Control variable adjustment for flow cytometry waveform acquisition

Abstract

The flow cytometer adjusts one or more control variables of the light source, the fluidic system, and the optical system based on the first set of values. The flow cytometer obtains a sequence of waveform data from particles flowing through the light beam in the interrogation zone at a first set of values of one or more control variables. The flow cytometer adjusts the one or more control variables at predetermined intervals to obtain an additional sequence of waveform data using different sets of values of the one or more control variables.

Description

[0001] This application was filed on January 22, 2024 as a PCT International Application and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 481,293, filed on January 24, 2023, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0002] Flow cytometry is a technique used to detect and analyze the chemical and physical properties of cells or particles in a fluid sample. For example, flow cytometers can be used to evaluate cells from blood, bone marrow, tumors, or other body fluids. Typically, the sample is passed through a fluid nozzle that aligns the particles in a single file within a sheath fluid. As the particles pass through the single file, a laser beam illuminates the particles, generating radiation that includes forward scattered light, side scattered light, and fluorescence. This radiation can then be detected and analyzed to determine one or more characteristics of the particles. Summary of the Invention

[0003] In general, the present disclosure relates to analyzing particles using flow cytometry. In one possible configuration, one or more control variables are automatically adjusted to have different values ​​for a sequence of acquiring waveform data. Various aspects are described in the present disclosure, including but not limited to the following aspects.

[0004] One aspect relates to a flow cytometer for analyzing particles, the flow cytometer comprising: a light source that generates a light beam directed toward an interrogation zone; a fluid system that causes particles to flow through the light beam in the interrogation zone; an optical system including a detector for detecting radiated light from particles flowing through the light beam in the interrogation zone; and a processing circuit system having a non-transitory computer-readable storage medium storing instructions that, when executed by the processing circuit system, cause the processing circuit system to: adjust one or more control variables of the light source, the fluid system, and the optical system based on a first set of values; acquire a sequence of waveform data from particles flowing through the light beam in the interrogation zone under the first set of values ​​of the one or more control variables; and adjust the one or more control variables at predetermined intervals to acquire additional sequences of waveform data using different sets of values ​​of the one or more control variables.

[0005] Another aspect relates to a method for analyzing particles flowing through an interrogation zone of a flow cytometer, the method comprising: adjusting one or more control variables based on a first set of values, the one or more control variables being used to control operation of at least one of: a light source that generates a light beam toward the interrogation zone; a fluidic system that causes particles to flow through the light beam in the interrogation zone; and an optical system for detecting one or more characteristics of the particles flowing through the light beam in the interrogation zone; acquiring a sequence of waveform data from the particles flowing through the light beam in the interrogation zone under the first set of values ​​of the one or more control variables; and adjusting the one or more control variables at predetermined intervals to acquire additional sequences of waveform data using different sets of values ​​of the one or more control variables.

[0006] Another aspect relates to a non-transitory computer-readable medium comprising program instructions that, when executed by a processor, cause the processor to perform the following operations: adjust one or more control variables based on a first set of values, the one or more control variables being used to control the operation of at least one of: a light source that generates a light beam toward an interrogation zone; a fluid system that causes particles to flow through the light beam in the interrogation zone; and an optical system for detecting one or more characteristics of the particles flowing through the light beam in the interrogation zone; acquire a sequence of waveform data from the particles flowing through the light beam in the interrogation zone under a first set of values ​​for the one or more control variables; and adjust the one or more control variables at predetermined intervals to acquire additional sequences of waveform data using different sets of values ​​for the one or more control variables.

[0007] Various additional aspects will be described in the following description. These aspects may relate to individual features and combinations of features. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative and do not limit the broad inventive concepts on which the embodiments disclosed herein are based. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The following drawings, which form a part of this application, are illustrative of the described technology and are not meant to limit the scope of the present disclosure in any way.

[0009] Figure 1 An example of a flow cytometer system is schematically shown.

[0010] Figure 2A Shows the entry Figure 1 Example of a particle in the interrogation zone of a flow cytometer system.

[0011] Figure 2B Shown by Figure 2A Example of particles in the central area of ​​the interrogation zone.

[0012] Figure 2C Shows leaving Figure 2A Example of particles in the interrogation zone.

[0013] Figure 3 Schematically shows Figure 1 Example of a waveform analysis device for a flow cytometer system.

[0014] Figure 4 Schematically shows the Figure 1 An example of a method for performing flow cytometry experiments using a flow cytometer system.

[0015] Figure 5 The graph shows the Figure 4 An example of the waveform data that can be generated after the method is completed.

[0016] Figure 6 shows that can be used by waveform analysis equipment Figure 5 Example of a graphical user interface generated from waveform data.

[0017] Figure 7 Shows that it can be Figure 1 Example of a graphical user interface generated by the waveform acquisition device of a flow cytometry system.

[0018] Figure 8 Shown in Figure 7 An example of a graphical user interface generated by a waveform acquisition device after selection of an icon on the graphical user interface.

