Phase correction for imaging flow cytometry

Abstract

Aspects of the present invention include methods for phase correcting signals from a light detection system (e.g., in a flow cytometer). Methods according to certain embodiments include detecting light from a sample having particles in a flow stream using a light detection system, the light detection system including a brightfield photodetector configured to generate a brightfield data signal and a fluorescence detector configured to generate a fluorescence data signal, and calculating a phase correction for the fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal. A system is also described having a processor having a memory operably coupled to the processor, the processor having instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction for the fluorescence detector based on the brightfield data signal and the fluorescence data signal from the fluorescence detector. An integrated circuit device (e.g., a field programmable gate array) having programming for implementing the method is also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is related to U.S. Provisional Patent Application No. 62 / 847,631, filed May 14, 2019; the disclosure of which is incorporated herein by reference.

[0003] introduction

[0004] Characterization of analytes in biological fluids has become an integral part of medical diagnostics and assessment of a patient's overall health. Detection of analytes in biological fluids, such as human blood or blood-derived products, can provide results that play a role in determining treatment regimens for patients suffering from various disease conditions.

[0005] Flow cytometry is a technique used to characterize and typically classify biological material, such as cells in a blood sample or particles of interest in other types of biological or chemical samples. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer delivers particles (including cells) in the fluid sample as a cell stream to a flow chamber, while also delivering sheath fluid to the flow chamber. To characterize the composition of the flow stream, the flow stream is exposed to light. Changes in the material in the flow stream, such as morphology or the presence of fluorescent markers, can result in changes in the observed light, which allow characterization and separation.

[0006] To characterize the composition of a flowing stream, light must impinge on it and be collected. The light source for a flow cytometer can be a broad-spectrum lamp, a light-emitting diode, or a single-wavelength laser. The light source is aligned with the flowing stream, and the optical response of the illuminated particles is collected and quantified. Summary of the Invention

[0007] Aspects of the present disclosure include methods for phase correcting signals from a light detection system (e.g., in a flow cytometer). Methods according to specific embodiments include detecting light from a sample having particles in a flow stream with a light detection system, the light detection system including a bright field photodetector configured to generate a bright field data signal and a fluorescence detector configured to generate a fluorescence data signal, and calculating a phase correction of the fluorescence detector based on the relative phase between the bright field data signal and the fluorescence data signal. A system having a processor is also described, the processor having a memory operably coupled to the processor having instructions stored thereon, the instructions, when executed by the processor, causing the processor to calculate a phase correction of the fluorescence detector based on the bright field data signal and the fluorescence data signal from the fluorescence detector. An integrated circuit device (e.g., a field programmable gate array) having programming for practicing the subject method is also provided.

[0008] In an embodiment, light from a sample with particles in an interrogation area of ​​the flow stream is detected. In some embodiments, the particles are calibration beads. In other embodiments, the particles are cells. The method according to the embodiment includes detecting light with a bright field photodetector to generate a bright field data signal, and detecting light with a fluorescence detector to generate a fluorescence data signal. In some embodiments, the method also includes detecting light scattering using, for example, a forward scattered light detector or a side scattered light detector. The phase correction of the fluorescence detector is determined based on the relative phase between the bright field data signal and the fluorescence data signal from the fluorescence detector. In some embodiments, the subject light detection system includes two or more fluorescence detectors, and the phase correction of each fluorescence detector is calculated based on the relative phase between the bright field data signal and the fluorescence data signal from each fluorescence detector.

[0009] In some embodiments, the method includes detecting light from the sample using a brightfield photodetector to generate a brightfield data signal, detecting light from the sample using a first fluorescence photodetector operating at a first voltage to generate a first fluorescence data signal, and detecting light from the sample using a second fluorescence photodetector operating at a second voltage to generate a second fluorescence data signal. A phase correction for the first fluorescence detector is calculated based on the relative phase between the brightfield data signal and the first fluorescence data signal, and a phase correction for the second fluorescence detector is calculated based on the relative phase between the brightfield data signal and the second fluorescence data signal. To correct the light detection system according to a specific embodiment, the operating voltages of the first fluorescence detector and the second fluorescence detector are changed (e.g., gradually increased), and a phase correction for each of the first fluorescence detector and the second fluorescence detector is calculated based on the relative phase between the fluorescence data signal and the brightfield data signal at each operating voltage. In some embodiments, to perform phase correction of the light detection system, the method includes calculating a lifetime phase correction for each fluorescence detector at each operating voltage based on the phase correction determined for each fluorescence detector and the fluorescence lifetime of the fluorophore in the sample.

[0010] In certain embodiments, the subject light detection system includes a brightfield photodetector and a plurality of fluorescence detectors. In these embodiments, the method includes calculating a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector. To perform phase correction of the light detection system, the operating voltage of each fluorescence detector is changed (e.g., increased), and the phase correction for each fluorescence detector at each operating voltage is calculated. In some cases, the lifetime phase correction for each of the plurality of fluorescence detectors in the light detection system is determined based on the phase correction determined for each fluorescence detector and the fluorescence lifetime of the fluorophore of the sample.

[0011] In some embodiments, the method further comprises generating frequency-encoded fluorescence data from particles in the sample using fluorescence detectors of the light detection system, and performing a transformation on the frequency-encoded fluorescence data to calculate phase-corrected spatial data of the particles by utilizing the calculated phase correction of each fluorescence detector. In one instance, multiple positions across the flow stream (horizontal axis) are irradiated by a laser comprising a local oscillator beam and multiple radio frequency offset laser beams, such that different positions across the flow stream are irradiated by one of the local oscillator beam and the radio frequency offset frequency shifted beam. In some cases, the local oscillator beam is a frequency-shifted beam of light from a laser. In such an instance, each spatial position across the particles in the flow stream is characterized by a different beat frequency corresponding to the difference between the frequency of the local oscillator beam and the frequency of the radio frequency offset frequency shifted beam at that position. In some embodiments, the frequency-encoded data from the particles comprises a beat frequency of the spatial encoding of the horizontal axis across the particles in the flow stream.

[0012] In practicing the subject methods according to particular embodiments, the frequency-encoded fluorescence data can be transformed by a Fourier transform of the frequency-encoded fluorescence data using a phase-corrected component. In some cases, the frequency-encoded fluorescence data is transformed by a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data using a phase-corrected component. In other cases, the phase-corrected spatial data is calculated by performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data using a phase correction. In other cases, the phase-corrected spatial data is calculated using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0013] Aspects of the present disclosure also include systems having a light detection system for characterizing particles of a sample in a flow stream (e.g., cells in a biological sample). Systems according to certain embodiments include: a light source configured to illuminate a sample having particles in a flow stream; a light detection system having a brightfield photodetector configured to generate a brightfield data signal and one or more fluorescence detectors configured to generate a fluorescence data signal; 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 phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector. In some embodiments, the light detection system includes a plurality of fluorescence detectors, and the phase correction for each fluorescence detector is calculated based on a relative phase between the brightfield data signal and the fluorescence data signal from each of the plurality of fluorescence detectors.

[0014] In some embodiments, the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction for a first fluorescence detector at a first voltage and to calculate a phase correction for a second fluorescence detector at a second voltage. In a specific embodiment, to calibrate the light detection system, the system is configured to change the operating voltage of the fluorescence detectors (e.g., gradually increase the voltage of each detector) and calculate the phase correction for each of the fluorescence detectors at each operating voltage based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector. In a specific embodiment, the system includes a memory having instructions stored thereon that, when executed by the processor, cause the processor to calculate a lifetime phase correction for each fluorescence detector at each operating voltage based on the phase correction determined for each fluorescence detector and the fluorescence lifetime of the fluorophore in the sample.

[0015] In some embodiments, the system is configured to generate frequency-encoded fluorescence data from particles in a sample. In some embodiments, the light source includes a beam generator component configured to generate at least a first frequency-shifted beam and a second frequency-shifted beam. The beam generator according to the specific case includes a laser (continuous wave laser) and an acousto-optic deflector (e.g., coupled to a direct digital synthesis RF comb generator). In some cases, the subject system includes a processor having a memory operably coupled to the processor so that the memory includes instructions stored thereon, which, when executed by the processor, causes the processor to perform a transformation on the frequency-encoded fluorescence data using the calculated phase correction of each fluorescence detector to calculate the phase-corrected spatial data of the particles. In an embodiment, the spatial data may include the horizontal dimension of the particle, the vertical dimension of the particle, the ratio of the particle size along two different dimensions, the size ratio of the particle components (e.g., the ratio of the horizontal dimension of the cell nucleus to the horizontal dimension of the cytoplasm). In these embodiments, to calculate the phase-corrected spatial data, the system is configured to perform a Fourier transform on the frequency-encoded fluorescence data using the calculated phase correction to generate the phase-corrected spatial data of the particles. In other embodiments, the system is configured to perform a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using the calculated phase correction to generate phase-corrected spatial data of the particles. In further embodiments, the system is configured to perform a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using the calculated phase correction. In further embodiments, the system is configured to calculate the phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0016] Aspects of the present disclosure also include integrated circuit devices that are programmed to calculate a phase correction for one or more fluorescence detectors based on the relative phase between the bright field data signal and the fluorescence data signal from each fluorescence detector. In some embodiments, the integrated circuit device is programmed to correct multiple fluorescence detectors in a light detection system. In these embodiments, the subject integrated circuit device is programmed to change (e.g., gradually increase) the operating voltage of each fluorescence detector in the light detection system, and calculate the phase correction of each fluorescence detector at each operating voltage based on the relative phase between the bright field data signal and the fluorescence data signal. The lifetime phase correction of each fluorescence detector at each operating voltage can also be determined based on the calculated phase correction and the fluorescence lifetime of the fluorophore in the sample. In particular aspects, 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).

[0017] In certain embodiments, the integrated circuit device is programmed to perform a transform on the frequency-encoded fluorescence data using the calculated phase correction to calculate phase-corrected spatial data for particles in the sample. In some cases, the integrated circuit device is programmed to perform a Fourier transform on the frequency-encoded fluorescence data using the calculated phase correction to generate phase-corrected spatial data for the particles. In other cases, the integrated circuit device is programmed to perform a discrete Fourier transform on the frequency-encoded fluorescence data using the calculated phase correction to generate phase-corrected spatial data for the particles. In other cases, the integrated circuit device is programmed to perform a short-time Fourier transform on the frequency-encoded fluorescence data using the calculated phase correction to generate phase-corrected spatial data for the particles. In other embodiments, the integrated circuit device is programmed to calculate the phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0018] Aspects of the present disclosure also include compositions for calculating the phase correction of one or more fluorescence detectors of a light detection system as described herein. In embodiments, the composition includes multiple particles and a fluorescent dye composition with a stable fluorescence lifetime. In some cases, the particle is a bead, such as a polymer bead (such as a porous or non-porous polymer bead). In some cases, the fluorescent dye composition includes a single dye. In other cases, the fluorescent dye composition includes two or more dyes. For example, the fluorescent dye composition can include Nile red dye or phycoerythrin cyanine dye (such as PE-Cy7). In specific embodiments, the fluorescent dye composition includes one or more polymer dyes. For example, the polymer dye can be a water-soluble conjugated polymer. In embodiments, the particle is stably combined with the fluorescent dye composition. In some cases, the fluorescent dye composition is covalently bonded to the particle.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A flow chart is described for calculating a phase correction for one or more detectors of a light detection system according to certain embodiments. DETAILED DESCRIPTION

[0021] Aspects of the present disclosure include methods for phase correcting signals from a light detection system (e.g., in a flow cytometer). Methods according to specific embodiments include detecting light from a sample having particles in a flow stream with a light detection system, the light detection system including a bright field photodetector configured to generate a bright field data signal and a fluorescence detector configured to generate a fluorescence data signal, and calculating a phase correction for the fluorescence detector based on the relative phase between the bright field data signal and the fluorescence data signal. A system having a processor is also described, the processor having a memory operably coupled to the processor having instructions stored thereon, the instructions, when executed by the processor, causing the processor to calculate a phase correction for the fluorescence detector based on the bright field data signal and the fluorescence data signal from the fluorescence detector. An integrated circuit device (e.g., a field programmable gate array) having programming for practicing the subject method is also provided.

[0022] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0023] Where a range of values ​​is provided, it is to be understood that unless the context clearly dictates otherwise, every intervening value (to the tenth of the unit of the lower limit) between the upper and lower limits of the range and any other specified value or intermediate value in the range is encompassed herein. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also encompassed herein, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or both limits, ranges excluding one or both of those included limits are also encompassed herein.

[0024] Certain ranges are given herein where a numerical value is preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as for a number that is near or approximately the number that the term precedes. In determining whether a number is near or approximately a specifically recited number, a number that is near or approximately an unrecited number may be a number that, in the context in which it appears, provides a substantial equivalent to the specifically recited number.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods similar or equivalent to any methods and materials described herein can be used in the practice or testing of the present invention, representative illustrative methods and materials are described herein.

[0026] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which need to be independently confirmed.

[0027] It should be noted that, as used herein and in the claims, unmodified quantifiers include plural referents unless the context clearly dictates otherwise. It should also be noted that claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as antecedent basis for using exclusive terminology such as "solely" or "only" or the use of a "negative" limitation when reciting claim elements.

[0028] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with features in any other several embodiments without departing from the scope or spirit of the invention. Any method described can be performed in the order of events recited or in any other order that is logically possible.

[0029] Although the apparatus and methods have been or will be described using functional interpretations for the sake of grammatical fluency, it is to be expressly understood that, except as expressly provided under 35 USC §112, the claims shall not be construed as necessarily limited by any interpretation of "means" or "step" limitation, but shall be given the full meaning and equivalent meaning of the definitions provided for in the claims under the doctrine of equivalent jurisdiction, and shall be given the full statutory equivalent rights under 35 USC §112 to the extent expressly stated in the claims under 35 USC §112.

[0030] As described above, the present disclosure provides systems and methods for characterizing (e.g., imaging) particles of a sample in a flow stream. To further describe embodiments of the present disclosure, methods for phase correcting signals from a light detection system and calculating phase-corrected spatial data for particles in a flow stream are first described in more detail. Next, a system for characterizing particles of a sample in a flow stream in real time using a phase-corrected signal is described. An integrated circuit device, such as a field programmable gate array, is also provided that is programmed to phase correct the signal from the light detection system and calculate phase-corrected spatial data for the particles.

[0031] Method for phase correction of a signal from a light detection system

[0032] Aspects of the present disclosure include methods for phase correcting signals from a light detection system. In practicing the methods according to certain embodiments, light from a sample having particles in a flowing stream is detected by the light detection system, the light detection system including a brightfield photodetector configured to generate a brightfield data signal in response to the detected light, and one or more fluorescence detectors configured to generate fluorescence data signals in response to the detected signal, and calculating a phase correction for each of the fluorescence detectors in the light detection system based on a relative phase between the brightfield data signal and the fluorescence data signal from each of the fluorescence detectors.

[0033] In practicing the subject method, a sample with particles (e.g., beads of a calibration composition described in more detail below) in a flowing stream is subjected to irradiation with light from a light source. In some embodiments, the light source is a broadband light source that emits light with a wide wavelength range, for example, across 50 nm or greater than 50 nm, for example, 100 nm or greater than 100 nm, for example, 150 nm or greater than 150 nm, for example, 200 nm or greater than 200 nm, for example, 250 nm or greater than 250 nm, for example, 300 nm or greater than 300 nm, for example, 350 nm or greater than 350 nm, for example, 400 nm or greater than 400 nm, and includes spanning 500 nm or greater than 500 nm. For example, a suitable broadband light source emits light with a wavelength of 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with a wavelength of 400 nm to 1000 nm. When the method includes irradiating with a broadband light source, protocols for the broadband light source of interest include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectrum, superluminescent 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.

