Method and system for modulating detector gain

Abstract

Aspects of the present disclosure include methods for modulating a photodetector in a light detection system, such as in a flow cytometer. A method according to certain embodiments includes irradiating a sample having particles in a flowing stream with a light source, detecting light from the particles in the sample with a photodetector, where a voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency, a data signal is generated from light from the particle detected at one or more of the first photodetector voltage and the second photodetector voltage, and one or more parameters of the particle are determined from the generated data signal. Systems and non-transitory computer-readable storage media configured to perform the subject methods are also provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 699,320, filed September 26, 2024, pursuant to 35 U.S. SC §119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a method and system for modulating detector gain. Background Technology

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

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

[0006] The separation of biological particles has been achieved by adding sorting or collection capabilities to flow cytometers. Particles detected in the separation stream as having one or more desired properties are individually separated from the sample stream by mechanical or electrical removal. A common flow cytometry sorting technique utilizes droplet sorting, in which a fluid stream containing linearly separated particles breaks into droplets. The droplet containing the particle of interest becomes charged and is deflected into a collection tube by passing through an electric field. Typically, linearly separated particles in the stream are characterized as they pass through an observation point located directly below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, the time it takes for it to reach the droplet break-off point and break into droplets from the stream can be predicted. Ideally, a brief charge is applied to the fluid stream before the droplet containing the selected particle breaks off from the stream, and then grounded immediately after the droplet breaks off. The droplet to be sorted retains its charge upon breaking off from the fluid stream, while all other droplets remain uncharged.

[0007] Flow cytometers use arbitrary units of measurement signal. They are capable of analyzing a wide range of cell types across large sizes and refractive indexes. For any flow cytometry experiment, the most common parameter for triggering data and initial optimization is light scattering. Optimal tuning of these parameters depends on the population of interest, often requires training, and individual differences can be subtle or subjective when trade-offs must be made due to the limited dynamic range of the detector. The optical detection system in a conventional flow cytometer has a dynamic range of approximately four orders of magnitude at a given setting, while the range of detectable cell sizes covered by flow cytometry applications spans at least seven orders of magnitude. The detection limits of photodetectors can vary by about 4.5 orders of magnitude across their available settings, meaning their total dynamic range can span as much as eight to nine orders of magnitude. Summary of the Invention

[0008] This disclosure includes methods for using a photodetector in a modulated light detection system (e.g., in a flow cytometer). The method according to some embodiments includes: illuminating a sample having particles in a flowing stream with a light source; detecting light from the particles in the sample with a photodetector, wherein the voltage of the photodetector is modulated at a modulation frequency between a first voltage and a second voltage; generating a data signal based on the light from the particles detected at one or more of the first and second photodetector voltages; and determining one or more parameters of the particles based on the generated data signal. A system and a non-transitory computer-readable storage medium configured to perform the subject method are also provided.

[0009] In some embodiments, the voltage of the photodetector is modulated between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some instances, the voltage of the photodetector is modulated between the first voltage and the second voltage at a frequency of 0.1 MHz to 1 MHz. In some instances, the voltage of the photodetector is maintained for 1 μs or less per cycle, for example, 0.5 μs or less per cycle. In some embodiments, the voltage of the first photodetector is higher than the voltage of the second photodetector. In some instances, the voltage of the first photodetector is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some instances, the voltage of the second photodetector is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0010] In some embodiments, the voltage of the photodetector is modulated between a square voltage variation and a first photodetector voltage and a second photodetector voltage. In some instances, the method includes generating a data signal based on light detected at the first photodetector voltage. In some instances, the method includes generating a data signal based on light detected at the second photodetector voltage. In some instances, the method includes generating data signals based on both light detected at the first photodetector voltage and light detected at the second photodetector voltage. In some instances, a data signal is generated based on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some instances, a data signal is generated only based on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some instances, the method includes detecting light with the photodetector only at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0011] In some embodiments, the method includes measuring baseline samples at a first photodetector voltage and a second photodetector voltage. In some instances, a data signal is also generated based on the baseline samples. In some instances, the method includes baseline sampling at a predetermined frequency. In some instances, the method includes measuring high-frequency baseline samples at a baseline frequency. In some instances, the method includes measuring baseline samples at a baseline frequency from 0.0001 MHz to 15 MHz. In some instances, the baseline sampling frequency is greater than the modulation frequency. In some instances, the baseline sampling frequency is less than the modulation frequency. In some instances, the baseline sampling frequency is equal to the modulation frequency. In some embodiments, the generated data signal is scaled. In some instances, the data signal is scaled on a single continuous scale and the generated data signal is divided by the photodetector gain at each photodetector voltage.

[0012] In some embodiments, one or more parameters of the particles in the sample are determined based on data signals generated from light detected at a first photodetector voltage. In some instances, one or more parameters of the particles in the sample are determined based on data signals generated from light detected at a second photodetector voltage. In some instances, one or more parameters of the particles in the sample are determined based on data signals generated from light detected at both the first and second photodetector voltages.

[0013] In some embodiments, light is detected in multiple photodetector channels. In some instances, the detected light is scattered light, such as forward-scattered light, side-scattered light, or a combination thereof. In some instances, the method includes illuminating a sample with a light source. In some instances, the light source includes a laser, such as multiple lasers.

[0014] This disclosure also includes systems for performing subject-matter methods, such as modulating a photodetector in a light detection system (e.g., in a flow cytometer). A system according to some embodiments includes: a light source configured to illuminate a sample having particles in a flowing stream; and a light detection system having a photodetector for detecting light from the particles in the sample, wherein the photodetector is configured to modulate between a first photodetector voltage and a second photodetector voltage, and to generate a data signal based on the light from the particles detected at one or more of the first and second photodetector voltages. In embodiments, the system further includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate the data signal in response to determining one or more parameters of the particles based on the generated data signal.

[0015] In some embodiments, the voltage of the photodetector is modulated between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some instances, the voltage of the photodetector is modulated between the first voltage and the second voltage at a frequency of 0.1 MHz to 1 MHz. In some instances, the photodetector is configured to maintain a voltage of 1 μs or less per cycle, for example, 0.5 μs or less per cycle. In some embodiments, the voltage of the first photodetector is higher than that of the second photodetector. In some instances, the voltage of the first photodetector is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some instances, the voltage of the second photodetector is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0016] In some embodiments, the light detection system is configured to modulate the voltage of the photodetector between a square voltage variation and a first photodetector voltage and a second photodetector voltage. In some instances, the photodetector is configured to generate a data signal based on light detected at the first photodetector voltage. In some instances, the photodetector is configured to generate a data signal based on light detected at the second photodetector voltage. In some instances, the photodetector is configured to generate a data signal based on both light detected at the first photodetector voltage and light detected at the second photodetector voltage. In some instances, the photodetector is configured to generate a data signal based on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some instances, the photodetector is configured to generate a data signal based only on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some instances, the photodetector is configured to detect light only at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0017] In some embodiments, the memory includes instructions that, when executed by a processor, cause the processor to measure baseline samples at a first photodetector voltage and a second photodetector voltage. In some instances, the memory includes instructions for generating a data signal based on the baseline samples. In some instances, the memory includes instructions for measuring the baseline samples at a baseline sampling frequency. In some instances, the memory includes instructions for measuring the baseline samples at frequencies from 0.0001 MHz to 15 MHz. In some instances, the memory includes instructions for measuring the baseline samples of the generated data signal at a frequency greater than the modulation frequency. In some instances, the memory includes instructions for measuring the baseline samples of the generated data signal at a frequency less than the modulation frequency. In some instances, the memory includes instructions for measuring the baseline samples of the generated data signal at a frequency equal to the modulation frequency. In some embodiments, the memory includes instructions for scaling the generated data signal. In some instances, the memory includes instructions for scaling the data signal on a single continuous scale and dividing the generated data signal by the photodetector gain at each photodetector voltage.

[0018] In some embodiments, the memory includes instructions for determining one or more parameters of particles in the sample based on data signals generated from light detected at a first photodetector voltage. In some instances, the memory includes instructions for determining one or more parameters of particles in the sample based on data signals generated from light detected at a second photodetector voltage. In some instances, the memory includes instructions for determining one or more parameters of particles in the sample based on data signals generated from light detected at both the first and second photodetector voltages.

[0019] In some embodiments, the light detection system includes a photodetector configured to detect scattered light. In some instances, the photodetector is a side-scattering photodetector. In some instances, the photodetector is a forward-scattering photodetector. In some instances, the system is a flow cytometer.

[0020] This disclosure also includes non-transitory computer-readable storage media, such as for practicing one or more computer-implemented methods described herein. In some embodiments, the non-transitory computer-readable storage medium includes: an algorithm for illuminating a sample comprising particles in a flowing stream with a light source; an algorithm for detecting light from the particles in the sample using a light detection system including a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency; an algorithm for generating a data signal based on the light from the particles detected at one or more of the first photodetector voltage and the second photodetector voltage; and an algorithm for determining one or more parameters of the particles based on the generated data signal.

[0021] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for modulating the voltage of a photodetector between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some instances, the non-transitory computer-readable storage medium includes an algorithm for modulating the voltage of a photodetector between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz. In some instances, the non-transitory computer-readable storage medium includes an algorithm for maintaining the voltage of the photodetector for 1 μs or less per cycle, for example, 0.5 μs or less per cycle. In some embodiments, the first photodetector voltage is higher than the second photodetector voltage. In some instances, the first photodetector voltage is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some instances, the second photodetector voltage is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0022] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for modulating a photodetector voltage between a first photodetector voltage and a second photodetector voltage with a square voltage variation. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light detected at the first photodetector voltage. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light detected at the second photodetector voltage. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on both light detected at the first photodetector voltage and light detected at the second photodetector voltage. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based solely on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some instances, the non-transitory computer-readable storage medium includes an algorithm for detecting light with a photodetector only at a second photodetector voltage when the light detected at a first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0023] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for measuring baseline samples of the generated data signal at each of a first photodetector voltage and a second photodetector voltage. In some instances, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on the baseline samples. In some instances, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples at a baseline sampling frequency. In some instances, the non-transitory computer-readable storage medium includes an algorithm for performing baseline sampling at frequencies from 0.0001 MHz to 15 MHz. In some instances, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples of the generated data signal at a frequency greater than the modulation frequency. In some instances, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples of the generated data signal at a frequency less than the modulation frequency. In some instances, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples of the generated data signal at a frequency equal to the modulation frequency. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for scaling the generated data signal. In some instances, the non-transitory computer-readable storage medium includes algorithms for scaling a data signal on a single continuous scale and dividing the resulting data signal by the photodetector gain at each photodetector voltage.

[0024] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample based on a data signal generated from light detected at a first photodetector voltage. In some instances, the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample based on a data signal generated from light detected at a second photodetector voltage. In some instances, the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample based on data signals generated from light detected at a first photodetector voltage and light detected at a second photodetector voltage. Attached Figure Description

[0025] This disclosure is best understood by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures:

[0026] Figure 1A A flowchart is depicted according to certain embodiments to determine one or more parameters of particles in a flowing stream based on light detected by a modulated photodetector. Figure 1B The image depicts the detection of light from particles in a sample using a modulated photodetector, according to certain embodiments.

[0027] Figure 2 A flow cytometry system according to certain embodiments is shown.

[0028] Figure 3 An image-enabled particle sorter according to certain embodiments is described.

[0029] Figure 4 A functional block diagram of a particle analysis system according to certain embodiments is depicted.

[0030] Figure 5 A functional block diagram of an example control system according to certain embodiments is depicted.

[0031] Figures 6A-6B A schematic diagram of a particle sorting system according to certain embodiments is depicted.

[0032] Figure 7 Aspects of a computer control system according to certain embodiments are described. Detailed Implementation

[0033] This disclosure includes methods for using a photodetector in a modulated light detection system (e.g., in a flow cytometer). The method according to some embodiments includes: illuminating a sample having particles in a flowing stream with a light source; detecting light from the particles in the sample with a photodetector, wherein the voltage of the photodetector is modulated at a modulation frequency between a first voltage and a second voltage; generating a data signal based on the light from the particles detected at one or more of the first and second photodetector voltages; and determining one or more parameters of the particles based on the generated data signal. A system and a non-transitory computer-readable storage medium configured to perform the subject method are also provided.

[0034] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, which may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure will be limited only by the appended claims.

[0035] When a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (accurate to one-tenth of the lower limit unit unless otherwise expressly specified by the context), as well as any other specified value or intermediate value within that range, is included in this invention. The upper and lower limits of these smaller ranges can be independently included in the smaller range and also in this invention, subject to any specific exclusions within the range. When the range contains one or both limit values, the range excluding any or both of the included limit values ​​is also included in this invention.

[0036] Certain ranges are presented in this document with the term "approximately" preceding the numerical value. The term "approximately" is used to provide textual support for the exact numerical value preceding it, as well as for values ​​that are close to or approximate to the preceding term. In determining whether a numerical value is close to or approximate to a specifically listed numerical value, the unlisted value that is close to or approximates should be a value that is substantially equivalent to the specifically listed value in the context presented.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, only representative exemplary methods and materials are described herein.

[0038] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were expressly and individually indicated to be incorporated herein by reference, and to disclose and describe technical methods and / or materials of interest in the referenced publications. Any reference to a publication is based on its publication date being earlier than the filing date of this application and should not be construed as an admission that the invention is not entitled to precede that publication by a prior disclosure. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0039] It should be noted that, unless the context clearly specifies otherwise, the use of an element in this document and the appended claims without a defined quantity means that at least one of that element is present. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a preliminary basis for the use of exclusive terms such as "unique," "only," etc., when referencing claim elements or using the "negative" limitation.

[0040] It will be apparent to those skilled in the art upon reading this invention that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any enumerated methods may be performed in the order of the enumerated events, or in any other logically feasible order.

[0041] Although the apparatus and method have been or will be described in conjunction with functional interpretation for grammatical fluency, it should be clearly understood that, unless expressly provided in 35 U.SC §112, the claims should not be construed in any way as necessarily limited to “apparatus” or “step”, but should be given the full range of meaning and equivalents provided by the definition of the claims in accordance with the principle of judicial equivalence, and if the claims are expressly provided in 35 U.SC §112, they should be given the full range of legal equivalents in accordance with 35 U.SC §112, etc.

[0042] Method for photodetectors in modulated light detection systems

[0043] This disclosure includes methods for modulating a photodetector in a light detection system (e.g., in a flow cytometer). In embodiments, modulating the photodetector improves sensitivity in detecting particles of different sizes and the brightness of fluorophores used for fluorescence measurements. In some embodiments, the subject method provides an extension of the dynamic range of the photodetector. The term dynamic range is used herein in its conventional sense, referring to the range of responses of the photodetector, such as the difference between its noise floor (lower threshold) and saturation intensity (upper threshold). In some embodiments, the subject method and system increase the dynamic range of the photodetector by 10% or more, for example, by 25% or more, for example, by 50% or more, for example, by 75% or more, for example, by 100% or more, for example, by 150% or more, for example, by 200% or more, for example, by 250% or more, and includes increasing the dynamic range of the photodetector by 300% or more. In some instances, as described herein, modulating the photodetector extends its dynamic range by 0.5 orders of magnitude or more, for example, 1 order of magnitude or more, 1.5 orders of magnitude or more, 2 orders of magnitude or more, 2.5 orders of magnitude or more, 3 orders of magnitude or more, 3.5 orders of magnitude or more, 4 orders of magnitude or more, 4.5 orders of magnitude or more, 5 orders of magnitude or more, 7.5 orders of magnitude or more, and including 10 orders of magnitude or more. In some instances, the total dynamic range of the photodetector in the system of interest is between 4 and 12 orders of magnitude, for example, between 6 and 11 orders of magnitude, between 7 and 10 orders of magnitude, and including 8 and 9 orders of magnitude. In some instances, the subject method and system reduce detector saturation when detecting light from an illuminated sample. In some instances, the subject method and system reduce photodetector noise in the data signal of light detected from an illuminated sample.

