Phase correction of radio frequency multiplexed signals

Abstract

A system for measuring particles in a flow stream. [Solution] The system includes a light beam generator, an acousto-optical device configured to receive the generated light beam and form a frequency-shifted beam of light to irradiate the particles in the flow stream, a light detection system to measure fluorescence of the irradiated particles, and a processor, the processor including a memory operatively coupled to the processor having instructions stored in the memory that, when executed by the processor, cause the processor to generate frequency-encoded fluorescence data based at least in part on the fluorescence of the irradiated particles and the frequency of the frequency-shifted light, and to calculate phase-corrected spatial data of the particles by performing a transformation of the frequency-encoded fluorescence data with a phase correction component.

Description

[Background technology]

[0001] Characterization of analytes in bodily fluids has become an essential part of medical diagnosis and evaluation of a patient's overall health. Detecting analytes in bodily fluids, such as human blood or blood-derived products, can provide results that can play a role in determining treatment protocols for patients with various medical conditions.

[0002] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells in a blood sample or other types of particles of interest in biological or chemical samples. A flow cytometer typically includes a sample container for receiving a fluid sample, such as a blood sample, and a sheath container containing a sheath fluid. The flow cytometer transports particles (including cells) in the fluid sample as a cell stream to a flow cell, while directing the sheath fluid toward the flow cell. To characterize the components of the flow stream, the flow stream is illuminated with light. Variations in the materials in the flow stream, such as morphology or the presence of fluorescent labels, can produce variations in the observed light, enabling characterization and separation.

[0003] To characterize the components in a flow stream, light must be applied to the flow stream and collected. The light source in a flow cytometer can vary, including broad spectrum lamps, light emitting diodes, and single wavelength lasers. The light source is aligned with the flow stream, and the optical response of the illuminated particles is collected and quantified. Summary of the Invention

[0004] Aspects of the present disclosure include methods for characterizing particles of a sample in a flowstream. The method, according to certain embodiments, includes generating frequency-encoded fluorescence data from particles of the sample in the flowstream and calculating phase-corrected spatial data of the particles by performing a transform of the frequency-encoded fluorescence data with a phase-correction component. In certain embodiments, the method includes generating an image of the particles in the flowstream based on the phase-corrected spatial data. Also described is a system having a processor, the processor having a memory operatively coupled to the processor, and instructions stored in the memory that, when executed by the processor, cause the processor to calculate phase-corrected spatial data from the frequency-encoded fluorescence data of particles in the flowstream. Additionally, an integrated circuit device (e.g., a field-programmable gate array) having programming for performing the method is also provided.

[0005] In embodiments, frequency-encoded fluorescence data from particles in a sample is generated from light detected in an examination region of the flowstream. In some embodiments, the particles are cells. In embodiments, the method includes detecting light emission (e.g., fluorescence) from the sample in the flowstream to generate frequency-encoded fluorescence data from the particles. In some embodiments, the method further includes detecting light absorption, light scattering, or a combination thereof. In some embodiments, particles having one or more fluorophores are illuminated with multiple frequency-shifted beams of light from a light beam generator to produce frequency-encoded fluorescence. In one example, multiple locations across the flowstream (in the horizontal axis) are illuminated with a laser beam including a local oscillator beam and multiple radio frequency shifted laser beams, such that different locations across the flowstream are illuminated with a local oscillator beam and one radio frequency shifted beam. In some cases, the local oscillator beam is a frequency-shifted light beam from a laser. In this example, each spatial location of a particle in the flowstream is characterized by a different beat frequency corresponding to the difference between the frequency of the local oscillator beam and the frequency of the radio frequency shifted beam at that location. In some embodiments, the frequency encoded data from the particles comprises a spatially encoded beat frequency in the horizontal axis of the particles in the flow stream.

[0006] In performing the method, light from a sample in the flow stream is detected in an examination region, and frequency-encoded data from particles in the sample is generated. In some embodiments, the particles detected in the examination region include cells. In some embodiments, the method includes detecting one or more of light absorption, light scattering, and light emission (e.g., fluorescence) from the sample in the flow stream. In some cases, phase-corrected spatial data of one or more particles in the sample is generated from detected light absorption (e.g., bright-field image data). In other cases, phase-corrected spatial data of one or more particles in the sample is generated from detected light scattering (e.g., forward-scatter image data, side-scatter image data). In yet other cases, phase-corrected spatial data of one or more particles in the sample is generated from detected fluorescence (e.g., fluorescent marker image data). In still other cases, phase-corrected spatial data of one or more particles in the sample is generated from a combination of two or more of detected light absorption, detected light scattering, and detected fluorescence.

[0007] In embodiments, frequency-encoded fluorescence data from particles in a flow stream are transformed with a phase-correction component to provide spatial data of the particles. In embodiments, the spatial data may include the horizontal size of the particle, the vertical size of the particle, the ratio of particle sizes along two different dimensions, or the ratio of particle component sizes (e.g., the ratio of the horizontal dimension of the cytoplasm to the horizontal dimension of the nucleus of a cell). In some embodiments, the frequency-encoded fluorescence data is transformed by a Fourier transform of the frequency-encoded fluorescence data with a phase-correction component. In some cases, the frequency-encoded fluorescence data is transformed by a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data with a phase-correction component. In other cases, the phase-corrected spatial data is calculated by performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data with the phase correction. In still other cases, the phase-corrected spatial data is calculated with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data.

[0008] In some embodiments, the method includes determining a phase correction component used to convert the frequency-encoded fluorescence data into phase-corrected spatial data. In some cases, the phase correction component includes a modified conversion coefficient. In certain embodiments, the phase correction component includes a first phase adjustment and a second phase adjustment. In some cases, the first phase adjustment includes an output signal from a light detection system. For example, the first phase adjustment may include an output signal from a bright-field light detector.

[0009] In some embodiments, the first phase adjustment is calculated by multiplying the output signal from the bright-field photodetector by a predetermined constant signal to generate a phase adjustment value, and then calculating the arctangent of the phase adjustment value to yield the first phase adjustment. In these embodiments, the phase adjustment value is the sum of all bins in the discrete Fourier transform of the frequency-encoded fluorescence data. In certain embodiments, the first phase adjustment is an interferometric phase adjustment. In these embodiments, the phase adjustment includes a phase shift induced by a light source for illuminating the sample in the flow stream. For example, the light source can be an optical beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. In certain cases, the optical beam generator includes a laser (e.g., a continuous wave laser) and an acousto-optic deflector (e.g., coupled to a direct digital synthesizer RF comb generator). In some embodiments, the interferometric phase adjustment includes a phase shift resulting from vibrations between components of the optical beam generator. In some embodiments, the second phase adjustment is based on the fluorescence lifetime of a fluorophore in the sample. In these embodiments, the second phase adjustment is calculated by acquiring signals from all fluorescence detectors and then calculating the phase shift. This can be calculated by determining the phase present in the signal and calculating a second phase adjustment from the fluorescence lifetime of the fluorophores.

[0010] Methods according to certain embodiments also include sorting one or more particles in the sample. In some embodiments, the particles are identified as single cells and sorted to a first sample component collection location. In other embodiments, the particles are identified as cell aggregates and sorted to a second sample component collection location. In some cases, the first sample component collection location includes a sample collection container and the second sample component collection location includes a waste collection container.

[0011] Aspects of the present disclosure also include a system for characterizing particles of a sample in a flowstream (e.g., cells in a biological sample). The system, according to certain embodiments, includes a light source, a light detection system, and a processor, where the light source is configured to illuminate a sample having particles in the flowstream, the light detection system having a photodetector, and the processor has a memory operably coupled to the processor, the memory having stored therein instructions that, when executed by the processor, cause the processor to generate frequency-encoded fluorescence data from particles in the flowstream and calculate phase-corrected spatial data for the particles by performing a transform of the frequency-encoded fluorescence data with a phase-correction component.

[0012] In embodiments, a system is configured to generate frequency-encoded fluorescence data from particles in a sample illuminated by a light source. In some embodiments, the light source includes a light beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. In certain cases, the light beam generator includes a laser (e.g., a continuous wave laser) and an acousto-optic deflector (e.g., coupled to a direct digital synthesizer RF comb generator). The system includes a light detection system configured to detect one or more of light absorption, light scattering, and light emission (e.g., fluorescence) from the sample in the flow stream. In some cases, the light detection system includes a light detector for detecting light absorption (e.g., a bright field light detector). In other cases, the light detection system includes a light detector for detecting light scattering (e.g., a forward scatter detector, a side scatter detector). In yet other cases, the light detection system includes a light detector for detecting fluorescence. In still other cases, the light detection system includes a combination of two or more of a light absorption detector, a light scattering detector, and an emitted light (e.g., fluorescence) detector.

[0013] In embodiments, the system includes a processor having a memory operatively coupled to the processor, the memory having instructions stored therein that, when executed by the processor, cause the processor to calculate phase-corrected spatial data for the particle by performing a transform of the frequency-encoded fluorescence data with a phase-correction component. In embodiments, the spatial data may include the particle's horizontal size, the particle's vertical size, the ratio of the particle's sizes along two different dimensions, or the ratio of particle component sizes (e.g., the ratio of the horizontal dimension of the cytoplasm to the horizontal dimension of the nucleus of a cell). In some embodiments, to calculate the phase-corrected spatial data, the system is configured to perform a Fourier transform of the frequency-encoded fluorescence data with the phase-correction component to generate phase-corrected spatial data for the particle. In other embodiments, the system is configured to perform a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data with the phase-correction component to generate phase-corrected spatial data for the particle. In yet other embodiments, the system is configured to perform a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data with the phase-correction component. In yet another embodiment, the system is configured to calculate phase-corrected spatial data with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data.

[0014] In some embodiments, the system includes a processor having a memory operatively coupled to the processor, the memory having instructions stored therein that, when executed by the processor, cause the processor to determine a phase correction component used to convert the frequency-encoded fluorescence data into phase-corrected spatial data. In some cases, the phase correction component includes a modified conversion coefficient. In some embodiments, the system is configured to determine the phase correction component by calculating a first phase adjustment and a second phase adjustment. In some cases, the first phase adjustment includes an output signal from an optical detection system. For example, the first phase adjustment may include an output signal from a bright-field optical detector.

[0015] In some embodiments, the system includes a processor having a memory operably coupled to the processor and instructions stored in the memory that, when executed by the processor, cause the processor to calculate a first phase adjustment by multiplying the output signal from the brightfield photodetector and a predetermined constant signal to generate a phase adjustment value and calculating the arctangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is a sum over all bins in a discrete Fourier transform of the frequency-encoded fluorescence data. In certain embodiments, the first phase adjustment is an interferometric phase adjustment. In other embodiments, the system includes a processor having a memory operably coupled to the processor and instructions stored in the memory that, when executed by the processor, cause the processor to calculate a second phase adjustment based on the fluorescence lifetimes of fluorophores in the sample. In these embodiments, the second phase adjustment can be calculated by the system by acquiring signals from all fluorescence detectors to determine the phases present in the signals and calculating the second phase adjustment from the fluorescence lifetimes of the fluorophores.

[0016] The subject systems are configured, in certain cases, to sort particles of a sample (e.g., a biological sample) in a flow stream. In some embodiments, the system further includes a particle sorting component having a sample fluid delivery subsystem, a sheath fluid delivery subsystem in fluid communication with an inlet of the particle sorting component, and one or more sample collection vessels for receiving sorted particles from the flow stream.

[0017] Aspects of the present disclosure also include integrated circuit devices programmed to generate frequency-encoded fluorescence data from particles in a flow stream and calculate phase-corrected spatial data for the particles by performing a transformation of the frequency-encoded fluorescence data with a phase-correction component. In some embodiments, the integrated circuit device is programmed to sort the particles, for example, into a sample collection container or a waste collection container. Targeted integrated circuit devices may, in certain cases, include field programmable gate arrays (FPGAs), application-specific integrated circuits (ASICs), or complex programmable logic devices (CPLDs).

[0018] According to certain embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from particles in a flow stream. In some embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from data signals from a light absorption detector (e.g., bright field image data). In other embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from data signals from a light scatter detector (e.g., forward scatter image data, side scatter image data). In yet other embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from data signals from a light emission detector (e.g., fluorescent marker image data). In yet other cases, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from a combination of two or more of detected light absorption, detected light scatter, and detected fluorescence.

[0019] In embodiments, the integrated circuit device is programmed to calculate phase-corrected spatial data of the particle by performing a transform of the frequency-encoded fluorescence data with a phase correction component. In some cases, the integrated circuit device is programmed to perform a Fourier transform of the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle. In other cases, the integrated circuit device is programmed to perform a discrete Fourier transform of the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle. In yet other cases, the integrated circuit device is programmed to perform a short-time Fourier transform of the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle. In yet other cases, the integrated circuit device is programmed to heterodyne and demultiplex the frequency-encoded fluorescence data and to calculate phase-corrected spatial data with a digital lock-in amplifier.

[0020] In some embodiments, the integrated circuit device is programmed to determine a phase correction component used to convert the frequency-encoded fluorescence data into phase-corrected spatial data. In some cases, the phase correction component includes a modified conversion coefficient. In some embodiments, the integrated circuit device is programmed to determine the phase correction component by calculating a first phase adjustment and a second phase adjustment. In some cases, the first phase adjustment includes an output signal from a light detection system. For example, the first phase adjustment may include an output signal from a bright-field light detector.

[0021] In some embodiments, the integrated circuit device is programmed to calculate the first phase adjustment by multiplying the output signal from the brightfield photodetector and a predetermined constant signal to generate a phase adjustment value and calculating the arctangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum over all bins in the discrete Fourier transform of the frequency-encoded fluorescence data. In certain embodiments, the first phase adjustment is an interferometric phase adjustment. In other embodiments, the integrated circuit device is programmed to calculate a second phase adjustment based on the fluorescence lifetimes of fluorophores in the sample. In these embodiments, the second phase adjustment can be calculated by the integrated circuit by acquiring signals from all fluorescence detectors to determine the phases present in the signals and calculating the second phase adjustment from the fluorescence lifetimes of the fluorophores.

