Phase Correction of RF Multiplexed Signals

Abstract

Aspects of the present disclosure include methods for characterizing sample particles in a flow stream. Methods according to certain embodiments include generating frequency-encoded fluorescence data based on sample particles in the flow stream; and calculating phase-corrected spatial data of the particles by transforming the frequency-encoded fluorescence data using a phase correction component. In certain embodiments, the method includes generating an image of the particles in the flow stream based on the phase-corrected spatial data. A system having a processor is also described, the processor having a memory operably coupled to the processor, the memory having instructions stored thereon, which when executed by the processor causes the processor to calculate phase-corrected spatial data based on the frequency-encoded fluorescence data of the particles of the flow stream. An integrated circuit device (e.g., a field programmable gate array) having programming for practicing the subject method is also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is related to U.S. Provisional Patent Application Serial No. 62 / 854,875 filed on May 30, 2019; the disclosure of which is incorporated herein by reference.

[0003] introduction

[0004] Characterization of analytes in biofluids has become an integral part of medical diagnostics and the assessment of a patient's overall health. Detection of analytes in biofluids, such as human blood or blood-derived products, can provide results that can play a role in determining treatment options for patients with a variety of diseases.

[0005] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells of a blood sample or particles of interest in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample such as a blood sample and a sheath reservoir containing a sheath fluid. A flow cytometer transports particles (including cells) in a fluid sample to a flow cell as a cell stream, while also directing a sheath fluid to the flow cell. In order to characterize the composition of this flow stream, the flow stream is illuminated with light. Changes in the material in the flow stream (such as morphology or the presence of a fluorescent marker) can result in changes in the observed light, and these changes can be characterized and separated.

[0006] To characterize the composition of a flowing stream, light must be projected onto the flowing stream and collected. The light source in a flow cytometer can vary between broad spectrum lamps, light emitting diodes, and single wavelength lasers. The light source is aligned with the flowing stream, and the optical response from the illuminated particles is collected and quantified. Summary of the invention

[0007] Aspects of the present disclosure include methods for characterizing sample particles in a flow stream. Methods according to certain embodiments include generating frequency-encoded fluorescence data based on sample particles in the flow stream, and calculating phase-corrected spatial data of the particles by transforming the frequency-encoded fluorescence data using a phase correction component. In certain embodiments, the method includes generating an image of the particles in the flow stream based on the phase-corrected spatial data. A system is also described, the system having a processor having a memory operably coupled to the processor, the memory having instructions stored thereon, which when executed by the processor cause the processor to calculate phase-corrected spatial data based on the frequency-encoded fluorescence data of the particles of the flow stream. An integrated circuit device (e.g., a field programmable gate array) having programming for practicing the subject method is also provided.

[0008] In an embodiment, frequency-encoded fluorescence data from particles in a sample is generated based on light detected in an interrogation region of a flow stream. In some embodiments, the particles are cells. In an embodiment, a method includes detecting light emission (e.g., fluorescence) from a sample in a flow stream to generate frequency-encoded fluorescence data based on the particles. In some embodiments, the method also includes detecting light absorption, light scattering, or a combination thereof. In some embodiments, a particle having one or more fluorophores is irradiated with a plurality of frequency-shifted beams from a beam generator to generate frequency-encoded fluorescence. In one example, a plurality of positions on a flow stream (of a horizontal axis) are irradiated by a laser beam, the laser beam comprising a local oscillator beam and a plurality of radio frequency-shifted laser beams, such that different positions on the flow stream are irradiated by one of the local oscillator beam and the radio frequency-shifted beam. In some instances, the local oscillator is a frequency-shifted beam from a laser. In this example, each spatial position on a particle in the flow stream is characterized by a different beat frequency, which corresponds to the difference between the frequency of the local oscillator beam and the frequency of the radio frequency-shifted beam at the position. In some embodiments, the frequency-encoded data from the particle includes a beat frequency of spatial encoding on the horizontal axis of the particle in the flow stream.

[0009] In the implementation of the subject method, light from a sample in a flow stream is detected in an interrogation region and frequency-encoded data from particles in the sample is generated. In some embodiments, the particles detected in the interrogation 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 a sample in a flow stream. In some instances, spatial data for phase correction of one or more particles in a sample is generated based on the detected light absorption (e.g., bright field image data). In other instances, spatial data for phase correction of one or more particles in a sample is generated based on the detected light scattering (e.g., forward scattering image data, side scattering image data). In other instances, spatial data for phase correction of one or more particles in a sample is generated based on the detected fluorescence (e.g., image data of a fluorescent marker). In other instances, spatial data for phase correction of one or more particles in a sample is generated based on a combination of two or more of the detected light absorption, detected light scattering, and detected fluorescence.

[0010] In an embodiment, frequency-encoded fluorescence data from particles in a flow stream are transformed using a phase correction component to give spatial data of the particles. In an embodiment, the spatial data may include the horizontal dimension of the particle, the vertical dimension of the particle, the ratio of the particle size along two different dimensions, the ratio of the size of the particle composition (e.g., the ratio of the horizontal dimension of the nucleus to the horizontal dimension of the cytoplasm). In some embodiments, the frequency-encoded fluorescence data is transformed by Fourier transforming the frequency-encoded fluorescence data using a phase correction component. In some instances, the frequency-encoded fluorescence data is transformed by performing a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using a phase correction component. In other instances, the phase-corrected spatial data is calculated by performing a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using a phase correction component. In other instances, a digital lock-in amplifier is used to calculate the phase-corrected spatial data so that the frequency-encoded fluorescence data produces heterodyne and demultiplexed frequency-encoded fluorescence data.

[0011] In some embodiments, the method includes determining a phase correction component used to transform frequency-encoded fluorescence data into phase-corrected spatial data. In some instances, the phase correction component includes a correction transformation coefficient. In certain embodiments, the phase correction component includes a first phase adjustment and a second phase adjustment. In some instances, 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.

[0012] In some embodiments, the first phase adjustment is calculated by: multiplying the output signal from the bright field photodetector with a predetermined constant signal to produce a phase adjustment value; and calculating the inverse tangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum of all frequency 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 caused by a light source used to illuminate the sample in the flow stream. For example, the light source can be a beam generator component configured to generate at least a first frequency-shifted beam and a second frequency-shifted beam. The beam generator according to some examples 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 caused by vibrations between components of the 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 can be calculated by acquiring signals from all fluorescence detectors to determine the phase present in the signal, and calculating the second phase adjustment based on the fluorescence lifetime of the fluorophore.

[0013] The method according to certain embodiments also includes sorting one or more particles in the sample. In some embodiments, the particles are identified as single cells and sorted to a first sample composition collection location. In other embodiments, the particles are identified as cell aggregates and sorted to a second sample composition collection location. In some instances, the first sample composition collection location includes a sample collection container, and the second sample composition collection location includes a waste collection container.

[0014] Aspects of the present disclosure include systems for characterizing sample particles (e.g., cells in a biological sample) in a flow stream. Systems according to certain embodiments include a light source configured to illuminate a sample having particles in the flow stream, a light detection system having a photodetector, and a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following operations: generate frequency-encoded fluorescence data from particles in the flow stream; and calculate phase-corrected spatial data of the particles by transforming the frequency-encoded fluorescence data using a phase correction component.

[0015] In an embodiment, the system is configured to generate frequency-encoded fluorescence data based on particles in a sample irradiated with a light source. In some embodiments, the light source includes a beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. The beam generator according to certain instances 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 subject system includes a light detection system configured to detect one or more of light absorption, light scattering, and light emission (e.g., fluorescence) from a sample in a flow stream. In some instances, the light detection system includes a photodetector (e.g., a bright field photodetector) for detecting light absorption. In other instances, the light detection system includes a photodetector (e.g., a forward scattering detector, a side scattering detector) for detecting light scattering. In other instances, the light detection system includes a photodetector for detecting fluorescence. In other instances, the light detection system includes a combination of two or more of the following: a light absorption detector, a light scattering detector, and a light detector for emission (e.g., fluorescence).

[0016] In an embodiment, the subject system includes a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate phase-corrected spatial data of a particle by transforming frequency-encoded fluorescence data using a phase-corrected component. In an embodiment, the spatial data may include a horizontal size dimension of the particle, a vertical size dimension of the particle, a ratio of particle sizes along two different dimensions, a ratio of sizes of particle components (e.g., a ratio of a horizontal dimension of a cell nucleus to a horizontal dimension of a cytoplasm). In some embodiments, to calculate the phase-corrected spatial data, the system is configured to Fourier transform the frequency-encoded fluorescence data using the phase-corrected component to generate the phase-corrected spatial data of the particle. In other embodiments, the system is configured to perform a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using the phase-corrected component to generate the phase-corrected spatial data of the particle. In other embodiments, the system is configured to perform a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using the phase-corrected component. In other embodiments, the system is configured to calculate the phase-corrected spatial data using a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data.

[0017] In some embodiments, a system includes a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine a phase correction component for transforming frequency-encoded fluorescence data into phase-corrected spatial data. In some instances, the phase correction component includes a correction transformation 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 a light detection system. For example, the first phase adjustment may include an output signal from a bright field photodetector.

[0018] In some embodiments, a system includes a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a first phase adjustment by: multiplying an output signal from a bright field photodetector by a predetermined constant signal to produce a phase adjustment value; and calculating an inverse tangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum of all frequency 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, a system includes a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a second phase adjustment based on a fluorescence lifetime of a fluorophore in the sample. In these embodiments, the second phase adjustment may be calculated by the subject system by: acquiring signals from all fluorescence detectors to determine the phase present in the signals, and calculating the second phase adjustment based on the fluorescence lifetime of the fluorophore to calculate the second phase adjustment.

[0019] In some instances, the system of interest is configured to sort particles of a sample (e.g., a biological sample) in a flow stream. In some embodiments, the system further comprises a particle sorting component having a sample fluid delivery subsystem and a sheath fluid delivery subsystem in fluid communication with an inlet of the particle sorting component and one or more sample collection containers for receiving sorted particles from the flow stream.

[0020] Aspects of the present disclosure also include an integrated circuit device programmed to perform the following operations: generate frequency-encoded fluorescence data from particles in a flow stream; calculate phase-corrected spatial data of the particles by transforming the frequency-encoded fluorescence data using 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. In some instances, the integrated circuit device of interest may include a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), or a complex programmable logic device (CPLD).

[0021] The integrated circuit device according to certain embodiments is programmed to generate frequency-encoded fluorescence data based on particles in the flow stream. In some embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data based on 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 based on data signals from a light scattering detector (e.g., forward scattering image data, side scattering image data). In other embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data based on data signals from a light emission detector (e.g., fluorescent marker image data). In other instances, the integrated circuit device is programmed to generate frequency-encoded fluorescence data based on a combination of two or more of detected light absorption, detected light scattering, and detected fluorescence.

[0022] In embodiments, the subject integrated circuit device is programmed to compute phase-corrected spatial data for a particle by transforming frequency-encoded fluorescence data using a phase-correction component. In some instances, the integrated circuit device is programmed to Fourier transform the frequency-encoded fluorescence data using the phase-correction component to generate phase-corrected spatial data for the particle. In other instances, the integrated circuit device is programmed to discrete Fourier transform the frequency-encoded fluorescence data using the phase-correction component to generate phase-corrected spatial data for the particle. In other instances, the integrated circuit device is programmed to short-time Fourier transform the frequency-encoded fluorescence data using the phase-correction component to generate phase-corrected spatial data for the particle. In other instances, the integrated circuit device is programmed to compute the phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0023] In some embodiments, the integrated circuit device is programmed to determine a phase correction component used to transform the frequency encoded fluorescence data into phase corrected spatial data. In some instances, the phase correction component includes a correction transformation 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 instances, 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.

[0024] 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 produce a phase adjustment value; and calculating the inverse tangent of the phase adjustment value to produce the first phase adjustment value. In these embodiments, the phase adjustment value is the sum of all frequency points in the discrete Fourier transform of the frequency encoded fluorescence data. In some embodiments, the first phase adjustment is an interferometric phase adjustment. In other embodiments, the integrated circuit device is programmed to calculate the second phase adjustment based on the fluorescence lifetime of the fluorophore in the sample. In these embodiments, the second phase adjustment can be calculated by the subject integrated circuit by acquiring the signals from all fluorescence detectors to determine the phase present in the signals, and calculating the second phase adjustment based on the fluorescence lifetime of the fluorophore. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention may be best understood from the following detailed description when read in conjunction with the accompanying drawings, which include the following figures:

[0026] Figure 1 Depicted is a flow chart for generating frequency-encoded fluorescence data and calculating phase-corrected spatial data from the frequency-encoded fluorescence data in accordance with certain embodiments.

