阅读说明：本技术 射频复用信号的相位校正 (Phase correction of RF multiplex signals ) 是由 乔纳森·林 基根·奥斯利 马修·巴尔 于 2020-05-29 设计创作，主要内容包括：本公开的各方面包括用于表征流动流中的样品颗粒的方法。根据某些实施例的方法包括根据流动流中的样品颗粒生成频率编码的荧光数据；以及通过利用相位校正分量对频率编码的荧光数据进行变换来计算颗粒的相位校正的空间数据。在某些实施例中,方法包括基于相位校正的空间数据生成流动流中的颗粒的图像。还描述了具有处理器的系统,该处理器具有可操作地耦合到该处理器的存储器,该存储器具有存储在其上的指令,该指令在由该处理器执行时使处理器根据流动流的颗粒的频率编码的荧光数据来计算相位校正的空间数据。还提供了具有编程用于实践主题方法的集成电路设备(例如现场可编程门阵列)。(Aspects of the present disclosure include methods for characterizing sample particles in a flow stream. A method according to some embodiments includes generating frequency-encoded fluorescence data from sample particles in a flow stream; and calculating phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with the phase correction component. In certain embodiments, the method includes generating an image of the particle in the flow stream based on the phase corrected spatial data. A system is also described having a processor with a memory operatively coupled to the processor, the memory having instructions stored thereon that, when executed by the processor, cause the processor to calculate phase corrected spatial data from frequency encoded fluorescence data of particles of a flow stream. An integrated circuit device (e.g., a field programmable gate array) having programming for practicing the subject methods is also provided.)

1. A method, comprising:

generating frequency-encoded fluorescence data from particles of a sample in a flow stream; and

calculating phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with a phase correction component.

2. The method of claim 1, wherein the spatial data is calculated by fourier transforming the frequency encoded fluorescence data with the phase correction component or with a digital lock-in amplifier to heterodyne the frequency encoded fluorescence data and demultiplex the frequency encoded fluorescence data.

3. The method of claim 2, wherein the spatial data is calculated by discrete fourier transforming the frequency encoded fluorescence data with the phase correction component or Short Time Fourier Transforming (STFT) the frequency encoded fluorescence data with the phase correction component.

4. The method of any of claims 1 to 3, wherein the phase correction component comprises a modified transform coefficient used to transform the frequency encoded fluorescence data into the phase corrected spatial data.

5. The method of any one of claims 1 to 4, wherein generating the frequency-encoded fluorescence data from the particles comprises detecting light from particles in the sample with a light detection system.

6. The method of claim 5, wherein the light detected from the particles comprises light absorption, light scattering, emitted light, or a combination thereof.

7. The method of any of claims 1-6, wherein the phase correction component comprises a first phase adjustment and a second phase adjustment.

8. The method of claim 7, wherein the first phase adjustment comprises an output signal from the light detection system, such as an output signal from a bright field photodetector, the method further comprising calculating the first phase adjustment by:

multiplying an output signal from the bright field photodetector with a predetermined constant signal to produce a phase adjustment value; and

calculating an arctangent of the phase adjustment value to generate the first phase adjustment.

9. The method of any of claims 8, wherein the first phase adjustment is an interferometric phase adjustment.

10. The method of any of claims 9, wherein the interferometric phase adjustment comprises a phase shift caused by vibration between components of a light source.

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

12. The method of any one of claims 1 to 11, 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 12, further comprising generating an image of the particle from the phase corrected spatial data.

14. A system, comprising:

a light source configured to illuminate a sample comprising particles in a flow stream;

a light detection system; and

a processor comprising a memory operably 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 from particles in the flow stream;

calculating phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with a phase correction component.

15. An integrated circuit programmed to perform the following operations:

generating frequency-encoded fluorescence data from particles in the flow stream;

calculating phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with a phase correction component.

Disclosure of Invention

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

In an embodiment, frequency-encoded fluorescence data from particles in the sample is generated from light detected in an interrogation zone of the flow stream. In some embodiments, the particle is a cell. 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 from particles. In some embodiments, the method further comprises detecting light absorption, light scattering, or a combination thereof. In some embodiments, particles having one or more fluorophores are illuminated with a plurality of frequency-shifted beams from a beam generator to generate frequency-encoded fluorescence. In one example, a plurality of locations on the (horizontal axis) flow stream are illuminated by laser beams, including a local oscillator beam and a plurality of radio frequency shifted laser beams, such that different locations on the flow stream are illuminated by one of the local oscillator beam and the radio frequency shifted beams. In some examples, the local oscillator is a frequency-shifted beam from a laser. In this example, each spatial location 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 that location. In some embodiments, the frequency encoded data from the particle comprises a spatially encoded beat frequency on a horizontal axis of the particle in the flow stream.

In practicing the subject methods, light from a sample in a flow stream is detected in an interrogation zone and frequency encoded data from particles in the sample is generated. In some embodiments, the particles detected in the interrogation zone comprise cells. In some embodiments, the method includes detecting one or more of light absorption, light scattering, light emission (e.g., fluorescence) from the sample in the flow stream. In some examples, phase-corrected spatial data of one or more particles in the sample is generated from detected light absorption (e.g., bright field image data). In other examples, phase-corrected spatial data for one or more particles in the sample is generated from detected light scatter (e.g., forward scatter image data, side scatter image data). In other examples, phase-corrected spatial data of one or more particles in the sample is generated from detected fluorescence (e.g., fluorescently labeled image data). In other examples, phase-corrected spatial data for one or more particles in the sample is generated from a combination of two or more of the detected light absorption, the detected light scattering, and the detected fluorescence.

In an embodiment, frequency-encoded fluorescence data from a particle in the flow stream is transformed with a phase correction component to give spatial data of the particle. In an embodiment, the spatial data may include a horizontal dimension of the particle, a vertical dimension of the particle, a ratio of particle sizes along two different dimensions, a size ratio of particle composition (e.g., a ratio of a horizontal dimension of the nucleus to a horizontal dimension of the cytoplasm). In some embodiments, the frequency encoded fluorescence data is transformed by fourier transforming the frequency encoded fluorescence data with the phase correction component. In some examples, the frequency-encoded fluorescence data is transformed by Discrete Fourier Transforming (DFT) the frequency-encoded fluorescence data with the phase correction component. In other examples, the phase corrected spatial data is calculated by Short Time Fourier Transforming (STFT) the frequency encoded fluorescence data with the phase correction component. In other examples, the phase corrected spatial data is calculated using a digital lock-in amplifier to heterodyne and demultiplex the frequency encoded fluorescence data.

In some embodiments, the method includes determining a phase correction component used to transform the frequency encoded fluorescence data into phase corrected spatial data. In some examples, the phase correction component includes a modified transform coefficient. In certain embodiments, the phase correction component includes a first phase adjustment and a second phase adjustment. In some examples, 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.

In some embodiments, the first phase adjustment is calculated by: multiplying an output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and calculating an arctangent of the phase adjustment value to generate a first phase adjustment. In these embodiments, the phase adjustment value is the sum of all frequency bins (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 may be a light beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. The light 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 vibration between components of the beam generator. In some embodiments, the second phase adjustment is based on a fluorescence lifetime of a fluorophore in the sample. In these embodiments, the second phase adjustment may be calculated as follows: signals from all fluorescence detectors are acquired to determine the phase present in the signal and a second phase adjustment is calculated based on the fluorescence lifetime of the fluorophore.

Methods according to certain embodiments further comprise 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 examples, the first sample composition collection location comprises a sample collection container and the second sample composition collection location comprises a waste collection container.

Aspects of the present disclosure include systems for characterizing sample particles (e.g., cells in a biological sample) in a flow stream. A system according to some embodiments includes a light source configured to illuminate a sample having particles in a flow stream, a light detection system having a photodetector and a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to: generating frequency-encoded fluorescence data from particles in the flow stream; and calculating phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with the phase correction component.

In an embodiment, the system is configured to generate frequency-encoded fluorescence data from particles in the sample illuminated with the light source. In some embodiments, the light source comprises a light beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. The optical beam generator according to some examples includes a laser (e.g., a continuous wave laser) and an acoustic optical deflector (e.g., coupled to a direct digital synthesizer RF comb generator). The subject systems include a light detection system configured to detect one or more of light absorption, light scattering, light emission (e.g., fluorescence) from a sample in a flow stream. In some examples, the light detection system includes a photodetector (e.g., a bright field photodetector) for detecting light absorption. In other examples, the light detection system includes a photodetector (e.g., a forward scatter detector, a side scatter detector) for detecting light scatter. In other examples, the light detection system includes a photodetector for detecting fluorescence. In other examples, the light detection system comprises a combination of two or more of: light absorption detectors, light scattering detectors, and light detectors that emit (e.g., fluorescence).

In an embodiment, the subject system includes a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with the phase correction component. In an embodiment, the spatial data may include a horizontal dimension of the particle, a vertical dimension of the particle, a ratio of particle sizes along two different dimensions, a size ratio of particle composition (e.g., a ratio of a horizontal dimension of the nucleus to a horizontal dimension of the cytoplasm). In some embodiments, to calculate the phase corrected spatial data, the system is configured to fourier transform the frequency encoded fluorescence data with the phase correction component to generate 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 with the phase correction 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 with the phase correction component. In other embodiments, the system is configured to calculate phase corrected spatial data using a digital lock-in amplifier to heterodyne and demultiplex frequency encoded fluorescence data.

In some embodiments, the system includes a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine a phase correction component used to transform the frequency encoded fluorescence data into phase corrected spatial data. In some examples, the phase correction component includes a modified transform 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 the light detection system. For example, the first phase adjustment may include an output signal from a bright field photodetector.

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 the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and calculating an arctangent of the phase adjustment value to generate a 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 other embodiments, the 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 the 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: signals from all fluorescence detectors are acquired to determine the phase present in the signal and a second phase adjustment is calculated from the fluorescence lifetime of the fluorophore to calculate the second phase adjustment.

In certain examples, the system of interest is configured for sorting 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 the inlet of the particle sorting component and one or more sample collection containers for receiving the sorted particles from the flow stream.

Aspects of the present disclosure also include an integrated circuit device programmed to perform the following operations: generating frequency-encoded fluorescence data from particles in the flow stream; phase corrected spatial data of the particles is calculated by transforming the frequency encoded fluorescence data with the phase correction component. In some embodiments, the integrated circuit device is programmed to sort the particles, for example, to 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).

