Method and system for detector gain modulation

Abstract

In photodetection systems, this reduces saturation or noise in the photodetector. [Solution] The invention includes a method for modulating a photodetector in a photodetection system (for example, in a flow cytometer). A method according to a particular embodiment includes irradiating a sample having particles in a flow stream with a light source, detecting light from the particles in the sample with a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency, generating a data signal from the light detected from the particles at one or more of the first and second photodetector voltages, and determining one or more parameters of the particles from the generated data signal. A system and a non-temporary computer-readable storage medium configured to perform the method in question are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications In accordance with 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 699,320, filed on 26 September 2024, and the disclosure of this U.S. Provisional Patent Application is incorporated herein by reference in its entirety. [Background technology]

[0002] Introduction The characterization of biological fluid analytes is a crucial part of biological research, medical diagnosis, and the assessment of a patient's overall health and wellness. Detecting biological fluid analytes, such as human blood or blood-derived products, can yield results that can play a role in determining treatment protocols for patients with various disease conditions.

[0003] Flow cytometry is a technique used to characterize and frequently sort biological materials, such as cells in a blood sample or particles of interest contained in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir for containing sheath fluid. The flow cytometer directs the sheath fluid to the flow cell while transporting particles (including cells) in the fluid sample as a cell stream to the flow cell. Light is shone into the flow stream to characterize its components. Variations in the material in the flow stream, such as morphology or the presence of fluorescent labels, can cause variations in the observed light, and these variations enable characterization and separation. To characterize the components of the flow stream, light must strike and collect from the flow stream. The light source of a flow cytometer can vary and may include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the shone particles is collected and quantified.

[0004] The isolation of biological particles has been achieved by adding sorting or recovery capabilities to flow cytometers. Particles in a segmented stream are detected if they possess one or more desirable characteristics, and these are individually isolated from the sample stream by mechanical or electrical removal. Common flow sorting techniques utilize droplet sorting, where a fluid stream containing linearly segmented particles is divided into droplets. Droplets containing the desired particles are charged and deflected towards a recovery tube by passing through an electric field. Typically, the linearly segmented particles in the stream have their characteristics determined as they pass through an observation point positioned directly below the nozzle tip. Once a particle is identified as meeting one or more desirable criteria, the time at which it reaches the droplet's breakoff point and separates from the stream can be predicted. Ideally, the stream is briefly charged just before the droplet containing the selected particles separates from the fluid stream, and then grounded immediately after the droplet breaks off. The sorted droplet retains its charge when it breaks off from the fluid stream, while all other droplets remain uncharged.

[0005] Flow cytometers measure signals using arbitrary units. Flow cytometers are capable of analyzing a wide range of cell types across large sizes and refractive indices. For any flow cytometry experiment, the most common parameter to trigger data and optimize first is light scattering. Optimal tuning of these parameters generally requires training depending on the target population(s) and can be subtly different or subjective, as trade-offs must be made due to the limited dynamic range of the detector. While conventional flow cytometer photodetector systems have a dynamic range of approximately 4 decades in a given setting, the range of detectable cell sizes addressed by flow cytometry applications extends to at least 7 decades. Across their available settings, photodetectors can differ in their detection limits by approximately 4.5 decades, meaning their total dynamic range can extend to 8-9 decades. [Overview of the Initiative]

[0006] Aspects of the present disclosure include methods for modulating a photodetector in a photodetection system (for example, in a flow cytometer). A method according to a particular embodiment includes irradiating a sample having particles in a flow stream with a light source, detecting light from the particles in the sample with a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency, generating a data signal from the light detected from the particles at one or more of the first and second photodetector voltages, and determining one or more parameters of the particles from the generated data signal. A system and a non-temporary computer-readable storage medium configured to perform the method in question are also provided.

[0007] In some embodiments, the photodetector voltage is modulated between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some cases, the photodetector voltage is modulated between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz. In some cases, the photodetector voltage is maintained for a period of 1 μs or less, for example, 0.5 μs or less. In some embodiments, the first photodetector voltage is higher than the second photodetector voltage. In some cases, the first photodetector voltage is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some cases, the second photodetector voltage is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0008] In some embodiments, the photodetector voltage is modulated between a first photodetector voltage and a second photodetector voltage with a rectangular voltage change. In some cases, the method includes generating a data signal from light detected at the first photodetector voltage. In some cases, the method includes generating a data signal from light detected at the second photodetector voltage. In certain specific cases, the method includes generating a data signal from light detected at both the first and second photodetector voltages. In some cases, the data signal is generated from 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 data signal is generated only from 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 method includes detecting light by 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.

[0009] In some embodiments, the method includes measuring a baseline sample at a first photodetector voltage and a second photodetector voltage. In some cases, a data signal is further generated based on the baseline sample. In some cases, the method includes baseline sampling at a predetermined frequency. In some cases, the method includes measuring a high-frequency baseline sample at a baseline frequency. In some cases, the method includes measuring a baseline sample at a baseline frequency of 0.0001 MHz to 15 MHz. In some cases, the baseline sampling frequency is greater than the modulation frequency. In some cases, the baseline sampling frequency is less than the modulation frequency. In some cases, the baseline sampling frequency is equal to the modulation frequency. In some embodiments, the generated data signal is scaled. In some cases, 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.

[0010] In some embodiments, one or more parameters of particles in a sample are determined from a data signal generated from light detected at a first photodetector voltage. In some cases, one or more parameters of particles in a sample are determined from a data signal generated from light detected at a second photodetector voltage. In a particular case, one or more parameters of particles in a sample are determined from a data signal generated from light detected at both the first and second photodetector voltages.

[0011] In some embodiments, light is detected by multiple photodetector channels. In some cases, the detected light is scattered light, such as forward scattered light, side scattered light, or a combination thereof. In some cases, the method involves irradiating the sample with a light source. In some cases, the light source includes lasers, such as multiple lasers.

[0012] Aspects of the present disclosure also include systems for carrying out the methods covered, for example, systems for modulating a photodetector in a photodetector system (such as in a flow cytometer). A system according to a particular embodiment includes a light source configured to irradiate a sample having particles in a flow stream, and a photodetector for detecting light from particles in the sample, wherein the photodetector is configured to modulate between a first photodetector voltage and a second photodetector voltage to generate a data signal from the light detected from the particles at one or more of the first and second photodetector voltages. In embodiments, the system also includes a processor having memory operably coupled to the processor, the memory storing instructions, which, when executed by the processor, cause the processor to generate a data signal in response, and to determine one or more parameters of particles from the generated data signal.

[0013] In some embodiments, the photodetector voltage is modulated between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some cases, the photodetector voltage is modulated between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz. In some cases, the photodetector is configured to maintain a voltage for a period of 1 μs or less, for example, 0.5 μs or less. In some embodiments, the first photodetector voltage is higher than the second photodetector voltage. In some cases, the first photodetector voltage is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some cases, the second photodetector voltage is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0014] In some embodiments, the photodetector system is configured to modulate the voltage of the photodetector between a first photodetector voltage and a second photodetector voltage with a rectangular voltage change. In some cases, the photodetector is configured to generate a data signal from light detected at the first photodetector voltage. In some cases, the photodetector is configured to generate a data signal from light detected at the second photodetector voltage. In certain cases, the photodetector is configured to generate a data signal from light detected at both the first and second photodetector voltages. In some cases, the photodetector is configured to generate a data signal from 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 photodetector is configured to generate a data signal only from 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 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.

[0015] In some embodiments, the memory includes instructions that, when executed by the process, cause the processor to measure a baseline sample at a first photodetector voltage and a second photodetector voltage. In some cases, the memory includes instructions for generating a data signal based on the baseline sample. In some cases, the memory includes instructions for measuring the baseline sample at a baseline sampling frequency. In some cases, the memory includes instructions for measuring the baseline sample at frequencies from 0.0001 MHz to 15 MHz. In some cases, the memory includes instructions for measuring the baseline sample of the generated data signal at a frequency greater than the modulation frequency. In some cases, the memory includes instructions for measuring the baseline sample of the generated data signal at a frequency less than the modulation frequency. In some cases, the memory includes instructions for measuring the baseline sample 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 cases, the memory includes instructions for scaling the data signal on a single continuous scale and for dividing the generated data signal by the photodetector gain at each photodetector voltage.

[0016] In some embodiments, the memory includes instructions for determining one or more parameters of particles in a sample from a data signal generated from light detected at a first photodetector voltage. In some cases, the memory includes instructions for determining one or more parameters of particles in a sample from a data signal generated from light detected at a second photodetector voltage. In a particular case, the memory includes instructions for determining one or more parameters of particles in a sample from data signals generated from light detected at a first photodetector voltage and light detected at a second photodetector voltage.

[0017] In some embodiments, the photodetector system includes a photodetector configured to detect scattered light. In some cases, the photodetector is a side-scattering light detector. In some cases, the photodetector is a forward-scattering light detector. In some cases, the system is a flow cytometer.

[0018] Aspects of the present disclosure also include non-temporary computer-readable storage media for carrying out, for example, one or more computer implementation methods described herein. In some embodiments, the non-temporary computer-readable storage media includes an algorithm for irradiating a sample containing particles in a flow stream with a light source; an algorithm for detecting light from particles in the sample with a photodetector system, 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 from the light detected from the particles at one or more of the first and second photodetector voltages; and an algorithm for determining one or more parameters of the particles from the generated data signal.

[0019] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for modulating the photodetector voltage between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some cases, the non-temporary computer-readable storage medium includes an algorithm for modulating the photodetector voltage between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz. In some cases, the non-temporary computer-readable storage medium includes an algorithm for maintaining the photodetector voltage for a period of 1 μs or less, for example, 0.5 μs or less. In some embodiments, the first photodetector voltage is higher than the second photodetector voltage. In some cases, the first photodetector voltage is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some cases, the second photodetector voltage is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0020] In some embodiments, the non - transitory computer - readable storage medium includes an algorithm for modulating the voltage of the photodetector between a first photodetector voltage and a second photodetector voltage with a square - shaped voltage change. In some cases, the non - transitory computer - readable storage medium includes an algorithm for generating a data signal from the 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 from the light detected at the second photodetector voltage. In a particular case, the non - transitory computer - readable storage medium includes an algorithm for generating a data signal from the light detected at the first photodetector voltage and the 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 from the light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold. In some cases, the non - transitory computer - readable storage medium includes an algorithm for generating a data signal only from the light detected at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold. In some cases, the non - transitory computer - readable storage medium includes an algorithm for detecting light by the photodetector only at the second photodetector voltage when the light detected at the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold.

[0021] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a first photodetector voltage and a second photodetector voltage, respectively. In some cases, the non-temporary computer-readable storage medium includes an algorithm for generating a data signal based on the baseline sample. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring the baseline sample at a baseline sampling frequency. In some cases, the non-temporary computer-readable storage medium includes an algorithm for baseline sampling at frequencies from 0.0001 MHz to 15 MHz. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a frequency greater than the modulation frequency. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a frequency less than the modulation frequency. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a frequency equal to the modulation frequency. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for scaling the generated data signal. In some cases, non-temporary computer-readable storage media include algorithms for scaling data signals on a single continuous scale and dividing the generated data signals by the photodetector gain at each photodetector voltage.

[0022] In some embodiments, the non - transient computer - readable storage medium includes an algorithm for determining one or more parameters of particles in a sample from a data signal generated from light detected at a first photodetector voltage. In some cases, the non - transient computer - readable storage medium includes an algorithm for determining one or more parameters of particles in a sample from a data signal generated from light detected at a second photodetector voltage. In a particular case, the non - transient computer - readable storage medium includes an algorithm for determining one or more parameters of particles in a sample from data signals generated from light detected at a first photodetector voltage and light detected at a second photodetector voltage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] This disclosure may be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures.

[0024] [Figure 1A] A flowchart for determining one or more parameters of particles of a sample in a flow stream based on light detected by a modulated photodetector according to a particular embodiment is shown. [Figure 1B] Detection of light from particles of a sample using a modulated photodetector according to a particular embodiment is shown. [Figure 2] A flow cytometry system according to a particular embodiment is presented. [Figure 3A] An image - corresponding particle sorter according to a particular embodiment is shown. [Figure 3B] An image - corresponding particle sorter according to a particular embodiment is shown (continuation of FIG. 3A). [Figure 4] A functional block diagram of a particle analysis system according to a particular embodiment is shown. [Figure 5] A functional block diagram of an example of a control system according to a particular embodiment is shown. [Figure 6A] A schematic diagram of a particle sorter system according to a particular embodiment is shown. [Figure 6B]A schematic diagram of a particle sorting system according to a specific embodiment is shown. [Figure 7] This shows an embodiment of a computer-controlled system according to a particular example. [Modes for carrying out the invention]

[0025] Aspects of the present disclosure include methods for modulating a photodetector in a photodetection system (for example, in a flow cytometer). A method according to a particular embodiment includes irradiating a sample having particles in a flow stream with a light source, detecting light from the particles in the sample with a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency, generating a data signal from the light detected from the particles at one or more of the first and second photodetector voltages, and determining one or more parameters of the particles from the generated data signal. A system and a non-temporary computer-readable storage medium configured to perform the method in question are also provided.

[0026] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and can therefore naturally vary. It should also be understood that the scope of this disclosure is limited only by the appended claims, and that the terms used herein are intended solely to describe and not to limit the specific embodiments.

[0027] Where a range of values ​​is presented, unless explicitly indicated in the context, each value between the upper and lower limits of that range, up to one-tenth of the lower limit, and any other stated or in-between values ​​within that stated range are understood to be included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in this disclosure, subject to any specifically excluded limits of the stated range. If the stated range includes one or both limits, the range excluding one or both of those limits is also included in this disclosure.

[0028] In this specification, certain ranges are presented with the term “approximately” preceding the number. The term “approximately” is used herein to provide verbatim support for the exact number preceding the term, and for numbers that are close to or nearly the number preceding the term. In determining whether a number is close to or approximates a specifically stated number, the close or approximate unstated number may be a number that, in the context in which it is presented, presents a substantial equivalent of the specifically stated number.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but representative exemplary methods and materials are described below.

[0030] All publications and patents cited herein are incorporated herein by reference, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the manner and / or materials by which the publications are cited. Any citation of a publication is for the purpose of making that disclosure prior to the filing date, and this disclosure should not be construed as an acknowledgment that such publication has no prior rights due to a prior disclosure. Furthermore, the publication dates provided may differ from the actual publication dates and may need to be independently verified.

[0031] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. It should also be noted that the claims may be drafted to exclude any of their elements. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as “alone,” “only,” etc., in relation to the enumeration of elements of the claims or the use of “negative” limitations.

[0032] As will be obvious to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any of the listed methods can be carried out in the order of the listed events, or in any other logically possible order.

[0033] While systems and methods are described or may be described for grammatical fluidity with functional descriptions, claims should not necessarily be construed as being limited by constructing a limitation of “means” or “steps” unless explicitly formulated under 35 U.S. SC § 112, and the meaning of the definitions and the full scope of equivalents provided by the claims should be given under the doctrine of equivalents, and if the claims are explicitly formulated under 35 U.S. SC § 112, the full legal equivalents should be given under 35 U.S. SC § 112.

[0034] A method for modulating a photodetector in a photodetection system. Aspects of the present disclosure include methods for modulating a photodetector in a photodetector system (for example, in a flow cytometer). In embodiments, modulating the photodetector increases the sensitivity in detecting particles of various sizes and luminances of fluorophores used in fluorescence measurements. In some embodiments, the methods in question provide an expansion of the dynamic range of the photodetector. The term dynamic range is used herein in its conventional sense and refers to the response range of the photodetector, such as the difference between its noise floor (lower threshold) and saturation intensity (upper threshold). In some embodiments, the methods and systems in question increase the dynamic range of the photodetector by 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 100% or more, e.g., 150% or more, e.g., 200% or more, e.g., 250% or more, and include cases where the dynamic range of the photodetector is increased by 300% or more. In some cases, modulating the photodetector described herein expands the dynamic range of the photodetector by more than 0.5 decades, e.g., more than 1 decade, e.g., more than 1.5 decades, e.g., more than 2 decades, e.g., more than 2.5 decades, e.g., more than 3 decades, e.g., more than 3.5 decades, e.g., more than 4 decades, e.g., more than 4.5 decades, e.g., more than 5 decades, e.g., more than 7.5 decades, and e.g., more than 10 decades. In some cases, the total dynamic range of the photodetector in the system of interest is in the range of 4 to 12 decades, e.g., 6 to 11 decades, e.g., 7 to 10 decades, and e.g., 8 to 9 decades. In some cases, the method and system of interest reduces detector saturation when detecting light from an irradiated sample. In some cases, the method and system of interest reduces photodetector noise in the data signal from the light detected from an irradiated sample.

