monitoring the flow rate of a flow stream in a flow cytometer. Aspects of the present disclosure include methods for monitoring the flow rate of a flow stream (e.g., in a flow cytometer). In embodiments, the flow rate of the flow stream is monitored to determine whether there is a change in the flow rate, and optionally, whether such a change is caused by a sudden failure of the sample fluid control system of the system. As described in more detail below, the subject method for monitoring the flow rate verifies whether the flow rate is within a stable range (e.g., between an upper absolute flow rate threshold and a lower absolute flow rate threshold) and whether the flow rate is changing relative to the moving window. In embodiments, monitoring the flow rate of the flow stream can accurately and early detect errors in the propagation of the sample in the system. Optionally, monitoring the flow rate according to the subject method can detect errors in the sample fluid control system caused by sudden changes in flow rate, such as blockage or air ingress in the fluid control system of a flow cytometer.

[0038] In some embodiments, the subject method and system provide recognition of flow rate patterns, enabling the determination of the cause of an error, for example, by distinguishing between clogging of the fluid control system, leakage from the sample line or sheath fluid line, completion of sample propagation through the flowstream, and the introduction of gas into the flowstream (e.g., due to error or failure or separation between samples). In some cases, when a change in flow rate is detected, an alert is generated, and the sample flow is stopped upon error detection to reduce sample loss. For example, sample loss can be reduced by 5%, 10%, 25%, 50%, 75%, 90%, or 99%. In some cases, the flow rate of the flowstream is monitored as described herein to prevent any sample loss.

[0039] In one embodiment, the flow rate for sample investigation is optimized by monitoring the flow rate of the flow stream, for example, by determining an optimized laser delay for irradiating sample particles in the flow stream. In some cases, the accuracy of the laser delay timing can be increased by more than 10%, for example more than 25%, for example more than 50%, for example more than 75%, for example more than 100%.

[0040] In some cases, error alerts can be generated to stop the sample flow when an error is detected, improving the reliability of flow cytometry data. In some cases, the method described in the subject can improve the reliability of data generated by irradiating the sample in the monitored flow stream by, for example, more than 10%, more than 25%, more than 50%, more than 75%, more than 100%, more than 150%, more than 200%, more than 250%, and for example, more than 300%.

[0041] When implementing the method described in the subject, the flow rate of the flow stream (e.g., in a flow cytometer) is measured. In some cases, the flow rate is measured continuously, for example by monitoring the flow rate in real time. In some cases, the flow rate is measured at discrete intervals, for example every 0.00001 seconds or more, for example every 0.00005 seconds or more, for example every 0.0001 seconds or more, for example every 0.0005 seconds or more, for example every 0.001 seconds or more, for example every 0.005 seconds or more, for example every 0.01 seconds or more, for example every 0.05 seconds or more, for example every 0.1 seconds or more, for example every 0.5 seconds or more, for example every 1 second or more, for example every 2 seconds or more, for example every 3 seconds or more, for example every 4 seconds or more, for example every 5 seconds or more, for example every 6 seconds or more, for example every 7 seconds or more, for example every 8 seconds or more, for example every 9 seconds or more, for example every 10 seconds or more, for example every 15 seconds or more, for example every 30 seconds or more, for example every 60 seconds or more.

[0042] In some embodiments, the flow rate of the flow stream may be 1 μL / min or more, for example 2 μL / min or more, for example 3 μL / min or more, for example 5 μL / min or more, for example 10 μL / min or more, for example 15 μL / min or more, for example 25 μL / min or more, for example 50 μL / min or more, for example 100 μL / min or more, and in some cases, the flow rate of the flow stream may be 1 μL / second or more, for example 2 μL / second or more, for example 3 μL / second or more, for example 5 μL / second or more, for example 10 μL / second or more, for example 15 μL / second or more, for example 25 μL / second or more, for example 50 μL / second or more, for example 100 μL / second or more. In one embodiment, the flow rate of the flow stream may be 25 μL / sec or more, for example 50 μL / sec or more, for example 75 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, for example 1000 μL / sec or more, for example 2500 μL / sec or more.

[0043] The flow rate of the flowstream may be measured by any convenient protocol, and in some cases, the flow rate may be measured based on the measured temperature of the flowstream. In some cases, the flow rate may be measured based on the measured viscosity of the flowstream. In some cases, the flow rate may be measured at one or more detection positions along the flowstream, for example, 2 or more, for example, 3 or more, for example, 4 or more, for example, 5 or more, for example, 6 or more, for example, 7 or more, for example, 8 or more, for example, 9 or more detection positions, and for example, 10 or more detection positions along the flowstream. In some cases, the detection positions for measuring the flow rate of the flowstream may extend to 0.001 mm or more of the flowstream, 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 for example, 10 mm or more of the flowstream. In some cases, the flow rate of the flowstream is measured at two or more detection positions along the flowstream based on the temperature change of the flowstream, for example, at 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 positions, and for example, at ten or more different detection positions along the flowstream based on the temperature change of the flowstream. In some cases, the flow rate of the flowstream is measured at two or more different detection positions along the flowstream based on the viscosity change of the flowstream, for example, at 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 different detection positions, and for example, at ten or more detection positions along the flowstream based on the viscosity change of the flowstream.

[0044] In some cases, the flow rate is measured using a temperature sensor flow meter. In some cases, the flow sensor has a controllable heating element located in the central part of a pressure-stabilizing membrane, and temperature sensors are mounted upstream and downstream (e.g., symmetrically) with respect to the direction of fluid flow by the flow stream. In some cases, heat is transferred to the downstream temperature sensor as the flow passes through, and the downstream temperature sensor generates a measurable signal due to the resulting temperature difference. In some cases, a microthermal flow sensor is integrated into a silicon chip flow meter type sensor. In some cases, the flow sensor has a temperature sensor that generates a signal that compensates for the effect of temperature. In some cases, the flow sensor has a fluid flow sensor such as the commercially available flow sensor product line from Sensiron (Steffa, Switzerland). In some cases, the flow rate is measured using a flow sensor such as those described in U.S. Patent Application Publication No. 2023 / 0324270, U.S. Patent No. 10942139, European Patent No. 3187881, and European Patent No. 3404373, whose disclosures are incorporated herein by reference.

[0045] In some embodiments, the flowstream includes a sheath fluid flowstream and a sample core fluid flowstream. In some embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid, for example, together with the sample fluid, into the internal chamber of the flow cell, thereby generating a layered flowstream of sheath fluid surrounding the sample core fluid flowstream. In some cases, the sheath fluid flowstream forms a laminar flow stream surrounding the sample core fluid flowstream. In some cases, the flow rate of the sheath fluid flowstream is measured by a method. In some cases, the flow rate of the sheath fluid flowstream is 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, e.g., 2500 μL / sec or more. In some cases, the flow rate of the sample core fluid flowstream is measured by a method. In some cases, the flow rate of the sample core fluid flow stream is 1 μL / min or more, e.g., 2 μL / min or more, e.g., 3 μL / min or more, e.g., 5 μL / min or more, e.g., 10 μL / min or more, e.g., 15 μL / min or more, e.g., 25 μL / min or more, e.g., 50 μL / min or more, e.g., 100 μL / min or more, and in some cases, the flow rate of the sample delivered to the flow cell chamber by the sample injection port is 1 μL / second or more, e.g., 2 μL / second or more, e.g., 3 μL / second or more, e.g., 5 μL / second or more, e.g., 10 μL / second or more, e.g., 15 μL / second or more, e.g., 25 μL / second or more, e.g., 50 μL / second or more, e.g., 100 μL / second or more. In some cases, the flow rate of the sheath fluid flow stream and the flow rate of the sample core fluid flow stream are measured by method.

[0046] In some cases, the flow rate of the sheath fluid flow stream is compared with the flow rate of the sample core fluid flow stream. In some cases, the flow rate of the sheath fluid flow stream is determined to be the same as the flow rate of the sample core fluid flow stream. In some cases, the flow rate of the sheath fluid flow stream is determined to be smaller than the flow rate of the sample core fluid flow stream by, for example, 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, or 50% or more. In some cases, the flow rate of the sheath fluid flow stream is determined to be greater than the flow rate of the sample core fluid flow stream by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%. If the relative flow rates of the sheath fluid flow stream and the sample core fluid flow stream are determined to exceed a predetermined threshold, the method may adjust one or more of the flow rates of the sheath fluid flow stream and the sample core fluid flow stream. In some cases, the method adjusts the flow rates of one or more of the sheath fluid flow stream and the sample core fluid flow stream if the relative flow rates of the sheath fluid flow stream and the sample core fluid flow stream exceed a threshold of 1%, for example, 2%, for example, 3%, for example, 4%, for example, 5%, for example, 10%, for example, 15%, for example, 20%, for example, 25%, or for example, 50%.

[0047] In some embodiments, flow measurement of the flowstream begins when a stable flow rate is detected. In some cases, the flow rate is measured when the flow rate has a fluctuation rate of 5% or less, e.g., 4% or less, e.g., 3% or less, e.g., 2% or less, e.g., 1% or less, e.g., 0.5% or less, e.g., 0.1% or less, e.g., 0.05% or less, e.g., 0.01% or less (e.g., showing a change that can be measured immediately), e.g., when the flow rate shows a change of 0.0001% or less. If the flow rate shows fluctuations that exceed the stabilization threshold, the system may be set to standby mode until the fluctuations fall below the stabilization threshold for a predetermined duration, e.g., 1 second or more, e.g., 5 seconds or more, e.g., 10 seconds or more, e.g., 15 seconds or more, e.g., 30 seconds or more, e.g., 60 seconds or more. Once it is determined that the flowstream is sufficiently stable for monitoring, the flow measurement may be moved to the monitoring phase. In some embodiments, if the flow rate shows instability or fluctuations that exceed the stabilization threshold during the monitoring phase, the system may be moved back to standby mode and the monitoring phase is stopped until the flowstream flow rate shows sufficient stability.

[0048] In some embodiments, the absolute flow rate has upper and lower thresholds. The term "absolute flow rate" refers to the flow rate of the flow stream measured at any point in time during the transport of the flow stream. In some cases, the absolute flow rate may be the real-time flow rate measured by a flow sensor. In other cases, the absolute flow rate is the flow rate at a specific point in the experiment during which the flow rate of the flow stream is recorded. In some cases, the absolute flow rate is measured (and possibly recorded) continuously. In some cases, the measured flow rate of the flow stream is continuously compared to an upper absolute flow rate threshold. In some cases, an error alert is generated if the measured flow rate is determined to be greater than the upper absolute flow rate threshold by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%. In some cases, an error alert is generated if the measured flow rate is determined to be less than the lower absolute flow rate threshold by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%. When continuously measuring the flow rate of a flowstream, an error alert may be generated if 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 more, for example, 0.0005 seconds or more, for example, 0.001 seconds or more, for example, 0.005 seconds or more, for example, 0.01 seconds or more, for example, 0.05 seconds or more, for example, 0.1 seconds or more, for example, 0.5 seconds or more, for example, 1 second or more, for example, 2 seconds or more, for example, 3 seconds or more, for example, 4 seconds or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 15 seconds or more, for example, 30 seconds or more, for example, 45 seconds or more, for example, 60 seconds or more.In some cases, if the flow rate exceeds the upper limit of the absolute flow rate threshold for a period of 0.0001 seconds or more, for example 0.0005 seconds or more, for example 0.001 seconds or more, for example 0.005 seconds or more, for example 0.01 seconds or more, for example 0.05 seconds or more, for example 0.1 seconds or more, for example 0.5 seconds or more, for example 1 second or more, for example 2 seconds or more, for example 3 seconds or more, for example 4 seconds or more, for example 5 seconds or more, for example 10 seconds or more, for example 15 seconds or more, for example 30 seconds or more, for example 45 seconds or more, for example 60 seconds or more, an error alert will be generated. In some cases, if the flow rate is less than the lower limit of the absolute flow rate threshold for 0.0001 seconds or more, for example 0.0005 seconds or more, for example 0.001 seconds or more, for example 0.005 seconds or more, for example 0.01 seconds or more, for example 0.05 seconds or more, for example 0.1 seconds or more, for example 0.5 seconds or more, for example 1 second or more, for example 2 seconds or more, for example 3 seconds or more, for example 4 seconds or more, for example 5 seconds or more, for example 10 seconds or more, for example 15 seconds or more, for example 30 seconds or more, for example 45 seconds or more, for example 60 seconds or more, an error alert will be generated.

[0049] In some cases, the measured flow rate of the flowstream is compared to an absolute flow rate threshold at discrete intervals, for example, every 0.00001 seconds or more, every 0.00005 seconds or more, every 0.0001 seconds or more, every 0.0005 seconds or more, every 0.001 seconds or more, every 0.005 seconds or more, every 0.01 seconds or more, every 0.05 seconds or more, every 0.1 seconds or more, every 0.5 seconds or more, every 1 second or more, every 2 seconds or more, every 3 seconds or more, every 4 seconds or more, every 5 seconds or more, every 6 seconds or more, every 7 seconds or more, every 8 seconds or more, every 9 seconds or more, every 10 seconds or more, every 15 seconds or more, every 30 seconds or more, and every 60 seconds or more. In some cases, if the measured flow rate exceeds the absolute flow rate threshold at each discrete comparison interval, an error alert is generated. In other cases, if the measured flow rate exceeds the absolute flow rate threshold, an error alert is generated when the measured flow rate exceeds the absolute flow rate threshold at discrete comparison intervals greater than 1, for example, 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more discrete comparison intervals.

[0050] In the embodiment, the method compares the measured flow rate of a flowstream with a moving window flow rate threshold. The term moving window flow rate refers to a predetermined time interval (i.e., duration) of the flowstream flow rate. For example, the moving window flow rate may be the moving average flow rate of the flowstream over a specific time interval, e.g., 0.1 seconds or longer, e.g., 0.5 seconds or longer, e.g., 1 second or longer, e.g., 2 seconds or longer, e.g., 3 seconds or longer, e.g., 4 seconds or longer, e.g., 5 seconds or longer, e.g., 10 seconds or longer. In some cases, the moving window flow rate is a snapshot of the measured flowstream flow rate over a specific duration, e.g., 0.1 seconds or longer, e.g., 0.5 seconds or longer, e.g., 1 second or longer, e.g., 2 seconds or longer, e.g., 3 seconds or longer, e.g., 4 seconds or longer, e.g., 5 seconds or longer, e.g., 10 seconds or longer. In the embodiment, the moving window flow rate may be the flow rate during a time interval of 0.1 seconds to 10 seconds, for example 0.5 seconds to 9.5 seconds, for example 1 second to 9 seconds, for example 1.5 seconds to 8.5 seconds, for example 2 seconds to 8 seconds, for example 2.5 seconds to 7.5 seconds, for example 3 seconds to 7 seconds. In some cases, the measured flow rate is compared with the moving window flow rate threshold one or more times, for example two or more times, for example three or more times, for example four or more times, for example five or more times, for example ten or more times, for example 25 or more times, for example 50 or more times, for example 100 or more times, for example 250 or more times.

[0051] In some embodiments, each moving window flow threshold has an upper and lower threshold. In some cases, the measured flow rate of the flow stream is compared to the upper threshold of the moving window flow threshold. In some cases, an error alert is generated if it is determined that the measured flow rate is greater than the upper threshold of the moving window flow threshold by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%. In some cases, an error alert is generated if the measured flow rate is determined to be smaller than the lower limit threshold of the moving window flow rate threshold by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%.

[0052] An error alert may be generated if 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 more, for example, 0.0005 seconds or more, for example, 0.001 seconds or more, for example, 0.005 seconds or more, for example, 0.01 seconds or more, for example, 0.05 seconds or more, for example, 0.1 seconds or more, for example, 0.5 seconds or more, for example, 1 second or more, for example, 2 seconds or more, for example, 3 seconds or more, for example, 4 seconds or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 15 seconds or more, for example, 30 seconds or more, for example, 45 seconds or more, for example, 60 seconds or more. In some cases, if the flow rate exceeds the upper threshold of the moving window flow rate threshold by 0.0001 seconds or more, for example 0.0005 seconds or more, for example 0.001 seconds or more, for example 0.005 seconds or more, for example 0.01 seconds or more, for example 0.05 seconds or more, for example 0.1 seconds or more, for example 0.5 seconds or more, for example 1 second or more, for example 2 seconds or more, for example 3 seconds or more, for example 4 seconds or more, for example 5 seconds or more, for example 10 seconds or more, for example 15 seconds or more, for example 30 seconds or more, for example 45 seconds or more, for example 60 seconds or more, an error alert will be generated. In some cases, if the flow rate is less than the lower limit threshold of the moving window flow rate threshold for 0.0001 seconds or more, for example 0.0005 seconds or more, for example 0.001 seconds or more, for example 0.005 seconds or more, for example 0.01 seconds or more, for example 0.05 seconds or more, for example 0.1 seconds or more, for example 0.5 seconds or more, for example 1 second or more, for example 2 seconds or more, for example 3 seconds or more, for example 4 seconds or more, for example 5 seconds or more, for example 10 seconds or more, for example 15 seconds or more, for example 30 seconds or more, for example 45 seconds or more, for example 60 seconds or more, an error alert will be generated.