An integrated circuit device according to some embodiments is programmed to generate frequency encoded fluorescence data from particles in a flow stream. In some embodiments, the integrated circuit device is programmed to generate frequency encoded fluorescence data from a data signal (e.g., bright field image data) from the light absorption detector. In other embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from data signals (e.g., forward scatter image data, side scatter image data) from the light scatter detector. In other embodiments, the integrated circuit device is programmed to generate frequency encoded fluorescence data from data signals (e.g., fluorescence label image data) from the light emission detector. In other examples, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from a combination of two or more of the detected light absorption, the detected light scattering, and the detected fluorescence.

In an embodiment, the subject integrated circuit device is programmed to calculate phase corrected spatial data of the particles by transforming the frequency encoded fluorescence data with the phase correction component. In some examples, the integrated circuit device is programmed to fourier transform the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle. In other examples, the integrated circuit device is programmed to generate phase corrected spatial data of the particle by discrete fourier transforming the frequency encoded fluorescence data with the phase correction component. In other examples, the integrated circuit device is programmed to generate phase corrected spatial data of the particle by short time fourier transforming the frequency encoded fluorescence data with the phase correction component. In other examples, the integrated circuit device is programmed to calculate phase corrected spatial data using a digital lock-in amplifier to heterodyne and demultiplex frequency encoded fluorescence data.

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 examples, the phase correction component includes a modified transform 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 examples, 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.

In some embodiments, the integrated circuit device is programmed to calculate the first phase adjustment by: multiplying an output signal from the bright field photodetector by a predetermined constant signal to generate a phase adjustment value; and calculating an arctangent of the phase adjustment value to generate a first phase adjustment value. 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 other embodiments, the integrated circuit device is programmed to calculate the 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 integrated circuit by: signals from all fluorescence detectors are acquired to determine the phase present in the signal and a second phase adjustment is calculated based on the fluorescence lifetime of the fluorophore.

Drawings

The invention is best understood from the following detailed description when read with the accompanying drawing figures. Included in the drawing are the following figures:

FIG. 1 depicts a flow chart for generating frequency encoded fluorescence data and calculating phase corrected spatial data from the frequency encoded fluorescence data according to certain embodiments.

Fig. 2 depicts generating an image of a particle using phase corrected spatial data in comparison to an image of spatial data without phase correction according to an embodiment.

Detailed Description

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

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

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

Certain ranges are presented herein as numerical values preceded by the term "about". As used herein, the term "about" is used to provide literal support for the exact number following it, as well as numbers that are close or approximate to the number following the term. In determining whether a number is near or approximate to a specifically recited number, the near or approximate non-recited number may be a number that provides substantial equivalents of the specifically recited number in the context in which it is presented.

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

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and were set forth in its entirety herein to disclose and describe the methods and/or materials in connection with which the publications were cited. 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. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," "the," and the like include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, 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.

It will be apparent to those skilled in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

Although the apparatus and method have or will be described using functional explanations for grammatical fluidity, it is to be expressly understood that unless a claim is expressly formulated under 35 u.s.c. § 112, it is not to be construed as being limited in any way by the construction of "means" or "steps" limitations, but is to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the doctrine of equivalents, and in the case where the claim is expressly formulated under 35 u.s.c. § 112 is to be accorded full statutory equivalents under 35 u.s.c. § 112.

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, a method for generating frequency encoded fluorescence data from a sample particle in a flow stream and calculating phase corrected spatial data of the particle is 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 having programming for generating frequency encoded fluorescence data from sample particles in a flow stream and calculating phase corrected spatial data for the particles.

Method for characterizing particles in a sample

Various aspects of the present disclosure include methods for characterizing sample particles (e.g., cells in a biological sample). In practicing methods according to certain embodiments, a sample having cells in a flow stream is illuminated with a light source and light from the sample is detected to generate frequency-encoded fluorescence data from the particles and to calculate phase-corrected spatial data for the particles by transforming the frequency-encoded fluorescence data with a phase correction component. In some embodiments, the sample is a biological sample. The term "biological sample" is used in its conventional sense to refer to a subset of whole biological, plant, fungal, or animal tissue, cells, or components, which in some cases may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amnion cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to a natural organism or a subset of its tissues as well as homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, skin slices, respiratory tract, gastrointestinal tract, cardiovascular, genitourinary 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 a derivative thereof, such as plasma, tears, urine, semen, etc., wherein in some instances the sample is a blood sample comprising whole blood, such as blood obtained from venipuncture or fingertip blood collection (wherein the blood may or may not be mixed with any reagents (e.g., preservatives, anticoagulants, etc.) prior to testing).

In certain embodiments, the sample source is a "mammal" or "mammalian", where these terms are used broadly to describe organisms of the class mammalia, including the orders carnivora (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some examples, the subject is a human. The methods can be applied to samples obtained from human subjects that are amphoteric and at any stage of development (i.e., neonatal, infant, juvenile, adolescent, adult), wherein in certain embodiments the human subject is a juvenile, adolescent, or adult. While the invention can be applied to samples from human subjects, it is to 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).

In practicing the subject methods, a sample (e.g., a cell in a flow stream of a flow cytometer) having particles is illuminated with light from a light source. In some embodiments, the light source is a broadband light source that emits light having a broad wavelength range, for example, a span of 50nm or more, for example 100nm or more, for example 150nm or more, for example 200nm or more, for example 250nm or more, for example 300nm or more, for example 350nm or more, for example 400nm or more, and including a span of 500nm or more. For example, a suitable broadband light source emits light having a wavelength of 200nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having a wavelength of 400nm to 1000 nm. Where the method includes illumination with a broadband light source, the broadband light source protocol of interest may include, but is not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with continuous spectrum, ultra-bright light emitting diodes, semiconductor light emitting diodes, broad spectrum LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof.

In other embodiments, the method comprises illuminating with a narrow band light source emitting a specific wavelength or narrow range of wavelengths, for example with a light source emitting light of a narrow range of wavelengths, for example 50nm or less, for example 40nm or less, for example 30nm or less, for example 25nm or less, for example 20nm or less, for example 15nm or less, for example 10nm or less, for example 5nm or less, for example 2nm or less and comprising a light source emitting light of a specific wavelength (i.e. monochromatic light). Where the method includes illumination 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.

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 may be pulsed or continuous wave lasers. For example, the laser may be a gas laser (e.g., helium-neon, argon, krypton, xenon, nitrogen, CO)₂Lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers or xenon-fluorine (XeF) excimer lasers or combinations thereof), dye lasers (e.g., stilbene, coumarin or rhodamine lasers), metal vapor lasers (e.g., helium cadmium (HeCd) lasers, helium mercury (HeHg) excimer lasers, and combinations thereof)A laser, a helium selenium (HeSe) laser, a helium silver (HeAg) laser, a strontium laser, a neon copper (NeCu) laser, a copper laser, or a gold laser, and combinations 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: YVO₄Laser, Nd: YCa₄O(BO₃)₃Laser, Nd: YCOB laser, Ti sapphire laser, Tm YAG laser, Yb₂O₃Lasers or cerium-doped lasers and combinations thereof), semiconductor diode lasers, Optically Pumped Semiconductor Lasers (OPSL) or any of the above implemented as frequency or frequency tripled lasers.

The sample in the flow stream may be illuminated with one or more of the light sources described above (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 sources may comprise any combination of types of light sources. For example, in some embodiments, a method includes illuminating a sample in a flow stream with an array of lasers (e.g., an array having one or more gas lasers, one or more dye lasers, and one or more solid state lasers).

The sample may be illuminated with wavelengths from 200nm to 1500nm (e.g. 250nm to 1250nm, such as 300nm to 1000nm, such as 350nm to 900nm and including 400nm to 800 nm). For example, in the case where the light source is a broadband light source, the sample may be irradiated with a wavelength of 200nm to 900 nm. In other examples, where the light source comprises a plurality of narrow band light sources, the sample may be illuminated with a particular wavelength of 200nm to 900 nm. For example, the light source may be a plurality of narrow band LEDs (1nm-25nm), each LED independently emitting light in the wavelength range of 200nm to 900 nm. In other embodiments, the narrow band light source comprises one or more lasers (e.g., a laser array) and illuminates with a specific wavelength of 200nm to 700nm, for example 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.

Where more than one light source is employed, the sample may be illuminated with the light sources simultaneously or sequentially or a combination thereof. For example, the sample may be illuminated with each light source simultaneously. In other embodiments, the flow stream is illuminated sequentially with each light source. Where more than one light source is employed to sequentially illuminate the sample, the time for which each light source illuminates the sample may 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 comprise illuminating the sample with a light source (e.g. 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 the sample is illuminated sequentially with two or more light sources, the duration of time each light source illuminates the sample may be the same or different.

The time period between illumination by each light source may also vary as desired, with the time periods being 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 illumination by each light source may 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 certain embodiments, the time period between illumination by each light source is 10 microseconds. In embodiments where the sample is illuminated sequentially by more than two (i.e., 3 or more) light sources, the delay between illumination by each light source may be the same or different.

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

Depending on the light source, the sample may be illuminated from a distance which varies, for example 0.01mm or more, for example 0.05mm or more, for example 0.1mm or more, for example 0.5mm or more, for example 1mm or more, for example 2.5mm or more, for example 5mm or more, for example 10mm or more, for example 15mm or more, for example 25mm or more and including 50mm or more. Furthermore, the illumination angle may also vary, being 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 illuminating at a 90 ° angle.

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

The light from the sample may 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.

Light in one or more wavelength ranges of 200nm-1200nm may be collected. In some examples, the method includes measuring light from the sample in a wavelength range, such as 200nm to 1200nm, such as 300nm to 1100nm, such as 400nm to 1000nm, such as 500nm to 900nm, and including 600nm to 800 nm. In other examples, the method includes measuring light collected at one or more specific wavelengths. For example, light collected 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 may be measured. In certain embodiments, the method comprises measuring the wavelength of light corresponding to the fluorescence peak wavelength of certain fluorophores.

The collected light may be measured continuously or at discrete intervals. In some examples, the method includes continuously measuring light. In other examples, 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, and including every 1000 milliseconds or some other interval.