[0022] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a flowchart for generating frequency-encoded fluorescence data and calculating phase-corrected spatial data from the frequency-encoded fluorescence data, according to certain embodiments. [Figure 2] FIG. 10 illustrates a comparison of an image of a particle generated using phase-corrected spatial data with an image in which the spatial data is not phase-corrected, according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0024] Aspects of the present disclosure include methods for characterizing particles of a sample in a flowstream. The method, according to certain embodiments, includes generating frequency-encoded fluorescence data from particles of the sample in the flowstream and calculating phase-corrected spatial data of the particles by performing a transform of the frequency-encoded fluorescence data with a phase-correction component. In certain embodiments, the method includes generating an image of the particles in the flowstream based on the phase-corrected spatial data. Systems are also described that include a processor, the processor having a memory operatively coupled to the processor, and instructions stored in the memory that, when executed by the processor, cause the processor to calculate phase-corrected spatial data from the frequency-encoded fluorescence data of particles in the flowstream. Additionally, integrated circuit devices (e.g., field-programmable gate arrays) having programming for performing the methods are also provided.

[0025] Before describing the present invention in detail, it is to be understood that the invention is not limited to particular embodiments described, as such embodiments may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0026] Where a range of values ​​is given, unless the context clearly indicates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and these values ​​are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0027] In this specification, certain ranges are presented with "about" before the numerical values. In this specification, the term "about" is used to describe the numerical value that follows it, as well as a number that is close to or approximately the numerical value that follows it. Whether a numerical value is close to or approximately the specifically stated numerical value depends on whether the unstated close or approximately numerical value can have a substantially equivalent effect to the specifically stated numerical value in the context in which it is presented.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0029] All publications and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publication is cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date of the present application and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0030] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like, or for use of a "negative" limitation in connection with the recitation of claim elements.

[0031] As will be apparent to those skilled in the art upon reading the description disclosed herein, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the invention. Any method described can be carried out in the order of events described or in any other order which is logically possible.

[0032] Although the apparatus and methods are described above and below in a grammatically fluid manner using functional descriptions, the claims are not to be construed as being limited in any way by construction of "means" or "step" limitations, except as expressly set forth in 35 U.S.C. 112, but are expressly understood to conform to the full range of meanings and equivalents of the definitions provided by the claims under the common law doctrine of equivalents, and where a claim is expressly set forth in 35 U.S.C. 112, it is to be expressly understood to conform to the full statutory equivalents under 35 U.S.C. 112.

[0033] As summarized above, the present disclosure provides systems and methods for characterizing (e.g., imaging) particles of a sample in a flowstream. In further describing embodiments of the present disclosure, methods for generating frequency-encoded fluorescence data from particles of a sample in a flowstream and calculating phase-corrected spatial data for the particles are first described in detail. Systems for characterizing particles in a flowstream and separating particles in a sample in real time are then described. Also provided are integrated circuit devices, such as field-programmable gate arrays, having programming for generating frequency-encoded fluorescence data from particles of a sample in a flowstream and calculating phase-corrected spatial data for the particles.

[0034] Methods for characterizing particles in a sample Aspects of the present disclosure include methods for characterizing particles in a sample (e.g., cells in a biological sample). Performing a method according to certain embodiments involves illuminating a sample having cells in a flow stream with a light source, detecting light from the sample to generate frequency-encoded fluorescence data from the particles, and calculating phase-corrected spatial data for the particles by performing a transformation of the encoded fluorescence data with a phase-correction component. In some embodiments, the sample is a biological sample. The term "biological sample" is used in its conventional sense to refer to a whole organism, plant, fungus, or a subset of animal tissues, cells, or components found in certain cases in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to both a natural organism or a subset of its tissues, and to homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymphatic fluid, portions of skin, respiratory, gastrointestinal, cardiovascular, genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. A biological sample can be any type of biological tissue and can include both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, a biological sample is a liquid sample such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, etc.; in some cases, the sample is a blood sample, including whole blood, e.g., blood obtained from a venipuncture or finger prick (which may or may not be combined with reagents such as preservatives, anticoagulants, etc., prior to analysis).

[0035] In certain embodiments, the source of the sample is a "mammal" or "mammalian animal," terms used broadly to describe organisms belonging to the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method is applicable to samples obtained from human subjects of both genders and at any developmental stage (i.e., newborn, infant, juvenile, adolescent, adult); in certain embodiments, the human subject is a juvenile, adolescent, or adult. While the present invention is applicable to samples from human subjects, it should be understood that the method can also be performed on samples from other animal subjects (i.e., "non-human subjects"), such as those described below. Other animal subjects include, but are not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0036] In carrying out this method, a sample containing particles (e.g., cells in a flow stream in a flow cytometer) is illuminated by light from a light source. In some embodiments, the light source is a broadband light source that emits light having a wide wavelength range of, for example, 50 nm or more, and the wavelength range may be, for example, 100 nm or more, for example, 150 nm or more, for example, 200 nm, for example, 250 nm or more, for example, 300 nm or more, for example, 350 nm or more, for example, 400 nm or more, or may be, for example, 500 nm or more. For example, one suitable broadband light source emits light having a wavelength of 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having a wavelength of 400 nm to 1000 nm. Where the method includes illumination with a broadband light source, the broadband light source protocol of interest may include, but is not limited to, a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a stabilized fiber-coupled broadband light source, a continuous spectrum broadband LED, a superluminescent diode, a semiconductor light emitting diode, a broad spectrum LED white light source, a multi-LED integrated white light source, other broadband light sources, or any combination thereof.

[0037] In other embodiments, the method includes irradiating with a narrowband light source that emits a specific wavelength or a narrow range of wavelengths. The light source emits light in a narrow wavelength range, such as a range of 50 nm or less. The wavelength range may be, for example, 40 nm or less, for example, 30 nm or less, for example, 25 nm or less, for example, 20 nm or less, for example, 15 nm or less, for example, 10 nm or less, for example, 5 nm or less, for example, 2 nm or less. The light source may be a light source that emits light of a specific wavelength (i.e., monochromatic light). The method includes irradiating with a narrowband light source. Narrowband light source protocols of interest may include, but are not limited to, narrow wavelength LEDs, laser diodes, or broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0038] In certain embodiments, the method includes irradiating the flowstream with one or more lasers. The type and number of lasers vary depending on the sample and the desired light collected, and can be pulsed or continuous wave lasers. The lasers can be, for example, gas lasers, dye lasers, metal vapor lasers, solid-state lasers, semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs), or lasers operating at double or triple the frequency of any of the above lasers. The gas lasers can be, for example, helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO2 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. The dye lasers can be, for example, stilbene, coumarin, or rhodamine lasers. The metal vapor laser may be, for example, 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. The solid-state laser may be, for example, a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, or a combination thereof.

[0039] The sample in the flowstream can be illuminated by one or more of the aforementioned light sources. The light sources can be, 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, or even ten or more light sources. The light sources can include any combination of light sources. For example, in some embodiments, the method includes illuminating the sample in the flowstream with a laser array. The array can include, for example, one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0040] The sample can be illuminated with a wavelength in the range of 200 nm to 1500 nm. The wavelength range can be, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, or 400 nm to 800 nm. For example, if the light source is a broadband light source, the sample can be illuminated with a wavelength in the range of 200 nm to 900 nm. In other cases, if the light source includes multiple narrowband light sources, the sample can be illuminated with a specific wavelength in the range of 200 nm to 900 nm. For example, the light source can be multiple narrowband LEDs (1 nm-25 nm), each independently emitting light 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), for example, a laser array including a gas laser, an excimer laser, a dye laser, a metal vapor laser, and a solid-state laser, as described above, to illuminate the sample with a specific wavelength in the range of 200 nm to 700 nm.

[0041] When more than one light source is used, the sample can be irradiated by the light sources simultaneously, sequentially, or a combination thereof. For example, the sample can be irradiated by each light source simultaneously. In other embodiments, the flow stream is irradiated sequentially by each light source. When more than one light source is used to irradiate the sample sequentially, the time for which each light source irradiates the sample can be 0.001 microseconds or more. The time can be, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, or even 60 microseconds or more. For example, the method can include irradiating the sample with a light source (e.g., a laser) for a duration ranging from 0.001 microseconds to 100 microseconds. The range of the duration can be, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microsecond to 25 microseconds, or 5 microseconds to 10 microseconds. In embodiments, when the sample is illuminated sequentially with two or more light sources, the duration for which the sample is illuminated by each light source may be the same or different.

[0042] The period between illumination by each light source can also be varied, as needed, so that they are separated by a delay of 0.001 microseconds or more. The delay can be, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 15 microseconds or more, for example, 30 microseconds or more, or even 60 microseconds or more. For example, the period between illumination by each light source can range from 0.001 microseconds to 60 microseconds, for example, from 0.01 microseconds to 50 microseconds, for example, from 0.1 microseconds to 35 microseconds, for example, from 1 microsecond to 25 microseconds, or even from 5 microseconds to 10 microseconds. In certain embodiments, the period between illumination by each light source is 10 microseconds. In embodiments, when the sample is illuminated sequentially with more than two (e.g., three or more) light sources, the delay between illumination by each light source can be the same or different.

[0043] The sample can be illuminated continuously or at discrete intervals. In some cases, the method includes continuously illuminating the sample with the light source. In other cases, the sample is illuminated by the light source at discrete intervals, such as every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, 1000 milliseconds, or other intervals.

[0044] Depending on the light source, the sample can be illuminated from various distances, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, or even 50 mm or more. The angle of illumination can also vary from 10° to 90°, such as from 15° to 85°. , for example 20° to 80°, for example 25° to 75°, for example 30° to 60°, for example an angle of 90°.

[0045] In practicing the method, light from the illuminated sample is measured, for example, by collecting light from the sample over a range of wavelengths (e.g., 200 nm to 1000 nm). In embodiments, the method may include one or more of measuring light absorbed by the sample (e.g., brightfield light data), measuring scattered light (e.g., forward or side scatter data), and measuring light emitted from the sample (e.g., fluorescence data).

[0046] Light from the sample may be measured at one or more wavelengths, for example light collected at 5 or more different wavelengths, such as 10 or more different wavelengths, for example 25 or more different wavelengths, for example 50 or more different wavelengths, for example 100 or more different wavelengths, for example 200 or more different wavelengths, for example 300 or more different wavelengths, or light collected at 400 or more different wavelengths may be measured.

[0047] Light can be collected over one or more wavelength ranges from 200 nm to 1200 nm. In some cases, the method includes measuring light from the sample over a range of wavelengths. The wavelength range can be, for example, 200 nm to 1200 nm, for example, 300 nm to 1100 nm, for example, 400 nm to 1000 nm, for example, 500 nm to 900 nm, or 600 nm to 800 nm. In other cases, the method includes measuring the collected light at one or more specific wavelengths. For example, the collected light can be measured at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, or any combination thereof. In certain embodiments, the method includes measuring light at a wavelength corresponding to the fluorescence peak wavelength of a particular fluorophore.

[0048] The collected light can be measured continuously or at discrete intervals. In some cases, the method includes measuring the light continuously. In other cases, the light is measured at discrete intervals, such as every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, 1000 milliseconds, or other intervals.

[0049] Measurement of the collected light can be performed one or more times in the present method. The number of measurements can be, for example, two or more, for example, three or more, for example, five or more, or even ten or more. In certain embodiments, the light from the sample is measured two or more times, and in certain cases, the data is averaged.

[0050] In some embodiments, the method includes further conditioning the light from the sample before detecting it. For example, the light from the sample source may pass through one or more lenses, mirrors, pinholes, slits, gratings, optical refractors, or any combination thereof. In some cases, the collected light passes through one or more focusing lenses to reduce the light profile. In other cases, the light emitted from the sample passes through one or more collimators to reduce the divergence of the light beam.

[0051] In certain embodiments, the method includes illuminating the 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 shifting the frequency of the laser light can be used. In these embodiments, the method includes illuminating the acousto-optical device with a laser. Depending on the desired wavelength of light generated in the output laser beam (e.g., the wavelength of light for illuminating the sample in the flow stream), the laser can have a specific wavelength that varies from 200 nm to 1500 nm. The wavelength variation range can be, for example, from 250 nm to 1250 nm, for example, from 300 nm to 1000 nm, for example, from 350 nm to 900 nm, or even from 400 nm to 800 nm. The acousto-optical device can be illuminated by one or more lasers. The lasers can be, for example, two or more lasers, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, or even ten or more lasers. The lasers can include any combination of lasers. For example, in some embodiments, the method includes illuminating the acousto-optical device with an array of lasers. The array of lasers is, for example, an array having one or more gas lasers, one or more dye lasers, and one or more solid state lasers.

[0052] When more than one laser is used, the lasers can irradiate the acousto-optical device simultaneously, sequentially, or a combination thereof. For example, each laser can irradiate the acousto-optical device simultaneously. In other embodiments, each laser can irradiate the acousto-optical device sequentially. When more than one laser is used to irradiate the acousto-optical device sequentially, the duration of irradiation of the acousto-optical device with each laser can be 0.001 microseconds or more. The irradiation time can be, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, or even 60 microseconds or more. For example, the method can include irradiating the acousto-optical device with a laser for a duration ranging from 0.001 microseconds to 100 microseconds. The duration can range from, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microsecond to 25 microseconds, or 5 microseconds to 10 microseconds. In embodiments, when two or more lasers sequentially illuminate the acousto-optical device, the duration for which each laser illuminates the acousto-optical device may be the same or different.

