Method and system for modulation and simultaneous detection in a flow cytometer

Abstract

Methods for determining parameters of particles in a flow stream from scattered light (e.g., in a particle analyzer of a flow cytometer) are described. Methods according to certain embodiments include illuminating particles in a flow stream with a frequency-modulated laser beam modulated at a reference frequency, detecting scattered light from the particles with a photodetector, generating a frequency-encoded data signal from the detected scattered light, synchronizing the frequency-encoded data signal to the reference frequency, and determining one or more parameters of the particles based on the synchronized frequency-encoded data signal. Systems for practicing the subject methods and non-transitory computer-readable storage media having instructions for practicing the subject methods are also provided.

Description

[0001] Cross Reference to Related Applications

[0002] This application is related to U.S. Provisional Patent Application Serial No. 63 / 017,365, filed April 29, 2020; the disclosure of which is incorporated herein by reference. BACKGROUND

[0003] Light detection is commonly used to characterize components of a sample (e.g., a biological sample), for example when the sample is used for the diagnosis of a disease or condition. When a sample is illuminated, light can be scattered by the sample, transmitted through the sample, and emitted by the sample (e.g., by fluorescence). Changes in sample components, such as morphology, absorbance, and the presence of fluorescent tags, can cause changes in the light scattered, transmitted, or emitted by the sample. To quantify these changes, light is collected and directed to the surface of a detector.

[0004] One technique that utilizes light detection to characterize components in a sample is flow cytometry. Using data generated from the detected light, properties of the components can be recorded, and desired materials can be sorted. A flow cytometer typically includes a sample vessel for receiving a fluid sample (e.g., a blood sample) and a sheath vessel containing sheath fluid. The flow cytometer transports particles (including cells) in the fluid sample as a cell stream to a flow cell, while also directing sheath fluid to the flow cell. Within the flow cell, a liquid sheath is formed around the cell stream, thereby imparting a substantially uniform velocity to the cell stream. The flow cell hydrodynamically focuses the cells in the stream to pass through the center of a light source in the flow cell. Light from the light source can be detected as a scatter or transmission spectrum, or can be absorbed by one or more components in the sample and re-emitted as luminescence. SUMMARY

[0005] Aspects of the present disclosure include methods for determining parameters of particles in a flow stream (e.g., cells in a biological sample) from scattered light. Methods according to certain embodiments include illuminating particles in a flow stream with a frequency-modulated laser beam modulated at a reference frequency, detecting scattered light from the particles with a photodetector, generating a frequency-encoded data signal from the detected scattered light, synchronizing the frequency-encoded data signal to the reference frequency, and determining one or more parameters of the particles based on the synchronized frequency-encoded data signal. Systems for practicing the subject methods and non-transitory computer-readable storage media having instructions for practicing the subject methods are also provided.

[0006] In embodiments, the particles in the flow stream are illuminated with frequency modulated laser light. In some embodiments, the method includes generating frequency modulated light by illuminating a light modulator with one or more lasers to generate a frequency modulated light beam. In some embodiments, the light modulator is an electro-optical modulator. In certain embodiments, the electro-optical modulator is a piezoelectric optical modulator. In other embodiments, the light modulator is an acousto-optical modulator. In some cases, generating frequency modulated laser light includes illuminating the light modulator with a laser through an input polarizer to generate a polarized frequency modulated light beam. In embodiments, a reference frequency signal is generated (e.g., using a controller) and the reference frequency signal is passed to the light modulator. In some embodiments, the light modulator is configured to generate a reference frequency. In some embodiments, the frequency modulated laser light has an oscillation frequency at the reference frequency.

[0007] In embodiments, the scattered light is detected with one or more scattered light photodetectors. In some embodiments, the light is detected with a side scatter photodetector. In other embodiments, the light is detected with a forward scatter photodetector. In yet other embodiments, the light is detected with a back scatter photodetector. In some embodiments, the scattered light is passed to the scattered light photodetector through an output polarizer. In certain cases, the scattered light is passed to the scattered light photodetector through a quarter wave plate. In certain cases, the scattered light is passed to the scattered light photodetector through a quarter wave plate and an output polarizer. In certain embodiments, the scattered light is detected in two different detector channels. In certain cases, the scattered light is detected in two different side scatter detector channels. In some embodiments, the side scatter detector channels include a first polarizer having a first polarization and a second polarizer having a second polarization perpendicular to the first polarization.

[0008] The frequency-encoded data signal is generated from the detected scattered light by a photodetector. In some embodiments, generating the frequency-encoded data signal includes detecting an amplitude of light scattered by particles oscillating at a reference frequency. In some embodiments, the reference frequency is a reference waveform. In embodiments, the frequency-encoded data signal is synchronized with the reference frequency signal. In some embodiments, synchronizing the frequency-encoded data signal with the reference frequency signal includes multiplying the frequency-encoded data signal with the reference waveform. In certain embodiments, the frequency-encoded data signal is synchronized with the reference frequency signal by a lock-in amplifier. In certain cases, the lock-in amplifier is implemented on a field programmable gate array (FPGA). In other embodiments, the frequency-encoded data signal is digitally synchronized with the reference frequency signal, for example where the particle analyzer includes a processor having a memory operably coupled to the processor, where the memory includes instructions stored thereon that, when executed by the processor, cause the processor to multiply the frequency-encoded data signal with the reference waveform. In some embodiments, the lock-in amplification includes applying a low pass filter to the synchronized frequency-encoded data signal to generate a data signal distribution. In certain cases, the data signal distribution generated by applying the low pass filter has a Gaussian signal distribution. In other cases, the data signal distribution generated by applying the low pass filter has a super-Gaussian signal distribution.

[0009] In some embodiments, one or more of the synchronized frequency-encoded data signal and the generated data signal distribution are used to determine one or more parameters of the particles in the flow stream. In some cases, the method includes identifying a particle in the flow stream based on the determined parameters. In other cases, the method includes sorting the particle.

[0010] Aspects of the present disclosure also include a system for determining one or more parameters of particles in a flow stream. The system according to certain embodiments includes: a light source having a laser and a light modulator component configured to generate a frequency-modulated laser beam at a reference frequency; a light detection system having a light scattering photodetector; and a processor having a memory operably coupled to the processor, where the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate a frequency-encoded data signal from the detected scattered light, synchronize the frequency-encoded data signal with the reference frequency, and determine one or more parameters of the particles based on the synchronized frequency-encoded data signal. In some embodiments, the system is a particle analyzer, for example a particle analyzer incorporated into a flow cytometer. In some embodiments, the system includes a particle sorter for sorting one or more particles from the flow stream.

[0011] In embodiments, the system includes an optical modulator for generating the frequency-modulated light beam by a laser. In some embodiments, the optical modulator is an electro-optical modulator. In certain embodiments, the electro-optical modulator is a piezoelectric optical modulator. In other embodiments, the optical modulator is an acousto-optical modulator. In some embodiments, the light source includes an input polarizer for polarizing the laser beam prior to illumination of the optical modulator. In some embodiments, the system includes a controller for applying a frequency drive signal to the optical modulator. In some embodiments, the controller is configured to apply a reference frequency drive signal to the optical modulator. In other embodiments, the optical modulator is configured to generate the reference frequency.

[0012] The system includes an optical detection system having one or more photodetectors for detecting scattered light. In some embodiments, the photodetectors are configured to detect side scatter light. In other embodiments, the photodetectors are configured to detect forward scatter light. In yet other embodiments, the photodetectors are configured to detect backscatter light. In some cases, the optical detection system includes an output polarizer in optical communication with the photodetectors. In these embodiments, the scattered light is transmitted through the output polarizer to the scattered photodetectors. In certain embodiments, the output polarizer component additionally includes a quarter wave plate. In some embodiments, the optical detection system includes two different detector channels. In some cases, the optical detection channels include two different side scatter detector channels. In some embodiments, the side scatter detector channels include a first polarizer having a first polarization and a second polarizer having a second polarization perpendicular to the first polarization.

[0013] The photodetector of the target light detection system is placed to generate a frequency encoded data signal in response to the detected scattered light. In some embodiments, the photodetector is configured to detect the amplitude of the scattered light oscillating at the reference frequency produced by the controller or light modulator. In some embodiments, the controller is configured to generate a reference frequency waveform. In embodiments, the frequency encoded data signal is synchronized to the reference frequency signal. In some embodiments, the system includes a processor including a memory operably coupled to the processor, wherein the memory has instructions stored thereon that, when executed by the processor, cause the processor to: detect the amplitude of the light scattered by the particles oscillating at the reference frequency and multiply the frequency encoded data signal by the reference waveform. In some embodiments, the system includes a lock-in amplifier in operative communication with the controller and the photodetector, the lock-in amplifier configured to synchronize the frequency encoded data signal to the reference waveform. In some embodiments, the system includes an FPGA programmed to perform lock-in amplification. In other embodiments, the memory includes instructions stored thereon for digitally synchronizing the frequency encoded data signal to the reference waveform. In certain embodiments, the system is configured to apply a low pass filter with a lock-in amplifier or digitally apply a low pass filter to the synchronized frequency encoded data signal. In certain cases, applying a low pass filter to the synchronized frequency encoded data signal generates a data signal distribution, such as a Gaussian data signal distribution or a super-Gaussian data signal distribution.

[0014] Aspects of the present disclosure also include non-transitory computer readable storage media for practicing the subject methods. The non-transitory computer readable storage media according to certain embodiments include algorithms for illuminating particles in a flow stream with a frequency modulated laser beam modulated at a reference frequency, algorithms for detecting scattered light from the particles with a photodetector, algorithms for generating a frequency encoded data signal from the detected scattered light, algorithms for synchronizing the frequency encoded data signal to the reference frequency, and algorithms for determining one or more parameters of the particles based on the synchronized frequency encoded data signal.

[0015] In some embodiments, the non-transitory computer readable storage media includes algorithms for illuminating an electro-optical modulator with a laser to produce a frequency modulated laser beam. In some cases, the non-transitory computer readable storage media includes algorithms for illuminating an electro-optical modulator with a laser through an input polarizer to generate a polarized frequency modulated laser beam. In some embodiments, the non-transitory computer readable storage media includes algorithms for generating a reference frequency signal. In some embodiments, the non-transitory computer readable storage media includes algorithms for generating a reference frequency waveform.

[0016] In embodiments, the non-transitory computer-readable storage medium includes an algorithm for synchronizing the frequency-encoded data signal with a reference frequency. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for synchronizing the frequency-encoded data signal with a reference frequency by multiplying the frequency-encoded data signal with a reference frequency waveform. In some cases, the non-transitory computer-readable storage medium includes an algorithm for synchronizing the frequency-encoded data signal with a reference frequency using a lock-in amplifier. In other embodiments, the non-transitory computer-readable storage medium includes an algorithm for digitally synchronizing the frequency-encoded data signal, for example with an FPGA having a program for multiplying the frequency-encoded data signal with a reference frequency waveform. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for applying a low-pass filter to the synchronized frequency-encoded data signal to generate a data signal distribution. In certain cases, the data signal distribution generated by applying the low-pass filter has a Gaussian signal distribution. In other cases, the data signal distribution generated by applying the low-pass filter has a super-Gaussian signal distribution.

[0017] Kits including one or more components of the subject systems are also provided. Kits according to certain embodiments include one or more lasers and optical modulators. In some embodiments, the kits include electro-optical modulators, for example piezoelectric optical modulators. In some embodiments, the kits further include one or more optical polarizers. In other embodiments, the kits include quarter-wave plates. In some embodiments, the kits can include one or more photodetectors. In certain cases, the kits include a lock-in amplifier. In certain embodiments, the kits include an integrated circuit device programmed to perform lock-in amplification, for example to synchronize a frequency-encoded data signal with a reference frequency. In some embodiments, the integrated circuit device is a field-programmable gate array (FPGA). In other embodiments, the integrated circuit device is an application-specific integrated circuit (ASIC). In yet other embodiments, the integrated circuit device is a complex programmable logic device (CPLD). In certain embodiments, the subject kits can further include light collection components, for example lenses, mirrors, or optical fibers, for propagating light from a flow stream to a photodetector. BRIEF DESCRIPTION OF DRAWINGS

[0018] The application can best be understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures:

[0019] Figure 1A A particle analyzer according to certain embodiments is depicted. Figure 1B Optical modulation components of a light source according to certain embodiments are depicted.

[0020] Figure 2A A flowchart for determining parameters of particles in a flow stream according to certain embodiments is depicted.Figure 2B Depiction of illumination generation and processing data signal distribution from particles in a flow stream according to certain embodiments.

[0021] Figure 3A Depiction of detection and recovery of Gaussian data signal distribution according to certain embodiments. Figure 3B Depiction of detection and recovery of super-Gaussian data signal distribution according to certain embodiments.

[0022] Figure 4A Depiction of functional block diagram of a particle analysis system for computed-based sample analysis and particle characterization according to certain embodiments. Figure 4B Depiction of a flow cytometer according to certain embodiments.

[0023] Figure 5 Depiction of functional block diagram of one example of a particle analyzer control system according to certain embodiments.

[0024] Figure 6 Depiction of block diagram of a computing system according to certain embodiments. DETAILED DESCRIPTION

[0025] Methods for determining parameters of particles in a flow stream (e.g., in a particle analyzer of a flow cytometer) from scattered light are described. Methods according to certain embodiments include illuminating particles in a flow stream with a frequency-modulated laser beam modulated at a reference frequency, detecting scattered light from the particles with a photodetector, generating a frequency-encoded data signal from the detected scattered light, synchronizing the frequency-encoded data signal with the reference frequency, and determining one or more parameters of the particles based on the synchronized frequency-encoded data signal. Systems and non-transitory computer-readable storage media having instructions for practicing the subject methods are also provided.

[0026] Before the present application is described in detail, it is to be understood that this application is not limited to the particular embodiments described, as such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present application will be limited only by the appended claims.

[0027] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the application.

[0028] Certain ranges are presented herein with numerical values having the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number approximately or nearly equivalent to the exact number that it precedes. When determining whether a number is approximately or nearly equivalent to a particular cited number, it is intended that numbers be taken as approximately or nearly equivalent if they are within 10% of the particular cited number, 5% of the particular cited number, 1% of the particular cited number, or within statistical experimentation error, Whichever is greater.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative illustrative methods and materials are now described.

[0030] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0031] Note that, as used in this document, and in the appended claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology in the claims to define scope of the technical field, without inviting a determination of equivalency that is due to accommodate its drafting to exclude any element disclosed in this document.

[0032] As will be apparent to those of ordinary skill in the art in the art in light of the present disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the present application. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0033] While devices and methods have been or will be described for grammatical flow and functional explanation, it should be expressly understood that claims are not to be necessarily limited to any means by construction or step limitation unless expressly recited under 35 U.S.C. § 112, but rather the claims are to be afforded all of the broadening definitions and equivalents under the doctrine of equivalents, and in the event claims are expressly recited under 35 U.S.C. § 112, are to be afforded the full statutory equivalency of 35 U.S.C. § 112.

[0034] As described above, the present disclosure provides methods for determining parameters of particles in a flow stream (e.g., cells in a biological sample) from detected scattered light. In further describing embodiments of the present disclosure, the following method is first described in more detail: the method for illuminating particles in a flow stream with a frequency-modulated laser beam modulated at a reference frequency, detecting scattered light from the particles with a photodetector, generating a frequency-encoded data signal from the detected scattered light, and synchronizing the frequency-encoded data signal with the reference frequency. Next, a system having a laser, an optical modulator, a scatter detector, and a controller for synchronizing the frequency-encoded data signal with the reference frequency signal is described. A non-transitory computer-readable storage medium having instructions for practicing the subject methods is also provided.