[0035] In some embodiments, modulating the photodetector described herein increases the detectable particle (e.g., cell) size by 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 1.5 times or more, e.g., 2 times or more, e.g., 5 times or more, and e.g., 10 times or more. In certain cases, simultaneous detection of very dark fluorophores and very bright fluorophores (e.g., in a sample having different types of particles and fluorescent markers) is provided, in which case, for example, the signal intensity of the detected fluorescence differs between the types of fluorophores by 50% or more, e.g., 75% or more, e.g., 100% or more, e.g., 1.5 times or more, e.g., 2 times or more, e.g., 3 times or more, e.g., 4 times or more, e.g., 5 times or more, e.g., 6 times or more, e.g., 7 times or more, e.g., 8 times or more, e.g., 9 times or more, e.g., 10 times or more, e.g., 25 times or more, e.g., 50 times or more, and e.g., 100 times or more.

[0036] In certain embodiments, the method in question provides optimized photodetector system performance, such as an increase in the signal-to-noise ratio of the photodetector system. For example, the signal-to-noise ratio of the photodetector system can be increased by 5% or more, e.g., 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more, and e.g., 99% or more. In certain cases, the method in question increases the signal-to-noise ratio by more than two times, e.g., three times or more, e.g., four times or more, e.g., five times or more, and e.g., ten times or more. In some embodiments, the method in question increases the consistency of the output from the photodetector of the photodetector system by 5% or more, e.g., 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more, and e.g., 99% or more.

[0037] When performing the method in question, a sample containing particles (for example, in the flow stream of a flow cytometer) is irradiated with light from a light source. In some embodiments, the light source is a broadband light source that emits light with a wide range of wavelengths, including, for example, 50 nm and above, 100 nm and above, 150 nm and above, 200 nm and above, 250 nm and above, 300 nm and above, 350 nm and above, 400 nm and above, and 500 nm and above. 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. If the method involves illumination with a broadband light source, the broadband light source protocol of interest may include, but is not limited to, other broadband light sources such as halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with a continuous spectrum, superluminescent light-emitting diodes, semiconductor light-emitting diodes, wide-spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof.

[0038] In other embodiments, the method includes irradiating with a narrowband light source that emits a specific wavelength or a narrow range of wavelengths, for example, a light source that emits light with wavelengths such as 40 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, or 2 nm or less, and a light source that emits a specific wavelength of light (i.e., monochromatic light). Where the method includes irradiating with a narrowband light source, the narrowband light source protocol of interest may include, but is not limited to, a narrow-wavelength LED, laser diode, or broadband light source coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0039] In certain embodiments, the method includes irradiating a sample with one or more lasers. As discussed above, the type and number of lasers vary depending on the sample and the desired light to be collected, and may be gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other cases, the method includes irradiating a flow stream with dye lasers such as stilbene, coumarin, or rhodamine lasers. In further cases, the method involves irradiating a flowstream with a metallic 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 a combination thereof. In yet another case, the method involves irradiating a flowstream with a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:yCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium-sapphire laser, a trimmyrm YAG laser, a ytterbium YAG laser, a ytterbium-2O3 laser or a cerium-doped laser, or a combination thereof.

[0040] The sample may be irradiated with one or more of the above-mentioned light sources, for example, two or more light sources, for example, three or more light sources, for example, four or more light sources, for example, five or more light sources, and for example, ten or more light sources. The light sources may include any combination of light sources of any type. For example, in some embodiments, the method includes irradiating the sample in a flow 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.

[0041] The sample may be irradiated with wavelengths in the range of 200 nm to 1500 nm, for example 250 nm to 1250 nm, for example 300 nm to 1000 nm, for example 350 nm to 900 nm, and for example 400 nm to 800 nm. For example, if the light source is a broadband light source, the sample may be irradiated with wavelengths in the range of 200 nm to 900 nm. In other cases where the light source includes multiple narrowband light sources, the sample may be irradiated with specific wavelengths in the range of 200 nm to 900 nm. For example, the light source may be multiple narrowband LEDs (1 nm to 25 nm) each independently emitting light having a wavelength range of 200 nm to 900 nm. In other embodiments, the narrowband light source includes one or more lasers (e.g., a laser array), and the sample is irradiated with specific wavelengths in the range of 200 nm to 700 nm using a laser array, such as the gas laser, excimer laser, dye laser, metal vapor laser, and solid-state laser described above.

[0042] When two or more light sources are used, the sample may be irradiated by the light sources simultaneously, sequentially, or in combination thereof. For example, each light source may irradiate the sample simultaneously. In other embodiments, the flowstream is irradiated sequentially by each light source. When two or more light sources are used to sequentially irradiate the sample, the duration for which each light source irradiates the sample can be independently 0.001 microseconds or more, e.g., 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 30 microseconds or more, and e.g., 60 microseconds or more. For example, the method may include irradiating the sample with a light source (e.g., a laser) for durations ranging from 0.001 microseconds to 100 microseconds, e.g., 0.01 microseconds to 75 microseconds, e.g., 0.1 microseconds to 50 microseconds, e.g., 1 microsecond to 25 microseconds, and e.g., 5 microseconds to 10 microseconds. In embodiments in which the sample is sequentially irradiated by two or more light sources, the duration for which the sample is irradiated by each light source may be the same or different.

[0043] The interval between irradiations from each light source may also be independently and variable as needed, by delays of 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 15 microseconds or more, e.g., 30 microseconds or more, and e.g., 60 microseconds or more. For example, the interval between irradiations from each light source may be in the range of 0.001 microseconds to 60 microseconds, e.g., 0.01 microseconds to 50 microseconds, e.g., 0.1 microseconds to 35 microseconds, e.g., 1 microsecond to 25 microseconds, and e.g., 5 microseconds to 10 microseconds. In a particular embodiment, the interval between irradiations from each light source is 10 microseconds. In embodiments in which the sample is sequentially irradiated by more than two (i.e., three or more) light sources, the delays between irradiations from each light source may be the same or different.

[0044] The sample may be irradiated continuously or at discrete intervals. In some cases, the method involves continuously irradiating the sample with a light source. In other cases, the sample is irradiated by 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, for example, every 1000 milliseconds, or at any other interval.

[0045] Depending on the light source, the sample may be irradiated from various distances, such as 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 2.5 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 15 mm or more, e.g., 25 mm or more, and e.g., 50 mm or more. The angle or irradiation may also be variable within the range of 10° to 90°, e.g., 15° to 85°, e.g., 20° to 80°, e.g., 25° to 75°, and e.g., 30° to 60°, e.g., 90°.

[0046] In certain embodiments, the method includes irradiating a sample with two or more frequency-shifted light beams. As described above, a light beam generator component having a laser and an acousto-optical device for frequency-shifting the laser light may be used. In these embodiments, the method includes irradiating the acousto-optical device with a laser. Depending on the desired wavelength of light produced by the output laser beam (for example, for use when irradiating a sample in a flow stream), the laser may have specific wavelengths such as 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, and e.g., 400 nm to 800 nm. The acousto-optical device may be irradiated with one or more lasers, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, and e.g., ten or more lasers. The lasers may include any combination of lasers. For example, in some embodiments, the method includes irradiating the acousto-optical 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.

[0047] When two or more lasers are used, the acousto-optical device may be irradiated by the lasers simultaneously, sequentially, or in combination thereof. For example, the acousto-optical device may be irradiated simultaneously by each of the lasers. In other embodiments, the acousto-optical device is irradiated sequentially by each of the lasers. When two or more lasers are used to sequentially irradiate the acousto-optical device, the time each laser irradiates the acousto-optical device may be independently 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 30 microseconds or more, and e.g., 60 microseconds or more. For example, the method may include irradiating the acousto-optical device with lasers for durations ranging from 0.001 microseconds to 100 microseconds, e.g., 0.01 microseconds to 75 microseconds, e.g., 0.1 microseconds to 50 microseconds, e.g., 1 microsecond to 25 microseconds, and e.g., 5 microseconds to 10 microseconds. In an embodiment in which an acoustic-optical device is sequentially irradiated by two or more lasers, the duration for which the acoustic-optical device is irradiated by each laser may be the same or different.

[0048] The interval between irradiations by each laser may also be independently and variable as needed, by delays of 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 15 microseconds or more, e.g., 30 microseconds or more, and e.g., 60 microseconds or more. For example, the interval between irradiations by each light source may be in the range of 0.001 microseconds to 60 microseconds, e.g., 0.01 microseconds to 50 microseconds, e.g., 0.1 microseconds to 35 microseconds, e.g., 1 microsecond to 25 microseconds, and e.g., 5 microseconds to 10 microseconds. In a particular embodiment, the interval between irradiations by each laser is 10 microseconds. In embodiments in which the acousto-optical device is sequentially irradiated by more than two (i.e., three or more) lasers, the delays between irradiations by each laser may be the same or different.

[0049] Acousto-optical devices can be irradiated continuously or at discrete intervals. In some cases, the method involves continuously irradiating the acousto-optical device with a laser. In other cases, the acousto-optical 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, for example, every 1000 milliseconds, or at any other interval.

[0050] Depending on the laser, the acousto-optic device may be illuminated from various distances, such as 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 2.5 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 15 mm or more, e.g., 25 mm or more, and e.g., 50 mm or more. The angle or illumination may also be variable within the range of 10° to 90°, e.g., 15° to 85°, e.g., 20° to 80°, e.g., 25° to 75°, and e.g., 30° to 60°, e.g., 90°.

[0051] In some embodiments, the method includes applying a high-frequency drive signal to an acoustic-optical device to generate an angularly deflected laser beam. Two or more high-frequency drive signals, for example, three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals, and for example, one hundred or more high-frequency drive signals may be applied to the acoustic-optical device to generate an output laser beam having a desired number of angularly deflected laser beams.

[0052] Each angle-deflected laser beam produced by a high-frequency drive signal has an intensity based on the amplitude of the applied high-frequency drive signal. In some embodiments, the method includes applying a high-frequency drive signal having an amplitude sufficient to produce an angle-deflected laser beam having a desired intensity. In some cases, each of the applied high-frequency drive signals independently has an amplitude of about 0.001V to about 500V, e.g., about 0.005V to about 400V, e.g., about 0.01V to about 300V, e.g., about 0.05V to about 200V, e.g., about 0.1V to about 100V, e.g., about 0.5V to about 75V, e.g., about 1V to 50V, e.g., about 2V to 40V, e.g., 3V to about 30V, and e.g., about 5V to about 25V. In some cases, each of the applied high-frequency drive signals independently has an amplitude of approximately 0.001V to 100V, for example, approximately 0.001V to 200V, for example, 0.001V to 300V, for example, 0.001V to 400V, and for example, 0.001V to 500V. In some embodiments, each of the applied high-frequency drive signals has a frequency of approximately 0.001MHz to approximately 500MHz, for example, approximately 0.005MHz to approximately 400MHz, for example, approximately 0.01MHz to approximately 300MHz, for example, approximately 0.05MHz to approximately 200MHz, for example, approximately 0.1MHz to approximately 100MHz, for example, approximately 0.5MHz to approximately 90MHz, for example, approximately 1MHz to approximately 75MHz, for example, approximately 2MHz to approximately 70MHz, for example, approximately 3MHz to approximately 65MHz, for example, approximately 4MHz to approximately 60MHz, and for example, approximately 5MHz to approximately 50MHz. In some embodiments, each of the applied high-frequency drive signals has a frequency of approximately 0.001 MHz to approximately 100 MHz, for example 0.001 MHz to 200 MHz, for example 0.001 MHz to 300 MHz, for example 0.001 MHz to 400 MHz, for example 0.001 MHz to 500 MHz.

[0053] In these embodiments, the angle-deflected laser beams within the output laser beam are spatially separated. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams may be separated by distances of 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, e.g., 5000 μm or more, and e.g., 5000 μm or more. In some embodiments, the angle-deflected laser beams overlap with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam, etc. The overlap between adjacent angle-deflected laser beams (such as beam spot overlap) may be 0.001 μm or larger, for example 0.005 μm or larger, for example 0.01 μm or larger, for example 0.05 μm or larger, for example 0.1 μm or larger, for example 0.5 μm or larger, for example 1 μm or larger, for example 5 μm or larger, for example 10 μm or larger, and for example 100 μm or larger.

[0054] In certain cases, see Diebold, et al. Nature Photonics Vol.7(10);806-810(2013) and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, and As described in Patent Nos. 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 (these disclosures are incorporated herein by reference), a flowstream is irradiated with multiple frequency-shifted light beams to image particles in the flowstream.

[0055] When the method of the subject is performed, light from each particle is detected by a photodetector system. In embodiments, the photodetector system includes a photodetector comprising one or more, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, and e.g., ten or more photodetectors. The photodetector for performing the method of the subject may be any convenient photodetector protocol, including but not limited to, other photodetectors, including, but also including, photosensitive photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), sensitized charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes and combinations thereof. In certain embodiments, the photodetector is 0.01 cm 2 ~10cm 2, for example, 0.05 cm 2 ~9 cm 2 , for example, 0.1 cm 2 ~8 cm 2 , for example, 0.5 cm 2 ~7 cm 2 , and for example, 1 cm 2 ~5 cm 2 It is a photomultiplier tube such as a photomultiplier tube having an active detection surface area of each region within the range of. Light from the irradiated sample is detected in two or more, for example three or more, for example four or more, for example five or more, for example six or more, for example seven or more, for example eight or more, for example nine or more, for example ten or more, for example twelve or more, for example sixteen or more, for example twenty-four or more, for example twenty-four or more, for example thirty-two or more, for example sixty-four or more, for example one hundred and twenty-eight or more, for example two hundred and fifty-six or more photodetector channels, and including five hundred and twelve or more photodetector channels.

[0056] Light can be measured by a photodetector, including measuring light from particles in the flow stream at one or more wavelengths, for example two or more wavelengths, for example five or more different wavelengths, for example ten or more different wavelengths, for example twenty-five or more different wavelengths, for example fifty or more different wavelengths, for example one hundred or more different wavelengths, for example two hundred or more different wavelengths, for example three hundred or more different wavelengths, and at four hundred or more different wavelengths. Light can be measured continuously or at discrete intervals. In some cases, the detector of interest is configured to continuously acquire measurements of light. In other cases, the detector of interest is configured to make measurements at discrete intervals, such as measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and for example every 1000 milliseconds, or at some other interval.

[0057] In certain embodiments, the light detected from a sample is scattered light. The term “scattered light” is used herein in its conventional sense and refers to the propagation of light energy from particles in a sample (e.g., flowing through a flowstream) that have been deflected from the incident beam path by reflection, refraction, or deflection of the light beam. In certain cases, the scattered light detected from particles in a flowstream is forward scattered light (FSC). In other cases, the scattered light detected from particles in a flowstream is side scattered light (SSC). In yet another case, the scattered light detected from particles in a flowstream is back scattered light (BSC).