[0044] In some embodiments, the photodetector, as described in this disclosure, is modulated to increase the size of detectable particles (e.g., cells) by 10% or more, for example, 25% or more, 50% or more, 75% or more, 1.5 times or more, 2 times or more, 5 times or more, and including 10 times or more. In some instances, simultaneous detection of very dark fluorophores and very bright fluorophores (e.g., in samples with different types of particles and fluorescent markers) is provided, for example, the detected fluorescence signal intensity differs between fluorophore types by 50% or more, for example, 75% or more, 100% or more, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 25 times or more, 50 times or more, and including 100 times or more.

[0045] In some embodiments, the subject method provides optimized photodetector system performance, such as improving the signal-to-noise ratio (SNR) of the optical detection system. For example, the SNR of the optical detection system may be improved by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including improvements of 99% or more. In some instances, the subject method improves the SNR by 2x or more, such as 3x or more, such as 4x or more, such as 5x or more, and including improvements of 10x or more. In some embodiments, the subject method improves the output consistency of the photodetector in the optical detection system by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including improvements of 99% or more.

[0046] In practicing the subject method, a sample containing particles is illuminated with light from a light source (e.g., in a flow stream of a flow cytometer). In some embodiments, the light source is a broadband light source that emits light with a wide range of wavelengths, such as spanning 50 nm or more, for example 100 nm or more, for example 150 nm or more, for example 200 nm or more, for example 250 nm or more, for example 300 nm or more, for example 350 nm or more, for example 400 nm or more, and including spanning 500 nm or more. For example, a suitable broadband light source emits light with wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with wavelengths from 400 nm to 1000 nm. When the method involves illumination with a broadband light source, the broadband light source schemes of interest may include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with a continuous spectrum, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof.

[0047] In other embodiments, the method includes illumination with a narrowband light source emitting a specific wavelength or a narrow range of wavelengths, such as illumination with a light source emitting a narrow wavelength range (e.g., 50 nm or less, e.g., 40 nm or less, e.g., 30 nm or less, e.g., 25 nm or less, e.g., 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less, e.g., 5 nm or less, e.g., 2 nm or less), and also includes illumination with a light source emitting a specific wavelength of light (i.e., monochromatic light). When the method includes illumination with a narrowband light source, the narrowband light source scheme of interest may include, but is not limited to, narrow-wavelength LEDs, laser diodes, or broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0048] In some embodiments, the method includes irradiating the sample with one or more lasers. As described above, the type and number of lasers will vary depending on the sample and the light to be collected, and can be gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other instances, the method includes irradiating the flow with a dye laser, such as a stilbene dye laser, a coumarin dye laser, or a rhodamine dye laser. In still other instances, the method includes irradiating the flow with a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, or combinations thereof. In other instances, the method involves irradiating the flow with a solid-state laser, such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:yCa4O(BO3)3 laser, an Nd:YCOB laser, a titanite sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium₂O₃ laser, or a cerium-doped laser, or combinations thereof.

[0049] The sample can be illuminated with one or more of the aforementioned light sources, such as two or more light sources, three or more light sources, four or more light sources, five or more light sources, and including ten or more light sources. The light sources can include combinations of any type of light source. For example, in some embodiments, the method includes illuminating the sample in the flowing stream with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0050] The sample can be illuminated with light ranging from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including 400 nm to 800 nm. For example, when the light source is a broadband light source, the sample can be illuminated with light ranging from 200 nm to 900 nm. In other instances, when the light source comprises multiple narrowband light sources, the sample can be illuminated with specific wavelengths in the range of 200 nm to 900 nm. For example, the light source can be multiple narrowband LEDs (1 nm-25 nm), each independently emitting light with a wavelength range between 200 nm and 900 nm. In other embodiments, the narrowband light source comprises one or more lasers (e.g., a laser array) and illuminates the sample with specific wavelengths ranging from 200 nm to 700 nm, for example using a laser array having gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers as described above.

[0051] When using more than one light source, the sample can be illuminated simultaneously or sequentially, or a combination thereof. For example, the sample can be illuminated simultaneously with each light source. In other embodiments, the flow stream is illuminated sequentially with each light source. When illuminating the sample sequentially with more than one light source, the duration of illumination for each light source can be independently 0.001 microseconds or longer, for example, 0.01 microseconds or longer, for example, 0.1 microseconds or longer, for example, 1 microsecond or longer, for example, 5 microseconds or longer, for example, 10 microseconds or longer, for example, 30 microseconds or longer, and includes 60 microseconds or longer. For example, the method can include illuminating the sample with a light source (e.g., a laser) for a duration from 0.001 microseconds to 100 microseconds, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microsecond to 25 microseconds, and includes 5 microseconds to 10 microseconds. In embodiments where the sample is illuminated sequentially with two or more light sources, the duration of illumination for each light source can be the same or different.

[0052] The time interval between each light source illumination can also vary as needed, independently spaced by a delay of 0.001 microseconds or longer, such as 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 15 microseconds or longer, 30 microseconds or longer, and including 60 microseconds or longer. For example, the time interval between each light source illumination can range from 0.001 microseconds to 60 microseconds, such as from 0.01 microseconds to 50 microseconds, such as from 0.1 microseconds to 35 microseconds, such as from 1 microsecond to 25 microseconds, and including from 5 microseconds to 10 microseconds. In some embodiments, the time interval between each light source illumination is 10 microseconds. In embodiments where the sample is sequentially illuminated by more than two (i.e., three or more) light sources, the delay between each light source illumination can be the same or different.

[0053] The sample can be illuminated continuously or at discrete intervals. In some instances, the method involves illuminating the sample continuously with a light source. In other instances, the sample is illuminated with a light source at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including once every 1000 milliseconds, or some other interval.

[0054] Depending on the light source, the sample can be illuminated from varying distances, such as 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. Furthermore, the illumination angle can also vary, ranging from 10° to 90°, such as from 15° to 85°, from 20° to 80°, from 25° to 75°, and including from 30° to 60°, such as at a 90° angle.

[0055] In some embodiments, the method includes illuminating a sample with two or more frequency-shifted beams of light. As described above, a beam generator assembly having a laser and an acousto-optic device for frequency-shifting the laser can be employed. In these embodiments, the method includes illuminating the acousto-optic device with a laser. Depending on the desired wavelength of the light generated in the output laser beam (e.g., for illuminating a sample in a flowing stream), the laser can have a specific wavelength varying from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including from 400 nm to 800 nm. The acousto-optic device can be illuminating with one or more lasers, for example two or more lasers, for example three or more lasers, for example four or more lasers, for example five or more lasers, and including ten or more lasers. The lasers can include any type of laser combination. For example, in some embodiments, the method includes illuminating the acousto-optic device with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0056] When using more than one laser, the lasers can be used to irradiate the acousto-optic device simultaneously, sequentially, or in combination. For example, each laser can be used to irradiate the acousto-optic device simultaneously. In other embodiments, each laser is used to irradiate the acousto-optic device sequentially. When using more than one laser to irradiate the acousto-optic device sequentially, the duration for which each laser irradiates the acousto-optic device can be independently 0.001 microseconds or longer, for example, 0.01 microseconds or longer, for example, 0.1 microseconds or longer, for example, 1 microsecond or longer, for example, 5 microseconds or longer, for example, 10 microseconds or longer, for example, 30 microseconds or longer, and includes 60 microseconds or longer. For example, the method can include irradiating the acousto-optic device with lasers for a duration from 0.001 microseconds to 100 microseconds, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microsecond to 25 microseconds, and includes 5 microseconds to 10 microseconds. In embodiments where two or more lasers are used to sequentially irradiate an acousto-optic device, the duration of irradiation by each laser on the acousto-optic device may be the same or different.

[0057] The time interval between each laser irradiation can also vary as needed, independently spaced by a delay of 0.001 microseconds or longer, such as 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 15 microseconds or longer, 30 microseconds or longer, and including 60 microseconds or longer. For example, the time interval between each light source irradiation can range from 0.001 microseconds to 60 microseconds, such as from 0.01 microseconds to 50 microseconds, such as from 0.1 microseconds to 35 microseconds, such as from 1 microsecond to 25 microseconds, and including from 5 microseconds to 10 microseconds. In some embodiments, the time interval between each laser irradiation is 10 microseconds. In embodiments where more than two (i.e., three or more) lasers are sequentially irradiated into the acousto-optic device, the delay between each laser irradiation can be the same or different.

[0058] The acousto-optic device can be irradiated continuously or at discrete intervals. In some instances, the method involves irradiating the acousto-optic device continuously with a laser. In other instances, the acousto-optic device is irradiated with a laser at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including once every 1000 milliseconds, or some other interval.

[0059] Depending on the laser, the acousto-optic device can be irradiated from varying distances, such as 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. Furthermore, the irradiation angle can also vary, ranging from 10° to 90°, such as from 15° to 85°, from 20° to 80°, from 25° to 75°, and including from 30° to 60°, such as at a 90° angle.

[0060] In some embodiments, the method includes applying radio frequency (RF) drive signals to an acousto-optic device to generate an angle-deflected laser beam. Two or more RF drive signals may be applied to the acousto-optic device to generate an output laser beam having a desired number of angle-deflected laser beams, such as three or more RF drive signals, four or more RF drive signals, five or more RF drive signals, six or more RF drive signals, seven or more RF drive signals, eight or more RF drive signals, nine or more RF drive signals, ten or more RF drive signals, fifteen or more RF drive signals, twenty-five or more RF drive signals, fifty or more RF drive signals, and including one hundred or more RF drive signals.

[0061] Each angle-deflecting laser beam generated by a radio frequency (RF) drive signal has an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal with a sufficient amplitude to generate an angle-deflecting laser beam with a desired intensity. In some instances, each applied RF drive signal independently has an amplitude of about 0.001V to about 500V, for example, about 0.005V to about 400V, for example, about 0.01V to about 300V, for example, about 0.05V to about 200V, for example, about 0.1V to about 100V, for example, about 0.5V to about 75V, for example, about 1V to 50V, for example, about 2V to 40V, for example, 3V to about 30V, and includes about 5V to about 25V. In some instances, each applied radio frequency drive signal independently has an amplitude of about 0.001V to 100V, for example, about 0.001V to 200V, for example, 0.001V to 300V, for example, 0.001V to 400V, and includes 0.001V to 500V. Each applied radio frequency drive signal in some embodiments has a frequency of about 0.001MHz to about 500MHz, for example, about 0.005MHz to about 400MHz, for example, about 0.01MHz to about 300MHz, for example, about 0.05MHz to about 200MHz, for example, about 0.1MHz to about 100MHz, for example, about 0.5MHz to about 90MHz, for example, about 1MHz to about 75MHz, for example, about 2MHz to about 70MHz, for example, about 3MHz to about 65MHz, for example, about 4MHz to about 60MHz, and includes about 5MHz to about 50MHz. Each applied radio frequency drive signal has a frequency of about 0.001 MHz to about 100 MHz in some embodiments, such as 0.001 MHz to 200 MHz, such as 0.001 MHz to 300 MHz, such as 0.001 MHz to 400 MHz, such as 0.001 MHz to 500 MHz.

[0062] In these embodiments, the angle-deflecting laser beams in the output laser beam are spatially separated. Depending on the applied RF drive signal and the desired illumination profile of the output laser beam, the angle-deflecting laser beams may be separated by 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, and including 5000 μm or more. In some embodiments, the angle-deflecting laser beams overlap, for example, with adjacent angle-deflecting laser beams on the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, and includes 100 μm or more.

[0063] In some instances, a flow is illuminated with multi-beam frequency-shifted light, and particles in the flow are imaged, as described in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013), and as described in U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,036,699; 10,078,045; 10,222,316; 10,288,546; 10,324,019; 10,408,758; 10,451,538; 10,620,111; 10,684 The disclosures of these patents are described in ,211; 10,845,295; 10,935,482; 10,935,485; 11,105,728; 11,280,718; 11,327,016; 11,366,052; 11,371,937; 11,692,926; 11,630,053; 11,774,343; 11,940,369; and 11,946,851; the disclosures of these patents are incorporated herein by reference.

[0064] During the execution of the subject method, light from each particle is detected by a photodetector system. In embodiments, the photodetector system includes one or more photodetectors, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, and including ten or more photodetectors. The photodetectors used to execute the subject method can be any convenient light detection scheme, including but not limited to photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-mode charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or combinations thereof, and other photodetectors. In some embodiments, the photodetector is a photomultiplier tube, for example, having an effective detection surface area per region ranging from 0.01 cm². 2 up to 10cm 2 Photomultiplier tubes, for example, from 0.05 cm 2 up to 9cm 2 For example, from 0.1cm 2 up to 8cm 2 For example, from 0.5cm 2 up to 7cm 2 And including from 1cm 2 up to 5cm 2 Detects light from the illuminated sample in two or more photodetector channels, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 24 or more, 32 or more, 64 or more, 128 or more, 256 or more, and includes 512 or more photodetector channels.

[0065] Light can be measured using a photodetector at one or more wavelengths, such as at two or more wavelengths, at five or more different wavelengths, at ten or more different wavelengths, at 25 or more different wavelengths, at 50 or more different wavelengths, at 100 or more different wavelengths, at 200 or more different wavelengths, at 300 or more different wavelengths, and including at 400 or more different wavelengths. Light can be measured continuously or at discrete intervals. In some instances, the detector of interest is configured to measure light continuously. In other instances, the detector of interest is configured to perform measurements at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, and including every 1000 milliseconds, or some other interval.

[0066] In some embodiments, the light detected from the sample is scattered light. The term "scattered light" is used herein in its conventional sense, referring to light energy propagating from particles in the sample (e.g., flowing in a flow stream) that is deflected from the path of the incident beam, for example, by reflection, refraction, or deflection. In some instances, the scattered light detected from particles in the flow stream is forward scattered light (FSC). In other instances, the scattered light detected from particles in the flow stream is side scattered light (SSC). And in still other instances, the scattered light detected from particles in the flow stream is backscattered light (BSC).

[0067] In some embodiments, the light detected from each particle in the sample is transmitted light, such as light detected using a bright-field detector. In other embodiments, the light detected from each particle in the sample is emitted light, such as particle luminescence (i.e., fluorescence or phosphorescence). In these embodiments, each particle may include one or more fluorophores that emit fluorescence in response to illumination from two or more light sources. For example, each particle may include two or more fluorophores, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, and ten or more fluorophores. In some instances, each particle includes a first fluorophore that emits fluorescence in response to illumination from a first laser, and a second fluorophore that emits fluorescence in response to illumination from a second laser. In some embodiments, the fluorophore of interest may include, but is not limited to, dyes suitable for analytical applications (e.g., flow cytometry, imaging, etc.), such as acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone imine dyes, azazine dyes, uronine dyes, saffron dyes, indigoamine, indophenol dyes, fluorane dyes, oxazine dyes, oxazolone dyes, thiazine dyes, thiazolium dyes, xanthan dyes, fluorene dyes, pyranine dyes, fluorane dyes, rhodamine dyes, phenanthridine dyes, as well as dyes combining two or more of the above dyes (e.g., tandem dyes), polymer dyes having one or more monomer dye units, and mixtures of two or more of the above dyes. A wide variety of 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 may include 4-acetamido-4'-isothiocyanate stilbene-2,2'-disulfonic acid; acridine and its derivatives, such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, polydinocyanin-chlorophyll, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalenedicarboximide-3,5-disulfonic acid (Lucifer YellowVS); N-(4-anilino-1-naphthyl)maleimide; aminoanisinolamide; brilliant yellow; coumarins and their derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 120). 151); cyanine and its derivatives, such as cyanin, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-diamidinyl-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfophthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriaminepentaacetic acid ester; 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl) chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and its derivatives, such as eosin and eosin isothiocyanate; erythrosine and its derivatives, such as erythrosine B and erythrosine isothiocyanate; ethidium bromide; fluorescein and its derivatives, such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein and QFITC ​​(XRITC); fluorescent amine; IR144; IR1446; green fluorescent protein (GFP); reef coral fluorescent protein (RCFP); lissamine. TM Rhodamine lysamide, Lucifer yellow; Malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; paracinon; Nile red; Oregon green; phenol red; β-phycoerythrin; o-phthalaldehyde; pyrene and its derivatives, such as pyrene, pyrenebutyric acid and succinimide-1-pyrenebutyric acid; Reactive Red 4 (Cibacron) TMBrilliant Red 3B-A); Rhodamine and its derivatives, such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine sulfonylamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulfonylrhodamine B, sulfonylrhodamine 101, and sulfonylrhodamine 101 sulfonyl chloride derivatives (Texas) Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosehip acid and terbium chelate derivatives; xanthones; dye conjugated polymers (i.e., polymer-attached dyes), such as fluorescein isothiocyanate-dextran and dyes combining two or more dyes (e.g., tandem dyes), polymer dyes having one or more monomer dye units, and mixtures or combinations of the above two or more dyes.