[0035] Method for determining parameters of particles in an illuminated sample in a flow stream

[0036] Aspects of the present disclosure also include methods for determining parameters of particles from scattered light of an illuminated flow stream. In practicing the methods according to certain embodiments, a sample having particles (e.g., a biological sample having cells) is illuminated in a flow stream with a modulated laser beam. The term “modulated” is used herein in its conventional meaning to refer to imposing a change to one or more properties of the laser beam, such as imposing a change to the phase, frequency, amplitude, or polarization of the laser beam. As described above, the methods according to the present disclosure include illuminating a sample in a flow stream with a frequency-modulated laser beam. The frequency of the laser beam can be modulated using any convenient protocol, such as by illuminating and passing the laser through an optical modulator. In some embodiments, the modulator is an electro-optical modulator. In some cases, the electro-optical modulator includes a component (e.g., a non-linear optical material, such as a non-linear organic polymer or crystalline lithium niobate) that changes refractive index in response to the application of a direct current, and illuminating the electro-optical modulator with a laser is sufficient to output a frequency-modulated beam from the laser. In other cases, the electro-optical modulator includes a component that changes refractive index in response to the application of a low-frequency electric field, and illuminating the electro-optical modulator with a laser is sufficient to output a frequency-modulated beam from the laser. In certain embodiments, the methods include generating a frequency-modulated laser beam by illuminating an acousto-optical modulator, such as a piezoelectric light modulator, with a laser.

[0037] As described in greater detail below, in some embodiments, the optical modulator is operatively coupled to a controller that transmits a frequency signal to the optical modulator to generate the frequency-modulated laser beam. The frequency applied in generating the frequency-modulated laser beam can vary, for example, from about 0.001 MHz to about 500 MHz, for example, from about 0.005 MHz to about 400 MHz, for example, from about 0.01 MHz to about 300 MHz, for example, from about 0.05 MHz to about 200 MHz, for example, from about 0.1 MHz to about 100 MHz, for example, from about 0.5 MHz to about 90 MHz, for example, from about 1 MHz to about 75 MHz, for example, from about 2 MHz to about 70 MHz, for example, from about 3 MHz to about 65 MHz, for example, from about 4 MHz to about 60 MHz, and including from about 5 MHz to about 50 MHz.

[0038] In some embodiments, the controller is configured to apply a current or an electric field to the optical modulator. In certain embodiments, the controller is configured with an electric pulse generator. In other embodiments, the controller is configured to apply a radio frequency drive signal, for example, the controller includes a direct digital synthesizer (DDS) or an arbitrary waveform generator (AWS). In some embodiments, the method includes generating a reference frequency signal waveform with the controller. The reference frequency signal waveform generated according to certain embodiments has an oscillation frequency, for example, from about 0.001 MHz to about 500 MHz, for example, from about 0.005 MHz to about 400 MHz, for example, from about 0.01 MHz to about 300 MHz, for example, from about 0.05 MHz to about 200 MHz, for example, from about 0.1 MHz to about 100 MHz, for example, from about 0.5 MHz to about 90 MHz, for example, from about 1 MHz to about 75 MHz, for example, from about 2 MHz to about 70 MHz, for example, from about 3 MHz to about 65 MHz, for example, from about 4 MHz to about 60 MHz, and including from about 5 MHz to about 50 MHz.

[0039] In some embodiments, the light modulator is illuminated with a laser through an input polarizer to generate a polarized beam of frequency-modulated laser light. The term "polarizer" is used herein in its conventional sense to refer to an optical conditioning component configured to pass light having a predetermined polarization and block light waves having other polarizations. The input polarizer can be any convenient optical polarizer, including but not limited to linear polarizers, absorptive polarizers, beam-splitting polarizers, birefringent polarizers, thin-film polarizers, wire-grid polarizers, circular polarizers, and optical polarizers configured for polarization by Fresnel reflection. The laser can pass through one or more input polarizers, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, and including 5 or more input polarizers, before being transmitted through the light modulator. The input polarizer can be illuminated with the laser at a distance that can vary, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 2.5 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 15 mm or more, e.g., 25 mm or more, and including 50 mm or more. In addition, the angle of illumination can also vary, e.g., 10° to 90°, e.g., 15° to 85°, e.g., 20° to 80°, e.g., 25° to 75°, and including 30° to 60°, e.g., 90° angle, relative to the planar surface of the polarizer.

[0040] In embodiments, the method includes illuminating the light modulator with a laser. The laser of interest can include a pulsed laser or a continuous wave laser. The type and number of lasers used in the subject method can vary and can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2laser, 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 combinations thereof. In other cases, the method includes illuminating the light modulator with a dye laser, such as a diphenylstilbene, coumarin, or rhodamine laser. In other cases, the method includes illuminating the light modulator with 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 (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet other cases, the method includes illuminating the light modulator with a solid-state laser, such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4laser, a Nd:YCa4O(BO3)3laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium oxide laser, or a cerium-doped laser, and combinations thereof. In yet other cases, the method includes illuminating the light modulator with a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a second or third harmonic implementation of any of the above lasers.

[0041] Depending on the desired wavelength of light produced in the output laser beam (e.g., for illuminating a sample in a flow stream), the laser can have a particular wavelength that varies from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm, and including 400 nm to 800 nm. The light modulator can be illuminated with one or more lasers, such as 2 or more lasers, such as 3 or more lasers, such as 4 or more lasers, such as 5 or more lasers, and including 10 or more lasers. The lasers can include any combination of laser types. For example, in some embodiments, the method includes illuminating the light modulator with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0042] In cases where more than one laser is used, multiple lasers can be used to illuminate the light modulator simultaneously or in sequence or a combination of lasers can be used to illuminate the light modulator. For example, the light modulator can be illuminated simultaneously with each of a plurality of lasers. In other embodiments, the light modulator is illuminated in sequence with each of a plurality of lasers. In cases where more than one laser is used to illuminate the light modulator in sequence, the time each laser illuminates the light modulator can independently be 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 30 microseconds or more, and including 60 microseconds or more. For example, the method 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 two or more lasers are used to illuminate the light modulator in sequence, the duration of time the light modulator is illuminated by each laser can be the same or different.

[0043] The period of time between illumination by each laser can also vary as desired, separated by a delay of 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 15 microseconds or more, such as 30 microseconds or more, and including 60 microseconds or more. For example, the period of time between illumination by each light source can be 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, such as 0.1 microseconds to 35 microseconds, such as 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In certain embodiments, the period of time between illumination by each laser is 10 microseconds. In embodiments where the light modulator device is illuminated in sequence by more than two lasers (i.e., 3 or more lasers), the delay between illumination by each laser can be the same or different.

[0044] The light modulator can be illuminated continuously or at discrete intervals. In some cases, the method includes continuously illuminating the light modulator with the laser. In other cases, the light modulator is illuminated with the laser at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0045] According to the laser, the light modulator can be illuminated according to varying distances, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more and including 50 mm or more. Further, the angle or illumination can also vary, such as 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 90° angle.

[0046] In certain embodiments, the method includes illuminating the sample with two or more frequency-shifted beams. In these embodiments, the beam generator components can include a laser and an acousto-optic device (e.g., an acousto-optic modulator) for frequency shifting the laser. In these embodiments, the method includes illuminating the acousto-optic device with the laser (e.g., through an input polarizer as described above). The laser can have a particular wavelength that varies from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm and including from 400 nm to 800 nm, depending on the desired wavelength of light produced in the output laser beam (e.g., for illuminating the sample in a flow stream). One or more lasers can be used to illuminate the acousto-optic device, such as 2 or more lasers, such as 3 or more lasers, such as 4 or more lasers, such as 5 or more lasers and including 10 or more lasers. The lasers can include any combination of laser types. For example, in some embodiments, the method includes illuminating the acousto-optic device with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers and one or more solid state lasers.

[0047] In cases where more than one laser is used, the multiple lasers can be used to irradiate the acousto-optic device simultaneously or sequentially or using a combination of lasers. For example, the acousto-optic device can be irradiated simultaneously with each of the multiple lasers. In other embodiments, the acousto-optic device is irradiated sequentially with each of the multiple lasers. In cases where more than one laser is used to irradiate the acousto-optic device sequentially, the time each laser irradiates the acousto-optic device can independently be 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 30 microseconds or more, and including 60 microseconds or more. For example, the method can include irradiating 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 irradiated sequentially by two or more lasers, the duration of irradiation of the acousto-optic device by each laser can be the same or different.

[0048] The time period between irradiation by each laser can also vary as desired, separated by a delay of 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 15 microseconds or more, such as 30 microseconds or more, and including 60 microseconds or more. For example, the time period between irradiation by each light source can be 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, such as 0.1 microseconds to 35 microseconds, such as 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In certain embodiments, the time period between irradiation by each laser source is 10 microseconds. In embodiments where the acousto-optic device is irradiated sequentially by more than two (i.e., 3 or more) lasers, the delay between irradiation by each laser can be the same or different.

[0049] The acousto-optic device can be irradiated continuously or at discrete intervals. In some cases, the method includes irradiating the acousto-optic device continuously with the laser. In other cases, the acousto-optic device is irradiated with the laser at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0050] Depending on the laser, the acousto-optic device can be illuminated at varying distances, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, and including 50 mm or more. Further, the angle or illumination can also vary, such as 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.

[0051] In embodiments, 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 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and including 100 or more radio frequency drive signals.

[0052] The angularly deflected laser beams produced by the radio frequency drive signals each have an intensity based on the amplitude of the applied radio frequency drive signal. In some embodiments, the method includes applying a radio frequency drive signal having an amplitude sufficient to produce an angularly deflected laser beam having a desired intensity. In some cases, each applied radio frequency drive signal independently has an amplitude of about 0.001 V to about 500 V, such as about 0.005 V to about 400 V, such as about 0.01 V to about 300 V, such as about 0.05 V to about 200 V, such as about 0.1 V to about 100 V, such as about 0.5 V to about 75 V, such as about 1 V to 50 V, such as about 2 V to 40 V, such as 3 V to about 30 V, and including about 5 V to about 25 V. In some embodiments, each applied radio frequency drive signal has a frequency of about 0.001 MHz to about 500 MHz, 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.

[0053] In these embodiments, the angularly deflected laser beams in the output laser beam are spatially separated. The angularly deflected laser beams can be separated by 0.001 pm or more, such as 0.005 pm or more, such as 0.01 pm or more, such as 0.05 pm or more, such as 0.1 pm or more, such as 0.5 pm or more, such as 1 pm or more, such as 5 pm or more, such as 10 pm or more, such as 100 pm or more, such as 500 pm or more, such as 1000 pm or more, and including 5000 pm or more, depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam. In some embodiments, the angularly deflected laser beams overlap, such as along the horizontal axis of the output laser beam with adjacent angularly deflected laser beams. The overlap, such as the overlap of beam spots, between adjacent angularly deflected laser beams can be 0.001 pm or more of overlap, such as 0.005 pm or more of overlap, such as 0.01 pm or more of overlap, such as 0.05 pm or more of overlap, such as 0.1 pm or more of overlap, such as 0.5 pm or more of overlap, such as 1 pm or more of overlap, such as 5 pm or more of overlap, such as 10 pm or more of overlap, and including 100 pm or more of overlap.

[0054] In certain cases, the light source includes a beam generator, such as described in Diebold et al., Nature Photonics, Vol. 7(10) 806-810 (2013); and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, and U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, the disclosures of which are incorporated herein by reference.

[0055] In embodiments, the particles in the flow stream that are illuminated can be cells, e.g., the sample in the flow stream is a biological sample. The term "biological sample" is used in its conventional sense to refer to a subset of an entire organism, plant, fungus, or animal tissue, cell, or constituent part that can be found in some instances in blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, umbilical cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers both to a natural organism or subset of tissue thereof, as well as to a homogenate, lysate, or extract prepared from an organism or subset of tissue thereof, including but not limited to, for example, blood plasma, serum, spinal fluid, lymphatic fluid, skin biopsy, respiratory, gastrointestinal, cardiovascular, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. The biological sample can be any type of organic tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample, e.g., blood or a blood derivative, such as plasma, tears, urine, semen, etc., in some cases the sample is a blood sample, including whole blood, e.g., blood obtained from a venipuncture or a finger prick (the blood can or can not be mixed with any reagents (e.g., preservatives, anticoagulants, etc.) prior to the assay).

[0056] In certain embodiments, the source of the sample is a "mammal" or "mammalian," which terms are used broadly to describe organisms that belong to the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The methods can be applied to samples obtained from human subjects of both sexes and at any stage of development (i.e., neonates, infants, juveniles, adolescents, adults), in certain embodiments, the human subject is a juvenile, adolescent, or adult. While the present application can be applied to samples from human subjects, it will be appreciated that the methods can also be performed on samples from other animal subjects (i.e., in "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0057] In practicing the subject methods, one or more photodetectors are used to detect scattered light from the sample. In embodiments, the scattered photodetectors can be side scatter photodetectors, forward scatter photodetectors, back scatter photodetectors, and combinations thereof. The term "light scattering" is used herein in its conventional sense to refer to the propagation of light energy from a particle (e.g., flowing in a flow stream) in a sample that is deflected from the path of the incident light beam, e.g., by reflection, refraction, or deflection of the beam, from the path of the incident light. In some embodiments, the scattered light is not luminescence from a component (e.g., a fluorophore) of the particle. In embodiments, the scattered light according to the present disclosure is not fluorescence or phosphorescence. In certain embodiments, the scattered light detected from the particle in the flow stream includes Mie scattering. In other embodiments, the scattered light detected from the particle in the flow stream includes Rayleigh scattering. In yet other embodiments, the scattered light detected from the particle in the flow stream includes Mie scattering and Rayleigh scattering.

[0058] In embodiments, scattered light can be detected by each photodetector at an angle relative to the incident beam illumination, e.g., at an angle of 1° or greater (e.g., 10° or greater, e.g., 15° or greater, e.g., 20° or greater, e.g., 25° or greater, e.g., 30° or greater, e.g., 45° or greater, e.g., 60° or greater, e.g., 75° or greater, e.g., 90° or greater, e.g., 135° or greater, e.g., 150° or greater), and including cases where the scattered light detector is configured to detect light from a particle in the sample at an angle of 180° or greater relative to the incident beam illumination. In certain cases, the light scattering photodetector comprises a side scatter photodetector, e.g., the photodetector is positioned to detect scattered light propagating at 30° to 120° relative to the incident beam illumination, e.g., at 45° to 105° and including at 60° to 90°. In certain cases, the light scattering detector is a side scatter photodetector positioned at a 90° angle relative to the incident beam illumination. In other cases, the light scattering detector is a forward scatter detector, e.g., the detector is positioned to detect scattered light propagating at 120° to 240° relative to the incident beam illumination, e.g., at 100° to 220°, e.g., at 120° to 200° and including at 140° to 180° relative to the incident beam illumination. In certain cases, the light scattering detector is a forward scatter photodetector positioned to detect scattered light propagating at a 180° angle relative to the incident beam illumination. In yet other cases, the light scattering detector is a backscatter photodetector positioned to detect scattered light propagating at 1° to 30° relative to the incident beam illumination, e.g., at 5° to 25° and including at 10° to 20° relative to the incident beam illumination. In certain cases, the scattered light is detected by a backscatter photodetector positioned to detect scattered light propagating at a 30° angle relative to the incident beam illumination.

[0059] The methods of the present disclosure include detecting scattered light using one or more photodetectors. In some embodiments, scattered light is detected using 2 or more side scatter photodetectors, for example 3 or more side scatter photodetectors, for example 4 or more side scatter photodetectors, for example 5 or more side scatter photodetectors, for example 6 or more side scatter photodetectors, for example 7 or more side scatter photodetectors, for example 8 or more side scatter photodetectors, for example 9 or more side scatter photodetectors and including 10 or more side scatter photodetectors. In other embodiments, scattered light is detected using a side scatter photodetector and a forward scatter photodetector, for example 2 or more side scatter photodetectors and forward scatter photodetectors, for example 3 or more side scatter photodetectors and forward scatter photodetectors, for example 4 or more side scatter photodetectors and forward scatter photodetectors, for example 5 or more side scatter photodetectors and forward scatter photodetectors, for example 6 or more side scatter photodetectors and forward scatter photodetectors, for example 7 or more side scatter photodetectors and forward scatter photodetectors, for example 8 or more side scatter photodetectors and forward scatter photodetectors, for example 9 or more side scatter photodetectors and forward scatter photodetectors and including 10 or more side scatter photodetectors and forward scatter photodetectors. In yet other embodiments, scattered light is detected using a side scatter photodetector and a back scatter photodetector, for example 2 or more side scatter photodetectors and back scatter photodetectors, for example 3 or more side scatter photodetectors and back scatter photodetectors, for example 4 or more side scatter photodetectors and back scatter photodetectors, for example 5 or more side scatter photodetectors and back scatter photodetectors, for example 6 or more side scatter photodetectors and back scatter photodetectors, for example 7 or more side scatter photodetectors and back scatter photodetectors, for example 8 or more side scatter photodetectors and back scatter photodetectors, for example 9 or more side scatter photodetectors and back scatter photodetectors and including 10 or more side scatter photodetectors and back scatter photodetectors.In yet other embodiments, scattered light is detected using a side scatter photodetector, a forward scatter photodetector, and a backscatter photodetector, for example 2 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, for example 3 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, for example 4 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, for example 5 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, for example 6 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, for example 7 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, for example 8 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, for example 9 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors, and including 10 or more side scatter photodetectors, forward scatter photodetectors, and backscatter photodetectors.