One or more measurements, such as 2 or more, such as 3 or more, such as 5 or more and including 10 or more, of the collected light may be made during the practice of the subject methods. In certain embodiments, light from the sample is measured 2 or more times, and in certain instances the data is averaged.

In some embodiments, the method comprises further modulating light from the sample prior to detecting the light. For example, light from the sample source may pass through one or more lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. In some examples, the collected light passes through one or more focusing lenses, e.g., to reduce the distribution of the light. In other examples, emitted light from the sample passes through one or more collimators to reduce beam divergence.

In some embodiments, the method includes illuminating the sample with two or more beams of frequency-shifted light. As mentioned above, a light beam generator means having a laser and an acousto-optic device may be employed for frequency shifting the laser light. In these embodiments, the method includes illuminating an acousto-optic device with a laser. Depending on the desired wavelength of light generated in the output laser beam (e.g., for illuminating a sample in a flow stream), the laser may have a specific wavelength ranging 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 800 nm. The acousto-optic device may be illuminated 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 lasers may include any combination of types of lasers. For example, in some embodiments, the method includes illuminating an acousto-optic device with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid state lasers.

Where more than one laser is employed, the acousto-optic device may be illuminated with laser light either simultaneously or sequentially or a combination thereof. For example, each laser may be used to illuminate an acousto-optic device simultaneously. In other embodiments, the acousto-optic device is illuminated sequentially with each laser. Where more than one laser is employed to sequentially illuminate the acousto-optic device, the time for which each laser illuminates the acousto-optic device may independently be 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 30 microseconds or more and including 60 microseconds or more. 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 used to sequentially illuminate the acousto-optic device, the duration of time each laser illuminates the acousto-optic device may be the same or different.

The time period between illumination by each laser may also vary as desired, with the time periods being 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 illumination by each light source may 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 certain embodiments, the time period between the illumination by each laser is 10 microseconds. In embodiments where the acousto-optic device is illuminated sequentially by more than two (i.e. 3 or more) lasers, the delay between the illumination by each laser may be the same or different.

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

Depending on the laser, the acousto-optic device may be illuminated from a distance that varies, for example 0.01mm or more, for example 0.05mm or more, for example 0.1mm or more, for example 0.5mm or more, for example 1mm or more, for example 2.5mm or more, for example 5mm or more, for example 10mm or more, for example 15mm or more, for example 25mm or more and including 50mm or more. Furthermore, the illumination angle may also vary from 10 ° to 90 °, for example 15 ° to 85 °, for example 20 ° to 80 °, for example 25 ° to 75 ° and including 30 ° to 60 °, for example illuminating at a 90 ° angle.

In an embodiment, the 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, e.g., 3 or more radio frequency drive signals, e.g., 4 or more radio frequency drive signals, e.g., 5 or more radio frequency drive signals, e.g., 6 or more radio frequency drive signals, e.g., 7 or more radio frequency drive signals, e.g., 8 or more radio frequency drive signals, e.g., 9 or more radio frequency drive signals, e.g., 10 or more radio frequency drive signal drive signals, e.g., 15 or more radio frequency drive signals, e.g., 25 or more radio frequency drive signals, e.g., 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 the desired number of angular deflections.

The angularly deflected laser beams produced by the rf drive signal each have an intensity that is based on the amplitude of the applied rf drive signal. In some embodiments, the method includes applying a radio frequency drive signal having an amplitude of the laser beam sufficient to produce the angular deflection of the desired intensity. In some examples, 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 0.001MHz to about 500MHz, such as about 0.005MHz to about 400MHz, such as about 0.01MHz to about 300MHz, such as about 0.05MHz to about 200MHz, such as about 0.1MHz to about 100MHz, such as about 0.5MHz to about 90MHz, such as about 1MHz to about 75MHz, such as about 2MHz to about 70MHz, such as about 3MHz to about 65MHz, such as about 4MHz to about 60MHz, and including about 5MHz to about 50 MHz.

In some embodiments, to generate frequency encoded fluorescence data, a sample in a flow stream is illuminated with output laser beams from an acousto-optic device, the output laser beams including laser beams each having an angular deflection of intensity based on the amplitude of an applied radio frequency drive signal. For example, the output laser beam used to irradiate the particles in the flow stream may comprise 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 radio frequency.

Each angularly deflected laser beam is also spatially displaced from each other. Depending on the applied radio frequency 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, e.g., overlap with adjacent angularly deflected laser beams along a horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of the beam spots) may 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.

When a particle passes through a portion of an excitation beam formed by the superposition of two beamlets, the particle is exposed to the superposition of electric fields of the two beamlets. The fluorescence emitted by the particles is frequency encoded with a beat frequency corresponding to the difference between the optical frequencies of the incident sub-beams. For example, a frequency-encoded fluorescent light emitted through a particle at the left horizontal edge of an excitation beam formed by the superposition of a first sub-beam and a second sub-beam will exhibit a beat frequency (i.e., f) corresponding to the difference between the frequency of the second sub-beam and the frequency of the first sub-beam_{First sub-beam}-f_{Second sub-beam}The beat frequency of). In this way, the position of particles passing through the excitation beam may be encoded by the RF beat frequency associated with the radiation emitted by these particles. In some embodiments, such encoding of the position of particles may be used to normalize the intensity of detected radiation emitted by the particles with respect to changes in the intensity of the beam (e.g., in its horizontal direction).

In some embodiments, the frequency encoded fluorescence emitted by the particle is the frequency (f) corresponding to the local oscillator beam_LO) And the beat frequency of the difference between the frequencies of the radio frequency shifted sub-beams. For example, frequency-encoded fluorescence data packetsDraw f_LO-f_{RF-shifted sub-beams}The beat frequency of (c). Where the illumination of the flow stream includes a local oscillator spanning the width (e.g., the entire horizontal axis) of the flow stream, the frequency-encoded fluorescence data includes a frequency (f) corresponding to the local oscillator beam_LO) And the frequency (f) of each RF-shifted sub-beam₁、f₂、f₃、f₄、f₅、f₆Etc.) of the difference between the beat frequencies. In these embodiments, the frequency encoded fluorescence data may include a plurality of beat frequencies that each correspond to a location on the horizontal axis of the flow stream.

As discussed in more detail below, in one mode of operation, particles in a flow stream may be simultaneously irradiated with 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 irradiated 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 one of the reference beam and the radio-frequency shifted beam irradiates each sample location to excite a fluorophore of interest (if present) at that location. In some embodiments, the reference local oscillator may be generated via radio frequency displacement of an optical beam (e.g., from a laser such as a continuous wave laser). In these embodiments, each spatial position of a particle in the flow stream illuminated with light is "marked" with a different beat frequency, which corresponds to the difference between the frequency of the reference beam and the frequency of one of the radio frequency shifted beams. In these examples, the fluorescence radiation emitted by the fluorophore will spatially encode the beat frequency.

In some examples, the flow stream is illuminated with a plurality of frequency-shifted beams, and cells in the flow stream are imaged by fluorescence imaging using radio frequency marker emission (FIRE) to generate frequency-encoded images, such as in Natural Photonics (Nature Photonics) Vol.7(10) of Diebold et al; 806-810(2013) and those described in U.S. patent nos. 9423353, 9784661, and 10006852 and U.S. patent publication nos. 2017/0133857 and 2017/0350803, the disclosures of which are incorporated herein by reference.

In an embodiment, the frequency encoded fluorescence data is generated by detecting light from a particle in the flow stream. The 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, 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 examples, one or more data components of the frequency encoded fluorescence data from the sample are generated from light absorption detected from the sample (e.g., from a bright field light detector). As described in more detail below, the phase correction component may include a signal from a bright field detector that is used in certain embodiments to generate phase corrected spatial data that is an interpretation of interferometric phase adjustments to spatial data calculated from frequency encoded fluorescence data. In other examples, one or more data components of the frequency encoded fluorescence data from the sample are generated from 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).

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

In some examples, the phase correction component is used to generate modified transform coefficients (i.e., for transforming frequency encoded data into spatial data, as described below). For example, the phase correction component may comprise 2 or more modified transform coefficients, for example 3 or more, for example 4 or more and comprising 5 or more modified transform coefficients. In the case where the spatial data is calculated by performing a fourier transform (as described below), in certain embodiments, the phase correction component comprises a modified transform coefficient in which the fourier transform generates only real number mathematically calculated components (i.e., does not generate imaginary mathematically calculated components).

In certain embodiments, the phase correction component includes a first phase adjustment and a second phase adjustment. Each phase adjustment may 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 may include an output signal from a bright field photodetector. In some embodiments, the first phase adjustment is calculated by: multiplying an output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and calculating an arctangent of the phase adjustment value to generate a 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 may be a light beam generator means configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam. The optical beam generator according to some examples includes a laser (e.g., a continuous wave laser) and an acoustic optical 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 vibration between components of the beam generator.

In some embodiments, the second phase adjustment is based on a fluorescence lifetime of a fluorophore in the sample. In these embodiments, the second phase adjustment may be calculated by: signals from all fluorescence detectors are acquired to determine the phase present in the signal and a second phase adjustment is calculated based on the fluorescence lifetime of the fluorophore. Depending on the particular type of fluorophore and the number of fluorophores present, one or more fluorescence lifetimes may be calculated, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, and including 5 or more fluorescence lifetimes may 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.

In an embodiment, the method further comprises calculating phase corrected spatial data by transforming the frequency encoded fluorescence data with the phase correction component determined above. In some embodiments, the method includes calculating spatial data from the frequency encoded fluorescence data from the target. In some examples, calculating the spatial data of the target includes 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 performing a Discrete Fourier Transform (DFT) on 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 computed using a digital lock-in amplifier to heterodyne and demultiplex the frequency encoded fluorescence data. By considering the phase correction component prior to transforming the frequency encoded data into spatial data, the output of the transformation is less computationally complex than transforming the original frequency data into spatial data (i.e., without considering the phase first). In some embodiments, the method includes transforming the frequency encoded fluorescence data without any mathematical imaginary calculations (i.e., performing only calculations for mathematical real calculations of the transformation) to generate spatial data from the frequency encoded fluorescence data.