[0053] The period between irradiations by each laser can also be varied, as needed, so that they are separated by a delay of 0.001 microseconds or more. The delay can be, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 15 microseconds or more, for example, 30 microseconds or more, or even 60 microseconds or more. For example, the period between irradiations by each light source can range from 0.001 microseconds to 60 microseconds. The range can be, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microsecond to 25 microseconds, or even 5 microseconds to 10 microseconds. In certain embodiments, the period between irradiations by each laser is 10 microseconds. In embodiments, when the acousto-optical device is sequentially irradiated by more than two (e.g., three or more) lasers, the delay between irradiations by each laser can be the same or different.

[0054] The acousto-optical device can be illuminated continuously or at discrete intervals. In some cases, the method includes illuminating the acousto-optical device continuously with a laser. In other cases, the acousto-optical device is illuminated with a laser at discrete intervals, such as every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, 1000 milliseconds, or other intervals.

[0055] Depending on the laser, the acousto-optical device can be illuminated from various distances, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, or even 50 mm or more. The angle of illumination can also vary from 10° to 90°, such as 15°. to 85°, for example 20° to 80°, for example 25° to 75°, may be 30° to 60°, for example an angle of 90°.

[0056] In an embodiment, a method includes applying a radio frequency drive signal to an acousto-optical device to generate an angularly deflected laser beam. Two or more radio frequency drive signals can be applied to the acousto-optical device to generate an output laser beam having a desired number of angularly deflected laser beams. For example, three or more radio frequency drive signals, for example, four or more radio frequency drive signals, for example, five or more radio frequency drive signals, for example, six or more radio frequency drive signals, for example, seven or more radio frequency drive signals, for example, eight or more radio frequency drive signals, for example, nine or more radio frequency drive signals, for example, ten or more radio frequency drive signals, for example, fifteen or more radio frequency drive signals, for example, twenty-five or more radio frequency drive signals, for example, fifty or more radio frequency drive signals, or even one hundred or more radio frequency drive signals can be applied.

[0057] Each angularly deflected laser beam generated by the radio frequency drive signal has an intensity based on the amplitude of the applied radio frequency drive signal. In some embodiments, the method includes applying a radio frequency drive signal having an amplitude sufficient to generate an angularly deflected laser beam having a desired intensity. In some cases, each applied radio frequency drive signal has an amplitude of about 0.001 V to about 500 V. The amplitude may be, for example, about 0.005 V to about 400 V, for example, about 0.01 V to about 300 V, for example, about 0.05 V to about 200 V, for example, about 0.1 V to about 100 V, for example, about 0.5 V to about 75 V, for example, about 1 V to about 50 V, for example, about 2 V to about 40 V, for example, 3 V to about 30 V, or about 5 V to about 25 V. In some embodiments, each applied radio frequency drive signal has a frequency of about 0.001 MHz to about 500 MHz. The frequency may be, for example, from about 0.005 MHz to about 400 MHz, for example, from about 0.01 MHz to about 300 MHz, for example, from about 0.05 MHz to about 200 MHz, for example, from about 0.1 MHz to about 100 MHz, for example, from about 0.5 MHz to about 90 MHz, for example, from about 1 MHz to about 75 MHz, for example, from about 2 MHz to about 70 MHz, for example, from about 3 MHz to about 65 MHz, for example, from about 4 MHz to about 60 MHz, or may be from about 5 MHz to about 50 MHz.

[0058] In some embodiments, to generate frequency-encoded fluorescence data, a sample in a flow stream is irradiated with an output laser beam from an acousto-optical device, the output laser beam including angularly polarized laser beams, each having an intensity based on the amplitude of an applied radio frequency drive signal. For example, the output laser beam used to irradiate particles in a flow stream may include two or more angularly polarized laser beams. The angularly polarized laser beams may be, 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, or even 25 or more. In embodiments, each of the angularly polarized laser beams has a different frequency, which is shifted from the frequency of the input laser beam by a predetermined radio frequency.

[0059] Additionally, each angularly deflected laser beam is spatially shifted relative to one another. Depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam, the angularly deflected laser beams may be spaced apart by 0.001 μm or more. The angularly deflected laser beams may be spaced apart by, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, or even 5000 μm or more. In some embodiments, the angularly deflected laser beams overlap with adjacent angularly deflected laser beams, for example, along the horizontal axis of the output laser beam. The overlap (e.g., beam spot overlap) between adjacent angularly deflected laser beams may be 0.001 μm or more. For example, an overlap of 0.005 μm or more, e.g., For example, the overlap may be 0.01 μm or more, for example, an overlap of 0.05 μm or more, for example, an overlap of 0.1 μm or more, for example, an overlap of 0.5 μm or more, for example, an overlap of 1 μm or more, for example, an overlap of 5 μm or more, for example, an overlap of 10 μm or more, or even an overlap of 100 μm or more.

[0060] When a particle passes through a portion of the excitation beam formed by the superposition of two beamlets, it is subjected to the superposition of their electric fields. The fluorescence emitted by the particle is frequency-encoded with a beat frequency corresponding to the difference between the optical frequencies of the incident beamlets. As an example, the frequency-encoded fluorescence emitted by a particle passing through the left horizontal edge of the excitation beam formed by the superposition of the first and second beamlets will have a beat frequency corresponding to the difference between the frequencies of the second and first beamlets, i.e., f 第1ビームレット -f 第2ビームレットshows the beat frequency of the RF beat frequency. In this way, the positions of particles passing through the excitation beam can be encoded by the RF beat frequency associated with the radiation emitted by these particles. In some embodiments, such encoding of particle positions can be used to normalize the intensity of the detected radiation emitted by these particles for variations in beam intensity, for example in the horizontal direction.

[0061] In some embodiments, the frequency-encoded fluorescence emitted by the particles is measured at the frequency of the local oscillator beam (f LO ) and the frequency of the radio-frequency-shifted beamlet. For example, frequency-encoded fluorescence data can be expressed as f LO -f RFシフトされたビームレット If the illumination of the flow stream includes a local oscillator beam that spans the width of the flow stream (e.g., the entire horizontal axis), the frequency-encoded fluorescence data will be generated at the frequency of the local oscillator beam (f LO ) and the frequency of each radio frequency shifted beamlet (f1, f2, f3, f4, f5, f6, etc.). In these embodiments, the frequency-encoded fluorescence data may include multiple beat frequencies, each corresponding to a position on the horizontal axis of the flow stream.

[0062] As discussed in more detail below, in one mode of operation, particles in a flow stream can be simultaneously illuminated with multiple excitation frequencies, each of which can be obtained, for example, by shifting the center frequency of a laser beam. More specifically, multiple sample locations can be simultaneously illuminated with a laser beam generated by mixing a reference laser beam (e.g., a local oscillator beam) with multiple radio frequency-shifted laser beams. As a result, each sample location is illuminated with the reference beam and one radio frequency-shifted beam to excite the fluorophore of interest (if present) at that location. In some embodiments, the reference local oscillator beam can be generated by radio frequency shifting of a light beam (e.g., a light beam from a laser such as a continuous wave laser). In these embodiments, each spatial location of a particle in the flow stream illuminated by the light is "tagged" with a different beat frequency corresponding to the difference between the frequency of the reference beam and the frequency of one of the radio frequency-shifted beams. In these cases, the fluorescent radiation emitted by the fluorophore spatially encodes the beat frequency.

[0063] In certain cases, the flow stream is illuminated with multiple beams of frequency-shifted light, and cells in the flow stream are imaged by fluorescence imaging using radio frequency tagged emission (FIRE) to generate a frequency-encoded image. Such techniques are described in Diebold et al., Nature Photonics Vol. 7(10), 806-810 (2013), and U.S. Patent Nos. 9,423,353, 9,784,661, 10,006,852, 2017 / 0133857, and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0064] In embodiments, frequency-encoded fluorescence data is generated by detecting light from particles in the flow stream. The fluorescence data can be generated from one or more fluorescence detectors (e.g., one or more detection channels). The number of fluorescence detectors (detection channels) can be, for example, two or more, three or more, four or more, five or more, six or more, or even eight or more. In some embodiments, the frequency-encoded fluorescence data includes data components obtained (or derived) from light from other detectors, such as detected light absorption or light scattering. In some cases, one or more data components of the frequency-encoded fluorescence data from a sample are generated from light absorption detected from the sample, for example, by a bright-field photodetector. As described in more detail below, a phase correction component can include a signal from a bright-field detector. In certain embodiments, the signal is used to generate phase-corrected spatial data that accounts for interference phase adjustments to spatial data calculated from the frequency-encoded fluorescence data. In other cases, one or more data components of the frequency-encoded fluorescence data from a sample are generated from light scattering detected from the sample, for example, by a side scatter detector, a forward scatter detector, or a combination of side scatter detectors and forward scatter detectors.

[0065] In embodiments, the method includes calculating spatial data from the frequency-encoded fluorescence data. The spatial data according to embodiments of the present disclosure is phase-corrected by performing a transform of the frequency-encoded fluorescence data with a phase correction component. In some embodiments, the spatial data includes a horizontal size of the particle, a vertical size of the particle, a ratio of particle sizes along two different dimensions, or a ratio of particle component sizes (e.g., the ratio of the horizontal dimension of the cytoplasm to the horizontal dimension of the nucleus of a cell).

[0066] In some cases, the phase correction component is used to generate modified transform coefficients (i.e., transform coefficients for converting frequency-encoded data to spatial data, as described below). For example, the phase correction component may include two or more modified transform coefficients. The number of modified transform coefficients may be, for example, three or more, such as four or more, or even five or more. The spatial data is calculated by performing a Fourier transform (as described below), and in certain embodiments, the phase correction component includes modified transform coefficients, which Fourier transform generates only real mathematical components (i.e., does not generate imaginary mathematical components).

[0067] In certain embodiments, the phase correction component includes a first phase adjustment and a second phase adjustment. Each phase adjustment can be the result of a different phase source in the frequency-encoded fluorescence data. In one example, the first phase adjustment includes an output signal from a light detection system. For example, the first phase adjustment can include an output signal from a bright-field photodetector. In some embodiments, the first phase adjustment is calculated by multiplying the output signal from the bright-field photodetector by a predetermined constant signal to generate a phase adjustment value, and then calculating the arctangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum over all bins in the discrete Fourier transform of the frequency-encoded fluorescence data.

[0068] In certain embodiments, the first phase adjustment is an interferometric phase adjustment. In these embodiments, the phase adjustment includes a phase shift induced by a light source for illuminating the sample in the flow stream. For example, the light source can be a component of an optical beam generator configured to generate at least a first frequency-shifted optical beam and a second frequency-shifted optical beam. In certain cases, the optical beam generator includes a laser (e.g., a continuous wave laser) and an acousto-optic deflector (e.g., coupled to a direct digital synthesizer RF comb generator). In some embodiments, the interferometric phase adjustment includes a phase shift resulting from vibrations between components of the optical beam generator.

[0069] In some embodiments, the second phase adjustment is based on the fluorescence lifetime of a fluorophore in the sample. In these embodiments, the second phase adjustment may be calculated by acquiring signals from all fluorescence detectors, determining the phases present in the signals, and calculating the second phase adjustment from the fluorescence lifetimes of the fluorophores. Depending on the particular type of fluorophore and the number of fluorophores present, one or more fluorescence lifetimes may be calculated, and the number of calculable fluorescence lifetimes may be, for example, two or more, such as three or more, such as four or more, or even five or more. In some embodiments, each fluorescence lifetime is calculated at the peak emission wavelength of the fluorophore. In these embodiments, each fluorophore lifetime may be determined and calculated using signals from different detection channels.

[0070] In embodiments, the method also includes calculating phase-corrected spatial data by performing a transform of the frequency-encoded fluorescence data with the determined phase correction component. In some embodiments, the method includes calculating spatial data from the frequency-encoded fluorescence data from the object. In some cases, calculating the spatial data of the object includes performing a transform of the frequency-encoded fluorescence data. In one example, the spatial data is calculated by performing a Fourier transform (FT) of the frequency-encoded fluorescence data. In another example, the spatial data is calculated by performing a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data. In yet another example, the spatial data is calculated by performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data. In yet another example, the spatial data is calculated with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data. By considering the phase correction component before performing the transform of the frequency-encoded data to spatial data, the output of the transform has reduced computational complexity compared to performing a transform of raw frequency data to spatial data (i.e., not initially considering the phase). In some embodiments, the method includes performing a transform of the frequency-encoded fluorescence data without performing imaginary mathematical calculations (i.e., performing only real mathematical calculations of the transform) to generate spatial data from the frequency-encoded fluorescence data.

[0071] In some embodiments, the method includes generating an image of particles in the flow stream from the frequency-encoded fluorescence. In some embodiments, the image of the particles can be generated from the frequency-encoded fluorescence in conjunction with detected light absorption, detected light scattering, or a combination thereof. In certain cases, the image of the particles is generated from frequency-encoded fluorescence alone. In other cases, the image of the object is generated from frequency-encoded fluorescence and detected light absorption from the sample, e.g., by a bright-field photodetector. In still other cases, the image of the particles is generated from frequency-encoded fluorescence and detected light scattering from the sample, e.g., by a side scatter detector, a forward scatter detector, or a side scatter detector and a forward scatter detector. In yet other cases, the image of the particles is generated from frequency-encoded fluorescence and a combination of detected light absorption, detected light scattering, and detected light emission.

[0072] One or more images of the particle can be generated from the frequency-encoded fluorescence data. In some embodiments, a single image of the particle is generated from the frequency-encoded fluorescence data. In other embodiments, two or more images of the particle are generated from the frequency-encoded fluorescence data. The number of images can be, for example, three or more, for example, four or more, for example, five or more, ten or more, or a combination thereof.