[0027] Figure 2 Depicted is an image of a particle generated using phase-corrected spatial data compared to an image in which the spatial data is not phase-corrected, according to an embodiment. DETAILED DESCRIPTION

[0028] Some aspects of the present disclosure include methods for characterizing sample particles in a flow stream. Methods according to certain embodiments include generating frequency-encoded fluorescence data based on sample particles in the flow stream; and calculating phase-corrected spatial data of the particles by transforming the frequency-encoded fluorescence data using a phase correction component. In certain embodiments, the method includes generating an image of the particles in the flow stream based on the phase-corrected spatial data. A system having a processor is also described, the processor having a memory operably coupled to the processor, the memory having instructions stored thereon, which when executed by the processor causes the processor to calculate phase-corrected spatial data based on the frequency-encoded fluorescence data of the particles of the flow stream. An integrated circuit device (e.g., a field programmable gate array) having programming for practicing the subject method is also provided.

[0029] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, and therefore variations are possible. It should also be understood that the terms used in this article are only used for the purpose of describing specific embodiments, and are not intended to be limiting, because the scope of the present invention will only be limited by the appended claims.

[0030] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range to one tenth of the unit of the lower limit, and any other specified value or intermediate value in the specified range, are included in the present invention unless the context clearly provides otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also included in the present invention, subject to any specific exclusions in the specified range. Where a specified range includes one or two limits, ranges excluding one or both of those included limits are also included in the present invention.

[0031] In this article, certain ranges are presented with the term "about" preceding the numerical value. In this article, the term "about" is used to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number can be a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented.

[0032] 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 the 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.

[0033] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated as incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0034] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," "the," etc. include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0035] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any described method can be carried out in the order of events described or in any other order that is logically possible.

[0036] Although the apparatus and methods are described or will be described using functional descriptions for the sake of grammatical fluency, it should be expressly understood that unless the claims are expressly formulated under 35 U.S.C. §112, the claims should not be interpreted as necessarily being limited in any way to the construction of "means" or "step" limitations, but rather be given the full meaning and scope of equivalents provided by the definitions in the claims in accordance with the doctrine of equivalents, and to be given full legal equivalents under 35 U.S.C. §112 if the claims are expressly formulated under 35 U.S.C. §112.

[0037] As described above, the present disclosure provides systems and methods for characterizing (e.g., imaging) sample particles in a flow stream. In further described embodiments of the present disclosure, methods for generating frequency-encoded fluorescence data based on sample particles in a flow stream and calculating phase-corrected spatial data of the particles are first described in more detail. Next, a system for real-time characterization of particles in a flow stream and real-time separation of particles in a sample is described. An integrated circuit device (e.g., a field programmable gate array) is also provided, which has a programming for generating frequency-encoded fluorescence data based on sample particles in a flow stream and calculating phase-corrected spatial data of the particles.

[0038] Methods for characterizing particles in a sample

[0039] Various aspects of the present disclosure include methods for characterizing sample particles (e.g., cells in biological samples). In practicing methods according to certain embodiments, a sample having cells in a flow stream is irradiated with a light source, and light from the sample is detected to generate frequency-encoded fluorescence data according to the particles and the phase-corrected spatial data of the particles is calculated by transforming the frequency-encoded fluorescence data using 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 subset of whole organisms, plants, fungi, or animal tissues, cells, or components, which in some cases can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Therefore, a "biological sample" refers to a subset of a natural organism or its tissues and a homogenate, lysate, or extract prepared from an organism or its tissues, including, but not limited to, for example, plasma, serum, spinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, urogenital tract, tears, saliva, milk, blood cells, tumors, organs. The biological sample can be any type of organ tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some instances, the sample is a blood sample including whole blood, such as blood obtained from venipuncture or finger stick (wherein the blood may or may not be mixed with any reagents (e.g., preservatives, anticoagulants, etc.) before testing).

[0040] In certain embodiments, the sample source is "mammal" or "mammalian", wherein these terms are widely used to describe organisms of the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subject is a human. The method can be applied to samples obtained from human subjects of both sexes and at any stage of development (i.e., newborns, infants, teenagers, adolescents, adults), wherein, in certain embodiments, the human subject is a teenager, adolescent, or adult. Although the present invention can be applied 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, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses).

[0041] When practicing the subject method, the sample with particles (e.g., cells in the flow stream of a flow cytometer) is illuminated with light from a light source. In some embodiments, the light source is a broadband light source, which emits light having, for example, a span of 50 nm or more, for example, 100 nm or more, for example, 150 nm or more, for example, 200 nm or more, for example, 250 nm or more, for example, 300 nm or more, for example, 350 nm or more, for example, 400 nm or more, and a wide wavelength range including a span of 500 nm or more. For example, a 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 having a wavelength of 400 nm to 1000 nm. In the case where the method includes irradiation 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 stable fiber-coupled broadband light source, a broadband LED with a continuous spectrum, an ultra-bright light emitting diode, a semiconductor light emitting diode, a wide spectrum LED white light source, a multi-LED integrated white light source, and other broadband light sources or any combination thereof.

[0042] In other embodiments, the method includes irradiating with a narrowband light source emitting a specific wavelength or a narrow wavelength range, such as using a light source emitting light in a narrow wavelength range, the narrow wavelength range is, for example, 50nm or less, such as 40nm or less, such as 30nm or less, such as 25nm or less, such as 20nm or less, such as 15nm or less, such as 10nm or less, such as 5nm or less, such as 2nm or less and including a light source emitting light of a specific wavelength (i.e., monochromatic light). Where the method includes irradiating with a narrowband light source, the narrowband light source protocol of interest may include, but is not limited to, a narrow wavelength LED, a laser diode, or a broadband light source coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0043] In certain embodiments, the method includes irradiating the flow stream with one or more lasers. The type and number of lasers will vary depending on the sample and the desired light collected, and can be pulsed lasers or continuous wave lasers. For example, the laser can be a gas laser (e.g., a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof), a dye laser (e.g., a stilbene, coumarin, or rhodamine laser), a metal vapor laser (e.g., 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 (N The present invention relates to a laser beam having a wavelength of 400 nm and a wavelength of 500 nm. The laser beam may be a laser beam having a wavelength of 400 nm and a wavelength of 200 nm. The laser beam may be a solid-state laser (e.g., 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, an ytterbium YAG laser, a Yb2O3 laser or a cerium-doped laser and a combination thereof), a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or any of the above lasers implemented as frequency doubled or frequency tripled.

[0044] The sample in the flow stream can be irradiated using one or more of the above light sources (e.g., 2 or more light sources, e.g., 3 or more light sources, e.g., 4 or more light sources, e.g., 5 or more light sources, and including 10 or more light sources). The light source can include a combination of any type of light sources. For example, in some embodiments, the method includes irradiating the sample in the flow stream using a laser array (e.g., an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers).

[0045] The sample can be irradiated with a wavelength of 200nm to 1500nm (e.g., 250nm to 1250nm, for example, 300nm to 1000nm, for example, 350nm to 900nm, and including 400nm to 800nm). For example, in the case where the light source is a broadband light source, the sample can be irradiated with a wavelength of 200nm to 900nm. In other examples, in the case where the light source includes a plurality of narrow-band light sources, the sample can be irradiated with a specific wavelength of 200nm to 900nm. For example, the light source can be a plurality of narrow-band LEDs (1nm-25nm), each LED independently emitting light in the wavelength range of 200nm to 900nm. In other embodiments, the narrow-band light source includes one or more lasers (e.g., a laser array) and irradiates with a specific wavelength of 200nm to 700nm, for example, irradiated with a laser array having a gas laser, an excimer laser, a dye laser, a metal vapor laser, and a solid-state laser as described above.

[0046] In the case of using more than one light source, the light source can be used to irradiate the sample simultaneously or sequentially or in a combination thereof. For example, each light source can be used to irradiate the sample simultaneously. In other embodiments, each light source is used to irradiate the flow stream sequentially. In the case of using more than one light source to sequentially irradiate the sample, the time for each light source to irradiate the sample can be independently 0.001 microseconds or longer, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microsecond or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 30 microseconds or longer and including 60 microseconds or longer. For example, the method can include irradiating the sample with a light source (such as a laser) for a duration of 0.001 microseconds to 100 microseconds, such as 0.01 microseconds to 75 microseconds, such as 0.1 microseconds to 50 microseconds, such as 1 microsecond to 25 microseconds and including 5 microseconds to 10 microseconds. In the embodiment of using two or more light sources to sequentially irradiate the sample, the duration of each light source irradiating the sample can be the same or different.

[0047] The time period between the irradiation performed by each light source can also vary as needed, and the time period is independently spaced apart by a delay of 0.001 microseconds or longer, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microsecond or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 15 microseconds or longer, such as 30 microseconds or longer, and including 60 microseconds or longer. For example, the time period between the irradiation performed by each light source can be 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, such as 0.1 microseconds to 35 microseconds, such as 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In some embodiments, the time period between the irradiation performed by each light source is 10 microseconds. In an embodiment where the sample is sequentially irradiated by more than two (i.e., 3 or more) light sources, the delay between the irradiation performed by each light source can be the same or different.

[0048] The sample may be illuminated continuously or at discrete intervals. In some instances, the method includes illuminating the sample in the sample continuously with a light source. In other instances, the sample is illuminated at discrete intervals with a light source, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds or some other interval.

[0049] Depending on the light source, the sample can be illuminated from a distance that varies, 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, and including 50 mm or more. Moreover, the illumination angle can also vary, and the illumination angle is 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including 30° to 60°, such as irradiation at a 90° angle.

[0050] In practicing the subject method, light from the illuminated sample is measured, for example, by collecting light from the sample within a certain wavelength range (e.g., 200nm-1000nm). In embodiments, the method may include measuring one or more of light absorption of the sample (e.g., bright field light data), measuring light scattering (e.g., forward or side scattered light data), and measuring light emission of the sample (e.g., fluorescence light data).

[0051] Light from the sample can be measured at one or more wavelengths, for example at 5 or more different wavelengths, for example at 10 or more different wavelengths, for example at 25 or more different wavelengths, for example at 50 or more different wavelengths, for example at 100 or more different wavelengths, for example at 200 or more different wavelengths, for example at 300 or more different wavelengths, and including measuring light collected at 400 or more different wavelengths.

[0052] Light in one or more 200nm-1200nm wavelength ranges can be collected. In some instances, method includes measuring the light from sample in a certain wavelength range, and this wavelength range is for example 200nm to 1200nm, for example 300nm to 1100nm, for example 400nm to 1000nm, for example 500nm to 900nm and including 600nm to 800nm. In other instances, method includes measuring the light collected at one or more specific wavelengths. For example, light collected at one or more places in 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, 617nm and any combination thereof can be measured. In certain embodiments, methods include measuring wavelengths of light corresponding to peak fluorescence wavelengths of certain fluorophores.

[0053] The collected light can be measured continuously or at discrete intervals. In some instances, the method includes measuring the light continuously. In other instances, the light is measured at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds or some other intervals.

[0054] During implementation of the subject method, the collected light can be measured once or more, such as 2 or more times, such as 3 or more times, such as 5 or more times and including 10 or more times. In some embodiments, the light from the sample is measured 2 or more times, and the data is averaged in some instances.

[0055] In some embodiments, the method includes further conditioning the light from the sample before detecting the light. For example, the light from the sample source can pass through one or more lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. In some instances, the collected light passes through one or more focusing lenses, for example, to reduce the distribution of the light. In other instances, the emitted light from the sample passes through one or more collimators to reduce beam divergence.

[0056] In some embodiments, the method includes irradiating the sample with two or more frequency-shifted light beams. As described above, a beam generator component having a laser and an acousto-optic device can be used to frequency-shift the laser. In these embodiments, the method includes irradiating the acousto-optic device with a laser. Depending on the desired wavelength of the light generated in the output laser beam (e.g., for irradiating a sample in a flowing stream), the laser can have a specific wavelength varying from 200nm to 1500nm, such as from 250nm to 1250nm, such as from 300nm to 1000nm, such as from 350nm to 900nm and including from 400nm to 800nm. The acousto-optic device can be irradiated with one or more lasers (e.g., 2 or more lasers, such as 3 or more lasers, such as 4 or more lasers, such as 5 or more lasers and including 10 or more lasers). The laser can include a combination of any type of laser. For example, in some embodiments, a method includes illuminating an acousto-optic device with a laser array, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0057] In the case of using more than one laser, the laser can be used to illuminate the acoustic-optical device simultaneously or sequentially or in a combination thereof. For example, the acoustic-optical device can be illuminated by each laser simultaneously. In other embodiments, the acoustic-optical device is illuminated by each laser sequentially. In the case of using more than one laser to illuminate the acoustic-optical device sequentially, the time for each laser to illuminate the acoustic-optical device can independently be 0.001 microseconds or longer, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microsecond or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 30 microseconds or longer and including 60 microseconds or longer. For example, the method may include illuminating the acoustic device with a laser for a duration of 0.001 microseconds to 100 microseconds, such as 0.01 microseconds to 75 microseconds, such as 0.1 microseconds to 50 microseconds, such as 1 microsecond to 25 microseconds and including 5 microseconds to 10 microseconds. In embodiments where two or more lasers are utilized to sequentially illuminate the acousto-optic device, the duration for which each laser illuminates the acousto-optic device may be the same or different.