[0068] In some instances, the fluorophore (i.e., the dye) is a fluorescent polymer dye. The fluorescent polymer dyes used in the subject methods and systems are diverse. In some instances of the methods, polymer dyes include conjugated polymers. Conjugated polymers (CPs) are characterized by a delocalized electronic structure comprising a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated bonds (e.g., single bonds), where π electrons can move from one bond to another. Therefore, the conjugated backbone can endow polymer dyes with extended linear structures and finite bond angles between polymer repeating units. For example, proteins and nucleic acids, while also polymers, do not form extended rod-like structures in some cases, but rather fold into more advanced three-dimensional shapes. Furthermore, CPs may form “rigid rod” polymer backbones, and experience finite torsional (e.g., twist) angles between monomer repeating units along the polymer backbone. In some instances, polymer dyes include CPs with rigid rod structures. As mentioned above, the structural properties of polymer dyes can influence the fluorescence properties of the molecule.

[0069] Any readily available polymer dye can be used in the subject methods and systems. In some instances, the polymer dye is a polychromatic with a structure capable of collecting light to amplify the fluorescence output of a fluorophore. In some instances, the polymer dye is capable of collecting light and efficiently converting it into emitted light at longer wavelengths. In some cases, the polymer dye has a light-collecting polychromatic system that can efficiently transfer energy to a nearby luminescent species (e.g., a “signal chromatic”). Energy transfer mechanisms include, for example, resonant energy transfer (e.g., Forster (or fluorescent) resonant energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some instances, these energy transfer mechanisms are relatively short-range; that is, the close proximity of the light-collecting polychromatic system to the signal chromatic provides efficient energy transfer. Under conditions of efficient energy transfer, the emission of the signal chromatic is amplified when the number of individual chromatics in the light-collecting polychromatic system is large; that is, the emission of the signal chromatic is more intense when the incident light (“excitation light”) is at a wavelength absorbed by the light-collecting polychromatic system than when the signal chromatic is directly excited by pump light.

[0070] Polychromatic groups can be found in conjugated polymers. Conjugated polymers (CPs) are characterized by delocalized electronic structures and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is significantly shorter than the polymer chain length, the backbone contains a large number of closely spaced conjugated segments. Therefore, conjugated polymers are efficient for light harvesting and can achieve optical amplification through energy transfer.

[0071] In some instances, polymers can be used as direct fluorescent reporters, such as fluorescent polymers with high extinction coefficients, high brightness, etc. In other instances, polymers can be used as strong chromophores, where color or optical density serves as an indicator.

[0072] Polymer dyes of interest include, but are not limited to, those described in Gaylord et al., U.S. Publications 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986, 20130190193, and 20160025735, the disclosures of which are incorporated herein by reference in their entirety; and Gaylord et al., J. Am. Chem. Soc., 2001, 123(26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133(32), pp 6417-6418. The dyes described in 12600-12607 are incorporated herein by reference in their entirety.

[0073] In embodiments, the method includes modulating the voltage of a photodetector between at least a first voltage and a second voltage. In some instances, the photodetector is modulated between three or more voltages, such as four or more, five or more, six or more, seven or more, and including eight or more different voltages. In some instances, the photodetector is modulated (e.g., oscillated) between a first photodetector voltage and a second photodetector voltage. In some embodiments, the voltage of the photodetector is modulated (e.g., oscillated) between the first photodetector voltage and the second photodetector voltage at a frequency of 0.0001 MHz to 15 MHz, such as 0.0005 MHz to 14 MHz, such as 0.001 MHz to 13 MHz, such as 0.005 MHz to 12 MHz, such as 0.01 MHz to 11 MHz, such as 0.05 MHz to 10 MHz, such as 0.1 MHz to 9 MHz, such as 0.5 MHz to 8 MHz, such as 1 MHz to 7 MHz, such as 1 MHz to 6 MHz, and including 1 MHz to 5 MHz. In some instances, the voltage of the photodetector oscillates between the first photodetector voltage and the second photodetector voltage at a frequency of 0.1 MHz to 1 MHz. In some instances, the photodetector voltage is modulated (e.g., oscillated) at frequencies from 0.0001 MHz to 5 MHz, such as 0.0005 MHz to 6 MHz, such as 0.001 MHz to 7 MHz, such as 0.005 MHz to 8 MHz, such as 0.01 MHz to 9 MHz, such as 0.05 MHz to 10 MHz, such as 0.1 MHz to 11 MHz, such as 0.5 MHz to 12 MHz, such as 1 MHz to 13 MHz, such as 1 MHz to 14 MHz, and including 1 MHz to 15 MHz. In some instances, the photodetector voltage is modulated at frequencies from 0.0001 MHz to 1 MHz, for example, 0.0001 MHz to 2 MHz, for example, 0.0001 MHz to 3 MHz, for example, 0.0001 MHz to 4 MHz, for example, 0.0001 MHz to 5 MHz, for example, 0.0001 MHz to 6 MHz, for example, 0.0001 MHz to 7 MHz, for example, 0.0001 MHz to 8 MHz, for example, 0.0001 MHz to 9 MHz, for example, 0.0001 MHz to 10 MHz, for example, 0.0001 MHz to 11 MHz, for example, 0.0001 MHz to 12 MHz, for example, 0.0001 MHz to 13 MHz, for example, 0.0001 MHz to 14 MHz, and including 0.0001 MHz to 15 MHz.

[0074] In some embodiments, the voltage of the photodetector is maintained at that voltage for 25 μs or less in each cycle, for example, 20 μs or less, for example, 15 μs or less, for example, 10 μs or less, for example, 9 μs or less, for example, 8 μs or less, for example, 7 μs or less, for example, 6 μs or less, for example, 5 μs or less, for example, 4 μs or less, for example, 3 μs or less, for example, 2 μs or less, for example, 1 μs or less, for example, 0.9 μs or less, for example, 0.8 μs or less, for example, 0.7 μs or less, for example, 0.6 μs or less, for example, 0.5 μs or less, for example, 0.4 μs or less, for example, 0.3 μs or less, for example, 0.2 μs or less, for example, 0.1 μs or less, and including 0.05 μs or less. In some instances, the voltage of the photodetector is maintained at the first photodetector voltage in each modulation cycle. The term "modulation period" in this document refers to each period between the voltages of the first and second photodetectors. In some instances, the voltage of the photodetector is maintained at the first photodetector voltage for 25 μs or less in each period, for example, 20 μs or less, for example, 15 μs or less, for example, 10 μs or less, for example, 9 μs or less, for example, 8 μs or less, for example, 7 μs or less, for example, 6 μs or less, for example, 5 μs or less, for example, 4 μs or less, for example, 3 μs or less, for example, 2 μs or less, for example, 1 μs or less, for example, 0.9 μs or less, for example, 0.8 μs or less, for example, 0.7 μs or less, for example, 0.6 μs or less, for example, 0.5 μs or less, for example, 0.4 μs or less, for example, 0.3 μs or less, for example, 0.2 μs or less, for example, 0.1 μs or less, and includes 0.05 μs or less. In some instances, the voltage of the photodetector is maintained at the voltage of the second photodetector for 25 μs or less per cycle, for example, 20 μs or less, for example, 15 μs or less, for example, 10 μs or less, for example, 9 μs or less, for example, 8 μs or less, for example, 7 μs or less, for example, 6 μs or less, for example, 5 μs or less, for example, 4 μs or less, for example, 3 μs or less, for example, 2 μs or less, for example, 1 μs or less, for example, 0.9 μs or less, for example, 0.8 μs or less, for example, 0.7 μs or less, for example, 0.6 μs or less, for example, 0.5 μs or less, for example, 0.4 μs or less, for example, 0.3 μs or less, for example, 0.2 μs or less, for example, 0.1 μs or less, and including 0.05 μs or less. In some embodiments, the duration for which the voltage of the photodetector is maintained at the first photodetector voltage is the same as the duration for which it is maintained at the second photodetector voltage. In other embodiments, the duration for which the voltage of the photodetector is maintained at the first photodetector voltage is different from the duration for which it is maintained at the second photodetector voltage.When the duration for which the voltage of the photodetector is maintained at the first photodetector voltage differs from that at the second photodetector voltage, one or more modulation cycles can be maintained at different voltages, such as two or more, three or more, four or more, five or more, ten or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, and including 1000 or more modulation cycles.

[0075] Depending on the type of photodetector (e.g., PMT, photodiode, etc.), in some embodiments, the first photodetector voltage is 250mV to 2500mV, for example 300mV to 2400mV, for example 400mV to 2300mV, for example 500mV to 2200mV, for example 500mV to 2100mV, for example 500mV to 2000mV, for example 500mV to 1900mV, for example 500mV to 1800mV, for example 500mV to 1700mV, for example 500mV to 1600mV, for example 500mV to 1500mV, for example 500mV to 1400mV, for example 500mV to 1300mV, for example 500mV to 1200mV, for example 500mV to 1100mV, and includes 500mV to 1000mV. In some instances, the second photodetector voltage is from 0.0001mV to 1000mV, for example 0.0005mV to 900mV, for example 0.001mV to 800mV, for example 0.005mV to 700mV, for example 0.01mV to 600mV, for example 0.05mV to 500mV, for example 0.1mV to 500mV, for example 0.5mV to 500mV, and includes 1mV to 500mV. In some instances, the second photodetector voltage is 50mV or less, for example 40mV or less, for example 30mV or less, for example 20mV or less, for example 10mV or less, and includes 5mV or less.

[0076] The voltage of the photodetector can be modulated between the first and second photodetector voltages by any convenient waveform variation, such as a sinusoidal voltage variation. In some embodiments, the voltage of the photodetector is modulated between the first and second photodetector voltages by a square voltage variation. In some embodiments, each modulation cycle presents a symmetrical voltage variation (e.g., a full square voltage variation). In other embodiments, one or more modulation cycles present different types of voltage variation waveforms, such as a square voltage variation during one or more modulation cycles and a sinusoidal voltage variation during another. In some instances, the method includes changing the shape of the voltage variation at predetermined intervals, such as after every two or more modulation cycles, for example, after three or more, four or more, five or more, ten or more, 25 or more, 50 or more, 100 or more, and including every 500 or more modulation cycles.

[0077] In embodiments, data signals are generated based on detected light at one or more photodetector voltages. In some instances, two or more data signals are generated at each photodetector voltage during each modulation cycle, such as three or more, four or more, five or more, ten or more, 25 or more, 50 or more, and including generating 100 or more data signals at each photodetector voltage during each modulation cycle. In some instances, light is detected at a photodetector voltage, and the data signals are averaged. In some instances, a data signal is generated at a first photodetector voltage during each modulation cycle. In some instances, a data signal is generated at a second photodetector voltage during each modulation cycle. In some instances, a data signal is generated at both the first and second photodetector voltages during each modulation cycle.

[0078] In some instances, data signals are generated only at the first photodetector voltage during each modulation cycle. For example, two or more data signals are generated only at the first photodetector voltage during each modulation cycle, such as three or more, four or more, five or more, and including generating ten or more data signals at the first photodetector voltage during each modulation cycle. In some instances, data signals are generated only at the second photodetector voltage during each modulation cycle. For example, two or more data signals are generated only at the second photodetector voltage during each modulation cycle, such as three or more, four or more, five or more, and including generating ten or more data signals at the second photodetector voltage during each modulation cycle.

[0079] In some embodiments, the method includes generating a data signal at a second photodetector voltage when light detected at a first photodetector voltage exceeds a saturation threshold of the photodetector, for example, the detected light exceeds the saturation threshold by 5% or more, such as 10% or more, 15% or more, 25% or more, or 50% or more, and when the light detected at the first photodetector voltage exceeds the saturation threshold of the photodetector by 75% or more. In some instances, when light detected at the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold, the method includes generating a data signal only based on light detected at the second photodetector voltage. In some instances, when light detected at the first photodetector voltage exceeds the saturation threshold, an alarm may be generated to indicate that saturation has been exceeded or is approaching a level where the data signal is unreliable or inaccurate. In these instances, the method may also include switching the photodetector voltage to a second photodetector voltage in response to an alarm.

[0080] In some embodiments, the method includes generating a data signal at a first photodetector voltage when the data signal generated at a second photodetector voltage exceeds a detected noise threshold (e.g., the signal-to-noise ratio drops below a predetermined threshold). In some instances, the noise of the photodetector at the second photodetector voltage is sufficient to mask or reduce the reliability of the data signal generated at the second photodetector voltage. In some instances, the method includes generating a data signal at a first photodetector voltage when the data signal generated at the second photodetector voltage is 5% or more below the noise threshold of the photodetector, for example, 10% or more, 15% or more, 25% or more, 50% or more, and when the data signal generated at the second photodetector voltage is 75% or more below the noise threshold of the photodetector. In some instances, when the data signal generated at the second photodetector voltage exhibits excessive noise (e.g., the signal-to-noise ratio drops below a threshold), the method includes generating a data signal solely based on light detected at the first photodetector voltage. In some instances, when the data signal generated at the second photodetector voltage is below a noise threshold, an alarm may be generated to indicate that the signal-to-noise ratio of the data signal generated at the second photodetector voltage is too low or close to a level where the data signal is unreliable or inaccurate. In these examples, the method may also include switching the photodetector voltage to a first photodetector voltage in response to an alarm.

[0081] In some embodiments, the method includes measuring baseline samples at a first photodetector voltage and a second photodetector voltage. In some instances, a data signal is also generated based on the baseline samples. In some instances, baseline noise is calculated at each photodetector voltage during each modulation period. In some instances, the baseline noise is calibrated at each photodetector voltage. In some instances, baseline samples are measured at each photodetector voltage at a baseline sampling frequency. In some instances, the method includes measuring baseline samples at a frequency greater than the modulation frequency. In some instances, the method includes measuring baseline samples at a frequency less than the modulation frequency. In some instances, the method includes measuring baseline samples at a frequency equal to the modulation frequency. In some instances, the method includes measuring a baseline sample at a baseline sampling frequency, such as 0.0001 MHz to 15 MHz, such as 0.0005 MHz to 14 MHz, such as 0.001 MHz to 13 MHz, such as 0.005 MHz to 12 MHz, such as 0.01 MHz to 11 MHz, such as 0.05 MHz to 10 MHz, such as 0.1 MHz to 9 MHz, such as 0.5 MHz to 8 MHz, such as 1 MHz to 7 MHz, such as 1 MHz to 6 MHz, and including 1 MHz to 5 MHz.

[0082] In some instances, the baseline is explained under the first and second photodetector voltages (i.e., dual-mode operation) through fixed-time-dependent analysis. In some instances, the baseline is explained under the first and second photodetector voltages through dynamic frequency domain derivation. In some embodiments, the generated data signal is scaled. In some instances, the data signal is scaled on a continuous scale. In some instances, the data signal is scaled on a single continuous scale, and the data signal generated under one or more of the first and second photodetector voltages is divided by the gain of the detector setting used for modulation. In some instances, the first and second photodetector voltages are scaled as two separate parameters.

[0083] In some embodiments, one or more parameters of the particles in the sample are determined based on data signals generated from light detected at a first photodetector voltage. In some instances, one or more parameters of the particles in the sample are determined based on data signals generated from light detected at a second photodetector voltage. In some instances, one or more parameters of the particles in the sample are determined based on data signals generated from light detected at both the first and second photodetector voltages. In some embodiments, the particle parameters include parameters summarized in Table 1, for example, for generating a gating strategy.