[0053] In some embodiments, the generated error alert indicates a malfunction in the flow cytometer. In some cases, the malfunction is a blockage in the flow stream. In some cases, the malfunction is the ingress of gas (e.g., air) into the flow stream or sample line. In some cases, the error alert is generated in real time. In some cases, the cause of the malfunction is determined based on the generated error alert. In some cases, the cause of the malfunction is determined by analyzing the flow pattern based on the measured flow rate. In some cases, the flow rate increases and then decreases to one or more of the absolute flow rate threshold and the moving window flow rate threshold, and it is determined that gas (e.g., bubbles) has ingressed into the flow stream. For example, the flow rate may increase by 0.1%, e.g., 0.5%, e.g., 1%, e.g., 2%, e.g., 3%, e.g., 4%, e.g., 5%, e.g., 10%, or more, and then decrease to one or more of the absolute flow rate threshold and the moving window flow rate threshold. In some cases, the flow rate increases above one or more upper thresholds of the absolute flow threshold and the moving window flow threshold, and then decreases below the lower threshold of the absolute flow threshold or the moving window flow threshold, indicating that gas has entered the flowstream. In some cases, a blockage in the flowstream is determined when the flow rate of the flowstream decreases below one or more of the absolute flow threshold and the moving window flow threshold.

[0054] In some embodiments, the method modifies one or more parameters of the flow cytometer in response to a generated error alert. In some cases, the flow rate of the flow stream is adjusted in response to a generated error alert. In some cases, the flow rate is increased in response to a generated error alert, for example, by 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, 25% or more, for example, by 50% or more. In some cases, the flow rate is decreased in response to a generated error alert, for example, by 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, 25% or more, for example, by 50% or more. In some cases, the flow rate is stopped in response to a generated error alert. In some cases, in response to a generated error alert, the flow rate of the flowstream is stopped for a predetermined time interval, for example, 0.1 seconds or more, 0.5 seconds or more, 1 second or more, 2 seconds or more, 3 seconds or more, 4 seconds or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 30 seconds or more, 45 seconds or more, or 60 seconds or more. In some cases, in response to a generated error alert, the experimental or calibration protocol is terminated. In some cases, in response to a generated error alert, the flowstream is released, for example, by releasing the flowstream with a predetermined amount of fluid buffer. In some cases, in response to a generated error alert (for example, if it is determined that there is a blockage or bubbles in the flowstream), the flowstream is released for a predetermined time interval, for example, by releasing the flowstream for 5 seconds or more, for example, 15 seconds or more, for example, 30 seconds or more, for example, 60 seconds or more, for example, 300 seconds or more, or for example, 600 seconds or more.

[0055] As described in more detail below, in some cases, the sample in the flowstream is irradiated using a light source. In some cases, the light source for irradiating the flowstream is adjusted in response to the generated error alert. In some cases, the light source is turned off in response to the generated error alert. In some cases, the light configured to irradiate the flowstream is blocked in response to the generated error alert. In some cases, the light source is adjusted (e.g., blocked or turned off) while the flow rate indicates a generated error, for example, while the flowstream flow rate is unstable, or while the flow rate exceeds one or more of the absolute flow threshold and moving window flow threshold. In some cases, the flow cytometer is set to standby mode without measuring the flow rate in response to the generated error alert. In some cases, the method adjusts one or more parameters of the photodetector system in response to the generated error alert. In some cases, one or more photodetector channels are turned off in response to the generated error alert. In some cases, light to one or more photodetectors of the photodetector system is blocked.

[0056] Figure 1A is a flowchart illustrating the process of monitoring the flow rate of a flow stream in a flow cytometer according to one embodiment. In step 101, the flow rate of the flow stream is measured. In some cases, the flow rate is measured continuously. In other cases, the flow rate is measured at discrete intervals. The flow rate may be measured using a flow sensor, such as a flow sensor that determines the flow rate based on the temperature of the flow stream passing through the sensor. In step 102a, the measured flow rate of the flow stream is compared to an absolute flow rate threshold. The absolute flow rate threshold may have an upper threshold and a lower threshold, and the measured flow rates are compared to determine whether the flow rate at any given time is greater than the upper threshold of the absolute flow rate threshold or less than the lower threshold of the absolute flow rate threshold. The measured flow rate is further compared (in step 102b) to a moving window flow rate threshold, for example, by comparing the flow rate to the threshold within a moving window time interval. For example, the moving window flow rate threshold may be the upper and lower limits of the flow rate over the past second. The moving window flow threshold may have an upper and lower threshold, for example, by comparing the measured flow rates during a time interval (e.g., the past second) to determine whether the flow rate during this time window is greater than the upper threshold of the moving window flow threshold or less than the lower threshold of the moving window flow threshold.

[0057] If the measured flow rate exceeds one or more upper thresholds of the absolute flow rate threshold or the moving window flow rate threshold, or falls below one or more lower thresholds of the absolute flow rate threshold or the moving window flow rate threshold, an error alert is generated in step 103. The error alert may indicate a malfunction in the flow cytometer, for example, a blockage or air ingress (e.g., bubbles) in the flow stream. In some cases, the flow rate is analyzed, for example, based on the flow rate pattern, to identify the cause of the generated error alert. In some cases, one or more parameters of the flow cytometer are adjusted (in step 104) in response to the generated error alert. In some cases, the flow rate is adjusted in step 105a, and the flow rate adjustment may include flushing the flow stream (e.g., with a fluid buffer) in step 106a, completely stopping the flow stream in step 106b, or increasing or decreasing the flow rate of the flow stream in step 106c. In some embodiments, one or more parameters of the photodetection system may be adjusted in step 105b in response to the generated error alert. In some cases, when adjusting the photodetector system, the gain of the photodetector, the signal amplitude of the photodetector, one or more photodetectors of the photodetector system, or one or more photodetector channels may be turned off. In some embodiments, the light source of the flow cytometer may be adjusted by, for example, turning off or blocking the light source, as shown in step 105c.

[0058] Figure 1B is a diagram showing the results of monitoring the flow rate of a flow stream in a flow cytometer according to one embodiment. The screenshot shows, for example, a situation where air enters the sample line of the flow stream when the sample source is empty. The code currently divides the monitoring phase into four different steps or states. The code defines the acquisition-related fluid modes that the algorithm monitors. 1) When the algorithm executed by the firmware starts, the algorithm completes the standby step and moves to the fluid control system start step (at 0 seconds). Next, the algorithm moves to the waiting step and waits for 10 seconds. In some cases, the nominal value acquisition step may be a further waiting step (e.g., a dummy step) that waits for 3 seconds or more. At this stage, additional checks of the flow stream (e.g., flow rate stability) can be completed before moving to the monitoring step. If immediate execution is desired, the further waiting step is omitted. A recording mode may be performed before the monitoring or standby step. The recording mode is a mode that records the flow rate from the start of acquisition until a specified time. In recording mode, any errors or changes in the fluid control system are ignored, and only flow rate data for observation is recorded.

[0059] During flowstream monitoring (at 13 seconds in Figure 1B), two different checks are completed: absolute flow rate and relative flow rate. If the average flow buffer value exceeds the absolute flow rate threshold (greater than or less than the absolute flow rate threshold), a general fluid control system error may be reported. In low mode, due to the long pattern development waiting time, a general fluid control system error is reported if the relative flow rate check fails. If the relative flow rate check fails in non-low mode, the algorithm generates an alert and records the timestamp of the relative check failure, the maximum and minimum flow rates (at 22 seconds in Figure 1B). Once this alert is generated, the algorithm performs the pattern check function over another time interval (e.g., 15 seconds). 1) If the flow rate exceeds the flow rate before the relative check failure (maximum value of the relative check error buffer), it indicates that air is present in the sample line. An air inclusion (e.g., bubbles) error alert may be communicated to the user via the user interface (at 32 seconds in Figure 1B). 2) If the flow rate drops to a very low value, or significantly below the moving window flow rate threshold, it indicates a blockage in the flow stream. A blockage error alert may be communicated to the user via the user interface. 3) If neither Pattern 1 nor Pattern 2 is detected within the pattern check interval, the algorithm may report a general error alert and transition to the error detection function. At this point, an error in the fluid control system is detected, and the defined error is notified to the user. The next function (error detection function) may handle internal state and variable resets, invoke the fluid control system mode to stop the sample, and flush the sample line. In some cases, the state reset may reset the algorithm to standby mode.

[0060] In some embodiments, a sample containing particles is transported through a flow stream by a method. In some cases, the sample is transported into the flow stream after resolving one or more errors in the flow rate of the flow stream. In some cases, the sample is transported while measuring the flow rate of the flow stream, as described above. In some embodiments, the sample in the flow stream is irradiated with light from a light source. In some embodiments, the light source is a broadband light source that emits light having a wide range of wavelengths, for example, 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, for example 500 nm or more. For example, a suitable broadband light source emits light having wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having wavelengths from 400 nm to 1000 nm. When irradiating using a broadband light source in this method, the broadband light source protocol of interest may include, but is not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, superluminescent light-emitting diodes, semiconductor light-emitting diodes, broad-spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof.

[0061] In other embodiments, the method involves irradiating with a narrowband light source that emits light at a specific wavelength or a narrow range of wavelengths, for example, using a light source that emits light at a narrow range of wavelengths such as 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, or 2 nm or less, or using a light source that emits light at a specific wavelength (i.e., monochromatic light). When the method uses a narrowband light source, the narrowband light source protocol of interest includes, 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.

[0062] In one embodiment, the sample is irradiated using one or more lasers in the method. As described above, the type and number of lasers vary depending on the sample and the desired light to be collected, and may 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 cases, the flowstream is irradiated using dye lasers such as stilbene lasers, coumarin lasers, or rhodamine lasers in the method. In other cases, the flowstream is irradiated using 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 yet other cases, the flowstream is irradiated using a solid-state laser such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium-sapphire laser, a thulium-YAG laser, a ytterbium-YAG laser, a Yb2O3 laser, or a cerium-doped laser, or combinations thereof.

[0063] The sample may be irradiated using one or more of the above-described light sources, for example, two or more light sources, for example, three or more light sources, for example, four or more light sources, for example, five or more light sources, for example, ten or more light sources. The light sources may include any combination of light source types. For example, in some embodiments, the method uses a laser array to irradiate the sample in the flow stream using an array having, for example, one or more gas lasers, one or more dye lasers and one or more solid-state lasers.

[0064] The sample may be irradiated using wavelengths within the range of 200nm to 1500nm, for example, 250nm to 1250nm, for example, 300nm to 1000nm, for example, 350nm to 900nm, or for example, 400nm to 800nm. For example, if the light source is a broadband light source, the sample may be irradiated using wavelengths of 200nm to 900nm. In other cases, if the light source has multiple narrowband light sources, the sample may be irradiated using specific wavelengths within the range of 200nm to 900nm. For example, the light source may be multiple narrowband LEDs (1nm to 25nm) each independently emitting light with wavelengths within the range of 200nm to 900nm. In other embodiments, the narrowband light source includes one or more lasers (e.g., a laser array), and the sample is irradiated with specific wavelengths within the range of 200nm to 700nm using a laser array having, for example, a gas laser, an excimer laser, a dye laser, a metal vapor laser, and a solid-state laser as described above.

[0065] When more than one light source is used, the sample may be irradiated simultaneously, sequentially, or in combination thereof using the light sources. For example, the sample may be irradiated simultaneously using each of the light sources. In other embodiments, the flow stream is irradiated sequentially using each of the light sources. When more than one light source is used to irradiate the sample sequentially, the time each light source spends irradiating the sample may be independently 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 30 microseconds or more, e.g., 60 microseconds or more. For example, the method may irradiate the sample with a light source (e.g., a laser) for a duration within the range of 0.001 microseconds to 100 microseconds, e.g., 0.01 microseconds to 75 microseconds, e.g., 0.1 microseconds to 50 microseconds, e.g., 1 microsecond to 25 microseconds, e.g., 5 microseconds to 10 microseconds. In an embodiment in which a sample is sequentially irradiated using two or more light sources, the duration of irradiation of the sample by each light source may be the same or different.

[0066] The time between irradiations from each light source may further vary as desired, and may be independently separated by delays of 0.001 microseconds or more, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 15 microseconds or more, for example, 30 microseconds or more, for example, 60 microseconds or more. For example, the time between irradiations from each light source may be in the range of 0.001 microseconds to 60 microseconds, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microseconds to 25 microseconds, for example, 5 microseconds to 10 microseconds. In one embodiment, the time between irradiations from each light source is 10 microseconds. In embodiments in which the sample is sequentially irradiated by more than two (i.e., three or more) light sources, the delays between irradiations from each light source may be the same or different.

[0067] The sample may be irradiated continuously or at discrete intervals. In some cases, the method involves irradiating the sample continuously using a light source. In other cases, the sample is irradiated at discrete intervals using a light source, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 1000 milliseconds, every 1000 milliseconds, or at other intervals.

[0068] Depending on the light source, the sample may be irradiated 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, or 50 mm or more. Furthermore, the angle or irradiation may vary within the range of 10° to 90°, for example 15° to 85°, for example 20° to 80°, for example 25° to 75°, for example 30° to 60°, or for example, an angle of 90°.

[0069] In one embodiment, the method irradiates the sample with two or more beams of frequency-shifted light. As described above, a light beam generator having a laser and an acousto-optic device for frequency shifting of the laser light may be used. In these embodiments, the method irradiates the acousto-optic device using a laser. Depending on the desired wavelength of the light produced by the output laser beam (for use when irradiating a sample in a flow stream, for example), the laser may have a specific wavelength in the range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. The acousto-optic device may be irradiated using one or more lasers, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers. The lasers may include any combination of laser types. For example, in some embodiments, the method irradiates the acousto-optic device using an array of lasers, e.g., an array having one or more gas lasers, one or more dye lasers and one or more solid-state lasers.

[0070] When more than one laser is used, the lasers may be used to irradiate the acousto-optic device simultaneously, sequentially, or in a combination thereof. For example, the acousto-optic device may be irradiated simultaneously using each of the lasers. In other embodiments, the acousto-optic device is irradiated sequentially using each of the lasers. When more than one laser is used to sequentially irradiate the acousto-optic device, the time each laser spends irradiating the acousto-optic device may be independently 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 30 microseconds or more, e.g., 60 microseconds or more. For example, the acousto-optic device may be irradiated using lasers for durations within the range of 0.001 microseconds to 100 microseconds, e.g., 0.01 microseconds to 75 microseconds, e.g., 0.1 microseconds to 50 microseconds, e.g., 1 microsecond to 25 microseconds, e.g., 5 microseconds to 10 microseconds. In an embodiment in which an acousto-optical device is sequentially irradiated using two or more lasers, the duration for which the acousto-optical device is irradiated by each laser may be the same or different.

[0071] The time between irradiations by each laser may further vary as desired, and may be independently separated by delays of 0.001 microseconds or more, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 15 microseconds or more, for example, 30 microseconds or more, for example, 60 microseconds or more. For example, the time between irradiations by each light source may be in the range of 0.001 microseconds to 60 microseconds, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microseconds to 25 microseconds, for example, 5 microseconds to 10 microseconds. In one embodiment, the time between irradiations by each laser is 10 microseconds. In embodiments in which an acousto-optical device is sequentially irradiated by more than two (i.e., three or more) lasers, the delays between irradiations by each laser may be the same or different.

[0072] Acousto-optical devices may be irradiated continuously or at discrete intervals. In some cases, a laser is used to continuously irradiate the acousto-optical device. In other cases, a laser is used to irradiate the acousto-optical device at discrete intervals, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, for example, every 1000 milliseconds, or at other intervals.

[0073] Depending on the laser, the acoustic-optical device may be irradiated 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, or 50 mm or more. Furthermore, the angle or irradiation may vary within the range of 10° to 90°, for example 15° to 85°, for example 20° to 80°, for example 25° to 75°, for example 30° to 60°, and may be, for example, 90°.

[0074] In some embodiments, the method involves applying a high-frequency drive signal to an acoustic-optical device to generate an angularly deflected laser beam. Two or more high-frequency drive signals, for example, three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals, for example, one hundred or more high-frequency drive signals may be applied to the acoustic-optical device to generate an output laser beam containing a desired number of angularly deflected laser beams.

[0075] The angle-deflected laser beams generated by the high-frequency drive signals each have an intensity based on the amplitude of the applied high-frequency drive signal. In some embodiments, the method applies a high-frequency drive signal having an amplitude sufficient to generate an angle-deflected laser beam of a desired intensity. In some cases, each applied high-frequency drive signal independently has an amplitude within the range of about 0.001 V to about 500 V, e.g., about 0.005 V to about 400 V, e.g., about 0.01 V to about 300 V, e.g., about 0.05 V to about 200 V, e.g., about 0.1 V to about 100 V, e.g., about 0.5 V to about 75 V, e.g., about 1 V to about 50 V, e.g., about 2 V to about 40 V, e.g., about 3 V to about 30 V, e.g., about 5 V to about 25 V. In some cases, each applied high-frequency drive signal independently has an amplitude within the range of approximately 0.001V to 100V, for example, approximately 0.001V to 200V, for example, 0.001V to 300V, for example, 0.001V to 400V, for example, 0.001V to 500V. In some embodiments, each applied high-frequency drive signal has a frequency within the range of approximately 0.001MHz to approximately 500MHz, for example, approximately 0.005MHz to approximately 400MHz, for example, approximately 0.01MHz to approximately 300MHz, for example, approximately 0.05MHz to approximately 200MHz, for example, approximately 0.1MHz to approximately 100MHz, for example, approximately 0.5MHz to approximately 90MHz, for example, approximately 1MHz to approximately 75MHz, for example, approximately 2MHz to approximately 70MHz, for example, approximately 3MHz to approximately 65MHz, for example, approximately 4MHz to approximately 60MHz, for example, approximately 5MHz to approximately 50MHz. Each applied high-frequency drive signal has a frequency of approximately 0.001 MHz to approximately 100 MHz, for example 0.001 MHz to 200 MHz, for example 0.001 MHz to 300 MHz, for example 0.001 MHz to 400 MHz, for example 0.001 MHz to 500 MHz, in some embodiments.