[0058] In some embodiments, the light detected from each particle in the sample is transmitted light, such as light detected by a bright-field photodetector. In other embodiments, the light detected from each particle in the sample is emitted light, such as particle emission (i.e., fluorescence or phosphorescence). In these embodiments, each particle may include one or more fluorophores that emit fluorescence in response to irradiation by two or more light sources. For example, each particle may include two or more fluorophores, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, and e.g., ten or more fluorophores. In some cases, each particle includes a first fluorophore that emits fluorescence in response to irradiation by a first laser and a second fluorophore that emits fluorescence in response to irradiation by a second laser. In some embodiments, the fluorophore of interest may include, but are not limited to, dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.), such as acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinoneimine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, as well as dyes combining two or more of the aforementioned dyes (e.g., in tandem), polymer dyes having one or more monomer dye units, and mixtures of two or more of the aforementioned dyes. Numerous dyes are produced by companies such as Molecular Probes (Eugene, OR), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, MO), Sirigen, Inc. (Santa Barbara, CA), and Exciton (Dayton,Fluorophores are commercially available from various suppliers such as OH). For example, fluorophores include 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine, and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, peridinin-chlorophyll protein, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl [Lu]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; brilliant yellow; coumarin, and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcruarin (coumaran 151); cyanine, and derivatives such as cyanosine, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-diaminidino- 2-Phenylindole (DAPI); 5',5”-Dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-Diethylamino-3-(4'-Isothiocyanatophenyl)-4-methylcoumarin; Diethylaminocoumarin; Diethylenetriaminepentaacetate; 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1- Sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin, and derivatives such as eosin and eosin isothiocyanate; erythrosine, and derivatives such as erythrosine B and erythrosine isothiocyanate; ethidium; fluorescein, and 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 derivatives such as QFITC ​​(XRITC); fluorescein; IR144; IR1446; green fluorescent protein (GFP); coral fluorescent protein (RCFP); Lisamin™; Lisamin Rhodamine, Lucifer Yellow; malachite green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararoseaniline; Nile Red; Oregon Green; phenol Red; β-phycoerythrin; o-phthalidaldehyde; pyrene, and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-pyrenebutyrate; Reactive Red4 (Cibacron® Brilliant Red 3B-A); Rhodamine, as well as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lysamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulforhodamine 101 sulfonyl chloride derivatives (Texas This may include pigment-conjugated polymers (i.e., polymer-bound pigments) such as red, N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthenes; fluorescein isothiocyanate-dextran; as well as pigments combining two or more pigments (e.g., in tandem), polymer pigments having one or more monomer pigment units, and mixtures of two or more of the aforementioned pigments or combinations thereof.

[0059] In some cases, the fluorophore (i.e., dye) is a fluorescent polymer dye. A variety of fluorescent polymer dyes are found to be used in the methods and systems of interest. In some cases of this method, the polymer dyes include conjugated polymers. Conjugated polymers (CPs) are characterized by a delocalized electronic structure containing an alternating backbone of unsaturated (e.g., double and / or triple) and saturated (e.g., single) bonds, where π electrons can move from one bond to the other. Thus, the conjugated backbone can confer an elongated linear structure on the polymer dye, where the bond angles between the repeating units of the polymer are restricted. For example, proteins and nucleic acids are polymers, but in some cases do not form elongated rod structures, but rather fold into higher-order three-dimensional shapes. Furthermore, CPs can form a “rigid rod” polymer backbone, experiencing a limited torsional (e.g., twist) angle between monomer repeating units along the polymer backbone chain. In some cases, the polymer dyes include CPs with rigid rod structures. As summarized above, the structural features of the polymer dye can influence the fluorescence properties of the molecule.

[0060] Any suitable polymer dye can be used in the method and system of interest. In some cases, the polymer dye is a multichromophore having a structure that can collect light to amplify the fluorescence output of a fluorophore. In some cases, the polymer dye can collect light and efficiently convert it into emission of longer wavelengths. In some cases, the polymer dye has a light-gathering multichromophore system that can efficiently transfer energy to a nearby emission species (e.g., a "signal-transferring chromophore"). Mechanisms for energy transfer include, for example, resonance energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, Fret), quantum charge exchange (Dexter energy transfer), etc. In some cases, these energy transfer mechanisms are over relatively short distances, i.e., proximity of the light-gathering multichromophore system to the signal-transferring chromophore provides efficient energy transfer. Under conditions for efficient energy transfer, amplification of emission from the signal-transferring chromophore occurs when there are a large number of individual chromophores in the light-gathering multichromophore system. In other words, the emission from a signal-transmitting chromophore is stronger when the incident light ("excitation light") is at a wavelength absorbed by a light-gathering multichromophore system than when the signal-transmitting chromophore is directly excited by pump light.

[0061] Multiple chromophores can be conjugated polymers. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is significantly shorter than the length of the polymer chain, the backbone contains numerous closely spaced conjugated segments. Thus, conjugated polymers are efficient at light focusing and enable light amplification via energy transfer.

[0062] In some cases, polymers can be used as direct fluorescent reporters, such as fluorescent polymers with high absorption coefficients or high brightness. In other cases, polymers can be used as potent chromophores, where color or optical density is used as an indicator.

[0063] The polymer dyes of interest are those disclosed by Gaylord et al. in U.S. Patent Applications Publications 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986, 20130190193 and 20160025735 (these disclosures are incorporated herein by reference in their entirety), as well as 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. This includes, but is not limited to, the dyes described in al., J.Am.Chem.Soc., 2011, 133(32), pp12600-12607 (these disclosures are incorporated herein by reference in their entirety).

[0064] In embodiments, the method includes modulating the voltage of a photodetector between at least a first voltage and a second voltage. In some cases, the photodetector is modulated between three or more voltages, e.g., four or more, five or more, six or more, seven or more, and e.g., eight or more different voltages. In certain cases, 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 a first photodetector voltage and a second photodetector voltage at frequencies of 0.0001MHz to 15MHz, e.g., 0.0005MHz to 14MHz, e.g., 0.001MHz to 13MHz, e.g., 0.005MHz to 12MHz, e.g., 0.01MHz to 11MHz, e.g., 0.05MHz to 10MHz, e.g., 0.1MHz to 9MHz, e.g., 0.5MHz to 8MHz, e.g., 1MHz to 7MHz, e.g., 1MHz to 6MHz, and e.g., 1MHz to 5MHz. In certain cases, the photodetector voltage oscillates between the first and second photodetector voltages at frequencies of 0.1 MHz to 1 MHz. In some cases, the photodetector voltage is modulated (e.g., oscillated) at frequencies of 0.0001 MHz to 5 MHz, e.g., 0.0005 MHz to 6 MHz, e.g., 0.001 MHz to 7 MHz, e.g., 0.005 MHz to 8 MHz, e.g., 0.01 MHz to 9 MHz, e.g., 0.05 MHz to 10 MHz, e.g., 0.1 MHz to 11 MHz, e.g., 0.5 MHz to 12 MHz, e.g., 1 MHz to 13 MHz, e.g., 1 MHz to 14 MHz, and e.g., 1 MHz to 15 MHz. In some cases, the photodetector voltage is modulated at frequencies of 0.0001MHz to 1MHz, e.g., 0.0001MHz to 2MHz, e.g., 0.0001MHz to 3MHz, e.g., 0.0001MHz to 4MHz, e.g., 0.0001MHz to 5MHz, e.g., 0.0001MHz to 6MHz, e.g., 0.0001MHz to 7MHz, e.g., 0.0001MHz to 8MHz, e.g., 0.0001MHz to 9MHz, e.g., 0.0001MHz to 10MHz, e.g., 0.0001MHz to 11MHz, e.g., 0.0001MHz to 12MHz, e.g., 0.0001MHz to 13MHz, e.g., 0.0001MHz to 14MHz, and e.g., 0.0001MHz to 15MHz.

[0065] In some embodiments, the photodetector voltage is maintained at that voltage for periods of 25 μs or less per period, e.g., 20 μs or less per period, e.g., 15 μs or less per period, e.g., 10 μs or less per period, e.g., 9 μs or less per period, e.g., 8 μs or less per period, e.g., 7 μs or less per period, e.g., 6 μs or less per period, e.g., 5 μs or less per period, e.g., 4 μs or less per period, e.g., 3 μs or less per period, e.g., 2 μs or less per period, e.g., 1 μs or less per period, e.g., 0.9 μs or less per period, e.g., 0.8 μs or less per period, e.g., 0.7 μs or less per period, e.g., 0.6 μs or less per period, e.g., 0.5 μs or less per period, e.g., 0.4 μs or less per period, e.g., 0.3 μs or less per period, e.g., 0.2 μs or less per period, e.g., 0.1 μs or less per period, and e.g., 0.05 μs or less per period. In some cases, the photodetector voltage is maintained at a first photodetector voltage during each modulation cycle. The term “modulation cycle” is referred to herein as each cycle between the first photodetector voltage and the second photodetector voltage. In some cases, the photodetector voltage is maintained at the first photodetector voltage for periods of 25 μs or less, e.g., 20 μs or less, e.g., 15 μs or less, e.g., 10 μs or less, e.g., 9 μs or less, e.g., 8 μs or less, e.g., 7 μs or less, e.g., 6 μs or less, e.g., 5 μs or less, e.g., 4 μs or less, e.g., 3 μs or less, e.g., 2 μs or less, e.g., 1 μs or less, e.g., 0.9 μs or less, e.g., 0.8 μs or less, e.g., 0.7 μs or less, e.g., 0.6 μs or less, e.g., 0.5 μs or less, e.g., 0.4 μs or less, e.g., 0.3 μs or less, e.g., 0.2 μs or less, e.g., 0.1 μs or less, and e.g., 0.05 μs or less.In some cases, the photodetector voltage is maintained at the second photodetector voltage for periods of 25 μs or less per period, e.g., 20 μs or less per period, e.g., 15 μs or less per period, e.g., 10 μs or less per period, e.g., 9 μs or less per period, e.g., 8 μs or less per period, e.g., 7 μs or less per period, e.g., 6 μs or less per period, e.g., 5 μs or less per period, e.g., 4 μs or less per period, e.g., 3 μs or less per period, e.g., 2 μs or less per period, e.g., 1 μs or less per period, e.g., 0.9 μs or less per period, e.g., 0.8 μs or less per period, e.g., 0.7 μs or less per period, e.g., 0.6 μs or less per period, e.g., 0.5 μs or less per period, e.g., 0.4 μs or less per period, e.g., 0.3 μs or less per period, e.g., 0.2 μs or less per period, e.g., 0.1 μs or less per period, and e.g., 0.05 μs or less per period. In some embodiments, the photodetector voltage is maintained at the first photodetector voltage for the same duration as the second photodetector voltage. In other embodiments, the photodetector voltage is maintained at the first photodetector voltage for a different duration than the second photodetector voltage. If the photodetector voltage is maintained at the first photodetector voltage for a different duration than the second photodetector voltage, the voltage may be maintained at different voltages over one or more modulation cycles, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., 100 or more modulation cycles, and e.g., 100 or more modulation cycles.

[0066] In some embodiments, depending on the type of photodetector (e.g., PMT, photodiode, etc.), the first photodetector voltage is 250mV to 2500mV, e.g., 300mV to 2400mV, e.g., 400mV to 2300mV, e.g., 500mV to 2200mV, e.g., 500mV to 2100mV, e.g., 500mV to 2000mV, e.g., 500mV These ranges are ~1900mV, for example 500mV~1800mV, for example 500mV~1700mV, for example 500mV~1600mV, for example 500mV~1500mV, for example 500mV~1400mV, for example 500mV~1300mV, for example 500mV~1200mV, for example 500mV~1100mV, and for example 500mV~1000mV. In some cases, the second photodetector voltage is between 0.0001mV and 1000mV, e.g., 0.0005mV and 900mV, e.g., 0.001mV and 800mV, e.g., 0.005mV and 700mV, e.g., 0.01mV and 600mV, e.g., 0.05mV and 500mV, e.g., 0.1mV and 500mV, e.g., 0.5mV and 500mV, and e.g., 1mV and 500mV. In some cases, the second photodetector voltage is 50mV or less, e.g., 40mV or less, e.g., 30mV or less, e.g., 20mV or less, e.g., 10mV or less, and e.g., 5mV or less.

[0067] The photodetector voltage may be modulated between the first and second photodetector voltages by any convenient waveform change, for example, in this case the voltage is modulated by a sinusoidal voltage change. In certain embodiments, the photodetector voltage is modulated between the first and second photodetector voltages by a square voltage change. In some embodiments, each modulation cycle exhibits a symmetrical voltage change (e.g., all square voltage changes). In other embodiments, one or more modulation cycles exhibit different types of voltage change waveforms, such as one or more modulation cycles exhibiting a square voltage change and one or more modulation cycles exhibiting a sinusoidal voltage change. In some cases, the method includes changes in the shape of the voltage change at predetermined intervals, such as every two or more modulation cycles, for example, every three or more, for example, four or more, for example, five or more, for example, ten or more, for example, 25 or more, for example, 50 or more, for example, 100 or more, and for example, every 500 or more modulation cycles.

[0068] In some embodiments, the data signal is generated from the detected light at one or more photodetector voltages. In some cases, this includes generating two or more data signals at each photodetector voltage during each modulation cycle, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and generating 100 or more data signals at each photodetector voltage during each modulation cycle. In some specific cases, the light is detected at the photodetector voltage and the data signal is averaged. In some cases, the data signal is generated at a first photodetector voltage during each modulation cycle. In some cases, the data signal is generated at a second photodetector voltage during each modulation cycle. In some cases, the data signal is generated at both the first and second photodetector voltages during each modulation cycle.

[0069] In some cases, the data signal is generated only at the first photodetector voltage during each modulation cycle, for example, in this case, two or more data signals are generated only at the first photodetector voltage during each modulation cycle, and ten or more data signals are generated at the first photodetector voltage during each modulation cycle. In some cases, the data signal is generated only at the second photodetector voltage during each modulation cycle, for example, in this case, two or more data signals are generated only at the first photodetector voltage during each modulation cycle, and ten or more data signals are generated at the first photodetector voltage during each modulation cycle.

[0070] In some embodiments, the method includes generating a data signal at a second photodetector voltage if the light detected at a first photodetector voltage exceeds the photodetector's saturation threshold, for example, if the detected light exceeds the saturation threshold by 5% or more, e.g., 10% or more, e.g., 15% or more, e.g., 25% or more, e.g., 50% or more, and including cases where the data signal at the first photodetector voltage exceeds the photodetector's saturation threshold by 75% or more. In some cases, when the 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 from the light detected at the second photodetector voltage. In some cases, if the light detected at the first photodetector voltage exceeds the saturation threshold, a warning may be generated indicating that saturation has been exceeded or that the data signal is approaching an unreliable or inaccurate level. In these cases, the method may also include switching the photodetector voltage to the second photodetector voltage in response to the warning.

[0071] In some embodiments, the method includes generating a data signal at the first photodetector voltage when the data signal generated at the second photodetector voltage exceeds a detected noise threshold, such as when the signal-to-noise ratio falls below a predetermined threshold. In some cases, the noise of the photodetector at the second photodetector voltage is sufficient to obscure or reduce the reliability of the data signal generated at the second photodetector voltage. In some cases, the method includes generating a data signal at the first photodetector voltage when the data signal generated at the second photodetector voltage is more than 5%, e.g., more than 10%, e.g., more than 15%, e.g., more than 25%, e.g., more than 50% below the photodetector noise threshold, and includes cases where the data signal at the second photodetector voltage is more than 75% below the photodetector noise threshold. In some cases, when the data signal generated at the second photodetector voltage exhibits excessive noise (e.g., the signal-to-noise ratio falls below a threshold), the method includes generating a data signal only from the light detected at the first photodetector voltage. In some cases, if the data signal generated at the second photodetector voltage falls below a noise threshold, a warning may be generated indicating that the signal-to-noise ratio of the data signal generated at the second photodetector voltage is too low, or that the data signal is approaching an unreliable or inaccurate level. In these cases, the method may also include switching the photodetector voltage to the first photodetector voltage in response to the warning.

[0072] In some embodiments, the method includes measuring a baseline sample at a first photodetector voltage and a second photodetector voltage. In some cases, a data signal is further generated based on the baseline sample. In some cases, baseline noise is calculated at each photodetector voltage during each modulation cycle. In certain cases, baseline noise is calibrated at each photodetector voltage. In some cases, the baseline sample is measured at each photodetector voltage at the baseline sampling frequency. In some cases, the method includes measuring the baseline sample at a frequency higher than the modulation frequency. In some cases, the method includes measuring the baseline sample at a frequency lower than the modulation frequency. In certain cases, the method includes measuring the baseline sample at a frequency equal to the modulation frequency. In some cases, the method involves measuring a baseline sample at a baseline sampling frequency of, for example, 0.0001MHz to 15MHz, 0.0005MHz to 14MHz, 0.001MHz to 13MHz, 0.005MHz to 12MHz, 0.01MHz to 11MHz, 0.05MHz to 10MHz, 0.1MHz to 9MHz, 0.5MHz to 8MHz, 1MHz to 7MHz, 1MHz to 6MHz, and 1MHz to 5MHz.