In some embodiments, the method includes generating an image of a particle in the flow stream from the frequency encoded fluorescence. In some embodiments, an image of the particle may be generated from the frequency encoded fluorescence in combination with the detected light absorption, the detected light scattering, or a combination thereof. In some examples, the image of the particle is generated from only the frequency encoded fluorescence. In other examples, an image of the target is generated from the frequency-encoded fluorescence and the light absorption detected from the sample (e.g., from a bright field light detector). In other examples, an image of the particle is generated from the frequency encoded fluorescence and 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). In other examples, an image of the particle is generated from the frequency encoded fluorescence and a combination of the detected light absorption, the detected light scattering, and the detected light emission.

One or more images of the particle may be generated from the frequency encoded fluorescence data. In some embodiments, a single image of the particle is generated from the frequency encoded fluorescence data. In other embodiments, two or more images of the particle are generated from the frequency encoded fluorescence data, such as 3 or more, such as 4 or more, such as 5 or more and including 10 or more images or combinations thereof.

As described above, the method of the present disclosure further comprises sorting the particles. In embodiments, particles may be sorted based on frequency encoded fluorescence data, calculated spatial data, generated images, one or more determined characteristics of the particles determined from the calculated spatial data or generated images (e.g., size, centroid, eccentricity), or some combination thereof. The term "sort" is used herein in its conventional sense to refer to separating components of a sample (e.g., droplets comprising cells, droplets comprising non-cellular particles (e.g., biological macromolecules)) and, in some instances, transporting the separated components to one or more sample collection vessels. For example, the method may comprise sorting 2 or more components of the sample, such as 3 or more components, such as 4 or more components, such as 5 or more components, such as 10 or more components, such as 15 or more components and including sorting 25 or more components of the sample. In some examples, the first sample composition collection location comprises a sample collection container and the second sample composition collection location comprises a waste collection container.

In sorting particles from a sample in a flow stream, the method includes, for example, using a computer to collect data (e.g., fluorescence data), analyze (determine frequency-encoded fluorescence data, determine phase correction components, calculate a transformation of the frequency-encoded data to phase-corrected spatial data), and record, wherein multiple data channels record data from each detector (e.g., scatter detector, bright field photodetector, or fluorescence detector). In these embodiments, the analysis includes classifying and counting the particles such that each particle exists as a set of digitized parameter values. The subject system (described below) may be arranged to trigger on selected parameters to distinguish particles of interest from background and noise.

Specific subpopulations (e.g., single cells) of interest can then be further analyzed by "gating" based on frequency-encoded fluorescence data collected for the entire population. To select the appropriate threshold, the data is plotted to obtain the best separation of the possible subgroups. This process may be performed by plotting moments of the image or one or more determined characteristics (e.g., size, center of mass, eccentricity). In other embodiments, the method includes plotting forward light scatter (FSC) versus lateral (i.e., orthogonal) light scatter (SSC) on the two-dimensional plot. In other embodiments, the method includes plotting one or more determined characteristics (e.g., size, centroid, eccentricity) for one or more of forward light scattering (FSC) and lateral (i.e., orthogonal) light scattering (SSC). In other embodiments, the method includes gating the population of particles for forward light scattering (FSC) and lateral (i.e., orthogonal) light scattering (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 the population of particles based on one or more determined characteristics (e.g., size, center of mass, eccentricity) based on an image of the target, and then gating the population of particles for forward light scattering (FSC) and lateral (i.e., orthogonal) light scattering (SSC).

A subgroup of the targets (i.e. those single cells within the threshold) is then selected and particles not within the threshold are excluded. Where desired, a cursor on a computer screen may be used to draw lines around the desired subgroup to select the threshold. Only those particles within the threshold are then further analyzed by plotting other parameters of these particles (e.g. fluorescence). Where desired, the analysis can be configured to produce a count of particles of interest in the sample.

In some embodiments, methods for sorting sample compositions include sorting particles (e.g., cells in a biological sample) using a particle sorting module having a deflector plate, such as described in U.S. patent publication No.2017/0299493 filed 3/28/2017, the disclosure of which is incorporated herein by reference. In certain embodiments, cells of a sample are sorted using a sort decision module having a plurality of sort decision units, such as those described in U.S. provisional patent application No.62/803264 filed on 8.2.2019, the disclosure of which is incorporated herein by reference.

FIG. 1 illustrates a flow chart for generating frequency encoded fluorescence data and calculating phase corrected spatial data from the frequency encoded fluorescence data according to certain embodiments. 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 (e.g., frequency data for each spatial location along a horizontal axis) for the particle is generated. At step 103, phase correction components, such as an interference phase component and a fluorescence lifetime phase component, 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, an image may be generated using the spatial data. Then, at step 106, an image mask may be generated using the image mask. At step 107, co-localization of one or more features of the cell (e.g., organelles) can be calculated using the two or more images, or at step 108, co-localization can be calculated using an image mask.

FIG. 2 illustrates generating an image of a particle using phase corrected spatial data in comparison to an image of spatial data without phase correction according to some embodiments. As shown in group a, the frequency-encoded fluorescence data is transformed into spatial data by using a Fast Fourier Transform (FFT), for example, without phase correction. The resulting image shows a lower resolution, the particles being masked by background noise. In group B, the frequency-encoded fluorescence data is phase-corrected with the phase adjusting means in conjunction with FFT to generate phase-corrected spatial data. The phase corrected spatial data provides enhanced resolution particle imaging that is not obscured by background noise.

System for characterizing particles in a sample

As described above, various aspects of the present disclosure include systems for characterizing sample particles (e.g., cells in a biological sample). A system according to some embodiments includes a light source configured to illuminate a sample having particles in a flow stream and a light detection system having a photodetector and a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to be configured to: generating frequency-encoded fluorescence data from particles in the flow stream; and calculating phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with the phase correction component.

The system of interest includes a light source configured to illuminate the sample in the flow stream. In embodiments, the light source may be any suitable broadband or narrowband light source. Depending on the composition (e.g., cells, beads, non-cellular particles, etc.) in the sample, the light source may be configured to emit light of varying wavelengths ranging from 200nm to 1500nm, such as 250nm to 1250nm, such as 300nm to 1000nm, such as 350nm to 900nm, and including 400nm to 800 nm. For example, the light source may include a broadband light source that emits light having a wavelength of 200nm to 900 nm. In other examples, the light source includes a narrow band light source emitting wavelengths of 200nm to 900 nm. For example, the light source may be a narrow band LED (1nm-25nm) emitting light at wavelengths of 200nm to 900 nm.

In some implementationsIn an example, 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., helium-neon, argon, krypton, xenon, nitrogen, CO)₂A 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 cadmium helium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and a combination thereof), a solid-state laser (e.g., a ruby laser, Nd: YAG laser, NdCrYAG laser, Er: YAG laser, Nd: YLF laser, Nd: YVO₄Laser, Nd: YCa₄O(BO₃)₃Laser, Nd: YCOB laser, Ti sapphire laser, Tm YAG laser, Yb₂O₃Lasers or cerium-doped lasers and combinations thereof), semiconductor diode lasers, Optically Pumped Semiconductor Lasers (OPSL) or any of the above implemented as frequency or frequency tripled lasers.

In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to halogen lamps, deuterium arc lamps, xenon arc lamps, light emitting diodes, such as broadband LEDs with continuous spectrum, super bright light emitting diodes, semiconductor light emitting diodes, broad spectrum LED white light sources, multi-LED integration. In some examples, 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.

In certain embodiments, the light source is a light beam generator configured to generate two or more frequency-shifted light beams. In some examples, the optical beam generator comprises a laser, a radio frequency generator configured to apply a radio frequency drive signal to the acousto-optic device to generate the two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous wave laser. E.g. in a light beam generator of interestThe laser may be a gas laser (e.g. helium-neon, argon, krypton, xenon, nitrogen, CO)₂Lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers or xenon-fluorine (XeF) excimer lasers or combinations thereof), dye lasers (e.g., stilbene, coumarin or rhodamine lasers), metal vapor lasers (e.g., cadmium helium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper or gold Nd lasers and combinations thereof), solid-state lasers (e.g., ruby lasers, Nd: YAG lasers, NdCrYAG lasers, Er: YAG lasers, YLF lasers, Nd: YVO lasers)₄Laser, Nd: YCa₄O(BO₃)₃Laser, Nd: YCOB laser, Ti sapphire laser, Tm YAG laser, Yb₂O₃Lasers or cerium-doped lasers and combinations thereof).

The acousto-optic device may be any convenient acousto-optic protocol configured to frequency shift the laser using an applied acoustic wave. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam from light from the laser and an applied radio frequency drive signal. The rf drive signal may 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.

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

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

In certain embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam with a desired intensity profile. For example, the memory may comprise instructions for generating two or more angularly deflected laser beams having the same intensity, for example 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 comprise 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 with different intensities, for example 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 with different intensities.

In certain embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with an intensity that increases along the horizontal axis from an edge to a center of the output laser beam. In these examples, the intensity of the angularly deflected laser beam at the center of the output beam may 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%, of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, 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 with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with an intensity that increases along a horizontal axis from an edge to a center of the output laser beam. In these examples, the intensity of the angularly deflected laser beam at the edge of the output beam may 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%, of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, and including from 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 with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity distributed along the horizontal axis in a gaussian distribution. In other embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having a top hat (top hat) intensity profile along a horizontal axis.

In an embodiment, the light beam generator of interest may be configured to generate spatially separated angularly deflected laser beams in the output laser beam. Depending on the applied radio frequency drive signal and the desired 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 system is configured to produce an angularly deflected laser beam in the output laser beam that overlaps, for example, an adjacent angularly deflected laser beam along a horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of the beam spots) may 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.

In some cases, a light beam generator configured to generate two or more frequency-shifted light beams includes a laser excitation module as described in U.S. patent nos. 9423353, 9784661, and 10006852 and U.S. patent publication nos. 2017/0133857 and 2017/0350803, the disclosures of which are incorporated herein by reference.

In an embodiment, the system includes a light detection system having one or more photodetectors for detecting and measuring light from the sample. The photodetector of interest may be configured to measure light absorption from the sample (e.g., light absorption for bright field light data), light scattering (e.g., forward or side scattered light data), light emission (e.g., fluorescence data), or a combination thereof. Photodetectors of interest may include, but are 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, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof) and other photodetectors. In certain embodiments, light from the 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.

In some embodiments, the light detection system of interest comprises a plurality of photodetectors. In some examples, the light detection system includes a plurality of solid state detectors, such as photodiodes. In certain examples, the light detection system includes an array of photodetectors, such as an array of photodiodes. 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.