[0073] In summary, the disclosed methods also include sorting particles. In embodiments, particles may be sorted based on frequency-encoded fluorescence data, calculated spatial data, generated images, one or more particle characteristics (e.g., size, center of mass, eccentricity) determined from the calculated spatial data or generated images, or some combination thereof. The term "sorting" is used in its conventional sense to refer to separating components of a sample (e.g., droplets containing cells, droplets containing non-cellular particles such as biopolymers) and, in some cases, sending the separated components to one or more sample collection containers. For example, the method may include sorting two or more components of a sample. The number of sorted components of a sample may be, for example, three or more, for example, four or more, for example, five or more, for example, ten or more, for example, fifteen or more, or even twenty-five or more. In some cases, a first sample component collection location includes a sample collection container, and a second sample component collection location includes a waste collection container.

[0074] For sorting particles from a sample in a flow stream, the method includes data (e.g., fluorescence data) acquisition, analysis (determining frequency-encoded fluorescence data, determining phase correction components, and calculating a transformation of the frequency-encoded data to phase-corrected spatial data), and recording (e.g., using a computer whose multiple data channels record data from each detector (e.g., scatter detector, bright-field detector, or fluorescence detector)). In these embodiments, analysis includes sorting and counting particles such that each particle is represented as a set of digitized parameter values. The system (described below) can be configured to trigger on selected parameters to distinguish particles of interest from background and noise.

[0075] Specific subpopulations of interest (e.g., single cells) can then be further analyzed by "gating" based on the frequency-encoded fluorescence data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible subpopulation separation. This procedure can be performed by plotting image moments or one or more determined properties (e.g., size, centroid, eccentricity). In other embodiments, the method involves plotting forward light scatter (FSC) versus side (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot plot. In still other embodiments, the method involves plotting one or more determined properties (e.g., size, centroid, eccentricity) against one or more of forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC). In yet other embodiments, the method involves gating a population of particles for forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC), followed by gating based on one or more properties (e.g., size, centroid, eccentricity) determined based on images of the objects. In yet other embodiments, the method includes gating a population of particles based on one or more properties (e.g., size, center of gravity, eccentricity) determined based on an image of the object, and subsequently gating the population of particles for forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC).

[0076] Next, a subpopulation of objects (i.e., single cells within the gate) is selected, and particles not within the gate are excluded. Optionally, a gate can be selected by drawing a line around the desired subpopulation using the cursor on the computer screen. Then, only those particles within the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. Optionally, the above analysis can be configured to obtain a count of particles of interest in the sample.

[0077] In some embodiments, a method for sorting components of a sample includes sorting particles (e.g., cells in a biological sample) with a particle sorting module having a polarizer. Such particle sorting modules are described, for example, in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, cells of the sample are sorted with a sorting determination module having a plurality of sorting determination units. Such sorting determination modules are described, for example, in U.S. Provisional Patent Application No. 62 / 803,264, filed February 8, 2019, the disclosure of which is incorporated herein by reference.

[0078] FIG. 1 is a flowchart for generating frequency-encoded fluorescence data and calculating phase-corrected spatial data from the frequency-encoded fluorescence data, according to certain embodiments. In step 101, light (absorbed, scattered, or emitted light) from particles (e.g., cells) in a flow stream is detected. In step 102, frequency-encoded fluorescence data (e.g., frequency data from each spatial position along a horizontal axis) of the particles is generated. In step 103, phase correction components, such as an interference phase component and a fluorescence lifetime phase component, are determined. In step 104, the phase-corrected spatial data is calculated by performing a transform (e.g., a discrete Fourier transform) of the frequency-encoded fluorescence data. The spatial data can be used to generate an image in step 105. An image mask can be used to generate an image mask in the following step 106. Two or more images can be used to calculate colocalization of one or more features of a cell (e.g., organelles) in step 107. Alternatively, colocalization can be calculated using the image mask in step 108.

[0079] FIG. 2 illustrates a comparison of an image of particles generated using phase-corrected spatial data with an image in which the spatial data is not phase-corrected, according to certain embodiments. As shown in FIG. 2A, frequency-encoded fluorescence data is converted to spatial data, for example, by using a fast Fourier transform (FFT) without phase correction. The image generated has low resolution because the particles are obscured by background noise. In FIG. 2B, the frequency-encoded fluorescence data is phase-corrected with a phase adjustment component along with the FFT to generate phase-corrected spatial data. The phase-corrected spatial data provides a resolution-enhanced particle image that is not obscured by background noise.

[0080] System for characterizing particles in a sample As previously mentioned, aspects of the present disclosure include a system for characterizing particles in a sample (e.g., cells in a biological sample). The system, according to certain embodiments, includes a light source, a light detection system, and a processor, where the light source is configured to illuminate a sample having particles in a flowstream, the light detection system having a photodetector, and the processor has a memory operably coupled to the processor, the memory having stored therein instructions that, when executed by the processor, cause the processor to generate frequency-encoded fluorescence data from particles in the flowstream and calculate phase-corrected spatial data for the particles by performing a transform of the frequency-encoded fluorescence data with a phase-correction component.

[0081] The subject system includes a light source configured to illuminate a sample in a flow stream. In embodiments, the light source can be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, beads, non-cellular particles, etc.), the light source can be configured to emit light at wavelengths ranging from 200 nm to 1500 nm. The wavelength range can be, for example, from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, or even from 400 nm to 800 nm. For example, the light source can include a broadband light source that emits light at wavelengths between 200 nm and 900 nm. In other cases, the light source can include a narrowband light source that emits light at wavelengths between 200 nm and 900 nm. For example, the light source can be a narrowband LED (1 nm to 25 nm) that emits light at wavelengths ranging from 200 nm to 900 nm.

[0082] In some embodiments, the light source is a laser. Lasers of interest may include pulsed or continuous-wave lasers. For example, the laser may be a gas laser, a dye laser, a metal vapor laser, a solid-state laser, a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a laser operating at double or triple the frequency of any of the above lasers. The gas laser may be, for example, a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO2 laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. The dye laser may be, for example, a stilbene, coumarin, or rhodamine laser. The metal vapor laser may be, for example, 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. The solid-state laser may be, for example, a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, or a combination thereof.

[0083] 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, a xenon arc lamp, a light emitting diode, a broadband continuous spectrum LED, a superluminescent light emitting diode, a semiconductor light emitting diode, a broad spectrum LED white light source, a multi-LED integrated light source, etc. In some cases, the non-laser light source is a stabilized fiber-coupled broadband light source, a white light source, other light sources, or any combination thereof.

[0084] In certain embodiments, the light source is an optical beam generator configured to generate two or more frequency-shifted optical beams. In some cases, the optical beam generator includes a laser, a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optical device to generate two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous wave laser. For example, the laser in the optical beam generator may be a gas laser, a dye laser, a metal vapor laser, or a solid-state laser. The gas laser may be, for example, a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO2 laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. The dye laser may be, for example, a stilbene, coumarin, or rhodamine laser. The metal vapor laser may be, for example, 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. The solid-state laser may be, for example, a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, or a combination thereof.

[0085] The acousto-optical device may be any suitable acousto-optical protocol configured to frequency-shift laser light using applied acoustic waves. In certain embodiments, the acousto-optical device is an acousto-optical deflector. The acousto-optical device in this system is configured to generate an angularly deflected laser beam from light from a laser and an applied radio frequency drive signal. The radio frequency drive signal may be applied to the acousto-optical device by a suitable radio frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0086] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optical device to generate a desired number of angularly deflected laser beams in the output laser beam, for example, configured to apply 3 or more radio frequency drive signals, for example 4 or more radio frequency drive signals, for example 5 or more radio frequency drive signals, for example 6 or more radio frequency drive signals, for example 7 or more radio frequency drive signals, for example 8 or more radio frequency drive signals, for example 9 or more radio frequency drive signals, for example 10 or more radio frequency drive signals, for example 15 or more radio frequency drive signals, for example 25 or more radio frequency drive signals, for example 50 or more radio frequency drive signals, and may be configured to apply 100 or more radio frequency drive signals.

[0087] In some cases, to generate an angularly deflected laser beam intensity profile in the output laser beam, the controller is configured to apply a radio frequency drive signal having an amplitude that varies, for example, from about 0.001 V to about 500 V, and the amplitude may vary, for example, from about 0.005 V to about 400 V, for example, from about 0.01 V to about 300 V, for example, from about 0.05 V to about 200 V, for example, from about 0.1 V to about 100 V, for example, from about 0.5 V to about 75 V, for example, from about 1 V to 50 V, for example, from about 2 V to 40 V, for example, from 3 V to about 30 V, or may vary from about 5 V to about 25 V. In some embodiments, each applied radio frequency drive signal has a frequency of from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, or may be from about 5 MHz to about 50 MHz.

[0088] In certain embodiments, the controller includes a processor having a memory operatively coupled to the processor, the memory having instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam, the output laser beam being an angularly deflected laser beam having a desired intensity profile. For example, the memory may include instructions for generating two or more angularly deflected laser beams having 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., twenty-five or more, e.g., fifty or more. The memory may include instructions for generating one hundred or more angularly deflected laser beams having the same intensity. In other embodiments, the memory may include instructions for generating two or more angularly deflected laser beams having different intensities, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more. The memory may include instructions for generating one hundred or more angularly deflected laser beams having different intensities.

[0089] In certain embodiments, the controller includes a processor having a memory operatively coupled to the processor, the memory having stored therein instructions that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the center to the edges of the output laser beam along a horizontal axis. In these examples, the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis can be in a range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edges of the output laser beam. The range can be, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, or for example, about 8% to about 55%. Along the horizontal axis, the intensity of the angularly deflected laser beam at the center of the output laser beam may be about 10% to about 50% of the intensity of the angularly deflected laser beam at the ends of the output laser beam. In other embodiments, the controller includes a processor having a memory operatively coupled to the processor and instructions stored in the memory that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the ends of the output laser beam to the center along the horizontal axis. In these examples, along the horizontal axis, the intensity of the angularly deflected laser beam at the ends of the output laser beam may be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam. The range may be, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%. Along the horizontal axis, the intensity of the angularly deflected laser beam at the edges of the output laser beam may be about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam.In yet another embodiment, the controller comprises a processor having a memory operatively coupled to the processor and instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having a Gaussian intensity profile along a horizontal axis. In yet another embodiment, the controller comprises a processor having a memory operatively coupled to the processor and instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0090] In embodiments, the subject optical beam generator can be configured to generate spatially separated angularly deflected laser beams in the output laser beam. Depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam, the angularly deflected laser beams can be spaced apart by 0.001 μm or more. The spacing can be, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, or even 5000 μm or more. In some embodiments, the system is configured to generate overlapping angularly deflected laser beams in the output laser beam, such as adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., overlap between beam spots) can be 0.001 μm or more. The overlap may be, for example, an overlap of 0.005 μm or more, for example, an overlap of 0.01 μm or more, for example, an overlap of 0.05 μm or more, for example, an overlap of 0.1 μm or more, for example, an overlap of 0.5 μm or more, for example, an overlap of 1 μm or more, for example, an overlap of 5 μm or more, for example, an overlap of 10 μm or more, or may be an overlap of 100 μm or more.

[0091] In certain cases, the optical beam generator configured to generate two or more frequency-shifted optical beams includes a laser excitation module described in U.S. Patent Nos. 9,423,353, 9,784,661, 10,006,852, U.S. Patent Application Publication No. 2017 / 0133857, and U.S. Patent Application Publication No. 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0092] In embodiments, the system includes a light detection system having one or more light detectors for detecting and measuring light from the sample. The light detectors may be configured to measure light absorption (e.g., brightfield light data), light scattering (e.g., forward or side-scattered light data), light emission (e.g., fluorescence data), or a combination thereof from the sample. The light detectors may include, but are not limited to, optical sensors. The optical sensors may be, for example, active pixel sensors (APS), avalanche photodiodes, imagers, charge-coupled devices (CDDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photodiodes, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or photodiodes, or combinations thereof, or other optical detectors. In certain embodiments, the light from the sample is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) imager, or an N-type metal-oxide-semiconductor (NMOS) imager.

[0093] In some embodiments, the subject light detection system comprises a plurality of photodetectors. In some cases, the light detection system comprises a plurality of solid-state detectors, such as photodiodes. In certain cases, the light detection system is a light detector array, such as an array of photodiodes. In these embodiments, the light detector array can comprise four or more light detectors. The light detector array can comprise, for example, 10 or more light detectors, for example, 25 or more light detectors, for example, 50 or more light detectors, for example, 100 or more light detectors, for example, 250 or more light detectors, for example, 500 or more light detectors, for example, 750 or more light detectors, or even 1000 or more light detectors. For example, the detector can be a photodiode array having four or more photodiodes. The photodiode array can comprise, for example, 10 or more photodiodes, for example, 25 or more photodiodes, for example, 50 or more photodiodes, for example, 100 or more photodiodes, for example, 250 or more photodiodes, for example, 500 or more photodiodes, for example, 750 or more photodiodes, or even 1000 or more photodiodes.

[0094] The photodetectors may be arranged in any geometric configuration as desired, including, but not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The photodetectors in the photodetector array may be oriented at angles relative to one another (as viewed in the XZ plane) ranging from 10° to 180°, for example, from 15° to 170°, for example, from 20° to 160°, for example, from 25° to 150°, for example, from 30° to 120°, or even from 45° to 90°. The photodetector array may be of any suitable shape, including rectilinear shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curvilinear shapes such as circles and ellipses, and irregular shapes such as a parabolic base joined to a flat top. In a particular embodiment, the photodetector array has a rectangular active surface.