[0034] In other embodiments, the method includes irradiating with a narrowband light source that emits a specific wavelength or a narrow wavelength range, for example, using a light source that emits light of a narrow wavelength range of 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less, and including a light source that emits light of a specific wavelength (i.e., monochromatic light). When the method includes irradiating with a narrowband light source, protocols for the narrowband light source of interest include, but are not limited to, narrow wavelength LEDs, laser diodes, broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0035] In certain embodiments, the method comprises irradiating the flow stream with one or more lasers. The type and number of lasers vary depending on the sample and the desired light to be collected, and can be pulsed lasers or continuous wave lasers. For example, the laser can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper ( solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs) or frequency doubled or tripled versions of any of the above lasers.

[0036] The sample in the flow stream can be illuminated by one or more of the light sources described above, such as two or more light sources, such as three or more light sources, such as four or more light sources, such as five or more light sources, and including ten or more light sources. The light sources can include any combination of light source types. For example, in some embodiments, the method includes illuminating the sample in the flow stream with a laser array, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0037] The sample can be irradiated with light of 200nm to 1500nm, for example 250nm to 1250nm, for example 300nm to 1000nm, for example 350nm to 900nm and including 400nm to 800nm. For example, when the light source is a broadband light source, the sample can be irradiated with a wavelength of 200nm to 900nm. In other cases, when the light source includes multiple narrowband light sources, the sample can be irradiated with light of a specific wavelength of 200nm to 900nm. For example, the light source can be multiple narrowband LDEs (1nm to 25nm), each independently emitting light of a wavelength of 200nm to 900nm. In other embodiments, the narrowband light source includes one or more than one laser (e.g., a laser array), and uses a specific wavelength of 200nm to 700nm to irradiate the sample, for example, using a laser array with a gas laser, an excimer laser, a dye laser, a metal vapor laser, and a solid-state laser as described above.

[0038] When using more than one light source, the sample can be irradiated by the light source simultaneously, irradiated by the light source in sequence, or a combination thereof. For example, the sample can be irradiated by each of the light sources simultaneously. In other embodiments, the flow stream is irradiated by each of the light sources in sequence. When more than one light source is used to irradiate the sample in sequence, the time for each light source to irradiate the sample can independently be 0.001 microsecond or greater than 0.001 microsecond, such as 0.01 microsecond or greater than 0.01 microsecond, such as 0.1 microsecond or greater than 0.1 microsecond, such as 1 microsecond or greater than 1 microsecond, such as 5 microseconds or greater than 5 microseconds, such as 10 microseconds or greater than 10 microseconds, such as 30 microseconds or greater than 30 microseconds, and including 60 microseconds or greater than 60 microseconds. For example, the method can include irradiating the sample with a light source (such as a laser) for a duration of 0.001 to 100 microseconds, such as 0.01 to 75 microseconds, such as 0.1 to 50 microseconds, such as 1 to 25 microseconds, and including 5 to 10 microseconds. In embodiments where the sample is illuminated sequentially by two or more light sources, the duration that the sample is illuminated by each light source may be the same or different.

[0039] The time period between being irradiated by each light source can be varied as desired, separated independently by a delay of 0.001 microsecond or greater than 0.001 microsecond, for example 0.01 microsecond or greater than 0.01 microsecond, for example 0.1 microsecond or greater than 0.1 microsecond, for example 1 microsecond or greater than 1 microsecond, for example 5 microseconds or greater than 5 microseconds, for example 10 microseconds or greater than 10 microseconds, for example 15 microseconds or greater than 15 microseconds, for example 30 microseconds or greater than 30 microseconds, and including 60 microseconds or greater than 60 microseconds. For example, the time period between being irradiated by each light source can be 0.001 microsecond to 60 microseconds, for example 0.01 microsecond to 50 microseconds, for example 0.1 microsecond to 35 microseconds, for example 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In a specific embodiment, the time period between being irradiated by each light source is 10 microseconds. In embodiments where the sample is illuminated sequentially by more than two (ie, more than 3) light sources, the delay between illumination by each light source may be the same or different.

[0040] The sample can be irradiated continuously or at discrete intervals. In some cases, the method includes irradiating the sample continuously with the light source. In other cases, the sample is irradiated with the light source at discrete intervals, such as once every 0.001 milliseconds, once every 0.01 milliseconds, once every 0.1 milliseconds, once every 1 millisecond, once every 10 milliseconds, once every 100 milliseconds, including once every 1000 milliseconds, or other intervals.

[0041] Depending on the light source, the sample can be illuminated from varying distances, such as 0.01 mm or greater, such as 0.05 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 2.5 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 15 mm or greater, such as 25 mm or greater and including 50 mm or greater. At the same time, the angle of illumination can also vary from 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75° and including 30° to 60°, such as a 90° angle.

[0042] In practicing the subject method, light from the illuminated sample is measured, for example, by collecting light from the sample in a specific wavelength range (e.g., 200 nm to 1000 nm). In embodiments, the method can include measuring one or more of light absorption (e.g., brightfield light data), light scattering (e.g., forward or side scattered light data), and light emission (e.g., fluorescence light data) of the sample.

[0043] As described above, a beam generator component having a laser and an acousto-optic device for frequency-shifting the laser can be used. In these embodiments, the method includes irradiating the acousto-optic device with a laser. Depending on the desired wavelength of light generated in the output laser beam (e.g., for irradiating a sample in a flowing stream), the specific wavelength of the laser can vary from 200nm to 1500nm, for example, 250nm to 1250nm, for example, 300nm to 1000nm, for example, 350nm to 900nm, and including 400nm to 800nm. The acousto-optic device can be irradiated by one or more lasers, for example, 2 or more lasers, such as 3 or more lasers, for example, 4 or more lasers, for example, 5 or more lasers, and including 10 or more lasers. The laser can include any combination of laser types. For example, in some embodiments, a method includes illuminating an acousto-optic device with a laser array, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0044] When more than one laser is used, the acousto-optic device can be illuminated by the lasers simultaneously, sequentially, or a combination thereof. For example, the acousto-optic device can be illuminated by each of the lasers simultaneously. In other embodiments, the acousto-optic device is illuminated by each of the lasers sequentially. When more than one laser is used to illuminate the acousto-optic device sequentially, the time that each laser illuminates the acousto-optic device can independently be 0.001 microseconds or greater, such as 0.01 microseconds or greater, such as 0.1 microseconds or greater, such as 1 microsecond or greater, such as 5 microseconds or greater, such as 10 microseconds or greater, such as 30 microseconds or greater, and including 60 microseconds or greater. For example, the method can include illuminating the acousto-optic 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 the acousto-optic device is illuminated sequentially by two or more lasers, the duration for which the acousto-optic device is illuminated by each laser can be the same or different.

[0045] The time period between being irradiated by each laser can be varied as desired, being independently separated by a delay of 0.001 microsecond or greater, for example 0.01 microsecond or greater, such as 0.1 microsecond or greater, such as 1 microsecond or greater, such as 5 microseconds or greater, such as 10 microseconds or greater, such as 15 microseconds or greater, such as 30 microseconds or greater, and including 60 microseconds or greater. For example, the time period between being irradiated by each light source can be 0.001 microsecond to 60 microseconds, such as 0.01 microsecond to 50 microseconds, such as 0.1 microsecond to 35 microseconds, such as 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In a specific embodiment, the time period between being irradiated by each laser is 10 microseconds. In embodiments where the acousto-optic device is sequentially illuminated by more than two (ie, more than three) lasers, the delay between illumination by each laser may be the same or different.

[0046] The acousto-optic device can be illuminated continuously or at discrete intervals. In some cases, the method includes continuously illuminating the acousto-optic device with the laser. In other cases, the acousto-optic device is illuminated by the laser at discrete intervals, such as once every 0.001 milliseconds, once every 0.01 milliseconds, once every 0.1 milliseconds, once every 1 millisecond, once every 10 milliseconds, once every 100 milliseconds, including once every 1000 milliseconds, or other intervals.

[0047] Depending on the laser, the acousto-optic device can be illuminated from varying distances, such as 0.01 mm or greater, such as 0.05 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 2.5 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 15 mm or greater, such as 25 mm or greater, and including 50 mm or greater. At the same time, the angle of illumination can also vary from 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including 30° to 60°, such as a 90° angle.

[0048] In an embodiment, the method includes applying a radio frequency drive signal to the acousto-optic device to generate an angularly deflected laser beam. Two or more radio frequency drive signals can be applied to the acousto-optic device to generate an output laser beam having a desired number of angularly deflected laser beams, such as three or more radio frequency drive signals, such as four or more radio frequency drive signals, such as five or more radio frequency drive signals, such as six or more radio frequency drive signals, such as seven or more radio frequency drive signals, such as eight or more radio frequency drive signals, such as nine or more radio frequency drive signals, such as ten or more radio frequency drive signals, such as fifteen or more radio frequency drive signals, such as twenty-five or more radio frequency drive signals, such as fifty or more radio frequency drive signals, and including one hundred or more radio frequency drive signals.

[0049] The angle-deflected laser beams generated by the RF drive signal each have an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal having an amplitude sufficient to generate an angle-deflected laser beam having a desired intensity. In some cases, 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 about 50V, such as about 2V to about 40V, such as about 3V to about 30V, and including about 5V to about 25V. In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0050] In some embodiments, a sample of a flowing stream is illuminated by an output laser beam from an acousto-optic device comprising angularly deflected laser beams, each angularly deflected laser beam having an intensity based on the amplitude of an applied radio frequency drive signal. For example, the output laser beam used to illuminate particles in the flowing stream can include two or more angularly deflected laser beams, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, and including 25 or more angularly deflected laser beams. In an embodiment, each of the angularly deflected laser beams has a different frequency, which is offset from the frequency of the input laser beam by a predetermined radio frequency.

[0051] Each angularly deflected laser beam is also spatially offset from one another. Depending on the applied RF drive signal and the desired illumination profile of the output laser beam, the angularly deflected laser beams can be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more, and including 5000 μm or more. In some embodiments, the angled deflected laser beam overlaps, for example, with an adjacent angled deflected laser beam along the horizontal axis of the output laser beam. The overlap between adjacent angled deflected laser beams (e.g., overlap of beam spots) can be 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, and including 100 μm or more.

[0052] When a particle passes through the portion of the excitation beam formed by the superposition of the two beams, it is exposed to the superposition of their electric fields. The fluorescence emitted by the particle is encoded by a beat frequency corresponding to the difference between the optical frequencies of the incident beams. For example, the frequency-coded fluorescence emitted by a particle passing through the left horizontal edge of the excitation beam formed by the superposition of the first and second beams will exhibit a beat frequency corresponding to the difference between the frequencies of the second and first beams, i.e., f 第一光束 -f第二光束 In this way, the positions of particles passing through the excitation beam can be encoded by the RF beat frequency associated with the radiation emitted by these particles. In some embodiments, this encoding of the positions of the particles can be used to normalize the intensity of the detected radiation emitted by these particles relative to variations in the intensity of the beam, for example, across its horizontal direction.

[0053] In some embodiments, the frequency-coded fluorescence emitted by the particle is a frequency corresponding to the local oscillator beam (f LO ) and the beat frequency of the RF offset beam. For example, frequency-encoded fluorescence data includes f LO -f RF偏移光束 When the illumination of the flow stream includes a local oscillator beam that spans the width of the flow stream (e.g., the entire horizontal axis), the frequency-encoded fluorescence data includes a beat frequency corresponding to the frequency of the local oscillator beam (f LO ) and the frequency of each RF offset beam (f1, f2, f3, f4, f5, f6, etc.). In these embodiments, the frequency-encoded fluorescence data can include multiple beat frequencies, each beat frequency corresponding to a position across the horizontal axis of the flow stream.

[0054] As discussed in more detail below, in one mode of operation, particles in a flow stream can be simultaneously illuminated by multiple excitation frequencies, each of which can be obtained, for example, by offsetting the center frequency of a laser beam. More specifically, multiple sample positions can be simultaneously illuminated by a laser beam formed by mixing a reference laser beam (e.g., a local oscillator beam) with multiple radio frequency offset laser beams, such that each sample position is illuminated by the reference laser beam and one of the radio frequency offset beams, thereby exciting the fluorophore of interest (if present) at that position. In some embodiments, the reference local oscillator beam can be obtained by radio frequency offsetting a beam (e.g., from a laser, such as a continuous wave laser). In these embodiments, each spatial position of the illuminated particle in the flow stream is "marked" by a different beat frequency, which corresponds to the difference between the frequency of the reference beam and the frequency of the radio frequency offset beam. In these cases, the fluorescence radiation emitted by the fluorophore will spatially encode the beat frequency.

[0055] In certain cases, the flow stream is illuminated by multiple frequency-shifted beams and cells in the flow stream are imaged by fluorescence imaging using radiofrequency labeled emission (FIRE) to generate frequency-encoded images, as described, for example, in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013), and as described in U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852, U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the entire contents of which are incorporated herein by reference.

[0056] When practicing the subject method, the light from the sample is detected by a light detection system. As described in more detail below, the light detection system includes a bright field photodetector and one or more fluorescence detectors. In specific aspects, the light detection system also includes a light scattering detector, such as a forward light scattering detector, a side light scattering detector, or a combination thereof. The collected light can be detected continuously or at discrete intervals. In some cases, the method includes continuously detecting the light. In other cases, the light is detected at discrete intervals, such as light is measured once every 0.001 millisecond, once every 0.01 millisecond, once every 0.1 millisecond, once every 1 millisecond, once every 10 milliseconds, once every 100 milliseconds, and including measurement every 1000 milliseconds, or other intervals.

[0057] During the method, the measurement of the detected light can be performed once or more than once, such as 2 times or more than 2 times, such as 3 times or more than 3 times, such as 5 times or more than 5 times, and including 10 times or more than 10 times. In certain embodiments, the light from the sample is measured 2 times or more, and in certain cases the data is averaged.

[0058] In some embodiments, the method further includes conditioning the light from the sample prior to detecting the light. For example, the light from the sample source can be passed through one or more lenses, mirrors, pinholes, slits, gratings, light deflectors, and any combination thereof. In some cases, the collected light is passed through one or more focusing lenses, for example, to reduce the profile of the light. In other cases, the light emitted by the sample is passed through one or more collimators to reduce beam divergence.

[0059] In an embodiment, the method includes detecting light with a brightfield photodetector to generate a brightfield data signal. Light from the sample can be detected with the brightfield photodetector at one or more wavelengths, such as 5 or more different wavelengths, such as 10 or more different wavelengths, such as 25 or more different wavelengths, such as 50 or more different wavelengths, such as 100 or more different wavelengths, such as 200 or more different wavelengths, such as 300 or more different wavelengths, and including detection at 400 or more different wavelengths. Light can be detected with the brightfield photodetector at one or more wavelengths in the range of 200 nm to 1200 nm. In some cases, the method includes detecting light from the sample with a brightfield photodetector over a wavelength range, such as 200 nm to 1200 nm, such as 300 nm to 1100 nm, such as 400 nm to 1000 nm, such as 500 nm to 900 nm, and including 600 nm to 800 nm.

[0060] The brightfield photodetector is configured to generate one or more brightfield data signals in response to the detected light, for example, generating two or more, for example, three or more, for example, four or more, for example, five or more, and including ten or more brightfield data signals in response to the detected light. When the brightfield photodetector is configured to detect light of multiple wavelengths (e.g., 400 nm to 800 nm), in some cases the method may include generating one or more brightfield data signals in response to each wavelength of the detected light. In other cases, a single brightfield data signal is generated in response to light detected by the brightfield photodetector across the entire wavelength range.