The photodetectors may be arranged in any geometric configuration as desired, with arrangements of interest including, but 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 may be oriented at an angle of 10 ° to 180 ° with respect to one another (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 any suitable shape, and can be a rectilinear shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curvilinear 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 photodetector array has a rectangular shaped active surface.

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 to 25 μm²To 10000 μm²E.g. 50 μm²To 9000 μm²E.g. 75 μm²To 8000 μm²E.g. 100 μm²To 7000 μm²E.g. 150 μm²To 6000 μm²And comprises 200 μm²To 5000 μm²。

The size of the photodetector array may vary depending on the amount and intensity of light, the number of photodetectors, and the desired sensitivity, and the length of the size of the photodetector array may be 0.01mm to 10mm0mm, such as 0.05mm to 90mm, such as 0.1mm to 80mm, such as 0.5mm to 70mm, such as 1mm to 60mm, such as 2mm to 50mm, such as 3mm to 40mm, such as 4mm to 30mm and including 5mm to 25 mm. The width of the photodetector array may also vary from 0.01mm to 100mm, for example 0.05mm to 90mm, for example 0.1mm to 80mm, for example 0.5mm to 70mm, for example 1mm to 60mm, for example 2mm to 50mm, for example 3mm to 40mm, for example 4mm to 30mm and including 5mm to 25 mm. Thus, the active surface of the photodetector array may be 0.1mm²To 10000mm²E.g. 0.5mm²To 5000mm²E.g. 1mm²To 1000mm²E.g. 5mm²To 500mm²And comprises 10mm²To 100mm²。

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 includes measuring light emitted by the sample in the flow stream at 400 or more different wavelengths.

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

The light detection system is configured to measure light continuously or at discrete intervals. In some instances, the photodetector of interest is configured to continuously measure the collected light. In other examples, 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, and including measuring light every 1000 milliseconds or at some other interval.

In an embodiment, the system is configured to generate the frequency-encoded fluorescence data by illuminating a sample having particles in a flow stream. In some embodiments, the light source includes a light generator component that generates a plurality of angularly deflected laser beams, each having an intensity that is based on the magnitude of an applied radio frequency drive signal (e.g., from a direct digital synthesizer coupled to an acousto-optic device). For example, the subject systems can 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, which is shifted from the frequency of the input laser beam by a predetermined radio frequency.

In accordance with certain embodiments, the subject system is configured to generate angularly deflected laser beams that are also spatially displaced from each other. Depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam, the subject system can be configured to generate an angularly deflected laser beam that is 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, e.g., overlap with adjacent angularly deflected laser beams along a horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of the beam spots) may 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.

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 generate frequency encoded fluorescence data by calculating a difference between optical frequencies of overlapping photon beams incident on the 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 location on a horizontal axis of a flow stream. In these embodiments, the frequency encoded fluorescent light emitted by the particles is the frequency (f) corresponding to the local oscillator beam_LO) And the beat frequency of the difference between the frequencies of the radio frequency shifted sub-beams. For example, the frequency encoded fluorescence data includes f_LO-f_{RF-shifted sub-beams}The beat frequency of (c). Where the illumination of the flow stream includes a local oscillator spanning the width (e.g., the entire horizontal axis) of the flow stream, the frequency-encoded fluorescence data includes a frequency (f) corresponding to the local oscillator beam_LO) And the frequency (f) of each RF-shifted sub-beam₁、f₂、f₃、f₄、f₅、f₆Etc.) of the difference between the beat frequencies. In these embodiments, the frequency encoded fluorescence data may include a plurality of beat frequencies that each correspond to a location on the horizontal axis of the flow stream.

In an embodiment, the system is configured to generate frequency-encoded fluorescence data from detected light from particles in the flow stream. The 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, 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 examples, the system is configured to generate one or more data components of the frequency-encoded fluorescence data from light absorption detected from the sample (e.g., from a bright field light detector). For example, the system may be configured to generate a phase correction component from a signal from the bright field detector. In certain embodiments, the system is configured to generate phase corrected spatial data that is an interpretation of interferometric phase adjustments to spatial data calculated from frequency encoded fluorescence data. In other examples, the system is configured to generate one or more data components of the frequency-encoded fluorescence data from 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).

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 from frequency encoded fluorescence data. Spatial data according to embodiments of the present disclosure is phase corrected by the system by transforming the frequency encoded fluorescence data with a phase correction component. In some embodiments, the spatial data includes a horizontal dimension of the particle, a vertical dimension of the particle, a ratio of particle sizes along two different dimensions, a size ratio of the particle composition (e.g., a ratio of a horizontal dimension of the nucleus to a horizontal dimension of the cytoplasm).

In some embodiments, the system is configured to calculate modified transform coefficients for transforming the frequency encoded fluorescence data into phase corrected spatial data. For example, the phase correction component may comprise 2 or more modified transform coefficients, for example 3 or more, for example 4 or more and comprising 5 or more modified transform coefficients. In the case of calculating spatial data by performing fourier transform, the phase correction component includes a modified transform coefficient in which fourier transform generates only a real number mathematical calculation component (i.e., an imaginary number mathematical calculation component is not generated).

In some examples, the system is configured to determine a phase correction component comprising a first phase adjustment and a second phase adjustment. Each phase adjustment may 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 may include an output signal from a bright field photodetector. 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 calculate a first phase adjustment by: multiplying an output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and calculating an arctangent of the phase adjustment value to generate a 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 other examples, the system is configured to calculate the second phase adjustment based on a fluorescence lifetime of a fluorophore in the sample. In these examples, the system is configured to calculate the second phase adjustment by: signals from all fluorescence detectors are acquired to determine the phase present in the signal and a second phase adjustment is calculated based on the fluorescence lifetime of the fluorophore. The subject systems can be configured to calculate fluorescence lifetimes using different detector channels, for example by calculating fluorescence lifetimes using 2 or more detector channels, e.g., 3 or more, e.g., 4 or more, and including 5 or more detector channels.

In an embodiment, the subject system includes a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with the phase correction component. In some embodiments, to calculate the phase corrected spatial data, the system is configured to fourier transform the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle. In other embodiments, the system is configured to perform a discrete fourier transform on the frequency encoded fluorescence data with the phase correction 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 with the phase correction component. In other embodiments, the system is configured to perform a Discrete Fourier Transform (DFT) on the frequency encoded fluorescence data with the phase correction component. In other embodiments, the system is configured to calculate phase corrected spatial data using a digital lock-in amplifier to heterodyne frequency encoded fluorescence data and demultiplex the frequency encoded fluorescence data.

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

The subject system may be configured to generate one or more images of particles in the flow stream from the frequency encoded fluorescence. In some embodiments, an image of the particle may be generated from the frequency encoded fluorescence in combination with the detected light absorption, the detected light scattering, or a combination thereof. In some examples, the image of the particle is generated from only the frequency encoded fluorescence. In other examples, an image of the target is generated from the frequency-encoded fluorescence and the light absorption detected from the sample (e.g., from a bright field light detector). In other examples, an image of the particle is generated from the frequency encoded fluorescence and 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). In other examples, an image of the particle is generated from the frequency encoded fluorescence and a combination of the detected light absorption, the detected light scattering, and the detected light emission.

A system according to some embodiments may include a display and an operator input device. For example, the operator input device may be a keyboard, mouse, or the like. The processing module includes a processor having access to a memory having instructions stored thereon for performing the steps of the subject method. The processing modules may include an operating system, a Graphical User Interface (GUI) controller, a system memory, a memory storage device, and input-output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or it may be one of the other processors that are or will be available. As is known in the art, the processor executes an operating system, which interacts with firmware and hardware in a well-known manner and facilitates the processor in coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages, such as Java, Perl, C + +, other high-level or low-level languages, and combinations thereof. An operating system typically cooperates with the processor to coordinate and perform 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 in accordance with known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that provide feedback control (e.g., negative feedback control).

The system memory may be any of a number of known or future memory storage devices. Examples include any conventional Random Access Memory (RAM), magnetic media (e.g., resident hard disks or magnetic tape), optical media (e.g., read-write optical disks), flash memory devices, or other memory storage devices. The memory storage device may be any of a number of known or future devices including an optical disk drive, a magnetic tape drive, a removable hard disk drive, or a floppy disk drive. Memory storage devices of this type typically read from and/or write to, respectively, a program storage medium (not shown), such as an optical disk, magnetic tape, removable hard or floppy disk. Any of these program storage media, or other program storage media now in use or later developed, may 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 called computer control logic) are typically stored in a system memory and/or in a program storage device used in conjunction with a memory storage device.

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 implemented primarily in hardware using, for example, a hardware state machine. Implementation of a hardware state machine to perform the functions described herein will be apparent to those skilled in the relevant art.

The memory may be any suitable device in which the processor is capable of storing and retrieving data, such as a magnetic, optical, or solid state storage device (including a magnetic or optical disk or tape or RAM or any other suitable fixed or portable device). The processor may comprise a general-purpose digital microprocessor suitably programmed via a computer readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communications channel or pre-stored in a computer program product (such as 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 may carry programming and can be read by a disk writer/reader. The system of the invention also comprises programming, for example in the form of a computer program product, an algorithm, for practicing the method described above. The programming according to the 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, magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; a portable flash memory drive; and hybrids of these categories, such as magnetic/optical storage media.

The processor may also access a communication channel to communicate with a user at a remote location. By remote location is meant that the user is not in direct contact with the system, but rather relays input information 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 telephone (i.e., a smart phone), to the input manager.

In some embodiments, a system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface comprises a receiver and/or transmitter for communicating with a network and/or another device. The communication interface may be configured for wired or wireless communication including, but not limited to, Radio Frequency (RF) communication (e.g., Radio Frequency Identification (RFID), Zigbee communication protocols, WiFi, infrared, wireless Universal Serial Bus (USB), Ultra Wideband (UWB)),Communication protocols) and cellular communications (e.g., Code Division Multiple Access (CDMA) or global system for mobile communications (GSM)).

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

In one embodiment, the communication interface is configured for infrared communication,Communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as computer terminals and/or networks, communication-enabled mobile telephones, personal digital assistants, or any other communication devices with which a user may be engaged.