[0076] In these embodiments, the angle-deflected laser beams of the output laser beam are spatially separated. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams may be separated by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, e.g., 50. In some embodiments, the angle-deflected laser beams overlap, for example, adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap of adjacent angle-deflected laser beams (e.g., beam spot overlap) may be 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.

[0077] In some cases, multiple beams of frequency-shifted light are irradiated onto the flow stream, as described in Diebold, et al. Nature Photonics Vol.7(10);806-810(2013) and U.S. Patent Nos. 9423353, 9784661, 9983132, 10006852, 10036699, 10078045, 10222316, 10288546, 10324019, 10408758, 10451538, 10620111, 10684211, 10845295, 10935482, and 10935485. Image particles in a flow stream as described in U.S. Patent No. 1,1105728, U.S. Patent No. 1,1280718, U.S. Patent No. 1,1327016, U.S. Patent No. 1,1366052, U.S. Patent No. 1,1371937, U.S. Patent No. 1,1692926, U.S. Patent No. 1,1630053, U.S. Patent No. 1,1774343, U.S. Patent No. 1,1940369, and U.S. Patent No. 1,1946851 (their disclosures are incorporated herein by reference).

[0078] When implementing the method of the subject matter, the light from each particle is detected by a light detection system. In an embodiment, the light detection system has one or more light detectors, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more light detectors. The light detectors for implementing the method of the subject matter can be any convenient light detection protocol among light sensors or light detectors, such as avalanche photodetectors (APDs), active pixel sensors (APSs), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), ICCDs (intensified charge-coupled devices), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof, but are not limited thereto. In an embodiment, the light detector is a photomultiplier tube, for example, 0.01 cm 2 ~10 cm 2 、 for example, 0.05 cm 2 ~9 cm 2 、 for example, 0.1 cm 2 ~8 cm 2 、 for example, 0.5 cm 2 ~7 cm 2 、 for example, 1 cm 2 ~5 cm 2 and is a photomultiplier tube having an active detection surface area in each region within the range. The light from the irradiated sample is detected by two or more light detector channels, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more, for example, twelve or more, for example, sixteen or more, for example, twenty-four or more, for example, twenty-four or more, for example, thirty-two or more, for example, sixty-four or more, for example, one hundred and twenty-eight or more, for example, two hundred and fifty-six or more, for example, five hundred and twelve or more light detector channels.

[0079] Light may be measured by a photodetector at one or more wavelengths, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, or light from particles in a flowstream may be measured at four hundred or more different wavelengths. Light may be measured continuously or at discrete intervals. In some cases, the detector of interest is configured to measure light continuously. In other cases, the detector of interest is configured to measure at discrete intervals, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, for example, every 1000 milliseconds or at other intervals.

[0080] In some embodiments, the light detected from a sample is scattered light. In this specification, the term “scattered light” is used in its conventional sense to refer to the propagation of light energy from particles in a sample that have been deflected from the incident beam path (e.g., flowing through a flowstream) due to reflection, refraction, or deflection of the light beam. In some cases, the scattered light detected from particles in a flowstream is forward scattered light (FSC). In other cases, the scattered light detected from particles in a flowstream is side scattered light (SSC). In yet another case, the scattered light detected from particles in a flowstream is back scattered light (BSC).

[0081] In some embodiments, the light detected from each particle in the sample is transmitted light, such as light detected by a bright-field photodetector. In other embodiments, the light detected from each particle in the sample is synchrotron radiation, such as particle emission (i.e., fluorescence or phosphorescence). In these embodiments, each particle may contain one or more fluorophores that fluoresce in response to irradiation by two or more light sources. For example, each particle may contain two or more fluorophores, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more fluorophores. In some cases, each particle may contain a first fluorophore that fluoresces in response to irradiation by a first laser and a second fluorophore that fluoresces in response to irradiation by a second laser. In some embodiments, the fluorophores of interest may include, but are not limited to, dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.), such as acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, and dyes formed by combining two or more of the aforementioned dyes (e.g., tandem dyes), polymer dyes having one or more monomer dye units, and mixtures of two or more of the aforementioned dyes. Numerous dyes are commercially available from various sources, such as Molecular Probes (Eugene, OR), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, MO), Sirigen, Inc. (Santa Barbara, CA), and Exciton (Dayton, OH). For example, fluorophores include 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine, and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, peridinin-chlorophyll protein, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; brilliant yellow; coumarin, and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanine, and derivatives such as cyanosine, Cy3, Cy3.5, Cy5, Cy5.5, Cy7; 4'6-diamidino-2-phenylindole (DAPI); 5' ,5”-Dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; Diethylaminocoumarin; Diethylenetriaminepentaacetate; 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride D(DNS, dancylkloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin, and derivatives such as eosin, eosin isothiocyanate; erythrosine, and derivatives such as erythrosine B, erythrosine isothiocyanate; ethidium; fluorescein, and 5-carboxyfluorescein (FAM), 5-(4,Derivatives such as 6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, QFITC ​​(XRITC); fluoresamine; IR144; IR1446; green fluorescent protein (GFP); coral reef fluorescent protein (RCFP); lysamin (trademark); lysaminlow Damin, Lucifer Yellow; Malachite Green Isothiocyanate; 4-Methylumbelliferone; Orthocresolphthalein; Nitrotyrosine; Pararose Aniline; Nile Red; Oregon Green; Phenol Red; β-Phycoerythrin; o-Phthaldaldehyde; Pyrene, and derivatives such as pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate; Reactive Red 4 (Sibacron® Brilliant Red 3B-A); Rhodamine, and 6-C Ruboxy-X-Rhodamine (ROX), 6-Carboxyrhodamine (R6G), 4,7-Dichlororhodamine Lysamine, Rhodamine B Sulfonyl Chloride, Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X Isothiocyanate, Sulforhodamine B, Sulforhodamine 101, Sulfonyl Chloride Derivative of Sulforhodamine 101 (Texas Red), N,N,N',N'-Tetramethyl-6-Carboxyrhodamine (TAMRA), Tetramethylrhodamine The materials may include pigment-conjugated polymers (i.e., pigments bonded to polymers) such as mine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosolic acid and terbium chelate derivatives, xanthene, fluorescein isothiocyanate dextran, and pigments formed by combining two or more pigments (e.g., tandem pigments), polymer pigments having one or more monomer pigment units, and mixtures of two or more of the aforementioned pigments, or combinations thereof.

[0082] In some cases, the fluorophore (i.e., dye) is a fluorescent polymer dye. The fluorescent polymer dyes used in the methods and systems of the subject matter are diverse. In some examples of the methods, the polymer dyes include conjugated polymers. Conjugated polymers (CPs) are characterized by a delocalized electronic structure in which unsaturated bonds (e.g., double and / or triple bonds) and saturated bonds (e.g., single bonds) alternate in a backbone, allowing π electrons to move from one bond to the other. Thus, the conjugated backbone can confer an extended linear structure to the polymer dye, with the bond angles between the polymer repeating units restricted. For example, proteins and nucleic acids are similarly macromolecules, but in some cases they do not form an extended rod structure, but rather fold into a higher-order three-dimensional shape. In addition, CPs may form a “rigid rod” polymer backbone, exhibiting restricted twist (e.g., torsion) angles between monomer repeating units along the polymer backbone chain. In some cases, the polymer dyes include CPs having a rigid rod structure. As summarized above, the structural properties of polymer dyes can affect the fluorescence properties of the molecules.

[0083] Any readily available polymer dye may be used in the subject method and system. In some cases, the polymer dye is a polyphosphoric system having a structure that can collect light to amplify the fluorescence output of a fluorophore. In some cases, the polymer dye can collect light and efficiently convert that light into longer wavelength synchrotron radiation. In some cases, the polymer dye has a light-gathering polyphosphoric system that can efficiently transfer energy to a nearby light-emitting species (e.g., a "signaling chromophore"). Mechanisms of energy transfer include, for example, resonance energy transfer (e.g., Förster (or fluorescence) resonance energy transfer (FRET)) and quantum charge exchange (Dexter energy transfer). In some cases, these energy transfer mechanisms are relatively short-range, i.e., efficient energy transfer is possible because the light-gathering polyphosphoric system is in close proximity to the signaling chromophore. Under conditions for efficient energy transfer, a large number of individual chromophores in a light-collecting polyphosphoric system amplifies the emission from the signaling chromophore. That is, the emission from the signaling chromophore is stronger when the incident light (excitation light) is at a wavelength absorbed by the light-collecting polyphosphoric system than when the signaling chromophore is directly excited by the pump light.

[0084] The polyphosphoric phosphodiester may be a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is substantially shorter than the polymer chain length, the backbone contains numerous conjugated segments in close proximity. Therefore, conjugated polymers are efficient at focusing light and allow for light amplification by energy transfer.

[0085] In some cases, polymers may be used directly as fluorescent reporters, for example, fluorescent polymers with high absorption coefficients and high brightness. In some cases, polymers may be used as strong chromophores, and their color or optical density may be used as an indicator.

[0086] The polymer dyes of interest include U.S. Patent Application Publication No. 2004 / 0142344, U.S. Patent Application Publication No. 2008 / 0293164, U.S. Patent Application Publication No. 2008 / 0064042, U.S. Patent Application Publication No. 2010 / 0136702, U.S. Patent Application Publication No. 2011 / 0256549, U.S. Patent Application Publication No. 2012 / 0028828, U.S. Patent Application Publication No. 2012 / 0252986, U.S. Patent Application Publication No. 2013 / 0190193, and U.S. Patent Application Publication No. 2016 / 0025735, whose entire disclosure is incorporated herein by reference, as well as Gaylord et al., J. Am. Chem. Soc., 2001, 123 (26), pp. This includes, but is not limited to, the dyes described in 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133 (32), pp 12600-12607.

[0087] In some cases, the sample is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungus, or subset of tissue, cells, or components of an animal, which may, in some cases, be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, or semen. Thus, “biological sample” refers to both a natural organism or a subset of its tissues, and homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, airways, gastrointestinal tract, cardiovascular, urogenital tract, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be tissue from any type of organism, including both healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, or semen, and in some cases, the sample is a blood sample containing whole blood, such as blood obtained by venipuncture or fingertip puncture (the blood may or may not be combined with any reagents such as preservatives and anticoagulants before the assay).

[0088] In some embodiments, the sample source is “mammal” or “mammal,” and these terms are used broadly to describe organisms belonging to 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 cases, the subject is human. The method may be applied to samples obtained from human subjects of both sexes and at any developmental stage (i.e., neonates, infants, young children, adolescents, and adults), and in some embodiments, the human subject is young children, adolescents, or adults. While this disclosure may be applied to samples from human subjects, it should be understood that the method may be further carried out on samples from other animal subjects (i.e., “non-human subjects”), including but not limited to birds, mice, rats, dogs, cats, livestock, and horses.

[0089] The cells of interest may be characterized according to various parameters, such as phenotypic characteristics identified by attaching a specific fluorescent label to the cells of interest. In some embodiments, the system is configured to deflect analysis droplets determined to contain target cells. Various cells may be characterized using the method of the subject. 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 erythrocytes. Target cells of interest include cells having a convenient cell surface marker or antigen that may be taken up by a convenient affinity agent or its conjugate, or to which a label may be affixed. For example, target cells may contain 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, disialoganglioside 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+) from whole blood, bone marrow, or umbilical cord blood.

[0090] When carrying out the subject method according to one embodiment, a certain amount of initial fluid sample is injected into a flow cytometer. The amount of sample injected into the particle sorting module may vary, for example, within the range of 0.001 mL to 1000 mL, for example 0.005 mL to 900 mL, for example 0.01 mL to 800 mL, for example 0.05 mL to 700 mL, for example 0.1 mL to 600 mL, for example 0.5 mL to 500 mL, for example 1 mL to 400 mL, for example 2 mL to 300 mL, for example 5 mL to 100 mL.

[0091] In some embodiments, the method counts and optionally sorts labeled particles (e.g., target cells) in the sample. When performing the method of the subject, first, a fluid sample containing particles is introduced into the system's flow nozzle. As the particles exit the flow nozzle, each particle passes through the sample investigation area, which is irradiated with a light source, substantially one at a time, and the results of light scattering parameter measurements, and optionally fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements), are recorded separately for each particle as desired. Depending on the characteristics of the flowstream being investigated, the light may be irradiated to a flowstream of 0.001 mm or more, for example, 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more. In some embodiments, the method irradiates a planar cross section of the flowstream within the sample investigation area with, for example, a laser (as described above). In other embodiments, the method involves irradiating a flow stream of a predetermined length within the sample investigation area to correspond to the irradiation profile of a diffuse laser beam or lamp.

[0092] In one embodiment, the flowstream is irradiated at or near the nozzle orifice of the flow cell by the method. For example, the flowstream may be irradiated at a position of about 0.001 mm or more from the nozzle orifice, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more from the nozzle orifice by the method. In one embodiment, the flowstream is irradiated right next to the nozzle orifice of the flow cell by the method.

[0093] In embodiments of the method, a detector such as a photomultiplier tube (PMT) is used to record the light passing through each particle (sometimes referred to as forward scattered light), the light reflected perpendicular to the direction of the particle flow passing through the detection area (sometimes referred to as orthogonal scattered light or side scattered light), and, if the particles are labeled with one or more fluorescent markers, the fluorescence emitted from the particles when they pass through the detection area and are irradiated by an energy source is recorded. Each of the forward scattered light (FSC), side scattered light (SSC), and fluorescence emission has separate parameters for each particle (or "event"). Thus, for example, two, three, or four parameters may be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle can be analyzed in real time as desired or stored in data storage and analysis means such as a computer.

[0094] In one embodiment, particles are detected and specifically identified by exposing them to excitation light as desired and measuring the fluorescence of each particle in one or more detection channels. The fluorescence emitted by the detection channels used to identify the particles and their associated binding complexes may be measured after excitation by a single light source, or individually after excitation by different light sources. When particle labels are excited using separate excitation light sources, the particle labels may be selected so that all particle labels can be excited by each of the excitation light sources used.

[0095] One embodiment of the method further includes, for example, computer-aided data acquisition, analysis, and recording, where multiple data channels record data from each detector regarding the light scattering and fluorescence emitted by each particle as it passes through the sample examination area of ​​the particle sorting module. In these embodiments, during analysis, particles are classified and counted such that each particle exists as a set of digitized parameter values. The system of the subject may be configured to trigger on selected parameters to distinguish the particle of interest from background and noise. "Trigger" refers to a preset threshold for parameter detection, which may be used as a means to detect when a particle has passed through a light source. When an event exceeding the threshold of the selected parameter is detected, the collection of light scattering and fluorescence data for the particle is triggered. Data for particles or other components in the analytical medium that cause a reaction below the threshold is not acquired. The trigger parameter may be the detection of forward scattered light produced when a particle passes through a light beam. In this way, the flow cytometer detects and collects light scattering and fluorescence data for the particle.

[0096] Subsequently, a specific subpopulation of interest is further analyzed by "gating" based on data collected for the entire population. The data is plotted to separate the subpopulation as best as possible in order to select the appropriate gate. This procedure may be performed by plotting forward scattered light (FSC) versus side-scattered (i.e., orthogonal) scattered light (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles (i.e., cells within the gate) is selected, and particles not within the gate are excluded. If desired, the gate may be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Only those particles within the gate are then further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to calculate the number of particles of interest in the sample.

[0097] In the methods of interest, particles may also be used in research, clinical trials, or treatment. In some embodiments, the methods of the subject matter obtain individual cells prepared from biological samples of a target fluid or tissue. For example, the methods of the subject matter obtain cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods of the subject matter obtain cells from fluid or tissue samples used for therapeutic purposes. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissues, is prepared and introduced into a subject as a therapeutic procedure. Conditions that can be treated by administration of flow cytometry-selected samples include, but are not limited to, blood disorders, immune system disorders, and organ damage.

[0098] A typical cell therapy protocol may include steps such as sample collection, cell isolation, genetic modification, culture, in vitro growth, cell harvesting, sample volume reduction, sample washing, in vivo preservation, storage, and introduction of cells into the subject. The protocol may be initiated by collecting viable cells and tissues from the subject's tissue source to produce a cell and / or tissue sample. Samples may be collected by any appropriate procedure, such as administering a cell recruiter to the subject, drawing blood from the subject, or removing bone marrow from the subject. After sample collection, cell enrichment may be performed by several methods, such as centrifugation-based methods, filter-based methods, elutriation, magnetic separation, or fluorescence-activated cell sorting (FACS). In some cases, the enriched cells may be genetically modified by any convenient method, such as nuclease-mediated gene editing. Genetically modified cells can be cultured, activated, and grown in vitro. In some cases, the cells may be preserved, for example, cryopreserved, and stored for future use. When ready for use, the cells may be thawed and then administered to the patient, for example, by injecting the cells into the patient.