[0060] In certain embodiments, scattered light is detected by a light detection system including a first side scatter photodetector positioned at a 90° angle relative to the incident beam illumination and a second side scatter photodetector positioned at an angle less than 90° relative to the incident beam illumination. In some cases, the first side scatter photodetector is configured to detect light scattered at an angle of 30° to 150° relative to the incident beam illumination, for example light scattered at an angle of 60° to 120° and including light scattered at a 90° angle relative to the incident beam illumination, and the second side scatter photodetector is configured to detect light scattered at an angle of 5 to 30 degrees relative to the incident beam illumination, for example light scattered at an angle of 10 to 30° relative to the incident beam illumination. In certain embodiments, the second side scatter photodetector is configured to detect both side scatter light and backscatter light. In these embodiments, backscatter light can be propagated from the flow stream to the detector by a mirror, for example by a mirror having an aperture (e.g., to propagate from the flow stream to the detector by illumination light from a light source).

[0061] In some embodiments, the scattered light from the flow stream is transmitted to the scattered light photodetector by one or more optical conditioning components. The term "optical conditioning" is used herein in its conventional sense to refer to optical components that change or condition the light propagating to the light scattering photodetector. For example, the optical conditioning can be to change the profile of the light beam, the focal point of the light beam, the direction of the light beam propagation, or to collimate the light beam. In certain embodiments, the scattered light is transmitted to the scattered light photodetector by one or more of a dichroic mirror and a bandpass filter. In certain cases, the scattered light from the particles in the flow stream is transmitted to each scattered light photodetector by a dichroic mirror and a bandpass filter.

[0062] The amount of light propagating to the light scattering photodetector through the optical conditioning components can also vary, in some embodiments, 50% or more of the collected light is transmitted to the light scattering photodetector, for example, 55% or more, for example 60% or more, for example 65% or more, for example 75% or more, for example 80% or more, for example 90% or more of the collected light is transmitted to the light scattering photodetector and including 95% or more of the light from the flow stream is transmitted to the light scattering photodetector through the optical conditioning components. For example, the amount of light propagating to the light scattering photodetector through the optical conditioning components can be from 25% to 99%, for example from 30% to 95%, for example from 35% to 90%, for example from 40% to 85%, for example from 45% to 80% and including from 50% to 75%.

[0063] The light scattering photodetector can be any suitable photodetector, among other types of photodetectors such as active pixel sensors (APSs), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), complementary metal-oxide-semiconductor (CMOS) image sensors or N-type metal-oxide-semiconductor (NMOS) image sensors, light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, combinations thereof, and other types of photodetectors. In embodiments, the light scattering photodetector can include 1 or more photodetectors, such as 2 or more, such as 3 or more, such as 5 or more, such as 10 or more photodetectors and including 25 or more photodetectors. In some cases, the light scattering photodetector is a photodetector array. The term “photodetector array” is used in its conventional sense to refer to an arrangement or series of two or more photodetectors configured to detect light. In embodiments, the photodetector array can include 2 or more photodetectors, such as 3 or more photodetectors, such as 4 or more photodetectors, such as 5 or more photodetectors, such as 6 or more photodetectors, such as 7 or more photodetectors, such as 8 or more photodetectors, such as 9 or more photodetectors, such as 10 or more photodetectors, such as 12 or more photodetectors and including 15 or more photodetectors. In certain embodiments, the photodetector array includes 5 photodetectors. 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 irregularly shaped configurations. The photodetectors in the light scattering photodetector array can be oriented at an angle of 10° to 180° relative to another photodetector (as referenced in the X-Z plane), such as 15° to 170°, such as 20° to 160°, such as 25° to 150°, such as 30° to 120° and including 45° to 90°.

[0064] The light scattering photodetectors of the present disclosure are configured to measure light collected at one or more wavelengths, for example at 2 or more wavelengths, for example at 5 or more different wavelengths, for example at 10 or more different wavelengths, for example at 25 or more different wavelengths, for example at 50 or more different wavelengths, for example at 100 or more different wavelengths, for example at 200 or more different wavelengths, for example at 300 or more different wavelengths, and include measuring light emitted by a sample in a flow stream at 400 or more different wavelengths.

[0065] In some embodiments, the subject photodetectors are configured to measure light collected within a wavelength range, for example, 200 nm to 1000 nm. In certain embodiments, the detectors of interest are configured to collect a spectrum of light within a wavelength range. For example, the system can include one or more detectors configured to collect a spectrum of light within one or more wavelength ranges in the 200 nm to 1000 nm wavelength range. In yet other embodiments, the detectors of interest are configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. In embodiments, the light detection system is configured to measure light continuously or at discrete intervals. In certain cases, the detectors of interest are configured to measure collected light continuously. In other cases, the light detection system is configured to measure at discrete intervals, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds, or some other interval.

[0066] In some embodiments, light scattered by particles in the flow stream is transmitted to a photodetector by one or more output polarizers. In certain embodiments, passing the scattered light through an output polarizer can generate polarized scattered light oscillating at the reference frequency. The output polarizer can be any convenient optical polarizer, including but not limited to linear polarizers, absorptive polarizers, beam-splitting polarizers, birefringent polarizers, thin-film polarizers, wire-grid polarizers, circular polarizers, and optical polarizers configured for polarization by Fresnel reflection. In certain embodiments, the output polarizer comprises a quarter-wave plate. Scattered light from the flow stream can be transmitted to a photodetector by one or more output polarizers, for example 2 or more, for example 3 or more, for example 4 or more and including 5 or more output polarizers. The output polarizers can be positioned at varying distances from the flow stream, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 2.5 mm or more, for example 5 mm or more, for example 10 mm or more, for example 15 mm or more, for example 25 mm or more and including 50 mm or more. Further, the angle of the polarizers relative to the flow stream can also vary, for example 10° to 90°, for example 15° to 85°, for example 20° to 80°, for example 25° to 75° and including 30° to 60°, for example a 90° angle relative to the planar surface of the polarizer.

[0067] In certain embodiments, the scattered light is detected in two or more different detector channels, for example the side scatter light is detected in two or more different detector channels. In some cases, where the scattered light is detected in two or more different detector channels, each detector channel is optically coupled to a polarizer. For example, the method can comprise detecting side scatter light in two different scattered detector channels, wherein light transmitted through a first polarizer having a first polarization is detected with a first side scatter detector and light transmitted through a second polarizer having a second polarization is detected with a second side scatter detector. In some cases, the first polarization and the second polarization differ from each other by 5° or more, for example by 10° or more, for example by 15° or more, for example by 20° or more, for example by 25° or more, for example by 30° or more, for example by 45° or more, for example by 60° or more, for example by 75° or more and including cases where the first polarizer has a polarization perpendicular to the second polarizer (i.e., the polarization of the first polarizer differs from the polarization of the second polarizer by 90°).

[0068] In embodiments, the method includes generating a frequency-encoded data signal from the scattered light using each of the plurality of photodetectors. In some embodiments, the frequency-encoded data signal is generated by detecting an amplitude of the scattered light oscillating at a reference frequency. In some cases, the method includes detecting an amplitude of a data signal from a photodetector having an oscillation frequency of 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.

[0069] In some embodiments, generating the frequency-encoded data signal is sufficient to reduce or eliminate a contribution from low frequency noise components of the photodetector data signal, such as detecting an amplitude of the scattered light oscillating at a reference frequency is capable of reducing a contribution from low frequency noise components of the photodetector data signal by 5% or more, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 30% or more, such as 50% or more, such as 60% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, and including cases where detecting an amplitude of the scattered light oscillating at a reference frequency is sufficient to eliminate any contribution of low frequency noise to the photodetector data signal. In other embodiments, detecting an amplitude of the scattered light oscillating at a reference frequency is capable of reducing a contribution of photodetector offset to the photodetector data signal, such as reducing by 5% or more, such as reducing by 10% or more, such as reducing by 15% or more, such as reducing by 20% or more, such as reducing by 25% or more, such as reducing by 30% or more, such as reducing by 50% or more, such as reducing by 60% or more, such as reducing by 75% or more, such as reducing by 90% or more, such as reducing by 95% or more, such as reducing by 97% or more, such as reducing by 99% or more, and including cases where detecting an amplitude of the scattered light oscillating at a reference frequency is sufficient to eliminate any contribution of photodetector offset to the photodetector data signal.

[0070] The generated frequency-encoded data signal is synchronized with the reference frequency signal. The term "synchronized" is used herein in its conventional sense to mean matching the modulation frequency of the reference signal (e.g., generated by a controller coupled to the optical modulator) to the oscillation frequency of the data signal generated by the photodetector in response to the detected scattered light. In some embodiments, the frequency-encoded data signal is synchronized with the reference data signal when the oscillation frequency of the frequency-encoded data signal differs from the frequency of the reference data signal by 10% or less, such as by 9% or less, such as by 8% or less, such as by 7% or less, such as by 6% or less, such as by 5% or less, such as by 4% or less, such as by 3% or less, such as by 2% or less, such as by 1% or less, such as by 0.5% or less, such as by 0.1% or less, such as by 0.01% or less, such as by 0.001% or less, such as by 0.0001% or less, and including the case where the oscillation frequency of the frequency-encoded data signal differs from the frequency of the reference data signal by 0.00001% or less. In certain embodiments, the frequency-encoded data signal is synchronized with the reference data signal when the oscillation frequency of the frequency-encoded data signal matches the frequency of the reference data signal.

[0071] In some embodiments, a lock-in amplifier is used to synchronize the frequency-encoded data signal with the reference data signal. In some cases, the frequency-encoded data signal is passed to the lock-in amplifier and synchronized with the reference waveform. In some embodiments, to synchronize the frequency-encoded data signal with the reference frequency waveform, each of the frequency-encoded data signal is multiplied by the reference frequency waveform. In certain cases, the unmodulated background signal is converted to a bipolar square wave by the lock-in amplification. In some embodiments, the lock-in amplification is performed on an integrated circuit device. In certain cases, the lock-in amplification is performed on a field programmable gate array (FPGA). In other cases, the lock-in amplification is performed on an application specific integrated circuit (ASIC). In yet other embodiments, the lock-in amplification is performed on a complex programmable logic device (CPLD). In certain embodiments, the frequency-encoded data signal is synchronized with the reference frequency digitally. For example, the frequency-encoded data signal can be processed on a processor having a memory operably coupled to the processor, where the memory has instructions stored thereon that, when executed by the processor, cause the processor to receive the frequency-encoded data signal from the photodetector and synchronize the reference signal with the frequency-encoded data signal. In certain cases, the reference frequency signal is stored on the memory. In other cases, the reference frequency signal is passed to the processor from a controller in communication with the optical modulator components.

[0072] In some embodiments, the method includes generating a data signal distribution from the synchronized frequency encoded data signal. In some cases, to generate the data signal distribution, a low pass filter is applied to the synchronized frequency encoded data signal. In some embodiments, the low pass filter is applied to the synchronized frequency encoded data signal using a lock-in amplifier. In other embodiments, the low pass filter is applied to the synchronized frequency encoded data signal digitally. In some cases, applying the low pass filter to the synchronized frequency encoded data signal can generate a Gaussian signal distribution. In other cases, applying the low pass filter to the synchronized frequency encoded data signal can generate a super-Gaussian signal distribution.

[0073] Figure 1A A particle analyzer according to certain embodiments is depicted. The particle analyzer 100 includes a laser 101 configured to generate a laser beam that is passed through an input polarizer 102 to an optical modulator 103 (e.g., electro-optical modulator) that receives a reference frequency signal from a controller 104. The output modulated frequency beam illuminates particles in a flow stream 105 originating from a flow cell 106. The illumination of the particles in the flow stream 105 produces fluorescence that is detected by fluorescence photodetectors 107a, 107b, 107c and scattered light that is detected by a side scatter photodetector 108 and a forward scatter photodetector 109. The scattered light from the particles is passed through an output polarizer 108a to the side scatter photodetector 108 and through an output polarizer 109a to the forward scatter photodetector 109. The scattered light photodetectors 108 and 109 are in communication with a lock-in amplifier 110 to synchronize the generated frequency encoded data signal with the reference frequency from the controller 104.

[0074] Figure 1B An optical modulation component of a light source according to certain embodiments is depicted. The optical modulation component includes an input polarizer for generating polarized laser light that is passed to a light receiving end of an electro-optical crystal for applying frequency modulation to the polarized laser beam. The modulated frequency laser light originating from the electro-optical crystal is passed through a quarter wave plate and an output polarizer to additionally apply polarization to the laser beam.

[0075] Figure 2AA flow chart for determining parameters of particles in a flow stream according to certain embodiments is depicted. At step 201, a reference frequency signal is generated for a light modulator that, when illuminated with a laser at step 202, imposes a frequency modulation on a laser beam, resulting in a frequency modulated laser beam with an oscillation frequency at the reference frequency. At step 203, the frequency modulated laser beam is used to illuminate particles in a flow stream. At step 204, scattered light from the particles is detected. In some embodiments, the scattered light is detected by an output polarizer. In some cases, the scattered light is detected in two different detector channels, where the detector channels have output polarizers with perpendicular polarizations. At step 205, a frequency encoded data signal is generated, where the data signal has an oscillation frequency at the reference frequency. At step 206, the generated data signal is synchronized to the reference frequency. The frequency encoded data signal can be digitally synchronized to a system processor or a lock-in amplifier. In certain embodiments, lock-in amplification is performed on an FPGA with programming to receive the reference frequency signal and synchronize the frequency encoded data signal to the reference frequency signal. In some embodiments, at step 208, one or more parameters (e.g., particle size, morphology) are determined based on the synchronized frequency encoded data signal. In other embodiments, at step 207, a data distribution is generated from the synchronized frequency encoded data signal. One or more parameters can also be determined based on the generated data distribution.

[0076] Figure 2B A data signal distribution is generated and processed from illumination of particles in a flow stream according to certain embodiments is depicted. A reference waveform with an oscillation frequency ω R is produced at panel 210. Scattered light from illuminated particles in a flow stream is detected and a modulated peak data signal is generated at panel 211. The modulated peak exhibits an oscillation at the reference frequency ω R . After polarization modulation, a background signal is introduced into the total signal depicted in panel 212. This signal in panel 212 is modeled as a random walk, which exhibits distortion of the data signal. At panel 213, the data signal generated by the scattered light photodetector is synchronized to the reference waveform by multiplying the frequency encoded data signal with the reference waveform using a lock-in amplifier. The unmodulated background signal is converted to a bipolar square wave. A low pass filter is applied to the synchronized data signal during lock-in amplification at panel 214 to generate a recovered data signal distribution.

[0077] Figure 3A and Figure 3B Gaussian data signal distributions ( Figure 3A ) and super-Gaussian data signal distributions ( Figure 3B) and recovery. Detection of the illuminated particles generates data points from the data signal and the background signal. The underlying baseline background signal is also depicted. The frequency-encoded data signal recovered by synchronizing the detected signal (and baseline) to the reference frequency waveform generates a recovered signal that eliminates any interference from the background signal, such as from low frequency noise components or detector offsets of the detection system. As Figure 3A and Figure 3B The recovered data signal distribution closely corresponds to the simulated underlying data signal, as shown.

[0078] In certain embodiments, the method further comprises determining one or more parameters of the particles in the flow stream. In some cases, the sample in the flow stream comprises cells, and the method comprises detecting the cells in the sample. In some embodiments, detecting the cells comprises identifying cell types in the sample. In other embodiments, the method comprises characterizing the cells of the sample. In other embodiments, the method comprises distinguishing cell types in the sample. In certain embodiments, the method comprises identifying and distinguishing cell types based on the synchronized frequency-encoded data signal or from the generated data signal distribution. In some cases, the method comprises generating an image of the flow stream and identifying cell types in the sample based on the synchronized frequency-encoded data signal or from the generated data signal distribution.