[0058] The time period between the illuminations performed by each laser can also vary as desired, with the time periods being independently spaced apart by delays of 0.001 microseconds or longer, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microsecond or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 15 microseconds or longer, such as 30 microseconds or longer, and including 60 microseconds or longer. For example, the time period between the illuminations performed by each light source can range from 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, such as 0.1 microseconds to 35 microseconds, such as 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In some embodiments, the time period between the illuminations performed by each laser is 10 microseconds. In embodiments where the acousto-optic device is sequentially illuminated by more than two (i.e., 3 or more) lasers, the delays between the illuminations performed by each laser can be the same or different.

[0059] The acousto-optic device may be illuminated continuously or at discrete intervals. In some examples, the method includes illuminating the acousto-optic device continuously with a laser. In other examples, the acousto-optic device is illuminated at discrete intervals with a laser, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds or some other interval.

[0060] Depending on the laser, the acousto-optic device may be illuminated from a distance that varies, 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, and including 50 mm or more. Moreover, the illumination angle may also vary from 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including 30° to 60°, such as irradiating at a 90° angle.

[0061] In an embodiment, a method includes applying a radio frequency drive signal to an acousto-optic device to generate an angularly deflected laser beam. Two or more radio frequency drive signals, such as 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and including 100 or more radio frequency drive signals, may be applied to the acousto-optic device to generate an output laser beam having a desired number of angularly deflected laser beams.

[0062] The angularly deflected laser beams generated by the RF drive signal each have an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal having an amplitude sufficient to generate an angularly deflected laser beam of a desired intensity. In some instances, each applied RF drive signal independently has an amplitude of about 0.001V to about 500V, such as about 0.005V to about 400V, such as about 0.01V to about 300V, such as about 0.05V to about 200V, such as about 0.1V to about 100V, such as about 0.5V to about 75V, such as about 1V to 50V, such as about 2V to 40V, such as 3V to about 30V, and including about 5V to about 25V. In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0063] In some embodiments, to generate frequency-encoded fluorescence data, a sample in a flowing stream is irradiated with an output laser beam from an acousto-optic device, the output laser beam comprising angularly deflected laser beams each having an intensity based on the amplitude of an applied RF drive signal. For example, the output laser beam used to irradiate particles in a flowing stream may include 2 or more angularly deflected laser beams, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, and including 25 or more angularly deflected laser beams. In an embodiment, each angularly deflected laser beam has a different frequency that is shifted by a predetermined RF from the frequency of the input laser beam.

[0064] Each angularly deflected laser beam is also spatially displaced from one another. Depending on the applied RF drive signal and the desired irradiation profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more and including 5000 μm or more. In some embodiments, the angularly deflected laser beams overlap, such as with adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of beam spots) can be 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, and including 100 μm or more.

[0065] When a particle passes through a portion of the excitation beam formed by the superposition of two beamlets, the particle is exposed to the superposition of the electric fields of the two beamlets. The fluorescence emitted by the particle is frequency coded with a beat frequency corresponding to the difference between the optical frequencies of the incident beamlets. For example, the frequency coded fluorescence emitted by a particle passing through the left horizontal edge of the excitation beam formed by the superposition of the first beamlet and the second beamlet will exhibit a beat frequency (i.e., f ) corresponding to the difference between the frequency of the second beamlet and the frequency of the first beamlet. 第一子束 -f 第二子束In this way, the positions of particles that pass through the excitation beam can be encoded by the RF beat frequency associated with the radiation emitted by these particles. In some embodiments, this encoding of the positions of the particles can be used to normalize the intensity of the detected radiation emitted by these particles relative to changes in the intensity of the beam (e.g., in its horizontal direction).

[0066] In some embodiments, the frequency-coded fluorescence emitted by the particle corresponds to the frequency of the local oscillator beam (f LO ) and the beat frequency of the RF-shifted beamlet. For example, frequency-encoded fluorescence data includes f LO -f RF移位子束 Where the illumination of the flow stream comprises a local oscillator that spans the width of the flow stream (e.g., across the horizontal axis), the frequency-encoded fluorescence data comprises a signal corresponding to the frequency (f LO ) and the frequency of each RF-shifted beamlet (f1, f2, f3, f4, f5, f6, etc.). In these embodiments, the frequency-encoded fluorescence data may include multiple beat frequencies that each correspond to a position on the horizontal axis of the flow stream.

[0067] As discussed in more detail below, in one mode of operation, particles in a flow stream may be simultaneously illuminated using multiple excitation frequencies, each of which may be obtained, for example, by shifting the center frequency of a laser beam. More specifically, multiple sample locations may be simultaneously illuminated with a laser beam generated by mixing a reference laser beam (e.g., a local oscillator) with multiple radio frequency shifted laser beams such that the reference beam and one of the radio frequency shifted beams illuminate each sample location to excite a fluorophore of interest (if present) at that location. In some embodiments, a reference local oscillator may be generated via radio frequency shifting of a beam (e.g., from a laser such as a continuous wave laser). In these embodiments, each spatial location of a particle in a flow stream that is illuminated with light is "marked" with a different beat frequency that corresponds to the difference between the frequency of the reference beam and the frequency of one of the radio frequency shifted beams. In these instances, the fluorescence radiation emitted by the fluorophore will spatially encode the beat frequency.

[0068] In some instances, cells in the flow stream are imaged using fluorescence imaging of radio frequency labeled emission (FIRE) using multiple frequency-shifted light beams to illuminate the flow stream to generate frequency-encoded images, such as those described in Diebold et al., Nature Photonics, Vol. 7(10); 806-810 (2013), and in U.S. Patent Nos. 9423353, 9784661, and 10006852, and in U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0069] In an embodiment, frequency-encoded fluorescence data is generated by detecting light from particles in a flow stream. Fluorescence data may be generated from one or more fluorescence detectors (e.g., one or more detection channels), such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, and including 8 or more fluorescence detectors (e.g., 8 or more detection channels). In some embodiments, frequency-encoded fluorescence data includes data components acquired (or derived) from light from other detectors, such as detected light absorption or detected light scattering. In some instances, one or more data components of frequency-encoded fluorescence data from a sample are generated based on light absorption detected from the sample (e.g., from a bright field light detector). As described in more detail below, a phase correction component may include a signal from a bright field detector, which in some embodiments is used to generate phase-corrected spatial data, which is an interpretation of interferometric phase adjustment of spatial data calculated based on frequency-encoded fluorescence data. In other examples, one or more data components of frequency-encoded fluorescence data from the sample are generated based on light scatter detected from the sample (e.g., from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector).

[0070] In an embodiment, a method includes calculating spatial data from the frequency-encoded fluorescence data. The spatial data according to an embodiment of the present disclosure is phase corrected by transforming the frequency-encoded fluorescence data using a phase correction component. In some embodiments, the spatial data includes a horizontal size dimension of the particle, a vertical size dimension of the particle, a ratio of particle sizes along two different dimensions, a ratio of sizes of particle components (e.g., a ratio of a horizontal dimension of a cell nucleus to a horizontal dimension of a cytoplasm).

[0071] In some examples, the phase correction component is used to generate modified transform coefficients (i.e., as described below, for transforming frequency-encoded data into spatial data). For example, the phase correction component may include 2 or more modified transform coefficients, such as 3 or more, such as 4 or more, and including 5 or more modified transform coefficients. In the case where the spatial data is calculated by performing a Fourier transform (as described below), in some embodiments, the phase correction component includes a modified transform coefficient for which the Fourier transform generates only real mathematical calculation components (i.e., does not generate imaginary mathematical calculation components).

[0072] In some 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 with a predetermined constant signal to generate a phase adjustment value; and calculating the inverse tangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum of all frequency points in the discrete Fourier transform of the frequency-encoded fluorescence data.

[0073] In some embodiments, the first phase adjustment is an interferometric phase adjustment. In these embodiments, the phase adjustment includes a phase shift caused by a light source used to illuminate the sample in the flow stream. For example, the light source can be a beam generator component configured to generate at least a first frequency-shifted beam and a second frequency-shifted beam. The beam generator according to some examples 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 caused by vibrations between components of the beam generator.

[0074] In some embodiments, the second phase adjustment is based on the fluorescence lifetime of the fluorophore in the sample. In these embodiments, the second phase adjustment can be calculated by acquiring signals from all fluorescence detectors to determine the phase present in the signal, and calculating the second phase adjustment based on the fluorescence lifetime of the fluorophore. Depending on the specific type of fluorophore and the number of fluorophores present, one or more fluorescence lifetimes can be calculated, for example, 2 or more, for example 3 or more, for example 4 or more and including 5 or more fluorescence lifetimes can be calculated. In some embodiments, each fluorescence lifetime is calculated at the peak emission wavelength of the fluorophore. In these embodiments, signals from different detector channels can be used to detect and calculate each fluorophore lifetime.

[0075] In an embodiment, the method further comprises calculating phase-corrected spatial data by transforming the frequency-encoded fluorescence data using the phase correction component determined above. In some embodiments, the method comprises calculating spatial data based on the frequency-encoded fluorescence data from the target. In some instances, calculating the spatial data of the target comprises transforming the frequency-encoded fluorescence data. In one example, the spatial data is calculated by Fourier transforming (FT) the frequency-encoded fluorescence data. In another example, the spatial data is calculated by discrete Fourier transforming (DFT) the frequency-encoded fluorescence data. In yet another example, the spatial data is calculated by short-time Fourier transforming (STFT) the frequency-encoded fluorescence data. In yet another example, the spatial data is calculated using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data. By considering the phase correction component before transforming the frequency-encoded data into spatial data, the output of the transform is less computationally complex than transforming the original frequency data into spatial data (i.e., without first considering the phase). In some embodiments, a method includes transforming the frequency-encoded fluorescence data without performing any imaginary mathematical calculations (ie, performing only real mathematical calculations for the transform) to generate spatial data from the frequency-encoded fluorescence data.

[0076] In some embodiments, the method includes generating an image of particles in a flow stream based on frequency-encoded fluorescence. In some embodiments, an image of a particle can be generated based on frequency-encoded fluorescence in combination with detected light absorption, detected light scattering, or a combination thereof. In some instances, an image of a particle is generated based on frequency-encoded fluorescence alone. In other instances, an image of a target is generated based on frequency-encoded fluorescence and light absorption detected from a sample (e.g., from a bright field light detector). In other instances, an image of a particle is generated based on frequency-encoded fluorescence and light scattering detected from a sample (e.g., from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector). In other instances, an image of a particle is generated based on frequency-encoded fluorescence and a combination of detected light absorption, detected light scattering, and detected light emission.

[0077] One or more images of the particle can be generated based on the frequency-encoded fluorescence data. In some embodiments, a single image of the particle is generated based on the frequency-encoded fluorescence data. In other embodiments, two or more images of the particle are generated based on the frequency-encoded fluorescence data, such as 3 or more, such as 4 or more, such as 5 or more, and including 10 or more images, or a combination thereof.

[0078] As described above, the method of the present disclosure also includes sorting particles. In an embodiment, particles can be sorted based on one or more determined characteristics (e.g., size, center of mass, eccentricity) of particles determined according to frequency-encoded fluorescence data, calculated spatial data, generated images, calculated spatial data or generated images, or some combination thereof. The term "sorting" is used in its conventional sense herein to refer to the composition of the separated sample (e.g., droplets containing cells, droplets containing non-cellular particles (e.g., biomacromolecules)), and in some instances, the separated composition is transported to one or more sample collection containers. For example, the method may include sorting 2 or more compositions of a sample, such as 3 or more compositions, such as 4 or more compositions, such as 5 or more compositions, such as 10 or more compositions, such as 15 or more compositions, and including sorting 25 or more compositions of a sample. In some instances, the first sample composition collection position includes a sample collection container, and the second sample composition collection position includes a waste collection container.