[0099] system Aspects of the present disclosure further include, for example, a system for carrying out a method of the subject for monitoring the flow rate of a flow stream (such as in a flow cytometer). A system according to one embodiment comprises a processor to which a memory storing instructions is operablely coupled, the memory including instructions for measuring the flow rate of a flow stream, instructions for comparing the measured flow rate of the flow stream with an absolute flow threshold and a moving window flow threshold, and instructions for generating an error alert if the measured flow rate of the flow stream exceeds the absolute flow threshold or the moving window flow threshold.

[0100] The flow sensor for measuring the flow rate of the flowstream may be any simple protocol, and in some cases, the flow sensor is configured to measure the flow rate based on the measured temperature of the flowstream. In some cases, the flow sensor is configured to measure the flow rate based on the measured viscosity of the flowstream. In some cases, the flow sensor is configured to measure the flow rate at one or more detection positions along the flowstream, for example, 2 or more, for example, 3 or more, for example, 4 or more, for example, 5 or more, for example, 6 or more, for example, 7 or more, for example, 8 or more, for example, 9 or more detection positions, and for example, it is configured to measure at 10 or more detection positions along the flowstream. In some cases, the detection positions for measuring the flow rate of the flowstream extend to 0.001 mm or more of the flowstream, 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 for example, 10 mm or more of the flowstream. In some cases, the flow sensor is configured to measure the flow rate of a flowstream at two or more detection positions in the flowstream based on temperature changes in the flowstream, 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 positions, for example, or to measure at ten or more different detection positions along the flowstream based on temperature changes in the flowstream. In some cases, the flow sensor is configured to measure the flow rate of a flowstream at two or more different detection positions in the flowstream based on viscosity changes in the flowstream, 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 different detection positions, for example, or to measure at ten or more detection positions along the flowstream based on viscosity changes in the flowstream.

[0101] In some cases, the flow sensor is a temperature sensor flow meter. In some cases, the flow sensor has a controllable heating element located in the central part of a pressure-stabilizing membrane, with temperature sensors mounted upstream and downstream (e.g. symmetrically) with respect to the direction of fluid flow by the flow stream. In some cases, heat is transferred to the downstream temperature sensor as the flow passes through, and the downstream temperature sensor generates a measurable signal due to the resulting temperature difference. In some cases, a microthermal flow sensor is integrated into a silicon chip flow meter type sensor. In some cases, the flow sensor has a temperature sensor that generates a signal that compensates for the effects of temperature. In some cases, the flow sensor has a fluid flow sensor such as the commercially available flow sensor product line from Sensiron (Steffa, Switzerland). In some cases, there are flow sensors such as those described in U.S. Patent Application Publication No. 2023 / 0324270, U.S. Patent No. 10942139, European Patent No. 3187881, and European Patent No. 3404373, whose disclosures are incorporated herein by reference.

[0102] In the embodiment, the system comprises a processor to which memory containing instructions is operablely coupled, and the memory contains instructions for measuring the flow rate of a flow stream. In some cases, the memory contains instructions for continuously measuring the flow rate, for example by monitoring the flow rate in real time. In some cases, the memory includes instructions for measuring the flow rate at discrete intervals, for example, every 0.00001 seconds or more, every 0.00005 seconds or more, every 0.0001 seconds or more, every 0.0005 seconds or more, every 0.001 seconds or more, every 0.005 seconds or more, every 0.01 seconds or more, every 0.05 seconds or more, every 0.1 seconds or more, every 0.5 seconds or more, every 1 second or more, every 2 seconds or more, every 3 seconds or more, every 4 seconds or more, every 5 seconds or more, every 6 seconds or more, every 7 seconds or more, every 8 seconds or more, every 9 seconds or more, every 10 seconds or more, every 15 seconds or more, every 30 seconds or more, every 60 seconds or more.

[0103] In some embodiments, the flow rate of the flow stream may be 1 μL / min or more, for example 2 μL / min or more, for example 3 μL / min or more, for example 5 μL / min or more, for example 10 μL / min or more, for example 15 μL / min or more, for example 25 μL / min or more, for example 50 μL / min or more, for example 100 μL / min or more, and in some cases, the flow rate of the flow stream may be 1 μL / second or more, for example 2 μL / second or more, for example 3 μL / second or more, for example 5 μL / second or more, for example 10 μL / second or more, for example 15 μL / second or more, for example 25 μL / second or more, for example 50 μL / second or more, for example 100 μL / second or more. In one embodiment, the flow rate of the flow stream may be 25 μL / sec or more, for example 50 μL / sec or more, for example 75 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, for example 1000 μL / sec or more, for example 2500 μL / sec or more.

[0104] In some embodiments, the flowstream includes a sheath fluid flowstream and a sample core fluid flowstream. In some embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid, for example, together with the sample fluid, into the internal chamber of the flow cell, thereby generating a layered flowstream of sheath fluid surrounding the sample core fluid flowstream. In some cases, the sheath fluid flowstream forms a laminar flow stream surrounding the sample core fluid flowstream. In some cases, the memory includes commands for measuring the flow rate of the sheath fluid flowstream. In some cases, the flow rate of the sheath fluid flowstream is 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, e.g., 2500 μL / sec or more. In some cases, the memory includes commands for measuring the flow rate of the sample core fluid flowstream. In some cases, the flow rate of the sample core fluid flow stream is 1 μL / min or more, e.g., 2 μL / min or more, e.g., 3 μL / min or more, e.g., 5 μL / min or more, e.g., 10 μL / min or more, e.g., 15 μL / min or more, e.g., 25 μL / min or more, e.g., 50 μL / min or more, e.g., 100 μL / min or more, and in some cases, the flow rate of the sample delivered to the flow cell chamber by the sample injection port is 1 μL / second or more, e.g., 2 μL / second or more, e.g., 3 μL / second or more, e.g., 5 μL / second or more, e.g., 10 μL / second or more, e.g., 15 μL / second or more, e.g., 25 μL / second or more, e.g., 50 μL / second or more, e.g., 100 μL / second or more. In some cases, the memory includes commands for measuring the flow rate of the sheath fluid flow stream and the flow rate of the sample core fluid flow stream.

[0105] In some cases, the memory includes instructions for comparing the flow rate of the sheath fluid flow stream with the flow rate of the sample core fluid flow stream. In some cases, the memory includes instructions for determining that the flow rate of the sheath fluid flow stream is the same as the flow rate of the sample core fluid flow stream. In some cases, the memory includes instructions for determining that the flow rate of the sheath fluid flow stream is 0.01% or more, e.g., 0.05% or more, e.g., 0.1% or more, e.g., 0.5% or more, e.g., 1% or more, e.g., 2% or more, e.g., 3% or more, e.g., 4% or more, e.g., 5% or more, e.g., 10% or more, e.g., 15% or more, e.g., 20% or more, e.g., 25% or more, e.g., 50% or more less than the flow rate of the sample core fluid flow stream. In some cases, the memory includes instructions for determining that the flow rate of the sheath fluid flow stream is 0.01% or more, e.g., 0.05% or more, e.g., 0.1% or more, e.g., 0.5% or more, e.g., 1% or more, e.g., 2% or more, e.g., 3% or more, e.g., 4% or more, e.g., 5% or more, e.g., 10% or more, e.g., 15% or more, e.g., 20% or more, e.g., 25% or more, e.g., 50% or more than the flow rate of the sample core fluid flow stream. If it is determined that the relative flow rates of the sheath fluid flow stream and the sample core fluid flow stream exceed a predetermined threshold, the memory includes instructions for adjusting one or more of the flow rates of the sheath fluid flow stream and the sample core fluid flow stream. In some cases, the memory includes instructions for adjusting the flow rates of one or more of the sheath fluid flow stream and the sample core fluid flow stream if the relative flow rates of the sheath fluid flow stream and the sample core fluid flow stream exceed a threshold of 1%, for example, 2%, for example, 3%, for example, 4%, for example, 5%, for example, 10%, for example, 15%, for example, 20%, for example, 25%, or for example, 50%.

[0106] In one embodiment, the memory includes an instruction to begin measuring the flow rate of the flow stream when a stable flow rate is detected. In some cases, the memory includes an instruction to measure the flow rate when the flow rate has a fluctuation rate of 5% or less, e.g., 4% or less, e.g., 3% or less, e.g., 2% or less, e.g., 1% or less, e.g., 0.5% or less, e.g., 0.1% or less, e.g., 0.05% or less, e.g., 0.01% or less (e.g., showing a change that can be measured immediately), e.g., when the flow rate shows a change of 0.0001% or less. When the flow rate shows a fluctuation that exceeds a stabilization threshold, the system may be set to standby mode until the fluctuation falls below the stabilization threshold for a predetermined duration, e.g., 1 second or more, e.g., 5 seconds or more, e.g., 10 seconds or more, e.g., 15 seconds or more, e.g., 30 seconds or more, e.g., 60 seconds or more. When it is determined that the flow stream is sufficiently stable for monitoring, the memory includes an instruction to move to a monitoring phase for measuring the flow rate. In some embodiments, if the flow rate exhibits instability or fluctuations exceeding a stabilization threshold during the monitoring phase, the system may be configured to re-enter standby mode and suspend the monitoring phase until the flow rate of the flowstream exhibits sufficient stability.

[0107] In some embodiments, the absolute flow rate has an upper and lower threshold. In some cases, the memory includes instructions for continuously measuring the absolute flow rate. In some cases, the memory includes instructions for continuously comparing the measured flow rate of the flow stream with the upper absolute flow rate threshold. In some cases, the memory includes instructions for generating an error alert if it is determined that the measured flow rate is greater than the upper absolute flow rate threshold by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%. In some cases, the memory includes instructions for generating an error alert if it is determined that the measured flow rate is less than a lower absolute flow rate threshold by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%. When continuously measuring the flow rate of a flow stream, the memory includes instructions for generating an error alert if 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 more, e.g., 0.0005 seconds or more, e.g., 0.001 seconds or more, e.g., 0.005 seconds or more, e.g., 0.01 seconds or more, e.g., 0.05 seconds or more, e.g., 0.1 seconds or more, e.g., 0.5 seconds or more, e.g., 1 second or more, e.g., 2 seconds or more, e.g., 3 seconds or more, e.g., 4 seconds or more, e.g., 5 seconds or more, e.g., 10 seconds or more, e.g., 15 seconds or more, e.g., 30 seconds or more, e.g., 45 seconds or more, e.g., 60 seconds or more.In some cases, the memory includes instructions to generate an error alert if the flow rate exceeds the upper threshold of the absolute flow rate threshold for 0.0001 seconds or more, e.g., 0.0005 seconds or more, e.g., 0.001 seconds or more, e.g., 0.005 seconds or more, e.g., 0.01 seconds or more, e.g., 0.05 seconds or more, e.g., 0.1 seconds or more, e.g., 0.5 seconds or more, e.g., 1 second or more, e.g., 2 seconds or more, e.g., 3 seconds or more, e.g., 4 seconds or more, e.g., 5 seconds or more, e.g., 10 seconds or more, e.g., 15 seconds or more, e.g., 30 seconds or more, e.g., 45 seconds or more, e.g., 60 seconds or more. In some cases, the memory includes instructions to generate an error alert if the flow rate is less than the lower limit threshold of the absolute flow rate threshold for 0.0001 seconds or more, e.g., 0.0005 seconds or more, e.g., 0.001 seconds or more, e.g., 0.005 seconds or more, e.g., 0.01 seconds or more, e.g., 0.05 seconds or more, e.g., 0.1 seconds or more, e.g., 0.5 seconds or more, e.g., 1 second or more, e.g., 2 seconds or more, e.g., 3 seconds or more, e.g., 4 seconds or more, e.g., 5 seconds or more, e.g., 10 seconds or more, e.g., 15 seconds or more, e.g., 30 seconds or more, e.g., 45 seconds or more, e.g., 60 seconds or more.

[0108] In some cases, the memory includes instructions for comparing the measured flow rate of a flow stream with an absolute flow threshold at discrete intervals, for example, every 0.00001 seconds or more, every 0.00005 seconds or more, every 0.0001 seconds or more, every 0.0005 seconds or more, every 0.001 seconds or more, every 0.005 seconds or more, every 0.01 seconds or more, every 0.05 seconds or more, every 0.1 seconds or more, every 0.5 seconds or more, every 1 second or more, every 2 seconds or more, every 3 seconds or more, every 4 seconds or more, every 5 seconds or more, every 6 seconds or more, every 7 seconds or more, every 8 seconds or more, every 9 seconds or more, every 10 seconds or more, every 15 seconds or more, every 30 seconds or more, every 60 seconds or more. In some cases, the memory includes instructions for generating an error alert if the measured flow rate exceeds the absolute flow threshold at each discrete comparison interval. In other cases, the memory includes instructions for generating an error alert when the measured flow rate exceeds the absolute flow rate threshold, or when the measured flow rate exceeds the absolute flow rate threshold at discrete comparison intervals greater than 1, 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 discrete comparison intervals.

[0109] In the embodiment, the memory includes instructions for comparing the measured flow rate of the flowstream with a moving window flow rate threshold. The predetermined time interval (i.e., duration) for each moving window may be the moving average flow rate of the flowstream over a specific time interval, e.g., 0.1 seconds or more, e.g., 0.5 seconds or more, e.g., 1 second or more, e.g., 2 seconds or more, e.g., 3 seconds or more, e.g., 4 seconds or more, e.g., 5 seconds or more, e.g., 10 seconds or more. In some cases, the moving window flow rate is a snapshot of the measured flow rate of the flowstream over a specific duration, e.g., 0.1 seconds or more, e.g., 0.5 seconds or more, e.g., 1 second or more, e.g., 2 seconds or more, e.g., 3 seconds or more, e.g., 4 seconds or more, e.g., 5 seconds or more, e.g., 10 seconds or more. In the embodiment, the moving window flow rate may be the flow rate during a time interval of 0.1 seconds to 10 seconds, for example 0.5 seconds to 9.5 seconds, for example 1 second to 9 seconds, for example 1.5 seconds to 8.5 seconds, for example 2 seconds to 8 seconds, for example 2.5 seconds to 7.5 seconds, for example 3 seconds to 7 seconds. In some cases, the memory includes instructions for comparing the measured flow rate with a moving window flow rate threshold one or more times, for example two or more times, for example three or more times, for example four or more times, for example five or more times, for example ten or more times, for example 25 or more times, for example 50 or more times, for example 100 or more times, for example 250 or more times.

[0110] In some embodiments, each moving window flow threshold has an upper and lower threshold. In some cases, the memory includes instructions for comparing the measured flow rate of the flow stream with the upper threshold of the moving window flow threshold. In some cases, the memory includes instructions for generating an error alert if it is determined that the measured flow rate is greater than the upper threshold of the moving window flow threshold by, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50%. In some cases, the memory includes instructions for generating an error alert if it is determined that the measured flow rate is less than, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50% less than the lower limit threshold of the moving window flow rate threshold.

[0111] In some embodiments, the memory includes instructions for generating an error alert if 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 more, for example, 0.0005 seconds or more, for example, 0.001 seconds or more, for example, 0.005 seconds or more, for example, 0.01 seconds or more, for example, 0.05 seconds or more, for example, 0.1 seconds or more, for example, 0.5 seconds or more, for example, 1 second or more, for example, 2 seconds or more, for example, 3 seconds or more, for example, 4 seconds or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 15 seconds or more, for example, 30 seconds or more, for example, 45 seconds or more, for example, 60 seconds or more. In some cases, the memory includes instructions to generate an error alert if the flow rate exceeds the upper threshold of the moving window flow rate threshold for 0.0001 seconds or more, e.g., 0.0005 seconds or more, e.g., 0.001 seconds or more, e.g., 0.005 seconds or more, e.g., 0.01 seconds or more, e.g., 0.05 seconds or more, e.g., 0.1 seconds or more, e.g., 0.5 seconds or more, e.g., 1 second or more, e.g., 2 seconds or more, e.g., 3 seconds or more, e.g., 4 seconds or more, e.g., 5 seconds or more, e.g., 10 seconds or more, e.g., 15 seconds or more, e.g., 30 seconds or more, e.g., 45 seconds or more, e.g., 60 seconds or more. In some cases, the memory includes instructions to generate an error alert if the flow rate is less than the lower limit threshold of the moving window flow rate threshold for 0.0001 seconds or more, e.g., 0.0005 seconds or more, e.g., 0.001 seconds or more, e.g., 0.005 seconds or more, e.g., 0.01 seconds or more, e.g., 0.05 seconds or more, e.g., 0.1 seconds or more, e.g., 0.5 seconds or more, e.g., 1 second or more, e.g., 2 seconds or more, e.g., 3 seconds or more, e.g., 4 seconds or more, e.g., 5 seconds or more, e.g., 10 seconds or more, e.g., 15 seconds or more, e.g., 30 seconds or more, e.g., 45 seconds or more, e.g., 60 seconds or more.