[0084] Table 1

[0085]

[0086]

[0087]

[0088] In some instances, an image of the particles is generated based on the data signal. In some instances, one or more image parameters are calculated based on the generated particle image. In some instances, centroid image parameters are calculated based on the generated image. In some instances, incremental centroid image parameters are calculated based on the generated image. In some instances, diffusivity image parameters are calculated based on the generated image. In some instances, eccentricity image parameters are calculated based on the generated image. In some instances, major axis moment image parameters are calculated based on the generated image. In some instances, maximum intensity image parameters are calculated based on the generated image. In some instances, radial moment image parameters are calculated based on the generated image. In some instances, minor axis moment image parameters are calculated based on the generated image. In some instances, particle size image parameters are calculated based on the generated image. In some instances, total intensity image parameters are calculated based on the generated image. In some instances, particle light loss image parameters are calculated based on the generated image. In some instances, forward scattered light image parameters are calculated based on the generated image. In some instances, side scattered light image parameters are calculated based on the generated image. In some instances, image moments are calculated based on the generated image. The term "image moment" is used in its conventional sense herein, referring to the weighted average of pixel intensities in an image. In some instances, the centroid can be calculated from the image moments of an image. In other instances, cell orientation can be calculated from the image moments of an image. Still in other instances, cell eccentricity can be calculated from the image moments of an image.

[0089] Figure 1AA flowchart illustrating, according to certain embodiments, the determination of one or more parameters of particles in a sample in a flowing stream based on light detected by a modulated photodetector. In step 101, a sample containing particles is illuminated in the flowing stream by a light source. In step 102a, the photodetector of a light detection system is modulated at a predetermined frequency under two different voltage settings. In embodiments, the system is modulated under a first higher voltage setting and a second lower voltage setting. In some instances, the voltage setting is modulated between the first and second photodetector voltages by a square voltage variation. In step 102b, light from the illuminated flowing stream is detected using a modulated photodetector. In step 103b, a data signal is generated based on the detected light under one or more photodetector voltage settings. In some instances, light is detected only under a lower photodetector voltage setting when detector saturation is observed at a higher photodetector voltage setting. In other instances, light is detected only under a higher photodetector voltage setting when the detector exhibits high noise values ​​in the generated data signal, for example, when the signal-to-noise ratio drops below a predetermined threshold. In some embodiments, the method includes measuring a baseline sample of the generated data signal at the first and second photodetector voltage settings (step 103a). In some instances, the baseline can be interpreted for dual-mode detection (under first and second photodetector voltage settings) through fixed-time-dependent analysis or dynamic frequency domain derivation. In step 104, one or more parameters, such as imaging parameters, can be calculated based on the generated data signal. In some instances, imaging parameters include lateral scattering parameters or forward scattering parameters. In some embodiments, the method (step 105) includes generating one or more gating strategies based on particle parameters and sorting the particles (e.g., into two or more different containers).

[0090] Figure 1BThe diagram depicts the detection of light from particles in a sample using a modulated photodetector according to certain embodiments. The photodetector is modulated at two different voltage settings: a high voltage setting and a low voltage setting, showing a square voltage variation between the two different voltage settings. Each figure shows the data signal of a bright signal generated by the photodetector at the high voltage setting (left) and the data signal of a dark signal generated by the photodetector at the low voltage setting (right). As shown in the bright signal diagram, the data signal generated at the low voltage setting is entirely within the detectable photodetector signal amplitude and does not exhibit any saturation. The data signal at the high voltage setting exhibits detector saturation for bright signals. On the other hand, for dark signals, the data signal generated at the low voltage setting exhibits a lower detectable photodetector signal amplitude and may exhibit a low signal-to-noise ratio. The data signal of the dark signal generated at the high voltage setting exhibits a detectable photodetector signal amplitude. Therefore, the modulated photodetector according to embodiments of the present disclosure provides accurate detection of both bright signals (no saturation at the lower voltage setting) and dark signals (sufficient signal-to-noise ratio at the higher voltage setting). Modulation of the photodetector voltage setting as described in this article can provide an increased dynamic range for the photodetector, thereby improving the detectable signal amplitude of the photodetector. Figure 1B As shown.

[0091] In some instances, the samples analyzed in this method are biological samples. The term "biological sample," used in its conventional sense, refers to a subset, cell, or component of a whole organism, plant, fungus, or animal tissue, which in some cases may be present in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Therefore, "biological sample" refers both to a natural organism or a subset of its tissues and to homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. Biological samples can be any type of organic tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some instances the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger prick (where the blood may or may not be bound to any reagents, such as preservatives, anticoagulants, etc., before testing).

[0092] In some embodiments, the source of the sample is "mammal" or "milk," terms that are broadly used to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subject is a human. The method can be applied to samples obtained from human subjects of both sexes and at any developmental stage (i.e., newborns, infants, toddlers, adolescents, and adults), wherein in some embodiments the human subject is a toddler, adolescent, or adult. While this disclosure is applicable to samples from human subjects, it should be understood that the method can also be implemented on other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0093] Cells of interest can be targeted and characterized based on various parameters, such as phenotypic features identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analytical droplets determined to contain target cells. A variety of cells can be characterized using a subject-specific approach. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest also include cells with readily available cell surface markers or antigens that can be captured or labeled by readily available affinity agents or conjugates thereof. For example, target cells may include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialiacoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) derived from whole blood, bone marrow, or umbilical cord blood.

[0094] When performing the subject method according to certain embodiments, a certain amount of initial fluid sample is injected into the flow cytometer. The sample volume injected into the particle sorting module may vary, for example, ranging from 0.001 mL to 1000 mL, for example from 0.005 mL to 900 mL, for example from 0.01 mL to 800 mL, for example from 0.05 mL to 700 mL, for example from 0.1 mL to 600 mL, for example from 0.5 mL to 500 mL, for example from 1 mL to 400 mL, for example from 2 mL to 300 mL, and includes samples from 5 mL to 100 mL.

[0095] In some embodiments, the method includes counting and optionally sorting labeled particles (e.g., target cells) in a sample. When performing the main method, a fluid sample containing particles is first introduced into a flow nozzle of the system. After exiting the flow nozzle, the particles pass through a sample detection region substantially one at a time, where each particle is illuminated by a light source, and measurements of light scattering parameters for each particle are recorded, and in some instances, fluorescence emission (e.g., measurements of two or more light scattering parameters and one or more fluorescence emissions) is recorded as needed. Depending on the characteristics of the detected flow, a flow of 0.001 mm or more can be illuminated, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, and includes flow flows of 1 mm or more that can be illuminated. In some embodiments, the method includes illuminating a planar cross-section of the flow in the sample detection region, for example, with a laser (as described above). In other embodiments, the method includes illuminating a jet flow of a predetermined length in the sample detection region, for example, corresponding to an illumination profile of a diffuse laser beam or lamp.

[0096] In some embodiments, the method includes atomizing the flow at or near the flow cell nozzle orifice. For example, the method may include atomizing the flow at a distance of approximately 0.001 mm or more from the nozzle orifice, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, and 1 mm or more from the nozzle orifice. In some embodiments, the method includes atomizing the flow immediately adjacent to the flow cell nozzle orifice.

[0097] In embodiments of this method, detectors, such as photomultiplier tubes (PMTs), are used to record the light passing through each particle (in some cases referred to as forward scattering), the light reflected orthogonally to the flow direction of the particle through the sensing region (in some cases referred to as orthogonal or lateral scattering), and, if the particle is labeled with a fluorescent marker, the fluorescence emitted from the particle as it passes through the sensing region and is illuminated by energy. Each of forward scattering (FSC), lateral scattering (SSC), and fluorescence emission includes a separate parameter for each particle (or each "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle can be analyzed in real time or stored in a data storage and analysis device, such as a computer, as needed.

[0098] In some embodiments, particles are detected and uniquely identified by exposing them to excitation light and measuring the fluorescence of each particle in one or more detection channels, as needed. The fluorescence emitted in the detection channels used to identify the particles and their associated binding complexes can be measured after excitation with a single light source, or individually after excitation with different light sources. If separate excitation sources are used to excite the particle tags, the tags can be selected such that all tags can be excited by each excitation source used.

[0099] In some embodiments, the method further includes data acquisition, analysis, and recording, for example using a computer, where multiple data channels record data from each detector for light scattering and fluorescence emitted by each particle as it passes through the sample detection area of ​​the particle sorting module. In these embodiments, the analysis includes classifying and counting particles such that each particle exists as a set of digitized parameter values. The subject system can be configured to trigger on selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for the detection parameter and can be used as a means of detecting particles passing through the light source. Detecting an event exceeding the selected parameter threshold triggers the acquisition of light scattering and fluorescence data for that particle. For particles or other components that elicit a response below the threshold in the test medium, no data is acquired. The trigger parameter could be the detection of forward scattered light caused by the particle passing through the light beam. Flow cytometry then detects and collects the light scattering and fluorescence data for that particle.

[0100] Then, specific subpopulations of interest are further analyzed by “gating” based on data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible subpopulation separation. This procedure can be performed by plotting forward light scattering (FSC) and lateral (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. The subpopulation of particles (i.e., those cells within the gate) is then selected, and particles not within the gate are excluded. If needed, gates can be selected by drawing lines around the desired subpopulation on the computer screen using the cursor. Those particles within the gate are then further analyzed only by plotting other parameters of these particles (e.g., fluorescence). If needed, the above analysis can be configured to produce a count of particles of interest in the sample.

[0101] Methods of interest may further include the use of particles in research, laboratory testing, or treatment. In some embodiments, the subject method includes obtaining single cells prepared from a targeted fluid or tissue biological sample. For example, the subject method includes obtaining cells from a fluid or tissue sample for use as a research or diagnostic specimen for diseases such as cancer. Similarly, the subject method includes obtaining cells from a fluid or tissue sample for therapeutic purposes. Cell therapy protocols are those that can prepare living cellular materials, including, for example, cells and tissues, and introduce them into a subject for therapeutic treatment. Conditions that can be treated by administering samples sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, organ damage, etc.

[0102] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, genetic modification, in vitro culture and expansion, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of cells into the subject. The protocol may begin with the collection of live cells and tissues from the subject's source tissue to produce cell and / or tissue samples. Samples can be collected through any suitable procedure, including, for example, administration of cell mobilizing agents to the subject, aspiration of blood from the subject, removal of bone marrow from the subject, etc. After sample collection, cells can be enriched using several methods, including, for example, centrifugation-based methods, filter-based methods, panning, magnetic separation methods, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells can be genetically modified using any convenient method, such as nuclease-mediated gene editing. Genetically modified cells can be cultured, activated, and expanded in vitro. In some cases, cells are preserved, such as cryopreserved, and stored for future use, where the cells are thawed and then administered to the patient, for example, cells can be infused into the patient.

[0103] system

[0104] This disclosure also includes systems for performing subject-specific methods, such as photodetectors in modulated light detection systems (e.g., in flow cytometers). Systems according to certain embodiments include a light source configured to illuminate label-free particles in a sample. In embodiments, the light source can be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, beads, non-cellular particles, etc.), the light source can be configured to emit light with varying wavelengths, ranging from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including from 400 nm to 800 nm. For example, the light source may include a broadband light source emitting light with wavelengths ranging from 200 nm to 900 nm. In other instances, the light source includes a narrowband light source emitting light with wavelengths ranging from 200 nm to 900 nm. For example, the light source may be a narrowband LED (1 nm–25 nm) emitting light with wavelengths ranging between 200 nm and 900 nm. In some embodiments, the light source is a laser. In some instances, the subject system includes gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO₂ lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other instances, the subject system includes dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers. In still other instances, lasers of interest include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, or combinations thereof. In other instances, the subject system includes solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:yCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers and combinations thereof.

[0105] In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to halogen lamps, deuterium arc lamps, xenon arc lamps, and light-emitting diodes (LEDs), such as broadband LEDs with a continuous spectrum, superluminescent LEDs, semiconductor LEDs, broadband LED white light sources, and multi-LED integrated light sources. In some instances, the non-laser light source is a stable fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0106] The light source can be positioned at any suitable distance from the sample (e.g., the flow stream in a flow cytometer), such as 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, and including distances of 100 mm or more. Furthermore, the light source can illuminate the sample at any suitable angle (e.g., relative to the vertical axis of the flow stream), such as angles ranging from 10° to 90°, from 15° to 85°, from 20° to 80°, from 25° to 75°, and including angles from 30° to 60°, such as at a 90° angle.

[0107] The light source can be configured to illuminate the sample continuously or at discrete intervals. In some instances, the system includes a light source configured to continuously illuminate the sample, such as continuously illuminating a flow stream at a detection point in a flow cytometer using a continuous-wave laser. In other instances, the system of interest includes a light source configured to illuminate the sample at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds, or some other interval. When the light source is configured to illuminate the sample at discrete intervals, the system may include one or more additional components to provide intermittent illumination of the sample by the light source. For example, the subject system in these embodiments may include one or more laser beam choppers, manually or computer-controlled beam stoppers, for blocking the sample and exposing it to the light source.

[0108] In some embodiments, the light source is a laser. Lasers of interest may include pulsed lasers or continuous-wave lasers. For example, the laser may be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, or a neon laser. Copper (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:yCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or frequency doubling or third harmonics of any of the above lasers.

[0109] In some embodiments, the light source is a beam generator configured to generate two or more beams of frequency-shifted light. In some instances, the beam generator includes a laser, a radio frequency (RF) generator configured to apply an RF drive signal to an acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser of interest in the beam generator of interest can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser or a combination thereof; a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, or a helium-cadmium (HeCd) laser. Selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:yCa4O(BO3)3 lasers, Nd:YCOB lasers, titania-sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers, and combinations thereof.

[0110] The acousto-optic device can be any convenient acousto-optic scheme configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angle-deflected laser beam based on light from the laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0111] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angle-deflected laser beams in the output laser beam, for example, to apply 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and includes being configured to apply 100 or more radio frequency drive signals.

[0112] In some instances, in order to generate the intensity distribution of the angle-deflected laser beam in the output laser beam, the controller is configured to apply an amplitude-varying radio frequency drive signal, for example, an amplitude-varying signal from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to 50V, for example from about 2V to 40V, for example from 3V to about 30V, and including from about 5V to about 25V. Each applied radio frequency drive signal has a frequency ranging from about 0.001 MHz to about 500 MHz in some embodiments, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and includes frequencies ranging from about 5 MHz to about 50 MHz.

[0113] In some embodiments, the controller has a processor with a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with an angle-deflected laser beam having a desired intensity distribution. For example, the memory may include instructions to generate two or more angle-deflected laser beams with the same intensity, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and including the memory may include instructions to generate 100 or more angle-deflected laser beams with the same intensity. In other embodiments, they may include instructions to generate two or more angle-deflected laser beams with different intensities, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and including the memory may include instructions to generate 100 or more angle-deflected laser beams with different intensities.

[0114] In some embodiments, the controller has a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with increasing intensity along a horizontal axis from edge to center. In these examples, the intensity of the angularly deflected laser beam at the center of the output beam can be from 0.1% to about 99%, for example 0.5% to about 95%, for example 1% to about 90%, for example about 2% to about 85%, for example about 3% to about 80%, for example about 4% to about 75%, for example about 5% to about 70%, for example about 6% to about 65%, for example about 7% to about 60%, for example about 8% to about 55%, and includes about 10% to about 50%. In other embodiments, the controller has a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with increasing intensity along a horizontal axis from edge to center. In these examples, the intensity of the angle-deflected laser beam at the edge of the output beam can be from 0.1% to about 99% of the intensity of the angle-deflected laser beam at the center of the output laser beam along the horizontal axis, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, and includes about 10% to about 50%. In other embodiments, the controller has a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to produce an output laser beam with a Gaussian intensity distribution along the horizontal axis. Still in other embodiments, the controller has a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to produce an output laser beam with a top-hat intensity distribution along the horizontal axis.