[0079] In some embodiments, the method comprises sorting one or more particles from the sample in the flow stream. The term "sorting" is used herein in its conventional meaning to refer to separating components of the sample (e.g., microdroplets containing cells, microdroplets containing non-cellular particles such as biological macromolecules) and, in some cases, delivering the separated components to one or more sample collection vessels. For example, particle classification can comprise a sorting gate for sorting 2 or more components of the sample, 2 or more components being, for example, 3 or more components, for example, 4 or more components, for example, 5 or more components, for example, 10 or more components, for example, 15 or more components and including 25 or more components of the sample. In sorting the particles, the method comprises data acquisition, analysis, and recording, for example, using a computer, wherein multiple data channels record data from each detector used. In these embodiments, the analysis can comprise spectrally resolving the light (e.g., by computing a spectral unmixing matrix). The analysis can be communicated to a sorting system configured to generate a set of digitized parameters based on the particle classification.

[0080] In some embodiments, methods for sorting components of a sample include sorting particles (e.g., cells in a biological sample), e.g., as described in U.S. Patent Nos. 3,960,449; 4,347,935; 4,667,830; 5,245,318; 5,464,581; 5,483,469; 5,602,039; 5,643,796; 5,700,692; 6,372,506; and 6,809,804, the disclosures of which are incorporated herein by reference. In some embodiments, the methods include sorting components of a sample with a particle sorting module, e.g., those described in U.S. Patent Nos. 9,551,643 and 10,324,019; U.S. Patent Publication Publication No. 2017 / 0299493; and International Patent Publication Publication No. WO / 2017 / 040151, the disclosures of which are incorporated herein by reference. In certain embodiments, cells of a sample are sorted using a sort decision module having a plurality of sort decision units, e.g., those described in U.S. Patent Application No. 16 / 725,756, filed December 23, 2019, the disclosure of which is incorporated herein by reference.

[0081] System for determining parameters of particles in an illuminated sample in a flow stream

[0082] Aspects of the present disclosure include systems for determining parameters of particles in an illuminated sample in a flow stream. A system according to certain embodiments includes a light source having a laser and a light modulator component configured to produce a frequency-modulated laser beam at a reference frequency, a light detection system having a light scattering photodetector, and a processor having a memory coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate a frequency-encoded data signal from the detected scattered light, synchronize the frequency-encoded data signal to the reference frequency, and determine one or more parameters of the particles based on the synchronized frequency-encoded data signal.

[0083] In embodiments, the system includes a light source configured to generate a modulated laser beam modulated at a reference frequency. The light source includes one or more lasers (as described in more detail below) and a light modulator component configured to receive light from the laser and generate the modulated laser beam. In embodiments, one or more properties of the laser can be changed by passing the laser through the light modulator component, including the phase, frequency, amplitude, or polarization of the laser beam. In certain embodiments, the light modulator is configured to generate a frequency-modulated light beam. In some embodiments, the modulator is an electro-optic modulator. In some cases, the electro-optic modulator includes a component that changes refractive index in response to an applied direct current (e.g., a nonlinear optical material, such as a nonlinear organic polymer or crystalline lithium niobate), and illuminating the electro-optic modulator with the laser is sufficient to output a frequency-modulated light beam from the laser. In other cases, the electro-optic modulator includes a component that changes refractive index in response to an applied low-frequency electric field, and illuminating the electro-optic modulator with the laser is sufficient to output a frequency-modulated light beam from the laser. In certain embodiments, the light modulator is an acousto-optic modulator, such as a piezoelectric light modulator with a laser.

[0084] In some embodiments, the light modulator is configured to modulate the frequency of the laser beam. In some cases, the light modulator is configured to modulate the frequency of the laser beam at a reference frequency, such as at an oscillation 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.

[0085] In some embodiments, the system includes a controller for applying a current or electric field to the optical modulator. In certain embodiments, the controller is configured with an electric pulse generator. In other embodiments, the controller is configured to apply a radio frequency drive signal, for example the controller includes a direct digital synthesizer (DDS) or an arbitrary waveform generator (AWS). In some embodiments, the controller is configured to generate a reference frequency signal waveform. The reference frequency signal waveform generated according to certain embodiments has an oscillation frequency of, for example, about 0.001 MHz to about 500 MHz, for example, about 0.005 MHz to about 400 MHz, for example, about 0.01 MHz to about 300 MHz, for example, about 0.05 MHz to about 200 MHz, for example, about 0.1 MHz to about 100 MHz, for example, about 0.5 MHz to about 90 MHz, for example, about 1 MHz to about 75 MHz, for example, about 2 MHz to about 70 MHz, for example, about 3 MHz to about 65 MHz, for example, about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0086] In some embodiments, the optical modulator is in optical communication with an input polarizer. In these embodiments, the input polarizer is positioned between the laser and the optical modulator such that the laser light is transmitted through the input polarizer to the light receiving end of the optical modulator. The input polarizer can be any convenient optical polarizer including, but not limited to, a linear polarizer, an absorption polarizer, a beam-splitting polarizer, a birefringent polarizer, a thin film polarizer, a wire grid polarizer, a circular polarizer, and an optical polarizer configured for polarization by Fresnel reflection. The light source can include one or more input polarizers, for example, 2 or more, for example, 3 or more, for example, 4 or more, and including 5 or more input polarizers. The input polarizer can be positioned 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2.5 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 15 mm or more, for example, 25 mm or more, and including 50 mm or more from the laser. Further, the input polarizer can be positioned at varying angles relative to the laser, for example, 10° to 90°, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and including 30° to 60°, for example, 90° angles relative to the planar surface of the polarizer.

[0087] In embodiments, the light source of the subject system includes one or more lasers, such as 2 or more lasers, such as 3 or more lasers, such as 4 or more lasers, such as 8 or more lasers, such as 12 or more lasers, such as 16 or more lasers, such as 24 or more lasers, such as 36 or more lasers, such as 48 or more lasers and including 60 or more lasers. The lasers of interest can include pulsed lasers or continuous wave lasers. The type and number of lasers in the subject light source can vary and can include gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other cases, the light source includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In yet other cases, the light source includes metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof. In yet other cases, the light source includes solid-state lasers, such as ruby lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4lasers, Nd:YCa4O(BO3)3lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium oxide lasers, or cerium-doped lasers, and combinations thereof. In yet other cases, the light source includes semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or second or third harmonic implementations of any of the above lasers.

[0088] Depending on the desired wavelength of light produced in the output laser beam (e.g., for illuminating a sample in a flow stream), the laser can have a particular wavelength that varies from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm and including wavelengths from 400 nm to 800 nm. The system can include any combination of laser types. For example, in some embodiments, the method includes illuminating the light modulator with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0089] In cases where more than one laser is used, the light source can be configured to use multiple lasers to illuminate the light modulator simultaneously or in sequence or a combination of lasers to illuminate the light modulator. For example, multiple lasers can be configured to illuminate the light modulator simultaneously. In other embodiments, multiple lasers are configured to illuminate the light modulator in sequence. In cases where the lasers are configured to illuminate the light modulator in sequence, each laser can be configured to independently illuminate the light modulator for 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 30 microseconds or more and including 60 microseconds or more. For example, the lasers can be configured to illuminate the electro-optical modulator 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, each laser can be configured to illuminate the light modulator for a duration that is the same or different from one another.

[0090] The time period between each laser illuminating can also vary as desired, separated by a delay of 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 15 microseconds or more, such as 30 microseconds or more and including 60 microseconds or more. For example, the time period between each light source illuminating can be 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, such as 0.1 microseconds to 35 microseconds, such as 1 microsecond to 25 microseconds and including 5 microseconds to 10 microseconds. In certain embodiments, the time period between each laser illuminating is 10 microseconds. In embodiments where the light modulator device is illuminated in sequence by more than two lasers (i.e., 3 or more lasers), the delay between each laser illuminating can be the same or different.

[0091] The lasers can be configured to illuminate the light modulator continuously or at discrete intervals. In some cases, each laser can be configured to illuminate the light modulator continuously. In other cases, each laser can be configured to illuminate the light modulator at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or some other interval.

[0092] The laser can be positioned in the subject light source at a varying distance from the light modulator, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, and including 50 mm or more. Further, the laser can be positioned at an angle to the light modulator, such as 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.

[0093] In certain embodiments, the light source is a beam generator configured to generate two or more beams of frequency-shifted light. In some cases, the beam generator includes a laser and a radio frequency generator configured to apply a radio frequency drive signal to an 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 a 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 CO2laser, 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 combinations thereof; a dye laser, such as a diphenylthioxanthene, 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 (NeCu) laser, a copper laser, or a gold laser, and combinations thereof; a solid-state laser, such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4laser, a Nd:YCa4O(BO3)3laser, a Nd:YCOB laser, a titanium sapphire laser, an erbium YAG laser, a ytterbium YAG laser, a ytterbium oxide laser, or a cerium-doped laser, and combinations thereof.

[0094] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency-shift a laser using an applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam from light from the 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.

[0095] In embodiments, the controller is configured to apply radio frequency drive signals 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 radio frequency drive signals, for example 4 or more radio frequency drive signals, for example 5 or more radio frequency drive signals, for example 6 or more radio frequency drive signals, for example 7 or more radio frequency drive signals, for example 8 or more radio frequency drive signals, for example 9 or more radio frequency drive signals, for example 10 or more radio frequency drive signals, for example 15 or more radio frequency drive signals, for example 25 or more radio frequency drive signals, for example 50 or more radio frequency drive signals and including configured to apply 100 or more radio frequency drive signals.

[0096] In some cases, to produce an intensity profile of angularly deflected laser beams in the output laser beam, the controller is configured to apply radio frequency drive signals having varying amplitudes of about 0.001 V to about 500 V, for example about 0.005 V to about 400 V, for example about 0.01 V to about 300 V, for example about 0.05 V to about 200 V, for example about 0.1 V to about 100 V, for example about 0.5 V to about 75 V, for example about 1 V to 50 V, for example about 2 V to 40 V, for example 3 V to about 30 V and including about 5 V to about 25 V. In some embodiments, each applied radio frequency drive signal has a frequency of about 0.001 MHz to about 500 MHz, for example about 0.005 MHz to about 400 MHz, for example about 0.01 MHz to about 300 MHz, for example about 0.05 MHz to about 200 MHz, for example about 0.1 MHz to about 100 MHz, for example about 0.5 MHz to about 90 MHz, for example about 1 MHz to about 75 MHz, for example about 2 MHz to about 70 MHz, for example about 3 MHz to about 65 MHz, for example about 4 MHz to about 60 MHz and including about 5 MHz to about 50 MHz.

[0097] In certain embodiments, the controller has a processor with 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 produce an output laser beam having angularly deflected laser beams having a desired intensity profile. For example, the memory can include instructions to produce two or more angularly deflected laser beams having the same intensity, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more angularly deflected laser beams, and including: the memory can include instructions to produce 100 or more angularly deflected laser beams having the same intensity. In other embodiments, instructions to produce two or more angularly deflected laser beams having different intensities can be included, 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 angularly deflected laser beams, and including: the memory can include instructions to produce 100 or more angularly deflected laser beams having different intensities.

[0098] In certain embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate a laser beam having an intensity that increases along a horizontal axis from an edge of the output laser beam to a center. 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%, 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 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 along a horizontal axis from a center to an edge of the output laser beam. 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%, 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 along a horizontal axis having a Gaussian distribution. 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 a top hat intensity along a horizontal axis.

[0099] In embodiments, the beam generator of interest can be configured to produce spatially separated angularly deflected laser beams in the output laser beam. The angularly deflected laser beams can be spaced 0.001 pm or more apart, such as 0.005 pm or more apart, such as 0.01 pm or more apart, such as 0.05 pm or more apart, such as 0.1 pm or more apart, such as 0.5 pm or more apart, such as 1 pm or more apart, such as 5 pm or more apart, such as 10 pm or more apart, such as 100 pm or more apart, such as 500 pm or more apart, such as 1000 pm or more apart and including 5000 pm or more apart, depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam. In some embodiments, the system is configured to produce angularly deflected laser beams in the output laser beam that, for example, overlap with adjacent angularly deflected laser beams along a horizontal axis of the output laser beam. The overlap (e.g., overlap of beam spots) between adjacent angularly deflected laser beams can be 0.001 pm or more of overlap, such as 0.005 pm or more of overlap, such as 0.01 pm or more of overlap, such as 0.05 pm or more of overlap, such as 0.1 pm or more of overlap, such as 0.5 pm or more of overlap, such as 1 pm or more of overlap, such as 5 pm or more of overlap, such as 10 pm or more of overlap and including 100 pm or more of overlap.

[0100] In certain instances, the beam generator configured to generate two or more frequency shifted beams includes a laser excitation module as described 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, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111 and U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, the disclosures of which are incorporated herein by reference.

[0101] Systems of interest include one or more photodetectors configured to detect scattered light from the sample in the flow stream. In embodiments, the scattered photodetector can be a side scatter photodetector, a forward scatter photodetector, a backscatter photodetector, and combinations thereof. In some embodiments, the scattered light detected by the photodetector is not luminescence from a component of the particle (e.g., a fluorophore). In embodiments, the scattered light detected by the photodetector is not fluorescence or phosphorescence. In certain embodiments, the photodetector is configured to detect Mie scattering. In other embodiments, the photodetector is configured to detect Rayleigh scattering. In yet other embodiments, the photodetector is configured to detect Mie scattering and Rayleigh scattering.

[0102] The photodetector can be positioned at an angle of 1° or greater relative to the flow stream, such as 10° or greater, such as 15° or greater, such as 20° or greater, such as 25° or greater, such as 30° or greater, such as 45° or greater, such as 60° or greater, such as 75° or greater, such as 90° or greater, such as 135° or greater, such as 150° or greater, and including cases where the scattered light detector is configured to detect light from a particle in the sample at an angle of 180° or greater relative to the incident beam illumination. In certain cases, the light scattering photodetector includes a side scatter photodetector, such as where the photodetector is positioned to detect scattered light propagating at an angle of 30° to 120° relative to the incident beam illumination, such as scattered light propagating at 45° to 105° and including scattered light propagating at 60° to 90°. In certain cases, the light scattering detector is a side scatter photodetector positioned at an angle of 90° relative to the incident beam illumination. In other cases, the light scattering detector is a forward scatter detector, such as where the detector is positioned to detect scattered light propagating at an angle of 120° to 240° relative to the incident beam illumination, such as scattered light propagating at 100° to 220°, such as 120° to 200° and including scattered light propagating at 140° to 180° relative to the incident beam illumination. In certain cases, the light scattering detector is a forward scatter photodetector positioned to detect scattered light propagating at an angle of 180° relative to the incident beam illumination. In yet other cases, the light scattering detector is a backscatter photodetector positioned to detect scattered light propagating at an angle of 1° to 30° relative to the incident beam illumination, such as scattered light propagating at 5° to 25° and including scattered light propagating at 10° to 20° relative to the incident beam illumination. In certain cases, the scattered light is detected by a backscatter photodetector positioned to detect scattered light propagating at an angle of 30° relative to the incident beam.