[0019] Figure 9 An exemplary architecture of a computing device that can be used to implement aspects of the present disclosure is shown. DETAILED DESCRIPTION

[0020] Various embodiments will be described in detail with reference to the accompanying drawings, wherein like reference numerals represent like parts and components throughout the several views. Reference to various embodiments does not limit the scope of the appended claims. Furthermore, any examples set forth in this specification are not intended to be limiting, but merely set forth some of the many possible embodiments of the appended claims.

[0021] Figure 1Schematically illustrated is an example of a flow cytometer system 100. In some cases, the flow cytometer system 100 can include aspects and features described in the following provisional patent applications: U.S. Provisional Patent Application No. 63 / 410,984, filed on September 28, 2022, entitled Flow Cytometry Waveform Processing, U.S. Provisional Patent Application No. 63 / 481,289, filed on January 24, 2023, entitled Threshold Logic for Flow Cytometry Waveform Analysis, and U.S. Provisional Patent Application No. 63 / 481,298, filed on January 24, 2023, entitled Doublet Analysis in Flow Cytometry, which are incorporated herein by reference in their entireties.

[0022] In general, flow cytometry is a technique for measuring and analyzing the properties of particles or cells as they flow in a fluid stream. Data from millions of particles or cells can be collected by the flow cytometer system 100 in minutes and displayed in a variety of formats. Illustrative applications of flow cytometry include performing phenotypic analysis to identify and count specific cell types in a population, analyzing the DNA or RNA content within a cell, determining the presence of antigens on or within a cell, and assessing cell health.

[0023] like Figure 1 As shown in the illustrative example of FIG, a flow cytometer system 100 generally includes three major component subsystems: a fluidics system 110, an optics system 120, and an electronics system 130. The fluidics system 110 includes a nozzle 112 that receives a sample comprising particles or cells suspended in a fluid. The nozzle 112 produces a fluid stream 114 that arranges the particles or cells in a single file. Each particle or cell passes through one or more light beams generated by a light source 102. The point where a particle or cell intersects the one or more light beams of the light source 102 is referred to as an interrogation zone 116. In some examples, the light source 102 includes one or more lasers.

[0024] Optical system 120 includes light source 102, optical element 122, and detector 124. At interrogation zone 116, light from light source 102 strikes particles or cells in fluid stream 114 and scatters. Optical element 122 directs the scattered light toward detector 124. Detector 124 may include a forward scatter (FSC) detector for measuring scatter in the path of light source 102, a side scatter (SSC) detector for measuring scatter at a ninety-degree angle relative to light source 102, and one or more fluorescence detectors (FL1, FL2, FL3, ..., FLn) for measuring emitted fluorescence intensity at different wavelengths of light.

[0025] Typically, FSC intensity is proportional to the size or diameter of the particle due to the diffraction of light around the particle. Therefore, FSC can be used to distinguish particles by size. On the other hand, SSC is produced by light refracted or reflected by the internal structure of the particle, and therefore can provide information about the internal complexity or granularity of the particle. By adding fluorescent markers to the sample, different fluorescent signals / channels (e.g., green, orange, and red) can be analyzed for the functional properties of the cell. For example, since T cells have CD3 binding sites, samples containing T cells can be "stained" with anti-CD3 antibodies conjugated to fluorescent molecules. When these cells pass through the interrogation zone 116, the light from the light source excites the fluorescent label or fluorescent dye to emit photons at a wavelength that can be detected by a fluorescence detector. Therefore, the detector 124 can measure several parameters simultaneously, and enables the particles to be classified by the function of the particles based on the wavelength of the detected light.

[0026] The electronic system 130 includes a waveform acquisition device 140 and a waveform analysis device 150. The waveform acquisition device 140 is communicatively coupled to the detector 124 to receive the analog waveform data 126 generated by the detector 124. The waveform acquisition device 140 includes an analog-to-digital converter (ADC) 142 configured to digitize the analog waveform data 126. The waveform acquisition device 140 may also include a graphical user interface (GUI) 144 for receiving user input. The user input received through the GUI 144 may be used to control one or more control variables of the fluid system 110 and the optical system 120 for analyzing particles in the fluid stream 114.

[0027] The waveform analysis device 150 is configured to receive digital waveform data and display it to a user of the flow cytometer system 100. In some embodiments, the waveform analysis device 150 comprises a computing device communicatively coupled to the flow cytometer 101, such as via a network. The flow cytometer 101 can include a fluidics system 110, an optical system 120, and a waveform acquisition device 140. In other embodiments, the waveform analysis device 150 is integrated with the flow cytometer 101.

[0028] Current flow cytometers use a field programmable gate array (FPGA) in the waveform acquisition device to obtain information about individual particles passing through the light beam. The waveform acquisition device uses a single threshold to determine when the output of the detector begins to convert from analog to digital. Only a single threshold can be used for a single run of a sample through a current flow cytometer. The threshold is a constant value and can be referred to as a voltage threshold. Therefore, if the detector output exceeds the voltage value of the threshold or when the detector output exceeds the voltage value of the threshold, digitization begins and the digital value is sent to the FPGA. As the waveform data is digitized, the FPGA calculates the height, width, and area of ​​each pulse. In addition to these parameters, other data related to the waveform (including data that does not exceed the voltage threshold) is not captured, stored, or otherwise used for analysis by current flow cytometers. In addition, if the user wants to adjust the threshold, the experiment must be rerun with the new threshold, which results in a cost in resources and time.