[0115] In embodiments, the beam generator of interest may be configured to generate spatially separated angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam, the angle-deflected laser beams may be separated by 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, and including 5000 μm or more. In some embodiments, the system is configured to generate overlapping angle-deflected laser beams within the output laser beam, for example, overlapping with adjacent angle-deflected laser beams on the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, and includes 100 μm or more.

[0116] In some instances, beam generators configured to generate two or more frequency-shifted beams include laser excitation modules as described by Diebold et al. in Nature Photonics, Vol. 7 (10); 806-810 (2013), and U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,036,699; 10,078,045; 10,222,316; 10,288,546; 10,324,019; 10,408,758; 10,45 The laser excitation modules described in patents 1,538; 10,620,111; 10,684,211; 10,845,295; 10,935,482; 10,935,485; 11,105,728; 11,280,718; 11,327,016; 11,366,052; 11,371,937; 11,692,926; 11,630,053; 11,774,343; 11,940,369; and 11,946,851; the disclosures of these patents are incorporated herein by reference.

[0117] In one embodiment, the system includes a light detection system with a photodetector configured to detect light emitted by irradiated particles. As described in detail above, the photodetector is modulated at a predetermined frequency between at least two different voltage settings (e.g., a high voltage setting and a low voltage setting).

[0118] In some embodiments, the light detection system is configured to detect scattered light. In some instances, the light detection system includes a side-scattering light detector. In some instances, the light detection system includes a forward-scattering light detector. In other embodiments, the light detection system includes a plurality of scattered light detectors, such as two or more, three or more, four or more, and including five or more. In some embodiments, the subject light detection system also includes a fluorescence detector configured to detect one or more fluorescence wavelengths. In other embodiments, the light detection system includes a plurality of fluorescence detectors, such as two or more, three or more, four or more, five or more, ten or more, fifteen or more, and including twenty or more fluorescence detectors.

[0119] Detectors of interest may include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-mode charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors, or combinations thereof, and other detectors. In some embodiments, the collected light is measured using a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), 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, having an effective detection surface area per region ranging from 0.01 cm². 2 up to 10cm 2 Photomultiplier tubes, for example, from 0.05 cm 2 up to 9cm 2 For example, from 0.1cm 2 up to 8cm 2 For example, from 0.5cm 2 up to 7cm 2 And including from 1cm 2 up to 5cm 2 .

[0120] When a subject system includes multiple fluorescence detectors, each fluorescence detector can be identical, or the set of fluorescence detectors can be a combination of detectors of different types. For example, when the subject system includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector (or imaging sensor) is a CMOS-type device. In other embodiments, both the first and second fluorescence detectors are CCD-type devices. In yet another embodiment, both the first and second fluorescence detectors are CMOS-type devices. Still in other embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector is a photomultiplier tube (PMT). Still in other embodiments, the first fluorescence detector is a CMOS-type device and the second fluorescence detector is a photomultiplier tube. In yet another embodiment, both the first and second fluorescence detectors are photomultiplier tubes.

[0121] In embodiments of this disclosure, the fluorescence detector of interest is configured to measure collected light at one or more wavelengths, such as at two or more wavelengths, such as at five or more different wavelengths, such as at ten or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and includes measuring light emitted by a sample in the flow stream at 400 or more different wavelengths. In some embodiments, two or more detectors in the module described herein are configured to measure collected light at the same or overlapping wavelengths.

[0122] In some embodiments, the fluorescence detector of interest is configured to measure collected light within a wavelength range (e.g., 200 nm–1000 nm). In some embodiments, the detector of interest is configured to collect a spectrum within a wavelength range. For example, a flow cytometer may include one or more detectors configured to collect a spectrum within one or more wavelength ranges of 200 nm–1000 nm. In still other embodiments, the detector of interest is configured to measure light emitted by a sample in a flowing stream at one or more specific wavelengths. For example, the module may include one or more detectors configured to measure light at one or more wavelengths of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In some embodiments, one or more detectors may be configured to pair with a specific fluorophore, such as those used with the sample in a fluorescence assay.

[0123] In embodiments, the system is configured to modulate the voltage of a photodetector between at least a first voltage and a second voltage. In some instances, the system is configured to modulate the photodetector between three or more voltages, such as four or more, five or more, six or more, seven or more, and including eight or more different voltages. In some instances, the system is configured to modulate the voltage of a photodetector between a first photodetector voltage and a second photodetector voltage. In some embodiments, the voltage of the photodetector is modulated between the first photodetector voltage and the second photodetector voltage at a frequency of 0.0001 MHz to 15 MHz, for example, 0.0005 MHz to 14 MHz, for example, 0.001 MHz to 13 MHz, for example, 0.005 MHz to 12 MHz, for example, 0.01 MHz to 11 MHz, for example, 0.05 MHz to 10 MHz, for example, 0.1 MHz to 9 MHz, for example, 0.5 MHz to 8 MHz, for example, 1 MHz to 7 MHz, for example, 1 MHz to 6 MHz, and including 1 MHz to 5 MHz. In some instances, the voltage of the photodetector oscillates between a first photodetector voltage and a second photodetector voltage at a frequency of 0.1 MHz to 1 MHz. In some instances, the photodetector voltage is modulated at a frequency of 0.0001 MHz to 5 MHz, for example 0.0005 MHz to 6 MHz, for example 0.001 MHz to 7 MHz, for example 0.005 MHz to 8 MHz, for example 0.01 MHz to 9 MHz, for example 0.05 MHz to 10 MHz, for example 0.1 MHz to 11 MHz, for example 0.5 MHz to 12 MHz, for example 1 MHz to 13 MHz, for example 1 MHz to 14 MHz, and including 1 MHz to 15 MHz. In some instances, the photodetector voltage is modulated at frequencies from 0.0001 MHz to 1 MHz, for example, 0.0001 MHz to 2 MHz, for example, 0.0001 MHz to 3 MHz, for example, 0.0001 MHz to 4 MHz, for example, 0.0001 MHz to 5 MHz, for example, 0.0001 MHz to 6 MHz, for example, 0.0001 MHz to 7 MHz, for example, 0.0001 MHz to 8 MHz, for example, 0.0001 MHz to 9 MHz, for example, 0.0001 MHz to 10 MHz, for example, 0.0001 MHz to 11 MHz, for example, 0.0001 MHz to 12 MHz, for example, 0.0001 MHz to 13 MHz, for example, 0.0001 MHz to 14 MHz, and including 0.0001 MHz to 15 MHz.

[0124] In some embodiments, the system is configured to maintain the voltage of the photodetector at that voltage for 25 μs or less in each cycle, for example, 20 μs or less, for example, 15 μs or less, for example, 10 μs or less, for example, 9 μs or less, for example, 8 μs or less, for example, 7 μs or less, for example, 6 μs or less, for example, 5 μs or less, for example, 4 μs or less, for example, 3 μs or less, for example, 2 μs or less, for example, 1 μs or less, for example, 0.9 μs or less, for example, 0.8 μs or less, for example, 0.7 μs or less, for example, 0.6 μs or less, for example, 0.5 μs or less, for example, 0.4 μs or less, for example, 0.3 μs or less, for example, 0.2 μs or less, for example, 0.1 μs or less, and including 0.05 μs or less. In some instances, the system is configured to maintain the voltage of the photodetector at the first photodetector voltage during each modulation cycle. In some instances, the system is configured to maintain the voltage of the photodetector at the first photodetector voltage for 25 μs or less in each cycle, for example, 20 μs or less, for example, 15 μs or less, for example, 10 μs or less, for example, 9 μs or less, for example, 8 μs or less, for example, 7 μs or less, for example, 6 μs or less, for example, 5 μs or less, for example, 4 μs or less, for example, 3 μs or less, for example, 2 μs or less, for example, 1 μs or less, for example, 0.9 μs or less, for example, 0.8 μs or less, for example, 0.7 μs or less, for example, 0.6 μs or less, for example, 0.5 μs or less, for example, 0.4 μs or less, for example, 0.3 μs or less, for example, 0.2 μs or less, for example, 0.1 μs or less, and including 0.05 μs or less. In some instances, the system is configured to maintain the voltage of the photodetector at the second photodetector voltage for 25 μs or less in each cycle, for example, 20 μs or less, for example, 15 μs or less, for example, 10 μs or less, for example, 9 μs or less, for example, 8 μs or less, for example, 7 μs or less, for example, 6 μs or less, for example, 5 μs or less, for example, 4 μs or less, for example, 3 μs or less, for example, 2 μs or less, for example, 1 μs or less, for example, 0.9 μs or less, for example, 0.8 μs or less, for example, 0.7 μs or less, for example, 0.6 μs or less, for example, 0.5 μs or less, for example, 0.4 μs or less, for example, 0.3 μs or less, for example, 0.2 μs or less, for example, 0.1 μs or less, and including 0.05 μs or less. In some embodiments, the system is configured to maintain the voltage of the photodetector for the same duration as maintaining the voltage of the photodetector at the first photodetector voltage for the same duration as maintaining the voltage of the photodetector at the second photodetector voltage. In other embodiments, the system is configured to maintain the voltage of the photodetector for a different duration than maintaining the voltage of the photodetector at the second photodetector voltage.When the voltage of the photodetector is maintained at a different duration than the voltage of the first photodetector, the voltage can be maintained at a different voltage for one or more modulation cycles, such as two or more, three or more, four or more, five or more, ten or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, and including 100 or more modulation cycles.

[0125] In some embodiments, the system is configured to set the voltage of the first photodetector to 250mV to 2500mV, for example 300mV to 2400mV, for example 400mV to 2300mV, for example 500mV to 2200mV, for example 500mV to 2100mV, for example 500mV to 2000mV, for example 500mV to 1900mV, for example 500mV to 1800mV, for example 500mV to 1700mV, for example 500mV to 1600mV, for example 500mV to 1500mV, for example 500mV to 1400mV, for example 500mV to 1300mV, for example 500mV to 1200mV, for example 500mV to 1100mV, and including 500mV to 1000mV. In some instances, the system is configured to set the second photodetector voltage to 0.0001mV to 1000mV, such as 0.0005mV to 900mV, such as 0.001mV to 800mV, such as 0.005mV to 700mV, such as 0.01mV to 600mV, such as 0.05mV to 500mV, such as 0.1mV to 500mV, such as 0.5mV to 500mV, and including 1mV to 500mV. In some instances, the system is configured to set the second photodetector voltage to 50mV or less, such as 40mV or less, such as 30mV or less, such as 20mV or less, such as 10mV or less, and including 5mV or less.

[0126] The voltage of the photodetector can be modulated between the first and second photodetector voltages by any convenient waveform variation, such as a sinusoidal voltage variation. In some embodiments, the system is configured to modulate the photodetector voltage between the first and second photodetector voltages with a square voltage variation. In some embodiments, the system is configured to modulate the photodetector voltage with a symmetrical voltage variation (e.g., a full square voltage variation) between each modulation cycle. In other embodiments, one or more modulation cycles present different types of voltage variation waveforms, such as a square voltage variation during one or more modulation cycles and a sinusoidal voltage variation during another. In some instances, the system is configured to change the shape of the voltage variation at predetermined intervals, such as after every two or more modulation cycles, for example, after three or more, four or more, five or more, ten or more, 25 or more, 50 or more, 100 or more, and including changing the shape of the voltage variation after every 500 or more modulation cycles.

[0127] In embodiments, the light detection system is configured to generate data signals based on detected light at one or more photodetector voltages. In some instances, the light detection system is configured to generate two or more data signals at each photodetector voltage during each modulation cycle, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and includes generating 100 or more data signals at each photodetector voltage during each modulation cycle. In some instances, light is detected at the photodetector voltage, and the data signals are averaged. In some instances, the system is configured to generate a data signal at a first photodetector voltage during each modulation cycle. In some instances, the system is configured to generate a data signal at a second photodetector voltage during each modulation cycle. In some instances, the system is configured to generate a data signal at both the first and second photodetector voltages during each modulation cycle.

[0128] In some instances, the system is configured to generate data signals only at the first photodetector voltage during each modulation cycle, for example, generating two or more data signals, such as three or more, four or more, five or more, and including generating ten or more data signals at the first photodetector voltage during each modulation cycle. In some instances, the system is configured to generate data signals only at the second photodetector voltage during each modulation cycle, for example, generating two or more data signals, such as three or more, four or more, five or more, and including generating ten or more data signals at the second photodetector voltage during each modulation cycle.

[0129] In some embodiments, the system is configured to generate a data signal at a second photodetector voltage when the light detected at the first photodetector voltage exceeds a saturation threshold of the photodetector, for example, the detected light exceeds the saturation threshold by 5% or more, such as 10% or more, 15% or more, 25% or more, or 50% or more, and including when the light detected at the first photodetector voltage exceeds the saturation threshold of the photodetector by 75% or more. In some instances, when the light detected at the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold, the system is configured to generate a data signal only based on the light detected at the second photodetector voltage. In some instances, when the light detected at the first photodetector voltage exceeds the saturation threshold, the system may be configured to generate an alarm to indicate that saturation has been exceeded or is approaching a level where the data signal is unreliable or inaccurate. In these instances, the system may also be configured to switch the photodetector voltage to the second photodetector voltage in response to an alarm.

[0130] In some embodiments, the system is configured to generate a data signal at the first photodetector voltage when the data signal generated at the second photodetector voltage exceeds a detected noise threshold (e.g., the signal-to-noise ratio drops below a predetermined threshold). In some instances, the noise of the photodetector at the second photodetector voltage is sufficient to mask or reduce the reliability of the data signal generated at the second photodetector voltage. In some instances, the system is configured to generate a data signal at the first photodetector voltage when the data signal generated at the second photodetector voltage is 5% or more below the noise threshold of the photodetector, for example, 10% or more, 15% or more, 25% or more, or 50% or more, and includes generating a data signal at the first photodetector voltage when the data signal generated at the second photodetector voltage is 75% or more below the noise threshold of the photodetector. In some instances, when the data signal generated at the second photodetector voltage exhibits excessive noise (e.g., the signal-to-noise ratio drops below a threshold), the system is configured to generate a data signal solely based on the light detected at the first photodetector voltage. In some instances, when the data signal generated under the second photodetector voltage is below a noise threshold, the system is configured to generate an alarm to indicate that the signal-to-noise ratio of the data signal generated under the second photodetector voltage is too low or close to a level where the data signal is unreliable or inaccurate. In these instances, the system is configured to switch the photodetector voltage back to the first photodetector voltage in response to the alarm.

[0131] In some embodiments, the photodetector voltage is a second photodetector voltage. In embodiments, the system further includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to measure baseline samples at the first and second photodetector voltages. In some instances, the memory includes instructions for generating a data signal based on the baseline samples. In some instances, the memory includes instructions for calculating baseline noise at each photodetector voltage during each modulation period. In some instances, the memory includes instructions for calibrating baseline noise at each photodetector voltage. In some instances, the memory includes instructions for measuring baseline samples at each photodetector voltage at a baseline sampling frequency. In some instances, the memory includes instructions for measuring baseline samples at a frequency greater than the modulation frequency. In some instances, the memory includes instructions for measuring baseline samples at a frequency less than the modulation frequency. In some instances, the memory includes instructions for measuring baseline samples at a frequency equal to the modulation frequency. In some instances, the memory includes instructions for measuring baseline samples at a baseline sampling frequency, such as 0.0001MHz to 15MHz, such as 0.0005MHz to 14MHz, such as 0.001MHz to 13MHz, such as 0.005MHz to 12MHz, such as 0.01MHz to 11MHz, such as 0.05MHz to 10MHz, such as 0.1MHz to 9MHz, such as 0.5MHz to 8MHz, such as 1MHz to 7MHz, such as 1MHz to 6MHz, and including 1MHz to 5MHz.

[0132] In some instances, the memory includes instructions for interpreting the baseline at the first and second photodetector voltages (i.e., dual-mode operation) through fixed-time-dependent analysis. In some instances, the baseline is interpreted at the first and second photodetector voltages through dynamic frequency domain derivation. In some embodiments, the memory includes instructions for scaling the generated data signal. In some instances, the memory includes instructions for scaling the data signal on a continuous scale. In some instances, the memory includes instructions for scaling the data signal on a single continuous scale and dividing the data signal generated at one or more of the first and second photodetector voltages by the gain of the detector setting used for modulation. In some instances, the first and second photodetector voltages are scaled as two separate parameters.