In one embodiment, the communication interface is configured to provide a data transfer connection using Internet Protocol (IP) over a cellular telephone network, Short Message Service (SMS), 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.

In one embodiment, the subject system is configured to wirelessly communicate with the server device via a communication interface, e.g., using a wireless communication interface such as 802.11 orThe common standard of the RF protocol or the IrDA infrared protocol communicates wirelessly with the server device via a communication interface. The server device may be another portable device, such as a smart phone, a Personal Digital Assistant (PDA), or a laptop computer; or larger devices such as desktop computers, appliances, etc. In some embodiments, the server device has a display (e.g., a Liquid Crystal Display (LCD)) and an input device (e.g., a button, keyboard, mouse, or touch screen).

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.

The output controls may include controls for any of a variety of known display devices for presenting information to a user (whether human or machine, whether local or remote). If one of the display devices provides visual information, the information may be typically logically and/or physically organized as an array of picture elements. A Graphical User Interface (GUI) controller may comprise any of a number of known or future software programs for providing graphical input and output interfaces between the system and a user, and for processing user inputs. The functional elements of the computer may communicate with each other over a system bus. Some of these communications may be implemented in alternative embodiments using networks or other types of telecommunications. The output manager may also provide information generated by the processing module to a user at a remote location, for example, over the internet, a telephone or satellite network, in accordance with known techniques. The presentation of data by the output manager may be implemented according to a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include an internet URL address so that the user may retrieve additional SQL, HTML, XML, or other documents or data from a remote source. The one or more platforms present in the subject system may be any type of known computer platform or type to be developed in the future, although they typically belong to a class of computers commonly referred to as servers. However, they may also be mainframe computers, workstations or other computer types. They may be connected via any known or future type of cable or other communication system, including wireless systems, whether networked or otherwise. They may be in the same location or may be physically separated. Various operating systems may be employed on any computer platform, which may depend on the type and/or architecture of the computer platform selected. Suitable operating systems include Windows 10, Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, Ubuntu, Zorin OS, and the like.

In certain embodiments, the subject systems include one or more optical adjustment components for adjusting light, such as light impinging on the sample (e.g., from a laser) or light collected from the sample (e.g., fluorescence). For example, the optical adjustment may be to increase the dimensionality of the light, focus the light, or collimate the light. In some examples, the optical adjustment is a magnification protocol to increase the dimension of the light (e.g., 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, the optical adjustment includes focusing the light so as to reduce the dimension of the light, 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 a beam spot dimension by 75% or more. In some embodiments, the 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, collimating includes narrowing the spatial cross-section of the beam (e.g., reducing the beam profile of the laser).

In some embodiments, the optical adjustment component is a focusing lens having 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 examples, the focusing lens is a double achromatic demagnifying lens with a magnification of about 0.6. The focal length of the focusing lens may vary from 5mm to 20mm, for example 6mm to 19mm, for example 7mm to 18mm, for example 8mm to 17mm, for example 9mm to 16mm, and including 10mm to 15 mm. In some embodiments, the focal length of the focusing lens is about 13 mm.

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

In some embodiments, the subject systems include a flow cell nozzle having a nozzle orifice configured to flow a flow stream through the flow cell nozzle. The subject flow cell nozzle has an orifice that propagates 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 frustoconical portion terminating in a planar surface having a nozzle orifice transverse to the longitudinal axis. The length (measured along the longitudinal axis) of the proximal cylindrical portion may 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 8 mm. The length of the distal frustoconical portion (measured along the longitudinal axis) may also vary from 1mm to 10mm, such as from 2mm to 9mm, such as from 3mm to 8mm and including from 4mm to 7 mm. In some embodiments, the diameter of the flow cell nozzle chamber may vary from 1mm to 10mm, such as 2mm to 9mm, such as 3mm to 8mm and including 4mm to 7 mm.

In certain examples, 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 frusto-conical nozzle chamber (measured along a longitudinal axis transverse to the nozzle orifice) may be from 1mm to 15mm, such as from 1.5mm to 12.5mm, such as from 2mm to 10mm, such as from 3mm to 9mm and including from 4mm to 8 mm. The proximal portion of the frusto-conical nozzle chamber may have a diameter of from 1mm to 10mm, such as from 2mm to 9mm, such as from 3mm to 8mm and including from 4mm to 7 mm.

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 may be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curvilinear cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (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 may vary from 1 μm to 20000 μm, such as from 2 μm to 17500 μm, such as from 5 μm to 15000 μm, such as from 10 μm to 12500 μm, such as from 15 μm to 10000 μm, such as from 25 μm to 7500 μm, such as from 50 μm to 5000 μm, such as from 75 μm to 1000 μm, such as from 100 μm to 750 μm and including from 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

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 from the sample injection port to the flow cell nozzle chamber can be 1 μ L/sec or more, such as 2 μ L/sec or more, such as 3 μ L/sec or more, such as 5 μ L/sec or more, such as 10 μ L/sec or more, such as 15 μ L/sec or more, such as 25 μ L/sec or more, such as 50 μ L/sec or more, such as 100 μ L/sec or more, such as 150 μ L/sec or more, such as 200 μ L/sec or more, such as 250 μ L/sec or more, such as 300 μ L/sec or more, such as 350 μ L/sec or more, such as 400 μ L/sec or more, such as 450 μ L/sec or more, and including 500 μ L/sec or more. For example, the sample flow rate can be from 1 μ L/sec to about 500 μ L/sec, such as from 2 μ L/sec to about 450 μ L/sec, such as from 3 μ L/sec to about 400 μ L/sec, such as from 4 μ L/sec to about 350 μ L/sec, such as from 5 μ L/sec to about 300 μ L/sec, such as from 6 μ L/sec to about 250 μ L/sec, such as from 7 μ L/sec to about 200 μ L/sec, such as from 8 μ L/sec to about 150 μ L/sec, such as from 9 μ L/sec to about 125 μ L/sec, and including from 10 μ L/sec to about 100 μ L/sec.

The sample injection port may be an aperture located in the nozzle chamber wall or may be a conduit located at the proximal end of the nozzle chamber. Where the sample injection port is an aperture located in a wall of the nozzle chamber, the aperture of the sample injection port may be of any suitable shape, where cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curvilinear cross-sectional shapes (e.g., circular, elliptical, etc.), and irregular shapes (e.g., a parabolic bottom portion coupled to a planar top portion). In certain embodiments, the sample injection port has a circular aperture. The size of the orifice of the sample injection port may vary depending on the shape, and in certain examples the orifice has an opening of 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 1.5 mm.

In some examples, the sample injection port is a conduit located at the proximal end of the flow cell nozzle chamber. For example, the sample injection port may be a conduit positioned such that the orifice of the sample injection port is in line with the flow cell nozzle orifice. Where the sample injection port is a conduit positioned in line with the flow cell nozzle orifice, the cross-sectional shape of the sample injection tube may be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curvilinear cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., a parabolic bottom portion coupled to a planar top portion). The orifice of the conduit may vary according to shape, in certain examples 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 a 1.5mm opening. The tip shape of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may comprise a beveled tip having a beveled angle of 1 ° to 10 °, such as a beveled angle of 2 ° to 9 °, such as 3 ° to 8 °, such as 4 ° to 7 °, and comprising a beveled angle of 5 °.

In some embodiments, the flow cell nozzle further comprises a sheath fluid injection port configured to provide a sheath fluid to the flow cell nozzle. In an embodiment, the sheath fluid injection system is configured to provide a sheath fluid to the flow cell nozzle chamber, for example, to provide the sheath fluid to the flow cell nozzle chamber with the sample to create a laminar sheath fluid flow around the sample flow stream. Depending on the desired characteristics of the flow stream, the rate of delivery of the sheath fluid to the flow cell nozzle chamber can be 25 μ L/sec or more, such as 50 μ L/sec or more, such as 75 μ L/sec or more, such as 100 μ L/sec or more, such as 250 μ L/sec or more, such as 500 μ L/sec or more, such as 750 μ L/sec or more, such as 1000 μ L/sec or more, and including 2500 μ L/sec or more. For example, the sheath fluid flow rate can be from 1 μ L/sec to about 500 μ L/sec, such as from 2 μ L/sec to about 450 μ L/sec, such as from 3 μ L/sec to about 400 μ L/sec, such as from 4 μ L/sec to about 350 μ L/sec, such as from 5 μ L/sec to about 300 μ L/sec, such as from 6 μ L/sec to about 250 μ L/sec, such as from 7 μ L/sec to about 200 μ L/sec, such as from 8 μ L/sec to about 150 μ L/sec, such as from 9 μ L/sec to about 125 μ L/sec, and including from 10 μ L/sec to about 100 μ L/sec.

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 may be any suitable shape, wherein cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curvilinear cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., a parabolic bottom portion coupled to a planar top portion). In certain examples, the size of the orifice of the sample injection port may vary depending on the shape, having an opening of 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 including an opening of 1.25mm to 1.75mm, such as a 1.5mm opening.

In certain examples, the subject systems include a sample interrogation region in fluid communication with a flow cell nozzle orifice. In these examples, the sample flow stream flows out of an orifice at the distal end of the flow cell nozzle, and particles in the flow stream can be illuminated with a light source at the sample interrogation region. The size of the interrogation zone may vary depending on the characteristics of the flow nozzle (e.g., the size of the nozzle orifice and the size of the sample injection port). In embodiments, the width of the interrogation zone may be 0.01mm or greater, such as 0.05mm or greater, such as 0.1mm or greater, such as 0.5mm or greater, such as 1mm or greater, such as 2mm or greater, such as 3mm or greater, such as 5mm or greater and including 10mm or greater. The length of the interrogation zone may also vary, in some examples the length is 0.01mm or greater, such as 0.1mm or greater, for example 0.5mm or greater, such as 1mm or greater, such as 1.5mm or greater, such as 2mm or greater, such as 3mm or greater, such as 5mm or greater, such as 10 or greater, such as 15mm or greater, such as 20mm or greater, such as 25mm or greater and including 50mm or greater.