[0029] To address the above issues, the flow cytometer system 100 is improved using a graphics processing unit (GPU) 152. Figure 1 In the example shown in FIG, a GPU 152 is shown as being included as a component of the waveform analysis device 150. The GPU 152 processes the continuous digital stream generated by the waveform acquisition device 140. The digital stream is continuous because the waveform acquisition device 140 does not threshold the waveform data generated by the detector 124. In contrast to current flow cytometry technology, during an experiment, the waveform acquisition device 140 continuously digitizes the analog waveform data 126 at a high rate (e.g., 1 GHz) without thresholding. In some cases, the GPU 152 can remove the FPGA from the waveform acquisition device 140.

[0030] The waveform analysis device 150 receives a digitized version of the waveform data with added data points, and the waveform data for the experiment is displayed and made available to the GPU 152 for processing as a whole. In addition to having the ability to process large waveform data streams or waveform data files, the GPU 152 enables thresholding of the waveform at a post-processing step rather than at the waveform acquisition step. This in turn provides several technical benefits, including the ability to dynamically adjust thresholds and update graphical charts in real time without rerunning the experiment. The GPU 152 can also measure and extract relevant information other than the three parameters of height, width, and area presented in the waveform data. Further details of these advantages are discussed below.

[0031] The flow cytometer system 100 includes the elements shown and described for discussion purposes, and it should be understood that many variations of components and functions are possible. For example, the optical elements 122 may include a series of filters, dichroic mirrors, and / or beam splitters to select light of different wavelengths and provide the wavelengths to appropriate detectors. The detector 124 may include a photomultiplier tube (PMT) or an avalanche photodiode (APD) or a single photon counting device.

[0032] Figures 2A to 2C An example of waveform data generated by particle 201 as particle 201 passes through interrogation zone 116 is shown. As particle 201 passes through interrogation zone 116, one or more of detectors 124 detects a pulse.

[0033] Figure 2A An example of a particle 201 entering interrogation zone 116 is shown. As particle 201 begins to intersect interrogation zone 116, it begins to generate scattered light and a fluorescent signal. Detector 124 generates a current or voltage proportional to the scattered light and fluorescent signal. Due to the current flowing in detector 124, the output of detector 124 begins to rise as shown in graph 212.

[0034] Figure 2B An example of a particle 201 passing through the central region of interrogation zone 116 is shown. As particle 201 continues to move through interrogation zone 116, particle 201 becomes fully illuminated. Since the photon density is highest in the central portion of interrogation zone 116, the maximum amount of light signal is generated in this example. As shown in graph 232, the current or voltage of detector 124 reaches a peak when particle 201 passes through the central region of interrogation zone 116.

[0035] Figure 2C An example of a particle 201 exiting the interrogation zone 116 is shown. When the particle 201 exits the interrogation zone 116, the current or voltage output of the detector 124 returns to baseline. The generation of the pulse shown in the graph 252 is referred to as an event.

[0036] The height of graph 252 represents the maximum current / voltage output by detector 124, which can be proportional to the signal strength and size of the particle, the width of graph 252 represents the time it takes for the particle to pass through interrogation zone 116, and the area below graph 252 can represent the signal strength and size of the particle. Therefore, the height, width, and area of ​​graph 252 can be used to characterize the particle.

[0037] Figure 3An example of a waveform analysis device 150 is schematically shown. The waveform analysis device 150 receives, stores, and displays waveform data that has been continuously sampled without being thresholded upstream at the waveform acquisition device 140. The waveform analysis device 150 includes an interface 310 for receiving digitized raw waveform data 332, a persistent storage device 330 for storing the digitized raw waveform data 332, and a graphical user interface (GUI) 320 that may be used to display the digitized raw waveform data 332. The persistent storage device 330 may also store a plurality of dynamic threshold values ​​334 that allow for real-time updating and display of applied threshold values ​​and nonlinear thresholding. The persistent storage device 330 may include system memory such as random access memory (RAM) and / or long-term non-volatile storage such as a hard drive.

[0038] The waveform analysis device 150 may also include a cytometry analysis application 350, which includes a software application or a set of related software applications configured to instruct the GPU 152 to process the digitized raw waveform data 332. The cytometry analysis application 350 may be executed on one or more processors to provide the functionality described herein in conjunction with the GPU 152, such as receiving user input via the GUI 320. One or more components of the waveform analysis device 150 may reside in a cloud computing application in a network distributed system. In this regard, the waveform analysis device 150 may be any of a variety of computing devices, including but not limited to a personal computing device, a server computing device, or a distributed computing device.