[0103] The systems described in the present disclosure include one or more photodetectors. In some embodiments, the light detection system includes 2 or more side scatter photodetectors, such as 3 or more side scatter photodetectors, such as 4 or more side scatter photodetectors, such as 5 or more side scatter photodetectors, such as 6 or more side scatter photodetectors, such as 7 or more side scatter photodetectors, such as 8 or more side scatter photodetectors, such as 9 or more side scatter photodetectors, and includes 10 or more side scatter photodetectors. In other embodiments, the light detection system includes a forward scatter photodetector and a side scatter photodetector, such as a forward scatter photodetector and 2 or more side scatter photodetectors, such as a forward scatter photodetector and 3 or more side scatter photodetectors, such as a forward scatter photodetector and 4 or more side scatter photodetectors, such as a forward scatter photodetector and 5 or more side scatter photodetectors, such as a forward scatter photodetector and 6 or more side scatter photodetectors, such as a forward scatter photodetector and 7 or more side scatter photodetectors, such as a forward scatter photodetector and 8 or more side scatter photodetectors, such as a forward scatter photodetector and 9 or more side scatter photodetectors, and includes a forward scatter photodetector and 10 or more side scatter photodetectors. In other embodiments, the light detection system includes a backscatter photodetector and a side scatter photodetector, such as a backscatter photodetector and 2 or more side scatter photodetectors, such as a backscatter photodetector and 3 or more side scatter photodetectors, such as a backscatter photodetector and 4 or more side scatter photodetectors, such as a backscatter photodetector and 5 or more side scatter photodetectors, such as a backscatter photodetector and 6 or more side scatter photodetectors, such as a backscatter photodetector and 7 or more side scatter photodetectors, such as a backscatter photodetector and 8 or more side scatter photodetectors, such as a backscatter photodetector and 9 or more side scatter photodetectors, and includes a backscatter photodetector and 10 or more side scatter photodetectors.In yet other embodiments, the light detection system includes a side scatter photodetector, a forward scatter photodetector, and a backscatter photodetector, such as 2 or more side scatter photodetectors, a forward scatter photodetector, and a backscatter photodetector, such as a forward scatter photodetector, a backscatter photodetector, and 3 or more side scatter photodetectors, such as a forward scatter photodetector, a backscatter photodetector, and 4 or more side scatter photodetectors, such as a forward scatter photodetector, a backscatter photodetector, and 5 or more side scatter photodetectors, such as a forward scatter photodetector, a backscatter photodetector, and 6 or more side scatter photodetectors, such as a forward scatter photodetector, a backscatter photodetector, and 7 or more side scatter photodetectors, such as a forward scatter photodetector, a backscatter photodetector, and 8 or more side scatter photodetectors, such as a forward scatter photodetector, a backscatter photodetector, and 9 or more side scatter photodetectors, and including a forward scatter photodetector, a backscatter photodetector, and 10 or more side scatter photodetectors.

[0104] In certain embodiments, the light detection system includes a first side scatter photodetector positioned at a 90° angle relative to the incident light beam illumination and a second side scatter photodetector positioned at an angle less than 90° relative to the incident light beam illumination. In some cases, the first side scatter photodetector is configured to detect light scattered at an angle of 30° to 150° relative to the incident light beam illumination, such as light scattered at an angle of 60° to 120° and including light scattered at an angle of 90° relative to the incident light beam illumination, and the second side scatter photodetector is configured to detect light scattered at an angle of 5 to 30 degrees relative to the incident light beam illumination, such as light scattered at an angle of 10 to 30° relative to the incident light beam illumination. In certain embodiments, the second side scatter photodetector is configured to detect both side scatter light and backscatter light. In these embodiments, the backscatter light can be propagated from the flow stream to the detector by a mirror, such as by a mirror having an aperture (e.g., to propagate from the flow stream to the detector by illumination light from a light source).

[0105] In some embodiments, the light detection system includes one or more optical conditioning components configured to transmit scattered light from the flow stream into one or more of the photodetectors. For example, the optical conditioning can be to change the profile of the light beam, the focus of the light beam, the direction of the light beam propagation, or the optical conditioning can be to collimate the light beam. In certain embodiments, the light detection system includes one or more mirrors (e.g., dichroic mirrors) positioned to transmit scattered light from the flow stream into the photodetectors. In other embodiments, the light detection system includes one or more beam splitters positioned to transmit scattered light from the flow stream into the photodetectors. In yet another embodiment, the light detection system includes one or more lenses positioned to transmit scattered light from the flow stream into the photodetectors. In other embodiments, light from the flow stream is transmitted directly into the photodetectors without passing through optical conditioning components.

[0106] The light scattering photodetector can be any suitable photodetector, among other types of photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), complementary metal-oxide-semiconductor (CMOS), image sensors or N-type metal-oxide-semiconductor (NMOS) image sensors, light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, combinations thereof, and other types of photodetectors. In embodiments, the light scattering photodetector can include 1 or more photodetectors, such as 2 or more, such as 3 or more, such as 5 or more, such as 10 or more photodetectors and including 25 or more photodetectors. In some cases, the light scattering photodetector is a photodetector array. The term “photodetector array” is used in its conventional meaning to refer to an arrangement or series of two or more photodetectors configured to detect light. In embodiments, the photodetector array can include 2 or more photodetectors, such as 3 or more photodetectors, such as 4 or more photodetectors, such as 5 or more photodetectors, such as 6 or more photodetectors, such as 7 or more photodetectors, such as 8 or more photodetectors, such as 9 or more photodetectors, such as 10 or more photodetectors, such as 12 or more photodetectors and including 15 or more photodetectors. In certain embodiments, the photodetector array includes 5 photodetectors. 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 irregularly shaped configurations. The photodetectors in the light scattering photodetector array can be oriented at an angle of 10° to 180° relative to another photodetector (as referenced in the X-Z plane), such as 15° to 170°, such as 20° to 160°, such as 25° to 150°, such as 30° to 120° and including 45° to 90°.

[0107] The light scattering photodetectors of the present disclosure are configured to measure light collected at one or more wavelengths, for example at 2 or more wavelengths, for example at 5 or more different wavelengths, for example at 10 or more different wavelengths, for example at 25 or more different wavelengths, for example at 50 or more different wavelengths, for example at 100 or more different wavelengths, for example at 200 or more different wavelengths, for example at 300 or more different wavelengths and include measuring light emitted by a sample in a flow stream at 400 or more different wavelengths.

[0108] In some embodiments, the subject photodetectors are configured to measure light collected within a wavelength range (e.g., 200 nm to 1000 nm). In certain embodiments, the detectors of interest are configured to collect a spectrum of light within a wavelength range. For example, the system can include one or more detectors configured to collect a spectrum of light within one or more wavelength ranges in the 200 nm to 1000 nm wavelength range. In yet another embodiment, the detectors of interest are configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. In embodiments, the light detection system is configured to measure light continuously or at discrete intervals. In certain cases, the detectors of interest are configured to measure collected light continuously. In other cases, the light detection system is configured to measure at discrete intervals, for example every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or some other interval.

[0109] In some embodiments, the system includes one or more output polarizers. In some cases, as described above, the output polarizer is configured to generate polarized scattered light oscillating at the reference frequency. The output polarizer can be any convenient optical polarizer, including but not limited to a linear polarizer, an absorptive polarizer, a beam-splitting polarizer, a birefringent polarizer, a thin-film polarizer, a wire grid polarizer, a circular polarizer, and an optical polarizer configured for polarization by Fresnel reflection. In certain embodiments, the output polarizer includes a quarter-wave plate. The light detection system can include one or more output polarizers of photodetectors, such as 2 or more, for example 3 or more, for example 4 or more output polarizers and including 5 or more output polarizers. The output polarizer can be positioned at a varying distance from the flow stream, such as 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 2.5 mm or more, for example 5 mm or more, for example 10 mm or more, for example 15 mm or more, for example 25 mm or more and including 50 mm or more. Further, the angle of the polarizer relative to the flow stream can also vary, such as 10° to 90° relative to a planar surface of the polarizer, for example 15° to 85°, for example 20° to 80°, for example 25° to 75° and including 30° to 60°, for example a 90° angle.

[0110] In certain embodiments, the light detection system includes two or more scattered photodetectors, where scattered light is detected in two or more different detector channels, such as lateral scattered light is detected in two or more different detector channels. In some cases, each detector channel is optically coupled to a polarizer. For example, the system can include two different scattered detector channels having a first lateral scattered detector and a second lateral scattered detector, the first lateral scattered detector is optically coupled to a first polarizer having a first polarization, and the second lateral scattered detector is optically coupled to a second polarizer having a second polarization. In some cases, the polarization of the first polarizer and the polarization of the second polarizer differ from each other by 5° or more, such as by 10° or more, for example by 15° or more, for example by 20° or more, for example by 25° or more, for example by 30° or more, for example by 45° or more, for example by 60° or more, for example by 75° or more. In certain embodiments, the polarization of the first polarizer is perpendicular to the polarization of the second polarizer (i.e., the polarization of the first polarizer differs from the polarization of the second polarizer by 90°).

[0111] In embodiments, the system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate a frequency encoded data signal from the detected scattered light. In some embodiments, the memory includes instructions for detecting an amplitude of the scattered light oscillating at a reference frequency. In certain embodiments, the memory includes instructions for detecting an amplitude of a data signal from the photodetector having an oscillation frequency of 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.

[0112] In some embodiments, the processor includes instructions stored thereon that, when executed by the processor, cause the processor to synchronize the generated frequency encoded data signal with a reference frequency signal. In some embodiments, the memory includes instructions for synchronizing the frequency encoded data signal with the reference data signal when the oscillation frequency of the frequency encoded data signal differs from the frequency of the reference data signal by 10% or less (such as by 9% or less, such as by 8% or less, such as by 7% or less, such as by 6% or less, such as by 5% or less, such as by 4% or less, such as by 3% or less, such as by 2% or less, such as by 1% or less, such as by 0.5% or less, such as by 0.1% or less, such as by 0.01% or less, such as by 0.001% or less, such as by 0.0001% or less, and including when the oscillation frequency of the frequency encoded data signal differs from the frequency of the reference data signal by 0.00001% or less). In certain embodiments, the memory includes instructions for synchronizing the frequency encoded data signal with the reference data signal when the oscillation frequency of the frequency encoded data signal matches the frequency of the reference data signal.

[0113] In certain embodiments, the system includes a lock-in amplifier for synchronizing the reference data signal with the frequency encoded data signal. In some cases, the lock-in amplifier is configured to synchronize the frequency encoded data signal with a reference frequency waveform. In certain cases, the lock-in amplifier is configured to multiply the frequency encoded data signal with a reference frequency waveform. In certain embodiments, the system includes an integrated circuit device configured to perform lock-in amplification. In some embodiments, the integrated circuit is a field programmable gate array (FPGA). In other cases, the integrated circuit is an application specific integrated circuit (ASIC). In yet other embodiments, the integrated circuit is a complex programmable logic device (CPLD). In certain embodiments, the system is configured to digitally synchronize the frequency encoded data signal with a reference frequency. For example, the frequency encoded data signal can be processed on a processor having a memory operatively coupled to the processor, the memory having instructions stored thereon that, when executed by the processor, cause the processor to receive the frequency encoded data signal from the photodetector and synchronize the reference signal with the frequency encoded data signal. In certain cases, the reference frequency signal is stored on the memory. In other cases, the processor of the subject system is configured to receive the reference frequency signal from a controller in communication with the light modulator component.

[0114] In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate a data signal profile from the synchronized frequency encoded data signal. In some cases, the memory includes instructions for applying a low pass filter to the synchronized frequency encoded data signal to generate the data signal profile. In some embodiments, the lock-in amplifier is configured to apply the low pass filter to the synchronized frequency encoded data signal. In other embodiments, the memory includes instructions for digitally applying a low pass filter to the synchronized frequency encoded data signal. In certain cases, the memory includes instructions for applying a low pass filter to the synchronized frequency encoded data signal to generate a Gaussian signal profile. In other cases, the memory includes instructions for applying a low pass filter to the synchronized frequency encoded data signal to generate a super-Gaussian signal profile.

[0115] In certain embodiments, the system additionally includes a flow cell configured to propagate the sample in a flow stream. Any convenient flow cell that propagates a fluid sample to a sample interrogation region can be used, where in some embodiments the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion that terminates in a planar surface having an orifice transverse to the longitudinal axis. The length (as 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 frustoconical 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.

[0116] In certain cases, the flow cell does not include a cylindrical portion and the entire flow cell interior chamber is frustoconical. In these embodiments, the length of the frustoconical interior chamber (measured along a 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 interior 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.

[0117] In some embodiments, the sample flow originates from an orifice at the distal end of the flow cell. The flow cell orifice can be any suitable shape depending on the desired characteristics of the flow stream, 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 portion coupled to a planar top portion. In certain embodiments, the flow cell of interest has a circular orifice. In some embodiments, the nozzle orifice can vary in size from 1 pm to 20,000 pm, such as 2 pm to 17,500 pm, such as 5 pm to 15,000 pm, such as 10 pm to 12,500 pm, such as 15 pm to 10,000 pm, such as 25 pm to 7,500 pm, such as 50 pm to 5,000 pm, such as 75 pm to 1,000 pm, such as 100 pm to 750 pm, and including 150 pm to 500 pm. In certain embodiments, the nozzle orifice is 100 pm.

[0118] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable flow of sample to the interior chamber of the flow cell. The rate at which sample is delivered to the flow cell chamber through the sample injection port can be 1 pL / min or more, such as 2 pL / min or more, such as 3 pL / min or more, such as 5 pL / min or more, such as 10 pL / min or more, such as 15 pL / min or more, such as 25 pL / min or more, such as 50 pL / min or more and including 100 pL / min or more, where in some cases the rate at which sample is delivered to the flow cell chamber through the sample injection port is 1 pL / sec or more, such as 2 pL / sec or more, such as 3 pL / sec or more, such as 5 pL / sec or more, such as 10 pL / sec or more, such as 15 pL / sec or more, such as 25 pL / sec or more, such as 50 pL / sec or more and including 100 pL / sec or more.

[0119] The sample injection port can be an orifice located in the wall of the interior chamber or can be a conduit located at the proximal end of the interior chamber. In cases where the sample injection port is an orifice located in the wall of the interior chamber, the sample injection port 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, etc.; and irregular shapes such as a parabolic bottom portion coupled to a planar top portion. In certain embodiments, the sample injection port has a circular orifice. In some cases, the size of the sample injection port orifice can vary depending on the shape, with an opening of 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm, such as 1.5 mm.

[0120] In certain instances, the sample injection port is a conduit located proximal to the interior chamber of the flow cell. For example, the sample injection port can be a conduit positioned such that the orifice of the sample injection port is aligned with the orifice of the flow cell. In instances where the sample injection port is a conduit positioned to align with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, with cross-sectional shapes of interest including, but 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 portion coupled to a planar top portion. In certain instances, the orifice of the conduit can vary in shape with an opening of 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm, such as 1.5 mm. The shape of the tip of the sample injection port can be the same or different than the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port can include a beveled tip with a bevel of 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7° and including 5°.

[0121] In some embodiments, the flow cell further includes a sheath fluid injection port configured to provide sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to provide a sheath fluid flow to the interior chamber of the flow cell, such as to combine with the sample to create a layered sheath fluid flow around the sample flow stream. The rate at which the sheath fluid is delivered to the flow cell chamber can be 25 pL / sec or more, such as 50 pL / sec or more, such as 75 pL / sec or more, such as 100 pL / sec or more, such as 250 pL / sec or more, such as 500 pL / sec or more, such as 750 pL / sec or more, such as 1000 pL / sec or more and including 2500 pL / sec or more, depending on the desired characteristics of the flow stream.

[0122] In some embodiments, the sheath fluid injection port is an orifice in the wall of the interior chamber. The sheath fluid injection port orifice can be any suitable shape, with cross-sectional shapes of interest including, but 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 portion coupled to a planar top portion. In certain instances, the size of the sheath fluid injection port orifice can vary in shape with an opening of 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm, such as 1.5 mm.

[0123] In some embodiments, the system further comprises a pump in fluid communication with the flow cell to propagate the flow stream through the flow cell. Any convenient fluid pump protocol can be used to control the flow of the flow stream through the flow cell. In some cases, the system comprises a peristaltic pump, for example a peristaltic pump having a pulse damper. The pump in the subject system is configured to deliver fluid through the flow cell at a rate suitable for detecting light from a sample in the flow stream. In some cases, the rate of the sample stream in the flow cell is 1 uL / min (microliters per minute) or more, for example 2 uL / min or more, for example 3 uL / min or more, for example 5 uL / min or more, for example 10 uL / min or more, for example 25 uL / min or more, for example 50 uL / min or more, for example 75 uL / min or more, for example 100 uL / min or more, for example 250 uL / min or more, for example 500 uL / min or more, for example 750 uL / min or more and including a rate of 1000 uL / min or more. For example, the system can comprise a pump configured to flow the sample through the flow cell at a rate of 1 uL / min to 500 uL / min, for example 1 uL / min to 250 uL / min, for example 1 uL / min to 100 uL / min, for example 2 uL / min to 90 uL / min, for example 3 uL / min to 80 uL / min, for example 4 uL / min to 70 uL / min, for example 5 uL / min to 60 uL / min and including 10 uL / min to 50 uL / min. In some embodiments, the flow rate of the flow stream is 5 uL / min to 6 uL / min.