[0061] The method of the present invention can also include detecting the fluorescence from the sample with one or more fluorescence detectors. As described in more detail below, the light detection system can include one or more fluorescence detectors, such as 2 or more than 2, such as 3 or more than 3, such as 4 or more than 4, such as 5 or more than 5, such as 6 or more than 6, such as 7 or more than 7, such as 8 or more than 8, such as 9 or more than 9, such as 10 or more than 10, such as 15 or more than 15, and include 25 or more than 25 fluorescence detectors. In embodiments, each in the fluorescence detector is configured to generate fluorescence data signal. Fluorescence from sample can be detected by each fluorescence detector independently with one or more wavelengths in the wavelength range of 200nm to 1200nm. In some cases, the method includes detecting fluorescence from the sample at a certain wavelength range, for example, 200nm to 1200nm, for example, 300nm to 1100nm, for example, 400nm to 1000nm, for example, 500nm to 900nm, and including 600nm to 800nm. In other cases, the method includes detecting fluorescence at one or more specific wavelengths with each fluorescence detector. For example, depending on the number of different fluorescence detectors of the target light detection system, fluorescence can be detected at one or more than one of 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, 617nm or a combination thereof. In a specific embodiment, the method includes detecting a light wavelength corresponding to the wavelength of the fluorescence peak of a specific fluorophore present in the sample.

[0062] In practicing the subject method, a phase correction is calculated for one or more fluorescence detectors in a light detection system based on the relative phase between a brightfield data signal and a fluorescence data signal from each fluorescence detector. In one example, the method includes detecting light with a brightfield photodetector configured to generate a brightfield data signal in response to the detected light; detecting light with a first fluorescence detector configured to generate a first fluorescence data signal in response to the detected light, and detecting light with a second fluorescence detector configured to generate a second fluorescence data signal in response to the detected light; calculating a phase correction for the first fluorescence detector based on the relative phase between the brightfield data signal and the first fluorescence data signal; and calculating a phase correction for the second fluorescence detector based on the relative phase between the brightfield data signal and the second fluorescence data signal. In another example, the method includes detecting light with a brightfield photodetector configured to generate a brightfield data signal in response to the detected light; detecting light with a plurality of fluorescence detectors, and calculating a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each of the plurality of fluorescence detectors.

[0063] In certain embodiments, a phase correction is calculated for each of the fluorescence detectors based on the relative phase between the brightfield data signal and the fluorescence data signal according to the following formula:

[0064]

[0065] Where % represents the modulo function and all phases are expressed in radians.

[0066] In other embodiments, a phase correction is calculated for each of the fluorescence detectors based on the relative phase between the brightfield data signal and the fluorescence data signal according to the following formula:

[0067]

[0068] where all phases are expressed as complex-phase unit vectors.

[0069] In embodiments, light is detected by each fluorescence detector operating at a predetermined voltage. In some embodiments, the method includes calculating a phase correction for each fluorescence detector at a plurality of different operating voltages. In these embodiments, the method includes detecting light with each fluorescence detector at a first set of operating voltages to generate a first set of fluorescence data signals; changing the operating voltage of one or more of the fluorescence detectors to a second set of operating voltages; and detecting light with each fluorescence detector at the second set of operating voltages to generate a second set of fluorescence data signals. The operating voltage can be changed (i.e., increased or decreased) in any increment, such as 0.01VV or more, such as 0.01VV or more, such as 0.05VV or more, such as 0.1VV or more, such as 0.5V or more, such as 1V or more, such as 2V or more, such as 3V or more, such as 5V or more, such as 10V or more, such as 25V or more, such as 50V or more, such as 50V or more, such as 100V or more, such as 100V or more, such as 250V or more, such as 250V or more, such as 500V or more, and including 1000V or more.

[0070] In one example, the operating voltage of each fluorescence detector is increased by 0.01V or more than 0.01V, for example 0.05V or more than 0.05V, for example 0.1V or more than 0.1V, for example 0.5V or more than 0.5V, for example 1V or more than 1V, for example 2V or more than 2V, for example 3V or more than 3V, for example 5V or more than 5V, for example 10V or more than 10V, for example 25V or more than 25V, for example 50V or more than 50V, for example 100V or more than 100V, for example 250V or more than 250V, for example 500V or more than 500V, and includes an increase of 1000V or more than 1000V in the operating voltage of each fluorescence detector. In another example, the operating voltage of each fluorescence detector is reduced by 0.01V or more than 0.01V, for example 0.05V or more than 0.05V, for example 0.1V or more than 0.1V, for example 0.5V or more than 0.5V, for example 1V or more than 1V, for example 2V or more than 2V, for example 3V or more than 3V, for example 5V or more than 5V, for example 10V or more than 10V, for example 25V or more than 25V, for example 50V or more than 50V, for example 100V or more than 100V, for example 250V or more than 250V, for example 500V or more than 500V, and includes a reduction of the operating voltage of each fluorescence detector by 1000V or more than 1000V.

[0071] Depending on the need for phase correction of the optical detection system, fluorescence data signals can be collected from each fluorescence detector at any number of operating voltages, for example, at 2 or more different operating voltages, for example, at 3 or more different operating voltages, for example, at 5 or more different operating voltages, for example, at 10 or more 10 different operating voltages, for example, at 25 or more 25 different operating voltages, and including collecting fluorescence data signals from each fluorescence detector at 50 or more 50 different operating voltages.

[0072] The operating voltage of each fluorescence detector can be changed by the same or different amounts. In some cases, the operating voltage of each fluorescence detector in the light detection system is changed by the same amount. In other cases, the operating voltage of each fluorescence detector in the light detection system is changed by different amounts. In other cases, the operating voltage of two or more fluorescence detectors in the light detection system can be changed by the same amount, or the operating voltage of two or more fluorescence detectors in the light detection system can be changed by different amounts. At each operating voltage, a phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal.

[0073] As described in more detail below, systems of interest according to certain embodiments include a variable gain amplifier. In some cases, the variable gain amplifier can be configured to operate between -100 dB and 100 dB, such as -75 dB to 75 dB, such as -50 dB to 50 dB, such as -25 dB to 25 dB, and including 0 dB to 50 dB. In some embodiments, the method includes changing an electronic gain setting (e.g., the gain of the amplifier) ​​and calculating a phase correction for each fluorescence detector. In some cases, the method includes changing the gain of the amplifier, for example by 0.01 dB or more, for example 0.05 dB or more, for example 0.1 dB or more, for example 0.5 dB or more, for example 1 dB or more, for example 2 dB or more, for example 3 dB or more, for example 5 dB or more, for example 10 dB or more, and including 25 dB or more. In one instance, the method includes gradually increasing the gain of the amplifier and calculating the phase correction, for example, gradually increasing the gain of the amplifier by 0.01 dB or more, for example, 0.05 dB or more, for example, 0.1 dB or more, for example, 0.5 dB or more, for example, 1 dB or more, for example, 2 dB or more, for example, 3 dB or more, for example, 5 dB or more, for example, 10 dB or more, and including 25 dB or more. In another example, the method includes gradually reducing the gain of the amplifier and calculating the phase correction, for example, gradually reducing the gain of the amplifier by 0.01 dB or more, for example, 0.05 dB or more, for example, 0.1 dB or more, for example, 0.5 dB or more, for example, 1 dB or more, for example, 2 dB or more, for example, 3 dB or more, for example, 5 dB or more, for example, 10 dB or more, and including 25 dB or more.

[0074] In some embodiments, calculating the phase correction for each of the fluorescence detectors of the light detection system includes calculating a lifetime phase correction component. The calculation of the lifetime phase correction is based on the phase correction calculated at each operating voltage of the fluorescence detector as described above and the fluorescence lifetime of the fluorophore in the sample. Depending on the specific type of fluorophore and the number of fluorophores present, one or more fluorescence lifetimes can be used to calculate the lifetime phase correction component, such as 2 or more than 2, such as 3 or more than 3, such as 4 or more than 4, and including 5 or more than 5 different fluorescence lifetimes can be used to calculate the lifetime phase correction component. In some embodiments, each fluorescence lifetime is calculated at the peak emission wavelength of the fluorophore. In these embodiments, each fluorescence lifetime can be detected and each fluorescence lifetime can be calculated using signals from different detection channels.

[0075] In certain embodiments, the lifetime phase correction for each of the fluorescence detectors is calculated based on the fluorescence lifetime of the fluorophores in the sample according to the following formula:

[0076]

[0077] τ = fluorescence lifetime

[0078] f = frequency

[0079] Figure 1 A flow chart is described for calculating a phase correction for one or more fluorescence detectors of a light detection system according to certain embodiments. In step 101, light from particles in a flow stream is detected using a brightfield photodetector and one or more fluorescence detectors. In step 102, a brightfield data signal and data signals from the fluorescence detectors are generated. In step 103, a phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector. In step 104, a lifetime phase correction is calculated using the phase correction component calculated in step 103 and the lifetime of the fluorophores of the sample in the flow stream. (Step 106) Steps 101 and 104 can be repeated after changing the operating voltage of each fluorescence detector. In step 105, the calculated phase correction is applied to each fluorescence detector. In step 107, frequency-encoded data is generated, and in step 108, phase-corrected spatial data is calculated by performing a transformation of the frequency-encoded fluorescence data, such as using a discrete Fourier transform. In step 109, the spatial data can be used to generate an image.

[0080] In certain embodiments, the method may further include calculating phase-corrected spatial data for the particles from the frequency-encoded fluorescence data using the calculated phase correction for each fluorescence detector as described above. In these embodiments, the method further includes generating frequency-encoded fluorescence data from particles in the sample using one or more fluorescence detectors in the light detection system; and calculating phase-corrected spatial data for the particles by performing a transform on the frequency-encoded fluorescence data using the calculated phase correction for each fluorescence detector. In one instance, the phase-corrected spatial data is calculated by performing a Fourier transform (FT) on the frequency-encoded fluorescence data using the calculated phase correction. In another instance, the phase-corrected spatial data is calculated by performing a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using the calculated phase correction. In another instance, the phase-corrected spatial data is calculated by performing a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using the calculated phase correction. In another instance, the phase-corrected spatial data is calculated using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data. By considering phase calibration before performing the transformation of the frequency-encoded data to spatial data, the output of the transformation has lower computational complexity than performing the transformation of the raw frequency data to spatial data (i.e., without first considering phase). In some embodiments, the method includes performing the transformation of the frequency-encoded fluorescence data without performing any imaginary mathematical calculations (i.e., performing only real mathematical calculations of the transformation), thereby generating spatial data from the frequency-encoded fluorescence data.

[0081] In some embodiments, the spatial data includes the size of the horizontal dimension of the particle, the size of the vertical dimension of the particle, the ratio of the particle sizes along two different dimensions, the size ratio of the particle components (e.g., the ratio of the horizontal dimension of the nucleus to the horizontal dimension of the cytoplasm of a cell).

[0082] In certain embodiments, the method includes generating an image of a particle in a flow stream by frequency coded fluorescence. In some embodiments, an image of a particle can be generated by frequency coded fluorescence in combination with detected light absorption, detected light scattering, or a combination thereof. In certain cases, an image of a particle is generated by frequency coded fluorescence alone. In other cases, an image of a target is generated by frequency coded fluorescence and light absorption detected from a sample, such as from a bright field photodetector. In other cases, an image of a particle is generated by frequency coded fluorescence and light scattering detected from a sample, such as from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector. In other cases, an image of a particle can be generated by a combination of frequency coded fluorescence and detected light absorption, detected light scattering, and detected light emission.

[0083] One or more images of a particle can be generated from the frequency-encoded fluorescence data. In some embodiments, the frequency-encoded fluorescence data generates a single image of the particle. In other embodiments, the frequency-encoded fluorescence data generates two or more images of the particle, such as three or more, such as four or more, such as five or more, and including ten or more, or a combination thereof.

[0084] System for phase correction of a signal from a light detection system

[0085] As described above, aspects of the present disclosure also include a system having a light detection system for characterizing sample particles in a flow stream (e.g., cells in a biological sample). Systems according to certain embodiments include a light source configured to illuminate a sample having particles in the flow stream, a light detection system having a brightfield photodetector configured to generate a brightfield data signal and one or more fluorescence detectors configured to generate a fluorescence data signal, and a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction for each of the fluorescence detectors based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector.

[0086] System of interest includes the light source configured to the sample in the irradiation flow stream.In embodiments, light source can be any suitable broadband light source or narrowband light source.Based on the composition in sample (for example, cell, bead, non-cellular particle etc.), light source can be configured to emission 200nm to 1500nm, for example 250nm to 1250nm, for example 300nm to 1000nm, for example 350nm to 900nm and the light comprising the wavelength of 400nm to 800nm.For example, light source can include the broadband light source of the light with the wavelength of 200nm to 900nm.In other cases, light source includes the narrowband light source of the wavelength of emission 200nm to 900nm.For example, light source can be the narrowband LED (1nm to 25nm) of the light of the wavelength of emission 200nm to 900nm.

[0087] In some embodiments, the light source is a laser. The laser of interest can include a pulsed laser or a continuous wave laser. For example, the laser can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon laser, or a combination thereof; Copper (NeCu) lasers, copper lasers or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs) or frequency doubling or frequency tripling of any of the above lasers.

[0088] In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a light emitting diode, such as a broadband LED with a continuous spectrum, a superluminescent diode, a semiconductor light emitting diode, a broad spectrum LED white light source, a multi-LED integration. In some cases, the non-laser light source is a stabilized fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0089] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams of light. In some cases, the beam generator includes a laser, a radio frequency generator configured to apply a radio frequency drive signal to the acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous wave laser. For example, the laser in the beam generator of interest can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a chlorine-xenon (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HSE) laser, or a combination thereof. solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers or cerium-doped lasers and combinations thereof.

[0090] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency-shift a laser beam using an applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied radio frequency drive signal. The radio frequency drive signal can be applied to the acousto-optic device by any suitable radio frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0091] In an embodiment, the controller is configured to apply an RF drive signal to the acousto-optic device to produce a desired number of angularly deflected laser beams in the output laser beam, for example, configured to apply 3 or more RF drive signals, for example, 4 or more RF drive signals, for example, 5 or more RF drive signals, for example, 6 or more RF drive signals, for example, 7 or more RF drive signals, for example, 8 or more RF drive signals, for example, 9 or more RF drive signals, for example, 10 or more RF drive signals, for example, 15 or more RF drive signals, for example, 25 or more RF drive signals, for example, 50 or more RF drive signals, and including being configured to apply 100 or more RF drive signals.

[0092] In some cases, to produce an angularly deflected laser beam with a certain intensity distribution in the output laser beam, the controller is configured to apply a radio frequency drive signal having a certain amplitude, such as from about 0.001 V to about 500 V, such as from about 0.005 V to about 400 V, such as from about 0.01 V to about 300 V, such as from about 0.05 V to about 200 V, such as from about 0.1 V to about 100 V, such as from about 0.5 V to about 75 V, such as from about 1 V to 50 V, such as from about 2 V to 40 V, such as from 3 V to about 30 V and including from about 5 V to about 25 V. In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0093] In certain embodiments, the controller comprises a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam having a desired intensity distribution. For example, the memory may include instructions to generate two or more angularly deflected laser beams having the same intensity, such as three or more, such as four or more, such as five or more, such as ten or more, such as twenty-five or more, such as fifty or more, and the memory may include instructions to generate one hundred or more angularly deflected laser beams having the same intensity. In other embodiments, instructions may be included to generate 2 or more angularly deflected laser beams having different intensities, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, and the memory may include instructions to generate 100 or more angularly deflected laser beams having different intensities.