[0073] In some cases, the baseline is interpreted by a fixed time-dependent analysis of the first and second photodetector voltages (i.e., dual-mode operation). In some cases, the baseline is interpreted by a dynamic frequency-domain derivation of the first and second photodetector voltages. In some embodiments, the generated data signal is scaled. In some cases, the data signal is scaled on a continuous scale. In certain cases, the data signal is scaled on a single continuous scale, and the generated data signal at one or more of the first and second photodetector voltages is divided by the gain of the detector setting used for modulation. In certain cases, the first and second photodetector voltages are scaled as two distinct parameters.

[0074] In some embodiments, one or more parameters of particles in a sample are determined from a data signal generated from light detected at a first photodetector voltage. In some cases, one or more parameters of particles in a sample are determined from a data signal generated from light detected at a second photodetector voltage. In certain specific cases, one or more parameters of particles in a sample are determined from data signals generated from light detected at both the first and second photodetector voltages. In certain embodiments, the particle parameters include those summarized in Table 1, such as those used for generating a gating strategy. [Table 1-1] [Table 1-2]

[0075] In certain cases, an image of a particle is generated from the data signal. In some cases, one or more image parameters are calculated from the generated particle image. In some cases, the center of mass image parameter is calculated from the generated image. In some cases, the delta center of mass image parameter is calculated from the generated image. In some cases, the diffuse image parameter is calculated from the generated image. In some cases, the eccentricity image parameter is calculated from the generated image. In some cases, the long-axis moment image parameter is calculated from the generated image. In some cases, the maximum intensity image parameter is calculated from the generated image. In some cases, the radial moment image parameter is calculated from the generated image. In some cases, the short-axis moment image parameter is calculated from the generated image. In some cases, the particle size image parameter is calculated from the generated image. In some cases, the total intensity image parameter is calculated from the generated image. In some cases, the particle light loss image parameter is calculated from the generated image. In some cases, the forward scattered light image parameter is calculated from the generated image. In some cases, the side scattered light image parameter is calculated from the generated image. In some cases, image moments are calculated from the generated image. The term "image moment" is used herein in its conventional sense, referring to the weighted average of pixel intensities within the image. In some cases, the center of mass can be calculated from the image moment of an image. In other cases, the orientation of a cell can be calculated from the image moment of an image. In yet another case, the eccentricity of a cell can be calculated from the image moment of an image.

[0076] Figure 1A shows a flowchart for determining one or more parameters of particles in a sample in a flow stream based on light detected by a modulated photodetector, according to a particular embodiment. In step 101, the sample containing particles is irradiated with a light source in the flow stream. In step 102a, the photodetector of the photodetection system is modulated with two different voltage settings at a predetermined frequency. In embodiments, the system is modulated with a first higher voltage setting and a second lower voltage setting. In some cases, the voltage setting is modulated between the first and second photodetector voltages with a square voltage change. In step 102b, the light from the irradiated flow stream is detected by the modulated photodetector. A data signal from the detected light is generated in step 103b at one or more of the photodetector voltage settings. In some cases, when detector saturation is exhibited at the higher photodetector voltage setting, the light is detected only at the lower photodetector voltage setting. In other cases, when the detector exhibits high noise values ​​in the generated data signal, such as when the signal-to-noise ratio falls below a predetermined threshold, the light is detected only at higher photodetector voltage settings. In some embodiments, the method includes measuring a baseline sample of the generated data signal at a first photodetector voltage setting and a second photodetector voltage setting (step 103a). In certain cases, the baseline may be interpreted for dual-mode detection (at the first and second photodetector voltage settings) by fixed time-dependent analysis or dynamic frequency-domain derivation. One or more parameters, such as imaging parameters, may be calculated in step 104 based on the generated data signal. In some cases, the imaging parameters include side 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).

[0077] Figure 1B illustrates the detection of light from particles in a sample using a modulated photodetector according to a particular embodiment. The photodetector is modulated with two different voltage settings, namely a high-voltage setting and a low-voltage setting, indicated by a square voltage change between the two different voltage settings. The data signals generated by the photodetector with the high-voltage setting for the light signal (left) and the data signals generated by the photodetector with the low-voltage setting for the dark signal (right) are shown in each plot. As shown in the plot for the light signal, all data signals generated with the low-voltage setting are within a detectable photodetector signal amplitude and exhibit no saturation. The data signals with the high-voltage setting exhibit detector saturation for the light signal. On the other hand, for the dark signal, the data signals generated with the low-voltage setting exhibit a lower detectable photodetector signal amplitude and may exhibit a lower signal-to-noise ratio. The data signals for the dark signal generated with a higher voltage setting exhibit a detectable photodetector signal amplitude. Thus, modulating the photodetector according to embodiments of this disclosure provides accurate detection of both the light signal (no saturation at the lower voltage setting) and the dark signal (with a sufficient signal-to-noise ratio at the higher voltage setting). The modulation of the photodetector voltage setting described herein can provide an increase in the dynamic range of the photodetector, improving the detectable signal amplitude of the photodetector, as shown in Figure 1B.

[0078] In some cases, the samples analyzed in this method are biological samples. The term “biological sample” is used in its conventional sense and refers to an entire organism, an entire plant, an entire fungus, or, in certain cases, a subset of animal tissues, cells, or components that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to both a naturally occurring organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, such as plasma, serum, cerebrospinal fluid, lymph, skin, respiratory, gastrointestinal, cardiovascular, and urogenital tract sections, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be any type of living tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, and in some cases, the sample is a blood sample containing whole blood, such as blood obtained from a venipuncture or fingertip puncture (the blood may or may not be combined with any reagents, such as preservatives and anticoagulants, before the assay).

[0079] In certain embodiments, the source of the sample is “mammal” or “mammalian,” a term widely used to describe organisms belonging to the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys). In some cases, the subject is human. The method may be applied to samples obtained from subjects that are human of both sexes and at any developmental stage (i.e., neonatal, infant, juvenile, adolescent, or adult), and in certain embodiments, the human subject may be juvenile, adolescent, or adult. While this disclosure may be applied to samples derived from human subjects, it will be understood that the method may also be applied to samples from other animal subjects (i.e., “non-human subjects”), such as birds, mice, rats, dogs, cats, livestock, and horses, but is not limited to these.

[0080] The target cells can be targeted for characterization according to various parameters, such as phenotypic characteristics identified by attaching specific fluorescent labels to the target cells. In some embodiments, the system is configured to deflect analyzed droplets determined to contain target cells. Various cells can be characterized using the method of interest. 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 include cells having favorable cell surface markers or antigens that can be captured or labeled by favorable affinity factors or their conjugates. For example, target cells may contain cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0081] When performing the method of the subject according to a particular embodiment, a certain amount of initial fluid sample is injected into a flow cytometer. The amount of sample injected into the particle sorting module can vary, for example, in the range of 0.001 mL to 1000 mL, 0.005 mL to 900 mL, 0.01 mL to 800 mL, 0.05 mL to 700 mL, 0.1 mL to 600 mL, 0.5 mL to 500 mL, 1 mL to 400 mL, 2 mL to 300 mL, and 5 mL to 100 mL.

[0082] In some embodiments, the method includes counting labeled particles (e.g., target cells) in a sample and selectively sorting them. When performing the method in question, the fluid sample containing the particles is first introduced into the system's flow nozzle. Exiting the flow nozzle, the particles pass through the sample interrogation region substantially one at a time, where each particle is irradiated with a light source, and measurements of light scattering parameters and, in some cases, desired fluorescence emission (e.g., measurements of two or more light scattering parameters and one or more fluorescence emission) are recorded separately for each particle. Depending on the characteristics of the interrogated flowstream, the light may be irradiated to a portion of the flowstream of 0.001 mm or more, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, or to a flowstream of 1 mm or more. In certain embodiments, the method includes irradiating a planar cross section of the flowstream within the sample interrogation region with a laser (as described above). In other embodiments, the method includes irradiating a sample interrogation region with a predetermined length of flowstream, for example, a length corresponding to the irradiation profile of a diffuse laser beam or lamp.

[0083] In certain embodiments, the method includes irradiating a flowstream at or near the flow cell nozzle orifice. For example, the method may include irradiating a flowstream at a position of about 0.001 mm or more from the nozzle orifice, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, and e.g., 1 mm or more. In certain embodiments, the method includes irradiating a flowstream immediately adjacent to the flow cell nozzle orifice.

[0084] In embodiments of this method, a detector such as a photomultiplier tube (PMT) is used to record the light passing through each particle (called forward scattering in certain cases), the light reflected perpendicular to the direction of particle flow as it passes through the detection region (called orthogonal scattering or side scattering in some cases), and the fluorescence emitted from the particle when it passes through the detection region and is irradiated by an energy source, if the particle is labeled with a fluorescent marker. Each of forward scattering (FSC), side scattering (SSC), and fluorescence emission contains distinct parameters 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, if desired, be analyzed in real time or stored in data storage and analysis means such as a computer.

[0085] In certain 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 desired. The fluorescence emitted in the detection channels used to identify the particles and their associated binding complexes may be measured after excitation by a single light source or separately after excitation by individual light sources. If separate excitation light sources are used to excite particle labels, the labels may be selected so that all labels are excitable by each of the excitation light sources used.

[0086] In certain embodiments, the method includes data acquisition, analysis, and recording using a computer or the like, with multiple data channels recording data from each detector for the light scattering and fluorescence emitted by each particle as it passes through the sample interrogation area of ​​the particle sorting module. In these embodiments, the analysis includes sorting and counting the particles so that each particle is represented as a set of digitized parameter values. The system in question may be configured to trigger on selected parameters to distinguish the target particles from background and noise. "Triggering" refers to a preset threshold for the detection of a parameter, which may be used as a means to detect particles passing through a light source. Detecting an event exceeding the threshold for the selected parameter triggers the acquisition of light scattering and fluorescence data for the particle. For particles or other components in the assayed medium that cause a response below the threshold, no data is acquired. The trigger parameter may be the detection of forward scattered light caused by a particle passing through a ray. The flow cytometer then detects and collects the light scattering and fluorescence data for the particle.

[0087] Next, a specific subpopulation of interest is 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 may be carried out by plotting forward light scattering (FSC) versus side (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles is selected (i.e., their cells in the gate), and particles not in the gate are excluded. If desired, the gate may be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Then, only those particles in the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to yield a count of the desired particles in the sample.

[0088] The method of interest may further include the use of particles in research, laboratory testing, or treatment. In some embodiments, the method of interest includes obtaining individual cells prepared from a biological sample of a target fluid or tissue. For example, the method of interest includes obtaining cells from a fluid or tissue sample used as a research or diagnostic sample for a disease such as cancer. Similarly, the method of interest includes obtaining cells from a fluid or tissue sample used in treatment. A cell therapy protocol is a protocol in which viable cell material, including, for example, cells and tissues, is prepared and can be introduced to a subject as a therapeutic procedure. Conditions that can be treated by administration of flow cytometry-sorted samples include, but are not limited to, blood disorders, immune system disorders, and organ damage.

[0089] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, gene modification, culture and in vitro growth, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of cells into the subject. The protocol may begin with the recovery of viable cells and tissues from the subject's source tissue to produce cell and / or tissue samples. Samples may be recovered via any preferred procedure, including, for example, administering a cell recruiter to the subject, drawing blood from the subject, or extracting bone marrow from the subject. After sample recovery, cell enrichment may be performed via several methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, and fluorescence-activated cell sorting (FACS). In some cases, enriched cells may be genetically modified by any convenient method, such as nuclease-mediated gene modification. Genetically modified cells can be cultured, activated, and grown in vitro. In some cases, cells may be stored, for example by cryopreservation, and then thawed for future use, and subsequently administered to a patient, for example, by injecting the cells into the patient.

[0090] system Aspects of this disclosure also include systems for carrying out the methods covered, such as systems for modulating a photodetector in a photodetector system (such as in a flow cytometer). A system according to a particular embodiment includes a light source configured to irradiate unlabeled particles of a sample. In embodiments, the light source may be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, beads, non-cellular particles), the light source may be configured to emit light with wavelengths varying in the ranges of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, and e.g., 400 nm to 800 nm. For example, the light source may include a broadband light source that emits light having wavelengths in the range of 200 nm to 900 nm. In other cases, the light source may include a narrowband light source that emits wavelengths in the range of 200 nm to 900 nm. For example, the light source may be a narrowband LED (1 nm to 25 nm) that emits light having wavelengths in the range of 200 nm to 900 nm. In certain embodiments, the light source is a laser. In some cases, the system in question 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 cases, the system in question includes dye lasers such as stilbene, coumarin, or rhodamine lasers. In yet another case, the laser of interest includes metal vapor lasers such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof.In other cases, the systems in question include solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:yCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium-2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0091] In other embodiments, the light source is a non-laser light source such as a lamp including but not limited to a halogen lamp, a deuterium arc lamp, or a xenon arc lamp; a light-emitting diode such as a broadband LED having a continuous spectrum; a superluminescent light-emitting diode; a semiconductor light-emitting diode; a broadband LED white light source; or a multi-LED integrated light source. In some cases, the non-laser light source is a stabilized fiber-coupled broadband light source, a white light source, or any combination thereof, among other light sources.

[0092] The light source may be positioned at any suitable distance from the sample (e.g., in the flow stream in a flow cytometer), including, for example, distances of 0.001 mm or more from the flow stream, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 25 mm or more, and e.g., 100 mm or more. Furthermore, the light source irradiates the sample at any suitable angle (e.g., with respect to the vertical axis of the flow stream), e.g., angles in the range of 10° to 90°, e.g., 15° to 85°, e.g., 20° to 80°, e.g., 25° to 75°, and e.g., 30° to 60°, e.g., 90°.

[0093] The light source may be configured to irradiate the sample continuously or at discrete intervals. In some cases, the system includes a light source configured to irradiate the sample continuously, such as a continuous-wave laser that continuously irradiates a flow stream at an interrogation point in a flow cytometer. In other cases, the system of interest includes a light source configured to irradiate 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 every 1000 milliseconds, or any other interval. If the light source is configured to irradiate the sample at discrete intervals, the system may include one or more additional components to provide intermittent irradiation of the sample by the light source. For example, the system of interest in these embodiments may include one or more laser beam choppers, manual or computer-controlled beam stops for blocking the sample and exposing it to the light source.

[0094] In some embodiments, the light source is a laser. The laser of interest may include pulsed lasers or continuous wave lasers. For example, the laser may be a gas laser or a combination thereof, such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, xenon-fluorine (XeF) excimer laser; a dye laser such as a stilbene, coumarin or rhodamine laser; a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neo Metal vapor lasers such as n-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, titanium sapphire lasers, trimmygium YAG lasers, ytterbium YAG lasers, ytterbium 2O3 lasers, cerium-doped lasers, and combinations thereof; semiconductor diode lasers, photo-excited semiconductor lasers (OPSLs), or any of the above lasers in a double or triple frequency implementation form.

[0095] In certain embodiments, the light source is a light beam generator configured to produce two or more frequency-shifted light beams. In some cases, the light beam generator includes a laser and a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optical device to produce two or more angle-shifted laser beams. In these embodiments, the laser may be a pulsed laser or a continuous-wave laser. For example, the laser in the light beam generator of choice may be a gas laser or a combination thereof, such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, xenon-fluorine (XeF) excimer laser; a dye laser, such as a stilbene, coumarin or rhodamine laser; a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe Metal vapor lasers such as 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, titanium sapphire lasers, trimmygium YAG lasers, ytterbium YAG lasers, ytterbium 2O3 lasers, cerium-doped lasers, and combinations thereof.

[0096] The acousto-optic device may be any convenient acousto-optic protocol configured to frequency-shift laser light using applied sound waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the system of interest is configured to generate an angle-deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital combiner (DDS), arbitrary waveform generator (AWG), or electrical pulse generator.

[0097] In the embodiment, the controller is configured to apply high-frequency drive signals to an acousto-optical device to produce an output laser beam deflected by a desired number of angles, for example, by applying three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals (including being configured to apply one hundred or more high-frequency drive signals).