[0079] In sorting particles from a sample in a flowing stream, the method includes data acquisition (e.g., fluorescence data), analysis (determining frequency-encoded fluorescence data, determining phase-corrected components, calculating a transformation of frequency-encoded data to phase-corrected spatial data), and recording, for example, using a computer, wherein multiple data channels record data from each detector (e.g., a scattering detector, a bright-field photodetector, or a fluorescence detector). In these embodiments, the analysis includes classifying and counting the particles so that each particle is present in the form of a set of digitized parameter values. The subject system (described below) can be configured to trigger on selected parameters to distinguish particles of interest from background and noise.

[0080] The specific subgroups 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. In order to select the appropriate threshold, the data is plotted to obtain the best separation of possible subgroups. The process can be performed by plotting image moments or one or more determined characteristics (e.g., size, center of mass, eccentricity). In other embodiments, the method includes plotting forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot plot. In other embodiments, the method includes plotting one or more determined characteristics (e.g., size, center of mass, eccentricity) for one or more of the forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC). In other embodiments, the method includes gating the particle population for forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC), and then gating based on the image of the target, based on one or more determined characteristics (e.g., size, center of mass, eccentricity). In other embodiments, the method includes gating particle populations based on an image of the target, based on one or more determined characteristics (e.g., size, center of mass, eccentricity), and then gating the particle populations for forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC).

[0081] The subpopulation of interest (i.e., those individual cells within the threshold) is then selected and particles that are not within the threshold are excluded. Where desired, the threshold can be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Only those particles within the threshold are then further analyzed by plotting other parameters of these particles (e.g., fluorescence). Where desired, the above analysis can be configured to produce a count of particles of interest in the sample.

[0082] In some embodiments, a method for sorting sample components includes sorting particles (e.g., cells in a biological sample) using a particle sorting module with a deflector plate, such as described in U.S. Patent Publication No. 2017 / 0299493, filed on March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, cells of a sample are sorted using a sorting decision module with multiple sorting decision units, such as those described in U.S. Provisional Patent Application No. 62 / 803264, filed on February 8, 2019, the disclosure of which is incorporated herein by reference.

[0083] Figure 1A flow chart for generating frequency-encoded fluorescence data and calculating phase-corrected spatial data based on the frequency-encoded fluorescence data according to certain embodiments is shown. At step 101, light (light absorption, scattered light, or light emission) from particles (e.g., cells) in a flow stream is detected. At step 102, frequency-encoded fluorescence data of the particles (e.g., frequency data for each spatial position along a horizontal axis) is generated. At step 103, phase correction components, such as interference phase components and fluorescence lifetime phase components, are determined. At step 104, phase-corrected spatial data is calculated by transforming the frequency-encoded fluorescence data, for example, using a discrete Fourier transform. At step 105, the spatial data can be used to generate an image. Then, at step 106, an image mask can be used to generate an image mask. At step 107, two or more images can be used to calculate the co-localization of one or more features (e.g., organelles) of a cell, or at step 108, an image mask can be used to calculate co-localization.

[0084] Figure 2 A comparison of images of particles generated using phase-corrected spatial data and images where the spatial data is not phase-corrected according to certain embodiments is shown. As shown in Group A, frequency-encoded fluorescence data is transformed into spatial data using a fast Fourier transform (FFT), for example, without phase correction. The resulting image shows a lower resolution with particles obscured by background noise. In Group B, the frequency-encoded fluorescence data is phase corrected using a phase adjustment component in conjunction with an FFT to generate phase-corrected spatial data. The phase-corrected spatial data provides enhanced resolution imaging of particles that are not obscured by background noise.

[0085] Systems for characterizing particles in samples

[0086] As described above, various aspects of the present disclosure include systems for characterizing sample particles (e.g., cells in a biological sample). Systems according to certain embodiments include a light source and a light detection system, the light source configured to illuminate a sample having particles in a flow stream, the light detection system having a photodetector and a processor, the processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon, which when executed by the processor configure the processor to: generate frequency-encoded fluorescence data from particles in the flow stream; and calculate phase-corrected spatial data of the particles by transforming the frequency-encoded fluorescence data using a phase correction component.

[0087] The system of interest includes a light source configured to illuminate the sample in the flow stream. In an embodiment, the light source can be any suitable broadband or narrowband light source. Depending on the composition (such as cells, beads, non-cellular particles, etc.) in the sample, the light source can be configured to emit light with a wavelength variation of 200nm to 1500nm, such as 250nm to 1250nm, such as 300nm to 1000nm, such as 350nm to 900nm and including 400nm to 800nm. For example, the light source can include a broadband light source of a light of 200nm to 900nm emission wavelength. In other examples, the light source includes a narrowband light source of 200nm to 900nm emission wavelength. For example, the light source can be a narrowband LED (1nm-25nm) of a light of 200nm to 900nm emission wavelength.

[0088] In some embodiments, the light source is a laser. The laser of interest may include a pulsed laser or a continuous wave laser. For example, the laser may be a gas laser (e.g., a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof), a dye laser (e.g., a stilbene, coumarin, or rhodamine laser), a metal vapor laser (e.g., 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 (N The present invention relates to a laser diode or an optically pumped semiconductor laser (e.g., an optically pumped semiconductor laser (eCu) laser, a copper laser or a gold laser and a combination thereof), a solid-state laser (e.g., 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, an ytterbium YAG laser, a Yb2O3 laser or a cerium-doped laser and a combination thereof), a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or any of the above lasers implemented as frequency doubled or frequency tripled.

[0089] 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, such as a broadband LED with a continuous spectrum, an ultra-bright light emitting diode, a semiconductor light emitting diode, a wide spectrum LED white light source, and a multi-LED integration. In some instances, the non-laser light source is a stable fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0090] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted light beams. In some instances, the beam generator includes a laser, a radio frequency generator configured to apply a radio frequency drive signal to the acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous wave laser. For example, the laser in the beam generator of interest can be a gas laser (e.g., a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof), a dye laser (e.g., a stilbene, coumarin, or rhodamine laser), a metal vapor laser (e.g., a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeS) laser, or a combination thereof). The present invention relates to a laser beam having a laser beam having a wavelength of 400 nm and a wavelength of 500 nm. The laser beam has a wavelength of 400 nm and a wavelength of 800 nm. The laser beam has a wavelength of 200 nm and a wavelength of 100 nm. The laser beam has a wavelength of 200 nm and a wavelength of 500 nm.

[0091] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency shift the laser using an applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam based on light from a laser and an applied RF drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0092] In an embodiment, the controller is configured to apply an RF drive signal to the acousto-optic device to produce a desired number of angularly deflected laser beams in the output laser beam, for example, configured to apply 3 or more RF drive signals, for example 4 or more RF drive signals, for example 5 or more RF drive signals, for example 6 or more RF drive signals, for example 7 or more RF drive signals, for example 8 or more RF drive signals, for example 9 or more RF drive signals, for example 10 or more RF drive signals, for example 15 or more RF drive signals, for example 25 or more RF drive signals, for example 50 or more RF drive signals and including being configured to apply 100 or more RF drive signals.

[0093] In some examples, to produce an intensity distribution of an angularly deflected laser beam in the output laser beam, the controller is configured to apply a radio frequency drive signal having a varying amplitude, such as about 0.001V to about 500V, such as about 0.005V to about 400V, such as about 0.01V to about 300V, such as about 0.05V to about 200V, such as about 0.1V to about 100V, such as about 0.5V to about 75V, such as about 1V to 50V, such as about 2V to 40V, such as 3V to about 30V, and including about 5V to about 25V. In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0094] In some embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam of an angularly deflected laser beam having a desired intensity distribution. For example, the memory may include instructions for generating two or more angularly deflected laser beams having the same intensity, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, and the memory may include instructions for generating 100 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, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, and the memory may include instructions for generating 100 or more angularly deflected laser beams having different intensities.

[0095] In certain embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from an edge to a center 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 beam can range from 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and including about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from an edge to a center of the output laser beam along the horizontal axis. In these examples, the intensity of the angularly deflected laser beam at the edge of the output beam can range from 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and including about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity distributed along a horizontal axis with a Gaussian distribution. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having a top hat intensity distribution along a horizontal axis.

[0096] In an embodiment, the beam generator of interest can be configured to generate spatially separated angularly deflected laser beams in the output laser beam. Depending on the applied RF drive signal and the desired illumination distribution of the output laser beam, the angularly deflected laser beams can be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more and including 5000 μm or more. In some embodiments, the system is configured to generate an angularly deflected laser beam in the output laser beam, for example, overlapping with an adjacent angularly deflected laser beam along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of beam spots) can be an overlap of 0.001 μm or more, such as an overlap of 0.005 μm or more, such as an overlap of 0.01 μm or more, such as an overlap of 0.05 μm or more, such as an overlap of 0.1 μm or more, such as an overlap of 0.5 μm or more, such as an overlap of 1 μm or more, such as an overlap of 5 μm or more, such as an overlap of 10 μm or more and including an overlap of 100 μm or more.

[0097] In some cases, a beam generator configured to generate two or more frequency-shifted beams includes a laser excitation module as described in U.S. Patent Nos. 9,423,353, 9,784,661, and 1,000,852 and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0098] In an embodiment, the system includes a light detection system having one or more photodetectors for detecting and measuring light from a sample. The photodetector of interest can be configured to measure light absorption (e.g., light absorption of bright field 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 photodetector of interest may include, but is not limited to, optical sensors (e.g., active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultipliers, phototransistors, quantum dot photoconductors or photodiodes and combinations thereof) and other photodetectors. In certain embodiments, light from a sample is measured using a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor.

[0099] In some embodiments, the light detection system of interest includes a plurality of photodetectors. In some instances, the light detection system includes a plurality of solid-state detectors, such as photodiodes. In some instances, the light detection system includes a photodetector array, such as a photodiode array. In these embodiments, the photodetector array may include 4 or more photodetectors, such as 10 or more photodetectors, such as 25 or more photodetectors, such as 50 or more photodetectors, such as 100 or more photodetectors, such as 250 or more photodetectors, such as 500 or more photodetectors, such as 750 or more photodetectors, and including 1000 or more photodetectors. For example, the detector may be a photodiode array having 4 or more photodiodes, such as 10 or more photodiodes, such as 25 or more photodiodes, such as 50 or more photodiodes, such as 100 or more photodiodes, such as 250 or more photodiodes, such as 500 or more photodiodes, such as 750 or more photodiodes, and including 1000 or more photodiodes.

[0100] The photodetectors can be arranged in any geometric configuration as desired, where arrangements of interest include, but are not limited to, square configurations, rectangular configurations, trapezoidal configurations, triangular configurations, hexagonal configurations, heptagonal configurations, octagonal configurations, nonagonal configurations, decagonal configurations, dodecagonal configurations, circular configurations, elliptical configurations, and irregular pattern configurations. The photodetectors in the photodetector array can be oriented at an angle of 10° to 180° relative to another photodetector (as referenced in the XZ plane), such as 15° to 170°, such as 20° to 160°, such as 25° to 150°, such as 30° to 120°, and including 45° to 90°. The photodetector array can be of any suitable shape, and can be a rectilinear shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). In some embodiments, the photodetector array has a rectangular shaped active surface.

[0101] Each photodetector (e.g., photodiode) in the array may have an active surface having a width of 5 μm to 250 μm (e.g., 10 μm to 225 μm, e.g., 15 μm to 200 μm, e.g., 20 μm to 175 μm, e.g., 25 μm to 150 μm, e.g., 30 μm to 125 μm, and including 50 μm to 100 μm) and a length of 5 μm to 250 μm (e.g., 10 μm to 225 μm, e.g., 15 μm to 200 μm, e.g., 20 μm to 175 μm, e.g., 25 μm to 150 μm, e.g., 30 μm to 125 μm, and including 50 μm to 100 μm), wherein the surface area of ​​each photodetector (e.g., photodiode) in the array is 25 μm 2 To 10000μm 2 , for example 50 μm 2 To 9000μm 2 , for example 75 μm 2 Up to 8000μm 2 , for example 100 μm 2 To 7000μm 2 , for example 150 μm 2 To 6000μm 2 and including 200μm 2 To 5000μm 2 .

[0102] The size of the photodetector array can vary depending on the amount of light and the intensity of the light, the number of photodetectors and the desired sensitivity, and the length of the photodetector array can be 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm and including 5 mm to 25 mm. The width of the photodetector array can also vary from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm and including 5 mm to 25 mm. Therefore, the active surface of the photodetector array can be 0.1 mm 2 Up to 10000mm 2 , for example 0.5mm 2 Up to 5000mm 2 , for example 1mm 2 Up to 1000mm 2 , for example 5mm 2 Up to 500mm 2 And including 10mm 2 Up to 100mm 2 .