[0124] In certain embodiments, the subject system is a flow cytometer system that uses the above-described light detection system to detect light emitted by a sample in a flow stream. In certain embodiments, the subject system is a flow cytometer system. Suitable flow cytometer systems can 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 Vol. 91, Humana Press (1997); Practical Flow Cytometry, 3rdEd., 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 disclosures of which are incorporated herein by reference. In certain cases, flow cytometer systems of interest include BD Biosciences FACSCanto TM BD Accuri™ C6 Flow Cytometer TM BD Biosciences FACSCelesta Flow Cytometer TM BD Biosciences FACSLyric Flow Cytometer TM BD Biosciences FACSVerse Flow Cytometer TM BD Biosciences FACSymphony Flow Cytometer TM BD Biosciences LSRFortessa Flow Cytometer TM BD Biosciences LSRFortessa Flow Cytometer TM BD Biosciences X-20 Flow Cytometer and FACSCalibur TM BD Biosciences FACSCount Cell Sorter TM BD Biosciences FACSLyric Cell Sorter TM BD Biosciences Via Flow Cell Sorter TM BD Biosciences Influx Cell SorterTM Cell Sorter, BD Biosciences Jazz TM Cell Sorter, BD Biosciences Aria TM Cell Sorter and BD Biosciences FACSMelody TM Cell Sorter, etc.

[0125] In some embodiments, the subject particle sorting system is a flow cytometer system, such as those described in U.S. Patent Nos. 10,006,852; 9,952,076; 9,933,341; 9,784,661; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039, the disclosures of which are incorporated by reference in their entireties.

[0126] In certain cases, the subject system is a flow cytometer system configured for imaging particles in a flow stream by using fluorescence imaging of radiofrequency tagged emission (FIRE), such as described in Diebold et al. Nature Photonics vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546; 10,324,019; 10,408,758; 10,451,538; 10,620,111 and U.S. Patent Publication Nos. 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895; and 2019 / 0376894, the disclosures of which are incorporated by reference.

[0127] In certain embodiments, the subject system is configured to sort one or more of the particles (e.g., cells) of a sample. The term “sort” is used herein in its conventional sense to refer to separating components (e.g., cells, non-cellular particles, e.g., biological macromolecules) of a sample and, in some cases, delivering the separated components to one or more sample collection containers. For example, the subject system can be configured for sorting samples having 2 or more components, e.g., sorting samples having 3 or more components, e.g., 4 or more components, e.g., 5 or more components, e.g., 10 or more components, e.g., 15 or more components, and including sorting samples having 25 or more components. One or more of the sample components can be separated from the sample and delivered to a sample collection container, e.g., 2 or more sample components, e.g., 3 or more sample components, e.g., 4 or more sample components, e.g., 5 or more sample components, e.g., 10 or more sample components, and including: 15 or more sample components can be separated from the sample and delivered to a sample collection container.

[0128] In some embodiments, the particle sorting system of interest is configured to sort particles as described in U.S. Patent Nos. 3,960,449; 4,347,935; 4,667,830; 5,245,318; 5,464,581; 5,483,469; 5,602,039; 5,643,796; 5,700,692; 6,372,506; and 6,809,804, the disclosures of which are incorporated herein by reference. In some embodiments, the particle sorting system is configured to sort particles with the particle sorting modules attached, e.g., those described in U.S. Patent Nos. 9,551,643 and 10,324,019; U.S. Patent Publication No. 2017 / 0299493; and International Patent Publication No. WO / 2017 / 040151, the disclosures of which are incorporated herein by reference. In certain embodiments, the particles (e.g., cells) of a sample are sorted using a sort decision module having multiple sort decision units, e.g., those described in U.S. Patent Application No. 16 / 725,756, filed December 23, 2019, the disclosure of which is incorporated herein by reference.

[0129] In some embodiments, the system is a particle analyzer, wherein the particles can be used for analysis and characterization, 401( Figure 4A ) whether or not the particles are physically sorted into a collector. Figure 4AA functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, particle analysis system 401 is a flow system. For example, particle analysis system 401 shown in FIG. 4 can be configured to perform all or part of the methods described herein. Particle analysis system 401 includes a fluidics system 402. The flow system 402 may include a sample tube 405 or a moving fluid column coupled to the sample tube 405 and within the sample tube, in which particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0130] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as it passes through one or more detection stations along a common sample path. Detection station 408 typically refers to a monitoring area 407 along the common sample path. In some embodiments, detection may include detecting light or one or more other characteristics of particle 403 as it passes through monitoring area 407. Figure 4A The image shows a detection station 408 with a monitoring area 407. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations can monitor more than one area.

[0131] A signal value is assigned to each signal, thus forming a data point for each particle. As mentioned above, this data can be referred to as event data. The data point can be a multidimensional data point including values ​​of various properties measured for the particle. The detection system 404 is configured to collect a series of such data points within a first time interval.

[0132] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system shown may be operationally associated with the fluid flow system 402. The control system may be configured to generate a calculated signal frequency based on a Poisson distribution and the number of data points collected by the detection system 404 during at least a portion of the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may also compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0133] Figure 4BA system 400 for flow cytometry according to an illustrative embodiment of the application is shown. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-c, a focusing lens 420, a flow cell 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-g, one or more bandpass filters 450a-e, one or more longpass (“LP”) filters 455a-b, and one or more fluorescence detectors 460a-f.

[0134] The excitation lasers 115a-c emit light in the form of laser beams. In the example system, the wavelengths of the laser beams emitted from the excitation lasers 415a-c are 488 nm, 633 nm, and 325 nm, respectively. Figure 4B The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. The beam splitter 445b transmits UV light (light with a wavelength of 10 nm to 400 nm) and reflects light at 488 nm and 633 nm.

[0135] The laser beams are then directed to the focusing lens 420, which focuses the beams onto a portion of the flow stream within the flow cell 425 where sample particles are located. The flow cell is part of a fluidics system that directs particles in the flow (typically one at a time) to the focused laser beams for interrogation. The flow cell can include a flow cell in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0136] Light from the laser beams interacts with the particles in the sample by diffracting, refracting, reflecting, scattering, and absorbing in various different wavelengths as a function of the particle characteristics (e.g., particle size, internal structure, and presence of one or more fluorescent molecules attached to the particle or naturally present on or within the particle). The fluorescent emissions, as well as the diffracted, refracted, reflected, and scattered light, can be routed to one or more of the forward scatter detector 430, the side scatter detector 435, and the one or more fluorescence detectors 460a-f through one or more of the beam splitters 445a-g, the bandpass filters 450a-e, the longpass filters 455a-b, and the fluorescence collection lens 440.

[0137] A fluorescence collection lens 440 collects light emitted from the particle-laser beam interaction and routes the light toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Shortpass filters transmit light at or shorter than a particular wavelength. Longpass filters, such as longpass filters 455a-455b, transmit light at or longer than a particular wavelength of light. For example, longpass filter 455a, which is a 670 nm longpass filter, transmits light at or longer than 670 nm. The filters are typically chosen to optimize the specificity of the detector for a particular fluorescent dye. The filters can be configured such that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0138] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be characterized by filter properties, such as shortpass and longpass. For example, beam splitter 445g is a 620SP beam splitter, which means that the beam splitter 445g transmits light at or shorter than 620 nm and reflects light at wavelengths greater than 620 nm in a different direction. In one embodiment, beam splitters 445a-445g can include optical mirrors, such as dichroic mirrors.

[0139] A forward scatter detector 430 is positioned slightly off axis from the direct beam through the flow cell and is configured to detect diffracted light, i.e., excitation light that propagates primarily in a forward direction through or around the particle. The intensity of light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector can include a photodiode. A side scatter detector 435 is configured to detect refracted and reflected light from the surface and internal structure of the particle and tends to increase with increasing complexity of the particle structure. One or more fluorescence detectors 460a-460f can detect fluorescent emissions from fluorescent molecules associated with the particle. The side scatter detector 435 and the fluorescence detectors can include photomultiplier tubes. Signals detected at the forward scatter detector 430, the side scatter detector 435, and the fluorescence detectors can be converted to electronic signals (voltage) by the detectors. This data can provide information about the sample.

[0140] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present application is not limited to Figure 4B the depicted flow cytometer, but can include any flow cytometer known in the art. For example, the flow cytometer can have any number of lasers with various wavelengths and various different configurations of beam splitters, filters, and detectors.

[0141] In operation, cytometer operation is controlled by controller / processor 490, and measurement data from the detectors can be stored in memory 495 and processed by controller / processor 490. Although not explicitly shown, controller / processor 190 is coupled to the detectors to receive output signals therefrom, and controller / processor 190 can also be coupled to the electrical and electromechanical components of flow cytometer 400 to control the lasers, fluid flow parameters, etc. Input / output (I / O) capability 497 can also be provided in the system. Memory 495, controller / processor 490, and I / O 497 can be provided entirely as an integrated part of flow cytometer 410. In such embodiments, a display can also form part of I / O capability 497 for presenting experimental data to a user of cytometer 400. Alternatively, some or all of memory 495 and controller / processor 490 and I / O capability can be part of one or more external devices, such as a general purpose computer. In some embodiments, some or all of memory 495 and controller / processor 490 can be in wireless or wired communication with cytometer 410. Controller / processor 490, together with memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0142] Figure 4BThe illustrated system includes six different detectors that detect fluorescence in six different wavelength bands (which can be referred to herein as the "filter window" for a given detector) as defined by the configuration of filters and / or beam splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in a flow cytometry experiment will emit light in their own characteristic wavelength band. The particular fluorescent labels used in an experiment and the fluorescence emission bands associated with the fluorescent labels can be selected to generally coincide with the filter window of a detector. However, as more detectors are provided and more labels are used, perfect correspondence between the filter windows and the fluorescence emission spectra is not possible. Generally, while the peak of the emission spectrum of a particular fluorescent molecule can indeed lie within the filter window of a particular detector, some of the emission spectrum of that label will also overlap with the filter window of one or more other detectors. This can be referred to as spillover. The I / O 497 can be configured to receive data regarding a flow cytometry experiment having a set of fluorescent labels and a plurality of cell populations having a plurality of markers, each cell population having a subset of the plurality of markers. The I / O 497 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. The flow cytometry experiment data, such as label spectrum characteristics and flow cytometer configuration data, can also be stored in the memory 495. The controller / processor 490 can be configured to evaluate one or more assignments of labels to markers.

[0143] Figure 5 A functional block diagram showing one example of a particle analyzer control system (e.g., analysis controller 500) for analyzing and displaying biological events is shown. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0144] The particle analyzer 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 through the data communication channel.

[0145] The analysis controller 500 can be configured to receive biological event data from the particle analyzer 502. The biological event data received from the particle analyzer 502 can include flow cytometry event data. The analysis controller 500 can be configured to provide a graphical display including a first plot of the biological event data to the display device 506. The analysis controller 500 can additionally be configured to present a region of interest as a gate around a population of biological event data shown by the display device 506, e.g., overlaid on the first plot. In some embodiments, the gate can be a logical combination of one or more regions of interest plotted on a single parameter histogram or a bivariate plot. In some embodiments, the display can be used to display particle parameters or saturated detector data.

[0146] The analysis controller 500 can additionally be configured to display the biological event data within the gate on the display device 506 differently than other events in the biological event data outside the gate. For example, the analysis controller 500 can be configured to render a color of the biological event data contained within the gate different from a color of the biological event data outside the gate. The display device 506 can be implemented as a monitor, a tablet, a smartphone, or other electronic device configured to render a graphical interface.

[0147] The analysis controller 500 can be configured to receive a gate selection signal from a first input device identifying a gate. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a gate selection signal to the analysis controller 500 identifying a gate to be displayed on the display device 506 or to be manipulated by the display device 506 (e.g., by clicking on a desired gate when a cursor is positioned at the desired gate). In some implementations, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices can include multiple input functions. In such implementations, each of the input functions can be considered an input device. For example, as shown, the mouse 510 can include a mouse right button and a mouse left button, each of which can generate a trigger event. Figure 5

[0148] The trigger event can cause the analysis controller 500 to change the way the data is displayed (which portions of the data are actually displayed on the display device 506), and / or provide input for further processing (e.g., selecting a population of interest) for particle sorting.

[0149] ​In some embodiments, the analysis controller 500 can be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a particular distribution of the biological event data received by the analysis controller 500.

[0150] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometer event data from the analysis controller 500. The storage device 504 can be further configured to allow retrieval of biological event data, such as flow cytometer event data, by the analysis controller 500.

[0151] The display device 506 can be configured to receive display data from the analysis controller 500. The display data can include a plot of biological event data and a gated portion of the plot. The display device 506 can be further configured to change information presented in accordance with input received from the analysis controller 500 along with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0152] In some implementations, the analysis controller 500 can generate a user interface to receive example events for sorting. For example, the user interface can include a control for receiving example events or example images. The example events or images or example gates can be provided prior to collecting event data for a sample, or based on an initial set of events for a portion of the sample.

[0153] Computer control system

[0154] Aspects of the present disclosure also include a computer control system for practicing the subject methods, wherein the system further includes one or more computers for fully automated or partially automated practice of the system for practicing the methods described herein. In some embodiments, the system includes a computer having a computer readable storage medium having stored thereon a computer program, wherein the computer program, when loaded onto the computer, includes instructions for illuminating a particle in a flow stream with a frequency modulated laser beam modulated at a reference frequency, instructions for detecting scattered light from the particle with a photodetector, instructions for generating a frequency encoded data signal from the detected scattered light, and instructions for synchronizing the frequency encoded data signal to the reference frequency. In certain embodiments, the computer readable storage medium includes instructions for determining one or more parameters of the particle based on the synchronized frequency encoded data signal.

[0155] In certain cases, the system includes a computer having a computer readable storage medium, wherein the computer readable storage medium has stored thereon a computer program, wherein the computer program, when loaded onto the computer, further includes instructions for illuminating a light modulator (e.g., an electro-optical modulator, a piezoelectric optical modulator, or an acousto-optical modulator device) to generate a frequency-modulated laser beam. In some embodiments, the computer program includes instructions for applying a current or an electric field to the electro-optical modulator to generate the frequency-modulated laser beam. In certain embodiments, the computer program includes instructions for applying a current or an electric field to the electro-optical modulator to generate the frequency-modulated laser beam having an oscillation frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, for example about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, for example about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, for example about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, for example 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.

[0156] In some cases, the computer program includes instructions for generating a reference frequency signal for the light modulator, such as wherein the reference frequency signal has an oscillation frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, for example about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, for example about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, for example about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, for example 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.

[0157] In some embodiments, the system includes a computer having a computer readable storage medium having stored thereon a computer program, wherein the computer program, when loaded onto the computer, additionally includes instructions for synchronizing the frequency encoded data signal with the reference frequency. In some embodiments, the computer program includes instructions for synchronizing the frequency encoded data signal with the reference frequency by multiplying the frequency encoded data signal with the reference frequency waveform. In some cases, the computer program includes instructions for synchronizing the frequency encoded data signal with the reference frequency using a lock-in amplifier. In other embodiments, the computer program includes instructions for digitally synchronizing the frequency encoded data signal, such as instructions for digitally synchronizing the frequency encoded data signal with an FPGA having programming for multiplying the frequency encoded data signal with the reference frequency waveform. In some embodiments, the computer program includes instructions for applying a low pass filter to the synchronized frequency encoded data signal to generate a data signal distribution. In certain cases, the data signal distribution generated by applying the low pass filter has a Gaussian signal distribution. In other cases, the data signal distribution generated by applying the low pass filter has a super-Gaussian signal distribution.

[0158] In embodiments, the system includes an input module, a processing module, and an output module. The subject system can include both hardware components and software components, where the hardware components can take the form of one or more platforms, such as the form of a server, such that functional elements, i.e., those elements of the system that perform particular system tasks, such as managing the input and output of information, processing information, etc., can be implemented by executing software applications on and across one or more computer platforms representing the system.