[0094] In some embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam can be from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, for example, from 0.5% to about 95%, for example, from 1% to about 90%, for example, from about 2% to about 85%, for example, from about 3% to about 80%, for example, from about 4% to about 75%, for example, from about 5% to about 70%, for example, from about 6% to about 65%, for example, from about 7% to about 60%, for example, from about 8% to about 55%, and including from about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam can be from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, such as from 0.5% to about 95%, such as from 1% to about 90%, such as from about 2% to about 85%, such as from about 3% to about 80%, such as from about 4% to about 75%, such as from about 5% to about 70%, such as from about 6% to about 65%, such as from about 7% to about 60%, such as from about 8% to about 55%, 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 yet other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity distribution that is a Gaussian distribution along a horizontal axis. In yet other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity distribution that is a top-hat distribution along a horizontal axis.

[0095] In an embodiment, the beam generator of interest can be configured to generate spatially separated angularly deflected laser beams in the output laser beam. Depending on the applied RF drive signal and the desired illumination profile of the output laser beam, the angularly deflected laser beams can be separated by 0.001 μm or greater, such as 0.005 μm or greater, such as 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 5 μm or greater, such as 10 μm or greater, such as 100 μm or greater, such as 500 μm or greater, such as 1000 μm or greater, and including 5000 μm or greater. In some embodiments, the system is configured to generate an angularly deflected laser beam in the output laser beam that overlaps, for example, an adjacent angularly deflected laser beam along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., overlap of beam spots) can be 0.001 μm or greater, such as 0.005 μm or greater, such as 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 5 μm or greater, such as 10 μm or greater, and including 100 μm or greater.

[0096] In some cases, a beam generator configured to generate two or more frequency-shifted beams includes, for example, a laser excitation module as described in U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0097] In an embodiment, the system includes a light detection system having a bright field photodetector and one or more fluorescence detectors for detecting and measuring light from a sample. The subject of interest, bright field and fluorescence detectors may include, but are not limited to, optical sensors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCDs), enhanced charge coupled devices (ICCDs), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultipliers, phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other photodetectors. In certain embodiments, 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 is utilized to measure light from a sample. In certain embodiments, the bright field photodetector includes an avalanche photodiode (APD). In some cases, one or more fluorescence detectors are avalanche photodiodes.

[0098] In some embodiments, the light detection system of interest includes a plurality of fluorescence detectors. In some cases, the light detection system includes a plurality of solid-state detectors, such as photodiodes. In some cases, the light detection system includes a fluorescence photodetector array, such as a photodiode array. In these embodiments, the photodetector array can 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 500 or more photodetectors, such as 750 or more photodetectors and including 1000 or more photodetectors. For example, the detector can be a photodiode array having 4 or more photodiodes, for example 10 or more photodiodes, for example 25 or more photodiodes, for example 50 or more photodiodes, for example 100 or more photodiodes, for example 250 or more photodiodes, for example 500 or more photodiodes, for example 750 or more photodiodes and including 1000 or more photodiodes.

[0099] The photodetectors can 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 can be oriented at angles of 10° to 180°, 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°, relative to one another (as referred to in the XZ plane). The photodetector array can be of any suitable shape and can be a rectilinear shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom coupled to a planar top). In some embodiments, the photodetector array has a rectangular active surface.

[0100] Each photodetector (e.g., photodiode) in the array may have an active surface having a width of 5 μm to 250 μm, e.g., 10 μm to 225 μm, e.g., 15 μm to 200 μm, e.g., 20 μm to 175 μm, e.g., 25 μm to 150 μm, e.g., 30 μm to 125 μm, and including 50 μm to 100 μm, and a length of 5 μm to 250 μm, e.g., 10 μm to 225 μm, e.g., 15 μm to 200 μm, e.g., 20 μm to 175 μm, e.g., 25 μm to 150 μm, e.g., 30 μm to 125 μm, and including 50 μm to 100 μm, wherein the surface area of ​​each photodetector (e.g., photodiode) in the array is 25 μm. 2 Up to 10000μm 2 , for example 50 μm 2 Up to 9000μm 2 , for example 75 μm 2 Up to 8000μm 2 , for example 100 μm 2 Up to 7000μm 2 , for example 150 μm 2 Up to 6000μm 2 And including 200μm 2 Up to 5000μm 2 .

[0101] The size of the photodetector array can vary depending on the amount and intensity of light, the number of photodetectors and the required sensitivity, and can have a length of 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm and including 5 mm to 25 mm. The width of the photodetector array can also be 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm and including 5 mm to 25 mm. Thus, the area of ​​the active surface of the photodetector array can be 0.1 mm 2 Up to 10000mm 2 , for example 0.5mm 2 Up to 5000mm 2 , for example 1mm 2 Up to 1000mm 2 , for example 5mm 2 Up to 500mm 2 And including 10mm 2 Up to 100mm 2 .

[0102] The photodetector of interest is configured to measure collected light 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 a sample in a flowing stream at 400 or more different wavelengths.

[0103] In an embodiment, the light detection system includes a bright field photodetector configured to generate a bright field data signal. The bright field photodetector can be configured to detect light from the sample at one or more wavelengths, for example at 5 or more than 5 different wavelengths, for example at 10 or more than 10 different wavelengths, for example at 25 or more than 25 different wavelengths, for example at 50 or more than 50 different wavelengths, for example at 100 or more than 100 different wavelengths, for example at 200 or more than 200 different wavelengths, for example at 300 or more than 300 and including detecting light from the sample at 400 or more than 400 different wavelengths. The bright field photodetector can be configured to detect light at one or more wavelength ranges in the wavelength range of 200 nm to 1200 nm. In some cases, the method includes detecting light from the sample with a bright field photodetector over 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 800nm.

[0104] In some embodiments, the brightfield photodetector in the light detection system of interest is configured to generate one or more brightfield data signals in response to the detected light, such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, and including 10 or more brightfield data signals. When the brightfield photodetector is configured to detect light at multiple light wavelengths (e.g., 400 nm to 800 nm), in some cases, the method may include generating one or more brightfield data signals in response to each light wavelength detected. In other cases, a single brightfield data signal is generated in response to light detected by the brightfield photodetector over the entire wavelength range.

[0105] The light detection system includes one or more fluorescence detectors, for example, 2 or more, for example, 3 or more, for example, 4 or more, for example, 5 or more, for example, 6 or more, for example, 7 or more, for example, 8 or more, for example, 9 or more, for example, 10 or more, for example, 15 or more, and including 25 or more fluorescence detectors. In an embodiment, each of the fluorescence detectors is configured to generate a fluorescence data signal. Each fluorescence detector can independently detect fluorescence from the sample at one or more wavelengths in the wavelength range of 200 nm to 1200 nm. In some cases, one or more fluorescence detectors are configured to detect light from the sample over a wavelength range, such as 200 nm to 1200 nm, such as 300 nm to 1100 nm, such as 400 nm to 1000 nm, such as 500 nm to 900 nm and including 600 nm to 800 nm. In other cases, one or more fluorescence detectors are configured to detect light at one or more specific wavelengths. For example, fluorescence can be detected at one or more wavelengths of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof, depending on the number of different fluorescence detectors in the subject light detection system. In certain embodiments, one or more fluorescence detectors are configured to detect wavelengths of light corresponding to peak fluorescence wavelengths of certain fluorophores in the sample.

[0106] The light detection system is configured to measure light continuously or at discrete intervals. In some cases, the detector of the light detection system is configured to measure the collected light continuously. In other cases, the light detection system is configured to measure light at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds, or some other interval.

[0107] In some embodiments, the system is configured to generate frequency-encoded fluorescence data by illuminating a sample having particles in a flowing stream. In some embodiments, the light source includes a light generator assembly that generates a plurality of angularly deflected laser beams, the intensity of each laser beam being based on the amplitude of an applied radio frequency drive signal (e.g., from a direct digital synthesizer coupled to an acousto-optic device). For example, the subject system can include a light generator assembly that generates 2 or more, 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 10 angularly deflected laser beams and includes 25 or more angularly deflected laser beams. In an embodiment, each of the angularly deflected laser beams has a different frequency that is offset from the frequency of the input laser beam by a predetermined radio frequency.

[0108] According to certain embodiments, the subject system is configured to generate angularly deflected laser beams that are also spatially offset from one another. Depending on the applied RF drive signal and the desired irradiation profile of the output laser beams, the subject system can be configured to generate angularly deflected laser beams separated by 0.001 μm or greater, such as 0.005 μm or greater, such as 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 5 μm or greater, such as 10 μm or greater, such as 100 μm or greater, such as 500 μm or greater, such as 1000 μm or greater, and including 5000 μm or greater. In some embodiments, the angularly deflected laser beam overlaps, for example, with an adjacent angularly deflected laser beam along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., overlap of beam spots) can be 0.001 μm or greater, such as 0.005 μm or greater, such as 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 5 μm or greater, such as 10 μm or greater, and including 100 μm or greater.

[0109] In some embodiments, a system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate frequency-encoded fluorescence data by calculating the difference between the optical frequencies of incident overlapping beams on a flow stream. In one example, a system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a beat frequency for each position across a horizontal axis of the flow stream. In these embodiments, the frequency-encoded fluorescence emitted by the particle is a frequency-encoded fluorescence signal that is correlated with the local oscillator beam (f LO ) and the frequency of the RF offset beam. For example, frequency-encoded fluorescence data includes f LO -f RF偏移光束 Where the illumination of the flow stream comprises LO light spanning the width of the flow stream (e.g., the entire horizontal axis), the frequency-encoded fluorescence data comprises the beat frequency corresponding to the LO light beam (f LO ) and the frequency of each RF offset beam (f1, f2, f3, f4, f5, f6, etc.). In these embodiments, the frequency-encoded fluorescence data can include multiple beat frequencies, each beat frequency corresponding to a position across the horizontal axis of the flow stream.

[0110] In an embodiment, a system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction for each fluorescence detector based on a relative phase between a brightfield data signal and a fluorescence data signal from each fluorescence detector. In one example, the system is configured to detect light using a brightfield photodetector to generate a brightfield data signal, detect light using a first fluorescence detector configured to generate a first fluorescence data signal, and detect light using a second fluorescence detector configured to generate a second fluorescence data signal; calculate a phase correction for the first fluorescence detector based on a relative phase between the brightfield data signal and the first fluorescence data signal; and calculate a phase correction for the second fluorescence detector based on a relative phase between the brightfield data signal and the second fluorescence data signal. In another example, the system is configured to detect light using a brightfield photodetector configured to generate a brightfield data signal; detect light using a plurality of fluorescence detectors, and calculate a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and each of the fluorescence data signals from the plurality of fluorescence detectors.

[0111] In certain 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 phase correction for each of the fluorescence detectors based on the relative phase between the brightfield data signal and the fluorescence data signal according to the following formula:

[0112]

[0113] Where % represents the modulo function and all phases are expressed in radians.

[0114] In other embodiments, the system is configured to calculate a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal according to the following formula:

[0115]

[0116] where all phases are expressed as complex phase unit vectors.

[0117] The light detection system is configured to operate each of the detectors at a predetermined voltage. In some embodiments, the subject system is configured to calculate a phase correction for each fluorescence detector at a plurality of different operating voltages. The subject system is configured to phase correct the light detection system by detecting light with each fluorescence detector at a first set of operating voltages to generate a first set of fluorescence data signals; calculating a phase correction for each fluorescence detector at the first set of operating voltages based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector; changing the operating voltage of one or more fluorescence detectors to a second set of operating voltages; detecting light with each fluorescence detector at the second set of operating voltages to generate a second set of fluorescence data signals; and calculating a phase correction for each fluorescence detector at the second set of operating voltages based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector.

[0118] According to certain embodiments, the subject 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 change the operating voltage of each fluorescence detector and calculate the phase correction for each fluorescence detector at each operating voltage. In these embodiments, the system can be configured to change the operating voltage of each fluorescence detector to, for example, 0.01 V or higher, for example, 0.05 V or higher, for example, 0.1 V or higher, for example, 0.5 V or higher, for example, 1 V or higher, for example, 2 V or higher, for example, 3 V or higher, for example, 5 V or higher, for example, 10 V or higher, for example, 25 V or higher, for example, 50 V or higher, for example, 100 V or higher, for example, 250 V or higher, for example, 500 V or higher, and including 1000 V or higher. In one instance, the system is configured to gradually increase the operating voltage of each fluorescence detector and calculate the phase correction at each operating voltage, for example, gradually increasing the operating voltage by 0.01V or more, for example, 0.05V or more, for example, 0.1V or more, for example, 0.5V or more, for example, 1V or more, for example, 2V or more, for example, 3V or more, for example, 5V or more, for example, 10V or more, for example, 25V or more, for example, 50V or more, for example, 50V or more, for example, 100V or more, for example, 250V or more, for example, 500V or more, and including 1000V or more. In another example, the system is configured to gradually reduce the operating voltage of each fluorescence detector and calculate the phase correction at each operating voltage, for example, gradually reducing the operating voltage by 0.01V or more, for example, 0.05V or more, for example, 0.1V or more, for example, 0.5V or more, for example, 1V or more, for example, 2V or more, for example, 3V or more, for example, 5V or more, for example, 10V or more, for example, 25V or more, for example, 50V or more, for example, 100V or more, for example, 250V or more, for example, 500V or more, for example, 1000V or more, for example, 250V or more, for example, 500V or more, and including 1000V or more.

[0119] The system of interest can be configured to collect fluorescence data signals from each fluorescence detector at any number of operating voltages to perform phase correction on the light detection system, such as collecting fluorescence data signals from each fluorescence detector at 2 or more different operating voltages, such as 3 or more different operating voltages, such as 5 or more different operating voltages, such as 10 or more different operating voltages, such as 25 or more different operating voltages, and including collecting fluorescence data signals from each fluorescence detector at 50 or more different operating voltages. In some embodiments, the system is configured to calculate a phase correction for each fluorescence detector at each operating voltage used to collect light signals from a sample (e.g., a biological sample).

[0120] In an embodiment, the subject system can be configured to change the operating voltage of each fluorescence detector by the same or different amounts. In some cases, the system is configured to change the operating voltage of each fluorescence detector in the light detection system by the same amount. In other cases, the system is configured to change the operating voltage of each fluorescence detector in the light detection system by different amounts. In still other cases, the system is configured to change the operating voltage of two or more fluorescence detectors in the light detection system by the same amount and to change the operating voltage of two or more fluorescence detectors in the light detection system by different amounts. At each operating voltage, the system is configured to calculate a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal.

[0121] In some embodiments, the system of interest includes a variable gain amplifier. In some cases, the variable gain amplifier can be configured to operate at -100 dB to 100 dB, such as -75 dB to 75 dB, such as -50 dB to 50 dB, such as -25 dB to 25 dB, and including 0 dB to 50 dB. 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 change an electronic gain setting (e.g., the gain of the amplifier) ​​and calculate a phase correction for each fluorescence detector. In some embodiments, the system can be configured to change the gain of the amplifier, for example, by 0.01 dB or more, for example, by 0.05 dB or more, for example, by 0.1 dB or more, for example, by 0.5 dB or more, for example, by 1 dB or more, for example, by 2 dB or more, for example, by 3 dB or more, for example, by 5 dB or more, for example, by 10 dB or more, and including by 25 dB or more. In one instance, the system is configured to gradually increase the gain of the amplifier and calculate the phase correction, for example, gradually increasing the gain of the amplifier by 0.01 dB or more, for example, 0.05 dB or more, for example, 0.1 dB or more, for example, 0.5 dB or more, for example, 1 dB or more, for example, 2 dB or more, for example, 3 dB or more, for example, 5 dB or more, for example, 10 dB or more, and including 25 dB or more. In another instance, the system is configured to gradually reduce the gain of the amplifier and calculate the phase correction, such as gradually reducing the gain of the amplifier by 0.01 dB or more, such as 0.05 dB or more, such as 0.1 dB or more, such as 0.5 dB or more, such as 1 dB or more, such as 2 dB or more, such as 3 dB or more, such as 5 dB or more, such as 10 dB or more and including 25 dB or more.