[0112] In some embodiments, the generated error alert indicates a malfunction in the flow cytometer. In some cases, the malfunction is a blockage in the flow stream. In some cases, the malfunction is the ingress of gas (e.g., air) into the flow stream or sample line. In some cases, the memory includes instructions for generating an error alert in real time. In some cases, the memory includes instructions for determining the cause of the malfunction based on the generated error alert. In some cases, the memory includes instructions for determining the cause of the malfunction by analyzing the flow pattern based on the measured flow rate. In some cases, the flow rate increases and then decreases to one or more of the absolute flow rate threshold and the moving window flow rate threshold, and the memory includes instructions for determining that gas (e.g., bubbles) has ingressed into the flow stream. For example, the flow rate may increase by 0.1% or more, e.g., 0.5% or more, e.g., 1% or more, e.g., 2% or more, e.g., 3% or more, e.g., 4% or more, e.g., 5% or more, e.g., 10% or more, and then decrease to one or more of the absolute flow rate threshold and the moving window flow rate threshold. In some cases, if the flow rate increases above one or more upper thresholds of the absolute flow threshold and the moving window flow threshold, and then decreases below the lower threshold of the absolute flow threshold or the moving window flow threshold, the memory includes an instruction to determine that gas has entered the flow stream. In some cases, if the flow rate of the flow stream decreases below one or more of the absolute flow threshold and the moving window flow threshold, the memory includes an instruction to determine that there is a blockage in the flow stream.

[0113] In some embodiments, the memory includes instructions for changing one or more parameters of the flow cytometer in response to a generated error alert. In some cases, the memory includes instructions for adjusting the flow rate of the flow stream in response to a generated error alert. In some cases, the memory includes instructions for increasing the flow rate in response to a generated error alert, for example, by increasing the flow rate by 0.1% or more, e.g., 0.5% or more, e.g., 1% or more, e.g., 2% or more, e.g., 3% or more, e.g., 4% or more, e.g., 5% or more, e.g., 10% or more, e.g., 25% or more, e.g., by increasing the flow rate by 50% or more. In some cases, the memory includes instructions for decreasing the flow rate by 0.1% or more, e.g., 0.5% or more, e.g., 1% or more, e.g., 2% or more, e.g., 3% or more, e.g., 4% or more, e.g., 5% or more, e.g., 10% or more, e.g., 25% or more, e.g., by decreasing the flow rate by 50% or more. In some cases, the memory includes instructions for stopping the flow rate in response to a generated error alert. In some cases, in response to a generated error alert, the flow rate of the flowstream is stopped for a predetermined time interval, for example, 0.1 seconds or more, 0.5 seconds or more, 1 second or more, 2 seconds or more, 3 seconds or more, 4 seconds or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 30 seconds or more, 45 seconds or more, or 60 seconds or more. In some cases, the memory includes a command to abort the experiment or calibration protocol in response to a generated error alert. In some cases, the memory includes a command to flush the flowstream in response to a generated error alert, for example, by flushing the flowstream with a predetermined amount of fluid buffer. In some cases, the memory includes a command to flush the flowstream for a predetermined time interval in response to a generated error alert (for example, if it is determined that there is a blockage or bubbles in the flowstream), for example, by flushing the flowstream for 5 seconds or more, for example, 15 seconds or more, for example, 30 seconds or more, for example, 60 seconds or more, for example, 300 seconds or more, or for example, 600 seconds or more.

[0114] As described in more detail below, the system includes a light source for illuminating the flow stream. In some cases, the memory includes commands to adjust one or more parameters of the light source in response to generated error alerts. In some cases, the memory includes commands to turn off the light source in response to generated error alerts. In some cases, the memory includes commands to shut off the light source in response to generated error alerts. In some cases, the memory includes commands to adjust the light source while the flow rate indicates a generated error, for example, while the flow rate of the flow stream is unstable, or while the flow rate exceeds one or more of the absolute flow threshold and moving window flow thresholds. In some cases, the memory includes commands to stop measuring the flow rate in response to a generated error alert and set the flow cytometer to standby mode. In some cases, the memory includes commands to adjust one or more parameters of the photodetector system in response to generated error alerts. In some cases, the memory includes commands to turn off one or more photodetector channels in response to generated error alerts. In some cases, the memory includes commands to cut off light to one or more photodetectors of the photodetector system.

[0115] A system according to one embodiment includes a light source configured to irradiate a flow stream. In an embodiment, the light source may be any suitable broadband or narrowband light source. The light source may be configured to emit light of various wavelengths within the ranges of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. For example, the light source may have a broadband light source that emits light having wavelengths in the range of 200 nm to 900 nm. In other cases, the light source may have a narrowband light source that emits wavelengths in the range of 200 nm to 900 nm. For example, the light source may be a narrowband LED (1 nm to 25 nm) that emits light having wavelengths in the range of 200 nm to 900 nm. In one embodiment, the light source is a laser. In some cases, the system of the subject comprises a gas laser, such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, or xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the system of the subject comprises a dye laser, such as a stilbene laser, coumarin laser, or rhodamine laser. In yet other cases, the laser of interest includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In other cases, the subject system comprises solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, thulium-YAG lasers, ytterbium-YAG lasers, Yb2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0116] In other embodiments, the light source is a non-laser light source and includes, but is not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, light-emitting diodes, such as broadband LEDs with a continuous spectrum, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, and multi-LED integrated lamps. In some cases, the non-laser light source is a stabilized fiber-coupled broadband light source, a white light source, or any combination thereof.

[0117] The light source may be placed at any suitable distance from the flowstream, for example, at a distance of 0.001 mm or more, for example 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 5 mm or more, for example 10 mm or more, for example 25 mm or more, for example 100 mm or more. In addition, the light source illuminates the flowstream at any suitable angle (for example, with respect to the vertical axis of the flowstream), for example, at an angle within the range of 10° to 90°, for example 15° to 85°, for example 20° to 80°, for example 25° to 75°, for example 30° to 60°, for example 90°.

[0118] The light source may be configured to irradiate the sample continuously or at discrete intervals. In some cases, the system includes a light source configured to irradiate the sample continuously, for example, a continuous-wave laser that continuously irradiates the flow stream at the investigation point of a flow cytometer. In other cases, the system of interest includes a light source configured to irradiate the sample at discrete intervals, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, for example, every 1000 milliseconds, or other intervals. When the light source is configured to irradiate the sample at discrete intervals, the system may include one or more additional components for intermittently irradiating the sample using the light source. For example, the system of the subject in these embodiments may include one or more laser beam choppers, manual or computer-controlled beam stops for blocking and exposing the sample to the light source.

[0119] In some embodiments, the light source is a laser. The laser of interest may include pulsed lasers or continuous-wave lasers. For example, the laser may be a gas laser, e.g., helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser or xenon-fluorine (XeF) excimer laser or a combination thereof; a dye laser, e.g., stilbene laser, coumarin laser or rhodamine laser; a metal vapor laser, e.g., helium-cadmium (HeCd) laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser, copper laser or gold laser or a combination thereof; a solid-state laser, e.g., ruby ​​laser, Nd:YAG laser, NdCrYAG Lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, thulium-YAG lasers, ytterbium-YAG lasers, Yb2O3 lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, photo-excited semiconductor lasers (OPSLs), or lasers with the frequency of any of the above lasers doubled or tripled.

[0120] In one embodiment, the light source is a light beam generator configured to generate two or more beams of frequency-shifted light. In some cases, the light beam generator has a laser, a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optical device to generate two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous-wave laser. For example, the lasers of the light beam generator of interest include 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; dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers; metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; and solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers. Lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, or combinations thereof.

[0121] The acousto-optic device may be any simple acousto-optic protocol configured to frequency-shift laser light using applied sound waves. In one embodiment, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), arbitrary waveform generator (AWG), or electrical pulse generator.

[0122] In this embodiment, the controller is configured to apply high-frequency drive signals to an acoustic-optical device to generate a desired number of angularly deflected laser beams of the output laser beam, and is configured to apply, for example, 3 or more high-frequency drive signals, 4 or more high-frequency drive signals, 5 or more high-frequency drive signals, 6 or more high-frequency drive signals, 7 or more high-frequency drive signals, 8 or more high-frequency drive signals, 9 or more high-frequency drive signals, 10 or more high-frequency drive signals, 15 or more high-frequency drive signals, 25 or more high-frequency drive signals, 50 or more high-frequency drive signals, and is configured to apply, for example, 100 or more high-frequency drive signals.

[0123] In some cases, to generate an intensity profile of the angularly deflected laser beam of the output laser beam, the controller is configured to apply a high-frequency drive signal having various amplitudes within the following ranges: for example, approximately 0.001 V to approximately 500 V, approximately 0.005 V to approximately 400 V, for example, approximately 0.01 V to approximately 300 V, for example, approximately 0.05 V to approximately 200 V, for example, approximately 0.1 V to approximately 100 V, for example, approximately 0.5 V to approximately 75 V, for example, approximately 1 V to approximately 50 V, for example, approximately 2 V to approximately 40 V, for example, approximately 3 V to approximately 30 V, for example, approximately 5 V to approximately 25 V. In some embodiments, the applied high-frequency drive signal has frequencies within the ranges of approximately 0.001 MHz to approximately 500 MHz, for example, approximately 0.005 MHz to approximately 400 MHz, for example, approximately 0.01 MHz to approximately 300 MHz, for example, approximately 0.05 MHz to approximately 200 MHz, for example, approximately 0.1 MHz to approximately 100 MHz, for example, approximately 0.5 MHz to approximately 90 MHz, for example, approximately 1 MHz to approximately 75 MHz, for example, approximately 2 MHz to approximately 70 MHz, for example, approximately 3 MHz to approximately 65 MHz, for example, approximately 4 MHz to approximately 60 MHz, for example, approximately 5 MHz to approximately 50 MHz.

[0124] In one embodiment, the controller has a processor to which the memory is operablely connected, such that the memory stores instructions, when executed by the processor, for generating an output laser beam including an angle-deflected laser beam having a desired intensity profile. For example, the memory may have instructions for generating two or more angle-deflected laser beams of the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., different intensities, e.g., the memory may have instructions for generating 100 or more angle-deflected laser beams of different intensities.

[0125] In one embodiment, the controller has a processor to which the memory is operablely connected, such that the memory stores instructions for the processor to generate an output laser beam whose intensity increases from the center to the edge of the output laser beam along the horizontal axis when executed by the processor. In these cases, the intensity of the angular deflection laser beam at the center of the output beam may be in the range of 0.1% to about 99% of the intensity of the angular deflection laser beam at the edge of the output laser beam along the horizontal axis, for example, in the range of about 0.5% to about 95%, for example, about 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%, or for example, in the range of about 10% to about 50% of the intensity of the angular deflection laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor to which the memory is operablely connected, such that the memory stores instructions for the processor to generate an output laser beam whose intensity increases from the edge to the center of the output laser beam along the horizontal axis when executed by the processor. In these cases, the intensity of the angular deflection laser beam at the edge of the output beam may be in the range of 0.1% to about 99% of the intensity of the angular deflection laser beam at the center of the output laser beam along the horizontal axis, for example, in the range of about 0.5% to about 95%, for example, about 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%, or for example, in the range of about 10% to about 50% of the intensity of the angular deflection laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller has a processor to which the memory is operablely coupled, such that the memory stores instructions for the processor to generate an output laser beam having a Gaussian intensity profile along the horizontal axis when executed by the processor.In yet another embodiment, the controller has a processor to which the memory is operablely coupled, such that the memory stores instructions for the processor to generate an output laser beam having a top-hat intensity profile along the horizontal axis when executed by the processor.

[0126] In some embodiments, the light beam generator of interest may be configured to generate spatially separated angular deflection laser beams of the output laser beam. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angular deflection laser beams may be separated by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, e.g., 1000 μm or more, e.g., 5000 μm or more, e.g., 5000 μm or more. In some embodiments, the system is configured to generate an angular deflection laser beam of the output laser beam that overlaps, for example, adjacent angular deflection laser beams along the horizontal axis of the output laser beam. The overlap of adjacent angle-deflected laser beams (e.g., beam spot overlap) may be 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.

[0127] In some cases, a light beam generator configured to produce two or more beams of frequency-shifted light is described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9423353, 9784661, 9983132, 10006852, 10036699, 10078045, 10222316, 10288546, 10324019, 10408758, 10451538, 10620111, 10684211, 10845295, 10935482, and 10935485. The laser excitation module is as described in U.S. Patent No. 11105728, U.S. Patent No. 11280718, U.S. Patent No. 11327016, U.S. Patent No. 11366052, U.S. Patent No. 11371937, U.S. Patent No. 11692926, U.S. Patent No. 11630053, U.S. Patent No. 11774343, U.S. Patent No. 11940369, and U.S. Patent No. 11,946,851 (their disclosures are incorporated herein by reference).

[0128] In some embodiments, the system comprises a photodetector having a photodetector configured to detect light. In some embodiments, the photodetector is configured to detect scattered light. In some cases, the photodetector has a side-scatter light detector. In some cases, the photodetector has a forward-scatter light detector. In other embodiments, the photodetector has a plurality of scattered light detectors, for example, two or more, for example, three or more, for example, four or more, for example, five or more scattered light detectors. In some embodiments, the photodetector system of the subject further comprises a fluorescence detector configured to detect light of one or more fluorescence wavelengths. In other embodiments, the photodetector has a plurality of fluorescence detectors, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, ten or more, for example, fifteen or more, for example, twenty or more fluorescence detectors.

[0129] The detectors of interest may include, but are not limited to, light sensors or photodetectors, such as avalanche photodiodes (APDs), active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), ICCDs (intensified charge-coupled devices), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof. In some embodiments, the collected light is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), avalanche photodiode (APD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, for example, 0.01 cm². 2 ~10cm 2 For example, 0.05 cm 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm2 For example, 1cm 2 ~5cm 2 This is a photomultiplier tube having an active detection surface area in each region within the specified range.

[0130] When the system of the subject comprises multiple fluorescence detectors, each fluorescence detector may be identical, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, when the system of the subject comprises two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD device and the second fluorescence detector (or image sensor) is a CMOS device. In other embodiments, both the first and second fluorescence detectors are CCD devices. In yet another embodiment, both the first and second fluorescence detectors are CMOS devices. In yet another embodiment, the first fluorescence detector is a CCD device and the second fluorescence detector is a photomultiplier tube (PMT). In yet another embodiment, the first fluorescence detector is a CMOS device and the second fluorescence detector is a photomultiplier tube. In yet another embodiment, both the first and second fluorescence detectors are photomultiplier tubes.

[0131] In embodiments of this disclosure, the fluorescence detector of interest is configured to measure collected light at one or more wavelengths, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, 25 or more different wavelengths, for example, 50 or more different wavelengths, for example, 100 or more different wavelengths, for example, 200 or more different wavelengths, for example, 300 or more different wavelengths, and is configured to measure light emitted from a sample in a flow stream at, for example, 400 or more different wavelengths. In some embodiments, two or more detectors of a module as described herein are configured to measure collected light at the same or overlapping wavelengths.

[0132] In some embodiments, the fluorescence detector of interest is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In some embodiments, the detector of interest is configured to collect the spectrum of light over a range of wavelengths. For example, a flow cytometer may include one or more detectors configured to collect the spectrum of light over one or more wavelengths within the 200 nm to 1000 nm range. In yet another embodiment, the detector of interest is configured to measure light emitted from a sample in a flow stream at one or more specific wavelengths. For example, the module may have one or more detectors configured to measure light at one or more of the following wavelengths: 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, and 617nm, and any combination thereof. In one embodiment, one or more detectors may be configured to pair with a specific fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0133] The flow cytometer described herein may have any suitable one or more mechanisms for supplying sheath fluid and sample fluid to a sheath fluid input coupler and a sample fluid input coupler. For example, the sample fluid input coupler may be fluidically connected to a sample fluid line (e.g., tubing) that is fluidically connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidically connected to a sheath fluid line that is fluidically connected to a sheath fluid reservoir. Similarly, the flow cytometer may have any suitable one or more mechanisms for managing waste from the flow stream. The fluid output coupler may be fluidically connected to a waste line that is fluidically connected to a waste reservoir. A fluid control system that may be adapted for use in the subject flow cytometer is described in U.S. Patent Application Publication No. 2022 / 0341838, the entire disclosure of which is incorporated herein by reference.

[0134] In some embodiments, the flow cytometer has a flow cell. The flow cell of interest has a cuvette configured to carry particles in a flow stream. As used herein, “flow cell” is used in the conventional sense to refer to an element having a channel for a liquid flow stream to carry particles in a sheath fluid. The cuvette of interest has a passage (i.e., a channel) that penetrates the cuvette. The flow stream in which the channel is formed may contain a liquid sample injected from a sample tube. In some cases, the flow cell has a light-transmitting 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 irradiated with light from a light source at one or more investigation points. As used herein, “investigation point” refers to an area within the flow cell where particles are irradiated with light from a light source for analysis, for example. The size of the investigation points may vary as desired. For example, if 0 μm represents the axis of light emitted by the light source, the investigation points may be within the range of -50 μm to 50 μm, for example, -25 μm to 40 μm, or for example, -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.

[0135] In some embodiments, the flow cell has a sample injection port configured to supply a sample to the flow cell, or is configured to be used in conjunction with such a sample injection port. In embodiments, the sample injection system is configured to supply a suitable flow of the sample to the internal chamber (i.e., the flow path) of the flow cell. Depending on the desired characteristics of the flowstream, the flow rate of the sample delivered to the flow cell chamber by the sample injection port may be 1 μL / min or more, for example 2 μL / min or more, for example 3 μL / min or more, for example 5 μL / min or more, for example 10 μL / min or more, for example 15 μL / min or more, for example 25 μL / min or more, for example 50 μL / min or more, for example 100 μL / min or more, and in some cases, the flow rate of the sample delivered to the flow cell chamber by the sample injection port may be 1 μL / second or more, for example 2 μL / second or more, for example 3 μL / second or more, for example 5 μL / second or more, for example 10 μL / second or more, for example 15 μL / second or more, for example 25 μL / second or more, for example 50 μL / second or more, for example 100 μL / second or more.