[0159] The system can include a display and an operator input device. The operator input device can be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access a memory having instructions stored thereon for performing the steps of the subject methods. The processing module can include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input-output controller, a cache memory, a data backup unit, and many other devices. The processor can be a commercially available processor or it can be one of the other processors presently existing or which can become available. The processor executes the operating system and an interface of the operating system with firmware and hardware in a well-known manner and facilitates the processor in coordinating and executing the functions of various computer programs that can be written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system, often in cooperation with the processor, coordinates and executes functions of the other components in the computer. The operating system provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. The processor can be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align the light source with the flow stream based on the first light signal and the second light signal. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0160] The system memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media (such as a resident hard disk or magnetic tape), optical media (such as a read-write compact disk), 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 a compact disk drive, a tape drive, a removable hard disk drive, or a floppy disk drive. This type of memory storage device typically reads from and / or writes to a program storage medium (not shown), such as a compact disk, a magnetic tape, a removable hard disk, or a floppy disk, respectively. Any of these program storage media, or other presently available or future-developed program storage media, can be considered a computer program product. As will be appreciated, these program storage media generally store computer software and / or data. Computer software programs (also referred to as computer control logic) generally store in system memory and / or the program storage device used in conjunction with the memory storage device.

[0161] In some embodiments, a computer program product is described that includes a computer usable medium having control logic (computer software program, including program code) stored therein. The control logic, which when executed by the processor of a computer, causes the processor to carry out functions described herein. In other embodiments, some functions are carried out primarily in hardware. Implementation of the hardware state machine so as to carry out the functions described herein will be obvious to those skilled in the relevant arts.

[0162] The memory can be any suitable device in which the processor is able to 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 device, whether fixed or portable). The processor can include a general purpose digital microprocessor suitably programmed from a computer readable medium carrying the necessary program code. Programming can be provided to the processor remotely over a communication channel, or pre-stored in a computer program product, such as the memory or some other portable or fixed computer readable storage medium used in connection with the memory. For example, a magnetic or optical disk can carry the program and be read by a disk writer / reader. The system of the present invention also includes programming, such as a computer program product in the form of algorithms, for practicing the above-described methods. Programming in accordance with the present invention can be recorded on a computer readable medium, such as any medium that is directly readable and accessible 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-ROM; electrical storage media such as RAM and ROM; portable flash drive; and hybrids of these categories such as magnetic / optical storage media.

[0163] The processor can also access a communication channel to communicate with a user at a remote location. By remote location is meant that the user is not in direct contact with the system and relays input information from an external device, such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone) to the input manager.

[0164] In some embodiments, a 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 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®, Bluetooth Low Energy (BLE), and / or near field communication (NFC). Communication protocols and cellular communication, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0165] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces (e.g., USB ports, RS-232 ports, or any other suitable electrical connection ports) to allow data communication between the subject system and other external devices, such as a computer terminal configured for similar supplemental data communication (e.g., in a physician's office or hospital environment).

[0166] In one embodiment, the communication interface is configured for infrared communication, Bluetooth communication, or any other suitable wireless communication protocol, thereby enabling the subject system to communicate with other devices, such as a computer terminal and / or network, a cellular phone supporting communication, a personal digital assistant, or any other communication device that a user can employ in conjunction.

[0167] In one embodiment, the communication interface is configured to provide connectivity for data transmission using Internet Protocol (IP) through a cellular phone network, Short Message Service (SMS), a wireless connection with a personal computer (PC) connected to a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet through a WiFi hotspot.

[0168] In one embodiment, the subject system is configured to wirelessly communicate with a server device through the communication interface, for example, using a common standard such as 802.11 or RF protocol, or IrDA infrared protocol. The server device can be another portable device, such as a smartphone, a personal digital assistant (PDA), or a notebook computer; or a large 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 buttons, a keyboard, a mouse, or a touch screen.

[0169] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the subject system, such as data stored in the optional data storage unit, with a network or a server device using one or more of the communication protocols and / or mechanisms as described above.

[0170] The output controller can include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, whether local or remote. If one of the display devices provides visual information, that information can typically be logically and / or physically organized as an array of picture elements. The graphical user interface (GUI) controller can include any of a variety of known or future software programs for providing a graphical input and output interface between the system and a user and for processing user input. The functional elements of the computer can communicate with each other by a system bus. Some of these communications can be accomplished using a network or other type of remote communication in alternative embodiments. The output manager can also provide information generated by the processing module to a user located at a remote location, e.g., over the Internet, a telephone, or a satellite network, in accordance with known techniques. The presentation of data by the output manager can be accomplished in accordance with a variety of known techniques. As some examples, the data can include SQL, HTML, or XML documents, e-mail, or other files, or other forms of data. The data can include an Internet URL address so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more of the platforms present in the subject system can be any type of known computer platform or type to be developed in the future, although they are typically of a type commonly referred to as a server. However, they can also be mainframes, workstations, or other computer types. They can be connected by any known or future type of cable or other communication system, whether networked or otherwise. They can be in the same location or physically separated. A variety of operating systems can be employed on any of the computer platforms, which can depend on the type and / or make of the computer platform selected. Applicable operating systems include Windows Windows XP, Windows 7, Windows 8, iOS, Oracle Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, Ubuntu, Zorin OS, and the like.

[0171] Figure 6 The overall architecture of the computing device 600 depicted in accordance with certain embodiments is depicted. Figure 6 The overall architecture of the computing device 600 depicted in accordance with certain embodiments is depicted. Figure 6The elements of the computing device 600 as shown are more (or fewer) than those necessary to practice the disclosed embodiments. However, to provide an enabling disclosure, it is not necessary to show all of the typical components of a computing device. As shown, the computing device 600 includes a processing unit 610, a network interface 620, a computer-readable media drive 630, an input / output device interface 640, a display 650, and an input device 660, all of which can communicate with one another by way of a communication bus. The network interface 620 can provide connectivity to one or more networks or computing systems. The processing unit 610 can thus receive information and instructions from other computing systems or services over a network. The processing unit 610 can also communicate with the memory 670 and additionally provide output information to the optional display 650 through the input / output device interface 640. The input / output device interface 840 can also accept input from optional input devices 660 such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input devices.

[0172] The memory 670 can contain computer program instructions (grouped in modules or components in some embodiments) that the processing unit 610 executes to implement one or more embodiments. The memory 670 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. The memory 670 can store an operating system 672 that provides computer program instructions for use by the processing unit 610 in the overall management and operation of the computing device 600. The memory 670 can also include computer program instructions and other information for implementing various aspects of the present disclosure.

[0173] Non-transitory computer-readable storage medium

[0174] Aspects of the present disclosure also include non-transitory computer- readable storage media having instructions for practicing the subject methods. The computer-readable storage media can be used in one or more computers for full or partial automation of a system to practice the methods described herein. In certain embodiments, the instructions according to the methods described herein can be encoded onto a computer-readable medium in a "programmed" form, where the term "computer-readable medium" as used herein refers to any non-transitory storage medium that is involved in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state drives, and network attached storage (NAS), whether these devices are internal or external to the computer. A file containing information can be "stored" on a computer-readable medium, where "stored" means recorded so that it can be later accessed and retrieved by a computer. The computer-implemented methods described herein can be performed using programming that can be written in one or more of any number of computer programming languages. For example, these languages include Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as any of a number of other programming languages.

[0175] In some embodiments, a computer-readable storage medium of interest includes a computer program stored thereon, where the computer program, when loaded onto a computer, includes instructions having an algorithm for illuminating a particle in a flow stream with a frequency-modulated laser beam modulated at a reference frequency, an algorithm for detecting scattered light from the particle using a photodetector, an algorithm for generating a frequency-encoded data signal from the detected scattered light, and an algorithm for synchronizing the frequency-encoded data signal with the reference frequency. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of the particle based on the synchronized frequency-encoded data signal.

[0176] In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for illuminating a light modulator (e.g., an electro-optical modulator, a piezoelectric light modulator, or an acousto-optical modulator device) to generate a frequency-modulated laser beam. In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for applying a current or an electric field to an electro-optical modulator to generate a frequency-modulated laser beam. In certain embodiments, the non-transitory computer-readable storage medium comprises an algorithm for applying a current or an electric field to an electro-optical modulator to generate a frequency-modulated light beam having an oscillation frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, for example about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, for example about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, for example about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, for example 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.

[0177] In some cases, the non-transitory computer-readable storage medium comprises an algorithm for generating a reference frequency signal for a light modulator, such as where the reference frequency signal has an oscillation frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, for example about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, for example about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, for example about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, for example 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.

[0178] In some embodiments, the non-transitory computer readable storage medium includes an algorithm for synchronizing the frequency encoded data signal with a reference frequency. In some embodiments, the computer program includes instructions for synchronizing the frequency encoded data signal with a reference frequency by multiplying the frequency encoded data signal with a reference frequency waveform. In some cases, the non-transitory computer readable storage medium includes an algorithm for synchronizing the frequency encoded data signal with a reference frequency using a lock-in amplifier. In other embodiments, the non-transitory computer readable storage medium includes an algorithm for digitally synchronizing the frequency encoded data signal, for example, by an FPGA programmed to multiply the frequency encoded data signal with a reference frequency waveform. In some embodiments, the non-transitory computer readable storage medium includes an algorithm for applying a low pass filter to the synchronized frequency encoded data signal to generate a data signal distribution. In some cases, the data signal distribution generated by applying the low pass filter has a Gaussian signal distribution. In other cases, the data signal distribution generated by applying the low pass filter has a super-Gaussian signal distribution.

[0179] In some embodiments, the non-transitory computer readable storage medium includes an algorithm for determining one or more parameters of the illuminated particles in the flow stream from the synchronized frequency modulated data signal. In some embodiments, the non-transitory computer readable storage medium includes an algorithm for identifying the particles based on the determined one or more parameters of the particles. In other embodiments, the non-transitory computer readable storage medium includes an algorithm for sorting the particles based on the determined one or more parameters of the particles.

[0180] The non-transitory computer readable storage medium can be employed on one or more computer systems with displays and operator input devices. The operator input devices can be, for example, keyboards, mice, etc. The processing module includes a processor that can access a memory having instructions stored thereon for performing the steps of the subject methods. The processing module can include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input-output controllers, cache memory, data backup units, and many other devices. The processor can be a commercially available processor or it can be one of the other processors presently existing or which can become available. The processor executes the operating system and the operating system's interface 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 can be written in various programming languages such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system, often in cooperation with the processor, coordinates and executes functions of the other components in the computer. The operating system further provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0181] kits

[0182] Aspects of the invention also include kits, where the kits include a laser, an optical modulator, and one or more optical polarizers. In some embodiments, the optical modulator is an electro-optical modulator. In other embodiments, the optical modulator is a piezoelectric optical modulator. In yet other embodiments, the optical modulator is an acousto-optic modulator. The kits can also include optical conditioning components, such as mirrors or beam splitters for transmitting light from a flow stream to a scattered photodetector. In certain embodiments, the kits include a lock-in amplifier. In some cases, the kits also include an integrated circuit, such as a field programmable gate array with programming designed to synchronize a generated frequency encoded data signal with a reference frequency signal from the optical modulator. In certain embodiments, the kits include one or more components of a beam generator, such as a waveform pulse generator, a direct digital synthesizer, an acousto-optic deflector, a beam combining lens, and a Powell lens.

[0183] The various assay components of the kits can be present in separate containers, or some or all of them can be pre-combined. For example, in some cases, one or more components of the kits, such as one or more scattered photodetectors, a laser, an optical modulator, are present in a sealed pouch, such as a sterile foil pouch or envelope.

[0184] In addition to the components described above, the subject kits can also include (in certain embodiments) instructions for practicing the subject methods. These instructions can be present in the subject kits in a variety of forms, one or more of which can be present in the kit. One form in which such instructions can be present is as printed information on a suitable medium or substrate, e.g., a sheet of paper on which the instructions are printed, in the packaging of the kit, in a package insert of the kit, etc. Another form in which such instructions can be present is as electronic

[0185] Utility

[0186] The subject systems, methods, and computer systems can be used in a variety of applications in which it is desirable to analyze and sort particle components in a sample (e.g., a biological sample) in a fluid medium. The present disclosure also finds use in flow cytometry, where it is desirable to provide a flow cytometer with improved cell sorting precision, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In embodiments, the present disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In certain embodiments, the subject systems provide a fully automated protocol such that little, if any, manual input is required for adjustment of the flow cytometer during use.

[0187] The present disclosure also finds use in applications where cells prepared from a biological sample can be needed for research, laboratory testing, or for treatment. In some embodiments, the subject methods and apparatuses can facilitate obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods and systems facilitate obtaining cells from a fluid or tissue sample for use as a research or diagnostic specimen for a disease, such as cancer. Likewise, the subject methods and systems facilitate obtaining cells from a fluid or tissue sample for use in treatment. In comparison to traditional flow cytometer systems, the methods and apparatuses of the present disclosure allow for isolation and collection of cells from a biological sample (e.g., an organ, a tissue, a tissue segment, a fluid) with improved efficiency and at a low cost.

[0188] The present disclosure is also defined by the following clauses, despite the appended claims:

[0189] 1. A method for determining a parameter of a particle in a flow stream in a particle analyzer, the method comprising:

[0190] illuminating the particle in the flow stream with a frequency modulated laser beam modulated with a reference frequency;

[0191] detecting scattered light from the particle with a photodetector;

[0192] generating a frequency encoded data signal from the detected scattered light;

[0193] synchronizing the frequency encoded data signal with the reference frequency; and

[0194] determining one or more parameters of the particle based on the synchronized frequency encoded data signal.

[0195] 2. The method of clause 1, wherein the method comprises illuminating an electro-optical modulator with a laser to produce the frequency modulated laser beam.

[0196] 3. The method of clause 2, wherein the electro-optical modulator is a piezoelectric optical modulator.

[0197] 4. The method of any of clauses 2-3, wherein the method comprises illuminating the electro-optical modulator with a laser through an input polarizer to produce a polarized frequency-modulated laser beam.

[0198] 5. The method of any of clauses 1-4, wherein the scattered light comprises forward scattered light.

[0199] 6. The method of any of clauses 1-5, wherein the scattered light comprises side scattered light.

[0200] 7. The method of any of clauses 1-6, wherein the scattered light is transmitted to the photodetector through an output polarizer.

[0201] 8. The method of clause 7, wherein the output polarizer comprises a quarter wave plate.

[0202] 9. The method of any of clauses 6-8, wherein the side scattered light is detected in two detector channels.

[0203] 10. The method of clause 9, wherein the side scattered detector channels comprise a first polarizer having a first polarization and a second polarizer having a second polarization perpendicular to the first polarization.

[0204] 11. The method of any of clauses 1-10, wherein generating the frequency encoded data signal comprises detecting an amplitude of light scattered by particles oscillating at the reference frequency.

[0205] 12. The method of any of clauses 1-11, further comprising generating a reference frequency signal.

[0206] 13. The method of clause 12, wherein the reference frequency signal comprises a reference waveform.

[0207] 14. The method of clause 13, wherein synchronizing the frequency encoded data signal comprises multiplying the frequency encoded data signal with the reference waveform.

[0208] 15. The method of any of clauses 1-14, wherein the frequency encoded data signal is synchronized with the reference frequency by a lock-in amplifier.

[0209] 16. The method of clause 15, wherein the lock-in amplifier is implemented on a field programmable gate array (FPGA).

[0210] 17. The method of any of clauses 1-14, wherein the frequency encoded data signal is digitally synchronized with the reference frequency.

[0211] 18. The method of any one of clauses 1-17, wherein the method further comprises applying a low-pass filter to the synchronized frequency-encoded data signal to generate a data signal distribution.

[0212] 19. The method of clause 18, wherein applying a low-pass filter to the synchronized frequency-encoded data signal generates a Gaussian signal distribution.

[0213] 20. The method of clause 18, wherein applying a low-pass filter to the synchronized frequency-encoded data signal generates a super-Gaussian signal distribution.

[0214] 21. The method of any one of clauses 1-20, wherein the particle analyzer is part of a flow cytometer.

[0215] 22. The method of clause 21, wherein the method further comprises identifying the particle based on one or more of the determined parameters of the particle.

[0216] 23. The method of any one of clauses 21-22, wherein the method further comprises sorting the particle based on one or more of the determined parameters of the particle.

[0217] 24. A method comprising:

[0218] illuminating a particle in a flow stream with a frequency-modulated laser beam modulated with a reference frequency;

[0219] detecting scattered light from the particle with a photodetector;

[0220] generating a frequency-encoded data signal from the detected scattered light; and

[0221] synchronizing the frequency-encoded data signal with the reference frequency.

[0222] 25. The method of clause 24, wherein the method comprises illuminating an electro- optical modulator with a laser to generate the frequency-modulated laser beam.

[0223] 26. The method of clause 25, wherein the electro-optical modulator is a piezoelectric optical modulator.