[0039] In some cases, a flow cytometer user may be interested in configuring the flow cytometer to analyze particles using different sets of control variable values. In this case, the user needs to run multiple experiments for each set of control variable values. For example, the user will need to run a first experiment using a first set of control variable values, a second experiment using a second set of control variable values, a third experiment using a third set of control variable values, and so on, until data has been collected for all desired sets of control variable values. As an illustrative example, a flow cytometer user interested in using voltage values ​​of 500V, 1000V, and 1200V for the FL1 detector will need to run a first experiment with a value of 500V for the FL1 detector, edit the FL1 detector voltage to 1000V, then rerun the experiment, and edit the FL1 detector voltage to 1200V, then rerun the experiment. This is tedious and time-consuming, especially as the number of desired changes in control variable values ​​increases.

[0040] Figure 4Schematically illustrates an example of a method 400 for performing a flow cytometry experiment by the flow cytometer system 100. As will be described in more detail, the method 400 eliminates the need to run multiple experiments for different sets of control variable values. Instead, a single experiment is run using different sets of control variable values.

[0041] Method 400 includes receiving one or more sets of adjustable control variable values ​​and an experiment duration operation 402. In some examples, the one or more sets of adjustable control variable values ​​and the experiment duration may be received as user input via GUI 144 of waveform acquisition device 140.

[0042] Figure 7 An example of a graphical user interface (GUI) 700 that may be generated by the waveform acquisition device 140 is shown. The GUI 700 includes an experiment definition window 702 in which a user of the flow cytometer system 100 selects and / or enters one or more sets of control variable values. For example, the user may select a set of control variable values ​​for the FL1 detector that includes voltage values ​​of 500V, 1000V, and 1200V. Although the examples described herein relate to voltage values ​​for the FL1 detector, it is contemplated that various aspects described herein may be applied to the flow cytometer 101 (see FIG. Figure 1 ) included in any detector 124. For example, the aspects described herein can be similarly applied to a multi-color detector. In addition, the term "voltage" as used herein is interchangeable with "gain," such that the aspects described herein can also be applied to adjusting the gain of any amplifier, transducer, or detector of the flow cytometer 101.

[0043] In some examples, such as Figure 7 In the example shown in , the voltage values ​​for the FL1 detector are predefined, so that the user simply selects one or more of the voltage values ​​in the experiment definition window 702. In another example, the user can enter one or more customized voltage values ​​for the FL1 detector in the experiment definition window 702.

[0044] like Figure 7 As shown in FIG, a user can add additional voltage values ​​for the FL1 detector by selecting the “Add Voltage Value” icon to expand the available options for the FL1 detector. Furthermore, the user can add voltage values ​​for other detectors in the optical system 120 (e.g., detectors FSC through FLn). Furthermore, the user can select and / or enter additional control variable values, such as flow rate values ​​for the fluidic system 110 (e.g., a sheath fluid flow rate value and / or a sample fluid flow rate value), and / or a beam intensity value for the light source 102.

[0045] like Figure 7As shown in FIG, the user can define the experiment duration in window 704. In this example, window 704 defines the experiment duration based on the number of detection events. As described above, the detection event can be such as Figure 2C 252 . In this example, window 704 includes 15,000 events, 30,000 events, and 60,000 events as options for the user to select, and the duration of 30,000 events is shown as selected. Additional experiment durations are possible, so that these options are provided by way of illustrative examples. In addition, other types of units for defining the experiment duration can be specified in window 704, such as time measured in seconds, minutes, or hours.

[0046] The GUI 700 includes a start icon 706 that a user of the flow cytometer 101 can select to run an experiment based on one or more sets of control variable values ​​and the experiment duration selected in the experiment definition window 702 .

[0047] Return to reference Figure 4 , method 400 can include an operation 404 of determining an experimental protocol based on the one or more sets of adjustable control variable values ​​and a duration set for the experiment. In some examples, the experimental protocol is automatically determined by the flow cytometer 101 .

[0048] As an illustrative example, when the user selects voltage values ​​of 500v, 1000v, and 1200v and a duration of 30,000 events for the FL1 detector (see Figure 7 ), operation 404 may include defining an experimental protocol including three different phases, wherein 10,000 events are recorded when the FL1 detector uses a first voltage value (e.g., 500 V), 10,000 events are recorded when the FL1 detector uses a second voltage value (e.g., 1000 V), and 10,000 events are recorded when the FL1 detector uses a third voltage value (e.g., 1200 V).

[0049] In an alternative example, the user can define a custom experimental protocol, for example, by using the GUI 144 of the waveform acquisition device 140. As an illustrative example, when the user selects voltage values ​​of 500v, 1000v, and 1200v and a duration of 30,000 events for the FL1 detector (see Figure 7), the user may define a customized experimental protocol in which 18,000 events are recorded when the FL1 detector uses a first voltage value (e.g., 500 V), 6,000 events are recorded when the FL1 detector uses a second voltage value (e.g., 1,000 V), and 6,000 events are recorded when the FL1 detector uses a third voltage value (e.g., 1,200 V). Additional examples for determining the experimental protocol in operation 404 are possible.

[0050] Next, method 400 includes an operation 406 of adjusting one or more control variables based on the experimental protocol determined in operation 404. For example, operation 406 may include adjusting the control variables to have values ​​according to the first phase of the experimental protocol. Figure 7 In the illustrative example shown in and described above, operation 406 may include adjusting the FL1 detector to have a voltage value of 500V.