[0095] Each photodetector (e.g., photodiode) in the array may have an active surface with a width in the range of 5 μm to 250 μm and a length in the range of 5 μm to 250 μm. The width may be, for example, 10 μm to 225 μm, such as 15 μm to 200 μm, for example 20 μm to 175 μm, such as 25 μm to 150 μm, for example 30 μm to 125 μm, or may be 50 μm to 100 μm. The length may be, for example, 10 μm to 225 μm, such as 15 μm to 200 μm, for example 20 μm to 175 μm, such as 25 μm to 150 μm, for example 30 μm to 125 μm, or may be 50 μm to 100 μm, and the surface area of ​​each photodetector (e.g., photodiode) in the array may be less than 25 μm. 2 to 10,000 μm 2 The range is, for example, 50 μm 2 to 9000 μm 2 , e.g., 75 μm 2 to 8000 μm 2 , e.g., 100 μm 2 to 7000 μm 2 , e.g., 150 μm 2 to 6000 μm 2 may be 200 μm 2 to 5000 μm 2 may be.

[0096] The size of the photodetector array can vary depending on the amount and intensity of light, the number of photodetectors, and the desired sensitivity, and can have a length ranging from 0.01 mm to 100 mm. The length range can be, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, or 5 mm to 25 mm. The width of the photodetector array can also vary from 0.01 mm to 100 mm. The width range can be, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, or 5 mm to 25 mm. Thus, the active surface of the photodetector array can be 0.1 mm or less. 2 From 10,000 mm 2 The range can be, for example, 0.5 mm 2 to 5000mm 2 , e.g. 1 mm 2 to 1000mm 2 , e.g. 5mm 2 from 500mm 2 may be 10 mm 2 from 100mm 2 may be.

[0097] The subject photodetectors may be configured to measure the collected light at one or more wavelengths, such as 2 or more different wavelengths, such as 5 or more different wavelengths, for example 10 or more different wavelengths, such as 25 or more different wavelengths, for example 50 or more different wavelengths, for example 100 or more different wavelengths, such as 200 or more different wavelengths, for example 300 or more different wavelengths. The subject photodetectors may be configured to measure light emitted by the sample in the flowstream at 400 or more different wavelengths.

[0098] In some embodiments, the photodetector is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the photodetector is configured to collect a spectrum of light over a range of wavelengths. For example, the system may include one or more detectors configured to collect a spectrum of light over one or more wavelength ranges from 200 nm to 1000 nm. In still other embodiments, the detector is configured to measure light from the sample in the flowstream at one or more specific wavelengths. For example, the system 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.

[0099] The light detection system may be configured to measure light continuously or at discrete intervals. In some cases, the target light detector is configured to measure collected light continuously. In other cases, the light detection system may be configured to measure light at discrete intervals, such as every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, 1000 milliseconds, or other intervals.

[0100] In embodiments, the system is configured to generate frequency-encoded fluorescence data by illuminating a sample having particles in a flow stream. In some embodiments, the light source includes a light generator component that generates multiple angularly polarized laser beams, each having an intensity based on the amplitude of an applied radio frequency drive signal (e.g., from a direct digital synthesizer coupled to an acousto-optical device). For example, the system may include a light generator component that generates two or more angularly polarized laser beams. The number of angularly polarized laser beams may be, 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, or even 25 or more. In embodiments, each angularly polarized laser beam has a different frequency. These frequencies are shifted by a predetermined radio frequency from the frequency of the input laser beam.

[0101] The system, according to certain embodiments, is configured to generate angularly deflected laser beams that are also spatially shifted relative to one another. Depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam, the system can be configured to generate angularly deflected laser beams that are spaced apart by 0.001 μm or more. The angularly deflected laser beams can be spaced apart by, for example, 0. The angularly deflected laser beams may be spaced apart by 0.005 μm or more, for example 0.01 μm or more, for example 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm or more, for example 1 μm or more, for example 5 μm or more, for example 10 μm or more, for example 100 μm or more, for example 500 μm or more, for example 1000 μm or more, or even 5000 μm or more. In some embodiments, the angularly deflected laser beams overlap with adjacent angularly deflected laser beams, for example along the horizontal axis of the output laser beam. The overlap (e.g., beam spot overlap) between adjacent angularly deflected laser beams may be 0.001 μm or more. For example, the overlap may be 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, or even 100 μm or more.

[0102] In some embodiments, the system includes a processor having a memory operatively coupled to the processor and instructions stored in the memory that, when executed by the processor, cause the processor to generate frequency-encoded fluorescence data by calculating the difference between the optical frequencies of overlapping beamlets of light incident on the flow stream. In one example, the system includes a processor having a memory operatively coupled to the processor and instructions stored in the memory that, when executed by the processor, cause the processor to calculate a beat frequency at each position on the horizontal axis of the flow stream. In these embodiments, the frequency-encoded fluorescence emitted by the particles is measured by calculating the frequency (f LO ) and the frequency of the radio-frequency-shifted beamlet. For example, frequency-encoded fluorescence data can be expressed as f LO -f RFシフトされたビームレットIf the illumination of the flow stream includes a local oscillator beam that spans the width of the flow stream (e.g., the entire horizontal axis), the frequency-encoded fluorescence data will be generated at the frequency of the local oscillator beam (f LO ) and the frequency of each radio frequency shifted beamlet (f1, f2, f3, f4, f5, f6, etc.). In these embodiments, the frequency-encoded fluorescence data may include multiple beat frequencies, each corresponding to a position on the horizontal axis of the flow stream.

[0103] In embodiments, the system is configured to generate frequency-encoded fluorescence data from light detected from particles in the flow stream. The fluorescence data can be generated from one or more fluorescence detectors (e.g., one or more detection channels). The number of fluorescence detectors (detection channels) can be, for example, two or more, three or more, four or more, five or more, six or more, or even eight or more. In some embodiments, the frequency-encoded fluorescence data includes data components obtained (or derived) from light from other detectors that detect light absorption or light scattering, for example. In some cases, the system is configured to generate one or more data components of the frequency-encoded fluorescence data from light absorption detected from the sample, for example, by a bright-field photodetector. For example, the system can be configured to generate a phase-corrected component from a signal from a bright-field detector. In certain embodiments, the system is configured to generate phase-corrected spatial data that accounts for an interferometric phase adjustment to the spatial data calculated from the frequency-encoded fluorescence data. In other cases, the system is configured to generate one or more data components of the frequency-encoded fluorescence data from light scattering detected from the sample, for example, by a side scatter detector, a forward scatter detector, or a combination of side scatter detectors and forward scatter detectors.

[0104] In embodiments, the system includes a processor having a memory operatively coupled to the processor, the memory having stored therein instructions that, when executed by the processor, cause the processor to calculate spatial data from the frequency-encoded fluorescence data. The spatial data according to embodiments of the present disclosure is phase-corrected by the system performing a transform of the frequency-encoded fluorescence data with a phase-correction component. In some embodiments, the spatial data includes a horizontal size of the particle, a vertical size of the particle, a ratio of particle sizes along two different dimensions, or a size ratio of particle components (e.g., the ratio of the horizontal dimension of the cytoplasm to the horizontal dimension of the nucleus of a cell).

[0105] In some embodiments, the system is configured to calculate modified transform coefficients for converting the frequency-encoded fluorescence data into phase-corrected spatial data. For example, the phase correction component can include two or more modified transform coefficients. The number of modified transform coefficients can be, for example, three or more, such as four or more, or even five or more. The spatial data is calculated by performing a Fourier transform, and the phase correction component includes the modified transform coefficients, which produces only real mathematical components (i.e., does not produce imaginary mathematical components).

[0106] In some cases, the system is configured to determine a phase correction component including a first phase adjustment and a second phase adjustment. Each phase adjustment can be the result of a different phase source in the frequency-encoded fluorescence data. In one example, the first phase adjustment includes an output signal from a light detection system. For example, the first phase adjustment can include an output signal from a bright-field photodetector. In some embodiments, the system includes a processor, the processor having a memory operably coupled to the processor, the memory having instructions stored therein that, when executed by the processor, cause the processor to calculate the first phase adjustment as follows: multiply the output signal from the bright-field photodetector by a predetermined constant signal to generate a phase adjustment value; and calculate the arctangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum over all bins in a discrete Fourier transform of the frequency-encoded fluorescence data.

[0107] In other cases, the system is configured to calculate the second phase adjustment based on the fluorescence lifetimes of fluorophores in the sample. In these cases, the system is configured to calculate the second phase adjustment by acquiring signals from all fluorescence detectors, determining the phases present in the signals, and calculating the second phase adjustment from the fluorescence lifetimes of the fluorophores. The system can be configured to calculate the fluorescence lifetimes using different detection channels. The number of detection channels can be, for example, two or more, for example, three or more, for example, four or more, or even five or more.

[0108] In embodiments, the system includes a processor having a memory operatively coupled to the processor, the memory having instructions stored therein that, when executed by the processor, cause the processor to calculate phase-corrected spatial data of the particles by performing a transform of the frequency-encoded fluorescence data with a phase correction component. In some embodiments, to calculate the phase-corrected spatial data, the system is configured to perform a Fourier transform of the frequency-encoded fluorescence data with the phase correction component to generate phase-corrected spatial data of the particles. In other embodiments, the system is configured to perform a discrete Fourier transform of the frequency-encoded fluorescence data with the phase correction component to generate phase-corrected spatial data of the particles. In yet other embodiments, the system is configured to perform a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data with the phase correction component. In yet other embodiments, the system is configured to perform a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data with the phase correction component. In yet another embodiment, the system is configured to calculate the phase-corrected spatial data with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data.

[0109] In some embodiments, the system is configured to consider a phase correction component before performing a transformation of the frequency-encoded data to spatial data, such that the output of the transformation has reduced computational complexity compared to performing a transformation of the raw frequency data to spatial data (i.e., not first considering the phase). In some embodiments, the system is configured to perform a transformation of the frequency-encoded fluorescence data without performing imaginary mathematical calculations (i.e., performing only real mathematical calculations of the transformation) to generate spatial data from the frequency-encoded fluorescence data.

[0110] The system can be configured to generate one or more images of particles in the flow stream from the frequency-encoded fluorescence. In some embodiments, the image of the particle can be generated from frequency-encoded fluorescence and detected light absorption, detected light scattering, or a combination thereof. In certain cases, the image of the particle is generated from frequency-encoded fluorescence alone. In other cases, the image of the object is generated from frequency-encoded fluorescence and detected light absorption from the sample, for example, with a bright-field photodetector. In still other cases, the image of the particle is generated from frequency-encoded fluorescence and detected light scattering from the sample, for example, with a side scatter detector, a forward scatter detector, or a combination of side scatter detector and forward scatter detector. In yet other cases, the image of the particle is generated from frequency-encoded fluorescence and a combination of detected light absorption, detected light scattering, and detected light emission.

[0111] The system according to some embodiments 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 storing instructions for executing the method steps. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage, an input / output controller, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors available now or in the future. The processor executes an operating system, which interfaces with firmware and hardware as is well known, and assists the processor in coordinating and executing the functions of various computer programs written in various programming languages, such as Java, Perl, C++, or other high-level or low-level languages ​​known in the art, or combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, communication control, and related services in accordance with known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0112] System memory can be any of a variety of known or future memory storage devices. Examples include 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 can be any of a variety of known or future devices, including compact disk drives, tape drives, removable hard disk drives, or diskette drives. Such types of memory storage devices typically read from and / or write to program storage media (not shown), such as compact disks, magnetic tapes, removable hard disks, or floppy diskettes, respectively. Any of these program storage media, or others now in use or that may be developed in the future, may be considered computer program products. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0113] In some embodiments, a computer program product is described that includes a computer-usable medium having stored thereon control logic (a computer software program including program code). The control logic, when executed by a processor or computer, instructs the processor to perform the functions described herein. In other embodiments, some functions are performed primarily in hardware, for example, using hardware state machines. Implementation of a hardware state machine to perform the functions described herein will be apparent to one skilled in the relevant art.

[0114] The memory may be any suitable device capable of storing and retrieving data by the processor, such as a magnetic, optical, or solid-state storage device (e.g., a magnetic or optical disk or tape, RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor that is suitably programmed from a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communications channel or may be pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium, using any of the devices connected to the memory. For example, a magnetic or optical disk may carry the programming and be readable by a disk writer / reader. The system of the present invention also includes programming, e.g., algorithms used to implement the methods described above, in the form of a computer program product. The programming of the present invention may be recorded on a computer-readable medium, e.g., any medium that can be read and directly 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.

[0115] The processor may also utilize a communication channel to communicate with a remote user, meaning that the user does not have direct contact with the system, but instead relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, e.g., a mobile phone (i.e., smartphone).

[0116] In some embodiments, a system according to the present disclosure may be configured with a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or other devices. The communications interface may be configured for wired or wireless communications, such as, but not limited to, radio frequency (RF) communications (e.g., radio frequency identification (RFID), Zigbee® communications protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications, such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0117] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment), configured for similar complementary data communication.

[0118] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable communication between the system and other devices, such as a computer terminal and / or network, a communication-enabled mobile phone, a personal digital assistant, or any other communication device that a user may use in combination.

[0119] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing Internet Protocol (IP) via a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0120] In one embodiment, the system is configured to wirelessly communicate with a server device via a communications interface using common standards such as 802.11 or Bluetooth® RF protocols or IrDA infrared protocols. 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, appliance, etc. 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 touch screen.

[0121] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the system, e.g., the optional data storage unit, with a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0122] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When a display device 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 a variety of known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. The functional elements of the computer may 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 provide information generated by the processing module to a remote user, for example, using the Internet, telephone, or satellite network, in accordance with known techniques. Presentation of data by the output manager may be performed in accordance with a variety of known techniques. As some examples, the data may include SQL, HTML, XML documents, email or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the system can be any type of known or future-developed computer platform, but they are typically computers of a class commonly referred to as servers. However, they may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cable or other communication system, including networks or otherwise connected wireless systems. They may be co-located or physically separate. Various operating systems may be utilized on any of the computer platforms, possibly depending on the type and / or model of computer platform selected.Suitable operating systems include Windows 10, Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, Ubuntu, Zorin OS, and the like.