[0103] The photodetector of interest is configured to measure light collected at one or more wavelengths, for example at 2 or more wavelengths, for example at 5 or more different wavelengths, for example at 10 or more different wavelengths, for example at 25 or more different wavelengths, for example at 50 or more different wavelengths, for example at 100 or more different wavelengths, for example at 200 or more different wavelengths, for example at 300 or more different wavelengths, and including measuring light emitted by a sample in a flowing stream at 400 or more different wavelengths.

[0104] In some embodiments, the photodetector is configured to measure the light collected in a certain wavelength range (e.g., 200nm-1000nm). In certain embodiments, the photodetector of interest is configured to collect a spectrum within a certain wavelength range. For example, the system may include one or more detectors configured to collect one or more spectra within a wavelength range of 200nm-1000nm. In other embodiments, the detector of interest is configured to measure the light from the sample in the flow stream at one or more specific wavelengths. For example, the system may include one or more detectors configured to measure the light at one or more of 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, 617nm, and any combination thereof.

[0105] The light detection system is configured to measure light continuously or at discrete intervals. In some instances, the photodetector of interest is configured to measure the collected light continuously. In other instances, the light detection system is configured to measure at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds, or at some other intervals.

[0106] In an embodiment, the system is configured to generate frequency-encoded fluorescence data by irradiating a sample having particles in a flow stream. In some embodiments, the light source includes a light generator component that generates a plurality of angularly deflected laser beams, each angularly deflected laser beam having an intensity based on the amplitude of an applied RF drive signal (e.g., from a direct digital synthesizer coupled to an acousto-optic device). For example, the subject system may include a light generator component that generates 2 or more angularly deflected laser beams, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more and including 25 or more angularly deflected laser beams. In an embodiment, each angularly deflected laser beam has a different frequency that is shifted from the frequency of the input laser beam by a predetermined RF.

[0107] According to certain embodiments, the subject system is configured to generate angularly deflected laser beams that are also spatially displaced from one another. Depending on the applied RF drive signal and the desired irradiation profile of the output laser beam, the subject system can be configured to generate angularly deflected laser beams that are separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more, and including 5000 μm or more. In some embodiments, the angularly deflected laser beams overlap, such as with adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of beam spots) can be 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, and including 100 μm or more.

[0108] In some embodiments, a system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate frequency-encoded fluorescence data by calculating the difference between optical frequencies of overlapping photon beams incident on a flow stream. In one example, a system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a beat frequency at each position on a horizontal axis of the flow stream. In these embodiments, the frequency-encoded fluorescence emitted by the particle is a frequency corresponding to a local oscillator beam (f LO ) and the beat frequency of the RF-shifted beamlet. For example, frequency-encoded fluorescence data includes f LO -f RF移位子束 Where the illumination of the flow stream comprises a local oscillator that spans the width of the flow stream (e.g., across the horizontal axis), the frequency-encoded fluorescence data comprises a signal corresponding to the frequency (f LO ) and the frequency of each RF-shifted beamlet (f1, f2, f3, f4, f5, f6, etc.). In these embodiments, the frequency-encoded fluorescence data may include multiple beat frequencies that each correspond to a position on the horizontal axis of the flow stream.

[0109] In an embodiment, the system is configured to generate frequency-encoded fluorescence data based on light detected from particles in a flow stream. The fluorescence data can be generated from one or more fluorescence detectors (e.g., one or more detection channels), such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, and including 8 or more fluorescence detectors (e.g., 8 or more detection channels). In some embodiments, the frequency-encoded fluorescence data includes data components acquired (or derived) from light from other detectors, such as detected light absorption or detected light scattering. In some instances, the system is configured to generate one or more data components of frequency-encoded fluorescence data based on light absorption detected from a sample (e.g., from a bright field light detector). For example, the system can be configured to generate a phase correction component based on a signal from a bright field detector. In certain embodiments, the system is configured to generate phase-corrected spatial data, which is an interpretation of interferometric phase adjustment of spatial data calculated based on frequency-encoded fluorescence data. In other examples, the system is configured to generate one or more data components of frequency-encoded fluorescence data based on light scatter detected from the sample (e.g., from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector).

[0110] In an embodiment, a system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate spatial data based on frequency-encoded fluorescence data. The spatial data according to an embodiment of the present disclosure is phase-corrected by the system by transforming the frequency-encoded fluorescence data using a phase correction component. In some embodiments, the spatial data includes a horizontal size dimension of a particle, a vertical size dimension of a particle, a ratio of particle sizes along two different dimensions, a ratio of sizes of particle components (e.g., a ratio of a horizontal dimension of a nucleus to a horizontal dimension of a cytoplasm).

[0111] In some embodiments, the system is configured to calculate correction transform coefficients for transforming the frequency-encoded fluorescence data into phase-corrected spatial data. For example, the phase correction component may include 2 or more correction transform coefficients, such as 3 or more, such as 4 or more, and including 5 or more correction transform coefficients. In the case where the spatial data is calculated by performing a Fourier transform, the phase correction component includes correction transform coefficients for which the Fourier transform generates only real mathematical calculation components (i.e., does not generate imaginary mathematical calculation components).

[0112] In some instances, the system is configured to determine a phase correction component that 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 system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the first phase adjustment by: multiplying the output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and calculating the inverse tangent of the phase adjustment value to generate the first phase adjustment. In these embodiments, the phase adjustment value is the sum of all frequency points in a discrete Fourier transform of the frequency-encoded fluorescence data.

[0113] In other examples, the system is configured to calculate the second phase adjustment based on the fluorescence lifetime of the fluorophore in the sample. In these examples, the system is configured to calculate the second phase adjustment by acquiring signals from all fluorescence detectors to determine the phase present in the signals, and calculating the second phase adjustment based on the fluorescence lifetime of the fluorophore. The subject system can be configured to calculate the fluorescence lifetime using different detector channels, such as by using 2 or more detection channels, such as 3 or more, such as 4 or more, and including 5 or more detector channels to calculate the fluorescence lifetime.

[0114] In embodiments, the subject system includes a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate phase-corrected spatial data of a particle by transforming frequency-encoded fluorescence data using a phase-corrected component. In some embodiments, to calculate the phase-corrected spatial data, the system is configured to Fourier transform the frequency-encoded fluorescence data using the phase-corrected component to generate phase-corrected spatial data of the particle. In other embodiments, the system is configured to discrete Fourier transform the frequency-encoded fluorescence data using the phase-corrected component to generate phase-corrected spatial data of the particle. In other embodiments, the system is configured to perform a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using the phase-corrected component. In other embodiments, the system is configured to perform a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using the phase-corrected component. In other embodiments, the system is configured to calculate the phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0115] In some embodiments, the system is configured to consider the phase correction component before transforming the frequency-encoded data into spatial data, such that the output of the transform is less computationally complex than transforming the original frequency data into spatial data (i.e., without first considering the phase). In some embodiments, the system is configured to transform the frequency-encoded fluorescence data without performing any mathematical imaginary calculations (i.e., performing only mathematical real calculations for the transform) to generate spatial data from the frequency-encoded fluorescence data.

[0116] The subject system can be configured to generate one or more images of particles in a flow stream based on frequency-encoded fluorescence. In some embodiments, images of particles can be generated based on frequency-encoded fluorescence in combination with detected light absorption, detected light scattering, or a combination thereof. In some instances, images of particles are generated based on frequency-encoded fluorescence alone. In other instances, images of targets are generated based on frequency-encoded fluorescence and light absorption detected from a sample (e.g., from a bright field light detector). In other instances, images of particles are generated based on frequency-encoded fluorescence and light scattering detected from a sample (e.g., from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector). In other instances, images of particles are generated based on frequency-encoded fluorescence and a combination of detected light absorption, detected light scattering, and detected light emission.

[0117] The system according to some embodiments may include a display and an operator input device. For example, the operator input device may be a keyboard, a mouse, etc. The processing module includes a processor that can access a memory having instructions stored thereon for performing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input-output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are available or will be available. As is known in the art, the processor executes an operating system that interacts with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in a variety of programming languages ​​(e.g., Java, Perl, C++, other high-level or low-level languages, and combinations thereof). The operating system typically collaborates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all of which are performed according to known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes an analog electronic device that provides feedback control (e.g., negative feedback control).

[0118] The system memory can be any of a variety of known or future memory storage devices. Examples include any commonly used random access memory (RAM), magnetic media (such as a resident hard disk or tape), optical media (such as a read-write optical disk), flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including an optical drive, a tape drive, a removable hard drive, or a floppy disk drive. This type of memory storage device typically reads from and / or writes to a program storage medium (not shown), such as an optical disk, a tape, a removable hard disk, or a floppy disk. Any of these program storage media, or other program storage media currently in use or that may be developed later, can be considered a computer program product. It should be understood that these program storage media typically store computer software programs and / or data. Computer software programs (also referred to as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0119] In some embodiments, a computer program product is described that includes a computer usable medium having control logic (a computer software program including program code) stored therein. The control logic, when executed by a computer processor, causes the processor to perform the functions described herein. In other embodiments, some functions are primarily implemented in hardware using, for example, a hardware state machine. Implementing a hardware state machine to perform the functions described herein will be apparent to those skilled in the relevant art.

[0120] The memory may be any suitable device in which the processor can store and retrieve data, such as a magnetic, optical or solid-state storage device (which includes a magnetic or optical disk or tape or RAM or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor that can be appropriately programmed by a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely through a communication channel, or pre-stored in a computer program product (such as a memory or some other portable or fixed computer-readable storage medium) using any of those devices associated with the memory. For example, a magnetic or optical disk can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes programming for practicing the above-mentioned method, such as programming in the form of a computer program product, an algorithm. The programming according to the present invention can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROMs; portable flash memory drives; and hybrids of these categories, such as magnetic / optical storage media.

[0121] The processor may also access a communication channel to communicate with a user at a remote location. A remote location refers to a user who does not interact directly 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"), a telephone network, a satellite network, or any other suitable communication channel including a mobile phone (i.e., a smart phone).

[0122] In some embodiments, the system according to the present disclosure can be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface can be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), communications protocols) and cellular communications (such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM)).

[0123] 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 allow data communications between the subject system and other external devices such as computer terminals configured for similar complementary data communications (e.g., computer terminals in a doctor's office or hospital environment).

[0124] In one embodiment, the communication interface is configured for infrared communication, communications or any other suitable wireless communication protocol to enable the subject system to communicate with other devices such as computer terminals and / or networks, communications-enabled mobile phones, personal digital assistants, or any other communications devices that a user may use in conjunction with the subject system.

[0125] In one embodiment, the communication interface is configured to provide a data transfer connection using the Internet Protocol (IP) through a cellular telephone network, a short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0126] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, such as using a wireless communication protocol such as 802.11 or The server device may be another portable device, such as a smart phone, a personal digital assistant (PDA), or a laptop computer, or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as a button, a keyboard, a mouse, or a touch screen.

[0127] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the subject system (in the optional data storage unit) with a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0128] The output controller may include a controller for any of a variety of known display devices for presenting information to a user (whether a person or a machine, whether local or remote). If one of the display devices provides visual information, the information can generally be logically and / or physically organized as an array of picture elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. The functional elements of the computer can communicate with each other through a system bus. Some of these communications can be implemented using a network or other types of remote communications in an alternative embodiment. According to known techniques, the output manager can also provide information generated by the processing module to a user at a remote location, for example, through the Internet, telephone or satellite network. The presentation of data by the output manager can be implemented according to a variety of known techniques. As some examples, the data may include SQL, HTML or XML documents, emails or other files or other forms of data. The data may include an Internet URL address so that a user can retrieve additional SQL, HTML, XML or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or a type to be developed in the future, although they generally belong to a class of computers generally referred to as servers. However, they can also be host computers, workstations or other computer types. They can be connected via any known or future type of cable or other communication system (including wireless system), whether networked or otherwise. They can be in the same location or physically separated. Various operating systems can be used on any computer platform, which may depend on the type and / or structure of the selected computer platform. 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, etc.

