Dual excitation light beams for illuminating a sample in a fluid stream and methods of using the same

Abstract

Aspects of the present disclosure include methods for generating angularly deflected laser beams to illuminate a sample in a fluid stream. Methods according to certain embodiments include generating a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, causing the first set of angularly deflected laser beams to propagate along a different optical path than the second set of angularly deflected laser beams, combining the first set of angularly deflected laser beams with the second set of angularly deflected laser beams and directing the combined sets of laser beams onto a sample in a fluid stream, and detecting light from the sample. Systems having a laser, an acousto-optic device, and an optical conditioning assembly configured to generate a first set of angularly deflected laser beams and a second set of angularly deflected laser beams are also described.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 044,601, filed June 26, 2020, the disclosure of which is incorporated herein by reference. Background Technology

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

[0004] One technique for characterizing components in a sample using optical detection is flow cytometry. Using data generated from the detected light, the properties of the components can be recorded, and desired materials can be classified. A flow cytometer typically includes a sample container for receiving a fluid sample (e.g., blood) and a sheath fluid container to hold the sheath fluid. The flow cytometer transports particles (including cells) from the fluid sample into a flow cell as a cell stream, while simultaneously guiding the sheath fluid into the flow cell. Within the flow cell, a liquid sheath forms around the cell stream to impart a substantially uniform velocity to it. The flow cell hydrodynamically converges / centers the cells within the cell stream to pass through the center of the light source in the flow cell. The light from the light source can be detected as scattered light or by transmission spectroscopy, or it can be absorbed by one or more components in the sample and re-emitted as cold light / fluorescence. Summary of the Invention

[0005] This disclosure includes a method for generating an angle-deflecting laser beam to irradiate a sample in a fluid flow. According to some embodiments, the method includes: generating a first set of angle-deflecting laser beams and a second set of angle-deflecting laser beams; propagating the first set of angle-deflecting laser beams along an optical path different from that of the second set of angle-deflecting laser beams; combining the first set of angle-deflecting laser beams with the second set of angle-deflecting laser beams; directing the combined multiple sets of laser beams onto the sample in the fluid flow; and detecting the light from the sample. A system having a laser, an acousto-optic device, and an optical adjustment assembly configured to generate the first set of angle-deflecting laser beams and the second set of angle-deflecting laser beams is also described.

[0006] In embodiments implementing the subject methods, a plurality of angularly deflected laser beams are generated for illuminating a sample in a fluid stream. In some embodiments, the methods include generating a local oscillation beam and a plurality of radio frequency comb beams by irradiating an acousto-optic device with a laser. In some cases, the acousto-optic device is an acousto-optic deflector. In certain cases, the laser is a continuous wave laser. In certain embodiments, the plurality of sets of angularly deflected laser beams are generated by a single laser.

[0007] In some embodiments, the methods include generating a first set of angularly deflected laser beams including a first local oscillation (LO) beam and a first plurality of radio frequency comb beams and a second set of angularly deflected laser beams including a second local oscillation beam and a second plurality of radio frequency comb beams. In certain embodiments, the first set of angularly deflected laser beams and the second set of angularly deflected laser beams are generated by splitting an input one local oscillation beam and plurality of radio frequency comb beams into two different output beam sets, each output beam set having one local oscillation beam and plurality of radio frequency comb beams. In some embodiments, each local oscillation beam has a substantially constant intensity profile along a transverse axis (e.g., an axis orthogonal to a longitudinal axis of a fluid stream as described in more detail below). In some cases, the local oscillation beams have a top-hat intensity profile along the transverse axis. In certain cases, methods include generating a local oscillation beam having a constant intensity profile and propagating the local oscillation beam with each set of angularly deflected laser beams (e.g., using a beam splitter) such that the first set of angularly deflected laser beams and the second set of angularly deflected laser beams each include a local oscillation beam having a substantially constant intensity profile along a transverse axis. In some embodiments, the local oscillation beam of each set of angularly deflected laser beams has a Gaussian intensity profile along a longitudinal axis. In other embodiments, the local oscillation beam of each set of angularly deflected laser beams has a super-Gaussian intensity profile along a longitudinal axis. In some embodiments, the radio frequency comb beams of each set of angularly deflected laser beams have substantially the same intensity. In some cases, methods include modulating each radio frequency comb beam to have the same intensity. In embodiments, each angularly deflected laser beam is spatially separated along the transverse axis. In some cases, each angularly deflected laser beam in each output laser beam set at least partially overlaps another angularly deflected laser beam.