[0098] In some cases, to produce an intensity profile of an angularly deflected laser beam within an output laser beam, the controller is configured to apply a high-frequency drive signal having an amplitude that varies, for example, about 0.001V to about 500V, about 0.005V to about 400V, about 0.01V to about 300V, about 0.05V to about 200V, about 0.1V to about 100V, about 0.5V to about 75V, about 1V to 50V, about 2V to 40V, about 3V to about 30V, and about 5V to about 25V. In some embodiments, each of the applied high-frequency drive signals has a frequency of approximately 0.001 MHz to approximately 500 MHz, for example approximately 0.005 MHz to approximately 400 MHz, for example approximately 0.01 MHz to approximately 300 MHz, for example approximately 0.05 MHz to approximately 200 MHz, for example approximately 0.1 MHz to approximately 100 MHz, for example approximately 0.5 MHz to approximately 90 MHz, for example approximately 1 MHz to approximately 75 MHz, for example approximately 2 MHz to approximately 70 MHz, for example approximately 3 MHz to approximately 65 MHz, for example approximately 4 MHz to approximately 60 MHz, and for example approximately 5 MHz to approximately 50 MHz.

[0099] In one particular embodiment, the controller has a processor having memory operably coupled to the processor, the memory storing instructions, which, when executed by the processor, cause the processor to produce an output laser beam having an angle-deflected laser beam having a desired intensity profile. For example, the memory may include instructions for producing two or more angle-deflected laser beams of the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may include instructions for producing 100 or more angle-deflected laser beams of the same intensity. In another embodiment, the memory may include instructions for producing two or more angle-deflected laser beams of different intensities, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may include instructions for producing 100 or more angle-deflected laser beams of different intensities.

[0100] In certain embodiments, the controller has a processor having memory operably coupled to the processor, the memory storing instructions, which, when executed by the processor, cause the processor to produce an output laser beam whose intensity increases from the edge to the center along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., 2% to about 85%, e.g., 3% to about 80%, e.g., 4% to about 75%, e.g., 5% to about 70%, e.g., 6% to about 65%, e.g., 7% to about 60%, e.g., 8% to about 55% (including about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis). In other embodiments, the controller has a processor having memory operably coupled to the processor, the memory storing instructions, which, when executed by the processor, cause the processor to produce an output laser beam whose intensity increases from the edge to the center along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may be in the range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., In yet another embodiment, the controller has a processor having memory operably coupled to the processor, the memory storing instructions, which, when executed by the processor, cause the processor to produce an output laser beam having an intensity profile having a Gaussian distribution along the horizontal axis.In yet another embodiment, the controller has a processor having memory operably coupled to the processor, the memory storing instructions, which, when executed by the processor, cause the processor to produce an output laser beam having a top-hat intensity profile along the horizontal axis.

[0101] In some embodiments, the target light beam generator may be configured to produce an angle-deflected laser beam within a spatially separated output laser beam. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams may be separated by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, e.g., 5000 μm or more, e.g., 15000 μm or more. In some embodiments, the system is configured to produce an angle-deflected laser beam within an output laser beam that overlaps with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (such as beam spot overlap) may be 0.001 μm or larger, for example 0.005 μm or larger, for example 0.01 μm or larger, for example 0.05 μm or larger, for example 0.1 μm or larger, for example 0.5 μm or larger, for example 1 μm or larger, for example 5 μm or larger, for example 10 μm or larger, and for example 100 μm or larger.

[0102] In certain cases, a light beam generator configured to produce two or more frequency-shifted light beams is described in Diebold, et al. Nature Photonics Vol.7(10);806-810(2013) and U.S. Patents 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, and 10,845. This includes laser excitation modules as described in Nos. 295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,92, 11,630,053, 11,774,343, 11,940,369 and 11,946,851 (these disclosures are incorporated herein by reference).

[0103] In the embodiment, the system includes a photodetector having a photodetector configured to detect light emitted by an irradiated particle. As described in detail above, the photodetector is modulated between at least two different voltage settings (e.g., a high voltage setting and a low voltage setting) at a predetermined frequency.

[0104] In some embodiments, the photodetector system is configured to detect scattered light. In some cases, the photodetector system includes a side-scatter light detector. In some cases, the photodetector system includes a forward-scatter light detector. In other embodiments, the photodetector system includes multiple scattered light detectors, e.g., two or more, e.g., three or more, e.g., four or more, and e.g., five or more. In some embodiments, the photodetector system in question also includes a fluorescence detector configured to detect one or more fluorescence wavelengths of light. In other embodiments, the photodetector system includes multiple fluorescence detectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more, ten or more, fifteen or more, and e.g., twenty or more.

[0105] The target detector may include, but is not limited to, optical sensors or detectors such as active-pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the focused light is measured by a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active-pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the detector is 0.01 cm 2 ~10cm 2 For example, 0.05 cm 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm 2 , and for example, 1 cm 2 ~5cm 2This is a photomultiplier tube, such as a photomultiplier tube, having an activity detection surface area in each region within the specified range.

[0106] If the system in question includes multiple fluorescence detectors, each fluorescence detector may be the same, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, if the system in question includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD device and the second fluorescence detector (or imaging sensor) is a CMOS device. In other embodiments, both the first and second fluorescence detectors are CCD devices. In yet another embodiment, both the first and second fluorescence detectors are CMOS devices. In yet another embodiment, the first fluorescence detector is a CCD device and the second fluorescence detector is a photomultiplier tube (PMT). In yet another embodiment, the first fluorescence detector is a CMOS device and the second fluorescence detector is a photomultiplier tube. In yet another embodiment, both the first and second fluorescence detectors are photomultiplier tubes.

[0107] In embodiments of the present disclosure, the fluorescence detector of interest is configured to measure the collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths, and includes measuring the light emitted by the 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 the same or overlapping wavelengths of the collected light.

[0108] In some embodiments, the desired fluorescence detector is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the desired detector is configured to collect the spectrum of light over a range of wavelengths. For example, a flow cytometer may include one or more detectors configured to collect the spectrum of light over one or more wavelengths in the 200 nm to 1000 nm range. In yet another embodiment, the desired detector is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a module may include one or more detectors configured to measure light at one or more of the following wavelengths: 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 certain embodiments, one or more detectors may be configured to pair with specific fluorophores, such as those used with a sample in a fluorescence assay.

[0109] In some embodiments, the system is configured to modulate the voltage of the photodetector between at least a first voltage and a second voltage. In some cases, the system is configured to modulate the photodetector between three or more voltages, e.g., four or more, five or more, six or more, seven or more, and e.g., eight or more different voltages. In certain specific cases, the system is configured to modulate the voltage of the photodetector between a first photodetector voltage and a second photodetector voltage. In some embodiments, the voltage of the photodetector is modulated between a first photodetector voltage and a second photodetector voltage at frequencies of 0.0001MHz to 15MHz, e.g., 0.0005MHz to 14MHz, e.g., 0.001MHz to 13MHz, e.g., 0.005MHz to 12MHz, e.g., 0.01MHz to 11MHz, e.g., 0.05MHz to 10MHz, e.g., 0.1MHz to 9MHz, e.g., 0.5MHz to 8MHz, e.g., 1MHz to 7MHz, e.g., 1MHz to 6MHz, and e.g., 1MHz to 5MHz. In certain cases, the photodetector voltage oscillates between the first and second photodetector voltages at frequencies of 0.1 MHz to 1 MHz. In some cases, the photodetector voltage is modulated at frequencies of 0.0001 MHz to 5 MHz, e.g., 0.0005 MHz to 6 MHz, e.g., 0.001 MHz to 7 MHz, e.g., 0.005 MHz to 8 MHz, e.g., 0.01 MHz to 9 MHz, e.g., 0.05 MHz to 10 MHz, e.g., 0.1 MHz to 11 MHz, e.g., 0.5 MHz to 12 MHz, e.g., 1 MHz to 13 MHz, e.g., 1 MHz to 14 MHz, and e.g., 1 MHz to 15 MHz.In some cases, the photodetector voltage is modulated at frequencies of 0.0001MHz to 1MHz, e.g., 0.0001MHz to 2MHz, e.g., 0.0001MHz to 3MHz, e.g., 0.0001MHz to 4MHz, e.g., 0.0001MHz to 5MHz, e.g., 0.0001MHz to 6MHz, e.g., 0.0001MHz to 7MHz, e.g., 0.0001MHz to 8MHz, e.g., 0.0001MHz to 9MHz, e.g., 0.0001MHz to 10MHz, e.g., 0.0001MHz to 11MHz, e.g., 0.0001MHz to 12MHz, e.g., 0.0001MHz to 13MHz, e.g., 0.0001MHz to 14MHz, and e.g., 0.0001MHz to 15MHz.

[0110] In some embodiments, the system is configured to maintain the voltage of the photodetector at a duration of 25 μs or less per period, for example 20 μs or less per period, for example 15 μs or less per period, for example 10 μs or less per period, for example 9 μs or less per period, for example 8 μs or less per period, for example 7 μs or less per period, for example 6 μs or less per period, for example 5 μs or less per period, for example 4 μs or less per period, for example 3 μs or less per period, for example 2 μs or less per period, for example 1 μs or less per period, for example 0.9 μs or less per period, for example 0.8 μs or less per period, for example 0.7 μs or less per period, for example 0.6 μs or less per period, for example 0.5 μs or less per period, for example 0.4 μs or less per period, for example 0.3 μs or less per period, for example 0.2 μs or less per period, for example 0.1 μs or less per period, and for example 0.05 μs or less per period. In some cases, the system is configured to maintain the photodetector voltage at a first photodetector voltage during each modulation cycle. In some cases, the system is configured to maintain the photodetector voltage at a first photodetector voltage for periods of 25 μs or less, e.g., 20 μs or less, e.g., 15 μs or less, e.g., 10 μs or less, e.g., 9 μs or less, e.g., 8 μs or less, e.g., 7 μs or less, e.g., 6 μs or less, e.g., 5 μs or less, e.g., 4 μs or less, e.g., 3 μs or less, e.g., 2 μs or less, e.g., 1 μs or less, e.g., 0.9 μs or less, e.g., 0.8 μs or less, e.g., 0.7 μs or less, e.g., 0.6 μs or less, e.g., 0.5 μs or less, e.g., 0.4 μs or less, e.g., 0.3 μs or less, e.g., 0.2 μs or less, e.g., 0.1 μs or less, and e.g., 0.05 μs or less.In some cases, the system is configured to maintain the photodetector voltage at a second photodetector voltage for periods of 25 μs or less, e.g., 20 μs or less, e.g., 15 μs or less, e.g., 10 μs or less, e.g., 9 μs or less, e.g., 8 μs or less, e.g., 7 μs or less, e.g., 6 μs or less, e.g., 5 μs or less, e.g., 4 μs or less, e.g., 3 μs or less, e.g., 2 μs or less, e.g., 1 μs or less, e.g., 0.9 μs or less, e.g., 0.8 μs or less, e.g., 0.7 μs or less, e.g., 0.6 μs or less, e.g., 0.5 μs or less, e.g., 0.4 μs or less, e.g., 0.3 μs or less, e.g., 0.2 μs or less, e.g., 0.1 μs or less, and e.g., 0.05 μs or less. In some embodiments, the system is configured to maintain the voltage of the photodetector at a first photodetector voltage for the same duration as the second photodetector voltage. In other embodiments, the system is configured to maintain the voltage of the photodetector at a first photodetector voltage for a different duration than the second photodetector voltage. If the voltage of the photodetector is maintained at a first photodetector voltage for a different duration than the second photodetector voltage, the voltage may be maintained at different voltages over one or more modulation cycles, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., 100 or more modulation cycles, and e.g., 100 or more modulation cycles.

[0111] In some embodiments, the system is configured to set the first photodetector voltage to 250mV~2500mV, for example 300mV~2400mV, for example 400mV~2300mV, for example 500mV~2200mV, for example 500mV~2100mV, for example 500mV~2000mV, for example 500mV~1900mV, for example 500mV~1800mV, for example 500mV~1700mV, for example 500mV~1600mV, for example 500mV~1500mV, for example 500mV~1400mV, for example 500mV~1300mV, for example 500mV~1200mV, for example 500mV~1100mV, and for example 500mV~1000mV. In some cases, the system is configured to set the second photodetector voltage to 0.0001mV to 1000mV, e.g., 0.0005mV to 900mV, e.g., 0.001mV to 800mV, e.g., 0.005mV to 700mV, e.g., 0.01mV to 600mV, e.g., 0.05mV to 500mV, e.g., 0.1mV to 500mV, e.g., 0.5mV to 500mV, and e.g., 1mV to 500mV. In some cases, the system is configured to set the second photodetector voltage to 50mV or less, e.g., 40mV or less, e.g., 30mV or less, e.g., 20mV or less, e.g., 10mV or less, and e.g., 5mV or less.

[0112] The photodetector voltage may be modulated between the first and second photodetector voltages by any convenient waveform change, for example, in this case the voltage is modulated by a sinusoidal voltage change. In certain embodiments, the system is configured to modulate the photodetector voltage between the first and second photodetector voltages by a square voltage change. In some embodiments, the system is configured to modulate the photodetector voltage between each modulation cycle by a symmetrical voltage change (e.g., all square voltage changes). In other embodiments, one or more modulation cycles exhibit different types of voltage change waveforms, such as one modulation cycle exhibiting a square voltage change and one or more modulation cycles exhibiting a sinusoidal voltage change. In some cases, the system is configured to change the shape of the voltage change at predetermined intervals, such as every two or more modulation cycles, for example, every three or more, for example, four or more, for example, five or more, for example, ten or more, for example, 25 or more, for example, five and for example, five or more, for example, five or more modulation cycles.

[0113] In some embodiments, the photodetector system is configured to generate data signals from detected light at one or more photodetector voltages. In some cases, the photodetector system is configured to generate two or more data signals at each photodetector voltage during each modulation cycle, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and includes generating 100 or more data signals at each photodetector voltage during each modulation cycle. In some particular cases, light is detected at photodetector voltages and the data signals are averaged. In some cases, the system is configured to generate a data signal at a first photodetector voltage during each modulation cycle. In some cases, the system is configured to generate a data signal at a second photodetector voltage during each modulation cycle. In some cases, the system is configured to generate data signals at a first photodetector voltage and a second photodetector voltage during each modulation cycle.

[0114] In some cases, the system is configured to generate data signals only at a first photodetector voltage during each modulation cycle, for example, in which case two or more, e.g., three or more, e.g., four or more, e.g., five or more data signals are generated only at the first photodetector voltage during each modulation cycle, and ten or more data signals are generated at the first photodetector voltage during each modulation cycle. In some cases, the system is configured to generate data signals only at a second photodetector voltage during each modulation cycle, for example, in which case two or more, e.g., three or more, e.g., four or more, e.g., five or more data signals are generated only at the first photodetector voltage during each modulation cycle, and ten or more data signals are generated at the first photodetector voltage during each modulation cycle.

[0115] In some embodiments, the system is configured to generate a data signal at a second photodetector voltage if the light detected at a first photodetector voltage exceeds the photodetector's saturation threshold, for example, if the detected light exceeds the saturation threshold by 5% or more, e.g., 10% or more, e.g., 15% or more, e.g., 25% or more, e.g., 50% or more, including the case where the light detected at the first photodetector voltage exceeds the photodetector's saturation threshold by 75% or more. In some cases, 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 from the light detected at the second photodetector voltage. In some cases, if the light detected at the first photodetector voltage exceeds the saturation threshold, the system may be configured to generate a warning indicating that saturation has been exceeded or that the data signal is approaching an unreliable or inaccurate level. In these cases, the system may also be configured to switch the photodetector voltage to the second photodetector voltage in response to the warning.