[0051] Next, the method 400 includes an operation 408 of acquiring waveform data using the control variables adjusted in operation 406. For example, operation 408 may include operating the light source 102, the fluid system 110, and / or the optical system 120 using the control variable values ​​adjusted in operation 406. Figure 7 In the illustrative example shown in , operation 408 may include operating the FL1 detector at a voltage value of 500 V to record 10,000 events.

[0052] Next, method 400 includes an operation 410 of determining whether additional waveform data is needed based on the experimental protocol determined in operation 404. For example, operation 410 may include determining whether additional stages of the experimental protocol need to be completed. Figure 7 In the illustrative example shown in and described above, operation 410 may include determining whether additional events need to be recorded by the FL1 detector operating at different voltage values ​​(eg, 1000 V and 1200 V).

[0053] When it is determined in operation 410 that additional waveform data is needed (ie, "yes" in operation 410), method 400 may return to operation 406 to adjust the control variables to have values ​​according to another stage of the experimental protocol. Figure 7 In the illustrative example shown in and described above, operation 406 may include adjusting the FL1 detector to have a voltage value of 1000 V. Thereafter, method 400 may repeat operations 408 and 410 until all phases of the experimental protocol have been completed such that no additional waveform data is needed (i.e., "No" at operation 410). Thus, method 400 begins by acquiring waveform data using a predetermined set of one or more control variable values, and then, at predetermined intervals, changes the one or more control variable values ​​to acquire additional waveform data using a different set of control variable values.

[0054] When additional waveform data is not required (ie, "No" at operation 410), method 400 may proceed to operation 412 of storing the waveform data. In some examples, operation 412 includes storing the waveform data in a single flow cytometry standard (FCS) file. Figure 7 The illustrative example shown in , uses metadata to tag each event stored in a single FCS file to identify whether the event was detected at the voltage value of 500 V, 1000 V, or 1200 V used by the FL1 detector during the experimental protocol.

[0055] Figure 5 An example of a waveform 500 that can be generated after method 400 is completed is graphically illustrated. In this illustrative example, waveform 500 is generated based on data collected by the FL1 detector, with time on the x-axis and fluorescence intensity on the y-axis. A user of flow cytometer system 100 uses GUI 144 of waveform acquisition device 140 to specify that they want to collect data from the FL1 detector operating at voltage values ​​of 500 V, 1000 V, and 1200 V for a total duration of 150 seconds. Waveform 500 is generated based on the experimental protocol and includes a first sequence 502a of events measured by the FL1 detector using a voltage value of 500 V over a time interval of 0 to 50 seconds, a second sequence 502b of events measured by the FL1 detector using a voltage value of 1000 V over a time interval of 50 to 100 seconds, and a third sequence 502c of events measured by the FL1 detector using a voltage value of 1200 V over a time interval of 100 to 150 seconds.

[0056] A first sequence of events 502a is tagged with metadata in an FCS file that correlates these events measured by an FL1 detector operating at 500 V. Similarly, a second sequence of events 502b is tagged with metadata in an FCS file that correlates these events measured by an FL1 detector operating at 1000 V. A third sequence of events 502c is tagged with metadata in an FCS file that correlates these events measured by an FL1 detector operating at 1200 V.

[0057] In some examples, the single FCS file generated by method 400 is transmitted to waveform analysis device 150 for storage in persistent storage 330. Thus, waveform analysis device 150 receives a digitized version of the waveform data that has not been thresholded and is available in its entirety for processing by GPU 152. As discussed above, GPU 152 enables thresholding of waveform data at a post-processing step rather than at the waveform acquisition step. This enables waveform analysis device 150 to dynamically adjust thresholds and update graphical charts in real time without rerunning the experiment.

[0058] Figure 6 4 shows an example of a graphical user interface (GUI) 600 that may be generated by the waveform analysis device 150 using a single FCS file generated by the method 400. Figure 6 , the GUI 600 may include a gating drop-down menu 602 that includes different gating options for selection by a user of the flow cytometer system 100. In this example, the gating drop-down menu 602 includes the FL1 detector voltage, which is selected to cause a secondary drop-down menu 604 to display a list of values ​​for that control variable.

[0059] In this example, the secondary drop-down menu 604 displays voltage values ​​of 500v, 1000v, and 1200v, which the FL1 detector uses to detect Figure 5 As an illustrative example, a user of the flow cytometer system 100 can set the input gate of the scatter plot to "FL1 voltage 500v" to filter the events analyzed by the cytometry analysis application 350 of the waveform analysis device 150. In this example, only events measured by the FL1 detector operating at a voltage value of 500v during the time interval from 0 seconds to 50 seconds are analyzed by the cytometry analysis application 350. In addition, the events included in one or more graphs 606, 608 on the GUI 600 are filtered to include only events measured by the FL1 detector operating at a voltage value of 500v during the time interval from 0 seconds to 50 seconds.