[0123] In certain embodiments, the system includes one or more optical conditioning components, for example, to condition light illuminating the sample (e.g., light from a laser) or light collected from the sample (e.g., fluorescence). For example, the optical conditioning can be increasing the size of the light, the focus of the light, or collimating the light. In some cases, the optical conditioning is an expansion protocol to increase the size of the light (e.g., beam spot), for example, by increasing the size by 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, or even by 75% or more. In other embodiments, the optical conditioning includes focusing the light to decrease the size of the light, for example, by decreasing the size of the beam spot, for example, by 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, or even by 75% or more. In certain embodiments, the optical conditioning includes collimating the light. The term "collimate" is used in its conventional sense to refer to optically adjusting the collinearity of light propagation or reducing the divergence of light from a common propagation axis. In some cases, collimating involves narrowing the spatial cross-section of a light beam (eg, reducing the beam profile of a laser).

[0124] In some embodiments, the optical adjustment component is a focusing lens having a magnification of 0.1 to 0.95, and the focusing lens may have a magnification of, for example, 0.2 to 0.9, for example, 0.3 to 0.85, for example, 0.35 to 0.8, for example, 0.5 to 0.75, for example, 0.55 to 0.7, or for example, 0.6. For example, the focusing lens may be an achromatic, non-magnifying lens with a magnification of about 0.6. The focal length of the focusing lens may vary from 5 mm to 20 mm. The focal length range may be, for example, 6 mm to 19 mm, for example, 7 mm to 18 mm, for example, 8 mm to 17 mm, for example, 9 mm to 16 mm, or even 10 mm to 15 mm. In certain embodiments, the focusing lens has a focal length of about 13 mm.

[0125] In other embodiments, the optical adjustment component is a collimator. The collimator can be any convenient collimation protocol, such as one or more mirrors or curved lenses, or a combination thereof. For example, in certain cases, the collimator is a single collimating lens. In other cases, the collimator is a collimating mirror. In still other cases, the collimator includes two lenses. In still other cases, the collimator includes a mirror and a lens. When the collimator includes one or more lenses, the focal length of the collimating lens can vary from 5 mm to 40 mm. The focal length range can be, for example, from 6 mm to 37.5 mm, for example, from 7 mm to 35 mm, for example, from 8 mm to 32.5 mm, for example, from 9 mm to 30 mm, for example, from 10 mm to 27.5 mm, for example, from 12.5 mm to 25 mm, or even from 15 mm to 20 mm.

[0126] In some embodiments, the system includes a flow cell nozzle having a nozzle opening configured to direct a flow stream through the flow cell nozzle. The flow cell nozzle has an opening that delivers a fluid sample to a sample testing region. In some embodiments, the flow cell nozzle includes a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating in a flat surface having a nozzle opening perpendicular to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) can vary from 1 mm to 15 mm. The length can vary from, for example, 1.5 mm to 12.5 mm, for example, 2 mm to 10 mm, for example, 3 mm to 9 mm, or even 4 mm to 8 mm. The length of the distal frustoconical portion (measured along the longitudinal axis) can also vary from 1 mm to 10 mm. The length can vary from, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, or even 4 mm to 7 mm. The diameter of the flow cell nozzle chamber can vary in some embodiments from 1 mm to 10 mm, such as from 2 mm to 9 mm, such as from 3 mm to 8 mm, or from 4 mm to 7 mm.

[0127] In certain cases, the nozzle chamber does not include a cylindrical portion, and the entire nozzle chamber of the flow cell is frustoconical. In these embodiments, the length of the frustoconical nozzle chamber (measured along a longitudinal axis perpendicular to the nozzle opening) may be in the range of 1 mm to 15 mm. The range may be, for example, 1.5 mm to 12.5 mm, such as 2 mm to 10 mm, such as 3 mm to 9 mm, or 4 mm to 8 mm. The diameter of the proximal end portion of the frustoconical nozzle chamber may be in the range of 1 mm to 10 mm. The range may be, for example, 2 mm to 9 mm, such as 3 mm to 8 mm, or 4 mm to 7 mm.

[0128] In embodiments, the sample flow stream is discharged from an opening at the distal end of a flow cell nozzle. Depending on the desired characteristics of the flow stream, the flow cell nozzle opening can be any suitable shape. Target cross-sectional shapes include, but are not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal, curvilinear cross-sectional shapes, such as circular and elliptical, and irregular shapes, such as a parabolic base joined to a flat top. In certain embodiments, a target flow cell nozzle has a circular opening. The size of the nozzle opening can vary from 1 μm to 20,000 μm in some embodiments. The size may vary from, for example, 2 μm to 17,500 μm, for example, 5 μm to 15,000 μm, for example, 10 μm to 12,500 μm, for example, 15 μm to 10,000 μm, for example, 25 μm to 7,500 μm, for example, 50 μm to 5,000 μm, for example, 75 μm to 1,000 μm, for example, 100 μm to 750 μm, or 150 μm to 500 μm. In a specific embodiment, the nozzle opening is 100 μm.

[0129] In some embodiments, the flow cell nozzle includes a sample inlet configured to provide a sample to the flow cell nozzle. In embodiments, the sample injection system is configured to provide a suitable flow of sample to the flow cell nozzle chamber. Depending on the desired characteristics of the flow stream, the flow rate of the sample delivered by the sample inlet to the flow cell nozzle chamber can be 1 μL / sec or greater. The flow rate can be, for example, 2 μL / sec or greater, such as 3 μL / sec or greater, such as 5 μL / sec or greater, such as 10 μL / sec or greater, such as 15 μL / sec or greater, such as 25 μL / sec or greater, such as 50 μL / sec or greater, such as 100 μL / sec or greater, such as 150 μL / sec or greater, such as 200 μL / sec or greater, such as 250 μL / sec or greater, such as 300 μL / sec or greater, such as 350 μL / sec or greater, such as 400 μL / sec or greater, such as 450 μL / sec or greater, or even 500 μL / sec or greater. For example, the sample flow rate may be in the range of 1 μL / sec to about 500 μL / sec, such as 2 μL / sec to about 450 μL / sec, such as 3 μL / sec to about 400 μL / sec, such as 4 μL / sec to about 350 μL / sec, such as 5 μL / sec to about 300 μL / sec, such as 6 μL / sec to about 250 μL / sec, such as 7 μL / sec to about 200 μL / sec, such as 8 μL / sec to about 150 μL / sec, such as 9 μL / sec to about 125 μL / sec, or may be 10 μL / sec to about 100 μL / sec.

[0130] The sample inlet may be an opening disposed in the wall of the nozzle chamber or a conduit disposed at the proximal end of the nozzle chamber. When the sample inlet is an opening disposed in the wall of the nozzle chamber, the opening of the sample inlet may have any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal, curvilinear shapes, such as circular and elliptical, and irregular shapes, such as a parabolic base joined to a flat top. In certain embodiments, the sample inlet has a circular opening. The size of the opening of the sample inlet may vary depending on the shape, and in certain cases, the opening may range from 0.1 mm to 5.0 mm, for example, from 0.2 to 3.0 mm, for example, from 0.5 mm to 2.5 mm, for example, from 0.75 mm to 2.25 mm, for example, from 1 mm to 2 mm, from 1.25 mm to 1.75 mm, or for example, 1.5 mm.

[0131] In certain cases, the sample inlet is a conduit located at the proximal end of the flow cell nozzle chamber. For example, the sample inlet can be a conduit positioned so that its opening matches the opening of the flow cell nozzle. When the sample inlet is a conduit positioned so that its opening matches the opening of the flow cell nozzle, the cross-sectional shape of the sample inlet tube can be any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal, curvilinear shapes, such as circular and elliptical, and irregular shapes, such as a parabolic base joined to a flat top. The opening of the conduit can vary in shape, and in certain cases, has an opening ranging from 0.1 mm to 5.0 mm, for example, from 0.2 to 3.0 mm, for example, from 0.5 mm to 2.5 mm, for example, from 0.75 mm to 2.25 mm, for example, from 1 mm to 2 mm, or from 1.25 mm to 1.75 mm, for example, 1.5 mm. The shape of the tip of the sample inlet may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the opening of the sample inlet may include a beveled tip having a bevel angle ranging from 1° to 10°. The bevel angle may be, for example, from 2° to 9°, for example, from 3° to 8°, for example, from 4° to 7°, or may be 5°.

[0132] In some embodiments, the flow cell nozzle also includes a sheath fluid inlet configured to provide sheath fluid to the flow cell nozzle. In embodiments, the sheath fluid injection system is configured to provide a flow of sheath fluid to the flow cell nozzle chamber, e.g., to generate a laminar flow stream of sheath fluid in conjunction with the sample and surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of the sheath fluid delivered to the flow cell nozzle chamber can be 25 μL / sec or greater. The flow rate can be, for example, 50 μL / sec or greater, e.g., 75 μL / sec or greater, e.g., 100 μL / sec or greater, e.g., 250 μL / sec or greater, e.g., 500 μL / sec or greater, e.g., 750 μL / sec or greater, e.g., 1000 μL / sec or greater, or even 2500 μL / sec or greater. For example, the flow rate of the sheath fluid can range from 1 μL / sec to about 500 μL / sec. The range may be, for example, 2 μL / sec to about 450 μL / sec, for example, 3 μL / sec to about 400 μL / sec, for example, 4 μL / sec to about 350 μL / sec, for example, 5 μL / sec to about 300 μL / sec, for example, 6 μL / sec to about 250 μL / sec, for example, 7 μL / sec to about 200 μL / sec, for example, 8 μL / sec to about 150 μL / sec, for example, 9 μL / sec to about 125 μL / sec, or may be 10 μL / sec to about 100 μL / sec.

[0133] In some embodiments, the sheath fluid inlet is an opening disposed in the wall of the nozzle chamber. The opening of the sheath fluid inlet can have any suitable shape. Suitable cross-sectional shapes include, but are not limited to, rectilinear shapes, such as squares, rectangles, trapezoids, triangles, and hexagons; curvilinear shapes, such as circles and ellipses; and irregular shapes, such as a parabolic base joined to a flat top. The size of the opening of the sample inlet can vary depending on the shape, and in certain cases, the opening may range from 0.1 mm to 5.0 mm, for example, from 0.2 to 3.0 mm, for example, from 0.5 mm to 2.5 mm, for example, from 0.75 mm to 2.25 mm, for example, from 1 mm to 2 mm, from 1.25 mm to 1.75 mm, or for example, 1.5 mm.

[0134] The system, in certain cases, includes a sample testing region in fluid communication with the flow cell nozzle opening. In these cases, the sample flow stream is discharged from an opening at the distal end of the flow cell nozzle, and particles in the flow stream can be illuminated by a light source in the sample testing region. The size of the testing region can vary depending on characteristics of the flow nozzle, such as the size of the nozzle opening and the size of the sample injection port. In embodiments, the testing region can have a width of 0.01 mm or greater. The width can be, for example, 0.05 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 2 mm or greater, such as 3 mm or greater, such as 5 mm or greater, or even 10 mm or greater. The length of the testing region can also vary. In some cases, the length can be 0.01 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 1.5 mm or greater, such as 2 mm or greater, such as 3 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 15 mm or greater, such as 20 mm or greater, such as 25 mm or greater, or even 50 mm or greater.

[0135] The inspection region may be configured to facilitate illumination of a planar cross-section of the ejected flow stream, or may be configured to facilitate illumination of a diffuse field of a predetermined length (e.g., illumination by a diffuse laser or lamp). In some embodiments, the inspection region includes a transparent window that facilitates illumination of a predetermined length of the ejected flow stream. The length may be, for example, 1 mm or more, for example, 2 mm or more, for example, 3 mm or more, for example, 4 mm or more, for example, 5 mm or more, or even 10 mm or more. Depending on the light source used to illuminate the ejected flow stream (as described below), the inspection region may be configured to transmit light in the range of 100 nm to 1500 nm. The range may be, for example, 150 nm to 1400 nm, for example, 200 nm to 1300 nm, for example, 250 nm to 1200 nm, for example, 300 nm to 1100 nm, for example, 350 nm to 1000 nm, for example, 400 nm to 900 nm, or 500 nm to 800 nm. Thus, the inspection region can be formed from any transparent material that transmits the desired range of wavelengths, including, but not limited to, optical glass, borosilicate glass, Pyrex glass, UV quartz, IR quartz, sapphire, and plastics, such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics, such as PETG (glycol-modified polyethylene terephthalate), and other polymeric plastic materials, including polyesters.Polyesters of interest include poly(ethylene terephthalate) (PET), bottle-grade PET (a copolymer made based on monoethylene glycol, terephthalic acid, and other comonomers, e.g., isophthalic acid, cyclohexene dimethanol), poly(alkylene terephthalates), such as poly(butylene terephthalate) (PBT), and poly(hexamethylene terephthalate); poly(alkylene adipates), such as poly(ethylene adipate), poly(1,4-butylene adipate), and poly(hexamethylene adipate); poly(ethylene Poly(alkylene suberates) such as poly(suberic acid); poly(alkylene sebacates) such as poly(ethylene sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalates) such as poly(ethylene isophthalate); poly(alkylene 2,6-naphthalenedicarboxylates) such as poly(ethylene 2,6-naphthalenedicarboxylate); poly(alkylenesulfonyl-4,4'-dibenzoates) such as poly(ethylenesulfonyl-4,4'-dibenzoate); poly(p-phenyleneethylene dicarboxylates) poly(p-phenylene alkylene dicarboxylate) such as poly(trans-1,4-cyclohexanediyl alkylene dicarboxylate) such as poly(trans-1,4-cyclohexanediylethylene dicarboxylate) poly(1,4-cyclohexane-dimethylene alkylene dicarboxylate) such as poly(1,4-cyclohexane-dimethylene ethylene dicarboxylate) poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate) poly(octane-1,4-dimethylene alkylene dicarboxylate); lactic acid polymers and copolymers such as (S)-polylactide, (R,S)-polylactide, poly(tetramethylglycolide), and poly(lactide-co-glycolide); and polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, and 3,3',5,5'-tetramethylbisphenol A; polyamides such as poly(p-phenylene terephthalamide); polyethylene terephthalate (e.g., Mylar).TM The material may include, but is not limited to, polyesters such as polyethylene terephthalate (PET). In some embodiments, the system includes a cuvette disposed in the sample inspection region. In embodiments, the cuvette is capable of transmitting light in the range of 100 nm to 1500 nm. The range may be, for example, 150 nm to 1400 nm, for example, 200 nm to 1300 nm, for example, 250 nm to 1200 nm, for example, 300 nm to 1100 nm, for example, 350 nm to 1000 nm, for example, 400 nm to 900 nm, or 500 nm to 800 nm.