[0116] In some embodiments, the system is configured to generate a data signal at the first photodetector voltage if the data signal generated at the second photodetector voltage exceeds a detected noise threshold, such as when the signal-to-noise ratio falls below a predetermined threshold. In some cases, the noise of the photodetector at the second photodetector voltage is sufficient to obscure or reduce the reliability of the data signal generated at the second photodetector voltage. In some cases, the system is configured to generate a data signal at the first photodetector voltage if the data signal generated at the second photodetector voltage is more than 5%, e.g., more than 10%, e.g., more than 15%, e.g., more than 25%, e.g., more than 50%, and includes cases where the data signal at the second photodetector voltage is more than 75% below the photodetector noise threshold. In some cases, when the data signal generated at the second photodetector voltage exhibits excessive noise (e.g., the signal-to-noise ratio falls below a threshold), the system is configured to generate a data signal only from the light detected at the first photodetector voltage. In some cases, if the data signal generated by the second photodetector voltage falls below a noise threshold, the system is configured to generate a warning indicating that the signal-to-noise ratio of the data signal generated by the second photodetector voltage is too low, or that the data signal is approaching an unreliable or inaccurate level. In these cases, the system is configured to switch the photodetector voltage back to the first photodetector voltage in response to the warning.

[0117] In some embodiments, a photodetector voltage and a second photodetector voltage. In some embodiments, the system also includes a processor to which a memory is operablely coupled, the memory storing instructions, which, when executed by the processor, cause the processor to measure a baseline sample at a first photodetector voltage and a second photodetector voltage. In some cases, the memory includes instructions for generating a data signal based on the baseline sample. In some cases, the memory includes instructions for calculating baseline noise at each photodetector voltage during each modulation cycle. In certain cases, the memory includes instructions for calibrating the baseline noise at each photodetector voltage. In some cases, the memory includes instructions for measuring the baseline sample at each photodetector voltage at the baseline sampling frequency. In some cases, the memory includes instructions for measuring the baseline sample at a frequency higher than the modulation frequency. In some cases, the memory includes instructions for measuring the baseline sample at a frequency lower than the modulation frequency. In certain cases, the memory includes instructions for measuring the baseline sample at a frequency equal to the modulation frequency. In some cases, the memory includes instructions for measuring a baseline sample at baseline sampling frequencies such as 0.0001MHz to 15MHz, 0.0005MHz to 14MHz, 0.001MHz to 13MHz, 0.005MHz to 12MHz, 0.01MHz to 11MHz, 0.05MHz to 10MHz, 0.1MHz to 9MHz, 0.5MHz to 8MHz, 1MHz to 7MHz, 1MHz to 6MHz, and 1MHz to 5MHz.

[0118] In some cases, the memory includes instructions for interpreting the baseline at the first and second photodetector voltages (i.e., dual-mode operation) by a fixed time-dependent analysis. In some cases, the baseline is interpreted at the first and second photodetector voltages by a dynamic frequency-domain derivation. In some embodiments, the memory includes instructions for scaling the generated data signal. In some cases, the memory includes instructions for scaling the data signal on a continuous scale. In a particular case, 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 a particular case, the first and second photodetector voltages are scaled as two separate parameters.

[0119] In some embodiments, the memory includes instructions for determining one or more parameters of particles in a sample from a data signal generated from light detected at a first photodetector voltage. In some cases, the memory includes instructions for determining one or more parameters of particles in a sample from a data signal generated from light detected at a second photodetector voltage. In certain specific cases, the memory includes instructions for determining one or more parameters of data signals of particles in a sample generated from light detected at both the first and second photodetector voltages.

[0120] In certain cases, the memory contains instructions for generating an image of a particle from a data signal. In some cases, the memory contains instructions for calculating one or more image parameters from the generated image of the particle. In some cases, the center of mass image parameter is calculated from the generated image. In some cases, the delta center of mass image parameter is calculated from the generated image. In some cases, the diffuse image parameter is calculated from the generated image. In some cases, the eccentricity image parameter is calculated from the generated image. In some cases, the long-axis moment image parameter is calculated from the generated image. In some cases, the maximum intensity image parameter is calculated from the generated image. In some cases, the radial moment image parameter is calculated from the generated image. In some cases, the short-axis moment image parameter is calculated from the generated image. In some cases, the particle size image parameter is calculated from the generated image. In some cases, the total intensity image parameter is calculated from the generated image. In some cases, the particle light loss image parameter is calculated from the generated image. In some cases, the forward scattered light image parameter is calculated from the generated image. In some cases, the side scattered light image parameter is calculated from the generated image. In some cases, image moments are calculated from the generated image. The term "image moment" is used herein in its conventional sense, referring to the weighted average of pixel intensities within the image. In some cases, the center of mass can be calculated from the image moment of an image. In other cases, the orientation of a cell can be calculated from the image moment of an image. In yet another case, the eccentricity of a cell can be calculated from the image moment of an image.

[0121] In certain embodiments, the photodetection system described herein is part of a flow cytometer. The flow cytometer may include a sample fluid input coupler and any suitable mechanism for supplying the sheath fluid and sample fluid to the sample fluid input coupler. For example, the sample fluid input coupler may be fluid-connected to a sample fluid line (e.g., piping) which is fluid-connected to a sample fluid reservoir. Similarly, the sheath fluid inlet coupler may be fluid-connected to a sheath fluid line which is fluid-connected to a sheath fluid reservoir. Similarly, the flow cytometer may include any suitable mechanism for managing waste from the flow stream. The fluid output coupler may be fluid-connected to a waste line which is fluid-connected to a waste reservoir. A fluid management system that can be adapted for use in the flow cytometer in question is provided in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0122] 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 used herein, “flow cell” is described in its conventional sense, referring to a component that includes a flow channel for a liquid flow stream for transporting particles in a sheath fluid. The cuvette of interest has a passage (i.e., a flow channel) through which the flow channel is formed. The flow stream through which the flow channel is formed may include a liquid sample injected from a sample tube. In certain cases, the flow cell includes a light-accessible flow channel. The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed from silica, such as fused silica. In some cases, the flow cell is configured so that light from a light source is irradiated at one or more interrogation points. As used herein, “interrogation point” refers to a region within the flow cell where particles are irradiated by light from a light source, for example, for analysis. The size of the interrogation points may vary as needed. For example, if 0 μm represents the optical axis of light emitted by the light source, the interrogation points may be in the range of -50 μm to 50 μm, e.g., -25 μm to 40 μm, and e.g., -15 μm to 30 μm. Depending on certain considerations (e.g., the number and arrangement of lasers), multiple irradiation points may exist within the flow cell.

[0123] In some embodiments, the flow cell includes, or is configured to be used with, a sample injection port configured to supply a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the internal chamber (i.e., flow channel) of the flow cell. Depending on the desired characteristics of the flowstream, the rate at which the sample is transported to the flow cell chamber by the sample injection port may be 1 μL / min or more, e.g., 2 μL / min or more, e.g., 3 μL / min or more, e.g., 5 μL / min or more, e.g., 10 μL / min or more, e.g., 15 μL / min or more, e.g., 25 μL / min or more, e.g., 50 μL / min or more, and e.g., 100 μL / min or more. In some cases, the rate at which the sample is transported to the flow cell chamber by the sample injection port may be 1 μL / second or more, e.g., 2 μL / second or more, e.g., 3 μL / second or more, e.g., 5 μL / second or more, e.g., 10 μL / second or more, e.g., 15 μL / second or more, e.g., 25 μL / second or more, e.g., 50 μL / second or more, and e.g., 100 μL / second or more.

[0124] The sample injection port may be an orifice located in the wall of the internal chamber, or a conduit located at the proximal end of the internal chamber. If the sample injection port is an orifice located in the wall of the internal chamber, the sample injection port orifice may be any preferred shape, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as parabolic bottoms coupled to flat tops. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, and e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm.

[0125] In certain cases, the sample injection port is a conduit located at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a conduit positioned so that its orifice is aligned with the flow cell orifice. When the sample injection port is a conduit positioned in line with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, and the desired cross-sectional shape includes, but is not limited to, straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom joined to a flat top. In certain cases, the orifice of the conduit may have an opening of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, and e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm, and may vary depending on the shape. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a slanted tip having an inclination angle in the range of 1° to 10°, for example 2° to 9°, for example 3° to 8°, for example 4° to 7°, and for example 5°.

[0126] In some embodiments, the flow cell also includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid to the internal chamber of the flow cell, for example, together with the sample, to produce a layered sheath fluid flowstream that surrounds the sample flowstream. Depending on the desired characteristics of the flowstream, the rate at which the sheath fluid is delivered to the flow cell chamber by the sheath fluid injection port may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, and e.g., 2500 μL / sec or more.

[0127] In some embodiments, the sheath fluid injection port is an orifice located in the wall of the internal chamber. The sheath fluid injection port orifice may be any suitable shape, and the desired cross-sectional shape includes, but is not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom joined to a flat top. The size of the sheath fluid injection port orifice may vary depending on the shape, in certain cases ranging from 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, and e.g., 1.25 mm to 1.75 mm, e.g., having an opening of e.g., 1.5 mm.

[0128] In some embodiments, the system includes a flow cytometer or is operably coupled to a flow cytometer. Appropriate flow cytometry systems include Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997), Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997), Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995), Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt 1):17-28, Linden, et al., Semin Throm Hemost. 2004 Oct;30(5):502-11, Alison, et al. J Pathol, 2010 Dec;222(4):335-344, and Herbig, et al. (2007) Crit Rev Ther Drug Carrier This may include what is described in Syst.24(3):203-255 (these disclosures are incorporated herein by reference).In certain cases, the target flow cytometry system is the BD Biosciences FACSCanto® flow cytometer, BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Accuri® C6 Plus flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortessa® X-20 flow cytometer, BD Biosciences FACSPresto® flow cytometer, BD Biosciences FACSVia® flow cytometer, as well as the BD Biosciences FACSCalibur® cell sorter, BD Biosciences FACSCount® cell sorter, and BD Biosciences This includes FACSLyric® cell sorters, BD Biosciences Via® cell sorters, BD Biosciences Influx® cell sorters, BD Biosciences Jazz® cell sorters, BD Biosciences Aria® cell sorters, BD Biosciences FACSAria® II cell sorters, BD Biosciences FACSAria® III cell sorters, BD Biosciences FACSAria® Fusion cell sorters, and BD Biosciences FACSMelody® cell sorters, BD Biosciences FACSymphony® S6 cell sorters, BD Biosciences FACSDiscover® cell sorters, and others.

[0129] In some embodiments, the system in question is specified in U.S. Patent Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, and 10,302,54 No. 5, No. 10,145,793, No. 10,113,967, No. 10,006,852, No. 9,952,076, No. 9,933,341, No. No. 9,726,527, No. 9,453,789, No. 9,200,334, No. 9,097,640, No. 9,095,494, No. 9,092,034 No. 8,975,595, No. 8,753,573, No. 8,233,146, No. 8,140,300, No. 7,544,326, No. 7,20 1,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, Flow cytometry systems such as those described in Patent Nos. 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766 (these disclosures are incorporated herein by reference in their entirety).

[0130] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in a particular case, the system in question is based on Diebold, et al. Nature Photonics. Vol.7(10);806-810(2013), and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, and A flow cytometry system configured to image particles in a flow stream by fluorescence imaging using radiofrequency tagged emission (FIRE), as described in Patent Nos. 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 (these disclosures are incorporated herein by reference).

[0131] Figure 2 shows a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. Although the example in Figure 2 shows a single laser, it is understood that multiple lasers can also be used. The laser beam from laser 201 is guided to a focusing lens 202, which focuses the beam onto the portion of the fluid stream where the particles 211 of the sample in the flow cell 210 are located. The flow cell 210 is part of a fluid system that guides particles in the stream to the focused laser beam, typically one at a time, for interrogation. Alternatively, a nozzle top may be used if the flow cytometer is a stream-in-air cytometer.

[0132] As shown in Figure 2, the flow cell 210 is fluidly connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. The sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a conduit (i.e., sheath fluid line) 207. In addition, the sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a conduit (i.e., sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce the particles 211 into the interior of the flow cell 210. The particles 211 are hydrodynamically focused via the sheath fluid entering from the sheath fluid injection port 208 so that a flowstream 214 is formed downstream of the tapered portion 212 of the flow cell 210. The particles released at the distal end of the flow cell 210 can be disposed of and / or collected via any suitable protocol. For example, depending on the type of flow cytometry performed, the particles may be collected at the distal end of the flow cell 210, for example, via a waste line. Alternatively, the particles may be sorted.

[0133] Light from the laser beam interacts with particles 211 in the sample by diffraction, refraction, reflection, scattering, and absorption, accompanied by re-emission at various different wavelengths, depending on the particle's characteristics, such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, may be sent to one or more detectors. In particular, forward scatter (FSC) is sent to a forward scatter detector 223. The forward scatter detector 223 is positioned slightly off-axis from the direct beam passing through the flow cell 210 and is configured to detect diffracted light, i.e., excitation light that travels mainly forward through or around the particle. The intensity of the light detected by the forward scatter detector 223 depends on the overall size of the particle. The forward scatter detector may include, for example, a photodiode. An optical filter 221a and a scattering bar 222 are positioned between the forward scatter detector 223 and the forward scatter detector 223. The optical filter 221a may be configured to filter out non-FSC light of at least one wavelength, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scatter light detector 223.

[0134] Furthermore, side-scattered light (SSC) is detected by the side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of the particle 211, which tends to increase as the particle structure becomes more complex. In the example in Figure 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered light detector 224 and allow non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by the side-scattered light detector 224. Fluorescence detectors 225a-225c, each configured to detect fluorescence of different wavelengths, are also shown. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a and allow light of other wavelengths to pass through. The optical filter 221c may be configured to prevent at least one wavelength of light that does not correspond to a 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 and allow light of a third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. The optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to a second wavelength (or wavelength range) from being detected by the fluorescence detector 225b. Furthermore, the optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to a third wavelength (or wavelength range) from being detected by the fluorescence detector 225c.

[0135] Those skilled in the art will recognize that the flow cytometer according to the embodiments of this disclosure is not limited to the flow cytometer shown in Figure 2, but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations. For example, the embodiment in Figure 2 shows three fluorescence detectors for illustrative purposes, but it will be understood that any suitable number of fluorescence detectors can be used.

[0136] During operation, the cytometer's operation is controlled by the controller / processor 290, and measurement data from the detector can be stored in memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 may be coupled to the detector to receive output signals from the detector, and may also be coupled to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function 297 may also be provided within the system. The memory 295, controller / processor 290, and I / O 297 may be provided as a single integrated part of the flow cytometer. In such embodiments, a display may also form part of the I / O function 297 for presenting experimental data to the user of the cytometer 200. Alternatively, some or all of the memory 295, controller / processor 290, and I / O function may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 295 and controller / processor 290 can communicate with the cytometer 200 wirelessly or via a wired connection. In conjunction with memory 295 and I / O 297, the controller / processor 290 can be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0137] Different fluorescent molecules in a fluorescent dye panel used in a flow cytometer experiment emit light in their own characteristic wavelength bands. Specific fluorescent labels used in the experiment, and their associated fluorescence emission bands, may be selected to substantially match the detector's filter window. I / O297 can be configured to receive data from flow cytometer experiments with a panel of fluorescent labels, and data for multiple cell populations having multiple markers, each cell population having a subset of multiple markers. I / O297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 295. The controller / processor 290 can be configured to evaluate the assignment of one or more labels to the markers.

[0138] In some embodiments, the system in question is a particle sorting system configured to sort particles using a sealed particle sorting module, such as the one described in U.S. Patent Application Publication No. 2017 / 0299493, filed on 28 March 2017, whose disclosure is incorporated herein by reference. In certain embodiments, particles of a sample (e.g., cells) are sorted using a sorting decision module having multiple sorting decision units, such as the one described in U.S. Patent Application Publication No. 2020 / 0256781, filed on 23 December 2019, whose disclosure is incorporated herein by reference. In some embodiments, the system for sorting components of a sample includes a particle sorting module having deflection plates, such as the one described in U.S. Patent Application Publication No. 2017 / 0299493, filed on 28 March 2017, whose disclosure is incorporated herein by reference.