[0008] In embodiments, each set of angularly deflected laser beams propagates along a different optical path and is directed onto a fluid stream. In some embodiments, the sets of angularly deflected laser beams propagate along parallel optical paths onto the fluid stream. In some cases, an optical plane of the first set of angularly deflected laser beams is spatially separated from an optical plane of the second set of angularly deflected laser beams by a predetermined distance. In certain cases, methods include directing the first set of angularly deflected laser beams onto the fluid stream at a different spatial location than the second set of angularly deflected laser beams, e.g., where the two sets of angularly deflected laser beams illuminate different vertical locations of the fluid stream.

[0009] light from the sample in the fluid stream in response to illumination by the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In some embodiments, a first set of light signals is generated in response to illuminating the sample with the first set of angularly deflected laser beams, and a second set of light signals is generated in response to illuminating the sample with the second set of angularly deflected laser beams. In certain embodiments, light from the sample illuminated by the first set of angularly deflected laser beams is detected by a plurality of photomultiplier tubes and a first set of light signals is generated by the photomultiplier tubes, and light from the sample illuminated by the second set of angularly deflected laser beams is detected by a plurality of photodiodes (e.g., an array of avalanche photodiodes) and a second set of light signals is generated by the photodiodes. In some cases, the first set of light signals comprises time-domain fluorescence emission signals. In certain cases, the method comprises generating an image of particles in the sample based on the time-domain fluorescence emission signals. Generating an image according to certain embodiments includes, for example, frequency de-multiplexing each fluorescence emission signal by taking a Fourier transform of the fluorescence signal. In certain embodiments, the second set of light signals contains a spectrum from particles in the sample, and the method comprises determining a spectral abundance from particles in the sample based on the second set of light signals.

[0010] Aspects of the present disclosure also include systems for illuminating a sample in a fluid stream, where the subject system (e.g., a particle analyzer) includes a beam generator assembly for illuminating a sample in a fluid stream and a detection assembly for detecting light from the illuminated sample. In embodiments, the beam generator includes a laser, an acousto-optic device configured to generate an output laser beam comprising a plurality of angularly deflected laser beams in response to irradiation by the laser, a first optical conditioning assembly configured to generate two or more sets of angularly deflected laser beams, and a second optical conditioning assembly configured to direct each set of angularly deflected laser beams onto a sample in the fluid stream.

[0011] In some embodiments, the acousto-optic device is an acousto-optic deflector (AOD). In other embodiments, the acousto-optic device is an acousto-optic frequency shifter (AOFS). In yet other embodiments, the acousto-optic device is an acousto-optic modulator (AOM). In some cases, the beam generator includes a single laser, and the two or more sets of angularly deflected laser beams are generated by the single laser of the beam generator. In some cases, the laser is a continuous wave laser, such as a 488 nm continuous wave laser. In some embodiments, the beam generator is configured to generate one local oscillator beam and a plurality of radio frequency comb beams. In some cases, the beam generator is configured to generate a local oscillator beam having a substantially constant intensity profile along a transverse axis (e.g., an axis orthogonal to a longitudinal axis of the fluid flow). In certain cases, the local oscillator beam has a top-hat intensity profile along the transverse axis. In other cases, the beam generator is configured to generate a local oscillator beam having a Gaussian intensity profile along a longitudinal axis. In other embodiments, the local oscillator beam of each set of angularly deflected laser beams has a super-Gaussian intensity profile along the longitudinal axis. In some embodiments, the beam generator is configured to generate a plurality of radio frequency comb beams, each having substantially the same intensity. In embodiments, each angularly deflected laser beam generated by the beam generator is spatially separated along the transverse axis. In some cases, in each set of output laser beams, each angularly deflected laser beam at least partially overlaps another angularly deflected laser beam.