[0136] The sample injection port may be an orifice provided in the wall of the internal chamber, or a tube provided at the proximal end of the internal chamber. If the sample injection port is an orifice provided in the wall of the internal chamber, the sample injection port orifice may have any suitable shape, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal shapes, curved cross-sectional shapes such as circular and oval shapes, and irregular shapes such as a parabolic bottom joined to a planar top. In one embodiment, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and may have an opening in the range of 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, for example 1.25 mm to 1.75 mm, for example 1.5 mm.

[0137] In some cases, the sample injection port is a tube located at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a tube positioned such that its orifice is aligned with the orifice of the flow cell. If the sample injection port is a tube positioned so as to be aligned with the orifice of the flow cell, the cross-sectional shape of the sample injection tube may have any suitable shape, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curvilinear cross-sectional shapes such as circles and ovals, and irregular shapes such as a parabolic bottom joined to a planar top. The orifice of the tube may vary depending on its shape and may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may have a bevel angle in the range of 1° to 10°, for example 2° to 9°, for example 3° to 8°, for example 4° to 7°, or a bevel angle of 5°.

[0138] 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, for example, together with the sample, into the internal chamber of the flow cell, thereby creating a layered flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of sheath fluid delivered to the chamber of the flow cell may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, e.g., 2500 μL / sec or more.

[0139] In some embodiments, the sheath fluid injection port is an orifice provided in the wall of the internal chamber. The orifice of the sheath fluid injection port may have any suitable shape, and the cross-sectional shapes of interest include, but are not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ovals, and irregular shapes such as a parabolic bottom joined to a planar top. The size of the orifice of the sheath fluid injection port may vary depending on the shape, and may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm.

[0140] In some embodiments, the system includes a flow cytometer or is operably coupled to a flow cytometer. Appropriate flow cytometry systems are disclosed herein by reference in the following publications: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), FlowCytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt 1):17-28; Linden, et. al., Semin Throm Hemost. 2004 Oct;30(5):502-11; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. Flow cytometry systems described in 24(3):203-255 may be included, but are not limited to, those described.In some cases, the flow cytometry systems of interest include BD Biosciences FACSCanto(trademark) flow cytometer, BD Biosciences FACSCanto(trademark) II flow cytometer, BD Accuri(trademark) flow cytometer, BD Accuri(trademark) C6 Plus flow cytometer, BD Biosciences FACSCelesta(trademark) flow cytometer, BD Biosciences FACSLyric(trademark) flow cytometer, BD Biosciences FACSVerse(trademark) flow cytometer, BD Biosciences FACSymphony(trademark) flow cytometer, BD Biosciences LSRFortessa(trademark) flow cytometer, BD Biosciences LSRFortessa(trademark) X-20 flow cytometer, BD Biosciences FACSPresto(trademark) flow cytometer, BD Biosciences FACSVia(trademark) flow cytometer, and BD Biosciences FACSCalibur(trademark) cell sorter, BD Biosciences FACSCount(trademark) cell sorter, BD Biosciences FACSLyric(trademark) cell sorter, and BD This includes Biosciences Via (trademark) cell sorters, BD Biosciences Influx (trademark) cell sorters, BD Biosciences Jazz (trademark) cell sorters, BD Biosciences Aria (trademark) cell sorters, BD Biosciences FACSAria (trademark) II cell sorters, BD Biosciences FACSAria (trademark) III cell sorters, BD Biosciences FACSAria (trademark) Fusion cell sorters, BD Biosciences FACSMelody (trademark) cell sorters, BD Biosciences FACSymphony (trademark) S6 cell sorters, BD Biosciences FACSDiscover (trademark) cell sorters, and others.

[0141] In some embodiments, the subject system is incorporated herein by reference in its entirety by reference to U.S. Patent Nos. 10663476, 10620111, 10613017, 10605713, 10585031, 10578542, 10578469, 10481074, 10302545, 10145793, 10113967, 10006852, 9952076, 9933341, 9726527, 9453789, 9200334, 9097640, and 9095494. These are flow cytometry systems such as those described in U.S. Patent No. 9092034, U.S. Patent No. 8975595, U.S. Patent No. 8753573, U.S. Patent No. 8233146, U.S. Patent No. 8140300, U.S. Patent No. 7544326, U.S. Patent No. 7201875, U.S. Patent No. 7129505, U.S. Patent No. 6821740, U.S. Patent No. 6813017, U.S. Patent No. 6809804, U.S. Patent No. 6372506, U.S. Patent No. 5700692, U.S. Patent No. 5643796, U.S. Patent No. 5627040, U.S. Patent No. 5620842, U.S. Patent No. 5602039, U.S. Patent No. 4987086, and U.S. Patent No. 4498766.

[0142] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the system described in the subject is as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013), and U.S. Patent No. 9423353, U.S. Patent No. 9784661, and U.S. Patent No. 9983132. U.S. Patent No. 10006852, U.S. Patent No. 10036699, U.S. Patent No. 10078045, U.S. Patent No. 10222316, U.S. Patent No. 10288546, U.S. Patent No. 10324019, U.S. Patent No. 10408758, U.S. Patent No. 10451538, U.S. Patent No. 10620111, U.S. Patent No. 10684211, U.S. Patent No. 10845295, U.S. Patent No. 10935482, U.S. Patent No. 10935485, U.S. Patent No. 11105728, U.S. Patent No. 11280 A flow cytometry system configured to image particles in a flow stream by fluorescence imaging using radio frequency tag emission (FIRE), as described in U.S. Patent No. 718, U.S. Patent No. 11327016, U.S. Patent No. 11366052, U.S. Patent No. 11371937, U.S. Patent No. 11692926, U.S. Patent No. 11630053, U.S. Patent No. 11774343, U.S. Patent No. 11940369, and U.S. Patent No. 11946851 (their disclosures are incorporated herein by reference).

[0143] Figure 2 shows a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 comprises a laser 201 configured to irradiate particles 211 in a flow stream 214 at an investigation point 215 within a flow cell 210. Although one laser is shown in the example in Figure 2, it is understood that multiple lasers may be used. The laser beam from laser 201 is guided to a focusing lens 202, which focuses the laser beam onto the portion of the fluid stream where the particles 211 of the sample in the flow cell 210 are present. The flow cell 210 is part of a fluid control system that guides particles in the stream to the focused laser beam, usually one at a time, for investigation. Alternatively, a nozzle top may be used if the flow cytometer is a stream-in-air cytometer.

[0144] As shown in Figure 2, the flow cell 210 is fluidically connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. The sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a tube (i.e., sheath fluid line) 207. In addition, sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a tube (i.e., sample fluid line) 205. The sample injection port 206 is fluidically connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce particles 211 into the flow cell 210. The particles 211 are hydrodynamically focused via the sheath fluid flowing in from the sheath fluid injection port 208, so that a flowstream 214 is formed downstream of the tapered portion 212 of the flow cell 210. Particles emitted from the distal end of the flow cell 210 may be discarded and / or collected via any appropriate protocol. For example, depending on the type of flow cytometry performed, particles may be collected at the distal end of the flow cell 210, for example, via a waste line. Alternatively, the particles may be sorted.

[0145] Light from one or more laser beams interacts with particles 211 in the sample by diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various different wavelengths depending on the characteristics of the particles, such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles. The fluorescent emission, as well as the diffracted, refracted, reflected, and scattered light, may be sent to one or more detectors. In particular, forward scatter (FSC) is sent to a forward scatter detector 223. The forward scatter detector 223 is positioned slightly axially offset from the direct beam passing through the flow cell 210 and is configured to detect diffracted light, i.e., excitation light that travels mainly forward through or around the particles. The intensity of the light detected by the forward scatter detector 223 depends on the overall size of the particles. The forward scatter detector may have, for example, a photodiode. A scattering bar 222 is positioned between the forward scatter detector 223 and the optical filter 221a. The optical filter 221a may be configured to remove non-FSC light of at least one wavelength, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scatter light detector 223.

[0146] In addition, side-scattered light (SSC) is detected by a side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of the particle 211, which tends to increase as the complexity of the particle structure increases. In the example in Figure 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered 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-scattered light detector 224. Further fluorescence detectors 225a to 225c, each configured to detect fluorescence at different wavelengths, are shown. 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 at least one wavelength of light that does not correspond 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 the second wavelength (or wavelength range) to the fluorescence detector 225b, while allowing light of the third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. The optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to the second wavelength (or wavelength range) from being detected by the fluorescence detector 225b. In addition, the optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to the third wavelength (or wavelength range) from being detected by the fluorescence detector 225c.

[0147] Those skilled in the art will recognize that the flow cytometer according to the embodiments of this disclosure is not limited to the flow cytometer shown in Figure 2, but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations. For example, although three fluorescence detectors are shown for illustrative purposes in the embodiment of Figure 2, it will be understood that any suitable number of fluorescence detectors may be used.

[0148] During operation, the flow cytometer's operation is controlled by the controller / processor 290, and measurement data from the detector is stored in memory 295 and can be processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is connected to the detector to receive output signals from the detector and may be further connected to the flow cytometer's electrical and electromechanical components to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function unit 297 may be further provided in the system. The memory 295, controller / processor 290, and I / O function unit 297 may be provided entirely as an integrated part of the flow cytometer. In such embodiments, a display may further form part of the I / O function unit 297 to present experimental data to the user of the flow cytometer 200. Alternatively, part or all of the memory 295, controller / processor 290, and I / O function unit 297 may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 295 and the controller / processor 290 can communicate with the flow cytometer wirelessly or via a wired connection. Together with the memory 295 and the I / O function unit 297, the controller / processor 290 may be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0149] The various fluorescent molecules in the fluorescent dye panel used in flow cytometry experiments emit light in their respective characteristic wavelength bands. The specific fluorescent labels used in the experiment, and the associated fluorescence emission bands of the fluorescent labels, may be selected to roughly match the filter window of the detector. The I / O function unit 297 may be configured to receive data relating to a flow cytometry experiment having a panel of fluorescent labels and multiple cell populations having multiple markers (each cell population having a subset of multiple markers). The I / O function unit 297 may be further configured to receive biodata assigning one or more markers to one or more cell populations, marker concentration data, emission spectral data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, may be further stored in memory 295. The controller / processor 290 may be configured to evaluate one or more assignments of labels to markers.

[0150] In some embodiments, the subject system is a particle sorting system configured to sort particles using a sealed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed on 28 March 2017 (the disclosure of which is incorporated herein by reference). In some embodiments, particles of a sample (e.g., cells) are sorted using a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed on 23 December 2019 (the disclosure of which is incorporated herein by reference). In some embodiments, the system for sorting components of a sample comprises a particle sorting module having deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed on 28 March 2017 (the disclosure of which is incorporated herein by reference).

[0151] In one embodiment, the system is fluorescence imaging using a high-frequency tag emission image-compatible particle sorter, as shown in Figure 3. The particle sorter 300 includes a light irradiation section 300a containing a light source 301 (e.g., a 488 nm laser), the light source 301 generating an output beam of light 301a, which is split into beams 302a and 302b by a beam splitter 302. The light beam 302a is propagated through an acousto-optical device (e.g., an acousto-optic deflector (AOD)) 303 to generate an output beam 303a having one or more angularly deflected beams of light. In some cases, the output beam 303a generated from the acousto-optical device 303 includes a local oscillation beam and multiple high-frequency comb beams. The light beam 302b is propagated through an acousto-optical device (e.g., an acousto-optic deflector (AOD)) 304 to generate an output beam 304a having one or more angularly deflected beams of light. In some cases, the output beam 304a generated from the acousto-optical device 304 includes a local oscillation beam and multiple high-frequency comb beams. The output beams 303a and 304a generated from the acousto-optical devices 303 and 304, respectively, are combined in a beam combiner 305 to generate an output beam 305a, which is transmitted through an optical component 306 (e.g., an objective lens) to irradiate particles in the flow cell 307. In one embodiment, the acousto-optical device 303 (AOD) splits a single laser beam into an array of beamlets, each having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in the beam combiner 305. In some embodiments, a light irradiation system having a light source and an acoustic-optical device may further include those described in Schraivogel, et al. (“High-speed fluorescence image-enabled cell sorting” Science (2022), 375 (6578): 315-320) and U.S. Patent Application Publication No. 2021 / 0404943 (the disclosure of which is incorporated herein by reference).

[0152] The output beam 305a irradiates the sample particles 308 propagating through the flow cell 307 (for example, together with the sheath fluid 309) in the irradiation area 310. As shown in the irradiation area 310, multiple beams (for example, angle-bending high-frequency shift beams of light shown as dots across the irradiation area 310) overlap with the reference local oscillator beam (shown as a shaded line across the irradiation area 310). The overlapping beams exhibit beat behavior because they have different optical frequencies, with each beamlet having a different frequency f 1-n This transmits sine wave modulation.

[0153] Light from the irradiated sample is transmitted to a photodetector system 300b having multiple photodetectors. The photodetector system 300b includes a forward scatter photodetector 311 for generating a forward scatter image 311a and a side scatter photodetector 312 for generating a side scatter image 312a. The photodetector system 300b further includes a bright-field photodetector 313 for generating an optical loss image 313a. In some embodiments, the forward scatter photodetector 311 and the side scatter photodetector 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 further detected by fluorescence detectors 314-317. In some cases, the photodetectors 314-317 are photomultiplier tubes. Light from the irradiated sample is directed via the beam splitter 320 to the side-scatter detection channel 312 and the fluorescence detection channels 314-317. The photodetection system 300b includes bandpass optical components 321-324 (e.g., dichroic mirrors) for propagating light of predetermined wavelengths to the photodetectors 314-317, respectively. In some cases, optical component 321 is a 534 nm / 40 nm bandpass. In some cases, optical component 322 is a 586 nm / 42 nm bandpass. In some cases, optical component 323 is a 700 nm / 54 nm bandpass. In some cases, optical component 324 is a 783 nm / 56 nm bandpass. The first digit represents the center of the spectral band. The second digit represents the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on both sides from the center of the spectral band, i.e., from 500 nm to 520 nm.

[0154] Data signals generated in response to light detected by the forward scatter detection channel 311, the side scatter detection channel 312, the bright-field detection channel 313, and the fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Based on the data signals generated by processors 350 and 351, images 311a-317a can be generated in each light detection channel. Image-based sorting is performed in response to a sorting signal generated by the sorting trigger 352. The sorting unit 300c has deflection plates 331 for deflecting particles towards the sample container 332 or the waste stream 333. In some cases, the sorting unit 300c is configured to sort particles using a sealed particle sorting module, as described in U.S. Patent Application Publication No. 2017 / 0299493, filed on March 28, 2017 (the disclosure of which is incorporated herein by reference). In one embodiment, the sorting unit 300c has a sorting decision module having a plurality of sorting decision units, as described in U.S. Patent Application Publication No. 2020 / 0256781 (the disclosure of which is incorporated herein by reference).

[0155] In some embodiments, the system is a particle analyzer and can analyze and characterize particles using the particle analysis system 401 (Figure 4), with or without physically separating the particles into a collection container. Figure 4 is a functional block diagram of the particle analysis system for computational sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 includes a fluid control system 402. The fluid control system 402 has a sample tube 405 and a moving fluid column within the sample tube through which sample particles 403 (e.g., cells) move along a common sample path 409, or can be connected to the sample tube 405 and the moving fluid column.

[0156] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. During detection, in some embodiments, it is possible to detect light or one or more other properties of a particle 403 as it passes through the monitoring area 407. Figure 4 shows one detection station 408 with one monitoring area 407. In some embodiments of the particle analysis system 401, multiple detection stations may be provided. Furthermore, some detection stations may monitor two or more areas.

[0157] Each signal is assigned a signal value, generating a data point for each particle. As described above, this data may be referred to as event data. The data points may be multidimensional data points containing the values ​​of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0158] The particle analysis system 401 may further comprise a control system 406. The control system 406 may have one or more processors, amplitude control circuits, and / or frequency control circuits. The illustrated control system may be operably associated with the fluid control system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval, based on the Poisson distribution and number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may further compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0159] Figure 5 is a functional block diagram of an example of a particle analysis control system, including an analysis controller (i.e., processor) 500 for analyzing and displaying biological events. The analysis controller 500 may be configured to perform various processes for controlling the graphical display of biological events.

[0160] The particle analyzer or particle sorting system 502 may be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or particle sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel. The analysis controller 500 may be a processor configured to perform the method of the present invention by, for example, 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 and low-density clusters based on the density threshold, and classifying the analyte data based on the high-density and low-density clusters based on the size-based analyte feature space.

[0161] The analysis controller 500 may be configured to receive bioevent data from a particle analyzer or particle sorting system 502. The bioevent data received from the particle analyzer or particle sorting system 502 may include flow cytometry event data. The analysis controller 500 may be configured to provide a display device 506 with a graphical display including a first plot of bioevent data. The analysis controller 500 may be further configured to render regions of interest as gates around the collection of bioevent data displayed by the display device 506, for example, overlaid on the first plot. In some embodiments, the gates may be logical combinations of one or more illustrated regions of interest drawn on a histogram or bivariate plot of one parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0162] The analysis controller 500 may be further configured to display bio-event data on the display device 506 within the gate differently from other events in the bio-event data outside the gate. For example, the analysis controller 500 may be configured to render the colors of the bio-event data contained within the gate differently from the colors of the bio-event data outside the gate. The display device 506 may be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0163] The analysis controller 500 may be configured to receive gate selection signals from a first input device that identify gates. For example, the first input device may be implemented as a mouse 510. The mouse 510 can initiate gate selection signals to the analysis controller 500 that identify gates to be displayed or manipulated via the display device 506 (for example, by clicking on or inside a desired gate when the cursor is at the desired gate). In some embodiments, the first device may be implemented as a keyboard 508, or as other means for providing input signals to the analysis controller 500, such as a touchscreen, input pen, photodetector, or voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, as shown in Figure 5, the mouse 510 may include a right mouse button and a left mouse button, the right mouse button and the left mouse button may each generate trigger events.