[0139] In a particular embodiment, the system is fluorescence imaging using a radiofrequency tagged emission image-enabled particle sorter as shown in Figures 3A and 3B. The particle sorter 300 includes an optical illumination component 300a, which includes a light source 301 (e.g., a 488 nm laser) that generates an output optical beam 301a, which is split into beams 302a and 302b by a beam splitter 302. The optical beam 302a is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a having one or more angularly deflected optical beams. In some cases, the output beam 303a generated from the acousto-optical device 303 includes a local oscillator beam and multiple radiofrequency comb beams. The optical beam 302b is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a having one or more angularly deflected optical beams. In some cases, the output beam 304a generated from the acousto-optic device 304 includes a local oscillator beam and multiple high-frequency comb beams. The output beams 303a and 304a generated from the acousto-optic devices 303 and 304, respectively, are combined with a beam splitter 305 to produce an output beam 305a, which is transported through an optical component 306 (e.g., an objective lens) to irradiate particles in the flow cell 307. In a particular embodiment, the acousto-optic device 303 (AOD) splits a single laser beam into an array of beamlets, each having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which then overlaps with the array of beamlets in a beam combiner 305. In certain embodiments, the light irradiation system having a light source and an acoustic-optical device may also include those described in Schraivogel, et al. ("High-speed fluorescence image-enabled cell sorting," Science (2022), 375(6578):315-320) and U.S. Patent Application Publication No. 2021 / 0404943, the disclosures of which are incorporated herein by reference.

[0140] The output beam 305a irradiates sample particles 308 propagating through the flow cell 307 (e.g., using the sheath fluid 309) in the irradiation area 310. As shown in the irradiation area 310, multiple beams (e.g., angle-deflected high-frequency shifted light beams shown as dots across the irradiation area 310) overlap with the reference local oscillator beam (shown as diagonal lines across the irradiation area 310). Due to their different optical frequencies, the overlapping beams exhibit beat behavior, thereby giving each beamlet a distinct frequency f 1-n This is used to carry a sine wave modulation signal.

[0141] Light from the irradiated sample is delivered to a photodetection system 300b, which includes multiple photodetectors. The photodetection system 300b includes a forward scatter photodetector 311 for generating a forward scatter image 311a and a side scatter photodetector 312 for generating a side scatter image 312a. The photodetection system 300b also includes a bright-field photodetector 313 for generating an optical loss image 313a. In some embodiments, the forward scatter detector 311 and the side scatter detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the irradiated sample is also detected by fluorescence detectors 314-317. In some cases, the photodetectors 314-317 are photomultiplier tubes. Light from the irradiated sample is directed through a beam splitter 320 to the side scatter detection channel 312 and the fluorescence detection channels 314-317. The photodetector 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 is a 534 nm / 40 nm bandpass. In some cases, optical component 322 is a 586 nm / 42 nm bandpass. In some cases, optical component 323 is a 700 nm / 54 nm bandpass. In some cases, optical component 324 is a 783 nm / 56 nm bandpass. The first digit represents the center of the spectral band. The second digit indicates the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on both sides of the center of the spectral band, i.e., from 500 nm to 520 nm.

[0142] Data signals generated in response to light detected by scattered light detection channels 311 and 312, bright-field light detection channel 313, and fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated by processors 350 and 351. Image-responsive sorting is performed in response to sorting signals generated by sorting trigger 352. The sorting component 300c includes deflection plates 331 for deflecting particles into the sample container 332 or into the waste stream 333. In some cases, the sorting component 300c is configured to sort particles using a sealed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, the sorting component 300c includes a sorting decision module having a plurality of sorting decision units, as described in U.S. Patent Application Publication No. 2020 / 0256781, which is incorporated herein by reference.

[0143] In some embodiments, the system is a particle analyzer, and the particle analysis system 401 (Figure 4) can be used to analyze and characterize particles, whether or not the particles are physically sorted into a collection container. Figure 4 shows a functional block diagram of the particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 includes a fluid system 402. The fluid system 402 includes or can include a sample tube 405 and a moving fluid column in the sample tube through which sample particles 403 (e.g., cells) move along a common sample path 409.

[0144] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. In some implementations, detection may include detecting light or one or more other properties of a particle 403 as it passes through the monitoring area 407. Figure 4 shows one detection station 408 with one monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor two or more areas.

[0145] Each signal is assigned a signal value to form a data point for each particle. This data can be referred to as event data, as described above. The data points can be multidimensional data points containing the values ​​of each characteristic measured for each particle. The detection system 404 is configured to collect such data points sequentially at a first time interval.

[0146] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuits and / or frequency control circuits. The illustrated control system can be operably associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of a first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 during a 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 a portion of the first time interval. The control system 406 may further compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0147] Figure 5 shows a functional block diagram of an example particle analyzer control system, such as an analysis controller (e.g., processor) 500, 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.

[0148] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel. The analysis controller 500 may be a processor configured to perform the method of the present invention by, for example, applying a distance-based classification model to determine a density distinction threshold in a size-based analyte feature space, applying a density-based clustering algorithm to separate analyte data into high-density and low-density clusters based on the density threshold, and classifying the analyte data based on high-density and low-density clusters based on a size-based analyte feature space.

[0149] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry event data. The analysis controller 500 can be configured to provide a display device 506 with a graphic display including a first plot of the biological event data. The analysis controller 500 can be further configured to render a region of interest as a gate around the collection of biological event data shown by the display device 506, for example, superimposed on the first plot. In some embodiments, the gate can be a logical combination of one or more graphic regions of interest drawn on a histogram or bivariate plot of a single parameter. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0150] The analysis controller 500 can further be configured to display biological event data within the gate on the display device 506 in a different way than other events in the biological event data outside the gate. For example, the analysis controller 500 can be configured to render the colors of the biological event data contained within the gate differently from the colors of the biological event data outside the gate. The display device 506 can be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0151] The analysis controller 500 can be configured to receive gate selection signals for identifying gates from a first input device. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate gate selection signals to the analysis controller 500 for identifying gates displayed on or operated through the display device 506 (for example, by clicking the desired gate when the cursor is positioned there). In some implementations, 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, photodetector, or speech recognition system. Some input devices may include multiple input functions. In such implementations, each input function can be considered an input device. For example, as shown in Figure 5, the mouse 510 may include a right mouse button and a left mouse button, each capable of generating a trigger event.

[0152] The trigger event can provide input to the analysis controller 500 for further processing, such as changing how the data is displayed, which parts of the data are actually displayed on the display device 506, and / or selecting a population for the purpose of particle sorting.

[0153] In some embodiments, the analysis controller 500 can be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a specific distribution of biological event data received by the analysis controller 500.

[0154] 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.

[0155] The display device 506 can be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that outline sections of the plots. The display device 506 can be further configured to change the information presented according to input received from the analysis controller 500, along with input from the particle analyzer 502, the memory device 504, the keyboard 508, and / or the mouse 510.

[0156] In some implementations, the analysis controller 500 can generate a user interface for receiving exemplary events for sorting. For example, the user interface may include controls for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates may be provided before the collection of event data for the sample, or based on events from an initial set of parts of the sample.

[0157] Figure 6A 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. As shown in Figure 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to a nozzle 603, or may include a nozzle 603, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample solution 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned in a single file so as to cross a monitoring area 611 (e.g., where laser streams intersect) which is irradiated by an irradiation source 612 (e.g., a laser). The vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to decompose into multiple droplets 610, some of which contain particles 609.

[0158] During operation, a detection station 614 (e.g., an event detector) identifies when a target particle (or target cell) crosses the monitoring area 611. The detection station 614 is fed to a timing circuit 628, which is then fed to a flash charge circuit 630. At a droplet separation point indicated by a time-set dropping delay (Δt), a flash charge can be applied to the moving fluid column 608 so that the target droplet carries the charge. The target droplet may contain one or more particles or cells to be separated. The charged droplet can then be separated by activating a deflection plate (not shown) to deflect the droplet into a container such as a collection tube or a multi-well or microwell sample plate, and the well or microwell can be associated with a specific target droplet. As shown in Figure 6A, the droplets can be collected in a drain receptacle 638.

[0159] The detection system 616 (e.g., a droplet boundary detector) plays a role in automatically determining the phase of the droplet drive signal as the target particle passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 enables the instrument to accurately calculate the location of each detected particle in the droplet. The detection system 616 can supply input to amplitude signals 620 and / or phase signals 618, which are then supplied input (via amplifier 622) to amplitude control circuits 626 and / or frequency control circuits 624. The amplitude control circuits 626 and / or frequency control circuits 624 then control the droplet formation transducer 602. The amplitude control circuits 626 and / or frequency control circuits 624 may be included in a control system.

[0160] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the particle event data. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit, or they can be communicatively coupled so that either the detection system 616 or the detection station 614 can collect event measurements and provide them to non-collecting elements.

[0161] Figure 6B is a schematic diagram of a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in Figure 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream charging wire in a barb. This creates a stream of droplets 610 containing particles 609 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed by sorting electronics or other detection systems (not shown in Figure 6B). The deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, guiding the droplets toward a destination collection receptacle (e.g., any of 672, 674, 676, or 678). As shown in Figure 6B, deflection plates 652 and 654 can be used to direct particles toward receptacle 674 along a first path 662 or toward receptacle 678 along a second path 668. If the particles are not of interest (e.g., do not exhibit scattering or illumination information within the specified sorting range), the deflection plates may allow the particles to continue along the flow path 664. Such uncharged droplets can then enter the waste receptacle via an aspirator 670 or the like.

[0162] Sorting electronics may be included to initiate measurement data collection, receive fluorescence signals related to particles, and determine how to adjust the deflection plates to induce particle sorting. An exemplary implementation of the embodiment shown in Figure 6B includes the BD FACSAria® line of flow cytometers commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0163] Computer control system The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses memory containing instructions for carrying out steps of the method in question. 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, a data backup unit, and many other devices. The processor may be a commercially available processor or one of several other processors that are available or will be available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known way and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in various programming languages ​​such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically works with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0164] System memory may be any of the various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read-and-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices may be any of the various known or future devices, including compact disk drives, tape drives, or floppy disk drives. Such types of memory storage devices typically read from and / or write to program storage media (not shown), such as compact disks. Any of these program storage media, or others currently in use or that may be developed in the future, can be thought of as computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in program storage devices used in conjunction with system memory and / or memory storage devices.

[0165] In some embodiments, a computer program product is described that includes a computer-usable medium on which control logic (a computer software program including program code) is stored. When the control logic is executed by the computer's processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. It will be obvious to those skilled in the art that a hardware state machine can be implemented to perform the functions described herein.

[0166] Memory may be any suitable device on which the processor can store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including magnetic or optical disks, or tapes or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium carrying the required program code. The programming may be supplied remotely to the processor via a communication channel, or may be pre-stored in a computer program product such as memory, or any other portable or fixed computer-readable storage medium that uses any of those devices in relation to memory. For example, a magnetic or optical disk may carry a program that can be read by a disk writer / reader. The system of this disclosure also includes, for example, programming in the form of a computer program product, and algorithms for use in carrying out the above methods. The programming according to this disclosure may be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROM; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0167] The processor can also access communication channels to communicate with users in remote locations. Remote location means that the user is not in direct contact with the system and is relaying input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0168] In some embodiments, the systems 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 another device. The communication interface can be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0169] In one embodiment, the communication interface is configured to include one or more physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port that enables data communication between the system in question and other external devices, such as a computer terminal configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0170] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the system in question to communicate with computer terminals and / or networks, other devices such as communicable mobile phones, personal digital assistants, or any other communication devices that a user may use in conjunction with it.

[0171] In one embodiment, the communication interface is configured to provide a connection for data transfer using Internet Protocol (IP), Short Message Service (SMS), wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0172] In one embodiment, the system in question is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11 or Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or equipment. In some embodiments, the server device has a display such as a liquid crystal display (LCD), as well as input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0173] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in a target system, such as any data storage unit, with a network or server device using one or more of the above-described communication protocols and / or mechanisms.

[0174] The output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. A graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. Functional elements of the computer can communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also transmit information generated by processing modules to a user in a remote location, for example, via the internet, telephone, or satellite network, according to known techniques. Data presentation by the output manager can be carried out according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email, or other files, or other forms of data. The data may include an internet URL address so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the system under consideration may be any type of known computer platform or a type to be developed in the future, but they are typically computers of a class commonly referred to as servers. However, they may be mainframe computers, workstations, or other types of computers. They may be connected via any known or future type of cabling or other communication systems, including wireless systems, and may or may not be networked. They may be located in the same place or may be physically separated.Depending on the type and / or manufacturer of the selected computer platform, various operating systems can be used on any computer platform. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBM i®, Android®, SGI IRIX®, Oracle Solaris®, and others.

[0175] Figure 7 shows a general architecture of an exemplary computing device 700 according to a particular embodiment. The general architecture of the computing device 700 shown in Figure 7 includes the configuration of computer hardware and software components. However, it is not necessary to show all of these generally conventional elements in order to make a working 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 can provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 can receive information and instructions from other computing systems or services via the network. The processing unit 710 can also communicate with memory 770 and further provide output information to an optional display 750 via the input / output device interface 740. For example, analysis software (such as data analysis software or programs like FlowJo®) stored as executable instructions in the non-temporary memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 740 can also accept input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, speech recognition system, gamepad, accelerometer, gyroscope, or other input devices.

[0176] The memory 770 may include computer program instructions (grouped as modules or components in some embodiments) that the processing unit 710 executes to implement one or more embodiments. The memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. The memory 770 may store an operating system 772 that provides computer program instructions used by the processing unit 710 in the general management and operation of the computing device 700. Data may be stored in a data storage device 790. The memory 770 may further include computer program instructions and other information for implementing embodiments of the present disclosure.

[0177] Non-temporary computer-readable storage medium Aspects of this disclosure further include non-temporary computer-readable storage media having instructions for performing the methods described herein, such as for performing one or more computer implementation methods described herein. The computer-readable storage media may be used in one or more computers for the full or partial automation of a system for performing the methods described herein. In certain embodiments, instructions by the methods described herein may be coded on computer-readable media in the form of “programming,” and as used herein, the term “computer-readable media” refers to any non-temporary storage medium involved in providing instructions and data to a computer for execution and processing. Examples of preferred non-temporary 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 disks, and network-attached storage (NAS), whether such devices are inside or outside a computer. A file containing information may be “stored” on computer-readable media, and “stored” means recording information so that it can be accessed and retrieved by a computer at a later date. The computer implementations described herein can be executed using programming that 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.

[0178] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for irradiating a sample containing particles in a flow stream with a light source; an algorithm for detecting light from particles in the sample with a photodetector in a photodetector system, 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 from the light detected from the particles at one or more of the first and second photodetector voltages; and an algorithm for determining one or more parameters of the particles from the generated data signal.

[0179] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for modulating the photodetector voltage between a first voltage and a second voltage at a frequency of 0.005 MHz to 10 MHz. In some cases, the non-temporary computer-readable storage medium includes an algorithm for modulating the photodetector voltage between a first voltage and a second voltage at a frequency of 0.1 MHz to 1 MHz. In some cases, the non-temporary computer-readable storage medium includes an algorithm for maintaining the photodetector voltage for a period of 1 μs or less, for example, 0.5 μs or less. In some embodiments, the first photodetector voltage is higher than the second photodetector voltage. In some cases, the first photodetector voltage is 500 mV to 2000 mV, for example, 600 mV to 1800 mV. In some cases, the second photodetector voltage is 0.001 mV to 500 mV, for example, 1 mV to 400 mV.

[0180] In some embodiments, the non-transient computer-readable storage medium includes an algorithm for modulating the voltage of a photodetector between a first photodetector voltage and a second photodetector voltage with a rectangular voltage change. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating a data signal from light detected at the first photodetector voltage. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating a data signal from light detected at the second photodetector voltage. In certain specific cases, the non-transient computer-readable storage medium includes an algorithm for generating a data signal from light detected at both the first and second photodetector voltages. In some cases, the non-transient computer-readable storage medium includes an algorithm for generating a data signal from 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-transient computer-readable storage medium includes an algorithm for generating a data signal only from 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, a non-temporary computer-readable storage medium includes an algorithm for detecting light by a photodetector only at a second photodetector voltage when the light detected at a first photodetector voltage exhibits a detector saturation level greater than the saturation threshold.

[0181] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a first photodetector voltage and a second photodetector voltage, respectively. In some cases, the non-temporary computer-readable storage medium includes an algorithm for generating a data signal based on the baseline sample. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring the baseline sample at a baseline sampling frequency. In some cases, the non-temporary computer-readable storage medium includes an algorithm for baseline sampling at frequencies from 0.0001 MHz to 15 MHz. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a frequency greater than the modulation frequency. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a frequency less than the modulation frequency. In some cases, the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample of the generated data signal at a frequency equal to the modulation frequency. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for scaling the generated data signal. In some cases, non-temporary computer-readable storage media include algorithms for scaling data signals on a single continuous scale and dividing the generated data signals by the photodetector gain at each photodetector voltage.