[0122] In some embodiments, the system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a lifetime phase correction component. The lifetime phase correction is calculated based on the phase correction calculated at each operating voltage of the fluorescence detector as described above and the fluorescence lifetime of the fluorophore in the sample. Depending on the specific type of fluorophore and the number of fluorophores present, one or more, such as 2 or more, such as 3 or more, such as 4 or more, fluorescence lifetimes can be used to calculate the lifetime phase correction component, and 5 or more different fluorescence lifetimes can be used to calculate the lifetime phase correction component. In some embodiments, the system is configured to calculate each fluorescence lifetime at the peak emission wavelength of the fluorophore. In these embodiments, the lifetime of each fluorophore can be detected by a light detection system and calculated using signals from different detector channels.

[0123] In certain 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 lifetime phase correction for each fluorescence detector based on the fluorescence lifetime of a fluorophore in the sample according to the following formula:

[0124]

[0125] τ = fluorescence lifetime

[0126] f = frequency

[0127] In some embodiments, the system is further configured to calculate phase-corrected spatial data for the particles from the frequency-encoded fluorescence data using the phase correction calculated for each fluorescence detector as described herein. In some cases, the system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate phase-corrected spatial data by performing a Fourier transform (FT) on the frequency-encoded fluorescence data using the calculated phase correction. The spatial data according to an embodiment of the present disclosure is phase corrected by the system by performing a transform on the frequency-encoded fluorescence data using the calculated phase correction described herein. In some embodiments, the spatial data includes the horizontal dimension of the particle, the vertical dimension of the particle, the ratio of the particle size along two different dimensions, the size ratio of the particle components (e.g., the ratio of the horizontal dimension of the nucleus to the horizontal dimension of the cytoplasm).

[0128] In some embodiments, the subject system includes a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate phase-corrected spatial data for the particle by performing a Fourier transform on the frequency-encoded fluorescence data using a phase correction calculated for each fluorescence detector in the light detection system. In other embodiments, the system is configured to perform a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data using the calculated phase correction to generate phase-corrected spatial data for the particle. In still other embodiments, the system is configured to perform a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using the calculated phase correction. In still other embodiments, the system is configured to calculate the phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0129] In some embodiments, the system is configured to account for the calculated phase correction prior to performing the transformation of the frequency-encoded data into phase-corrected spatial data, such that the output of the transformation is computationally less complex than performing the transformation of the raw frequency data into spatial data (i.e., without first accounting for the calculated phase). In some embodiments, the system is configured to perform the transformation of the frequency-encoded fluorescence data to generate spatial data from the frequency-encoded fluorescence data without performing any mathematical imaginary calculations (i.e., performing only mathematical real calculations of the transformation).

[0130] The subject system can be configured to generate one or more images of particles in a flow stream from frequency-coded fluorescence. In some embodiments, an image of a particle can be generated by frequency-coded fluorescence in combination with detected light absorption, detected light scattering, or a combination thereof. In some cases, an image of a particle is generated solely by phase-corrected frequency-coded fluorescence. In other cases, an image of an object is generated by phase-corrected frequency-coded fluorescence and light absorption detected from a sample, such as light absorption detected from a brightfield photodetector. In still other cases, an image of a particle is generated by phase-corrected frequency-coded fluorescence and light scattering detected from a sample, such as from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector. In still other cases, an image of a particle is generated by a combination of phase-corrected frequency-coded fluorescence and detected light absorption, detected light scattering, and detected light emission.

[0131] Systems according to some embodiments may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, or the like. The processing module includes a processor that can access a memory having stored thereon instructions for executing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or it may be one of other processors currently available or that will become available. The processor executes the operating system and interfaces the operating system with firmware and hardware in a well-known manner, and facilitates the processor's coordination and execution of the functions of various computer programs that may be written in various programming languages ​​known in the art (e.g., Java, Perl, C++, other high-level or low-level languages, and combinations thereof). The operating system typically collaborates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, communication control, and related services according to known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0132] The system memory can be any of a variety of known or future memory storage devices. Examples include any commonly used random access memory (RAM), magnetic media (e.g., a resident hard drive or magnetic tape), optical media (e.g., a read-write optical disc), a flash memory device, or other memory storage device. The memory storage device can be any of a variety of known or future devices, including an optical disc drive, a magnetic tape drive, a removable hard drive, or a floppy disk drive. This type of memory storage device typically reads and / or writes a program storage medium (not shown), such as a compact disc, a magnetic tape, a removable hard drive, or a floppy disk. Any of these program storage media, or others currently in use or that may be developed in the future, can be considered a computer program product. As will be understood, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with the memory storage device.

[0133] In some embodiments, a computer program product is described that includes a computer-usable medium having stored therein control logic (a computer software program, including program code). The control logic, when executed by a processor of a computer, causes the processor to perform the functions described herein. In other embodiments, some functions are primarily implemented in hardware using, for example, a hardware state machine. Implementing a hardware state machine to perform the functions described herein will be apparent to those skilled in the relevant art.

[0134] The memory may be any suitable device in which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (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 by a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communication channel, or may be pre-stored in a computer program product such as a memory or some other portable or fixed computer-readable storage medium using any of those devices associated with the memory. For example, a magnetic or optical disk may carry the programming and be readable by a disk writer / reader. The system of the present invention also includes programming, such as an algorithm for practicing the above-described method in the form of a computer program product. The programming according to the present invention may be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROMs; electronic storage media such as RAM and ROM; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0135] The processor may also access a communication channel to communicate with a user at a remote location. A remote location refers to a user who is not directly interacting with the system and relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0136] In some embodiments, the system according to the present disclosure can be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface can be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (such as radio frequency identification (RFID)), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth communication protocol and cellular communication (such as code division multiple access (CDMA) or global system for mobile communications (GSM)).

[0137] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces (e.g., a USB port, an RS-232 port, or any other suitable electrical connection port) to allow data communication between the subject system and other external devices, such as computer terminals configured for similar supplemental data communications (e.g., in a doctor's office or hospital environment).

[0138] In one embodiment, the communication interface is configured for infrared communication, Bluetooth communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that a user may use in conjunction with.

[0139] In one embodiment, the communication interface is configured to provide a connection for data transmission using Internet Protocol (IP) through a cellular telephone network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0140] In one embodiment, the subject system is configured to communicate via a communication interface (e.g., using, for example, 802.11 or The server device may be another portable device, such as a smartphone, a personal digital assistant (PDA), or a notebook computer, or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as a button, a keyboard, a mouse, or a touch screen.

[0141] In some embodiments, the communication interface is configured to automatically or semi-automatically transfer data stored in the subject system (e.g., in the optional data storage unit) with a network or server device using one or more of the above-mentioned communication protocols and / or mechanisms.

[0142] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether a human or a machine, local or remote. If one of the display devices provides visual information, the information may typically be logically and / or physically organized as an array of graphic elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be accomplished using a network or other type of remote communication in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location using known techniques, such as via the Internet, telephone, or satellite networks. The presentation of data by the output manager may be implemented using a variety of known techniques. For example, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. The one or more platforms present in the subject system may be any type of known computer platform or one to be developed in the future, although they are typically a type of computer known as a server. However, they may also be mainframe computers, workstations, or other computer types. They may be connected by any known or future type of cable or other communication system (including wireless systems), whether networked or otherwise. They may be co-located or physically separate. A variety of operating systems may be used on any computer platform, depending on the type and / or brand of computer platform selected. Suitable operating systems include Windows 10, Windows Windows XP, Windows7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, SiemensReliant Unix, Ubuntu, Zorin OS, etc.

[0143] In certain embodiments, the subject system includes one or more optical adjustment components for adjusting light, such as light directed onto a sample (e.g., from a laser) or light collected from a sample (e.g., fluorescence). For example, the optical adjustment can be to increase the size of the light, the focus of the light, or to collimate the light. In some cases, the optical adjustment is an amplification protocol to increase the size of the light (e.g., a spot), such as by increasing the size by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, and including increasing the size by 75% or more. In other embodiments, the optical adjustment includes focusing the light to reduce the size 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 reducing the size of the spot by 75% or more. In certain embodiments, the optical adjustment includes collimating the light. The term "collimation" in its traditional sense refers to adjusting the collinearity of light propagation or reducing the divergence of light from a common propagation axis by optical means. In some cases, collimation involves narrowing the spatial cross-section of the light beam (e.g., reducing the beam profile of a laser).

[0144] 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 (e.g., a magnification of 0.6). For example, in some cases, the focusing lens is a bi-achromatic reduction lens with a magnification of approximately 0.6. The focal length of the focusing lens can be 5 mm to 20 mm, such as 6 mm to 19 mm, such as 7 mm to 18 mm, such as 8 mm to 17 mm, such as 9 mm to 16 mm, and including a focal length of 10 mm to 15 mm. In some embodiments, the focusing lens has a focal length of approximately 13 mm.

[0145] In other embodiments, the optical adjustment component is a collimator. The collimator can be any convenient collimating protocol, such as one or more reflectors or curved lenses or a combination thereof. For example, the collimator in some cases is a single collimating lens. In other cases, the collimator is a collimating lens. In still other cases, the collimator includes two lenses. In still other cases, the collimator includes a reflector and a lens. When the collimator includes one or more lenses, the focal length of the collimating lens can be 5 mm to 40 mm, such as 6 mm to 37.5 mm, such as 7 mm to 35 mm, such as 8 mm to 32.5 mm, such as 9 mm to 30 mm, such as 10 mm to 27.5 mm, such as 12.5 mm to 25 mm, and including a focal length of 15 mm to 20 mm.

[0146] In some embodiments, the subject system includes a flow cell nozzle having a nozzle orifice configured to cause a flow stream to flow through the flow cell nozzle. The subject flow cell nozzle has an orifice for propagating a fluid sample to a sample interrogation area, wherein in some embodiments, the flow cell nozzle includes a proximal cylindrical portion defining a longitudinal axis and a nozzle orifice transverse to the longitudinal axis that terminates in a distal truncated conical portion having a flat surface. The length (e.g., measured along the longitudinal axis) of the proximal cylindrical portion can be 1 mm to 15 mm, such as 1.5 mm to 12.5 mm, such as 2 mm to 10 mm, such as 3 mm to 9 mm, and including 4 mm to 8 mm. The length (as measured along the longitudinal axis) of the distal truncated conical portion can also be 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm, and including 4 mm to 7 mm. In some embodiments, the diameter of the flow cell nozzle chamber can be 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm, and including 4 mm to 7 mm.

[0147] In some cases, the nozzle chamber does not include a cylindrical portion and the entire flow cell nozzle chamber is frustoconical. In these embodiments, the length of the frustoconical nozzle chamber (e.g., measured along the longitudinal axis transverse to the nozzle orifice) can be 1 mm to 15 mm, such as 1.5 mm to 12.5 mm, such as 2 mm to 10 mm, such as 3 mm to 9 mm, and including 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical nozzle chamber can be 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm, and including 4 mm to 7 mm.

[0148] In an embodiment, the sample flow stream emanates from an orifice at the distal end of the flow cell nozzle. Depending on the desired characteristics of the flow stream, the flow cell nozzle orifice can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: straight cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved cross-sectional shapes, such as circular, elliptical; and irregular shapes, such as a parabolic bottom coupled to a flat top. In certain embodiments, the flow cell nozzle of interest has a circular orifice. In some embodiments, the size of the nozzle orifice can be 1 μm to 20,000 μm, such as 2 μm to 17,500 μm, such as 5 μm to 15,000 μm, such as 10 μm to 12,500 μm, such as 15 μm to 10,000 μm, such as 25 μm to 7,500 μm, such as 50 μm to 5,000 μm, such as 75 μm to 1,000 μm, such as 100 μm to 750 μm, and including 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0149] In some embodiments, the flow cell nozzle includes a sample injection port that is configured to provide a sample to the flow cell nozzle. In embodiments, 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 of sample delivered to the flow cell nozzle chamber through the sample injection port can be 1 μL / sec or higher, such as 2 μL / sec or higher, such as 3 μL / sec or higher, such as 5 μL / sec or higher, such as 10 μL / sec or higher, such as 15 μL / sec or higher, such as 25 μL / sec or higher, such as 50 μL / sec or higher, or, such as 100 μL / sec or higher. L / sec or higher than 100 μL / sec, for example 150 μL / sec or higher than 150 μL / sec, for example 200 μL / sec or higher than 200 μL / sec, for example 250 μL / sec or higher than 250 μL / sec, for example 300 μL / sec or higher than 300 μL / sec, for example 350 μL / sec or higher than 350 μL / sec, for example 400 μL / sec or higher than 400 μL / sec, for example 450 μL / sec or higher than 450 μL / sec and including 500 μL / sec or higher than 500 μL / sec. For example, the sample flow rate can be 1 μL / sec to about 500 μL / sec, for example 2 μL / sec to about 450 μL / sec, for example 3 μL / sec to about 400 μL / sec, for example 4 μL / sec to about 350 μL / sec, for example 5 μL / sec to about 300 μL / sec, for example 6 μL / sec to about 250 μL / sec, for example 7 μL / sec to about 200 μL / sec, for example 8 μL / sec to about 150 μL / sec, for example 9 μL / sec to about 125 μL / sec and including 10 μL / sec to about 100 μL / sec.

[0150] The sample injection port can be an orifice located in the wall of the nozzle chamber, or it can be a conduit located at the proximal end of the nozzle chamber. In the case where the sample injection port is an orifice located in the wall of the nozzle chamber, the sample injection port orifice can be any suitable shape, where the cross-sectional shapes of interest include, but are not limited to: straight cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as a parabolic bottom coupled to a flat top. In some embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on the shape, and in some cases, its opening ranges from 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm (e.g., 1.5 mm).

[0151] In some cases, the sample injection port is a conduit located proximal to the flow cell nozzle chamber. For example, the sample injection port can be a conduit positioned so that the orifice of the sample injection port is aligned with the orifice of the flow cell nozzle. In the case where the sample injection port is a conduit aligned with the flow cell nozzle orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: straight cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved cross-sectional shapes, such as circular, elliptical; and irregular shapes, such as a parabolic bottom coupled to a flat top. The orifice of the conduit can vary in shape, and in some cases, its opening ranges from 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, and including 1.25 mm to 1.75 mm (e.g., 1.5 mm). The shape of the tip of the sample injection port can be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip having a bevel angle of 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, and including 5°.