[0164] In response to a trigger event, the analysis controller 500 can provide input for further processing, such as changing how the data is displayed, which portion of the data is actually displayed on the display device 506, and / or selecting a population of interest for particle sorting.

[0165] In some embodiments, the analysis controller 500 may be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 may be further configured to automatically modify the plot visualization to facilitate gate processing. This modification can be made based on a specific distribution of biological event data received by the analysis controller 500.

[0166] The analysis controller 500 may be connected to a storage device 504. The storage device 504 may be configured to receive and store biological event data from the analysis controller 500. The storage device 504 may be further configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 may be further configured by the analysis controller 500 to enable retrieval of biological event data, such as flow cytometry event data.

[0167] The display device 506 may be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that show an overview of the divisions of the plots. The display device 506 may be further configured to change the information displayed in response to input received from the analysis controller 500, in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0168] In some embodiments, the analysis controller 500 can generate a user interface to receive exemplary events for sorting. For example, the user interface may include controls for receiving exemplary events or exemplary images. The exemplary events or images, or exemplary gates, may be provided before the collection of event data for the sample, or based on a first set of events for a portion of the sample.

[0169] Figure 6A is a schematic diagram showing a particle sorting system 600 (e.g., a particle analyzer or particle sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in Figure 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is connected to a fluid tube 601, which may be connected to a nozzle 603, may include a nozzle 603, or may be a nozzle 603. Within the fluid tube 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are arranged in a line and irradiated by an irradiation source 612 (e.g., a laser) across a monitoring area 611 (e.g., where the laser and stream intersect). The vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to split into multiple droplets 610, some of which contain particles 609.

[0170] During operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring area 611. The detection station 614 supplies input to a timing circuit 628, which then supplies input to a flash charge circuit 630. At droplet splitting points indicated by a timed droplet delay (Δt), a flash charge may be applied to the moving fluid column 608 so that the droplet of interest carries charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect them into a collection tube or a container such as a multi-well or microwell sample plate, and the wells or microwells may be associated with the droplet of interest in particular. As shown in Figure 6A, the droplets may be collected in a drain container 638.

[0171] A detection system 616 (e.g., a droplet boundary detector) plays a role in automatically determining the phase of the droplet driving signal as the particle of interest passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679039, the entirety of which is incorporated herein by reference. The detection system 616 enables the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 can supply an amplitude signal 620 and / or a phase signal 618, which are then supplied (via amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet-forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be located within a control system.

[0172] In some embodiments, the sorting electronic equipment (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory configured to store detected events and sorting decision results based on the detected events. The sorting decision results may be included in the event data for the particles. In some embodiments, the detection system 616 and detection station 614 may be implemented as a single detection unit, or they may be communicatively coupled so that event measurements can be collected by either the detection system 616 or the detection station 614 and provided to non-collecting elements.

[0173] Figure 6B is a schematic diagram showing a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in Figure 6B comprises deflection plates 652 and 654. Charge can be applied via stream-charging wires in barbs. This generates a stream of droplets 610 containing particles 609 for analysis. The particles can be irradiated using one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed by sorting electronic equipment (not shown in Figure 6B) or other detection systems. The deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets and guide the droplets toward a target collection container (e.g., one of 672, 674, 676, or 678). As shown in Figure 6B, deflection plates 652 and 654 can be controlled to guide particles toward container 674 along a first path 662 or toward container 678 along a second path 668. If the particles are not of interest (e.g., do not show scattering or irradiation information within a specified sorting range), the deflection plates may allow the particles to continue along the flow path 664. Such uncharged droplets may flow into the waste container via a suction device 670 or the like.

[0174] Sorting electronics may be included to initiate the collection of measurement data, receive fluorescence signals related to particles, and determine how to adjust the deflection plates to sort the particles. An exemplary embodiment of the embodiment shown in Figure 6B is a flow cytometer from the BD FACSAria® line, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0175] Computer control system The system may include a display and an operator input device. The operator input device may be, for example, a keyboard or mouse. The processing module has a processor that can access memory in which instructions for performing steps of the method of the subject are stored. The processing module may also have an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or will be available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs, which may be written in various programming languages ​​such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system generally works with the processor to coordinate and execute the functions of other components of the computer. The operating system further provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all according to known techniques. In some embodiments, the processor has analog electronic equipment that provides feedback control, such as negative feedback control.

[0176] System memory may be any of the various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices may be any of the various known or future devices, including compact disk drives, tape drives, or diskette drives. Such types of memory storage devices generally read from and / or write to program storage media such as compact disks (not shown). Any of these program storage media, or any other program storage media currently in use or to be developed in the future, may be considered computer program products. As is understood, these program storage media generally store computer software programs and / or data. Computer software programs, also called computer control logic, are generally stored in system memory and / or in program storage devices used in conjunction with memory storage devices.

[0177] In some embodiments, a computer program product is described that includes a computer-usable medium in which control logic (a computer software program including program code) is stored. When executed by a 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. Implementations of hardware state machines for performing the functions described herein will be apparent to those skilled in the art.

[0178] Memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic device, optical device, or solid-state storage device (including magnetic disks or optical disks or tapes or RAM or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium that stores the necessary program code. The program may be provided remotely to the processor via a communication channel, or may be pre-recorded in a computer program product such as memory, or on other portable or fixed computer-readable storage media using any of those devices connected to memory. For example, a magnetic disk or optical disk may store a program and may be read by a disk writer / reader. The system of this disclosure further includes, for example, a program in the form of a computer program product, and algorithms for use in carrying out the methods described above. The program of this disclosure may be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as magnetic disks, hard disk storage media and magnetic tapes; optical storage media such as CD-ROMs; electromechanical storage media such as RAM and ROMs; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0179] The processor may also access communication channels to communicate with the user at a remote location. A remote location means that the user does not have direct contact with the system, but rather relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0180] In some embodiments, the system relating 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 another device. The communication interface may be configured for wired or wireless communication, and wireless communication includes, but is not limited to, radio frequency (RF) communication, such as radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).

[0181] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces, e.g., USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, enabling data communication between the system of the subject and other external devices, such as computer terminals (e.g., in a clinic or hospital environment), configured for similar complementary data communication.

[0182] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the system of the subject to communicate with other devices, such as computer terminals and / or networks, communicative mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with it.

[0183] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet Protocol (IP) via a mobile phone 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 via a Wi-Fi hotspot.

[0184] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11, Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer; or a larger device, such as a desktop computer or electrical equipment. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as buttons, a keyboard, a mouse, or a touchscreen.

[0185] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network or server device using one or more of the communication protocols and / or communication mechanisms described above, for example, data stored in the subject system, such as any data storage unit.

[0186] The output controller may include a controller for any of the various 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, this information may generally be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input / 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. Some of these communications may be implemented using a network or other type of remote communication in alternative embodiments. The output manager may further provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone or satellite network, according to known techniques. The presentation of data by the output manager may be carried out according to various known techniques. As some examples, the data may include SQL, HTML or XML documents, email or other files, or other forms of data. The data may include an Internet URL address so that the user can retrieve additional SQL, HTML, XML or other documents or data from a remote source. One or more platforms present within the subject system are typically a class of computers commonly referred to as servers, but may be any type of known or future computer platform. However, the platforms may also be mainframe computers, workstations, or other computer types. The platforms may be connected via any type of known or future cabling, wireless or other communication systems, whether networked or not. The platforms may be located in the same location or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems may be used on any of the computer platforms. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBM i®, Android®, SGI IRIX®, Oracle Solaris®, and others.

[0187] Figure 7 shows a general configuration of an exemplary computing device 700 according to one embodiment. The general configuration of the computing device 700 shown in Figure 7 includes the arrangement of computer hardware and software components. However, it is not necessary to show all of these common conventional elements in order to provide a valid disclosure. As shown, the computing device 700 comprises a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which may communicate with each other via a communication bus. The network interface 720 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via the network. The processing unit 710 may further communicate with a memory 770 and may further provide output information for any display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or program, e.g., FlowJo®) stored as execution instructions in the non-temporary memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 740 may further accept input from any input device 760, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, speech recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0188] Memory 770 may include computer program instructions (grouped as modules or components in some embodiments) that are executed by the processing unit 710 to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. Data may be stored in a data storage device 790. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0189] Non-temporary computer-readable storage medium Aspects of this disclosure further include a non-temporary computer-readable storage medium having instructions for performing, for example, one or more of the methods performed by a computer as described herein, in order to perform the methods of the subject matter. The computer-readable storage medium may be used on one or more computers for the full or partial automation of a system for performing the methods described herein. In some embodiments, the instructions relating to the methods described herein may be coded on a computer-readable medium in the form of a “program,” and the term “computer-readable medium” as used herein refers to any non-temporary storage medium involved in providing instructions and data to a computer for execution and processing. Suitable examples of non-temporary storage mediums include magnetic disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray® disks, solid-state disks, and network-attached storage devices (NAS), whether such devices are located inside or outside a computer. A file containing information may be “stored” on a computer-readable medium, and “stored” means recording the information so that it can be accessed and retrieved at a later date by a computer. The computer implementations described herein may be executed using programs that can be written in 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.

[0190] In some embodiments, the non-temporary 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 threshold and a moving window flow threshold, and an algorithm for generating an error alert if the measured flow rate of the flow stream exceeds the absolute flow threshold or the moving window flow threshold.

[0191] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for continuously measuring the flow rate of a flow stream. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring the flow rate of a flow stream at discrete intervals, such as measuring the flow rate of a flow stream at intermittent intervals of a predetermined duration.

[0192] In some cases, the absolute flow rate has upper and lower thresholds. In some cases, the non-temporary computer-readable storage medium includes an algorithm for continuously comparing the measured flow rate of the flow stream with the upper and lower absolute flow rate thresholds. In some cases, the non-temporary computer-readable storage medium includes an algorithm for generating an error alert if the measured flow rate is determined to be less than the lower absolute flow rate threshold. In some cases, the non-temporary computer-readable storage medium includes an algorithm for generating an error alert if the measured flow rate is determined to be greater than the upper absolute flow rate threshold. In some cases, the non-temporary computer-readable storage medium includes an algorithm for generating an error alert if the measured flow rate is determined to be 1% or more greater than the upper absolute flow rate threshold, e.g., 3% or more, or 1% or more less than the lower absolute flow rate threshold, e.g., 3% or more.

[0193] In some embodiments, the moving window flow threshold has an upper threshold and a lower threshold. In some cases, the non-temporary computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow threshold at discrete intervals. In some cases, the non-temporary computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow threshold by determining whether the measured flow rate is greater than the upper moving window flow threshold during a given time interval. In some cases, the non-temporary computer-readable storage medium includes an algorithm for comparing the measured flow rate with each moving window flow threshold by determining whether the measured flow rate is less than the lower moving window flow threshold during a given time interval. In some cases, the time interval of the moving window for comparing the measured flow rate with the flow threshold is in the range of 0.1 seconds to 10 seconds, for example, 1 second to 5 seconds. In some cases, the time interval of the moving window is 1 second.

[0194] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for generating error alerts indicating a malfunction in the flow cytometer. In some cases, the malfunction is a blockage in the flow stream. In some cases, the malfunction is the ingress of gas (e.g., air) into the flow stream or sample line. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating error alerts in real time. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating an error alert if the measured flow rate continues to exceed an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration, e.g., 0.001 seconds or longer, e.g., 0.01 seconds or longer, e.g., 0.1 seconds or longer, e.g., 5 seconds or longer.

[0195] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for modifying one or more parameters of the flow cytometer in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for adjusting the flow rate of the flow stream in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for increasing the flow rate in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for decreasing the flow rate in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for stopping the flow rate in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for adjusting the light source for illuminating the flow stream in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for turning off the light source in response to generated error alerts. In some cases, the non-transient computer-readable storage medium includes an algorithm for blocking light configured to illuminate the flow stream in response to generated error alerts.

[0196] In some embodiments, the flowstream includes a sheath fluid flowstream and a sample core fluid flowstream. In some cases, a non-temporary computer-readable storage medium includes an algorithm for measuring the flow rate of the sheath fluid flowstream. In some cases, a non-temporary computer-readable storage medium includes an algorithm for measuring the flow rate of the sample core fluid flowstream. In some cases, a non-temporary computer-readable storage medium includes an algorithm for measuring the flow rates of the sheath fluid flowstream and the sample core fluid flowstream. When a sample containing particles is carried in the sample core fluid flowstream, the non-temporary computer-readable storage medium may include an algorithm for irradiating the sample with a light source and an algorithm for detecting light from the irradiated particles in the flowstream.

[0197] Non-temporary computer-readable storage media may be used in one or more computer systems having a display and operator input devices. Operator input devices may be, for example, a keyboard, a mouse, etc. A processing module has a processor that has access to memory in which instructions for performing steps of the method of the subject are stored. The processing module may have an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or one of other processors that are available or will become available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs, which may be written in various programming languages ​​such as those described above, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system generally works with the processor to coordinate and execute the functions of other components of the computer. The operating system further provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all according to known techniques.

[0198] kit Aspects of the present disclosure further include a kit, which comprises a storage medium, such as a magneto-optical disk, CD-ROM, CD-R, magnetic tape, non-volatile memory card, ROM, DVD-ROM, Blu-ray® disk, solid-state disk, and network-attached storage (NAS). Any of these programmable storage media, or other programmable storage media currently in use or to be developed in the future, may be included in the subject kit. In embodiments, the programmable storage medium includes instructions for monitoring the flow rate of a flow stream, as in a method, and instructions for use in the system described herein. In embodiments, instructions or a portion thereof contained in a computer-readable medium provided in the subject kit may be implemented as software components of software for analyzing data. In these embodiments, the computer control system according to the present disclosure may function as a software "plug-in" to an existing software package (e.g., FlowJo®).

[0199] In addition to the components described above, the subject kit may further include (in some embodiments) instructions for use. These instructions may be present in the subject kit in various forms, and one or more of these instructions may be present in the kit. One form in which these instructions may be provided is information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, the kit packaging, or accompanying documents. Yet another form of these instructions may be a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), or portable flash drive. Yet another form of these instructions may be a website address, which may be used via the Internet to access the information remotely.

[0200] usefulness The subject method, system, and computer system are used in a variety of applications where it is desirable to calibrate or optimize the analysis of particles in a flow stream. In some cases, monitoring the flow rate of the flow stream is used to maintain consistency and accuracy of the analytical data acquired by the subject system to minimize variability between samples and to prevent system errors during analysis. In some cases, the subject method optimizes the analysis of particles in a flow stream using a photodetector system (e.g., having a photodetector) within a particle analyzer. The subject method and system are further used in photodetector systems used to analyze and sort particulate components in samples in a fluid medium, such as biological samples. The disclosure is further used in flow cytometry where it is desirable to provide a flow cytometer with improved cell sorting accuracy, improved particle collection, reduced energy consumption, improved particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting. In some embodiments, the disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In some embodiments, the subject method and system provide a fully automated protocol so that adjustments to the flow cytometer in use require little to no human input, if any.

[0201] Notwithstanding the attached claims, this disclosure is also defined by the following addition:

[0202] Note 1. Measure the flow rate of the flow stream in the flow cytometer. The measured flow rate of the flowstream is compared with the absolute flow threshold and the moving window flow threshold. A method for generating an error alert when the measured flow rate of a flow stream exceeds an absolute flow threshold or a moving window flow threshold.

[0203] Note 2. The method described in Note 1 for continuously measuring the flow rate of a flow stream.

[0204] Note 3. The method described in Note 1, for measuring the flow rate of a flow stream at discrete intervals.

[0205] Note 4. The absolute flow threshold has an upper threshold and a lower threshold, as described in any one of Notes 1 to 3.

[0206] Appendix 5. The method according to any one of the appendices 1 to 4, which involves continuously comparing the measured flow rate of the flowstream with each absolute flow threshold.

[0207] Appendix 6. The method according to Appendix 4 or 5, which generates an error alert if the measured flow rate is determined to be less than the lower absolute flow rate threshold.

[0208] Appendix 7. The method according to Appendix 4 or 5, which generates an error alert if the measured flow rate is determined to be greater than the upper absolute flow rate threshold.

[0209] Appendix 8. The method according to Appendix 6 or 7, which generates an error alert if the measured flow rate is determined to be 1% or more greater or less than the absolute flow rate threshold.

[0210] Appendix 9. The method according to Appendix 6 or 7, which generates an error alert if the measured flow rate is determined to be 3% or more greater or less than the absolute flow rate threshold.

[0211] Note 10. The method according to any one of Notes 1 to 9, wherein the moving window flow threshold has an upper threshold and a lower threshold.

[0212] Appendix 11. The method according to any one of the appendices 1 to 9, which compares the measured flow rate with each moving window flow rate threshold at discrete intervals.