[0133] In some embodiments, the memory includes instructions for determining one or more parameters of particles in the sample based on data signals generated from light detected at a first photodetector voltage. In some instances, the memory includes instructions for determining one or more parameters of particles in the sample based on data signals generated from light detected at a second photodetector voltage. In some instances, the memory includes instructions for determining one or more parameters of particles in the sample based on data signals generated from light detected at both the first and second photodetector voltages.

[0134] In some instances, the memory includes instructions for generating particle images based on data signals. In some instances, the memory includes instructions for calculating one or more image parameters based on the generated particle images. In some instances, centroid image parameters are calculated based on the generated images. In some instances, incremental centroid image parameters are calculated based on the generated images. In some instances, diffusivity image parameters are calculated based on the generated images. In some instances, eccentricity image parameters are calculated based on the generated images. In some instances, major axis moment image parameters are calculated based on the generated images. In some instances, maximum intensity image parameters are calculated based on the generated images. In some instances, radial moment image parameters are calculated based on the generated images. In some instances, minor axis moment image parameters are calculated based on the generated images. In some instances, particle size image parameters are calculated based on the generated images. In some instances, total intensity image parameters are calculated based on the generated images. In some instances, particle light loss image parameters are calculated based on the generated images. In some instances, forward scattered light image parameters are calculated based on the generated images. In some instances, side scattered light image parameters are calculated based on the generated images. In some instances, image moments are calculated based on the generated images. The term "image moment" is used in its conventional sense herein, referring to the weighted average of pixel intensities in an image. In some instances, the centroid can be calculated from the image moments of an image. In other instances, cell orientation can be calculated from the image moments of an image. Still in other instances, cell eccentricity can be calculated from the image moments of an image.

[0135] In some embodiments, the optical detection system described herein is part of a flow cytometer. The flow cytometer may include any suitable mechanism for providing sheath fluid and sample fluid to a sample fluid input coupler and a sheath fluid input coupler. For example, the sample fluid input coupler may be fluidly connected to a sample fluid line (e.g., a conduit) that is fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidly connected to a sheath fluid line that is fluidly connected to a sheath fluid reservoir. Similarly, the flow cytometer may include any suitable mechanism for managing waste from the flow stream. A fluid output coupler may be fluidly connected to a waste line that is fluidly connected to a waste fluid reservoir. A fluid management system suitable for the subject flow cytometer is provided in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0136] In some embodiments, a flow cytometer includes a flow cell. The flow cell of interest includes a cuvette configured to transport particles in a flow stream. As described herein, a “flow cell” in its conventional sense refers to an assembly containing a flow channel for transporting particles in a sheath fluid. The cuvette of interest has a channel (i.e., a flow channel) extending through it. The flow stream configured by the flow channel may include a liquid sample injected from a sample tube. In some instances, the flow cell includes a light-accessible flow channel. The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed of silica, such as fused silica. In some cases, the flow cell is configured to be illuminated with light from a light source at one or more detection points. A “detection point” discussed herein refers to an area within the flow cell in which particles are illuminated by light from a light source, for example, for analysis. The size of the detection point may vary as needed. For example, 0 μm represents the optical axis of the light emitted by the light source, and the detection point range can be from -50 μm to 50 μm, for example from -25 μm to 40 μm, and includes -15 μm to 30 μm. Depending on certain considerations (such as the number and arrangement of lasers), there may be multiple irradiation points within the flow cell.

[0137] In some embodiments, the flow cell includes or is configured to use a sample injection port configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable sample flow to the flow cell chamber (i.e., the flow channel). Depending on the desired characteristics of the flow, the sample rate delivered from the sample injection port to the flow cell can be 1 μL / min or higher, for example, 2 μL / min or higher, for example, 3 μL / min or higher, for example, 5 μL / min or higher, for example, 10 μL / min or higher, for example, 15 μL / min or higher, for example, 25 μL / min or higher, for example, 50 μL / min or higher, and includes 100 μL / min or higher. In some cases, the sample rate delivered from the sample injection port to the flow cell is 1 μL / sec or higher, for example, 2 μL / sec or higher, for example, 3 μL / sec or higher, for example, 5 μL / sec or higher, for example, 10 μL / sec or higher, for example, 15 μL / sec or higher, for example, 25 μL / sec or higher, for example, 50 μL / sec or higher, and includes 100 μL / sec or higher.

[0138] The sample injection port can be an orifice located in the inner chamber wall or a conduit located proximal to the inner chamber. When the sample injection port is an orifice located in the inner chamber wall, the orifice can be of any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as a parabola with its base portion connecting to its top portion of a plane. In some embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on the shape, and in some cases, the opening ranges from 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0139] In some cases, the sample injection port is a conduit located proximal to the flow cell chamber. For example, the sample injection port can be a conduit positioned such that its orifice is aligned with the flow cell orifice. When the sample injection port is a conduit aligned with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including but not limited to: straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses; and irregular shapes, such as a parabola with its base connected to a flat top. The orifice of the conduit can vary depending on its shape, and in some cases, the opening ranges from 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm. The shape of the tip of the sample injection port can be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip with a bevel angle ranging from 1° to 10°, such as from 2° to 9°, such as from 3° to 8°, such as from 4° to 7°, and including a bevel angle of 5°.

[0140] 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 sheath flow stream to the flow cell chamber, for example, in combination with a sample to generate a laminated flow stream around the sample. Depending on the desired characteristics of the flow stream, the sheath fluid rate delivered from the sheath fluid injection port to the flow cell chamber can be 25 μL / sec or higher, for example 50 μL / sec or higher, for example 75 μL / sec or higher, for example 100 μL / sec or higher, for example 250 μL / sec or higher, for example 500 μL / sec or higher, for example 750 μL / sec or higher, for example 1000 μL / sec or higher, and includes 2500 μL / sec or higher.

[0141] In some embodiments, the sheath injection port is an orifice located in the inner chamber wall. The sheath injection port orifice can be of any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: straight cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as a parabola with its base connected to a flat top portion. The size of the sheath injection port orifice can vary depending on the shape, and in some cases, the opening ranges from 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0142] In some embodiments, the system includes or is operatively coupled to a flow cytometer. Suitable flow cytometry systems may include, but are not limited to, those in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012), Ann Clin Biochem, January; 49(pt 1):17-28; Linden et al., Seminar on Thrombosis and Hemostasis, October 2004; 30(5):502-11; Alison et al., Journal of Pathology (J Pathol, December 2010; 222(4):335-344; and Herbig et al. (2007) Crit Rev TherDrug CarrierSyst. 24(3):203-255; the contents of these publications are incorporated herein by reference. In some cases, flow cytometry systems of interest include BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSL FortessaTM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BDBiosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorting instrument, BDBiosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorting instrument, BDBiosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorting instrument, BD Biosciences FACSAria TM II Cell Sorter, BD Biosciences FACSAria TM III Cell Sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorting instrument, BDBiosciences FACSymphony TM S6 Cell Sorter, BD Biosciences FACSDiscover TM Cell sorting instruments, etc.

[0143] In some embodiments, the subject system is a flow cytometry system, such as U.S. Patent Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,0 Those described in 95,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766; the disclosures of these patents are incorporated herein by reference in their entirety.

[0144] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the subject system is a flow cytometry system configured to image particles in a flowing stream using fluorescence imaging with radio frequency labeled emission (FIRE), such as those described in Diebold et al., Nature Photonics, Vol. 7 (10); 806-810 (2013), and U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,036,699; 10,078,045; 10,222,316; 10,288,546; 10,324,019; Those described in 10,408,758; 10,451,538; 10,620,111; 10,684,211; 10,845,295; 10,935,482; 10,935,485; 11,105,728; 11,280,718; 11,327,016; 11,366,052; 11,371,937; 11,692,926; 11,630,053; 11,774,343; 11,940,369; and 11,946,851; the disclosures of these patents are incorporated herein by reference.

[0145] Figure 2A system 200 for flow cytometry according to an illustrative embodiment of this disclosure is shown. System 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at a detection point 215 within a flow cell 210. Although Figure 2 The example shows a single laser, but it should be understood that multiple lasers can also be used. The laser beam from laser 201 is directed to focusing lens 202, which focuses the beam onto the portion of the fluid flow within flow cell 210 where the sample particles 211 reside. Flow cell 210 is part of a fluid system that directs particles (typically one at a time) in the flow to the focused laser beam for detection. Alternatively, when the flow cytometer is an airflow cytometer, a nozzle top may be used.

[0146] like Figure 2 As shown, flow cell 210 is fluidly connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. Sheath fluid from sheath fluid reservoir 203 is supplied via conduit (i.e., sheath fluid line) 207 to at least one sheath fluid injection port 208. Furthermore, sample fluid containing particles 211 from sample fluid reservoir 204 is supplied via conduit (i.e., sample fluid line) 205 to sample injection port 206. Sample injection port 206 is fluidly connected to a sample syringe 213 (e.g., a sample injection needle) configured to introduce particles 211 into the interior of flow cell 210. Particles 211 are hydrodynamically focused by the sheath fluid entering from sheath fluid injection port 208, such that a flow stream 214 is formed downstream of the conical portion 212 of flow cell 210. Particles emitted at the distal end of flow cell 210 can be disposed of and / or collected by any suitable method. For example, depending on the type of flow cytometry performed, particles can be collected at the distal end of flow cell 210, for example, via a waste line. Alternatively, the particles can be sorted.

[0147] Light from one or more laser beams interacts with particles 211 in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various wavelengths depending on the characteristics of the particles, such as their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on the particles. Fluorescence emission, as well as diffracted, refracted, reflected, and scattered light, can be routed to one or more detectors. Specifically, forward scattered light (FSC) is routed to forward scattered light detector 223. Forward scattered light detector 223 is located slightly off-axis from the direct beam passing through flow cell 210 and is configured to detect diffracted light, i.e., excitation light that propagates primarily in the forward direction through or around the particles. The intensity of the light detected by forward scattered light detector 223 depends on the overall size of the particles. The forward scattered detector may include, for example, a photodiode. Between forward scattered light detectors 223 are an optical filter 221a and a scattering baffle 222. Optical filter 221a can be configured to filter out non-FSC light of at least one wavelength, while scattering baffle 222 can be configured to prevent the incident beam (i.e. non-scattered light) from laser 201 from being detected by forward scattering detector 223.

[0148] Furthermore, side-scattered light (SSC) is detected by side-scattered light detector 224. In other words, side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structures of particle 211, which tend to increase with increasing particle structure complexity. Figure 2 In the example, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to a side-scatter light detector 224 while transmitting non-SSC light (e.g., fluorescence). An optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by the side-scatter light detector 224. Fluorescence detectors 225a-225c are also shown, each configured to detect fluorescence of a different wavelength. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a while transmitting light of other wavelengths. The optical filter 221c may be configured to prevent light of at least one wavelength not corresponding to the first wavelength (or wavelength range) from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect FL light corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b while transmitting light of a third wavelength (or wavelength range) for detection by the fluorescence detector 225c. Optical filter 221d is configured to prevent light of at least one wavelength that does not correspond to the second wavelength (or wavelength range) from being detected by fluorescence detector 225b. Furthermore, optical filter 221e is configured to prevent light of at least one wavelength that does not correspond to the third wavelength (or wavelength range) from being detected by fluorescence detector 225c.

[0149] Those skilled in the art will recognize that the flow cytometer according to embodiments of this disclosure is not limited to... Figure 2 The flow cytometer described herein may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors, at various wavelengths and in various different configurations. For example, although Figure 2 The example shown is for illustrative purposes and features three fluorescence detectors, but it should be understood that any suitable number of fluorescence detectors may be used.

[0150] During operation, the cytometer is controlled by controller / processor 290, and measurement data from the detectors can be stored in memory 295 and processed by controller / processor 290. Although not explicitly shown, controller / processor 290 is coupled to detectors to receive output signals from them and may also be coupled to the electrical and electromechanical components of the flow cytometer to control laser 201, fluid flow parameters, etc. Input / output (I / O) capability 297 may also be provided in the system. Memory 295, controller / processor 290, and I / O 297 may be provided entirely as part of the flow cytometer. In such embodiments, a display may also form part of I / O capability 297 for presenting experimental data to the user of cytometer 200. Alternatively, memory 295 and controller / processor 290, along with some or all of the I / O capability, may be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, some or all of memory 295 and controller / processor 290 may communicate wirelessly or wired with cytometer 210. The controller / processor 290, combined with memory 295 and I / O 297, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0151] Different fluorescent molecules in the fluorescent dye panel used in flow cytometry experiments will emit light in their respective characteristic wavelength bands. Specific fluorescent labels used in the experiment and their associated fluorescence emission bands can be selected to typically coincide with the filter window of the detector. I / O 297 can be configured to receive data on a flow cytometry experiment having a fluorescently labeled panel and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O 297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, label density data, emission spectral data, data assigning labels to one or more markers, and flow cytometry configuration data. Flow cytometry experiment data, such as label spectral characteristics and flow cytometry configuration data, can also be stored in memory 295. Controller / processor 290 can be configured to evaluate one or more label-to-marker assignments.

[0152] In some embodiments, the subject system is a particle sorting system configured to sort particles using closed particle sorting modules, such as those described in U.S. Patent Publication No. 2017 / 0299493 (filed March 28, 2017), which is incorporated herein by reference. In some embodiments, particles (e.g., cells) of a sample are sorted using a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781 (filed December 23, 2019), which is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module with deflection plates, such as those described in U.S. Patent Publication No. 2017 / 0299493 (filed March 28, 2017), which is incorporated herein by reference.

[0153] In some embodiments, the system is an image-enabled particle sorter that uses radio frequency marker emission imaging, for example... Figure 3 As depicted, the particle sorter 300 includes an illumination assembly 300a comprising a light source 301 (e.g., a 488nm laser) that generates an output beam 301a, which is split into beams 302a and 302b by a beam splitter 302. Beam 302a propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a with one or more angle deflections. In some cases, the output beam 303a generated by the acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Beam 302b propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a with one or more angle deflections. In some cases, the output beam 304a generated by the acousto-optic device 304 includes a local oscillator beam and multiple radio frequency comb beams. Output beams 303a and 304a, generated by acousto-optic devices 303 and 304 respectively, are combined by beam splitter 305 to produce output beam 305a, which is transmitted through optical assembly 306 (e.g., objective lens) to illuminate particles in flow cell 307. In some embodiments, acousto-optic device 303 (AOD) splits a single laser beam into an array of beams, each beam having a different optical frequency and angle. A second AOD 304 tunes the optical frequency of a reference beam, which is then overlapped with the beam array at beam combiner 305. In some embodiments, the light illumination system having a light source and acousto-optic devices may also include those described in Schraivogel et al. (“High-speed fluorescence image-enabled cellsorting”, Science (2022), 375(6578): 315-320) and U.S. Patent Publication No. 2021 / 0404943, the disclosure of which is incorporated herein by reference.

[0154] Output beam 305a illuminates sample particles 308 (e.g., together with sheath fluid 309) propagating through flow cell 307 at irradiation region 310. As shown in irradiation region 310, multiple beams (e.g., angle-deflected radio frequency shift beams, depicted as dots on irradiation region 310) overlap with a reference local oscillator beam (depicted as shaded lines on irradiation region 310). Due to their different optical frequencies, the overlapping beams exhibit beat frequency behavior, resulting in each small beam operating at a different frequency f. 1-n Carrying sinusoidal modulation.