[0060] In another example, the user of the flow cytometer system 100 can select more than one value for the control variable in the secondary drop-down menu 604. For example, the user of the flow cytometer system 100 can set the input gates of the scatter plot to "FL1 Voltage 500 V" and "FL1 Voltage 1000 V" to filter the events analyzed by the cytometry analysis application 350. In this example, events measured by the FL1 detector operating at a voltage value of 500 V during the time interval from 0 seconds to 50 seconds and events measured by the FL1 detector operating at a voltage value of 1000 V during the time interval from 50 seconds to 100 seconds are analyzed. In addition, the events included in one or more graphs 606, 608 displayed in the GUI 600 are filtered to include only events measured by the FL1 detector operating at voltage values ​​of 500 V and 1000 V.

[0061] In further examples, a user of the flow cytometer system 100 may select voltage values ​​of 500v, 1000v, and 1200v for the FL1 detector voltage so that events marked with these voltage values ​​are included in one or more of the graphs 606 , 608 displayed in the GUI 600 .

[0062] In other examples, multiple values ​​may be selected for multiple control variables to obtain waveform data via the waveform acquisition device 140. For example, Figure 7 As shown in FIG, a user of the flow cytometer system 100 can request waveform data acquisition for FL1 voltage values ​​of 500V, 1000V, and 1200V, as well as waveform data acquisition for slow and fast flow rates (but not for medium flow rates) of the sheath fluid flow rate and / or sample fluid flow rate controlled by the fluidics system 110. Adjusting the flow rate can be useful in distinguishing small particles from optical noise because as the flow rate slows, the width of small particles will expand, while the width of optical noise (stray photons) will remain approximately constant. As the information in the signal increases and the noise remains constant, the signal-to-noise ratio of the waveform data acquisition can be improved.

[0063] In this example, when the user requests a total duration of 60,000 events, an experimental protocol can be generated that includes the following sets of control variable values, each set of control variable values ​​being used to detect 10,000 events: (1) FL1 voltage of 500 V, slow flow rate; (2) FL1 voltage of 1000 V, slow flow rate; (3) FL1 voltage of 1200 V, slow flow rate; (4) FL1 voltage of 500 V, fast flow rate; (5) FL1 voltage of 1000 V, fast flow rate; and (6) FL1 voltage of 1200 V, fast flow rate.

[0064] Additional examples of running a single experiment using multiple values ​​for multiple control variables are possible, so the foregoing is provided for illustrative purposes only. For example, the intensity of the light beam generated by light source 102 can be selected to adjust between different values. Thus, a user of flow cytometer system 100 can run a single experiment using different combinations of control variable values. This eliminates the need to run multiple experiments using different sets of control variable values. Instead, a single experiment is run using different sets of control variable values. This can save time and resources and improve the usability of flow cytometer system 100.

[0065] like Figure 7 As further shown in FIG, in some examples, a previous experiment with multiple values ​​of one or more control variables can be rerun by the user selecting icon 708. For example, after running a successful experiment, the user of the flow cytometer 101 can save the selected set of control variable values ​​and the experiment duration in the memory of the waveform acquisition device 140 for future repetitions of the experiment. Upon selecting icon 708, the waveform acquisition device 140 can display a graphical user interface (see FIG. Figure 8 ), the graphical user interface lists previous experiments for the user to select. In this way, the user does not need to re-enter or re-select the values ​​of the control variables and the experiment duration. Instead, the user can simply select a previous experiment to re-run it.

[0066] Figure 8 Shows the selected Figure 7 An example of a graphical user interface (GUI) 800 generated by the waveform acquisition device 140 following the icon 708 in the graphical user interface 700 of FIG. In this illustrative example, the GUI 800 lists previous experiments including Experiment A, Experiment B, Experiment C, and so on. Figure 8 In the example shown in FIG, Experiment B is expanded to show multiple values ​​selected for at least one control variable. In this example, FCS detector voltage 802a includes selections of 500V, 1000V, and 1200V, and flow rate 802b includes selections of slow and fast. Light source intensity 802c includes a selection of medium intensity, and experiment duration includes a selection of 60,000 events. The user of flow cytometer 101 can select Start icon 804 to rerun Experiment B without having to re-enter or reselect values ​​for the control variables, which can save time and resources.

[0067] In addition to the foregoing, in some cases, a previous experiment is associated with analyzing a particular particle or cell, or is used to identify a particular characteristic of a particle or cell. For example, a previous experiment may be considered ideal for analyzing a particular type of cancer cell. Advantageously, a user of the flow cytometer 101 can select a previous experiment identified as optimal for a particular application without having to re-enter or re-select values ​​for the experimental control variables.

[0068] In some further examples, the GUI may also include an edit icon 806 that allows the user to edit the selection of values ​​for the control variables of a given experiment. The edits may be saved so that the edited experiment can be rerun without the user having to re-enter or reselect the edited values ​​for the control variables of the given experiment.

[0069] Figure 9 An exemplary architecture of a computing device 900 is shown that may be used to implement aspects of the present disclosure, including aspects of the waveform acquisition device 140 and the waveform analysis device 150 as described above. Figure 9 The computing device 900 shown in FIG. 9 can be used to execute the operating systems, applications, and software modules (including software engines) described herein.