[0012] In some embodiments, the beam generator includes an optical conditioning assembly configured to generate a first set of angularly deflected laser beams and a second set of angularly deflected laser beams from an output of an acousto-optic device irradiated by a laser. In some cases, the generated first set of angularly deflected laser beams includes a first local oscillator (LO) beam and a first plurality of radio frequency comb beams, and the generated second set of angularly deflected laser beams includes a second local oscillator beam and a second plurality of radio frequency comb beams. In some embodiments, the optical conditioning assembly is a beam splitter configured to split an input one local oscillator beam and a plurality of radio frequency comb beams into two different output beam sets, each output beam set having one local oscillator beam and a plurality of radio frequency comb beams. In some embodiments, each local oscillator beam has a substantially constant intensity profile along a transverse axis. In some embodiments, the local oscillator beam of each set of angularly deflected laser beams has a Gaussian intensity profile along a longitudinal axis. In other embodiments, the local oscillator beam of each set of angularly deflected laser beams has a super-Gaussian intensity profile along the longitudinal axis. In some embodiments, the radio frequency comb beams of each set of angularly deflected laser beams have substantially the same intensity.

[0013] In some embodiments, the light beam generator comprises an optical conditioning assembly configured to direct the first set of angularly deflected laser beams and the second set of angularly deflected laser beams onto a sample in the fluid stream. In some cases, the optical conditioning assembly is configured to cause the first set of angularly deflected laser beams to propagate along a different optical plane than the second set of angularly deflected laser beams. In certain cases, the optical conditioning assembly is configured to direct the first set of angularly deflected laser beams to different spatial locations on the fluid stream than the second set of angularly deflected laser beams, e.g., different vertical locations on the fluid stream. In certain embodiments, the optical conditioning assembly is a telescopic lens system.

[0014] The system of the present disclosure comprises a light detection assembly for detecting light from the illuminated sample in the fluid stream. In some embodiments, the light detection assembly comprises a first set of photodetectors configured to detect light from the sample illuminated by the first set of angularly deflected laser beams and a second set of photodetectors configured to detect light from the sample illuminated by the second set of angularly deflected laser beams. In some embodiments, the photodetectors configured to detect light from the sample illuminated by the first set of angularly deflected laser beams comprise a plurality of photomultiplier tubes. In certain cases, the photodetectors configured to detect light from the sample illuminated by the second set of angularly deflected laser beams comprise a plurality of photodiodes. BRIEF DESCRIPTION OF DRAWINGS

[0015] The application can best be understood by reading the following detailed description in conjunction with the accompanying drawings. The following drawing(s) are included:

[0016] Figure 1 depicts a flowchart of illuminating a sample with a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, according to certain embodiments.

[0017] Figure 2 depicts a system for illuminating a sample in a fluid stream with a plurality of angularly deflected laser beams, according to certain embodiments.

[0018] Figure 3 depicts illuminating a fluid stream in a flow cell with two sets of angularly deflected laser beams, according to certain embodiments.

[0019] Figure 4A depicts a functional block diagram of a particle analysis system for computer-based sample analysis and particle characterization, according to certain embodiments. Figure 4B depicts a flow cytometer, according to certain embodiments.

[0020] Figure 5 depicts a functional block diagram of one example of a particle analyzer control system, according to certain embodiments.

[0021] Figure 6 A block diagram depicting a computing system in accordance with certain embodiments is depicted. DETAILED DESCRIPTION

[0022] Aspects of the present disclosure include methods for generating angularly deflected laser beams to illuminate a sample in a fluid stream. Methods in accordance with certain embodiments include generating a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, propagating the first set of angularly deflected laser beams along a different optical path than the second set of angularly deflected laser beams, combining the first set of angularly deflected laser beams with the second set of angularly deflected laser beams and directing the combined sets of laser beams onto a sample in a fluid stream, and detecting light from the sample. Systems having a laser, an acousto-optic device, and an optical conditioning assembly configured to generate a first set of angularly deflected laser beams and a second set of angularly deflected laser beams are also described.

[0023] Before the present application is described in detail, it is to be understood that this application is not limited to the particular embodiments described herein because variations of these particular embodiments can exist. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0024] 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. Unless otherwise stated, the lower limit of each range

[0025] Certain ranges presented herein are approximations. Unless otherwise indicated, the term "approximately" shall mean within 10% of the stated value. Unless otherwise indicated, the term "substantially" shall mean within 5% of the stated value. Unless otherwise indicated, the term "about" shall mean within 10% of the stated value.