[0152] In some embodiments, the flow cell nozzle further comprises a sheath liquid injection port configured to provide sheath liquid to the flow cell nozzle. In an embodiment, the sheath liquid injection system is configured to provide a sheath liquid flow to the flow cell nozzle chamber, for example, in combination with the sample to produce a laminar sheath liquid flow surrounding the sample flow. Depending on the desired characteristics of the flow stream, the rate of sheath liquid delivered to the flow cell nozzle chamber can be 25 μL / sec or higher, such as 50 μL / sec or higher, such as 75 μL / sec or higher, such as 100 μL / sec or higher, such as 250 μL / sec or higher, such as 500 μL / sec or higher, such as 750 μL / sec or higher, such as 1000 μL / sec or higher and including 2500 μL / sec or higher. For example, the sheath fluid flow rate can be 1 μL / sec to about 500 μL / sec, for example 2 μL / sec to about 450 μL / sec, for example 3 μL / sec to about 400 μL / sec, for example 4 μL / sec to about 350 μL / sec, for example 5 μL / sec to about 300 μL / sec, for example 6 μL / sec to about 250 μL / sec, for example 7 μL / sec to about 200 μL / sec, for example 8 μL / sec to about 150 μL / sec, for example 9 μL / sec to about 125 μL / sec and including 10 μL / sec to about 100 μL / sec.

[0153] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the nozzle chamber. The sheath fluid injection port orifice can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved cross-sectional shapes, such as circular, elliptical; and irregular shapes, such as a parabolic bottom coupled to a flat top. The size of the sample injection port orifice can vary depending on the shape, and in some cases, its opening ranges from 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm (e.g., 1.5 mm).

[0154] In some cases, the subject system includes a sample interrogation area that is fluidly connected to the flow cell nozzle hole. In these cases, the sample flow stream is emitted from the orifice at the far end of the flow cell nozzle, and the particles in the flow stream can be irradiated with a light source in the sample interrogation area. The size of the interrogation area may vary depending on the characteristics of the flow nozzle, such as the size of the nozzle hole and the size of the sample injection port. In embodiments, the width of the interrogation area can be 0.01 mm or greater than 0.01 mm, such as 0.05 mm or greater than 0.05 mm, such as 0.1 mm or greater than 0.1 mm, such as 0.5 mm or greater than 0.5 mm, such as 1 mm or greater than 1 mm, such as 2 mm or greater than 2 mm, such as 3 mm or greater than 3 mm, such as 5 mm or greater than 5 mm and including 10 mm or greater than 10 mm. In some cases, the length of the interrogation region may also be 0.01 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 1.5 mm or greater, such as 2 mm or greater, such as 3 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 15 mm or greater, such as 20 mm or greater, such as 25 mm or greater and including 50 mm or greater.

[0155] The interrogation area can be configured to facilitate illuminating a planar cross-section of the outgoing flow stream, or can be configured to facilitate illuminating a diffuse field of a predetermined length (e.g., using a diffuse laser or lamp). In some embodiments, the interrogation area includes a transparent window that facilitates illuminating an outgoing flow of a predetermined length, such as 1 mm or longer than 1 mm, such as 2 mm or longer than 2 mm, such as 3 mm or longer than 3 mm, such as 4 mm or longer than 4 mm, such as 5 mm or longer than 5 mm and including 10 mm or longer than 10 mm. Depending on the light source used to illuminate the outgoing flow (as described below), the interrogation area can be configured to pass light of 100 nm to 1500 nm, such as 150 nm to 1400 nm, such as 200 nm to 1300 nm, such as 250 nm to 1200 nm, such as 300 nm to 1100 nm, such as 350 nm to 1000 nm, such as 400 nm to 900 nm and including light of 500 nm to 800 nm.Thus, the interrogation region may be formed from any transparent material that passes the desired wavelength range, including but not limited to optical glass, borosilicate glass, Pyrex, UV quartz, IR quartz, sapphire, and plastics such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics (e.g., PETG (ethylene glycol-modified polyethylene terephthalate)), as well as other polymeric plastic materials, including polyesters, where polyesters of interest may include but are not limited to poly(alkylene terephthalates), such as poly(ethylene terephthalate glycol) (PET), bottle-grade PET (a copolymer based on (ethylene glycol, terephthalic acid and other comonomers such as isophthalic acid, cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT) and poly(hexamethylene terephthalate); poly(alkylene adipates) such as poly(ethylene adipate), poly(1,4-butylene adipate) and poly(hexamethylene adipate); poly(alkylene suberates) such as poly(ethylene suberate); poly(alkylene sebacates) such as poly(ethylene sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalates) such as poly(ethylene isophthalate); poly(2,6-alkylene naphthalate)s such as Such as poly (ethylene 2,6-naphthalate); poly (alkylene sulfonyl-4,4'-dibenzoate), such as poly (ethylene sulfonyl-4,4'-dibenzoate); poly (p-phenylene alkylene dicarboxylates), such as poly (p-phenylene ethylene dicarboxylates); poly (trans-1,4-cyclohexane dialkylene dicarboxylates), such as poly (trans-1,4-cyclohexane diethylene dicarboxylates); poly (1,4-cyclohexane-dimethylene ethylene dicarboxylates), such as poly (1,4-cyclohexane-dimethylene ethylene dicarboxylates); poly ([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylates), such as poly ( [2.2.2]-bicyclooctane-1,4-dimethyleneethylene dicarboxylate); lactic acid polymers and copolymers such as (S)-polylactide, (R,S)-polylactide, poly(tetramethyl glycolide) and poly(lactide-co-glycolide); and polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A; polyamides such as poly(terephthalamide); polyesters such as polyethylene terephthalate, such as Mylar™ polyethylene terephthalate, etc.; In some embodiments, the subject system includes a cuvette positioned in the sample interrogation area.In embodiments, the cuvette can pass light from 100 nm to 1500 nm, such as from 150 nm to 1400 nm, such as from 200 nm to 1300 nm, such as from 250 nm to 1200 nm, such as from 300 nm to 1100 nm, such as from 350 nm to 1000 nm, such as from 400 nm to 900 nm, and including from 500 nm to 800 nm.

[0156] In some embodiments, the subject system includes a particle sorting assembly for sorting particles (e.g., cells) of a sample. In some cases, the particle sorting assembly is a particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed on March 28, 2017, and U.S. Provisional Patent Application No. 62 / 752793, filed on October 30, 2018, the disclosures of which are incorporated herein by reference. In certain embodiments, the particle sorting assembly includes one or more droplet deflectors, such as those described in U.S. Patent Publication No. 2018 / 0095022, filed on June 14, 2017, the disclosure of which is incorporated herein by reference.

[0157] In some embodiments, the subject system is a flow cytometer system. Suitable flow cytometry systems may include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49 (pt 1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222 (4): 335-344 and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosure of which is incorporated herein by reference. In some cases, flow cytometer systems of interest include the BD Biosciences FACSCanto TMII flow cytometer, BD Accuri TM Flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortess TM X-20 flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter and BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter and BD Biosciences FACSMelody TM Cell sorting machine, etc.

[0158] In some embodiments, the subject particle sorting system is a flow cytometer system, such as those described in U.S. Pat. Nos. 10,006,852, 9,952,076, 9,933,341, 9,784,661, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,2331, 46, 8140300, 7544326, 7201875, 7129505, 6821740, 6813017, 6809804, 6372506, 5700692, 5643796, 5627040, 5620842, 5602039; the disclosures of the above patents are incorporated herein by reference in their entirety.

[0159] In some cases, the subject system is a flow cytometry system configured for characterizing and imaging particles in a flowing stream by using fluorescence imaging of radiofrequency tag emission (FIRE), such as those described in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013), and in U.S. Patent Nos. 9,423,353, 9,784,661, and 1,000,6852, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0160] integrated circuit devices

[0161] Aspects of the present disclosure also include integrated circuit devices programmed to perform phase correction on a light detection system having a brightfield photodetector and one or more fluorescence detectors. In an embodiment, the subject integrated circuit device is programmed to receive a brightfield data signal from a brightfield photodetector and a data signal from one or more fluorescence detectors, and calculates a phase correction for each fluorescence photodetector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector. In an embodiment, the integrated circuit device is programmed to receive data signals from one or more fluorescence detectors (e.g., one or more detection channels), e.g., 2 or more than 2, e.g., 3 or more than 3, e.g., 4 or more than 4, e.g., 5 or more than 5, e.g., 6 or more than 6 and including 8 or more fluorescence detectors (e.g., 8 or more detection channels). In some cases, 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).

[0162] In one embodiment, the integrated circuit device is programmed to receive a brightfield data signal from a brightfield photodetector, receive a first fluorescence data signal from a first fluorescence detector, and receive a second fluorescence data signal from a second fluorescence detector; calculate a phase correction for the first fluorescence detector based on a relative phase between the brightfield data signal and the first fluorescence data signal; and calculate a phase correction for the second fluorescence detector based on a relative phase between the brightfield data signal and the second fluorescence data signal. In another embodiment, the integrated circuit device is programmed to receive a brightfield data signal from a brightfield photodetector; receive data signals from a plurality of fluorescence detectors and calculate a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and each of the fluorescence data signals from the plurality of fluorescence detectors.

[0163] In some embodiments, the integrated circuit device is programmed to calculate a phase correction for each fluorescence detector at a plurality of different operating voltages. In these embodiments, the integrated circuit device is programmed to phase correct the light detection system by receiving a fluorescence data signal from each fluorescence detector at a first set of operating voltages; calculating a phase correction for each fluorescence detector at the first set of operating voltages based on a relative phase between a brightfield data signal and the fluorescence data signal from each fluorescence detector; changing the operating voltage of one or more fluorescence detectors to a second set of operating voltages; receiving a fluorescence data signal from each fluorescence detector at the second set of operating voltages to generate a second set of fluorescence data signals; and calculating a phase correction for each fluorescence detector at the second set of operating voltages based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector.

[0164] The subject integrated circuit device can be programmed to change the operating voltage of each fluorescence detector and calculate the phase correction of each fluorescence detector at each operating voltage. In these embodiments, the integrated circuit device can be programmed to change the operating voltage of each fluorescence detector, for example, 0.01V or more, for example, 0.05V or more, for example, 0.1V or more, for example, 0.5V or more, for example, 1V or more, for example, 2V or more, for example, 3V or more, for example, 5V or more, for example, 10V or more, for example, 25V or more, for example, 50V or more, for example, 100V or more, for example, 250V or more, for example, 500V or more, and including 1000V or more. In one embodiment, the integrated circuit device can be programmed to gradually increase the operating voltage of each fluorescence detector and calculate the phase correction at each operating voltage, for example, gradually increasing the operating voltage by 0.01V or more than 0.01V, for example 0.05V or more than 0.05V, for example 0.1V or more than 0.1V, for example 0.5V or more than 0.5V, for example 1V or more than 1V, for example 2V or more than 2V, for example 3V or more than 3V, for example 5V or more than 5V, for example 10V or more than 10V, for example 25V or more than 25V, for example 50V or more than 50V, for example 100V or more than 100V, for example 250V or more than 250V, for example 500V or more than 500V and including 1000V or more than 1000V. In another embodiment, the integrated circuit device can be programmed to gradually reduce the operating voltage of each fluorescence detector and calculate the phase correction at each operating voltage, for example, by gradually reducing the operating voltage by 0.01V or more, for example 0.05V or more, for example 0.1V or more, for example 0.5V or more, for example 1V or more, for example 2V or more, for example 3V or more, for example 5V or more, for example 10V or more, for example 25V or more, for example 50V or more, for example 50V or more, for example 100V or more, for example 250V or more, for example 500V or more, for example 1000V or more, for example 250V or more, for example 500V or more and including 1000V or more.

[0165] The integrated circuit device of interest can be programmed to collect fluorescence data signals from each fluorescence detector at any number of operating voltages to perform phase correction on the light detection system, such as collecting fluorescence data signals from each fluorescence detector at 2 or more different operating voltages, such as 3 or more different operating voltages, such as 5 or more different operating voltages, such as 10 or more different operating voltages, such as 25 or more different operating voltages, and including collecting fluorescence data signals from each fluorescence detector at 50 or more different operating voltages. In certain embodiments, the integrated circuit device is programmed to calculate the phase correction for each of the fluorescence detectors at each operating voltage used to collect light signals from a sample (e.g., a biological sample).

[0166] In an embodiment, the integrated circuit device can be programmed to change the operating voltage of each fluorescence detector by the same or different amounts. In some cases, the integrated circuit device is programmed to change the operating voltage of each fluorescence detector in the light detection system by the same amount. In other cases, the integrated circuit device is programmed to change the operating voltage of each fluorescence detector in the light detection system by different amounts. In other cases, the integrated circuit device is programmed to change the operating voltage of two or more fluorescence detectors in the light detection system by the same amount, and to change the operating voltage of two or more fluorescence detectors in the light detection system by different amounts. At each operating voltage, the integrated circuit device is programmed to calculate a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal.

[0167] In some embodiments, the integrated circuit device is programmed to calculate a lifetime phase correction component. The lifetime phase correction is calculated based on the phase correction calculated at each operating voltage of the fluorescence detector as described above and the fluorescence lifetime of the fluorophore in the sample. Depending on the specific type of fluorophore and the number of fluorophores present, one or more fluorescence lifetimes can be used to calculate the lifetime phase correction component, such as 2 or more than 2, such as 3 or more than 3, such as 4 or more than 4, and including 5 or more than 5 different fluorescence lifetimes can be used to calculate the lifetime phase correction component. In some embodiments, the integrated circuit device is programmed to calculate each fluorescence lifetime at the peak emission wavelength of the fluorophore. In these embodiments, the lifetime of each fluorophore can be detected by the light detection system and calculated using signals from different detector channels.

[0168] In some embodiments, the integrated circuit device is programmed to generate frequency-encoded fluorescence data from particles in a sample using fluorescence detectors; and phase-corrected spatial data for the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction for each fluorescence detector. In some cases, 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 cases, the system is configured to generate one or more data components of the frequency-encoded fluorescence data based on light absorption detected from the sample, such as from a brightfield photodetector. In some cases, one or more data components of the frequency-encoded fluorescence data are generated by light scattering detected from the sample, such as from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector.

[0169] In an embodiment, the subject integrated circuit device is programmed to calculate phase-corrected spatial data from the frequency-encoded fluorescence data. According to embodiments of the present disclosure, the spatial data is phase-corrected by transforming the frequency-encoded fluorescence data using the calculated phase correction described herein. In some embodiments, the spatial data includes the size of the horizontal dimension of the particle, the size of the vertical dimension of the particle, the ratio of the size of the particle along two different dimensions, the size ratio of the particle components (e.g., the ratio of the horizontal dimension of the nucleus to the horizontal dimension of the cytoplasm).

[0170] In an embodiment, the subject integrated circuit device is programmed to calculate phase-corrected spatial data for a particle by transforming the frequency-encoded fluorescence data with the calculated phase correction for each fluorescence detector in the light detection system. In some embodiments, to calculate the phase-corrected spatial data, the integrated circuit device is programmed to perform a Fourier transform on the frequency-encoded fluorescence data with the calculated phase correction to generate the phase-corrected spatial data for the particle. In other embodiments, the integrated circuit device is programmed to perform a discrete Fourier transform (DFT) on the frequency-encoded fluorescence data with the calculated phase correction to generate the phase-corrected spatial data for the particle. In other embodiments, the integrated circuit device is programmed to perform a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data with the calculated phase correction. In other embodiments, the integrated circuit device is programmed to calculate the phase-corrected spatial data using a digital lock-in amplifier to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0171] Composition for phase correction of light detection system

[0172] Aspects of the present disclosure include compositions for phase correction of light detection systems, such as by causing the subject composition to flow in a flow stream and calculating the phase correction of one or more fluorescence detectors of the light detection system according to the method described herein. The composition according to the embodiment includes multiple particles and a fluorescent dye component so that the fluorescent dye component has a stable fluorescence lifetime. In this article, the term "stable fluorescence lifetime" is used in its conventional sense to refer to a fluorophore with almost no change in fluorescence lifetime, such as in response to different experimental conditions (such as temperature, laser irradiation, gas composition, etc.). In some cases, the fluorescence lifetime of the fluorescent dye component of interest shows a fluorescence lifetime change of 1ns or shorter than 1ns, such as 0.5ns or shorter than 0.5ns, such as 0.1ns or shorter than 0.1ns, such as 0.05ns or shorter than 0.05ns, such as 0.01ns or shorter than 0.01ns, such as 0.005ns or shorter than 0.005ns, such as 0.001ns or shorter than 0.001ns and including 0.0001ns or shorter than 0.0001ns. In these cases, the fluorescent dye composition exhibits a fluorescence lifetime change of 5% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.05% or less, such as 0.01% or less, such as 0.005% or less, and including a fluorescence lifetime change of 0.001% or less.