[0070] The computing device 900 includes at least one processing device 902, such as a central processing unit (CPU). In this example, the computing device 900 also includes a system memory 904 and a system bus 906 that couples various system components, including the system memory 904, to the at least one processing device 902. The system bus 906 is one of any number of types of bus structures, including a memory bus or memory controller; a peripheral bus; and a local bus using any of a variety of bus architectures.

[0071] The system memory 904 includes read-only memory (ROM) 908 and random access memory (RAM) 910. A basic input / output system 912, containing the basic routines used to transfer information within the computing device 900, such as during startup, is typically stored in the read-only memory 908. In some examples, the system memory 904 has a large memory capacity, such as equal to or greater than 1 terabyte of RAM. The RAM can be used to load and subsequently analyze waveform data (e.g., raw waveform data, such as stored in a raw waveform data file, which may include digitized waveform data).

[0072] In some embodiments, the computing device 900 also includes a secondary storage device 914, such as a hard drive, for storing digital data. The secondary storage device 914 is connected to the system bus 906 via a secondary storage interface 916. In some examples, the secondary storage device 914 and its associated computer-readable media provide non-volatile storage of computer-readable instructions (including applications and program modules), data structures, and other data for the computing device 900.

[0073] While the exemplary environment described herein utilizes a hard drive as a secondary storage device, other types of computer-readable storage media may be used in other embodiments. Examples of these other types of computer-readable storage media include magnetic tape cassettes, flash memory cards, digital video disks, Bernoulli cassettes, compact disc read-only memories, digital versatile disc read-only memories, random access memories, or read-only memories. Some embodiments include non-transitory media. Additionally, such computer-readable storage media may include local storage or cloud-based storage.

[0074] Several program modules may be stored in the secondary storage device 914 or the system memory 904, including an operating system 918, one or more application programs 920, other program modules 922 (such as the software engines described herein), and program data 924. The computing device 900 may use any suitable operating system, such as Microsoft Windows TM , Google Chrome TM , Apple OS, and any other operating system for a computing device.

[0075] In some examples, a user provides input to the computing device 900 through one or more input devices 926. Examples of input devices 926 include a keyboard 928, a mouse 930, a microphone 932, and a touch sensor 934 (e.g., a touchpad or touch-sensitive display). Additional examples include additional types of input devices 926, or fewer types of input devices 926. The input devices 926 are connected to at least one processing device 902 via an input / output interface 936 coupled to the system bus 906. The input / output interface 936 may include any number of input / output interfaces, such as a parallel port, a serial port, a game port, or a universal serial bus. In some possible implementations, wireless coupling between the input device 926 and the input / output interface 936 is also possible, such as via infrared, 802.11a / b / g / n, cellular, or other RF communication systems.

[0076] In this example embodiment, a display device 942 (e.g., a monitor, liquid crystal display device, projector, or touch-sensitive display device) is also connected to the system bus 906 via the video adapter 940. In addition to the display device 942, the computing device 900 may also include various other peripheral devices (not shown), such as speakers or a printer.

[0077] When used in a local area networking environment or a wide area networking environment (e.g., the Internet), the computing device 900 is typically connected to the network, such as through a network interface 938 (e.g., an Ethernet interface). Other possible implementations use other communication devices. For example, some implementations of the computing device 900 include a modem for communicating across the network.

[0078] The computing device 900 typically includes at least some form of computer-readable media. Computer-readable media includes any available media that can be accessed by the computing device 900. By way of example, computer-readable media includes computer-readable storage media and computer-readable communication media.

[0079] Computer-readable storage media includes volatile and nonvolatile, removable and non-removable media implemented in any device configured to store information such as computer-readable instructions, data structures, program modules or other data. Computer-readable storage media includes, but is not limited to, random access memory, read-only memory, electrically erasable programmable read-only memory, flash memory, compact disc read-only memory, digital versatile disks or other optical storage devices, magnetic cassettes, magnetic tape, magnetic disk storage devices or other magnetic storage devices, or any other medium that can be used to store the desired information and can be accessed by a computing device. Computer-readable storage media does not include computer-readable communication media.

[0080] Computer-readable communication media typically embodies computer-readable instructions, data structures, program modules, or other data in the form of a modulated data signal, such as a carrier wave or other transport mechanism, and includes any information delivery media. The term "modulated data signal" refers to a signal that has one or more of its characteristics set or changed in such a way as to encode information in the signal. By way of example, computer-readable communication media include wired media, such as a wired network or direct-wired connection, and wireless media, such as acoustic, radio frequency, infrared, and other wireless media. Any combination of the above is also included within the scope of computer-readable media.

[0081] Figure 9 The computing device 900 shown in FIG. 1 is also an example of a programmable electronic device, which may include one or more such computing devices, and when multiple computing devices are included, such computing devices may be coupled together with a suitable data communication network to jointly perform various aspects disclosed herein.

[0082] Although specific embodiments are described herein, the scope of the present disclosure is not limited to those specific embodiments.The scope of the present disclosure is defined by the following claims and any equivalents thereof.