The interrogation region may be configured to facilitate illumination of a planar cross-section of the outgoing flow stream, or may be configured to facilitate illumination of a diffuse field of predetermined length (e.g., with a diffuse laser or lamp). In some embodiments, the interrogation zone comprises a transparent window that facilitates illumination of a predetermined length of the outgoing flow stream, such as 1mm or more, such as 2mm or more, such as 3mm or more, such as 4mm or more, such as 5mm or more and including 10mm or more. Depending on the light source used to illuminate the effluent flow stream (as described below), the interrogation region may be configured to 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 500nm to 800 nm. Thus, the interrogation region may be formed of any transparent material passing through the desired wavelength range including, but not limited to, optical glass, borosilicate glass, pyrex glass, ultraviolet quartz, infrared quartz, sapphire, and plastics, such as polycarbonates, polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides, polyimides, or copolymers of these thermoplastics, such as PETG (ethylene glycol modified polyethylene terephthalate), and other polymeric plastic materials including polyesters, where polyesters of interest may include, but are not limited to, polyalkylene terephthalates, such as: polyethylene terephthalate (PET), bottle grade PET (copolymers based on monoethylene glycol, terephthalic acid and other comonomers (e.g., such as isophthalic acid, cyclohexene dimethanol, and the like)), poly (butylene terephthalate) (PBT), and poly (hexamethylene terephthalate); poly (alkylene adipates) such as poly (vinyl adipate), poly (butylene-1, 4-adipate), and poly (hexamethylene adipate); poly (alkylene suberate) s, such as poly (ethylene suberate); poly (alkylene sebacates), for exampleSuch as poly (ethylene sebacate); poly (epsilon-caprolactone) and poly (beta-propiolactone); poly (alkylene isophthalates), such as poly (ethylene isophthalate); poly (alkylene 2, 6-naphthalene-dicarboxylate) such as poly (ethylene 2, 6-naphthalene-dicarboxylate); poly (alkylenesulfonyl-4, 4 '-dibenzoate) such as poly (ethylenesulfonyl-4, 4' -dibenzoate); poly (p-phenylene alkylene dicarboxylate) such as poly (p-phenylene ethylene dicarboxylate); poly (trans-1, 4-cyclohexanedialkylene dicarboxylate) such as poly (trans-1, 4-cyclohexanediethylene dicarboxylate); poly (1, 4-cyclohexane-dimethylenealkylene dicarboxylate) such as poly (1, 4-cyclohexane-dimethyleneethylene dicarboxylate); poly ([ 2.2.2)]Bicyclo-octane-1, 4-dimethylenealkylene dicarboxylates) such as poly ([ 2.2.2)]-bicyclooctane-1, 4-dimethyleneethylenedicarboxylate); lactic acid polymers and copolymers, such as (S) -polylactide, (R, S) -polylactide, poly (tetramethylglycolide), and poly (lactide-co-glycolide); and polycarbonates of bisphenol a, 3' -dimethyl bisphenol a, 3',5,5' -tetrachlorobisphenol a, 3',5,5' -tetramethyl bisphenol a; polyamides, such as polyterephthalamides; polyesters, e.g. polyethylene terephthalate, e.g. Mylar^TMPolyethylene terephthalate; and so on. In some embodiments, the subject systems include a cuvette located in the sample interrogation zone. In an embodiment, the cuvette may pass light of 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 500nm to 800 nm.

In some embodiments, the subject systems include a particle sorting component for sorting sample particles (e.g., cells). In certain examples, the particle sorting component is a particle sorting module, such as those described in U.S. patent publication No.2017/0299493 filed on 28.3.2017 and U.S. provisional patent application No.62/752793 filed on 30.10.2018, the disclosures of which are incorporated herein by reference. In certain embodiments, the particle sorting component comprises one or more drop deflectors, such as those described in U.S. patent publication No.2018/0095022 filed on 2017, 6/14, the disclosure of which is incorporated herein by reference.

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 (eds.), Flow Cytometry: A Practical Approach, Oxford university Press (1997); jarosszeski et al (eds.), Flow Cytometry Protocols, Methods in Molecular Biology, 91 st, Humana Press (1997); practical Flow Cytometry, 3 rd edition, Wiley-Liss (1995); virgo et al (2012) Ann Clin biochem.1 month; 49(pt 1) 17 to 28; linden et al, Semin from hemost.2004, month 10; 30, (5) 502-11; alison et al, J Pathol, 12 months 2010; 222(4) 335-; and those described in Herbig et al (2007) Crit Rev Therg Drug Carrier Syst.24(3): 203-; the disclosure of which is incorporated herein by reference. In certain examples, the flow cytometry system of interest comprises BD Biosciences facscan to^TMII flow cytometer, BD Accuri^TMFlow cytometer, BD Biosciences FACSCELEsta^TMFlow cytometer, BD Biosciences FACSLric^TMFlow cytometer, BD Biosciences FACSVerse^TMFlow cytometer, BD Biosciences FACSYMMhony^TMFlow cytometer, BD Biosciences LSRFortess^TMX-20 flow cytometer and BD Biosciences FACSCalibur^TMCell sorter, BD Biosciences FACSCount^TMCell sorter, BD Biosciences FACSLric^TMCell sorter and BD Biosciences Via^TMCell sorter BD Biosciences Influx^TMCell sorter, BD Biosciences Jazz^TM、BD Biosciences Aria^TMCell sorter and BD Biosciences FACCSmolody^TMCell sorters, and the like.

In some embodiments, the subject particle sorting system is a flow cytometry system, such as U.S. patent No. 10006852; no. 9952076; no. 9933341; no. 9784661; no. 9726527; no. 9453789; no. 9200334; no. 9097640; no. 9095494; no. 9092034; no. 8975595; no. 8753573; no. 8233146; no. 8140300; no. 7544326; no. 7201875; no. 7129505; no. 6821740; no. 6813017; no. 6809804; no. 6372506; no. 5700692; no. 5643796; no. 5627040; no. 5620842; those described in No. 5602039; the disclosures of which are incorporated herein by reference in their entirety.

In certain examples, the subject systems are flow cytometry systems configured to characterize and image particles in a flow stream by fluorescence imaging using radio frequency marker emission (FIRE), such as in Diebold et al, Nature Photonics volume 7 (10); 806-810(2013) and those described in U.S. Pat. Nos. 9423353, 9784661 and 10006852 and U.S. Pat. publication Nos. 2017/0133857 and 2017/0350803, the disclosures of which are incorporated herein by reference.

Integrated circuit device

Various aspects of the present disclosure also include an integrated circuit device programmed to: generating frequency-encoded fluorescence data from particles in the flow stream; phase corrected spatial data of the particles is calculated by transforming the frequency encoded fluorescence data with the phase correction component. In some embodiments, the integrated circuit device is programmed to sort the particles, for example, to 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).

In an embodiment, the integrated circuit device is programmed to generate frequency encoded fluorescence data. The fluorescence data may be generated from one or more fluorescence detectors (e.g., one or more detection channels), for example, 2 or more, e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 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 examples, the system is configured to generate one or more data components of the frequency-encoded fluorescence data from light absorption detected from the sample (e.g., from a bright field light detector). For example, the system may be configured to generate a phase correction component from a signal from the bright field detector. In certain embodiments, the system is configured to generate phase corrected spatial data that is an interpretation of interferometric phase adjustments to spatial data calculated from frequency encoded fluorescence data. In other examples, one or more data components of the frequency-encoded fluorescence data are generated from 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).

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

In some embodiments, the integrated circuit device is programmed to calculate modified transform coefficients for transforming the frequency encoded fluorescence data into phase corrected spatial data. For example, the phase correction component may comprise 2 or more modified transform coefficients, for example 3 or more, for example 4 or more and comprising 5 or more modified transform coefficients. In the case of calculating spatial data by performing a fourier transform, which generates only real mathematical calculation components (i.e., does not generate imaginary mathematical calculation components), the phase correction component may include a modified transform coefficient.

In some examples, the integrated circuit device is programmed to determine a phase correction component that includes a first phase adjustment and a second phase adjustment. Each phase adjustment may 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 may be programmed to determine the first phase adjustment based on an output signal from the bright field photodetector. In some embodiments, the integrated circuit device is programmed to calculate the first phase adjustment by: multiplying an output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and calculating an arctangent of the phase adjustment value to generate a first phase adjustment value. 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 other cases, the integrated circuit device is programmed to calculate a second phase adjustment based on the fluorescence lifetime of the fluorophore in the sample. In these examples, the integrated circuit device is programmed to calculate the second phase adjustment by: signals from all fluorescence detectors are acquired to determine the phase present in the signal and a second phase adjustment is calculated based on the fluorescence lifetime of the fluorophore. The subject integrated circuit devices can be programmed to calculate fluorescence lifetimes using different detector channels, for example by calculating fluorescence lifetimes using 2 or more detector channels, e.g., 3 or more, e.g., 4 or more, and including 5 or more detector channels.

In an embodiment, the subject integrated circuit device is programmed to calculate phase corrected spatial data of the particle by transforming the frequency encoded fluorescence data with the phase correction component. In some embodiments, to calculate the phase corrected spatial data, the system is configured to fourier transform the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle. In other embodiments, the integrated circuit device is programmed to discrete fourier transform the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle. In other embodiments, the integrated circuit device is programmed to perform a Short Time Fourier Transform (STFT) of the frequency encoded fluorescence data with the phase correction component. In other embodiments, the integrated circuit device is programmed to perform a Discrete Fourier Transform (DFT) on the frequency-encoded fluorescence data with the phase correction component. In other embodiments, the integrated circuit device is programmed to calculate phase corrected spatial data using a digital lock-in amplifier to heterodyne frequency encoded fluorescence data and demultiplex the frequency encoded fluorescence data.

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

External member

Various aspects of the present disclosure also include kits, wherein a 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, for example in the form of computer readable media (e.g., flash memory drives, USB memory, optical disks, DVDs, blu-ray disks, etc.) or instructions for downloading the programming from an internet network protocol or cloud server. The kit may also include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate (e.g., one or more sheets of paper having information printed thereon, in the packaging of the kit, in package inserts, etc.). Yet another form of these instructions is a computer-readable medium, such as a floppy disk, a Compact Disc (CD), a portable flash drive, or the like, on which information has been recorded. Yet another form of these instructions that may exist is a website address that may be used to access information at a remote site via the internet.