[0213] Appendix 12. The method according to Appendix 10 or 11, which determines whether the measured flow rate is greater than the upper limit moving window flow rate threshold during a predetermined time interval when comparing the measured flow rate with each moving window flow rate threshold.

[0214] Appendix 13. The method according to Appendix 10 or 11, which determines whether the measured flow rate is less than the lower limit moving window flow rate threshold during a predetermined time interval when comparing the measured flow rate with the moving window flow rate threshold.

[0215] Note 14. The method described in Note 12 or 13, wherein the specified time interval is within the range of 0.1 seconds to 10 seconds.

[0216] Note 15. The specified time interval of the moving window is within the range of 1 second to 5 seconds, as described in Note 14.

[0217] Note 16. The method described in Note 14 or 15, wherein the specified time interval is 1 second.

[0218] Note 17. The generated error alert indicates a malfunction in the flow cytometer, as described in any one of the methods in Notes 1-16.

[0219] Note 18. Failures include those described in Note 17, including blockages in the flowstream.

[0220] Note 19. Failures include the methods described in Note 17, including gas contamination of the flowstream.

[0221] Note 20. A method according to any one of the methods described in Notes 1 to 19 for generating error alerts in real time.

[0222] Appendix 21. The method according to Appendix 20, wherein an error alert is generated if the measured flow rate continues to exceed an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration.

[0223] Note 22. A method according to any one of Notes 1 to 21, which adjusts the flow rate of the flow stream in response to the generated error alert.

[0224] Note 23. The method described in Note 22, which increases the flow rate of the flow stream in response to the generated error alert.

[0225] Note 24. The method described in Note 22, which reduces the flow rate of the flow stream in response to the generated error alert.

[0226] Note 25. The method described in Note 22 for stopping the flow of the flow stream in response to the generated error alert.

[0227] Note 26. The method described in Note 22 for flushing the flow stream in response to the generated error alerts.

[0228] Appendix 27. The method according to any one of the appendices 1 to 26, wherein the flow rate of a flow stream is measured using a flow sensor based on one or more of the temperature and viscosity of the flow stream.

[0229] Note 28. The method according to any one of the notes 1 to 26, wherein the flowstream comprises a sheath fluid flowstream and a sample core fluid flowstream.

[0230] Appendix 29. The method described in Appendix 28 for measuring the flow rate of a sheath fluid flow stream.

[0231] Appendix 30. The method described in Appendix 28 for measuring the flow rate of the sample core fluid flow stream.

[0232] Appendix 31. The method according to any one of the appendices 28 to 30 for measuring the relative flow rates of the sheath fluid flow stream and the sample core fluid flow stream.

[0233] Appendix 32. The method according to any one of Appendix 1 to 31, for transporting a sample containing particles in a flowstream.

[0234] Appendix 33. The method according to Appendix 32, wherein a sample is irradiated using a light source and light from the irradiated particles in the flow stream is detected.

[0235] Note 34. A system for configuring a flow cytometer, It has a processor in which memory containing instructions is operablely linked, Memory is, Command to measure the flow rate of a flow stream in a flow cytometer. Commands for comparing the measured flow rate of a flow stream with the absolute flow threshold and the moving window flow threshold, and Command to generate an error alert if the measured flow rate of the flowstream exceeds the absolute flow threshold or the moving window flow threshold. A system that includes this.

[0236] Note 35. The memory is the system described in Note 34, which includes instructions for continuously measuring the flow rate of a flow stream.

[0237] Note 36. The memory is the system described in Note 34, which includes instructions for measuring the flow rate of a flow stream at discrete intervals.

[0238] Note 37. The absolute flow threshold has an upper threshold and a lower threshold, as described in any one of the systems in Notes 34 to 36.

[0239] Note 38. The system described in any one of Notes 34-37 includes a memory containing instructions for continuously comparing the measured flow rate of a flow stream with each absolute flow threshold.

[0240] Note 39. The system as described in Note 37 or 38, which includes a memory that contains instructions for generating an error alert if the measured flow rate is determined to be less than the lower absolute flow rate threshold.

[0241] Note 40. The system as described in Note 37 or 38, which includes a memory that contains instructions for generating an error alert if the measured flow rate is determined to be greater than the upper absolute flow rate threshold.

[0242] Note 41. The system as described in Note 39 or 40, which includes a memory that contains instructions for generating an error alert if the measured flow rate is determined to be 1% or more greater or less than the absolute flow rate threshold.

[0243] Note 42. The system as described in Note 39 or 40, which includes a memory that contains instructions for generating an error alert if the measured flow rate is determined to be 3% or more greater or less than the absolute flow rate threshold.

[0244] Note 43. The moving window flow threshold has an upper threshold and a lower threshold, as described in any one of Notes 34 to 42.

[0245] Note 44. The memory is a system described in any one of Notes 34 to 42, which includes instructions for comparing the measured flow rate with each moving window flow rate threshold at discrete intervals.

[0246] Note 45. The system according to Note 43 or 44, wherein the memory includes instructions for comparing the measured flow rate with each moving window flow threshold by determining whether the measured flow rate is greater than the upper moving window flow threshold during a predetermined time interval.

[0247] Note 46. The system according to Note 43 or 44, wherein the memory includes instructions for comparing the measured flow rate with a moving window flow threshold by determining whether the measured flow rate is less than the lower moving window flow threshold during a predetermined time interval.

[0248] Note 47. The system described in Note 45 or 46, wherein the specified time interval is within the range of 0.1 seconds to 10 seconds.

[0249] Note 48. The system described in Note 47, where the predetermined time interval of the moving window is within the range of 1 second to 5 seconds.

[0250] Note 49. The system described in Note 47 or 48, wherein the specified time interval is 1 second.

[0251] Note 50. The generated error alert indicates a malfunction in the flow cytometer, as described in any one of the systems listed in Notes 34-49.

[0252] Note 51. The failure is the system described in Note 50, including a blockage in the flowstream.

[0253] Note 52. The failure includes the system described in Note 50, including gas contamination of the flowstream.

[0254] Note 53. The memory is a system described in any one of Notes 34-52, which includes instructions for generating error alerts in real time.

[0255] Note 54. The system as described in Note 53, wherein the memory includes instructions for generating an error alert if the measured flow rate continues to exceed an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration.

[0256] Note 55. The memory is a system described in any one of Notes 34-54, which includes instructions for adjusting the flow rate of the flow stream in response to the generated error alerts.

[0257] Note 56. The system described in Note 55 includes memory containing instructions for increasing the flow rate of the flow stream in response to generated error alerts.

[0258] Note 57. The memory is the system described in Note 55, which includes instructions for reducing the flow rate of the flow stream in response to the generated error alerts.

[0259] Note 58. The memory is the system described in Note 55, which includes instructions to stop the flow of the flow stream in response to the generated error alert.

[0260] Note 59. The memory is the system described in Note 55, which includes instructions for flushing the flow stream in response to generated error alerts.

[0261] Appendix 60. The system according to any one of Appendix 34 to 59, comprising a flow sensor configured to measure the flow rate of a flow stream based on one or more of the temperature and viscosity of the flow stream.

[0262] Note 61. The flowstream is a system according to any one of the notes 34 to 60, comprising a sheath fluid flowstream and a sample core fluid flowstream.

[0263] Note 62. The memory is the system described in Note 61, which includes instructions for measuring the flow rate of a sheath fluid flow stream.

[0264] Note 63. The memory is the system described in Note 61, which includes instructions for measuring the flow rate of the sample core fluid flow stream.

[0265] Note 64. The system described in any one of Notes 61 to 63 includes a memory that contains instructions for measuring the relative flow rates of the sheath fluid flow stream and the sample core fluid flow stream.

[0266] Note 65. A system according to any one of Notes 34 to 64, configured to transport a sample containing particles within a flowstream.

[0267] Note 66. Light source for irradiating the sample in the flow stream, A photodetection system having a photodetector for detecting light from irradiated particles The system described in Appendix 65, which includes the features described therein.

[0268] Note 67. A non-temporary computer-readable storage medium for configuring a flow cytometer, An algorithm for measuring the flow rate of a flow stream in a flow cytometer. An algorithm for comparing the measured flow rate of a flowstream with an absolute flow threshold and a moving window flow threshold, and Algorithm for generating an error alert when the measured flow rate of a flow stream exceeds the absolute flow threshold or the moving window flow threshold. A non-temporary, computer-readable storage medium that stores instructions including [specific instructions].

[0269] Appendix 68. The non - transient computer - readable storage medium according to Appendix 67, including an algorithm for continuously measuring the flow rate of the frost stream.

[0270] Appendix 69. The non - transient computer - readable storage medium according to Appendix 67, including an algorithm for measuring the flow rate of the frost stream at discrete intervals.

[0271] Appendix 70. The non - transient computer - readable storage medium according to any one of Appendices 67 - 69, wherein the absolute flow rate threshold has an upper limit threshold and a lower limit threshold.

[0272] Appendix 71. The non - transient computer - readable storage medium according to any one of Appendices 67 - 70, including an algorithm for continuously comparing the measured flow rate of the frost stream with each absolute flow rate threshold.

[0273] Appendix 72. The non - transient computer - readable storage medium according to Appendix 70 or 71, including an algorithm for generating an error alert when it is determined that the measured flow rate is less than the lower absolute flow rate threshold.

[0274] Appendix 73. The non - transient computer - readable storage medium according to Appendix 70 or 71, including an algorithm for generating an error alert when it is determined that the measured flow rate is greater than the upper absolute flow rate threshold.

[0275] Appendix 74. The non - transient computer - readable storage medium according to Appendix 72 or 73, including an algorithm for generating an error alert when it is determined that the measured flow rate is greater than or less than the absolute flow rate threshold by 1% or more.

[0276] Appendix 75. The non - transient computer - readable storage medium according to Appendix 72 or 73, including an algorithm for generating an error alert when it is determined that the measured flow rate is greater than or less than the absolute flow rate threshold by 3% or more.

[0277] Appendix 76. A non - transient computer - readable storage medium according to any one of Appendices 67 - 75, wherein the moving window flow threshold has an upper threshold and a lower threshold.

[0278] Appendix 77. A non - transient computer - readable storage medium according to any one of Appendices 67 - 75, including an algorithm for comparing the measured flow rate with each moving window flow threshold at discrete intervals.

[0279] Appendix 78. A non - transient computer - readable storage medium according to Appendix 76 or 77, including an algorithm for comparing the measured flow rate with each moving window flow threshold by determining whether the measured flow rate is greater than the upper moving window flow threshold during a predetermined time interval.

[0280] Appendix 79. A non - transient computer - readable storage medium according to any one of Appendices 76 - 78, including an algorithm for comparing the measured flow rate with the moving window flow threshold by determining whether the measured flow rate is less than the lower moving window flow threshold during a predetermined time interval.

[0281] Appendix 80. A non - transient computer - readable storage medium according to Appendix 78 or 79, wherein the predetermined time interval is in the range of 0.1 seconds to 10 seconds.

[0282] Appendix 81. A non - transient computer - readable storage medium according to Appendix 80, wherein the predetermined time interval of the moving window is in the range of 1 second to 5 seconds.

[0283] Appendix 82. A non - transient computer - readable storage medium according to Appendix 80 or 81, wherein the predetermined time interval is 1 second.

[0284] Appendix 83. A non - transient computer - readable storage medium according to any one of Appendices 67 - 82, wherein the generated error alert indicates that there is a fault in the flow cytometer.

[0285] Appendix 84. A non - transient computer - readable storage medium according to Appendix 83, wherein the fault includes a blockage of the flow stream.

[0286] Note 85. Failure includes gas contamination of the flowstream, including non-temporary computer-readable storage media as described in Note 83.

[0287] Appendix 86. A non-temporary computer-readable storage medium as described in any one of Appendix 67-85, including an algorithm for generating error alerts in real time.

[0288] Appendix 87. A non-temporary computer-readable storage medium as described in Appendix 86, including an algorithm for generating an error alert if the measured flow rate continues to exceed an absolute flow rate threshold or a moving window flow rate threshold for a predetermined duration.

[0289] Appendix 88. A non-temporary computer-readable storage medium as described in any one of Appendix 67-87, including an algorithm for adjusting the flow rate of a flow stream in response to an error alert generated.

[0290] Appendix 89. A non-temporary computer-readable storage medium as described in Appendix 88, including an algorithm for increasing the flow rate of a flow stream in response to an error alert generated.

[0291] Appendix 90. A non-temporary computer-readable storage medium as described in Appendix 88, including an algorithm for reducing the flow rate of a flow stream in response to an error alert generated.

[0292] Appendix 91. A non-temporary computer-readable storage medium as described in Appendix 88, including an algorithm for stopping the flow of a flow stream in response to an error alert generated.

[0293] Appendix 92. A non-temporary computer-readable storage medium as described in Appendix 88, including an algorithm for flushing out the flow stream in response to generated error alerts.

[0294] Note 93. A non-temporary computer-readable storage medium according to any one of Notes 67 to 92, wherein the flowstream has a sheath fluid flowstream and a sample core fluid flowstream.

[0295] Appendix 94. A non-temporary computer-readable storage medium as described in Appendix 93, including an algorithm for measuring the flow rate of a sheath fluid flow stream.

[0296] Appendix 95. A non-temporary computer-readable storage medium as described in Appendix 93, including an algorithm for measuring the flow rate of a sample core fluid flow stream.

[0297] Appendix 96. A non-temporary computer-readable storage medium as described in any one of Appendix 93 to 95, including an algorithm for measuring the relative flow rates of the sheath fluid flow stream and the sample core fluid flow stream.

[0298] While the above disclosure is described in some detail as an illustration and example for ease of understanding, it will be readily apparent to those skilled in the art that, in view of the teachings of this disclosure, several changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0299] Therefore, the foregoing content merely shows the essence of the present disclosure. It is clear that those skilled in the art can devise various configurations that embody the essence of the present disclosure and are within the spirit and scope of the present disclosure but are not explicitly described or shown in this specification. Furthermore, all examples and conditional terms described in this specification are essentially intended to assist the reader in understanding the essence of the present disclosure and the concepts provided by the present disclosure for the advancement of the technical field, and should not be construed as limiting to the specifically described examples and conditions. Moreover, all descriptions in this specification that describe the essence, aspects, and embodiments of the present disclosure as well as specific examples of the present disclosure are intended to include both structural and functional equivalents of the present disclosure. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, that is, all developed elements that perform the same function regardless of structure. Furthermore, regardless of whether such disclosure is explicitly described in the claims, the disclosure in this specification is not generally intended to be publicly disclosed.

[0300] Therefore, it is intended that the scope of the present disclosure is not limited to the exemplary embodiments shown and described in this specification. Rather, the scope and spirit of the present disclosure are embodied by the appended claims. With respect to the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is clearly defined to be exercised only for limitations in a claim when the exact phrase "means for" or "step for" begins such limitation in the claim, and 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not exercised when such exact phrase is not used in the claim limitation.

[0301] Cross - reference to related applications This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 705166, filed on 9 October 2024, pursuant to Section 119(e) of the U.S. Patent Act, and the entire disclosure thereof is incorporated herein by reference.

Claims

1. Measure the flow rate of the flow stream in the flow cytometer. The measured flow rate of the flow stream is compared with the absolute flow threshold and the moving window flow threshold. A method for 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.

2. The method according to claim 1, wherein the flow rate of the flow stream is continuously measured.

3. The method according to claim 1, wherein the flow rate of the flow stream is measured at discrete intervals.

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

5. The method according to any one of claims 1 to 4, wherein the measured flow rate of the flow stream is continuously compared with each absolute flow rate threshold.

6. The method according to claim 4 or 5, wherein an error alert is generated if the measured flow rate is determined to be less than the lower absolute flow rate threshold or greater than the upper absolute flow rate threshold.

7. The method according to claim 6, wherein an error alert is generated if the measured flow rate is determined to be 1% or more greater or less than the absolute flow rate threshold.

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

9. The method according to any one of claims 1 to 7, wherein the measured flow rate is compared with each moving window flow rate threshold at discrete intervals.

10. A method according to any one of claims 1 to 9, which generates an error alert in real time.

11. The method according to claim 10, wherein an error alert is generated if the measured flow rate continues to exceed 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 to 10, wherein the flow rate of the flow stream is adjusted in response to the generated error alert.

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

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

15. The method according to any one of claims 1 to 14, comprising transporting a sample containing particles within the flowstream.

16. The method according to claim 15, wherein the sample is irradiated using a light source and light from the irradiated particles in the flow stream is detected.

17. A system for configuring a flow cytometer, It has a processor in which memory containing instructions is operablely linked, The aforementioned memory is Command for measuring the flow rate of the flow stream in the flow cytometer, Commands for comparing the measured flow rate of the flow stream with the absolute flow threshold and the moving window flow threshold, and If the measured flow rate of the flow stream exceeds the absolute flow threshold or the moving window flow threshold, an error alert is issued. A system that includes this.

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

19. A non-temporary computer-readable storage medium for configuring a flow cytometer, An algorithm for measuring the flow rate of a flow stream in the flow cytometer, An algorithm for comparing the measured flow rate of the flow stream with an absolute flow threshold and a moving window flow threshold, and Algorithm for 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 A non-temporary, computer-readable storage medium that stores instructions including [specific instructions].

20. A non-temporary computer-readable storage medium according to claim 19, comprising an algorithm for measuring the flow rate of the flow stream continuously or at discrete intervals.

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