[0129] In certain embodiments, the subject system includes one or more optical adjustment components for adjusting light, such as light irradiated onto a sample (e.g., from a laser) or light collected from a sample (e.g., fluorescence). For example, optical adjustment can be to increase the dimension of light, focus the light, or collimate the light. In some instances, the optical adjustment is an amplification protocol to increase the dimension of light (e.g., a beam spot), such as by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, and including by 75% or more. In other embodiments, optical adjustment includes focusing the light to reduce the dimension of the light, such as by reducing the dimension of the light by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, and including by reducing the beam spot dimension by 75% or more. In certain embodiments, optical adjustment includes collimating the light. The term "collimation" is used in its conventional sense to refer to optically adjusting the collimation of light propagation or reducing the divergence of light from a common propagation axis. In some examples, collimation includes narrowing the spatial cross-section of the light beam (eg, reducing the beam profile of a laser).

[0130] In some embodiments, the optical adjustment component is a focusing lens with a magnification of 0.1 to 0.95, such as a magnification of 0.2 to 0.9, such as a magnification of 0.3 to 0.85, such as a magnification of 0.35 to 0.8, such as a magnification of 0.5 to 0.75, and including a magnification of 0.55 to 0.7, such as a magnification of 0.6. For example, in some instances, the focusing lens is a bichromatic reduction lens with a magnification of about 0.6. The focal length of the focusing lens can vary from 5 mm to 20 mm, such as a focal length of 6 mm to 19 mm, such as a focal length of 7 mm to 18 mm, such as a focal length of 8 mm to 17 mm, such as a focal length of 9 mm to 16 mm, and including a focal length of 10 mm to 15 mm. In some embodiments, the focal length of the focusing lens is about 13 mm.

[0131] In other embodiments, the optical adjustment component is a collimator. The collimator can be any convenient collimation protocol, such as one or more reflectors or curved lenses or a combination thereof. For example, the collimator is a single collimating lens in some instances. In other instances, the collimator is a collimating reflector. In other instances, the collimator includes two lenses. In other instances, the collimator includes a reflector and a lens. In the case where the collimator includes one or more lenses, the focal length of the collimating lens can vary from 5mm to 40mm, such as a focal length of 6mm to 37.5mm, such as a focal length of 7mm to 35mm, such as a focal length of 8mm to 32.5mm, such as a focal length of 9mm to 30mm, such as a focal length of 10mm to 27.5mm, such as a focal length of 12.5mm to 25mm, and including a focal length of 15mm to 20mm.

[0132] In some embodiments, the subject system includes a flow cell nozzle having a nozzle orifice configured to allow a flow stream to flow through the flow cell nozzle. The subject flow cell nozzle has an orifice for propagating a fluid sample to a sample interrogation region, wherein in some embodiments, the flow cell nozzle includes a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminating in a flat surface having a nozzle orifice transverse to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) can vary from 1mm to 15mm, such as 1.5mm to 12.5mm, such as 2mm to 10mm, such as 3mm to 9mm, and including 4mm to 8mm. The length of the distal truncated conical portion (measured along the longitudinal axis) can also vary from 1mm to 10mm, such as 2mm to 9mm, such as 3mm to 8mm, and including 4mm to 7mm. In some embodiments, the diameter of the flow cell nozzle chamber can vary from 1mm to 10mm, such as 2mm to 9mm, such as 3mm to 8mm, and including 4mm to 7mm.

[0133] In some instances, the nozzle chamber does not include a cylindrical portion, and the entire flow cell nozzle chamber is frustoconical. In these embodiments, the length of the frustoconical nozzle chamber (measured along the longitudinal axis transverse to the nozzle orifice) can be 1mm to 15mm, such as 1.5mm to 12.5mm, such as 2mm to 10mm, such as 3mm to 9mm, and including 4mm to 8mm. The diameter of the proximal portion of the frustoconical nozzle chamber can be 1mm to 10mm, such as 2mm to 9mm, such as 3mm to 8mm, and including 4mm to 7mm.

[0134] In an embodiment, the sample flow stream flows out of an orifice at the distal end of the flow cell nozzle. Depending on the desired characteristics of the flow stream, the flow cell nozzle orifice can be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). In certain embodiments, the flow cell nozzle of interest has a circular orifice. In some embodiments, the size of the nozzle orifice can vary from 1 μm to 20,000 μm, such as 2 μm to 17,500 μm, such as 5 μm to 15,000 μm, such as 10 μm to 12,500 μm, such as 15 μm to 10,000 μm, such as 25 μm to 7,500 μm, such as 50 μm to 5,000 μm, such as 75 μm to 1,000 μm, such as 100 μm to 750 μm, and including 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0135] In some embodiments, the flow cell nozzle includes a sample injection port configured to provide a sample to the flow cell nozzle. In an embodiment, the sample injection system is configured to provide a suitable sample stream to the flow cell nozzle chamber. Depending on the desired characteristics of the flow stream, the rate at which the sample is delivered to the flow cell nozzle chamber by the sample injection port can be 1 μL / sec or higher, such as 2 μL / sec or higher, such as 3 μL / sec or higher, such as 5 μL / sec or higher, such as 10 μL / sec or higher, such as 15 μL / sec or higher, such as 25 μL / sec or higher, such as 50 μL / sec or higher, such as 100 μL / sec or higher, such as 150 μL / sec or higher, such as 200 μL / sec or higher, such as 250 μL / sec or higher, such as 300 μL / sec or higher, such as 350 μL / sec or higher, such as 400 μL / sec or higher, such as 450 μL / sec or higher and including 500 μL / sec or higher. For example, the sample flow rate can be 1 μL / sec to about 500 μL / sec, 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, and including 10 μL / sec to about 100 μL / sec.

[0136] The sample injection port can be an orifice located in the nozzle chamber wall or can be a conduit located at the proximal end of the nozzle chamber. In the case where the sample injection port is an orifice located in the nozzle chamber wall, the orifice of the sample injection port can be any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical, etc.), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). In certain embodiments, the sample injection port has a circular orifice. The size of the orifice of the sample injection port can vary according to the shape, and in certain instances, the orifice has an opening of 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, and includes an opening of 1.25 mm to 1.75 mm, such as 1.5 mm.

[0137] In some instances, the sample injection port is a conduit located at the proximal end of the flow cell nozzle chamber. For example, the sample injection port can be a conduit positioned so that the orifice of the sample injection port is in line with the orifice of the flow cell nozzle. In the case where the sample injection port is a conduit positioned in line with the orifice of the flow cell nozzle, the cross-sectional shape of the sample injection tube can be any suitable shape, wherein the cross-sectional shape of interest includes but is not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). The orifice of the conduit can vary according to the shape, and in some instances, the opening of the orifice is 0.1mm to 5.0mm, such as 0.2mm to 3.0mm, such as 0.5mm to 2.5mm, such as 0.75mm to 2.25mm, such as 1mm to 2mm, and includes an opening of 1.25mm to 1.75mm, such as an opening of 1.5mm. The tip shape of the sample injection port can be the same or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a bevel tip having a bevel angle of 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, and including a bevel angle of 5°.

[0138] In some embodiments, the flow cell nozzle further comprises a sheath liquid injection port configured to provide sheath liquid to the flow cell nozzle. In an embodiment, the sheath liquid injection system is configured to provide sheath liquid to the flow cell nozzle chamber, for example, to provide sheath liquid to the flow cell nozzle chamber together with the sample to generate a laminar sheath liquid flow around the sample flow stream. Depending on the desired characteristics of the flow stream, the rate of delivery of sheath liquid to the flow cell nozzle chamber can be 25 μL / sec or higher, such as 50 μL / sec or higher, such as 75 μL / sec or higher, such as 100 μL / sec or higher, such as 250 μL / sec or higher, such as 500 μL / sec or higher, such as 750 μL / sec or higher, such as 1000 μL / sec or higher and including 2500 μL / sec or higher. For example, the sheath fluid flow rate can be 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 and including 10 μL / sec to about 100 μL / sec.

[0139] In some embodiments, the sheath fluid injection port is an orifice located in the nozzle chamber wall. The orifice of the sheath fluid injection port can be any suitable shape, where the cross-sectional shapes of interest include, but are not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom portion coupled to a planar top portion). In some instances, the size of the orifice of the sample injection port can vary depending on the shape, with an opening of 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, and including an opening of 1.25 mm to 1.75 mm, such as an opening of 1.5 mm.

[0140] In some instances, the subject system includes a sample interrogation region that is fluidly connected to the flow cell nozzle orifice. In these instances, the sample flow stream flows out of the orifice at the far end of the flow cell nozzle, and a light source can be used to illuminate the particles in the flow stream at the sample interrogation region. The size of the interrogation region can vary according to the characteristics of the flow nozzle (e.g., the size of the nozzle orifice and the size of the sample injection port). In an embodiment, the width of the interrogation region can be 0.01 mm or greater, such as 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, and including 10 mm or greater. The length of the interrogation zone may also vary, and in some instances the length is 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 or greater, such as 15 mm or greater, such as 20 mm or greater, such as 25 mm or greater and including 50 mm or greater.

[0141] The interrogation region can be configured to facilitate illumination of a planar cross-section of the outgoing flow stream, or can be configured to facilitate illumination of a predetermined length of a diffuse field (e.g., using a diffuse laser or lamp). In some embodiments, the interrogation region includes a transparent window that facilitates illumination of a predetermined length of the outgoing flow stream, the predetermined length being, for example, 1 mm or longer, for example, 2 mm or longer, for example, 3 mm or longer, for example, 4 mm or longer, for example, 5 mm or longer, and including 10 mm or longer. Depending on the light source used to illuminate the outgoing flow stream (as described below), the interrogation region can be configured to pass light of 100 nm to 1500 nm, 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, and including 500 nm to 800 nm.Thus, the interrogation region may be formed of any transparent material that passes the desired wavelength range, including but not limited to optical glass, borosilicate glass, pyrex glass, ultraviolet quartz, infrared quartz, sapphire, and plastics such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics, such as PETG (ethylene glycol modified polyethylene terephthalate), and other polymeric plastic materials including polyesters, where the polyesters of interest may include but are not limited to polyalkylene terephthalates, such as polyethylene terephthalate (PET), bottle grade P ET (copolymers based on monoethylene glycol, terephthalic acid and other comonomers such as isophthalic acid, cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT) and poly(hexamethylene terephthalate); poly(alkylene adipates) such as poly(vinyl adipate), poly(1,4-butylene adipate) and poly(hexamethylene adipate); poly(alkylene suberates) such as poly(ethylene suberate); poly(alkylene sebacates) such as poly(ethylene sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalates) such as poly( isophthalate); poly(alkylene 2,6-naphthalene-dicarboxylates) such as poly(ethylene 2,6-naphthalene-dicarboxylate); poly(alkylene sulfonyl-4,4'-dibenzoate) such as poly(ethylene sulfonyl-4,4'-dibenzoate); poly(p-phenylene alkylene dicarboxylates) such as poly(p-phenylene ethylene dicarboxylates); poly(trans-1,4-cyclohexanediyl alkylene dicarboxylates) such as poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate); poly(1,4-cyclohexane-dimethylene alkylene dicarboxylates) such as poly(1,4-cyclohexane-dimethylene ethylene dicarboxylate); Poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylate) such as poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate); lactic acid polymers and copolymers, such as (S)-polylactide, (R,S)-polylactide, poly(tetramethyl glycolide) and poly(lactide-co-glycolide); and polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A; polyamides, such as polyterephthalamide; polyesters, such as polyethylene terephthalate, such as Mylar. TMPolyethylene terephthalate; etc. In some embodiments, the subject system includes a cuvette located in the sample interrogation area. In embodiments, the cuvette can pass light from 100nm to 1500nm, such as 150nm to 1400nm, such as 200nm to 1300nm, such as 250nm to 1200nm, such as 300nm to 1100nm, such as 350nm to 1000nm, such as 400nm to 900nm, and including light from 500nm to 800nm.

[0142] In some embodiments, the subject system includes a particle sorting component for sorting sample particles (e.g., cells). In some instances, the particle sorting component is a particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed on March 28, 2017, and U.S. Provisional Patent Application No. 62 / 752793, filed on October 30, 2018, the disclosures of which are incorporated herein by reference. In some embodiments, the particle sorting component includes one or more droplet deflectors, such as those described in U.S. Patent Publication No. 2018 / 0095022, filed on June 14, 2017, the disclosures of which are incorporated herein by reference.

[0143] In some embodiments, the subject system is a flow cytometry system. Suitable flow cytometry systems may include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (ed.), 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 Throm 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 disclosure of which is incorporated herein by reference. In certain examples, the flow cytometry system of interest includes the BD Biosciences FACSCanto TM II flow cytometer, BD AccuriTM Flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSR Fortess TM X-20 Flow Cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell Sorter and BD Biosciences Via TM Cell sorter BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM , BD Biosciences Aria TM Cell sorter and BD Biosciences FACSMelody TM Cell sorter, etc.