[0155] Light from the illuminated sample is transmitted to a light detection system 300b, which includes multiple photodetectors. The light detection system 300b includes a forward-scattering photodetector 311 for generating a forward-scattering image 311a and a side-scattering photodetector 312 for generating a side-scattering image 312a. The light detection system 300b also includes a bright-field photodetector 313 for generating a light loss image 313a. In some embodiments, the forward-scattering detector 311 and the side-scattering detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the illuminated sample is also detected by fluorescence photodetectors 314-317. In some cases, photodetectors 314-317 are photomultiplier tubes. Light from the illuminated sample is directed by a beamsplitter 320 to the side-scattering detection channel 312 and the fluorescence detection channels 314-317. The optical detection system 300b includes bandpass optical components 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optical component 321 has a 534nm / 40nm bandpass. In some cases, optical component 322 has a 586nm / 42nm bandpass. In some cases, optical component 323 has a 700nm / 54nm bandpass. In some cases, optical component 324 has a 783nm / 56nm bandpass. The first number indicates the center of the spectral band. The second number provides the range of the spectral band. Therefore, the 510 / 20 filter extends 10nm on each side of the center of the spectral band, or from 500nm to 520nm.

[0156] Data signals generated in response to light detected in scattered light detection channels 311 and 312, bright field light detection channel 313, and fluorescence detection channels 314-317 are digitally processed in real time by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated in processors 350 and 351. Image-enabled sorting is performed in response to a sorting signal generated in sorting trigger 352. Sorting assembly 300c includes a deflector plate 331 for deflecting particles into sample container 332 or waste flow stream 333. In some cases, sorting assembly 300c is configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493 (filed March 28, 2017), which is incorporated herein by reference. In some embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781, which is incorporated herein by reference.

[0157] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4 It can be used to analyze and characterize particles, whether or not the particles are physically sorted into a collection container. Figure 4 A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, particle analysis system 401 is a fluid system. Particle analysis system 401 includes a fluid system 402. Fluid system 402 may include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, wherein particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0158] The particle analysis system 401 includes a detection system 404 configured to collect signals as each particle passes through one or more detection stations along a common sample path. Detection station 408 typically refers to a monitored area 407 of the common sample path. In some embodiments, detection may include detecting light or one or more other characteristics of the particle as particle 403 passes through the monitored area 407. Figure 4 The image shows a detection station 408 with a monitored area 407. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor multiple areas.

[0159] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be referred to as event data. The data points can be multidimensional data points, including values ​​of various attributes measured for the particle. The detection system 404 is configured to collect a series of such data points within a first time interval.

[0160] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system may be operationally associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the number of data points collected by the detection system 404 during the first time interval. The control system 406 may be further configured to generate an experimental signal frequency based on the number of data points in that portion of the first time interval. The control system 406 may additionally compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0161] Figure 5 An example functional block diagram of a particle analyzer control system (e.g., an analysis controller (i.e., processor) 500) is shown for analyzing and displaying biological events. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0162] The particle analyzer or sorting system 502 may be configured to acquire biological event data. For example, a flow cytometer may generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to an analysis controller 500. A data communication channel may be included between the particle analyzer or 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, for example, by applying a distance-based classification model to determine a density discrimination threshold in a size-based analyte feature space, applying a density-based clustering algorithm to separate the analyte data into high-density clusters and low-density clusters based on the density threshold, and classifying the analyte data based on the high-density clusters and low-density clusters according to the size-based analyte feature space.

[0163] Analysis controller 500 may be configured to receive biological event data from particle analyzer or sorting system 502. The biological event data received from particle analyzer or sorting system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display of a first graph including the biological event data to display device 506. Analysis controller 500 may be further configured to render regions of interest as gates around the population of biological event data shown on display device 506, for example, overlaying them on the first graph. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest plotted on a single-parameter histogram or bivariate graph. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0164] The analysis controller 500 can be further configured to display the biological event data inside the door on the display device 506 in a manner different from other events in the biological event data outside the door. For example, the analysis controller 500 can be configured to render the colors of the biological event data inside the door differently from the colors of the biological event data outside the door. The display device 506 can be a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0165] The analysis controller 500 can be configured to receive a door selection signal from a first input device to identify a door. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a door selection signal to the analysis controller 500, identifying a door to be displayed on or operated via the display device 506 (e.g., clicking when the cursor is positioned inside or on the desired door). In some embodiments, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, optical detector, or voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function can be considered an input device. For example, such as... Figure 5 As shown, mouse 510 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0166] Triggering events can cause the analysis controller 500 to change the way data is displayed, the portion of data actually displayed on the display device 506, and / or provide input for further processing, such as selecting a group of interest for particle sorting.

[0167] In some embodiments, the analysis controller 500 may be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 may be further configured to automatically modify the graph visualization to facilitate the gating process. The modification may be based on a specific distribution of the biological event data received by the analysis controller 500.

[0168] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can be further configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0169] Display device 506 can be configured to receive display data from analysis controller 500. The display data may include gating of portions of graphs and delineations of biological event data. Display device 506 can be further configured to change the presented information based on input received from analysis controller 500, in conjunction with input received from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0170] In some implementations, the analysis controller 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include controls for receiving sample events or sample images. The sample events, images, or sample gates may be provided before collecting event data for the samples, or based on an initial set of events from a portion of the samples.

[0171] Figure 6A This is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Figure 6A As shown, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, include, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a flow). Within the moving fluid column 608, particles 609 (e.g., cells) align in a single file to pass through a monitored area 611 (e.g., where laser streams intersect) and are irradiated by an irradiation source 612 (e.g., a laser). Vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to break into multiple droplets 610, some of which contain particles 609.

[0172] In operation, a detection station 614 (e.g., an event detector) identifies when a particle of interest (or cell of interest) crosses a monitored area 611. The detection station 614 feeds into a timing circuit 628, which in turn feeds into a flash charge circuit 630. At the droplet disconnection point, notified by a timing droplet delay (Δt), a flash charge can be applied to a moving fluid column 608, causing the droplet of interest to carry a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect it into a container (e.g., a collection tube or a porous or microporous sample plate), where a pore or micropore can be associated with a specific droplet of interest. Figure 6A As shown, the droplets can be collected in the discharge container 638.

[0173] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a particle of interest passes through a monitored region 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 616 allows the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may be fed an amplitude signal 620 and / or a phase signal 618, which are then fed through an 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, in turn, control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0174] In some embodiments, sorting electronics (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory configured to store detected events and sorting decisions based thereon. Sorting decisions may be included in the event data of the particles. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or communication coupling, such that event measurements can be collected by one of detection system 616 or detection station 614 and provided to non-collecting elements.

[0175] Figure 6B This is a schematic diagram of a particle sorting system according to an embodiment of the present document. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via a current-charged wire in the barbs. This generates a droplet stream 610 containing particles 610 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The particle information is analyzed, for example, by sorting electronics or other detection systems. Figure 6B (Not shown in the image). Deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, thereby guiding the droplets toward a destination collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflector plates 652 and 654 can be controlled to guide particles to container 674 along a first path 662 or to container 678 along a second path 668. If the particles are of no interest (e.g., do not exhibit scattering or irradiation information within a specified sorting range), the deflector plates can allow the particles to continue moving along flow path 664. Such uncharged droplets can then enter a waste container, for example, via a suction device 670.

[0176] It can include sorting electronics to initiate the collection of measurement data, receive the fluorescence signal of particles, and determine how to adjust the deflection plate to sort the particles. Figure 6BThe example implementation shown includes BD FACSAria, commercially available from Becton Dickinson, Inc. (Franklin Lake, New Jersey). TM A series of flow cytometers.

[0177] Computer control system

[0178] The system may include a display and operator input devices. Operator input devices may be, for example, a keyboard, mouse, etc. The processing module includes a processor that accesses memory having instructions stored thereon for executing the subject method steps. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or other existing or future-available processors. The processor executes the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor's coordination and execution of various computer programs written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically collaborates with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0179] System memory can be any known or future memory storage device. Examples include any common random access memory (RAM), magnetic media (such as resident hard disks or magnetic tapes), optical media (such as optical discs), flash memory devices, or other memory storage devices. Memory storage devices can be any known or future devices, including optical disc drives, magnetic tape drives, or floppy disk drives. This type of memory storage device is typically read from and / or written to program storage media (not shown) (such as optical discs). Any of these program storage media, or other media currently in use or that may be developed in the future, can be considered a computer program product. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0180] In some embodiments, a computer program product is described, including a computer-usable medium in which control logic (computer software program, including program code) is stored. When executed by a computer's processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, such as using a hardware state machine. The implementation of a hardware state machine to perform the functions described herein will be apparent to those skilled in the art.

[0181] The memory can be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including disks, optical discs, magnetic tapes, RAM, or any other suitable device, whether fixed or portable). The processor may include a general-purpose digital microprocessor, appropriately programmed via a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel or pre-stored in a computer program product using any of these devices connected to the memory, such as memory or other portable or fixed computer-readable storage media. For example, a disk or optical disc may carry the programming and can be read by a disk writer / reader. The systems disclosed herein also include programming, such as in the form of a computer program product, algorithms for practicing the methods described above. The programming according to this disclosure can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media such as CD-ROMs; electronic storage media such as RAM and ROMs; portable flash drives; and mixtures of these categories, such as magneto-optical storage media.

[0182] The processor can also access communication channels to communicate with users in remote locations. A remote location refers to a user who does not directly interact with the system and relays input information from external devices (such as computers connected to a wide area network (WAN), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones)) to the input manager.

[0183] In some embodiments, the system according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or other devices. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., RFID, Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB)). Communication protocols and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0184] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces like USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject system and other external devices, such as computer terminals configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0185] In one embodiment, the communication interface is configured for infrared communication. Communication or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0186] In one embodiment, the communication interface is configured to provide connectivity for data transmission using the Internet Protocol (IP) via a cellular telephone network, a short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0187] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, such as using a common standard like 802.11 or... The protocol is RF, or IrDA infrared protocol. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, keyboard, mouse, or touchscreen.

[0188] In some embodiments, the communication interface is configured to automatically or semi-automatically transmit data stored in the subject system (e.g., in an optional data storage unit) to a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0189] The output controller may include controllers for any variety of known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of picture elements. The graphical user interface (GUI) controller may include any variety of known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of this communication may be implemented using a network or other types of remote communication in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location, such as via the Internet, telephone, or satellite network, according to known technologies. The output manager may present data according to various known technologies. For example, the data may include SQL, HTML, or XML documents, emails, or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or type to be developed in the future, although they will generally be computer categories commonly referred to as servers. However, they may also be mainframes, workstations, or other computer types. They can be connected via any known or future cable or other communication system (including wireless systems), whether networked or otherwise. They can be co-located or physically separated. Various operating systems are available for any computer platform, depending on the type and / or brand of the chosen platform. Suitable operating systems include... XP 7. 8. 10. Android TM SGI Oracle wait.

[0190] Figure 7 A general architecture of an example computing device 700 according to certain embodiments is described. Figure 7The general architecture of the computing device 700 depicted includes the arrangement of computer hardware and software components. However, it is not necessary to show all these generally conventional elements to provide an enabling disclosure. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. The network interface 720 provides connectivity to one or more networks or computing systems. The processing unit 710 can therefore receive information and instructions from other computing systems or services via the network. The processing unit 710 can also communicate with a memory 770 and further provide output information to an optional display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or programs, such as...) stored in the non-transitory memory of the analysis system... It can display flow cytometry event data to the user. The input / output device interface 740 can also accept input from optional input devices 760, such as keyboards, mice, digital pens, microphones, touch screens, gesture recognition systems, voice recognition systems, game controllers, accelerometers, gyroscopes, or other input devices.

[0191] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) that processing unit 710 executes to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store operating system 772, which provides computer program instructions for use by processing unit 710 in the general management and operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may further include computer program instructions and other information for implementing various aspects of this disclosure.

[0192] Non-transitory computer-readable storage medium

[0193] Various aspects of this disclosure further include non-transitory computer-readable storage media having instructions for practicing the subject methods, such as practicing one or more computer-implemented methods described herein. The computer-readable storage media can be used on one or more computers for systems that fully or partially automate the practice of the methods described herein. In some embodiments, instructions according to the methods described herein can be encoded in a “programmable” form onto a computer-readable medium, wherein the term “computer-readable medium” as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network-attached storage (NAS), whether such devices are internal or external to a computer. Files containing information can be “stored” on a computer-readable medium, where “stored” means recording the information so that it can be accessed and retrieved by a computer at a later time. The computer-implemented methods described herein can be executed using programming, which can be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Python, Java, JavaScript, C, C#, C++, Go, R, Swift, PHP, and many other languages.

[0194] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for illuminating a sample comprising particles in a flowing stream with a light source, an algorithm for detecting light from the particles in the sample using a light detection system including a photodetector, wherein the voltage of the photodetector is modulated at a modulation frequency between a first voltage and a second voltage, an algorithm for generating a data signal based on the light from the particles detected at one or more of the first photodetector voltage and the second photodetector voltage, and an algorithm for determining one or more parameters of the particles based on the generated data signal.

[0195] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for modulating a photodetector voltage between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for modulating a photodetector voltage between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for maintaining the photodetector voltage for 1 μs or less per cycle, for example, 0.5 μs or less per cycle. In some embodiments, the first photodetector voltage is higher than the second photodetector voltage. In some embodiments, the first photodetector voltage is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some embodiments, the second photodetector voltage is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0196] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for modulating a photodetector voltage between a first photodetector voltage and a second photodetector voltage with a square voltage variation. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light detected at the first photodetector voltage. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light detected at the second photodetector voltage. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on both light detected at the first photodetector voltage and light detected at the second photodetector voltage. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based solely on light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold. In some cases, the non-transitory computer-readable storage medium includes an algorithm for detecting light with a photodetector only at a second photodetector voltage when the light detected at a first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0197] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for measuring baseline samples of the generated data signal at each of a first photodetector voltage and a second photodetector voltage. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on the baseline samples. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples at a baseline sampling frequency. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for performing baseline sampling at a frequency from 0.0001 MHz to 15 MHz. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples of the generated data signal at a frequency greater than the modulation frequency. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples of the generated data signal at a frequency less than the modulation frequency. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for measuring the baseline samples of the generated data signal at a frequency equal to the modulation frequency. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for scaling the generated data signal. In some cases, non-transitory computer-readable storage media include algorithms for scaling data signals on a single continuous scale and dividing the resulting data signals by the photodetector gain at each photodetector voltage.

[0198] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample based on a data signal generated from light detected at a first photodetector voltage. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample based on a data signal generated from light detected at a second photodetector voltage. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample based on data signals generated from light detected at a first photodetector voltage and light detected at a second photodetector voltage.

[0199] Non-transitory computer-readable storage media can be used in one or more computer systems having a display and operator input devices. Operator input devices may be, for example, a keyboard, mouse, etc. A processing module includes a processor that can access memory having instructions stored thereon for performing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or other existing or future-available processors. The processor executes the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor's coordination and execution of various computer programs written in various programming languages, such as those mentioned above, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, storage management, and communication control and related services according to known technologies.

[0200] kit

[0201] Various aspects of this disclosure further include kits, wherein the kits include storage media such as magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network-attached storage (NAS). Any of these program storage media, or other media now in use or that may be developed in the future, may be included in the subject kit. In embodiments, the program storage media includes instructions for analyzing flow cytometry data, as described herein, and instructions for use with the systems described herein. In embodiments, instructions contained on a computer-readable medium provided in the subject kit, or portions thereof, may be implemented as software components of data analysis software. In these embodiments, a computer control system according to this disclosure may serve as an existing software package (e.g., ) software "plugins".

[0202] In addition to the components described above, the theme kit may further include (in some embodiments) instructions. These instructions may exist in various forms within the theme kit, with one or more of them present in the kit. One form of these instructions may be printed information on a suitable medium or substrate, such as printed paper, kit packaging, inserts, etc. Another form of these instructions is a computer-readable medium, such as a disk, optical disc (CD), portable flash drive, etc., on which information is recorded. Yet another form of these instructions may be a website address, accessible via the Internet for accessing information at a remote site.

[0203] practicality

[0204] The methods, systems, and computer systems described herein are useful in a variety of applications where calibration or optimization of optical detection systems (e.g., those with photodetectors) is required, for example, in particle analyzers. The methods and systems described herein are also used in optical detection systems for analyzing and sorting particulate components of samples in fluid media (e.g., biological samples). This disclosure is also applicable to flow cytometry, wherein it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In embodiments, this disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In some embodiments, the methods and systems described herein provide a fully automated scheme so that adjustments to the flow cytometer during use require little, if any, manual input.