[0173] In embodiments, the composition includes a fluorescent dye component. In some cases, the fluorescent dye component includes a detectable detectable portion or label based on, for example, fluorescence emission maximum, fluorescence polarization, fluorescence lifetime, or a combination thereof. In certain embodiments, the detectable label is a fluorophore (i.e., a fluorescent marker, a fluorescent dye, etc.). The fluorophore of interest can include, but is not limited to, dyes suitable for analytical applications (e.g., flow cytometry, imaging, etc.). For example, the fluorescent dye component can include compounds such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, dipyrromethene boron difluoride, naphthalimide, phycobiliprotein, polydinium chlorophyll protein, conjugates thereof, and combinations thereof. In certain embodiments, the fluorescent dye component includes Nile red dye.In other embodiments, the fluorescent dye component includes a dye selected from the group consisting of: 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and its derivatives, such as acridine, acridine orange, acridine yellow, acridine red, acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and its derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 120), 4-amino-5-methyl-1-oxolane-2-ol (Lucifer Yellow VS); 151); cyanine and its derivatives, such as cyanine, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-diamino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and its derivatives, such as eosin and Eosin isothiocyanate; erythrosine and its derivatives, such as erythrosine B and erythrosine isothiocyanate; ethidium; fluorescein and its derivatives, such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), chlorotriazinylfluorescein, naphthofluorescein and QFITC ​​(XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); ReefCoral Fluorescent Protein (RCFP); Lissamine. TM Lissamine rhodamine, fluorescein; malachite green molecule; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; para-fuchsin; Nile red; Oregon green; phenol red; beta-phycoerythrin (PE); o-phthalaldehyde; pyrene and its derivatives, such as pyrene, pyrene butyrate, and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron TMBrilliant Red 3B-A); rhodamine and its derivatives, such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, the sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; cresylic acid and terbium chelate derivatives; xanthenes; carotenoid-protein complexes, such as peridinin chlorophyll protein (PerCP); allophycocyanin (APC); or combinations thereof.

[0174] In some cases, fluorescent dye composition includes polymer dye (such as fluorescent polymer dye). In some cases, polymer dye includes conjugated polymer. Conjugated polymer (CP) is characterized by delocalized electronic structure, which includes the main chain of alternating unsaturated bonds (such as double bonds and / or triple bonds) and saturated (such as single bonds), wherein π electrons can move from one bond to another bond. Therefore, the conjugated main chain can give an extended linear structure on the polymer dye, with limited bond angles between the repeating units of the polymer. For example, although proteins and nucleic acids are also polymerized, they will not form extended rod structures in some cases, but will fold into higher-order three-dimensional shapes. In addition, CP may form a "rigid rod" polymer main chain and experience limited distortion (such as, torsion) angles between the monomer repeating units along the polymer main chain. In some cases, polymer dye includes the CP with a rigid rod structure. The structural characteristics of polymer dyes can have an impact on the fluorescent properties of molecules.

[0175] In certain embodiments, polymeric dyes of interest include, but are not limited to, those disclosed by Gaylord et al. in U.S. Patent Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, 201301 No. 90193, the disclosures of which are incorporated herein by reference in their entireties; and those described in Gaylord et al., J. Am. Chem. Soc., 2001, 123(26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419 and Traina et al., J. Am. Chem. Soc., 2011, 133(32), pp 12600-12607, the disclosures of which are incorporated herein by reference in their entireties.

[0176] The composition of interest also includes a plurality of particles. In some embodiments, the fluorescent dye component is stably bound to the particles. Stable association refers to that the fluorescent dye component is not easily dissociated from the particles, for example, when in contact with a liquid medium, such as an aqueous medium. Therefore, when present in a flow stream (for example, when used to calculate the phase correction of one or more fluorescence detectors as described herein), the fluorescent dye component remains bound to its particles. In some cases, the fluorescent dye component is covalently bonded to the particles.

[0177] In some embodiments, the particles in the subject compositions are beads, such as structures with diameters in the nanometer to micrometer range, such as 0.01 μm to 1000 μm in diameter, such as 0.1 μm to 100 μm in diameter, and including 1 μm to 100 μm in diameter, and for flow cytometry, including about 1 μm to 10 μm in diameter. These particles can be of any shape and in some cases are approximately spherical. These particles can be made of any suitable material (or combination thereof), including but not limited to polymers such as polystyrene; polystyrene containing other copolymers such as divinylbenzene; polymethyl methacrylate (PMMA); polyvinyl toluene (PVT); copolymers such as styrene / butadiene, styrene / vinyl toluene; latex; glass; or other materials such as silica (e.g., SiO2). In some embodiments, particles such as beads, such as glass beads, with low or no autofluorescence are of interest.

[0178] In some embodiments, the beads are metal organic polymer matrices, for example, organic polymer matrices having a backbone structure comprising a metal such as aluminum, barium, antimony, calcium, chromium, copper, erbium, germanium, iron, lead, lithium, phosphorus, potassium, silicon, tantalum, tin, titanium, vanadium, zinc, or zirconium. In some embodiments, the porous metal organic matrix is ​​an organosiloxane polymer, including but not limited to methyltrimethoxysilane, dimethyldimethoxysilane, tetraethoxysilane, methacryloxypropyltrimethoxysilane, bis(triethoxysilyl)ethane, bis(triethoxysilyl)butane, bis(triethoxysilyl)pentane, bis(triethoxysilyl)hexane, bis(triethoxysilyl)heptane, bis(triethoxysilyl)octane, and combinations thereof.

[0179] The particles in the composition of interest can be porous or non-porous. In some embodiments, the particles are non-porous. In other embodiments, they can be porous, for example, wherein the particles have pores with a diameter of 0.01 nm to 1000 nm, such as pores with a diameter of 0.05 nm to 750 nm, such as pores with a diameter of 0.1 nm to 500 nm, such as pores with a diameter of 0.5 nm to 250 nm, such as pores with a diameter of 1 nm to 100 nm, such as pores with a diameter of 5 nm to 75 nm, and including particles with pores with a diameter of 10 nm to 50 nm.

[0180] In certain embodiments, fluorescently labeled beads of interest include, but are not limited to, fluorescently labeled polystyrene beads, fluorescein beads, rhodamine beads, and other beads labeled with fluorescent dyes. Other examples of fluorescently labeled beads are described in U.S. Patent Nos. 6,350,619, 7,738,094, and 8,248,597, the disclosures of each of which are incorporated herein by reference in their entirety.

[0181] Kit

[0182] Aspects of the present disclosure also include kits, wherein the kit includes one or more than one of the above-mentioned components, such as integrated circuit devices, phase calibration compositions, etc., such as described herein. In some embodiments, the kit may also include a program for the subject system, such as in the form of a computer-readable medium (e.g., a flash drive, a USB storage device, an optical disc, a DVD, a Blu-ray disc, etc.) or an instruction for downloading a program from an Internet network protocol or a cloud server. The kit may also include one or more than one compositions for phase correction of the light detection system. The kit may also include instructions for practicing the subject method. These instructions may exist in the subject kit in various forms, wherein one or more than one form may exist in the kit. One form in which these instructions may exist is to be presented as printed information on a suitable medium or substrate, such as paper on which information is printed, in the packaging of the kit, in a packaging insert, etc. Another form of these instructions is a computer-readable medium, such as a disk, an optical disc (CD), a portable flash drive, etc., wherein information has been recorded thereon. Another form in which these instructions may exist is a website address, which can be used to access information on a site away from the Internet.

[0183] Practicality

[0184] The subject systems, methods, and computer systems can be used in a variety of applications where it is desirable to analyze and classify particle components in a sample in a fluid medium, such as a biological sample. In some embodiments, the systems and methods described herein can be used for flow cytometric characterization of biological samples labeled with fluorescent tags. In other embodiments, the systems and methods can be used for spectroscopy of emitted light. Additionally, the subject systems and methods can be used to increase the signal obtained from light collected from a sample (e.g., in a flowing stream). Embodiments of the present disclosure can be used in situations where it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting.

[0185] Embodiments of the present disclosure may also be used for cells prepared from biological samples that may need to be used for research, laboratory testing, or for treatment applications. In some embodiments, subject method and apparatus may contribute to obtaining single cells prepared from target fluid or tissue biological samples. For example, method and system of the present invention contribute to obtaining cells from fluid or tissue samples, to be used as research or diagnostic samples for diseases such as cancer. Equally, subject method and system may contribute to obtaining cells from fluid or tissue samples for treatment. Compared with traditional flow cytometry systems, method and apparatus of the present disclosure make it possible to separate and collect cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with improved efficiency and low cost.

[0186] Notwithstanding the appended claims, the present disclosure is defined by the following terms:

[0187] 1. A method comprising:

[0188] detecting light from a sample containing particles in a flow stream with a light detection system, the light detection system comprising:

[0189] a brightfield photodetector configured to generate a brightfield data signal in response to detected light; and

[0190] a fluorescence detector configured to generate a fluorescence data signal in response to the detected light;

[0191] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal.

[0192] 2. A method according to clause 1, wherein the phase correction of the fluorescence detector is calculated in the first configuration.

[0193] 3. The method according to any one of clauses 1-2, wherein the method further comprises:

[0194] In a second configuration, detecting light from the sample using a fluorescence detector; and

[0195] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector in the second configuration.

[0196] 4. The method according to clause 3, further comprising:

[0197] In a third configuration, light from the sample is detected using a fluorescence detector; and

[0198] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector in the third configuration.

[0199] 5. A method according to any of clauses 2-4, wherein the configuration is a voltage.

[0200] 6. The method according to clause 5, wherein the method comprises:

[0201] detecting light from the sample using a fluorescence detector at a first voltage;

[0202] calculating a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and a fluorescence data signal from the fluorescence detector at a first voltage;

[0203] detecting light from the sample using a fluorescence detector at a second voltage; and

[0204] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector at the second voltage.

[0205] 7. A method according to any of clauses 2-4, wherein the configuration is an electronic gain setting.

[0206] 8. The method according to clause 7, wherein the method comprises:

[0207] detecting light from the sample using a fluorescence detector at a first electronic gain setting;

[0208] calculating a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and a fluorescence data signal from the fluorescence detector at a first electronic gain setting;

[0209] detecting light from the sample using a fluorescence detector at a second electronic gain setting; and

[0210] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector at the second electronic gain setting.

[0211] 9. The method according to any one of clauses 1 to 8, further comprising:

[0212] A lifetime phase correction for the fluorescence detector is calculated based on the calculated phase correction for the fluorescence detector and the fluorescence lifetime of the fluorophores in the sample.

[0213] 10. The method of any of clauses 1-9, wherein the light detection system comprises a plurality of fluorescence detectors, wherein each fluorescence detector is configured to independently generate a fluorescence data signal in response to detected light.

[0214] 11. A method according to clause 10, wherein the method comprises calculating a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector.

[0215] 12. The method of clause 11, wherein the phase correction is calculated for each fluorescence detector comprising a predetermined configuration.

[0216] 13. The method of clause 12, wherein the predetermined configuration is a voltage.

[0217] 14. The method according to clause 13, wherein the method comprises:

[0218] detecting light from the sample using each fluorescence detector at a first voltage;

[0219] calculating a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at a first voltage;

[0220] detecting light from the sample using each fluorescence detector at a second voltage; and

[0221] A phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at the second voltage.

[0222] 15. The method of clause 14, wherein the predetermined voltage is the same for all fluorescence detectors in the light detection system.

[0223] 16. The method of clause 14, wherein the predetermined voltage is different for each of the fluorescence detectors.

[0224] 17. The method of clause 12, wherein the predetermined configuration is an electronic gain setting.

[0225] 18. The method according to clause 17, wherein the method comprises:

[0226] detecting light from the sample using each fluorescence detector at a first electronic gain setting;

[0227] calculating a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at a first electronic gain setting;

[0228] detecting light from the sample using each fluorescence detector at a second electronic gain setting; and

[0229] A phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at the second electronic gain setting.

[0230] 19. The method of clause 18, wherein the predetermined electronic gain setting is the same for all fluorescence detectors in the light detection system.

[0231] 20. The method of clause 18, wherein the predetermined electronic gain setting is different for each of the fluorescence detectors.

[0232] 21. The method of any of clauses 10-20, further comprising calculating a lifetime phase correction for each of the fluorescence detectors based on the calculated phase correction for each fluorescence detector and the fluorescence lifetime of the fluorophores in the sample.

[0233] 22. The method of any one of clauses 1 to 21, further comprising:

[0234] generating frequency-encoded fluorescence data from particles in the sample using a fluorescence detector; and

[0235] Phase-corrected spatial data for the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction for each fluorescence detector.

[0236] 23. The method of clause 22, wherein the spatial data is calculated by Fourier transforming the frequency encoded fluorescence data using the phase correction component.

[0237] 24. The method of clause 23, wherein the spatial data is calculated by discrete Fourier transforming the frequency encoded fluorescence data using a phase correction component.

[0238] 25. The method of clause 23, wherein the spatial data is calculated by performing a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data using a phase correction component.

[0239] 26. The method of clause 22, wherein the spatial data is calculated using a digital lock-in amplifier to heterodyne the frequency coded fluorescence data and demultiplex the frequency coded fluorescence data.

[0240] 27. The method of any of clauses 1-26, wherein the sample in the flow stream is illuminated with a light source.

[0241] 28. The method of clause 27, wherein the light source comprises a beam generator component configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light.

[0242] 29. The method of clause 28, wherein the light beam generator comprises an acousto-optic deflector.

[0243] 30. A method according to any of clauses 28-29, wherein the beam generator comprises a direct digital synthesizer (DDS) radio frequency comb generator.

[0244] 31. A method according to any of clauses 28-30, wherein the beam generator component is configured to generate a frequency-shifted local oscillator beam.

[0245] 32. A method according to any of clauses 27-31, wherein the light source comprises a laser.

[0246] 33. The method of clause 32, wherein the laser is a continuous wave laser.

[0247] 34. A method according to any of clauses 1 to 33, wherein the phase correction for each fluorescence detector is calculated by an integrated circuit device.

[0248] 35. The method of clause 34, wherein the integrated circuit device is a field programmable gate array (FPGA).

[0249] 36. The method of clause 34, wherein the integrated circuit device is an application specific integrated circuit (ASIC).

[0250] 37. The method of clause 34, wherein the integrated circuit device is a complex programmable logic device (CPLD).

[0251] 38. The method of any of clauses 1-37, further comprising generating an image of particles in the flow stream.

[0252] 39. A system comprising:

[0253] a light source configured to illuminate a sample comprising particles in a flowing stream;

[0254] Light detection system, including:

[0255] a brightfield photodetector configured to generate a brightfield data signal in response to detected light; and

[0256] a fluorescence detector configured to generate a fluorescence data signal in response to the detected light; and

[0257] A processor comprising a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0258] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal.