[0144] In some embodiments, the subject particle sorting system is a flow cytometry system, such as U.S. Patent Nos. 10,006,852; 9952,076; 9933,341; 9784,661; 9726,527; 9453,789; 9200,334; 9097,640; 9095,494; 9092,034; 8975,595; 8753,573; 8233 those described in 146; No.8140300; No.7544326; No.7201875; No.7129505; No.6821740; No.6813017; No.6809804; No.6372506; No.5700692; No.5643796; No.5627040; No.5620842; No.5602039; the disclosures of which are incorporated herein by reference in their entirety.

[0145] In some instances, the subject system is a flow cytometry system configured to characterize and image particles in a flow stream by fluorescence imaging using radio frequency labeled emission (FIRE), such as those described in Diebold et al., Nature Photonics, Vol. 7(10); pp. 806-810 (2013), and in U.S. Patent Nos. 9,423,353, 9,784,661, and 1,000,6852, and in U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0146] Integrated Circuit Equipment

[0147] Various aspects of the present disclosure also include an integrated circuit device programmed to: generate frequency-encoded fluorescence data from particles in a flow stream; and calculate phase-corrected spatial data of the particles by transforming the frequency-encoded fluorescence data using 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. In some instances, the integrated circuit device of interest may include a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), or a complex programmable logic device (CPLD).

[0148] In an embodiment, the integrated circuit device is programmed to generate frequency-encoded fluorescence data. The fluorescence data can be generated from one or more fluorescence detectors (e.g., one or more detection channels), the fluorescence detectors are, for example, 2 or more, for example, 3 or more, for example, 4 or more, for example, 5 or more, for example, 6 or more, and include 8 or more fluorescence detectors (e.g., 8 or more detection channels). 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 detected light scattering. In some instances, the system is configured to generate one or more data components of frequency-encoded fluorescence data based on light absorption detected from the sample (e.g., from a bright field light detector). For example, the system can be configured to generate a phase correction component based on a signal from a bright field detector. In certain embodiments, the system is configured to generate phase-corrected spatial data, which is an interpretation of interferometric phase adjustment of spatial data calculated based on frequency-encoded fluorescence data. In other examples, one or more data components of frequency encoded fluorescence data are generated based on light scatter detected from a sample (eg, from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector).

[0149] In an embodiment, the subject integrated circuit device is programmed to calculate spatial data from the frequency-encoded fluorescence data. The spatial data according to an embodiment of the present disclosure is phase-corrected by transforming the frequency-encoded fluorescence data using a phase-correction component. In some embodiments, the spatial data includes a horizontal size dimension of a particle, a vertical size dimension of a particle, a ratio of particle sizes along two different dimensions, a ratio of sizes of particle components (e.g., a ratio of a horizontal dimension of a cell nucleus to a horizontal dimension of a cytoplasm).

[0150] In some embodiments, the integrated circuit device is programmed to calculate correction transform coefficients for transforming frequency-encoded fluorescence data into phase-corrected spatial data. For example, the phase correction component may include 2 or more correction transform coefficients, such as 3 or more, such as 4 or more, and including 5 or more correction transform coefficients. In the case where the spatial data is calculated by performing a Fourier transform, the phase correction component may include the correction transform coefficients, wherein the Fourier transform generates only real mathematical calculation components (i.e., does not generate imaginary mathematical calculation components).

[0151] In some instances, 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 with a predetermined constant signal to generate a phase adjustment value; and calculating the inverse tangent of the phase adjustment value to generate the first phase adjustment value. In these embodiments, the phase adjustment value is the sum of all frequency points in the discrete Fourier transform of the frequency-encoded fluorescence data.

[0152] In other cases, the integrated circuit device is programmed to calculate a second phase adjustment based on the fluorescence lifetime of a fluorophore in the sample. In these examples, the integrated circuit device is programmed to calculate the second phase adjustment by acquiring signals from all fluorescence detectors to determine the phase present in the signals, and calculating the second phase adjustment based on the fluorescence lifetime of the fluorophore. The subject integrated circuit device can be programmed to calculate the fluorescence lifetime using different detector channels, such as by using 2 or more detection channels, such as 3 or more, such as 4 or more, and including 5 or more detector channels to calculate the fluorescence lifetime.

[0153] In embodiments, the subject integrated circuit device is programmed to calculate phase-corrected spatial data of a particle by transforming the frequency-encoded fluorescence data using a phase-corrected component. In some embodiments, to calculate the phase-corrected spatial data, the system is configured to Fourier transform the frequency-encoded fluorescence data using the phase-corrected component to generate the phase-corrected spatial data of the particle. In other embodiments, the integrated circuit device is programmed to discrete Fourier transform the frequency-encoded fluorescence data using the phase-corrected component to generate the phase-corrected spatial data of the particle. In other embodiments, the integrated circuit device is programmed to perform a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using the phase-corrected component. In other embodiments, the integrated circuit device is programmed to perform a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using the phase-corrected component. In other embodiments, the integrated circuit device is programmed to calculate the phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0154] In certain embodiments, the integrated circuit device is programmed to make sorting decisions (as described above) based on frequency-encoded fluorescence data, calculated spatial data, generated images, one or more determined characteristics of particles (e.g., size, center of mass, eccentricity) determined from calculated spatial data or generated images, or some combination thereof. In these embodiments, the analysis includes classifying and counting particles such that each particle exists as a set of digitized parameter values. The subject integrated circuit device can be programmed to trigger sorting components based on selected parameters so as to distinguish particles of interest from background and noise.

[0155] Kits

[0156] Various aspects of the present disclosure also include a kit, wherein the kit includes one or more of the integrated circuit devices described herein. In some embodiments, the kit may also include programming for the subject system, such as a computer-readable medium (e.g., flash memory drive, USB memory, optical disc, DVD, Blu-ray disc, etc.) or a form of instructions for downloading programming from an Internet network protocol or a cloud server. The kit may also include instructions for practicing the subject method. These instructions may exist in the subject kit in a variety of forms, and one or more of the forms may exist in the kit. One form in which these instructions may exist is as printed information on a suitable medium or substrate (e.g., one or more sheets of paper with information printed thereon, in the packaging of the kit, and in a package instruction, etc.). Another form of these instructions is a computer-readable medium on which information has been recorded, such as a floppy disk, an optical disc (CD), a portable flash drive, etc. Another form of these instructions that may exist is a website address, which may be used to access information at a remote site via the Internet.

[0157] application

[0158] The subject system, method and computer system are used for a variety of applications in which it is desired to analyze and classify the particle composition in a sample (e.g., a biological sample) in a fluid medium. In some embodiments, the system and method described herein can be used for flow cytometry characterization of biological samples labeled with fluorescent tags. In other embodiments, the system and method are used for spectroscopy of emitted light. In addition, the subject system and method are used to increase the signal that can be obtained from the light collected from a sample (e.g., a sample in a fluid stream). Embodiments of the present disclosure are expected to provide an application such as a flow cytometer with improved cell sorting accuracy, enhanced particle collection, particle loading efficiency, more accurate particle loading and enhanced particle deflection during cell sorting.

[0159] Embodiments of the present disclosure are also used to provide the following application, and it can be expected that the cell prepared from biological sample will be used for research, laboratory testing or for treatment.In certain embodiments, subject method and equipment can be conducive to obtaining individual cells prepared from target fluid or tissue biological sample.For example, subject method and system are conducive to obtaining cells from fluid or tissue sample to be used as research or diagnostic specimens of disease (such as cancer).Equally, subject method and system can be conducive to obtaining cells from fluid or tissue sample for treatment.Compared with traditional flow cytometry system, method and equipment of the present disclosure allow to separate and collect cells from biological sample (such as organ, tissue, tissue fragment, fluid) with improved efficiency and low cost.

[0160] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses:

[0161] 1. A method comprising:

[0162] generating frequency-encoded fluorescence data from sample particles in the flow stream; and

[0163] Phase-corrected spatial data of the particle is calculated by transforming the frequency-encoded fluorescence data with a phase correction component.

[0164] 2. A method according to clause 1, wherein the spatial data is calculated by Fourier transforming the frequency encoded fluorescence data with a phase correction component.

[0165] 3. A method according to clause 2, wherein the spatial data is calculated by discrete Fourier transforming the frequency encoded fluorescence data with a phase correction component.

[0166] 4. A method according to clause 2, wherein the spatial data is calculated by performing a short time Fourier transform (STFT) on the frequency encoded fluorescence data with a phase correction component.

[0167] 5. The method of clause 1, wherein the spatial data is calculated using a digital lock-in amplifier to heterodyne the frequency encoded fluorescence data and to demultiplex the frequency encoded fluorescence data.

[0168] 6. A method according to any one of clauses 1 to 5, wherein the phase correction component comprises correction transformation coefficients for transforming the frequency encoded fluorescence data into phase corrected spatial data.

[0169] 7. A method according to any one of clauses 1 to 6, wherein generating frequency encoded fluorescence data from the particles comprises detecting light from the particles in the sample using a light detection system.

[0170] 8. The method of clause 7, wherein the light detected from the particles comprises light absorption, light scattering, emitted light, or a combination thereof.

[0171] 9. The method of clause 8, wherein light absorption is detected using a bright field photodetector.

[0172] 10. The method according to any one of clauses 8 to 9, wherein the emitted light is detected using a fluorescence detector.

[0173] 11. A method according to any of clauses 1 to 10, wherein the phase correction component comprises a first phase adjustment and a second phase adjustment.

[0174] 12. The method of clause 11, wherein the first phase adjustment comprises an output signal from a light detection system.

[0175] 13. The method of clause 12, wherein the first phase adjustment comprises an output signal from a bright field photodetector.

[0176] 14. The method of clause 13, further comprising calculating the first phase adjustment by:

[0177] multiplying an output signal from a bright field photodetector by a predetermined constant signal to generate a phase adjustment value; and

[0178] An arc tangent of the phase adjustment value is calculated to generate a first phase adjustment.

[0179] 15. The method of clause 14, wherein the phase adjustment value is the sum of all frequency bins in a discrete Fourier transform of the frequency encoded fluorescence data.

[0180] 16. The method according to any of clauses 11 to 15, wherein the first phase adjustment is an interferometric phase adjustment.

[0181] 17. The method of clause 16, wherein the interferometric phase adjustment comprises a phase shift from a light source configured to illuminate the sample in the flow stream.

[0182] 18. The method according to clause 17, wherein the light source comprises a beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam.

[0183] 19. The method of clause 18, wherein the light beam generator comprises an acousto-optic deflector.

[0184] 20. A method according to any of clauses 18 to 19, wherein the beam generator comprises a direct digital synthesizer (DDS) RF comb generator.

[0185] 21. A method according to any one of clauses 18 to 20, wherein the beam generator component is configured to generate a frequency-shifted local oscillator beam.

[0186] 22. A method according to any one of clauses 17 to 21, wherein the light source comprises a laser.

[0187] 23. The method according to clause 22, wherein the laser is a continuous wave laser.

[0188] 24. A method according to any of clauses 17 to 23, wherein the interferometric phase adjustment comprises a phase shift caused by vibrations between components of the light source.

[0189] 25. The method of any one of clauses 11 to 24, further comprising calculating a second phase adjustment based on a fluorescence lifetime of a fluorophore in the sample.

[0190] 26. A method according to any one of clauses 1 to 25, wherein the phase-corrected spatial data of the particles is calculated from the frequency-encoded fluorescence data by an integrated circuit device.

[0191] 27. The method of clause 26, wherein the integrated circuit device is a field programmable gate array (FPGA).

[0192] 28. The method of clause 26, wherein the integrated circuit device is an application specific integrated circuit (ASIC).

[0193] 29. The method of clause 26, wherein the integrated circuit device is a complex programmable logic device (CPLD).

[0194] 30. The method of any one of clauses 1 to 29, further comprising illuminating the flow stream with a light source.

[0195] 31. A method according to clause 30, wherein the flow stream is illuminated with a light source having a wavelength of 200 nm to 800 nm.

[0196] 32. A method according to any of clauses 30 to 31, wherein the method comprises illuminating the flow stream with a first frequency shifted light beam and a second frequency shifted light beam.

[0197] 33. The method of clause 32, wherein the first frequency-shifted beam comprises a local oscillator (LO) beam and the second frequency-shifted beam comprises a radio frequency comb beam.

[0198] 34. A method according to any one of clauses 32 to 33, further comprising:

[0199] applying a radio frequency drive signal to the acousto-optic device; and

[0200] The acousto-optic device is illuminated with a laser to generate a first frequency-shifted light beam and a second frequency-shifted light beam.