Applications of

The subject systems, methods, and computer systems find use in a variety of applications where it is desirable to analyze and classify the composition of particles in a sample (e.g., a biological sample) in a fluid medium. In some embodiments, the systems and methods described herein can be used for flow cytometry characterization of biological samples labeled with fluorescent labels. In other embodiments, the systems and methods are used for spectroscopy of emitted light. In addition, the subject systems and methods are used to increase the signal that can be obtained from light collected from a sample (e.g., a sample in a fluid stream). Embodiments of the present disclosure desirably provide an application of a flow cytometer having improved cell sorting accuracy, enhanced particle collection, particle loading efficiency, more accurate particle loading, and enhanced particle deflection during cell sorting.

Embodiments of the present disclosure are also useful for providing applications where cells prepared from a biological sample may be desired for research, laboratory testing, or for therapy. In some embodiments, the subject methods and apparatus may facilitate obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods and systems facilitate obtaining cells from a fluid or tissue sample for use as a research or diagnostic specimen for a disease (e.g., cancer). Likewise, the subject methods and systems may facilitate obtaining cells from a fluid or tissue sample for treatment. The methods and apparatus of the present disclosure allow for the separation and collection of cells from a biological sample (e.g., organ, tissue debris, fluid) with improved efficiency and low cost compared to conventional flow cytometry systems.

The present disclosure is further defined by the following clauses notwithstanding the appended claims:

1. a method, comprising:

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

phase corrected spatial data of the particles is calculated by transforming the frequency encoded fluorescence data with the phase correction component.

2. The method according to clause 1, wherein the spatial data is calculated by fourier transforming the frequency encoded fluorescence data with the phase correction component.

3. The method according to clause 2, wherein the spatial data is calculated by discrete fourier transforming the frequency encoded fluorescence data with the phase correction component.

4. The method according to clause 2, wherein the spatial data is calculated by short-time fourier transforming (STFT) the frequency-encoded fluorescence data with the phase correction component.

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

6. The method according to any of clauses 1 to 5, wherein the phase correction component comprises a modified transform coefficient used to transform the frequency encoded fluorescence data into phase corrected spatial data.

7. The method according to any of clauses 1 to 6, wherein generating frequency-encoded fluorescence data from the particles comprises detecting light from the particles in the sample with a light detection system.

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

9. The method of clause 8, wherein detecting light absorption is performed using a bright field photodetector.

10. The method according to any of clauses 8 to 9, wherein the emitted light is detected with a fluorescence detector.

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

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

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

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

multiplying an output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and

an arctangent of the phase adjustment value is calculated to generate a first phase adjustment.

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

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

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.

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

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

20. The method of any of clauses 18 to 19, wherein the light beam generator comprises a Direct Digital Synthesizer (DDS) RF comb generator.

21. The method of any of clauses 18 to 20, wherein the beam generator component is configured to generate the frequency shifted local oscillator beam.

22. The method of any of clauses 17 to 21, wherein the light source comprises a laser.

23. The method of clause 22, wherein the laser is a continuous wave laser.

24. The method of any of clauses 17 to 23, wherein the interferometric phase adjustment comprises a phase shift caused by vibration between components of the light source.

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

26. The method according to any of clauses 1 to 25, wherein the phase corrected spatial data of the particles is calculated from the frequency encoded fluorescence data by the integrated circuit device.

27. The method of clause 26, wherein the integrated circuit device is a Field Programmable Gate Array (FPGA).

28. The method of clause 26, wherein the integrated circuit device is an Application Specific Integrated Circuit (ASIC).

29. The method of clause 26, wherein the integrated circuit device is a Complex Programmable Logic Device (CPLD).

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

31. The method of clause 30, wherein the flowing stream is illuminated with a light source having a wavelength of 200nm to 800 nm.

32. The method according to any of clauses 30 to 31, wherein the method comprises irradiating the flow stream with the first frequency-shifted beam and the second frequency-shifted beam.

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.

34. The method of any of clauses 32 to 33, further comprising:

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

an acousto-optic device is illuminated with a laser to generate a first frequency-shifted beam and a second frequency-shifted beam.

35. The method of clause 34, wherein the laser is a continuous wave laser.

36. The method according to any of clauses 1 to 35, further comprising generating an image of the particle from the phase corrected spatial data.

37. The method of clause 36, further comprising generating an image mask of the grain.

38. The method of any of clauses 1 to 37, further comprising sorting the particles.

39. A system, comprising:

a light source configured to illuminate a sample comprising particles in a flow stream;

a light detection system; and

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 from particles in the flow stream;

phase corrected spatial data of the particles is calculated by transforming the frequency encoded fluorescence data with the phase correction component.

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 with the phase correction component to generate phase corrected spatial data of the particle.

41. The system of clause 40, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform a discrete fourier transform of the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle.

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) of the frequency encoded fluorescence data with the phase correction component.

43. The system of clause 40, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the phase corrected spatial data using a digital lock-in amplifier to cause the frequency encoded fluorescence data to produce heterodyning and demultiplex the frequency encoded fluorescence data.

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

45. The system according to any 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.

46. The system according to clause 45, wherein the light detection system comprises a bright field photodetector.

47. The system according to any of clauses 39 to 46, wherein the light detection system comprises a fluorescence detector.

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 a first phase adjustment and a second phase adjustment.

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:

multiplying an output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and

an arctangent of the phase adjustment value is calculated to generate a first phase adjustment.

50. The system of clause 49, wherein the phase adjustment value is a sum of all frequency points in a discrete fourier transform of the frequency encoded fluorescence data.

51. The system of any of clauses 48-50, wherein the first phase adjustment is an interferometric phase adjustment.

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

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

54. The system of clause 53, wherein the light beam generator comprises an acousto-optic deflector.

55. The system of any of clauses 53 to 54, wherein the light beam generator comprises a Direct Digital Synthesizer (DDS) RF comb generator.

56. The system of any of clauses 53 to 55, wherein the beam generator component is configured to generate the frequency shifted local oscillator beam.

57. The system of any of clauses 39 to 56, wherein the light source comprises a laser.

58. The system of clause 57, wherein the laser is a continuous wave laser.

59. The system of any of clauses 51-58, wherein the interferometric phase adjustment comprises a phase shift caused by vibration between components of the light source.

60. The system according to 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.

61. The system of any of clauses 39 to 60, which includes an integrated circuit component programmed to:

generating frequency-encoded fluorescence data from sample particles in the flow stream;

phase corrected spatial data of the particles is calculated by transforming the frequency encoded fluorescence data with the phase correction component.

62. The system of clause 61, wherein the integrated circuit device is a Field Programmable Gate Array (FPGA).

63. The system of clause 61, wherein the integrated circuit device is an Application Specific Integrated Circuit (ASIC).

64. The system of clause 61, wherein the integrated circuit device is a Complex Programmable Logic Device (CPLD).

65. The system according to any of clauses 39 to 64, wherein the system is a flow cytometer.

66. The 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 from the phase corrected spatial data.

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 particle.

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

69. The system of clause 68, wherein the cell sorting component comprises a droplet deflector.

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

generating frequency-encoded fluorescence data from particles in the flow stream;

phase corrected spatial data of the particles is calculated by transforming the frequency encoded fluorescence data with the phase correction component.

71. The integrated circuit of clause 70, wherein the integrated circuit is programmed to fourier transform the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle.

72. The integrated circuit of clause 71, wherein the integrated circuit is programmed to perform a discrete fourier transform of the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle.

73. The integrated circuit according to clause 71, wherein the integrated circuit is programmed to perform a short time fourier transform of the frequency encoded fluorescence data with the phase correction component to generate phase corrected spatial data of the particle.

74. The integrated circuit of clause 70, wherein the integrated circuit is programmed to calculate the phase corrected spatial data using a digital lock-in amplifier to heterodyne and demultiplex the frequency encoded fluorescence data.

75. The integrated circuit according to any of clauses 70-74, wherein the integrated circuit is programmed to transform the frequency encoded fluorescence data into spatial data using a phase correction component comprising modified transform coefficients.

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

77. The integrated circuit of clause 76, wherein the integrated circuit is programmed to calculate the first phase adjustment by:

multiplying an output signal from the bright field photodetector with a predetermined constant signal to generate a phase adjustment value; and

an arctangent of the phase adjustment value is calculated to generate a first phase adjustment.

78. The integrated circuit of 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.

79. The integrated circuit of any of clauses 76 to 78, wherein the first phase adjustment is an interferometric phase adjustment.

80. The integrated circuit of clause 79, wherein the interferometric phase adjustment comprises a phase shift from a light source configured to illuminate the sample in the flow stream.

81. The integrated circuit of clause 80, wherein the light source comprises a light beam generator component configured to generate at least a first frequency-shifted light beam and a second frequency-shifted light beam.

82. The integrated circuit according to clause 81, wherein the light beam generator comprises an acousto-optic deflector.

83. The integrated circuit of any of clauses 81 to 82, wherein the light beam generator comprises a Direct Digital Synthesizer (DDS) RF comb generator.

84. The integrated circuit of any of clauses 81 to 83, wherein the beam generator means is configured to generate the frequency shifted local oscillator beam.

85. The integrated circuit of any of clauses 80-84, wherein the interferometric phase adjustment comprises a phase shift caused by vibration between components of the light source.

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

87. The integrated circuit of any of clauses 69 to 86, wherein the integrated circuit is a Field Programmable Gate Array (FPGA).

88. The integrated circuit according to any of clauses 69 to 86, wherein the integrated circuit device is an Application Specific Integrated Circuit (ASIC).

89. The integrated circuit according to any of clauses 69 to 86, wherein the integrated circuit device is a Complex Programmable Logic Device (CPLD).

90. The integrated circuit according to any of clauses 69 to 89, wherein the integrated circuit is programmed to generate the image of the particle from the phase corrected spatial data.

91. The integrated circuit of clause 90, wherein the integrated circuit is programmed to generate an image mask of the grain.

92. The integrated circuit of any of clauses 69 to 91, wherein the integrated circuit is programmed to generate the sorting decision based on the phase corrected spatial data.

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 readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the foregoing merely illustrates the principles of the invention. It will thus be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention is embodied by the appended claims. In the claims, 35 u.s.c. § 112(f) or 35 u.s.c. § 112(6) being expressly defined as being incorporated by reference only when the exact phrase "means for … …" or the exact phrase "step for … …" is referred to at the beginning of such limitations in the claims; 35 u.s.c. § 112(f) or 35 u.s.c. § 112(6) not cited if no such exact phrases are used within the limits of the claims.