Claims

1. A flow cytometer for analyzing particles, the flow cytometer comprising: a light source generating a light beam directed toward the interrogation zone; a fluidic system that flows the particles through the light beam in the interrogation zone; an optical system comprising a detector for detecting radiated light from the particles flowing through the light beam in the interrogation zone; as well as Processing circuitry having a non-transitory computer-readable storage medium storing instructions that, when executed by the processing circuitry, cause the processing circuitry to: adjusting one or more control variables of the light source, the fluid system, and the optical system based on a first set of values; acquiring a sequence of waveform data from the particles flowing through the light beam in the interrogation zone under the first set of values ​​of the one or more control variables; and The one or more control variables are adjusted at predetermined intervals to acquire additional sequences of waveform data using different sets of values ​​for the one or more control variables.

2. The flow cytometer according to claim 1, wherein The non-transitory computer-readable storage medium stores additional instructions that, when executed by the processing circuitry, further cause the processing circuitry to: The predetermined interval is determined based on an experiment duration and a number of the different value sets for the one or more control variables.

3. A flow cytometer according to any one of the preceding claims, wherein The non-transitory computer-readable storage medium stores additional instructions that, when executed by the processing circuitry, further cause the processing circuitry to: The waveform data is stored in a single flow cytometry standards file that labels each event in the waveform data with a predefined set of values ​​for the one or more control variables.

4. A flow cytometer according to any one of the preceding claims, wherein The non-transitory computer-readable storage medium stores additional instructions that, when executed by the processing circuitry, further cause the processing circuitry to: The waveform data is acquired as a continuous digital stream of data without thresholding.

5. A flow cytometer according to any one of the preceding claims, wherein The different sets of values ​​for the one or more control variables are received via a graphical user interface.

6. A flow cytometer according to any one of the preceding claims, wherein The experiment duration is based on the total number of events detected by the optical system.

7. A flow cytometer according to any one of the preceding claims, wherein The experimental duration is based on time.

8. A method for analyzing particles flowing through an interrogation zone of a flow cytometer, the method comprising: One or more control variables are adjusted based on the first set of values, the one or more control variables being used to control operation of at least one of: a light source generating a light beam directed toward the interrogation zone; a fluidic system that flows the particles through the light beam in the interrogation zone; as well as an optical system for detecting one or more characteristics of the particles flowing through the light beam in the interrogation zone; acquiring a sequence of waveform data from the particles flowing through the light beam in the interrogation zone under the first set of values ​​of the one or more control variables; and The one or more control variables are adjusted at predetermined intervals to acquire additional sequences of waveform data using different sets of values ​​for the one or more control variables.

9. The method according to claim 8, further comprising: The predetermined interval is determined based on an experiment duration and a number of the different value sets for the one or more control variables.

10. The method according to claim 8 or 9, further comprising: The waveform data is stored in a single flow cytometry standards file that labels each event in the waveform data with a predefined set of values ​​for the one or more control variables.

11. The method according to any one of claims 8 to 10, further comprising: The waveform data is acquired as a continuous digital stream of data without thresholding.

12. The method according to any one of claims 8 to 11, further comprising: The different sets of values ​​for the one or more control variables are received via a graphical user interface.

13. The method according to any one of claims 8 to 12, wherein The experiment duration is based on the total number of events detected by the optical system.

14. The method according to any one of claims 8 to 13, wherein The experimental duration is based on time.

15. A non-transitory computer-readable medium comprising program instructions that, when executed by a processor, cause the processor to: One or more control variables are adjusted based on the first set of values, the one or more control variables being used to control operation of at least one of: a light source generating a light beam directed toward the interrogation zone; a fluidic system that flows particles through the light beam in the interrogation zone; and an optical system for detecting one or more characteristics of the particles flowing through the light beam in the interrogation zone; acquiring a sequence of waveform data from the particles flowing through the light beam in the interrogation zone under the first set of values ​​of the one or more control variables; and The one or more control variables are adjusted at predetermined intervals to acquire additional sequences of waveform data using different sets of values ​​for the one or more control variables until the experiment duration ends.

16. The non-transitory computer readable medium of claim 15, further comprising additional program instructions that, when executed by a processor, further cause the processor to: The predetermined interval is determined based on the experiment duration and the number of the different value sets of the one or more control variables.

17. The non-transitory computer readable medium according to claim 15 or 16, further comprising program instructions that, when executed by a processor, further cause the processor to: The waveform data is stored in a single flow cytometry standards file that labels each event in the waveform data with a predefined set of values ​​for the one or more control variables.

18. The non-transitory computer readable medium according to any one of claims 15 to 17, further comprising program instructions that, when executed by a processor, further cause the processor to: The waveform data is acquired as a continuous digital stream of data without thresholding.

19. The non-transitory computer readable medium according to any one of claims 15 to 18, further comprising program instructions that, when executed by a processor, further cause the processor to: The different sets of values ​​for the one or more control variables are received via a graphical user interface.

20. The non-transitory computer readable medium of any one of claims 15 to 19, wherein: The experiment duration is based on time or the total number of events detected by the optical system.

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