[0136] In some embodiments, the system includes a particle sorting component for sorting particles (e.g., cells) of a sample. In certain cases, the particle sorting component is a particle sorting module, such as those described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, and U.S. Provisional Patent Application No. 62 / 752,793, filed October 30, 2018, the disclosures of which are incorporated herein by reference. In certain embodiments, the particle sorting component includes one or more droplet deflectors, such as those described in U.S. Patent Application Publication No. 2018 / 0095022, filed June 14, 2017, the disclosures of which are incorporated herein by reference.

[0137] In some embodiments, the system is a flow cytometry system. Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd Edition, Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. January; 49(pt 1):17-28; Linden et al., Semin Thromb Hemost. October 2004; 30(5):502-11; Alison et al., J Pathol. December 2010; 222(4):335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference.In certain cases, the flow cytometry systems of interest are a FACSCanto™ II flow cytometer (BD Biosciences), a BD Accuri™ flow cytometer, a FACSCelesta™ flow cytometer (BD Biosciences), a FACSLyric™ flow cytometer (BD Biosciences), a FACSVerse™ flow cytometer (BD Biosciences), a FACSymphony™ flow cytometer (BD Biosciences), an LSRFortessa™ flow cytometer (BD Biosciences), an LSRFortess™ X-20 flow cytometer (BD Biosciences), and a FACSCalibur™ cell sorter (BD Biosciences), a FACSCount™ cell sorter (BD Biosciences), a FACSLyric™ cell sorter (BD Biosciences), and a Via™ cell sorter (BD Biosciences), an Influx™ cell sorter (BD Biosciences), a Jazz™ cell sorter (BD Biosciences). Bi. Cell sorters include the Aria™ cell sorter (BD Biosciences), the FACSMelody™ cell sorter (BD Biosciences), and the like.

[0138] In some embodiments, the particle sorting system may be configured as described in, for example, U.S. Pat. Nos. 10,006,852, 9,952,076, 9,933,341, 9,784,661, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140, ​​8,140, ​​8,250, 8,300, 8,400, 8,500, 8,600, 8,750, 8,800, 8,900, 8,140, ​​8,140, ​​8,250, 8,300, 8,400, 8,500, 8,600, 8,750, 8,80 ...300, 8,400, 8,500, 8,600, 8,750, 8,800, 8,300, 8,400, 8,500, 8,600, 8,750, 8,800, 8,300, 8, Nos. 5,627,040, 5,620,842, and 5,602,039, the disclosures of which are incorporated herein by reference.

[0139] In certain cases, the system is a flow cytometry system configured to image and characterize particles in a flow stream by FIRE (Fluorescence Imaging using Radiofrequency-tagged Emission) techniques, such as those described in Diebold et al., Nature Photonics Vol. 7(10), 806-810 (2013), or in U.S. Patent Nos. 9,423,353, 9,784,661, 10,006,852, U.S. Patent Application Publication No. 2017 / 0133857, and U.S. Patent Application Publication No. 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0140] integrated circuit device Aspects of the present disclosure also include integrated circuit devices programmed to generate frequency-encoded fluorescence data from particles in a flow stream and calculate phase-corrected spatial data for the particles by performing a transformation of the frequency-encoded fluorescence data with a phase-correction component. In some embodiments, the integrated circuit device is programmed to sort the particles, for example, into a sample collection container or a waste collection container. Targeted integrated circuit devices may, in certain cases, include field programmable gate arrays (FPGAs), application-specific integrated circuits (ASICs), or complex programmable logic devices (CPLDs).

[0141] In embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data. The fluorescence data may be generated from one or more fluorescence detectors (e.g., one or more detection channels), such as two or more, such as three or more, such as four or more, such as five or more, such as six or more fluorescence photodetectors, or even eight or more fluorescence photodetectors (e.g., eight or more detection channels). In some embodiments, the frequency-encoded fluorescence data includes data components obtained (or derived) from light from other detectors (e.g., detected light absorption or detected light scattering). In some cases, the system is configured to generate one or more data components of the frequency-encoded fluorescence data from light absorption detected from the sample, for example, by a bright-field photodetector. For example, the system may be configured to generate a phase-corrected component from a signal from a bright-field detector. In certain embodiments, the system is configured to generate phase-corrected spatial data that accounts for an interferometric phase adjustment to the spatial data calculated from the frequency-encoded fluorescence data. In other cases, the system is configured to generate one or more data components of the frequency-encoded fluorescence data from light scatter detected from the sample, for example, by a side scatter detector, a forward scatter detector, or a combination of side scatter detector and forward scatter detector.

[0142] In embodiments, the integrated circuit device is programmed to calculate spatial data from the frequency-encoded fluorescence data. The spatial data according to embodiments of the present disclosure is phase-corrected by performing a transform of the frequency-encoded fluorescence data with a phase-correction component. In some embodiments, the spatial data includes a horizontal particle size, a vertical particle size, a ratio of particle sizes along two different dimensions, or a ratio of particle component sizes (e.g., the ratio of the horizontal dimension of a cell's cytoplasm to the horizontal dimension of its nucleus).

[0143] In some embodiments, the integrated circuit device is programmed to calculate modified transform coefficients for converting the frequency-encoded fluorescence data into phase-corrected spatial data. For example, the phase correction component can include two or more modified transform coefficients. The number of modified transform coefficients can be, for example, three or more, such as four or more, or even five or more. When the spatial data is calculated by performing a Fourier transform, the phase correction component can include modified transform coefficients, which generate only real mathematical components (i.e., do not generate imaginary mathematical components).

[0144] In some cases, the integrated circuit device is programmed to determine a phase correction component including a first phase adjustment and a second phase adjustment. Each phase adjustment can be the result of a different phase source in the frequency-encoded fluorescence data. In one example, the first phase adjustment includes an output signal from a light detection system. For example, the integrated circuit device can be programmed to determine the first phase adjustment based on an output signal from a bright-field photodetector. In some embodiments, the integrated circuit device is programmed to calculate the first phase adjustment by multiplying the output signal from the bright-field photodetector by a predetermined constant signal to generate a phase adjustment value and calculating the arctangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum over all bins in the discrete Fourier transform of the frequency-encoded fluorescence data.

[0145] In other cases, the integrated circuit device is programmed to calculate the second phase adjustment based on the fluorescence lifetime of the fluorophores in the sample. In these cases, the integrated circuit device is programmed to calculate the second phase adjustment by acquiring signals from all of the fluorescence detectors, determining the phase present in the signals, and calculating the second phase adjustment from the fluorescence lifetime of the fluorophores. The integrated circuit device can be programmed to calculate the fluorescence lifetime using different detection channels. The number of detection channels can be, for example, two or more, for example, three or more, for example, four or more, or even five or more.

[0146] In embodiments, the integrated circuit device is programmed to calculate phase-corrected spatial data of the particle by performing a transform of the frequency-encoded fluorescence data with a phase correction component. In some embodiments, to calculate the phase-corrected spatial data, the integrated circuit device is configured to perform a Fourier transform of the frequency-encoded fluorescence data with the phase correction component to generate phase-corrected spatial data of the particle. In other embodiments, the integrated circuit device is programmed to perform a discrete Fourier transform of the frequency-encoded fluorescence data with the phase correction component to generate phase-corrected spatial data of the particle. In yet other embodiments, the integrated circuit device is programmed to perform a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data with the phase correction component. In yet other embodiments, the integrated circuit device is programmed to perform a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data with the phase correction component. In yet another embodiment, the integrated circuit device is programmed to heterodyne and demultiplex the frequency-encoded fluorescence data and to calculate phase-corrected spatial data with a digital lock-in amplifier.

[0147] In certain embodiments, the integrated circuit device is programmed to make sorting decisions (as described above) based on the frequency-encoded fluorescence data, the calculated spatial data, the generated images, one or more determined characteristics of the particles (e.g., size, center of mass, eccentricity) determined from the calculated spatial data or the generated images, or some combination thereof. In these embodiments, the analysis involves classifying and counting particles such that each particle is represented as a set of digitized parameter values. The integrated circuit device can be programmed to trigger sorting components based on selected parameters to distinguish particles of interest from background and noise.

[0148] kit Aspects of the present disclosure further include kits, where the kits include one or more of the integrated circuit devices described herein. In some embodiments, the kits may further include programming for the system, for example, in the form of a computer-readable medium (e.g., a flash drive, USB storage, compact disc, DVD, Blu-ray disc, etc.) or instructions for downloading the programming from an Internet web protocol or cloud server. The kits may further include instructions for practicing the methods. These instructions may be included in the kit in a variety of forms, one or more of which may be included within the kit. One form in which these instructions may be provided is printed information on a suitable medium or substrate (e.g., one or more sheets of paper on which information is printed), such as in a package insert within the kit's packaging. Another form in which these instructions may be provided is a computer-readable medium having the information recorded thereon, such as a diskette, compact disc (CD), portable flash drive, etc. Another form in which these instructions may be provided is a website address that can be used to access the information at a remote site via the Internet.

[0149] usefulness The present systems, methods, and computer systems find use in a variety of applications where it is desirable to analyze and sort particle components in a sample (e.g., a biological sample) in a fluid medium. In some embodiments, the systems and methods described herein find use in flow cytometric characterization of biological samples labeled with fluorescent tags. In other embodiments, the systems and methods find use in spectroscopic analysis of emitted light. Additionally, the present systems and methods find use in increasing the signal obtainable from light collected from a sample (e.g., a sample in a flow stream). Embodiments of the present disclosure find use where it is desirable to provide a flow cytometer that exhibits improved cell sorting accuracy, enhanced particle collection, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting.

[0150] Embodiments of the present disclosure are also useful in applications where cells prepared from a biological sample may be desired for research, clinical testing, or therapeutic use. In some embodiments, the present methods and devices may facilitate the preparation of collected individual cells from a target fluid or tissue biological sample. For example, the present methods and systems may facilitate the collection of cells from a fluid or tissue sample to be used as a research or diagnostic specimen for diseases such as cancer. Similarly, the present methods and systems may facilitate the collection of cells from a fluid or tissue sample to be used in therapeutics. The presently disclosed methods and devices enable the separation and collection of cells from biological samples (e.g., organs, tissues, tissue slices, bodily fluids) with high efficiency and low cost compared to conventional flow cytometry systems.