[0182] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample from a data signal generated from light detected by a first photodetector voltage. In some cases, the non-temporary computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample from a data signal generated from light detected by a second photodetector voltage. In a particular case, the non-temporary computer-readable storage medium includes an algorithm for determining one or more parameters of particles in a sample from data signals generated from light detected by a first photodetector voltage and light detected by a second photodetector voltage.

[0183] Non-temporary 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, a mouse, etc. A processing module includes a processor that accesses memory containing instructions for carrying out steps of the method in question. A processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of several other processors that are available or will become available. The processor runs an operating system, which interfaces with firmware and hardware in well-known ways and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in various programming languages, such as those described above, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically works with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all according to known techniques.

[0184] kit Aspects of this disclosure further include kits, which 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 disks, and network-attached storage devices (NAS). Any of these programmable storage media, or others currently in use or to be developed in the future, may be included in the kits of this disclosure. In embodiments, the programmable storage media includes instructions for analyzing flow cytometer data in the methods described herein and for use in the systems described herein. In embodiments, instructions contained in a computer-readable medium provided in the kits of this disclosure or in part thereof may be implemented as software components of software for analyzing data. In these embodiments, the computer-controlled systems of this disclosure may function as software "plug-ins" of existing software packages (e.g., FlowJo®).

[0185] In addition to the components described above, the kit may further include instructions (in some embodiments). These instructions may be present in the kit in various forms, and one or more of them may be present in the kit. One possible form of these instructions is information printed on a suitable medium or substrate, e.g., one or more sheets of paper on which the information is printed, the kit's packaging, accompanying documentation, etc. Yet another form of these instructions is a computer-readable medium on which the information is recorded, e.g., a diskette, a compact disc (CD), a portable flash drive, etc. Yet another possible form of these instructions is a website address that can be used over the internet to access information at a remote site.

[0186] Utility The methods, systems, and computer systems described herein are used in a variety of applications where it is desirable to calibrate or optimize a photodetector (e.g., having a photodetector), such as in particle analyzers. The methods and systems described herein are also used in photodetectors used to analyze and sort particulate components in samples in fluid media, such as biological samples. The disclosure also finds use in flow cytometry where 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 improved particle deflection during cell sorting. In embodiments, the disclosure reduces the need for user input or manual adjustment during sample analysis by a flow cytometer. In certain embodiments, the methods and systems described herein provide a fully automated protocol such that little to no human input is required for the adjustment of the flow cytometer in use.

[0187] Notwithstanding the attached claims, this disclosure is also provided for by the following sections:

[0188] 1. A method, Irradiating a sample containing particles in a flowstream with a light source, The light from particles in a sample is detected by a photodetector equipped with a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency. To generate a data signal from light detected from a particle at one or more of the first and second photodetector voltages, Determining one or more parameters of a particle from the generated data signal, Methods that include...

[0189] 2. The method according to Clause 1, 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.

[0190] 3. The method according to Clause 2, 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.

[0191] 4. The method according to any one of the clauses 1 to 3, wherein the voltage of the photodetector is maintained for a period of 1 μs or less per unit of time.

[0192] 5. The method according to Clause 4, wherein the voltage of the photodetector is maintained for a period of 0.5 μs or less per unit of time.

[0193] 6. The method according to any one of the clauses 1 to 5, wherein the first photodetector voltage is higher than the second photodetector voltage.

[0194] 7. The method according to any one of the clauses 1 to 6, wherein the first photodetector voltage is 500mV to 2000mV.

[0195] 8. The method according to any one of the clauses 1 to 7, wherein the second photodetector voltage is 0.001 mV to 500 mV.

[0196] 9. The method according to any one of the claims 1 to 8, wherein the voltage of the photodetector is modulated between a first photodetector voltage and a second photodetector voltage by a rectangular voltage change.

[0197] 10. The method according to any one of the claims 1 to 9, wherein the method comprises generating a data signal from light detected at a first photodetector voltage and light detected at a second photodetector voltage.

[0198] 11. The method according to clause 10, wherein the data signal is generated from 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.

[0199] 12. The method according to clause 10, wherein the data signal is generated only from light detected at a second photodetector voltage when the light detected at a first photodetector voltage exhibits a detector saturation level greater than the saturation threshold.

[0200] 13. The method according to Clause 10, wherein the method includes detecting light by the 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.

[0201] 14. The method according to any one of the claims 1 to 13, further comprising measuring a baseline sample at a first photodetector voltage and a second photodetector voltage, wherein a data signal is further generated based on the baseline sample.

[0202] 15. The method according to clause 14, wherein the baseline sample is measured at the baseline sampling frequency.

[0203] 16. The method according to clause 15, wherein the baseline sampling frequency is 0.0001 MHz to 15 MHz.

[0204] 17. The method described in any one of the clauses 15 to 16, wherein the baseline sampling frequency is greater than the modulation frequency.

[0205] 18. The method described in any one of the clauses 15 to 16, wherein the baseline sampling frequency is less than the modulation frequency.

[0206] 19. The method according to any one of the clauses 15 to 16, wherein the baseline sampling frequency is equal to the modulation frequency.

[0207] 20. The method described in any one of the clauses 1 to 19, further comprising scaling the generated data signal.

[0208] 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.

[0209] 22. The method according to any one of the clauses 1 to 21, wherein the detected light includes scattered light from particles in the flowstream.

[0210] 23. The method according to Clause 22, wherein the detected light includes side-scattered light, forward-scattered light, or a combination thereof.

[0211] 24. A system, A light source for irradiating a sample containing flowstream particles, A photodetection system comprising a photodetector for detecting light from particles in a sample, wherein the photodetector Modulated between the first photodetector voltage and the second photodetector voltage, A data signal is generated from the light detected from the particle using one or more of the first and second photodetector voltages. A light detection system configured as follows, A processor including memory operably coupled to the processor, wherein the memory stores instructions, and when an instruction is executed by the processor, causes the processor to determine one or more parameters of a particle from the generated data signal. A system equipped with these features.

[0212] 25. The system described in Clause 24, wherein the photodetector includes a photomultiplier tube (PMT).

[0213] 26. The system described in Clause 24, wherein the photodetector includes a photodiode.

[0214] 27. The system according to any one of clauses 24 to 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.

[0215] 28. The system described in 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.

[0216] 29. The system described in any one of clauses 24 to 28, wherein the photodetector is configured to maintain a voltage for a period of 1 μs or less per unit of time.

[0217] 30. The system described in any one of clauses 24 to 28, wherein the photodetector is configured to maintain a voltage for a period of 0.5 μs or less per unit of time.

[0218] 31. The system described in any one of clauses 24 to 30, wherein the first photodetector voltage is higher than the second photodetector voltage.

[0219] 32. A system according to any one of clauses 24 to 31, wherein the first photodetector voltage is 500mV to 2000mV.

[0220] 33. The system described in any one of clauses 24 to 32, wherein the second photodetector voltage is between 0.001 mV and 500 mV.

[0221] 34. The system according to any one of clauses 24 to 33, wherein the photodetector is configured to modulate between a first photodetector voltage and a second photodetector voltage with a rectangular voltage change.

[0222] 35. The system according to any one of clauses 24 to 34, wherein the photodetector is configured to generate a data signal from light detected at a first photodetector voltage and light detected at a second photodetector voltage.

[0223] 36. The system according to Clause 35, wherein the photodetector is configured to generate a data signal from 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.

[0224] 37. The system according to Clause 35, wherein the photodetector is configured to generate a data signal only from 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.

[0225] 38. The system according to any one of Clauses 24 to 37, wherein memory stores instructions, and when an instruction is executed by the processor, the processor causes the processor to measure a baseline sample at a first photodetector voltage and a second photodetector voltage, and the memory generates a data signal based on the baseline sample.

[0226] 39. The system described in Clause 38, wherein memory stores instructions, and when an instruction is executed by the processor, the processor causes the processor to measure a baseline sample of the generated data signal at the baseline sampling frequency.

[0227] 40. The system as described in Clause 38, wherein memory stores instructions, and when an instruction is executed by the processor, the processor causes the processor to measure a baseline sample of the generated data signal at a frequency greater than the modulation frequency.

[0228] 41. The system described in Clause 38, wherein memory stores instructions, and when an instruction is executed by the processor, the processor causes the processor to measure a baseline sample of the generated data signal at a frequency lower than the modulation frequency.

[0229] 42. The system described in Clause 38, wherein memory stores instructions, and when an instruction is executed by the processor, the processor causes the processor to measure a baseline sample of the generated data signal at a frequency equal to the modulation frequency.

[0230] 43. A system as described in any one of clauses 24 to 42, wherein memory stores instructions, and when an instruction is executed by the processor, causes the processor to scale data signals.

[0231] 44. The system as described in Clause 43, wherein memory stores instructions, and when an instruction is executed by the processor, the processor causes the processor to scale the data signal on a continuous scale and divide the generated data signal by the photodetector gain at each photodetector voltage.

[0232] 45. A system as described in any one of clauses 24 to 44, wherein the photodetector includes a scattered light detector.

[0233] 46. ​​The system described in Clause 45, wherein the photodetector is a side-scatter photodetector.

[0234] 47. The system described in Clause 45, wherein the photodetector is a forward-scattering photodetector.

[0235] 48. A system described in any one of clauses 24 to 47, wherein the system is a flow cytometer.

[0236] 49. A non-temporary computer-readable storage medium that stores instructions, where the instructions are An algorithm for irradiating a sample containing particles in a flowstream with a light source, An algorithm for detecting light from particles in a sample using 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 from light detected from a particle at one or more of the first and second photodetector voltages, An algorithm for determining one or more parameters of a particle from the generated data signal, Non-temporary computer-readable storage media, including [specific type of storage medium].

[0237] 50. A non-temporary computer-readable storage medium as described in 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.

[0238] 51. A non-temporary computer-readable storage medium as described in 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.

[0239] 52. A non-temporary computer-readable storage medium as described in any one of clauses 49 to 51, wherein the voltage of the photodetector is maintained for a period of 1 μs or less per unit of time.

[0240] 53. A non-temporary computer-readable storage medium as described in Clause 52, wherein the voltage of the photodetector is maintained for a period of 0.5 μs or less per unit of time.

[0241] 54. A non-temporary computer-readable storage medium as described in any one of clauses 49 to 53, wherein the first photodetector voltage is higher than the second photodetector voltage.

[0242] 55. A non-temporary computer-readable storage medium as described in any one of clauses 49 to 54, wherein the first photodetector voltage is 500 mV to 2000 mV.

[0243] 56. A non-temporary computer-readable storage medium as described in any one of clauses 49 to 55, wherein the second photodetector voltage is 0.001 mV to 500 mV.

[0244] 57. A non-temporary computer-readable storage medium as described in any one of clauses 49 to 56, wherein the voltage of the photodetector is modulated between a first photodetector voltage and a second photodetector voltage in a rectangular voltage change.

[0245] 58. A non-temporary computer-readable storage medium according to any one of the clauses 49 to 57, wherein the non-temporary computer-readable storage medium includes an algorithm for generating data signals from light detected by a first photodetector voltage and a second photodetector voltage.

[0246] 59. A non-temporary computer-readable storage medium as described in Clause 58, comprising an algorithm for generating a data signal from 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.

[0247] 60. A non-temporary computer-readable storage medium according to Clause 59, comprising an algorithm for measuring light by 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.

[0248] 61. A non-temporary computer-readable storage medium according to any one of the clauses 49 to 60, comprising 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.

[0249] 62. A non-temporary computer-readable storage medium as described in Clause 61, comprising an algorithm for measuring a baseline sample of a generated data signal at a baseline sampling frequency.

[0250] 63. A non-temporary computer-readable storage medium as described in Clause 62, wherein the non-temporary computer-readable storage medium includes an algorithm for baseline sampling at frequencies between 0.0001 MHz and 15 MHz.

[0251] 64. A non-temporary computer-readable storage medium as described in Clause 62, wherein the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample at a frequency higher than the modulation frequency.

[0252] 65. A non-temporary computer-readable storage medium as described in Clause 62, wherein the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample at a frequency lower than the modulation frequency.

[0253] 66. A non-temporary computer-readable storage medium as described in Clause 62, wherein the non-temporary computer-readable storage medium includes an algorithm for measuring a baseline sample at a frequency equal to the modulation frequency.

[0254] 67. A non-temporary computer-readable storage medium as described in any one of clauses 49 to 66, wherein the non-temporary computer-readable storage medium includes an algorithm for scaling the generated data signals.

[0255] 68. A non-temporary computer-readable storage medium as described in Clause 67, comprising 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.

[0256] While the foregoing disclosure has been described in some detail as examples and illustrations to clarify understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of this disclosure, several changes and modifications can be made without departing from the spirit or scope of the attached claims.

[0257] Therefore, the foregoing is merely illustrative of the principles of the present disclosure. Those skilled in the art will understand that various configurations embodying the principles of the present disclosure and that fall within its spirit and scope can be devised, although not expressly described or shown herein. Furthermore, all examples and conditional statements listed herein are intended primarily to help the reader understand the principles of the present disclosure and the concepts that the disclosure has contributed to advancing the art, and should be construed as not being limited to such specifically listed examples and conditions. Moreover, all descriptions herein listing the principles, aspects and embodiments of the present disclosure and specific examples thereof are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any developed element that performs the same function regardless of its structure. Furthermore, nothing disclosed herein is intended to be made available to the public, whether such disclosure is expressly described in the claims or not.

[0258] Accordingly, the scope of this disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied in the appended claims. In the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) are expressly defined as being invoked for limitation in the claims only if the exact phrase “means” or the exact phrase “step” is stated at the beginning of such limitation in the claims, and if such exact phrase is not used in limitation in the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) are not invoked.

Claims

1. It is a method, Irradiating a sample containing particles in a flowstream with a light source, The light from the particles in the sample is detected by a photodetector equipped with a photodetector, wherein the voltage of the photodetector is modulated between a first voltage and a second voltage at a modulation frequency. A data signal is generated from the light detected from the particles using one or more of the first photodetector voltage and the second photodetector voltage. Determining one or more parameters of the particle from the generated data signal, Methods that include...

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 0.005 MHz to 10 MHz.

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 a period of 1 μs or less per unit of time.

5. The method according to claim 4, wherein the voltage of the photodetector is maintained for a period of 0.5 μs or less per unit of time.

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

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

8. The method according to any one of claims 1 to 7, wherein the second photodetector voltage is 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 between the first photodetector voltage and the second photodetector voltage in a rectangular voltage change.

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

11. (a) The data signal is generated from the light detected by the second photodetector voltage when the light detected by the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold, (b) The data signal is generated from only the light detected by the second photodetector voltage when the light detected by the first photodetector voltage exhibits a detector saturation level greater than the saturation threshold, or (c) The method according to claim 10, wherein the method includes detecting light by 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.

12. The method according to any one of claims 1 to 11, further comprising measuring a baseline sample with the first photodetector voltage and the second photodetector voltage, and further generating a data signal based on the baseline sample.

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

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

15. The baseline sampling frequency is greater than the modulation frequency, The baseline sampling frequency is smaller than the modulation frequency, or The method according to claim 13 or 14, wherein 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 according to claim 16, wherein the scaling includes continuous scaling, and comprises 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 includes scattered light from particles in the flow stream.

19. The method according to claim 18, wherein the detected light includes side-scattered light, forward-scattered light, or a combination thereof.

20. It is a system, A light source for irradiating a sample containing flowstream particles, A photodetection system comprising a photodetector for detecting light from the particles in the sample, wherein the photodetector is Modulated between the first photodetector voltage and the second photodetector voltage, A data signal is generated from the light detected from the particles using one or more of the first and second photodetector voltages. A light detection system configured as follows, A processor including memory operably coupled to the processor, wherein the memory stores instructions, and when an instruction is executed by the processor, the processor causes the processor to determine one or more parameters of the particle from the generated data signal. A system that includes these features.

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