[0259] 40. The system of clause 39, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction for the fluorescence detector in the first configuration.

[0260] 41. The system of clause 39, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0261] In a second configuration, detecting light from the sample using a fluorescence detector; and

[0262] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector in the second configuration.

[0263] 42. The system of clause 41, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0264] In a third configuration, light from the sample is detected using a fluorescence detector; and

[0265] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector in the third configuration.

[0266] 43. The system of any of clauses 40-42, wherein the configuration is a voltage.

[0267] 44. The system of clause 43, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0268] detecting light from the sample using a fluorescence detector at a first voltage;

[0269] calculating a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and a fluorescence data signal from the fluorescence detector at a first voltage;

[0270] detecting light from the sample using a fluorescence detector at a second voltage; and

[0271] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector at the second voltage.

[0272] 45. The system of any of clauses 40-42, wherein the configuration is an electronic gain setting.

[0273] 46. ​​The system of clause 45, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0274] detecting light from the sample using a fluorescence detector at a first electronic gain setting;

[0275] calculating a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and a fluorescence data signal from the fluorescence detector at a first electronic gain setting;

[0276] detecting light from the sample using a fluorescence detector at a second electronic gain setting; and

[0277] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector at the second electronic gain setting.

[0278] 47. The system of clause 39, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0279] A lifetime phase correction for the fluorescence detector is calculated based on the calculated phase correction for the fluorescence detector and the fluorescence lifetime of the fluorophores in the sample.

[0280] 48. The system of any of clauses 39-47, wherein the light detection system comprises a plurality of fluorescence detectors, wherein each fluorescence detector is configured to independently generate a fluorescence data signal in response to detected light.

[0281] 49. A system according to clause 48, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector.

[0282] 50. The system of clause 49, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to calculate a phase correction for each fluorescence detector comprising the predetermined configuration.

[0283] 51. The system of clause 50, wherein the predetermined configuration is a voltage.

[0284] 52. The system of clause 51, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0285] detecting light from the sample using each fluorescence detector at a first voltage;

[0286] calculating a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at a first voltage;

[0287] detecting light from the sample using each fluorescence detector at a second voltage; and

[0288] A phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at the second voltage.

[0289] 53. The system of clause 52, wherein the predetermined voltage is the same for all fluorescence detectors in the light detection system.

[0290] 54. The system of clause 52, wherein the predetermined voltage is different for each of the fluorescence detectors.

[0291] 55. The system of clause 50, wherein the predetermined configuration is an electronic gain setting.

[0292] 56. The system of clause 55, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0293] detecting light from the sample using each fluorescence detector at a first electronic gain setting;

[0294] calculating a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at a first electronic gain setting;

[0295] detecting light from the sample using each fluorescence detector at a second electronic gain setting; and

[0296] A phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at the second electronic gain setting.

[0297] 57. The system of clause 56, wherein the predetermined electronic gain setting is the same for all fluorescence detectors in the light detection system.

[0298] 58. The system of clause 56, wherein the predetermined electronic gain setting is different for each of the fluorescence detectors.

[0299] 59. A system according to any of clauses 48-58, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate a lifetime phase correction for each of the fluorescence detectors based on the calculated phase correction for each of the fluorescence detectors and the fluorescence lifetime of the fluorophores in the sample.

[0300] 60. A system according to any of clauses 39-59, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to:

[0301] generating frequency-encoded fluorescence data from particles in the sample using a fluorescence detector; and

[0302] Phase-corrected spatial data for the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction for each fluorescence detector.

[0303] 61. The system of clause 60, 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 a phase correction component to generate phase-corrected spatial data of the particle.

[0304] 62. A system according to claim 61, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform a discrete Fourier transform on the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle.

[0305] 63. The system of clause 61, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data with the phase correction component.

[0306] 64. A system according to claim 60, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to use a digital lock-in amplifier to calculate phase-corrected spatial data to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0307] 65. The system of any of clauses 39-64, wherein the light source comprises a beam generator component configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light.

[0308] 66. A system according to clause 65, wherein the light beam generator comprises an acousto-optic deflector.

[0309] 67. A system according to any of clauses 65-66, wherein the beam generator comprises a direct digital synthesizer (DDS) radio frequency comb generator.

[0310] 68. A system according to any of clauses 65-67, wherein the beam generator component is configured to generate a frequency-shifted local oscillator beam.

[0311] 69. The system of any of clauses 39-68, wherein the light source comprises a laser.

[0312] 70. The system of clause 69, wherein the laser is a continuous wave laser.

[0313] 71. A system according to any of clauses 39-70, comprising an integrated circuit component programmed to:

[0314] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal.

[0315] 72. The system of clause 71, wherein the integrated circuit device is a field programmable gate array (FPGA).

[0316] 73. The system of clause 71, wherein the integrated circuit device is an application specific integrated circuit (ASIC).

[0317] 74. The system of clause 71, wherein the integrated circuit device is a complex programmable logic device (CPLD).

[0318] 75. The system of any one of clauses 39-74, wherein the system is a flow cytometer.

[0319] 76. The system of any of clauses 39-75, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an image of particles in the sample.

[0320] 77. An integrated circuit programmed to calculate a phase correction for a fluorescence detector based on the relative phase between a brightfield data signal and a fluorescence data signal.

[0321] 78. The integrated circuit of clause 77, wherein the integrated circuit is in electrical communication to receive a data signal from a light detection system, the light detection system comprising:

[0322] a brightfield photodetector configured to generate a brightfield data signal in response to detected light; and

[0323] A fluorescence detector is configured to generate a fluorescence data signal in response to the detected light.

[0324] 79. An integrated circuit according to clause 77, wherein the integrated circuit is programmed to calculate a phase correction for the fluorescence detector in the first configuration.

[0325] 80. The integrated circuit of clause 79, wherein the integrated circuit is programmed to calculate a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector in the second configuration.

[0326] 81. The integrated circuit of clause 80, wherein the integrated circuit is programmed to calculate a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and a fluorescence data signal from the fluorescence detector in the third configuration.

[0327] 82. An integrated circuit as recited in any of clauses 79-81, wherein the configuration is a voltage.

[0328] 83. The integrated circuit of clause 82, wherein the integrated circuit is programmed to:

[0329] calculating a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector at the first voltage; and

[0330] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector at the second voltage.

[0331] 84. An integrated circuit according to any of clauses 79-81, wherein the configuration is an electronic gain setting.

[0332] 85. The integrated circuit of clause 84, wherein the integrated circuit is programmed to:

[0333] calculating a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and a fluorescence data signal from the fluorescence detector at a first electronic gain setting;

[0334] A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector at the second electronic gain setting.

[0335] 86. The integrated circuit of clause 77, wherein the integrated circuit is programmed to:

[0336] A lifetime phase correction for the fluorescence detector is calculated based on the calculated phase correction for the fluorescence detector and the fluorescence lifetime of the fluorophores in the sample.

[0337] 87. The integrated circuit of clause 78, wherein the light detection system comprises a plurality of fluorescence detectors, wherein each fluorescence detector is configured to independently generate a fluorescence data signal in response to detected light.

[0338] 88. An integrated circuit according to clause 87, wherein the integrated circuit is programmed to calculate a phase correction for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector.

[0339] 89. An integrated circuit according to clause 88, wherein the integrated circuit is programmed to calculate a phase correction for each fluorescence detector comprising a predetermined configuration.

[0340] 90. The integrated circuit of clause 89, wherein the predetermined configuration is a voltage.

[0341] 91. The integrated circuit of clause 90, wherein the integrated circuit is programmed to:

[0342] calculating a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at a first voltage; and

[0343] A phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at the second voltage.

[0344] 92. An integrated circuit according to clause 91, wherein the predetermined voltage is the same for all fluorescence detectors in the light detection system.

[0345] 93. An integrated circuit according to clause 91, wherein the predetermined voltage is different for each of the fluorescence detectors.

[0346] 94. The integrated circuit of clause 89, wherein the predetermined configuration is an electronic gain setting.

[0347] 95. The integrated circuit of clause 94, wherein the integrated circuit is programmed to:

[0348] calculating a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at a first electronic gain setting; and

[0349] A phase correction is calculated for each fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector at the second electronic gain setting.

[0350] 96. An integrated circuit according to clause 95, wherein the predetermined electronic gain setting is the same for all fluorescence detectors in the light detection system.

[0351] 97. An integrated circuit according to clause 95, wherein the predetermined electronic gain setting is different for each of the fluorescence detectors.

[0352] 98. An integrated circuit according to any of clauses 87-97, wherein the integrated circuit is programmed to calculate a lifetime phase correction for each of the fluorescence detectors based on the calculated phase correction for each fluorescence detector and the fluorescence lifetime of the fluorophores in the sample.

[0353] 99. An integrated circuit according to any of clauses 77-98, wherein the integrated circuit is programmed to:

[0354] generating frequency-encoded fluorescence data from particles in the sample using a fluorescence detector; and

[0355] Phase-corrected spatial data for the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction for each fluorescence detector.

[0356] 100. An integrated circuit according to clause 99, 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 particles.

[0357] 101. An integrated circuit according to clause 100, wherein the integrated circuit is programmed to perform a discrete Fourier transform on the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particle.

[0358] 102. An integrated circuit according to clause 101, wherein the integrated circuit is programmed to perform a short-time Fourier transform on the frequency-encoded fluorescence data with a phase correction component to generate phase-corrected spatial data of the particles.

[0359] 103. An integrated circuit according to claim 99, wherein the integrated circuit is programmed to use a digital lock-in amplifier to calculate phase-corrected spatial data to heterodyne the frequency-encoded fluorescence data and demultiplex the frequency-encoded fluorescence data.

[0360] 104. An integrated circuit as recited in any of clauses 77-103, wherein the integrated circuit device is a field programmable gate array (FPGA).

[0361] 105. An integrated circuit as recited in any of clauses 77-103, wherein the integrated circuit device is an application specific integrated circuit (ASIC).

[0362] 106. An integrated circuit as recited in any of clauses 77-103, wherein the integrated circuit device is a complex programmable logic device (CPLD).

[0363] 107. An integrated circuit according to any of clauses 77-106, wherein the integrated circuit is programmed to generate an image of the particle based on the phase-corrected spatial data.

[0364] 108. A composition for phase calibration of one or more fluorescence detectors of a flow cytometer, the composition comprising:

[0365] a plurality of particles; and

[0366] A fluorescent dye component, wherein the fluorescent dye component has a stable fluorescence lifetime.

[0367] 109. The composition of clause 108, wherein the fluorescent dye component comprises a single dye.

[0368] 110. The composition of clause 108, wherein the fluorescent dye component comprises two or more dyes.

[0369] 111. The composition of any of clauses 108-110, wherein the fluorescent dye component comprises Nile Red dye.

[0370] 112. The composition of any of clauses 108-111, wherein the fluorescent dye component comprises a phycoerythrin cyanine dye.

[0371] 113. A composition according to clause 112, wherein the fluorescent dye component comprises PE-Cy7.

[0372] 114. The composition of any of clauses 108-113, wherein the fluorescent dye component comprises one or more than one polymeric dye.

[0373] 115. The composition of clause 114, wherein the polymeric dye is a water-soluble conjugated polymer.

[0374] 116. The composition of any of clauses 108-115, wherein the fluorescent dye component is stably associated with the particle.

[0375] 117. The composition of clause 116, wherein the fluorescent dye component is covalently bonded to the particle.

[0376] 118. The composition of any of clauses 108-117, wherein the particles are porous.

[0377] 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 one skilled in the art, based on 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.

[0378] Therefore, the foregoing only illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to design various arrangements that, although not explicitly described or shown herein, embody the principles of the present invention and are included in its spirit and scope. In addition, all embodiments and conditional language cited herein are primarily intended to help the reader understand the principles of the present invention and the concepts contributed by the inventor to promote this area, and should be interpreted as not being limited to these specifically cited embodiments and conditions. In addition, all statements citing the principles, aspects and embodiments of the present invention and their specific embodiments herein are intended to cover their structural and functional equivalents. In addition, such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, any element that performs the same function, regardless of the structure. In addition, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is clearly stated in the claims.

[0379] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 USC § 112 (f) or 35 USC § 112 (6) is expressly defined as invoking the limitation in the claim only when the exact phrase "means for..." or the exact phrase "step for..." is cited at the beginning of such limitation in the claim; if such exact phrase is not used in the limitation of the claim, 35 USC § 112 (f) or 35 USC § 112 (6) is not invoked.

Claims

1. A method for phase correction of a signal from a light detection system, comprising: detecting light from a sample containing particles in a flow stream with a light detection system, the light detection system comprising: a brightfield photodetector configured to generate a brightfield data signal in response to detected light; and a fluorescence detector configured to generate a fluorescence data signal in response to the detected light; calculating a phase correction for the fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal; generating frequency-encoded fluorescence data from particles in the sample using a fluorescence detector; and Phase-corrected spatial data of the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction of the fluorescence detector.

2. The method of claim 1, wherein the phase correction of the fluorescence detector is calculated at a first configuration, wherein the configuration is a voltage or electronic gain setting.

3. The method according to claim 2, wherein the method further comprises: in a second configuration, detecting light from the sample using a fluorescence detector, wherein the configuration is a voltage or electronic gain setting; and A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector in the second configuration.

4. The method according to claim 3, further comprising: in a third configuration, detecting light from the sample using a fluorescence detector, wherein the configuration is a voltage or electronic gain setting; and A phase correction for the fluorescence detector is calculated based on the relative phase between the brightfield data signal and the fluorescence data signal from the fluorescence detector in the third configuration.

5. The method of claim 1 , wherein the light detection system comprises a plurality of fluorescence detectors, wherein each fluorescence detector is configured to independently generate a fluorescence data signal in response to detected light, wherein the method comprises calculating a phase correction for each fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal from each fluorescence detector.

6. The method according to claim 5, further comprising: generating frequency-encoded fluorescence data from particles in the sample using a fluorescence detector; and Phase-corrected spatial data for the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction for each fluorescence detector.

7. The method of claim 1, wherein the sample in the flow stream is illuminated with a light source, wherein the light source comprises a beam generator component configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light.

8. The method of claim 5, wherein the phase correction for each fluorescence detector is calculated by an integrated circuit device.

9. The method of claim 1, further comprising generating an image of particles in the flow stream.

10. A system having a light detection system for characterizing particles of a sample in a flowing stream, comprising: a light source configured to illuminate a sample comprising particles in a flowing stream; Light detection system, including: a brightfield photodetector configured to generate a brightfield data signal in response to detected light; and a fluorescence detector configured to generate a fluorescence data signal in response to the detected light; and A processor comprising a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to: calculating a phase correction for the fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal; generating frequency-encoded fluorescence data from particles in the sample using a fluorescence detector; and Phase-corrected spatial data of the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction of the fluorescence detector.

11. An integrated circuit device programmed to receive a brightfield data signal from a brightfield photodetector and a fluorescence data signal from a fluorescence detector, and to calculate a phase correction for the fluorescence detector based on a relative phase between the brightfield data signal and the fluorescence data signal; generating frequency-encoded fluorescence data from particles in the sample using a fluorescence detector; and Phase-corrected spatial data of the particles is calculated by transforming the frequency-encoded fluorescence data with the calculated phase correction of the fluorescence detector.

12. A kit comprising the system of claim 10 having a light detection system for characterizing particles of a sample in a flow stream and a composition for phase correction of the light detection system, the composition comprising: a plurality of particles; and A fluorescent dye component, wherein the fluorescent dye component has a stable fluorescence lifetime.

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