[0151] Notwithstanding the appended claims, the present disclosure is also defined by the following appendix. 1. generating frequency-encoded fluorescence data from a sample of particles in a flow stream; calculating phase-corrected spatial data of the particle by performing a transform of the frequency-encoded fluorescence data with a phase-correction component; A method for providing the above. 2. Spatial data is calculated by performing a Fourier transform of the frequency-encoded fluorescence data with a phase-corrected component; The method described in Appendix 1. 3. The method of claim 2, wherein the spatial data is calculated by performing a discrete Fourier transform of the frequency-encoded fluorescence data with a phase-corrected component. 4. The method of claim 2, wherein the spatial data is calculated by performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data with a phase-corrected component. 5. The method of claim 1, wherein the spatial data is calculated with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data. 6. The method of any one of claims 1-5, wherein the phase correction component comprises modified conversion coefficients for converting the frequency-encoded fluorescence data into phase-corrected spatial data. 7. The method of any one of claims 1-6, wherein generating frequency-encoded fluorescence data from the particles comprises detecting light from the particles in the sample with a light detection system. 8. The method of claim 7, wherein the light detected from the particle comprises absorbed light, scattered light, emitted light, or a combination thereof. 9. The method of claim 8, wherein light absorption is detected with a bright-field photodetector. 10. The method of claim 8 or 9, wherein the emitted light is detected with a fluorescence detector. 11. The method of any one of claims 1-10, wherein the phase correction component comprises a first phase adjustment and a second phase adjustment. 12. The method of claim 11, wherein the first phase adjustment comprises an output signal from the optical detection system. 13. The method of claim 12, wherein the first phase adjustment comprises an output signal from a bright field photodetector. 14. The method of claim 13, further comprising calculating a first phase adjustment by multiplying the output signal from the bright field photodetector and a predetermined constant signal to generate a phase adjustment value, and calculating the arctangent of the phase adjustment value to generate the first phase adjustment. 15. The method of claim 14, wherein the phase adjustment value is the sum over all bins in the discrete Fourier transform of the frequency-encoded fluorescence data. 16. The method of any one of claims 11-15, wherein the first phase adjustment is an interferometric phase adjustment. 17. The method of claim 16, wherein the interferometric phase adjustment comprises a phase shift from a light source configured to illuminate the sample in the flow stream. 18. The method of claim 17, wherein the light source comprises a light beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. 19. The method of claim 18, wherein the optical beam generator comprises an acousto-optic deflector. 20. The method of claim 18 or 19, wherein the optical beam generator comprises a direct digital synthesizer (DDS) RF comb generator. 21. The method of any one of claims 18-20, wherein the optical beam generator component is configured to generate a frequency-shifted local oscillator beam. 22. The method of any one of claims 17-21, wherein the light source comprises a laser. 23. The method of claim 22, wherein the laser is a continuous wave laser. 24. The method of any one of claims 17-23, wherein the interferometric phase adjustment comprises a phase shift resulting from vibration between components of the light source. 25. The method of any one of claims 11-24, further comprising calculating a second phase adjustment based on the fluorescence lifetime of a fluorophore in the sample. 26. The method of any one of claims 1-25, wherein the phase-corrected spatial data of the particles is calculated from the frequency-encoded fluorescence data by an integrated circuit device. 27. The method of claim 26, wherein the integrated circuit device is a field programmable gate array (FPGA). 28. The method of claim 26, wherein the integrated circuit device is an application specific integrated circuit (ASIC). 29. The method of claim 26, wherein the integrated circuit device is a complex programmable logic device (CPLD). 30. The method of any one of claims 1-29, further comprising illuminating the flow stream with a light source. 31. The method of claim 30, wherein the flow stream is illuminated by a light source at a wavelength between 200 nm and 800 nm. 32. The method of claim 30 or 31, comprising illuminating the flow stream with a first frequency-shifted light beam and a second frequency-shifted light beam. 33. The method of claim 32, wherein the first frequency-shifted optical beam comprises a local oscillator (LO) beam and the second frequency-shifted optical beam comprises a radio frequency comb beam. 34. Applying a radio frequency drive signal to an acousto-optic device; illuminating the acousto-optical device with a laser to generate a first frequency-shifted light beam and a second frequency-shifted light beam; 34. The method of claim 32 or 33, further comprising: 35. The method of claim 34, wherein the laser is a continuous wave laser. 36. The method of any one of claims 1-35, further comprising generating an image of the particle from the phase-corrected spatial data. 37. The method of claim 36, further comprising generating an image mask of the particles. 38. The method of any one of claims 1-37, further comprising sorting the particles. 39. A light source configured to illuminate a sample comprising particles in a flow stream; an optical detection system; a processor; The processor comprises a memory operatively coupled to the processor, the memory having stored therein instructions that, when executed by the processor, generating frequency-encoded fluorescence data from particles in the flow stream; calculating phase-corrected spatial data of the particle by performing a transform of the frequency-encoded fluorescence data with a phase-correction component; the processor, the system. 40. Instructions are stored in a memory, and when executed by a processor, the instructions: performing a Fourier transform of the frequency-encoded fluorescence data with the phase-corrected component to generate phase-corrected spatial data of the particles; 40. The system of claim 39, wherein the processor: 41. A memory stores instructions that, when executed by a processor, performing a discrete Fourier transform of the frequency-encoded fluorescence data with the phase-corrected component to generate phase-corrected spatial data of the particles; 41. The system of claim 40, wherein the processor: 42. A memory stores instructions that, when executed by a processor, Performing a short-time Fourier transform (STFT) of frequency-encoded fluorescence data with a phase-correction component 41. The system of claim 40, wherein the processor: 43. A memory stores instructions that, when executed by a processor, Calculating phase-corrected spatial data with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data. 41. The system of claim 40, wherein the processor: 44. Instructions are stored in a memory, and when executed by a processor, the instructions: Transforming frequency-encoded fluorescence data into spatial data with a phase correction component having modified transformation coefficients 44. The system of any one of claims 39-43, wherein the system causes a processor to: 45. A system described in any one of notes 39-44, wherein the optical detection system includes an optical detector configured to detect one or more of light absorption, light scattering, and fluorescence. 46. ​​The system of claim 45, wherein the optical detection system comprises a bright-field optical detector. 47. The system of any one of claims 39-46, wherein the optical detection system comprises a fluorescence detector. 48. A memory stores instructions that, when executed by a processor, Calculating a phase correction component comprising a first phase adjustment and a second phase adjustment 48. The system of any one of claims 44-47, wherein the system causes a processor to: 49. A memory stores instructions that, when executed by a processor, calculating a first phase adjustment by multiplying the output signal from the bright field photodetector and a predetermined constant signal to generate a phase adjustment value, and calculating the arctangent of the phase adjustment value to generate a first phase adjustment; 49. The system of claim 48, wherein the processor executes: 50. The system of claim 49, wherein the phase adjustment value is the sum over all bins in the discrete Fourier transform of the frequency-encoded fluorescence data. 51. The system of any one of statements 48-50, wherein the first phase adjustment is an interferometric phase adjustment. 52. The system of claim 51, wherein the interferometric phase adjustment comprises a phase shift from the light source. 53. The system of any one of notes 39-52, wherein the light source comprises a light beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. 54. The system of claim 53, wherein the optical beam generator comprises an acousto-optic deflector. 55. The system of claim 53 or 54, wherein the optical beam generator comprises a direct digital synthesizer (DDS) RF comb generator. 56. The system of any one of notes 53-55, wherein the optical beam generator component is configured to generate a frequency-shifted local oscillator beam. 57. The system of any one of notes 39-56, wherein the light source comprises a laser. 58. The system of claim 57, wherein the laser is a continuous wave laser. 59. The system of any one of notes 51-58, wherein the interferometric phase adjustment comprises a phase shift resulting from vibration between components of the light source. 60. The system of any one of claims 39-59, wherein the memory stores instructions that, when executed by the processor, cause the processor to calculate a second phase adjustment based on the fluorescence lifetime of a fluorophore in the sample. 61. Generating frequency-encoded fluorescence data from sample particles in a flow stream; Calculate the phase-corrected spatial data of the particle by performing a transformation of the frequency-encoded fluorescence data with a phase-correction component 61. The system of any one of Clauses 39-60, comprising an integrated circuit component programmed to: 62. The system of claim 61, wherein the integrated circuit device is a field programmable gate array (FPGA). 63. The integrated circuit device is an application specific integrated circuit (ASIC), Stem. 64. The system of claim 61, wherein the integrated circuit device is a complex programmable logic device (CPLD). 65. The system of any one of claims 39-64, wherein the system is a flow cytometer. 66. The system of any one of notes 39-65, wherein the memory stores instructions that, when executed by the processor, cause the processor to generate an image of the particle from the phase-corrected spatial data. 67. A memory stores instructions that, when executed by a processor, Generating an image mask of particles 67. The system of claim 66, wherein the processor: 68. The system of any one of claims 39-67, further comprising a cell sorting component configured to sort cells in the sample based on the calculated phase-corrected spatial data. 69. The system of claim 68, wherein the cell sorting component comprises a droplet deflector. 70. Generating frequency-encoded fluorescence data from particles in a flow stream; Calculate the phase-corrected spatial data of the particle by performing a transformation of the frequency-encoded fluorescence data with a phase-correction component An integrated circuit that is programmed to 71. The integrated circuit of claim 70, programmed to perform a Fourier transform of the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle. 72. The integrated circuit of claim 71, programmed to perform a discrete Fourier transform of the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particles. 73. The integrated circuit of claim 71, programmed to perform a short-time Fourier transform of the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle. 74. The integrated circuit of claim 70, programmed to calculate phase-corrected spatial data with a digital lock-in amplifier to heterodyne and demultiplex frequency-encoded fluorescence data. 75. The integrated circuit of any one of clauses 70-74, programmed to convert frequency-encoded fluorescence data to spatial data with a phase correction component having modified conversion coefficients. 76. The integrated circuit of any one of clauses 70-75, programmed to calculate a phase correction component comprising a first phase adjustment and a second phase adjustment. 77. Multiplying the output signal from the bright field photodetector and a predetermined constant signal to generate a phase adjustment value; calculating the arctangent of the phase adjustment value to generate a first phase adjustment; 77. The integrated circuit of claim 76, programmed to calculate a first phase adjustment. 78. The integrated circuit of claim 77, wherein the phase adjustment value is the sum over all bins in a discrete Fourier transform of the frequency-encoded fluorescence data. 79. The integrated circuit of any one of clauses 76-78, wherein the first phase adjustment is an interferometric phase adjustment. 80. The integrated circuit of claim 79, wherein the interferometric phase adjustment comprises a phase shift from a light source configured to illuminate the sample in the flow stream. 81. The integrated circuit of claim 80, wherein the light source comprises a light beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. 82. The integrated circuit of claim 81, wherein the optical beam generator comprises an acousto-optic deflector. 83. The integrated circuit of claim 81 or 82, wherein the optical beam generator comprises a direct digital synthesizer (DDS) RF comb generator. 84. The integrated circuit of any one of statements 81-83, wherein the optical beam generator component is configured to generate a frequency-shifted local oscillator beam. 85. The integrated circuit of any one of statements 80-84, wherein the interferometric phase adjustment comprises a phase shift resulting from vibration between components of the light source. 86. The integrated circuit of any one of clauses 76-85, programmed to calculate a second phase adjustment based on the fluorescence lifetime of a fluorophore in the sample. 87. The integrated circuit of any one of notes 69-86, which is a field programmable gate array (FPGA). 88. The integrated circuit of any one of clauses 69-86, which is an application specific integrated circuit (ASIC). 89. The integrated circuit of any one of notes 69-86, which is a complex programmable logic device (CPLD). 90. The integrated circuit of any one of clauses 69-89, programmed to generate an image of a particle from the phase-corrected spatial data. 91. The integrated circuit of claim 90, programmed to generate an image mask of particles. 92. The integrated circuit of any one of clauses 69-91, programmed to generate a sorting decision based on the phase-corrected spatial data.

[0152] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, those skilled in the art will readily appreciate that, in light of the teachings of this invention, certain changes and modifications can be made to the present disclosure without departing from the spirit or scope of the appended claims.

[0153] Thus, the foregoing merely illustrates the principles of the present invention. Although not explicitly described or shown herein, it is understood that those skilled in the art will be able to devise various configurations that embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language described herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts provided by the inventors to further the art, and should not be construed as being limited to the examples and conditions specifically described. Moreover, all statements herein that describe principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. Furthermore, such equivalents include both currently known equivalents and equivalents developed in the future; i.e., it is intended that any element that performs the same function, regardless of structure, be developed. Furthermore, nothing disclosed herein is intended to be disclosed to the public, regardless of whether such disclosure is expressly recited in the claims.

[0154] Accordingly, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly defined as being invoked for a limitation in a claim only when the precise phrase "means for" or the precise phrase "step for" is recited at the beginning of such limitation in the claim; if such precise phrases are not used in the limitation in the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0155] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Patent Application No. 62 / 854,875, filed May 30, 2019, the disclosure of which is incorporated herein by reference.

Claims

1. 1. A system for measuring particles in a flow stream, comprising: an optical beam generator; an acousto-optical device configured to receive the generated light beam and form a frequency-shifted beam of light to illuminate the particles in the flow stream; a light detection system that measures the fluorescence of the illuminated particles; Processor and the processor comprising a memory operatively coupled to the processor, the memory having stored therein instructions that, when executed by the processor, generating frequency-encoded fluorescence data based at least in part on the fluorescence of the illuminated particles and the frequency of the frequency-shifted light; calculating phase-corrected spatial data of the particle by performing a transform of the frequency-encoded fluorescence data with a phase correction component; The system causes the processor to:

2. The memory stores instructions that, when executed by the processor, performing a Fourier transform of the frequency-encoded fluorescence data with the phase correction component to generate the phase-corrected spatial data of the particle. The system of claim 1 , further comprising:

3. The memory stores instructions that, when executed by the processor, performing a discrete Fourier transform of the frequency-encoded fluorescence data with the phase correction component to generate the phase-corrected spatial data of the particle. The system of claim 2 , further comprising:

4. The memory stores instructions that, when executed by the processor, performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data with the phase correction component; The system of claim 2 , further comprising:

5. The memory stores instructions that, when executed by the processor, computing the phase-corrected spatial data with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data. The system of claim 2 , further comprising:

6. The memory stores instructions that, when executed by the processor, Transforming the frequency-encoded fluorescence data into the spatial data with a phase correction component including a modified transformation coefficient. The system of claim 1 , further comprising:

7. The system of claim 1 , wherein the light detection system comprises a light detector configured to detect one or more of light absorption, light scattering, and fluorescence.

8. The system of claim 7 , wherein the light detection system comprises a bright field light detector.

9. The system of claim 1 , wherein the optical beam generator comprises a direct digital synthesizer (DDS) RF comb generator.

10. The memory stores instructions that, when executed by the processor, Calculating a phase correction component including a first phase adjustment and a second phase adjustment The system of claim 6 , further comprising:

11. The memory stores instructions that, when executed by the processor, multiplying the output signal from the bright field photodetector and a predetermined constant signal to generate a phase adjustment value; calculating the arctangent of the phase adjustment value to generate the first phase adjustment; calculating the first phase adjustment by The system of claim 10 , further comprising:

12. The system of claim 11 , wherein the phase adjustment value is a sum over all bins in a discrete Fourier transform of the frequency-encoded fluorescence data.

13. The system of claim 11 , wherein the first phase adjustment is an interferometric phase adjustment.

14. The memory stores instructions that, when executed by the processor, Calculating a second phase adjustment based on the fluorescence lifetime of the fluorophores in the sample. The system of claim 1 , further comprising:

15. generating the frequency-encoded fluorescence data from the particles of a sample in the flowstream; Calculating phase-corrected spatial data for the particle by performing a transform of the frequency-encoded fluorescence data with a phase correction component.

10. The system of claim 1, comprising an integrated circuit component programmed to:

16. The system of claim 1 , wherein the system is a flow cytometer.

17. The memory stores instructions that, when executed by the processor, generating an image of the particle from the phase-corrected spatial data; The system of claim 1 , further comprising:

18. The memory stores instructions that, when executed by the processor, generating an image mask of said particles; The system of claim 17 , further comprising:

19. 10. The system of claim 1, further comprising a cell sorting component configured to sort cells in a sample based on the calculated phase-corrected spatial data.

20. 20. The system of claim 19, wherein the cell sorting component comprises a droplet deflector.

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