[0224] 27. The method of any one of clauses 25-26, wherein the method comprises illuminating the electro-optical modulator with a laser through an input polarizer to generate a polarized frequency-modulated laser beam.

[0225] 28. The method of any one of clauses 24-27, wherein the scattered light comprises forward scattered light.

[0226] 29. The method of any of clauses 24-28, wherein the scattered light comprises side scatter light.

[0227] 30. The method of any of clauses 24-29, wherein the scattered light is transmitted through an output polarizer to the photodetector.

[0228] 31. The method of clause 30, wherein the output polarizer comprises a quarter wave plate.

[0229] 32. The method of any of clauses 29-31, wherein the side scatter light is detected in two detector channels.

[0230] 33. The method of clause 32, wherein the side scatter detector channels comprise a first polarizer having a first polarization and a second polarizer having a second polarization perpendicular to the first polarization.

[0231] 34. The method of any of clauses 24-33, wherein generating the frequency encoded data signal comprises detecting an amplitude of light scattered by particles oscillating at the reference frequency.

[0232] 35. The method of any of clauses 24-34, further comprising generating a reference frequency signal.

[0233] 36. The method of clause 35, wherein the reference frequency signal comprises a reference waveform.

[0234] 37. The method of clause 36, wherein synchronizing the frequency encoded data signal comprises multiplying the frequency encoded data signal with the reference waveform.

[0235] 38. The method of any of clauses 24-37, wherein the frequency encoded data signal is synchronized with the reference frequency using a lock-in amplifier.

[0236] 39. The method of clause 38, wherein the lock-in amplifier is implemented on a field programmable gate array (FPGA).

[0237] 40. The method of any of clauses 24-37, wherein the frequency encoded data signal is digitally synchronized with the reference frequency.

[0238] 41. The method of any of clauses 24-40, wherein the method further comprises applying a low pass filter to the synchronized frequency encoded data signal to generate a data signal profile.

[0239] 42. The method of clause 41, wherein applying a low-pass filter to the synchronized frequency-encoded data signal generates a Gaussian signal distribution.

[0240] 43. The method of clause 41, wherein applying a low-pass filter to the synchronized frequency-encoded data signal generates a super-Gaussian signal distribution.

[0241] 44. A particle analyzer for determining one or more parameters of particles in a flow stream, the particle analyzer comprising:

[0242] a light source comprising:

[0243] a laser; and

[0244] a light modulator component configured to generate a frequency-modulated laser beam at a reference frequency;

[0245] a light detection system comprising a photodetector configured to detect scattered light from illuminated particles in the flow stream; and

[0246] a processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to:

[0247] generate a frequency-encoded data signal from the detected scattered light;

[0248] synchronize the frequency-encoded data signal with the reference frequency; and

[0249] determine the one or more parameters of the particles based on the synchronized frequency-encoded data signal.

[0250] 45. The particle analyzer of clause 44, wherein the particle analyzer is incorporated into a flow cytometer.

[0251] 46. The particle analyzer of any one of clauses 44-45, further comprising a particle sorter for sorting particles based on one or more of the determined parameters of the particles.

[0252] 47. The particle analyzer of any one of clauses 44-46, wherein the light modulator component comprises an electro-optical modulator.

[0253] 48. The particle analyzer of clause 47, wherein the electro-optical modulator is a piezoelectric optical modulator.

[0254] 49. The particle analyzer of any one of clauses 47-48, wherein the light modulator component further comprises an input polarizer.

[0255] 50. The particle analyzer of any one of clauses 44-49, wherein the photodetector comprises a forward scatter detector.

[0256] 51. The particle analyzer of any one of clauses 44-50, wherein the photodetector comprises a side scatter detector.

[0257] 52. The particle analyzer of any one of clauses 44-51, wherein the light detection system further comprises an output polarizer.

[0258] 53. The particle analyzer of clause 52, wherein the output polarizer comprises a quarter wave plate.

[0259] 54. The particle analyzer of any one of clauses 51-53, wherein the light detection system comprises two side scatter detectors.

[0260] 55. The particle analyzer of clause 54, wherein the light detection system comprises:

[0261] a first side scatter detector comprising a first polarizer having a first polarization; and

[0262] a second side scatter detector comprising a second polarizer having a second polarization perpendicular to the first polarization.

[0263] 56. The particle analyzer of any one of clauses 44-55, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to detect an amplitude of light scattered by a particle oscillating at a reference frequency.

[0264] 57. The particle analyzer of any one of clauses 44-56, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to generate a reference frequency signal.

[0265] 58. The particle analyzer of clause 57, wherein the reference frequency signal comprises a reference waveform.

[0266] 59. The particle analyzer of clause 58, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to synchronize the frequency encoded data signal by multiplying the frequency encoded data signal with the reference waveform.

[0267] 60. The particle analyzer of any one of clauses 44-59, wherein the particle analyzer is in communication with a lock-in amplifier configured to synchronize the frequency encoded data signal with the reference frequency.

[0268] 61. The particle analyzer of clause 60, further comprising an FPGA configured to synchronize the frequency-encoded data signal with the reference frequency by lock-in amplification.

[0269] 62. The particle analyzer of any one of clauses 60 to 61, wherein the lock-in amplifier is configured to apply a low-pass filter to the synchronized frequency-encoded data signal to generate a data signal distribution.

[0270] 63. The particle analyzer of any one of clauses 44 to 62, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to digitally synchronize the frequency-encoded data signal with the reference frequency.

[0271] 64. The particle analyzer of clause 63, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to apply a low-pass filter to the synchronized frequency-encoded data signal to generate a data signal distribution.

[0272] 65. The particle analyzer of any one of clauses 62 and 64, wherein the generated data signal distribution is a Gaussian signal distribution.

[0273] 66. The particle analyzer of any one of clauses 62 and 64, wherein the generated data signal distribution is a super-Gaussian signal distribution.

[0274] 67. A system comprising:

[0275] a light source comprising:

[0276] a laser; and

[0277] a light modulator component configured to generate a frequency-modulated laser beam at a reference frequency;

[0278] a light detection system comprising a photodetector configured to detect scattered light from illuminated particles in a flow stream; and

[0279] a processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to:

[0280] generate a frequency-encoded data signal from the detected scattered light; and

[0281] synchronize the frequency-encoded data signal with the reference frequency.

[0282] 68. The system of clause 67, wherein the light modulator component comprises an electro-optical modulator.

[0283] 69. The system of clause 68, wherein the electro-optical modulator is a piezoelectric optical modulator.

[0284] 70. The system of any one of clauses 68-69, wherein the light modulator component further comprises an input polarizer.

[0285] 71. The system of any one of clauses 67-70, wherein the photodetector comprises a forward scatter detector.

[0286] 72. The system of any one of clauses 67-70, wherein the photodetector comprises a side scatter detector.

[0287] 73. The system of any one of clauses 67-72, wherein the light detection system further comprises an output polarizer.

[0288] 74. The system of clause 73, wherein the output polarizer comprises a quarter wave plate.

[0289] 75. The system of any one of clauses 72-74, wherein the light detection system comprises two side scatter detectors.

[0290] 76. The system of clause 75, wherein the light detection system comprises:

[0291] a first side scatter detector comprising a first polarizer having a first polarization; and

[0292] a second side scatter detector comprising a second polarizer having a second polarization perpendicular to the first polarization.

[0293] 77. The system of any one of clauses 67-76, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to detect an amplitude of light scattered by particles oscillating at the reference frequency.

[0294] 78. The system of any one of clauses 67-77, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to generate a reference frequency signal.

[0295] 79. The system of clause 78, wherein the reference frequency signal comprises a reference waveform.

[0296] 80. The system of clause 79, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to synchronize the frequency encoded data signal by multiplying the frequency encoded data signal with the reference waveform.

[0297] 81. The system of any one of clauses 67-80, further comprising a lock-in amplifier configured to synchronize the frequency encoded data signal with the reference frequency.

[0298] 82. The system of clause 81, further comprising a FPGA configured to synchronize the frequency encoded data signal with the reference frequency by lock-in amplification.

[0299] 83. The system of any one of clauses 81-82, wherein the lock-in amplifier is configured to apply a low pass filter to the synchronized frequency encoded data signal to generate a data signal distribution.

[0300] 84. The system of any one of clauses 67-80, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to synchronize the frequency encoded data signal with the reference frequency digitally.

[0301] 85. The system of clause 84, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to apply a low pass filter to the synchronized frequency encoded data signal to generate a data signal distribution.

[0302] 86. The system of any one of clauses 83 and 85, wherein the generated data signal distribution is a Gaussian signal distribution.

[0303] 87. The system of any one of clauses 83 and 85, wherein the generated data signal distribution is a super-Gaussian signal distribution.

[0304] 88. A non-transitory computer readable storage medium comprising instructions stored thereon for determining a parameter of a particle in a flow stream in a particle analyzer, the instructions comprising:

[0305] an algorithm for illuminating a particle in a flow stream with a frequency modulated laser beam modulated at a reference frequency;

[0306] an algorithm for detecting scattered light from the particle using a photodetector;

[0307] an algorithm for generating a frequency encoded data signal from the detected scattered light;

[0308] An algorithm for synchronizing a frequency-encoded data signal with a reference frequency; and

[0309] An algorithm for determining one or more parameters of a particle based on a synchronized frequency-encoded data signal.

[0310] 89. The non-transitory computer-readable storage medium of clause 88, wherein the non-transitory computer-readable storage medium comprises an algorithm for illuminating an electro-optical modulator with a laser to generate the frequency-modulated laser beam.

[0311] 90. The non-transitory computer-readable storage medium of clause 89, wherein the electro-optical modulator is a piezoelectric optical modulator.

[0312] 91. The non-transitory computer-readable storage medium of any one of clauses 89-90, wherein the non-transitory computer-readable storage medium comprises an algorithm for illuminating the electro-optical modulator with a laser through an input polarizer to generate a polarized frequency-modulated laser beam.

[0313] 92. The non-transitory computer-readable storage medium of any one of clauses 88-91, wherein the non-transitory computer-readable storage medium comprises an algorithm for detecting forward scattered light

[0314] 93. The non-transitory computer-readable storage medium of any one of clauses 88-92, wherein the non-transitory computer-readable storage medium comprises an algorithm for detecting side scattered light.

[0315] 94. The non-transitory computer-readable storage medium of clause 93, wherein the non-transitory computer-readable storage medium comprises an algorithm for detecting side scattered light in two detector channels.

[0316] 95. The non-transitory computer-readable storage medium of any one of clauses 88-94, wherein the non-transitory computer-readable storage medium comprises an algorithm for generating the frequency-encoded data signal, the algorithm comprising detecting an amplitude of light scattered by a particle oscillating at a reference frequency.

[0317] 96. The non-transitory computer-readable storage medium of any one of clauses 88-95, wherein the non-transitory computer-readable storage medium comprises an algorithm for generating a reference frequency signal.

[0318] 97. The non-transitory computer-readable storage medium of clause 96, wherein the reference frequency signal comprises a reference waveform.

[0319] 98. The non-transitory computer-readable storage medium of clause 97, wherein the non-transitory computer-readable storage medium comprises an algorithm for multiplying the frequency encoded data signal with the reference waveform to generate the synchronized frequency encoded data signal.

[0320] 99. The non-transitory computer-readable storage medium of any one of clauses 88-98, wherein the non-transitory computer-readable storage medium comprises an algorithm for synchronizing the frequency encoded data signal with the reference frequency using a lock-in amplifier.

[0321] 100. The non-transitory computer-readable storage medium of any one of clauses 88-99, wherein the non-transitory computer-readable storage medium comprises an algorithm for applying a low pass filter to the synchronized frequency encoded data signal to generate a data signal distribution.

[0322] 101. The non-transitory computer-readable storage medium of clause 100, wherein the non-transitory computer-readable storage medium comprises an algorithm for generating a Gaussian signal distribution.

[0323] 102. The non-transitory computer-readable storage medium of clause 100, wherein the non-transitory computer-readable storage medium comprises an algorithm for generating a super-Gaussian signal distribution.

[0324] 103. A kit comprising:

[0325] a laser;

[0326] an optical modulator; and

[0327] one or more optical polarizers.

[0328] 104. The kit of clause 103, wherein the optical modulator is an electro-optical modulator.

[0329] 105. The kit of clause 104, wherein the electro-optical modulator is a piezoelectric optical modulator.

[0330] 106. The kit of any one of clauses 103-105, further comprising a lock-in amplifier.

[0331] 107. The kit of any one of clauses 103-106, further comprising a quarter wave plate.

[0332] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of the foregoing disclosure that various changes in form and detail can be made without departing from the spirit and scope thereof as defined by the appended claims.

[0333] Therefore, the foregoing merely illustrates the principles of the application. It will thus be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the application and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to be only for pedagogical purposes to aid the reader in understanding the principles of the application and the concepts contributed by the inventor to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the application, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, unless otherwise noted, the examples and conditions recited herein are not to be construed as limiting the scope of the application. Any skilled person will readily appreciate that the application is capable of further embodiments and of being practiced or being carried out in various ways.

[0334] Accordingly, the scope of the application is not intended to be limited to the examples described herein. Rather, the scope and spirit of the application is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined as being invoked regardless of the absence of the specific recitations "means for" or "steps for" in the claim limitations and should not be invoked with respect to the appended claims except where the claims expressly recite the exact language "means for" or "steps for".

Claims

1. A method for determining parameters of particles in a flow stream in a particle analyzer, the method comprising: generating a reference frequency signal with an electro-optical modulator; illuminating the electro-optical modulator with a laser through an input polarizer to generate a frequency modulated laser beam modulated in polarization at a reference frequency; illuminating particles in a flow stream in a particle analyzer with the frequency modulated laser beam modulated in polarization at the reference frequency; detecting scattered light from the particles transmitted through an output polarizer with a photodetector; generating a frequency encoded data signal from the detected polarized scattered light; synchronizing the frequency encoded data signal with the reference frequency signal, wherein the reference frequency signal comprises a reference waveform, wherein synchronizing the frequency encoded data signal comprises multiplying the frequency encoded data signal with the reference waveform; and determining one or more parameters of the particles based on the synchronized frequency encoded data signal.

2. The method of claim 1, wherein, The electro-optical modulator is a piezoelectric optical modulator.

3. The method of any one of claims 1-2, wherein, The scattered light comprises at least one of forward scattered light and side scattered light.

4. The method of claim 1, wherein, The output polarizer comprises a quarter wave plate.

5. The method of claim 3, wherein, The side scattered light is detected in two detector channels.

6. The method of claim 5, wherein, A side scatter detector channel comprises a first polarizer having a first polarization and a second polarizer having a second polarization perpendicular to the first polarization.

7. The method of any one of claims 1 to 2, wherein, Generating the frequency encoded data signal comprises detecting an amplitude of light scattered by a particle oscillating at the reference frequency.

8. A particle analyzer for determining one or more parameters of particles in a flow stream, the particle analyzer comprising: a light source comprising: a laser; and an input polarizer; and an electro-optical modulator configured to generate a frequency modulated laser beam at a reference frequency and to generate a reference frequency signal; a light detection system comprising a photodetector configured to detect scattered light from illuminated particles in the flow stream transmitted through an output polarizer; and a processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to: generate a frequency encoded data signal from the detected polarized scattered light; synchronize the frequency encoded data signal with the reference frequency signal, wherein the reference frequency signal comprises a reference waveform, wherein synchronizing the frequency encoded data signal comprises multiplying the frequency encoded data signal with the reference waveform; and determine one or more parameters of the particles based on the synchronized frequency encoded data signal.

9. A system comprising: a light source comprising: a laser; and an input polarizer; and an electro-optical modulator configured to generate a frequency modulated laser beam at a reference frequency and to generate a reference frequency signal; a light detection system comprising a photodetector configured to detect scattered light from illuminated particles in the flow stream transmitted through an output polarizer; and a processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to: generate a frequency encoded data signal from the detected polarized scattered light; and synchronizing the frequency encoded data signal with the reference frequency, wherein the reference frequency signal comprises a reference waveform, wherein synchronizing the frequency encoded data signal comprises multiplying the frequency encoded data signal with the reference waveform. synchronizing the frequency encoded data signal with the reference frequency, wherein the reference frequency signal comprises a reference waveform, wherein synchronizing the frequency encoded data signal comprises multiplying the frequency encoded data signal with the reference waveform.

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