[0205] Notwithstanding the appended claims, this disclosure is also defined by the following provisions:

[0206] 1. A method comprising:

[0207] Illuminate a sample containing particles in a flowing stream with a light source;

[0208] Light from particles in a sample is detected using a light detection system including a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency;

[0209] Based on the light generation data signal from the particle detected at one or more of the first and second photodetector voltages; and

[0210] One or more parameters of the particle are determined based on the generated data signal.

[0211] 2. The method according to Clause 1, wherein the voltage of the photodetector is modulated between the first voltage and the second voltage at a frequency of 0.005 MHz to 10 MHz.

[0212] 3. The method according to Clause 2, wherein the voltage of the photodetector is modulated between the first voltage and the second voltage at a frequency of 0.1 MHz to 1 MHz.

[0213] 4. The method according to any one of clauses 1-3, wherein the voltage of the photodetector is maintained per cycle.

[0214] 1μs or less.

[0215] 5. The method according to Clause 4, wherein the voltage of the photodetector is maintained for 0.5 μs or less per cycle.

[0216] 6. The method according to any one of clauses 1-5, wherein the voltage of the first photodetector is higher than the voltage of the second photodetector.

[0217] 7. The method according to any one of Clauses 1-6, wherein the voltage of the first photodetector is from 500mV to 2000mV.

[0218] 8. The method according to any one of Clauses 1-7, wherein the voltage of the second photodetector is from 0.001mV to 500mV.

[0219] 9. The method according to any one of clauses 1-8, wherein the voltage of the photodetector is modulated between the voltage of the first photodetector and the voltage of the second photodetector by a square voltage variation.

[0220] 10. The method according to any one of clauses 1-9, wherein the method includes generating a data signal based on light detected at a first photodetector voltage and light detected at a second photodetector voltage.

[0221] 11. The method according to Clause 10, wherein when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold, a data signal is generated based on the light detected at the second photodetector voltage.

[0222] 12. The method according to Clause 10, wherein when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold, the data signal is generated only based on the light detected at the second photodetector voltage.

[0223] 13. The method according to Clause 10, wherein the method includes detecting light with a photodetector only at a second photodetector voltage when the light detected at a first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0224] 14. The method according to any one of clauses 1-13, wherein the method further comprises measuring a baseline sample at a first photodetector voltage and a second photodetector voltage, and wherein a data signal is generated based on the baseline sample.

[0225] 15. The method according to Clause 14, wherein the baseline sample is measured at the baseline sampling frequency.

[0226] 16. The method according to Clause 15, wherein the baseline sampling frequency is from 0.0001 MHz to 15 MHz.

[0227] 17. The method according to any one of Clauses 15-16, wherein the baseline sampling frequency is greater than the modulation frequency.

[0228] 18. The method according to any one of Clauses 15-16, wherein the baseline sampling frequency is less than the modulation frequency.

[0229] 19. The method according to any one of Clauses 15-16, wherein the baseline sampling frequency is equal to the modulation frequency.

[0230] 20. The method according to any one of clauses 1-19 further includes scaling the generated data signal.

[0231] 21. The method according to Clause 20, wherein scaling includes continuous scaling and includes dividing the generated data signal by the photodetector gain at each photodetector voltage.

[0232] 22. The method according to any one of clauses 1-21, wherein the detected light comprises scattered light from particles in the flowing stream.

[0233] 23. The method according to Clause 22, wherein the detected light includes side-scattered light, forward-scattered light, or a combination thereof.

[0234] 24. A system comprising:

[0235] A light source used to illuminate a sample, including particles in a flowing stream;

[0236] A light detection system including a photodetector is used to detect light from particles in a sample, wherein the photodetector is configured as follows:

[0237] Modulation is performed between the voltage of the first photodetector and the voltage of the second photodetector; and

[0238] Based on the light generation data signal from the particle detected at one or more of the first and second photodetector voltages; and

[0239] The processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine one or more parameters of a particle based on a generated data signal.

[0240] 25. The system according to Clause 24, wherein the photodetector includes a photomultiplier tube (PMT).

[0241] 26. The system according to Clause 24, wherein the photodetector includes a photodiode.

[0242] 27. The system according to any one of clauses 24-26, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz.

[0243] 28. The system according to Clause 27, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz.

[0244] 29. The system according to any one of clauses 24-28, wherein the photodetector is configured to maintain a voltage of 1 μs or less per cycle.

[0245] 30. The system according to any one of clauses 24-28, wherein the photodetector is configured to maintain a voltage of 0.5 μs or less per cycle.

[0246] 31. The system according to any one of clauses 24-30, wherein the voltage of the first photodetector is higher than the voltage of the second photodetector.

[0247] 32. The system according to any one of clauses 24-31, wherein the voltage of the first photodetector is from 500mV to 2000mV.

[0248] 33. The system according to any one of clauses 24-32, wherein the voltage of the second photodetector is from 0.001mV to 500mV.

[0249] 34. The system according to any one of clauses 24-33, wherein the photodetector is configured to modulate between a square voltage variation between the voltage of the first photodetector and the voltage of the second photodetector.

[0250] 35. The system according to any one of clauses 24-34, wherein the photodetector is configured to generate a data signal based on light detected at a first photodetector voltage and light detected at a second photodetector voltage.

[0251] 36. The system according to Clause 35, wherein the photodetector is configured to generate a data signal based on the light detected at a second photodetector voltage when the light detected at a first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0252] 37. The system according to Clause 35, wherein the photodetector is configured to generate a data signal based solely on the light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0253] 38. The system according to any one of clauses 24-37, wherein the memory includes instructions stored thereon that, when executed by a processor, cause the processor to measure a baseline sample at a first photodetector voltage and a second photodetector voltage, and wherein the memory includes instructions for generating a data signal based on the baseline sample.

[0254] 39. The system according to Clause 38, wherein the memory includes instructions stored thereon that, when executed by a processor, cause the processor to measure baseline samples of the generated data signal at a baseline sampling frequency.

[0255] 40. The system according to Clause 38, wherein the memory includes instructions stored thereon that, when executed by a processor, cause the processor to measure a baseline sample of the generated data signal at a frequency greater than the modulation frequency.

[0256] 41. The system according to Clause 38, wherein the memory includes instructions stored thereon that, when executed by a processor, cause the processor to measure a baseline sample of the generated data signal at a frequency less than the modulation frequency.

[0257] 42. The system according to Clause 38, wherein the memory includes instructions stored thereon that, when executed by a processor, cause the processor to measure a baseline sample of the generated data signal at a frequency equal to the modulation frequency.

[0258] 43. The system according to any one of clauses 24-42, wherein the memory includes instructions stored thereon that, when executed by a processor, cause the processor to scale data signals.

[0259] 44. The system according to Clause 43, wherein the memory includes instructions stored thereon that, when executed by a processor, cause the processor to scale the data signal on a continuous scale and divide the resulting data signal by the photodetector gain at each photodetector voltage.

[0260] 45. The system according to any one of clauses 24-44, wherein the photodetector includes a scattered light detector.

[0261] 46. ​​The system according to Clause 45, wherein the photodetector is a side-scattering photodetector.

[0262] 47. The system according to Clause 45, wherein the photodetector is a forward-scattering photodetector.

[0263] 48. The system according to any one of clauses 24-47, wherein the system is a flow cytometer.

[0264] 49. A non-transitory computer-readable storage medium comprising instructions stored thereon, the instructions comprising:

[0265] An algorithm for illuminating a sample containing particles in a flowing stream with a light source;

[0266] An algorithm for detecting light from particles in a sample using a light detection system including a photodetector, wherein the voltage of the photodetector is modulated at a modulation frequency between a first voltage and a second voltage; and an algorithm for generating a data signal based on the light from the particles detected at one or more of the first photodetector voltage and the second photodetector voltage; and

[0267] An algorithm for determining one or more parameters of a particle based on the generated data signal.

[0268] 50. The non-transitory computer-readable storage medium according to Clause 49, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz.

[0269] 51. The non-transitory computer-readable storage medium according to Clause 49, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz.

[0270] 52. The non-transitory computer-readable storage medium according to any one of clauses 49-51, wherein the voltage of the photodetector is maintained for 1 μs or less per cycle.

[0271] 53. The non-transitory computer-readable storage medium as described in Clause 52, wherein the voltage of the photodetector is maintained for 0.5 μs or less per cycle.

[0272] 54. The non-transitory computer-readable storage medium according to any one of clauses 49-53, wherein the voltage of the first photodetector is higher than the voltage of the second photodetector.

[0273] 55. The non-transitory computer-readable storage medium according to any one of clauses 49-54, wherein the voltage of the first photodetector is from 500mV to 2000mV.

[0274] 56. The non-transitory computer-readable storage medium according to any one of clauses 49-55, wherein the voltage of the second photodetector is from 0.001mV to 500mV.

[0275] 57. The non-transitory computer-readable storage medium according to any one of clauses 49-56, wherein the voltage of the photodetector is modulated between the voltage of the first photodetector and the voltage of the second photodetector by a square voltage variation.

[0276] 58. The non-transitory computer-readable storage medium according to any one of clauses 49-57, wherein the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light detected under a first photodetector voltage and a second photodetector voltage.

[0277] 59. The non-transitory computer-readable storage medium according to Clause 58, wherein the non-transitory computer-readable storage medium includes an algorithm for generating a data signal based on light measured at a second photodetector voltage when light detected at a first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0278] 60. The non-transitory computer-readable storage medium according to Clause 59, wherein the non-transitory computer-readable storage medium includes an algorithm for measuring light with a photodetector only at a second photodetector voltage when light detected at a first photodetector voltage exhibits a detector saturation level greater than a saturation threshold.

[0279] 61. The non-transitory computer-readable storage medium according to any one of clauses 49-60, wherein the non-transitory computer-readable storage medium includes an algorithm for measuring a baseline sample at a first photodetector voltage and a second photodetector voltage, and an algorithm for generating a data signal based on the baseline sample.

[0280] 62. The non-transitory computer-readable storage medium as described in Clause 61, wherein the non-transitory computer-readable storage medium includes an algorithm for measuring baseline samples of the generated data signal at a baseline sampling frequency.

[0281] 63. The non-transitory computer-readable storage medium as described in Clause 62, wherein the non-transitory computer-readable storage medium includes an algorithm for baseline sampling at frequencies from 0.0001 MHz to 15 MHz.

[0282] 64. The non-transitory computer-readable storage medium as described in Clause 62, wherein the non-transitory computer-readable storage medium includes an algorithm for measuring baseline samples at a frequency greater than the modulation frequency.

[0283] 65. The non-transitory computer-readable storage medium as described in Clause 62, wherein the non-transitory computer-readable storage medium includes an algorithm for measuring baseline samples at a frequency less than the modulation frequency.

[0284] 66. The non-transitory computer-readable storage medium as described in Clause 62, wherein the non-transitory computer-readable storage medium includes an algorithm for measuring baseline samples at a frequency equal to the modulation frequency.

[0285] 67. The non-transitory computer-readable storage medium according to any one of clauses 49-66, wherein the non-transitory computer-readable storage medium includes an algorithm for scaling the generated data signal.

[0286] 68. The non-transitory computer-readable storage medium as described in Clause 67, wherein the non-transitory computer-readable storage medium includes an algorithm for scaling a data signal on a continuous scale and dividing the generated data signal by the photodetector gain at each photodetector voltage.

[0287] Although the foregoing disclosure has been described in detail by way of illustration and example for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of this disclosure, that some changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0288] Therefore, the foregoing only illustrates the principles of this disclosure. It will be understood that those skilled in the art will be able to design various arrangements that, while not explicitly described or shown herein, embody the principles of this disclosure and are included within its spirit and scope. Furthermore, all examples and conditional language cited herein are primarily intended to assist the reader in understanding the principles of this disclosure and the concepts it contributes to advancing the art, and should be construed as not being limited to these specifically cited examples and conditions. Moreover, all statements herein regarding the principles, aspects, and embodiments of this disclosure, as well as specific examples thereof, are intended to cover their structural and functional equivalents. Furthermore, it is intended that such equivalents include both currently known equivalents and future development equivalents, i.e., any development element performing the same function, regardless of its structure. Additionally, nothing disclosed herein is intended to be exclusive to the public, whether or not such disclosure is expressly stated in the claims.

[0289] Therefore, the scope of this disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied in the appended claims. In the claims, 35U.SC §112(f) or 35U.SC §112(6) expressly provides that the limitation is invoked only if the exact phrase “means for…” or the exact phrase “steps for…” is expressly recited at the beginning of the limitation in the claim; if such an exact phrase is not used in the limitation in the claim, 35U.SC §112(f) or 35U.SC §112(6) is not invoked.

Claims

1. A method comprising: Illuminate a sample containing particles in a flowing stream with a light source; Light from particles in a sample is detected using a light detection system including a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency; Based on the light generation data signal from the particle detected at one or more of the first photodetector voltage and the second photodetector voltage; as well as One or more parameters of the particle are determined based on the generated data signal.

2. The method according to claim 1, wherein, The voltage of the photodetector is modulated between the first voltage and the second voltage at a frequency of 0.005MHz to 10MHz.

3. The method according to claim 2, wherein, The voltage of the photodetector is modulated between the first voltage and the second voltage at a frequency of 0.1 MHz to 1 MHz.

4. The method according to any one of claims 1 to 3, wherein the voltage of the photodetector is maintained for 1 μs or less per cycle.

5. The method of claim 4, wherein the voltage of the photodetector is maintained for 0.5 μs or less per cycle.

6. The method according to any one of claims 1 to 5, wherein the voltage of the first photodetector is higher than the voltage of the second photodetector.

7. The method according to any one of claims 1 to 6, wherein the voltage of the first photodetector is from 500mV to 2000mV.

8. The method according to any one of claims 1 to 7, wherein the voltage of the second photodetector is from 0.001 mV to 500 mV.

9. The method according to any one of claims 1 to 8, wherein, The voltage of the photodetector is modulated by a square voltage change between the voltage of the first photodetector and the voltage of the second photodetector.

10. The method according to any one of claims 1 to 9, wherein, The method includes generating a data signal based on light detected at a first photodetector voltage and light detected at a second photodetector voltage.

11. The method of claim 10, wherein: (a) When the light detected at the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold, a data signal is generated based on the light detected at the second photodetector voltage; (b) When the light detected at the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold, a data signal is generated only based on the light detected at the second photodetector voltage; or (c) The method includes: when the light detected at the first photodetector voltage exhibits a detector saturation level greater than a saturation threshold, detecting the light only at the second photodetector voltage.

12. The method according to any one of claims 1 to 11, wherein the method further comprises: A baseline sample is measured at a first photodetector voltage and a second photodetector voltage, and a data signal is also generated based on the baseline sample.

13. The method according to claim 12, wherein, The baseline sample was measured at the baseline sampling frequency.

14. The method according to claim 13, wherein, The baseline sampling frequency is from 0.0001MHz to 15MHz.

15. The method according to any one of claims 13-14, wherein: The baseline sampling frequency is greater than the modulation frequency; The baseline sampling frequency is less than the modulation frequency; or The baseline sampling frequency is equal to the modulation frequency.

16. The method according to any one of claims 1 to 15, further comprising scaling the generated data signal.

17. The method of claim 16, wherein scaling includes continuous scaling and includes dividing the generated data signal by the photodetector gain at each photodetector voltage.

18. The method according to any one of claims 1 to 17, wherein the detected light comprises scattered light from particles in the flowing stream.

19. The method of claim 18, wherein the detected light comprises side-scattered light, forward-scattered light, or a combination thereof.

20. A system comprising: A light source used to illuminate a sample, including particles in a flowing stream; A light detection system including a photodetector is used to detect light from particles in a sample, wherein the photodetector is configured as follows: Modulation is performed between the voltage of the first photodetector and the voltage of the second photodetector; as well as Based on the light generation data signal from the particle detected at one or more of the first photodetector voltage and the second photodetector voltage; as well as A processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine one or more parameters of a particle based on a generated data signal.

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