[0201] 35. A method according to clause 34, wherein the laser is a continuous wave laser.

[0202] 36. A method according to any one of clauses 1 to 35, further comprising generating an image of the particle based on the phase-corrected spatial data.

[0203] 37. The method of clause 36, further comprising generating an image mask of the particles.

[0204] 38. A method according to any one of clauses 1 to 37, further comprising sorting the particles.

[0205] 39. A system comprising:

[0206] a light source configured to illuminate a sample including particles in a flow stream;

[0207] Light detection system; and

[0208] A processor comprising a memory operatively coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, configure the processor to:

[0209] Generate frequency-encoded fluorescence data based on particles in the flow stream;

[0210] Phase-corrected spatial data of the particle is calculated by transforming the frequency-encoded fluorescence data with a phase correction component.

[0211] 40. The system of clause 39, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to Fourier transform the frequency encoded fluorescence data using the phase correction component to generate phase-corrected spatial data of the particle.

[0212] 41. The system of clause 40, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to discrete Fourier transform the frequency encoded fluorescence data using the phase correction component to generate phase-corrected spatial data of the particle.

[0213] 42. The system of clause 40, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform a short time Fourier transform (STFT) on the frequency encoded fluorescence data using the phase correction component.

[0214] 43. A system according to clause 40, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency encoded fluorescence data and demultiplex the frequency encoded fluorescence data.

[0215] 44. A system according to any of clauses 39 to 43, wherein the memory includes instructions stored thereon which, when executed by the processor, cause the processor to transform the frequency encoded fluorescence data into spatial data using a phase correction component including a modified transform coefficient.

[0216] 45. The system of any one of clauses 39 to 44, wherein the light detection system comprises a photodetector configured to detect one or more of light absorption, light scattering, and fluorescence.

[0217] 46. ​​The system of clause 45, wherein the light detection system comprises a bright field photodetector.

[0218] 47. A system according to any one of clauses 39 to 46, wherein the light detection system comprises a fluorescence detector.

[0219] 48. The system of any of clauses 44 to 47, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction component comprising the first phase adjustment and the second phase adjustment.

[0220] 49. The system of clause 48, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the first phase adjustment by:

[0221] multiplying an output signal from a bright field photodetector by a predetermined constant signal to generate a phase adjustment value; and

[0222] An arc tangent of the phase adjustment value is calculated to generate a first phase adjustment.

[0223] 50. The system of clause 49, wherein the phase adjustment value is the sum of all frequency bins in a discrete Fourier transform of the frequency encoded fluorescence data.

[0224] 51. The system of any of clauses 48 to 50, wherein the first phase adjustment is an interferometric phase adjustment.

[0225] 52. The system of clause 51, wherein the interferometric phase adjustment comprises a phase shift from a light source.

[0226] 53. A system according to any of clauses 39 to 52, wherein the light source comprises a beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam.

[0227] 54. A system according to clause 53, wherein the light beam generator comprises an acousto-optic deflector.

[0228] 55. A system according to any of clauses 53 to 54, wherein the beam generator comprises a direct digital synthesizer (DDS) RF comb generator.

[0229] 56. A system according to any one of clauses 53 to 55, wherein the beam generator component is configured to generate a frequency-shifted local oscillator beam.

[0230] 57. A system according to any one of clauses 39 to 56, wherein the light source comprises a laser.

[0231] 58. A system according to clause 57, wherein the laser is a continuous wave laser.

[0232] 59. A system according to any of clauses 51 to 58, wherein the interferometric phase adjustment comprises a phase shift caused by vibrations between components of the light source.

[0233] 60. The system of any of clauses 39 to 59, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the second phase adjustment based on a fluorescence lifetime of a fluorophore in the sample.

[0234] 61. A system according to any one of clauses 39 to 60, comprising an integrated circuit component programmed to:

[0235] generating frequency-encoded fluorescence data from sample particles in a flow stream;

[0236] Phase-corrected spatial data of the particle is calculated by transforming the frequency-encoded fluorescence data with a phase correction component.

[0237] 62. The system of clause 61, wherein the integrated circuit device is a field programmable gate array (FPGA).

[0238] 63. The system of clause 61, wherein the integrated circuit device is an application specific integrated circuit (ASIC).

[0239] 64. The system of clause 61, wherein the integrated circuit device is a complex programmable logic device (CPLD).

[0240] 65. A system according to any one of clauses 39 to 64, wherein the system is a flow cytometer.

[0241] 66. A system according to any of clauses 39-65, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an image of the particle based on the phase-corrected spatial data.

[0242] 67. The system of clause 66, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an image mask of the particles.

[0243] 68. A system according to any one of clauses 39 to 67, further comprising a cell sorting component configured to sort cells in a sample based on the calculated phase-corrected spatial data.

[0244] 69. A system according to clause 68, wherein the cell sorting component includes a droplet deflector.

[0245] 70. An integrated circuit programmed to perform the following operations:

[0246] Generate frequency-encoded fluorescence data based on particles in the flow stream;

[0247] Phase-corrected spatial data of the particle is calculated by transforming the frequency-encoded fluorescence data with a phase correction component.

[0248] 71. An integrated circuit according to clause 70, wherein the integrated circuit is programmed to Fourier transform the frequency encoded fluorescence data with a phase correction component to generate phase corrected spatial data of the particle.

[0249] 72. An integrated circuit according to clause 71, wherein the integrated circuit is programmed to perform a discrete Fourier transform on the frequency encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle.

[0250] 73. An integrated circuit according to clause 71, wherein the integrated circuit is programmed to perform a short-time Fourier transform on the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particles.

[0251] 74. An integrated circuit according to clause 70, wherein the integrated circuit is programmed to utilize a digital lock-in amplifier to calculate the phase-corrected spatial data, to heterodyne the frequency-encoded fluorescence data and to demultiplex the frequency-encoded fluorescence data.

[0252] 75. An integrated circuit according to any of clauses 70 to 74, wherein the integrated circuit is programmed to transform the frequency encoded fluorescence data into spatial data using a phase correction component including a modified transform coefficient.

[0253] 76. An integrated circuit according to any of clauses 70 to 75, wherein the integrated circuit is programmed to calculate a phase correction component comprising a first phase adjustment and a second phase adjustment.

[0254] 77. An integrated circuit according to clause 76, wherein the integrated circuit is programmed to calculate the first phase adjustment by:

[0255] multiplying an output signal from a bright field photodetector by a predetermined constant signal to produce a phase adjustment value; and

[0256] An arc tangent of the phase adjustment value is calculated to generate a first phase adjustment.

[0257] 78. An integrated circuit according to clause 77, wherein the phase adjustment value is the sum of all frequency bins in a discrete Fourier transform of the frequency encoded fluorescence data.

[0258] 79. An integrated circuit according to any of clauses 76 to 78, wherein the first phase adjustment is an interferometric phase adjustment.

[0259] 80. An integrated circuit according to clause 79, wherein the interferometric phase adjustment comprises a phase shift from a light source configured to illuminate the sample in the flow stream.

[0260] 81. An integrated circuit according to clause 80, wherein the light source comprises a beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam.

[0261] 82. An integrated circuit according to clause 81, wherein the optical beam generator comprises an acousto-optic deflector.

[0262] 83. An integrated circuit according to any of clauses 81 to 82, wherein the beam generator comprises a direct digital synthesizer (DDS) RF comb generator.

[0263] 84. An integrated circuit according to any of clauses 81 to 83, wherein the beam generator component is configured to generate a frequency-shifted local oscillator beam.

[0264] 85. An integrated circuit according to any of clauses 80 to 84, wherein the interferometric phase adjustment comprises a phase shift caused by vibrations between components of the light source.

[0265] 86. An integrated circuit according to any one of clauses 76 to 85, wherein the integrated circuit is programmed to calculate the second phase adjustment based on the fluorescence lifetime of a fluorophore in the sample.

[0266] 87. An integrated circuit according to any of clauses 69 to 86, wherein the integrated circuit is a field programmable gate array (FPGA).

[0267] 88. An integrated circuit according to any one of clauses 69 to 86, wherein the integrated circuit device is an application specific integrated circuit (ASIC).

[0268] 89. An integrated circuit according to any one of clauses 69 to 86, wherein the integrated circuit device is a complex programmable logic device (CPLD).

[0269] 90. An integrated circuit according to any of clauses 69 to 89, wherein the integrated circuit is programmed to generate an image of the particle based on the phase-corrected spatial data.

[0270] 91. An integrated circuit according to clause 90, wherein the integrated circuit is programmed to generate an image mask of the particles.

[0271] 92. An integrated circuit according to any one of clauses 69 to 91, wherein the integrated circuit is programmed to generate a binning decision based on the phase-corrected spatial data.

[0272] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art based on the teachings of the invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0273] Therefore, the foregoing only illustrates the principles of the present invention. It should be understood that, although not explicitly described or shown herein, those skilled in the art will be able to design various arrangements that embody the principles of the present invention and are included in the spirit and scope of the present invention. In addition, all examples and conditional language described herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to promote this field, and should be interpreted as not being limited to such specific examples and conditions of narration. In addition, all statements describing the principles, aspects and embodiments of the present invention and their specific examples herein are intended to cover the equivalents of their structures and functions. In addition, such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, any element that performs the same function regardless of the structure. In addition, regardless of whether the disclosure is explicitly described in the claims, any content disclosed herein is not intended to be used by the public.

[0274] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 USC § 112 (f) or 35 USC § 112 (6) is expressly defined as being cited as a limitation to the claim only when the exact phrase "means for..." or the exact phrase "step for..." is cited at the beginning of such limitation in the claim; if such exact phrase is not used in the limitation of the claim, 35 USC § 112 (f) or 35 USC § 112 (6) is not cited.

Claims

1. A method for characterizing particles of a sample in a flowing stream, the method comprising: generating frequency-encoded fluorescence data based on particles of the sample in the flow stream; as well as Phase-corrected spatial data of the particle is calculated by transforming the frequency-encoded fluorescence data with a phase correction component including interferometric phase adjustment.

2. The method according to claim 1, wherein: The phase-corrected spatial data is calculated by Fourier transforming the frequency-encoded fluorescence data using the phase-corrected component or using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplexing the frequency-encoded fluorescence data.

3. The method of claim 2, wherein the phase-corrected spatial data is calculated by performing a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using the phase correction component or performing a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using the phase correction component.

4. The method according to any one of claims 1 to 3, wherein: The phase correction component includes correction transform coefficients used to transform the frequency encoded fluorescence data into the phase corrected spatial data.

5. The method according to any one of claims 1 to 3, wherein: Generating the frequency-encoded fluorescence data from the particles includes detecting light from particles in the sample using a light detection system.

6. The method according to claim 5, wherein: Light detected from the particles includes light absorption, light scattering, emitted light, or a combination thereof.

7. The method according to any one of claims 1 to 3, wherein: The phase correction component includes a first phase adjustment and a second phase adjustment.

8. The method according to claim 7, wherein: The first phase adjustment includes an output signal from a bright field photodetector from a light detection system, the method further comprising calculating the first phase adjustment by: multiplying the output signal from the bright field photodetector by a predetermined constant signal to produce a phase adjustment value; and An arc tangent of the phase adjustment value is calculated to generate the first phase adjustment.

9. The method according to claim 8, wherein: The first phase adjustment includes the interferometric phase adjustment.

10. The method according to claim 1, wherein: The interferometric phase adjustment includes phase shifts caused by vibrations between components of the light source.

11. The method of claim 7, further comprising calculating the second phase adjustment based on a fluorescence lifetime of a fluorophore in the sample.

12. The method according to any one of claims 1 to 3, wherein: Phase-corrected spatial data of the particle is calculated from the frequency-encoded fluorescence data by an integrated circuit device.

13. The method of any one of claims 1 to 3, further comprising generating an image of the particles based on the phase-corrected spatial data.

14. A system for characterizing particles of a sample in a flowing stream, the system comprising: a light source configured to illuminate a sample including particles in a flow stream; Light detection system; as well as A processor comprising a memory operatively coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to: generating frequency-encoded fluorescence data based on particles in the flow stream; Phase-corrected spatial data of the particle is calculated by transforming the frequency-encoded fluorescence data with a phase correction component including interferometric phase adjustment.

15. An integrated circuit programmed to perform the following operations: Generate frequency-encoded fluorescence data based on particles in the flow stream; Phase-corrected spatial data of the particle is calculated by transforming the frequency-encoded fluorescence data with a phase correction component including interferometric phase adjustment.

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