[0026] 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 described below.

[0027] 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 the citation of any publication or patent is not to be construed as an admission that the inventors are not entitled to antedate the content of the publication or patent. The citation of any publication or patent is for the purpose of disclosure only, which purpose it is intended to serve only in the jurisdiction where the application is filed. Nothing contained in the citation is to be construed as an admission that the inventors are not entitled to antedate the content of the publication or patent.

[0028] It should be noted that, as used in this document and the appended claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the use of such exclusive terminology as "solely", "only" and "exclusively" in connection with claims recital elements.

[0029] It will be apparent to those skilled in the art that, upon reading the present disclosure, each individual embodiment described and listed herein has layered components and features that can be quickly broken down or merged with features of any of several other embodiments without departing from the scope and spirit of the present invention. Any recited method can be practiced in the order of recited events or in any other order that is logically possible.

[0030] As described above, the present disclosure provides methods of illuminating a sample in a fluid stream with angularly deflected laser beams. In further describing embodiments of the present disclosure, the methods of illuminating a fluid stream with angularly deflected laser beams are first described in more detail. Next, systems for implementing the described methods are also described, the systems having a beam generator assembly with a laser, an acousto-optic device, and an optical conditioning assembly for generating sets of angularly deflected laser beams, and a light detection assembly for detecting light from an illuminated sample in the fluid stream. Kits having one or more components of the subject systems are also provided.

[0031] Methods of illuminating a fluid stream with angularly deflected laser beams

[0032] Aspects of the present disclosure include methods for generating angularly deflected laser beams to irradiate a sample in a fluid stream. In implementing the methods according to certain embodiments, a first set of angularly deflected laser beams and a second set of angularly deflected laser beams are generated and caused to propagate along two different optical paths. The term "angularly deflected laser beam" as used herein refers to a laser beam that has been produced via the interaction of an acoustic wave produced in an acousto-optic device by an applied radio frequency drive signal with a beam from a laser to produce one or more sub-beams having a shift in the optical frequency and a propagation angle deflection, in accordance with its conventional meaning. Each set of angularly deflected laser beams includes a plurality of laser sub-beams, e.g., 3 or more sub-beams, 4 or more sub-beams, 5 or more sub-beams, 6 or more sub-beams, 7 or more sub-beams, 8 or more sub-beams, 9 or more sub-beams, 10 or more sub-beams, 12 or more sub-beams, 16 or more sub-beams, 24 or more sub-beams, including 48 or more sub-beams.

[0033] In embodiments, the methods include irradiating the acousto-optic device with a laser. The target laser can include a pulsed laser or a continuous wave laser. The type and number of lasers used in the methods 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 methods include irradiating the acousto-optic device with a dye laser, such as a diphenylstilbene, coumarin, or rhodamine laser. In yet other cases, the methods include irradiating the acousto-optic device 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 still other cases, the methods include irradiating the acousto-optic device 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 YAG thulium laser, a YAG ytterbium laser, a diytterbium oxide laser, or a cerium-doped laser, and combinations thereof. In still other cases, the methods include irradiating the acousto-optic device with a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a frequency-doubled or frequency-tripled implementation of any of the above lasers.

[0034] Depending on the desired wavelength of light generated in the output laser beam (e.g., for irradiating a sample in a fluid stream), each laser can have a different specific wavelength, with a range of wavelengths from 200 nm to 1500 nm, e.g., from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, and including from 400 nm to 800 nm. One or more lasers can be used to irradiate the acousto-optic device, e.g., 2 or more lasers, 3 or more lasers, 4 or more lasers, 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 irradiating the acousto-optic device with an array of lasers, e.g., an array having one or more gas lasers, one or more dye lasers, and one or more solid state lasers.

[0035] In cases where more than one laser is employed, the lasers can be used to irradiate the acousto-optic device simultaneously, or sequentially, or a combination of both. For example, the acousto-optic device can be irradiated simultaneously with each of the lasers. In other embodiments, the acousto-optic device is irradiated sequentially with each of the lasers. When more than one laser is employed to irradiate the acousto-optic device sequentially, each laser can irradiate the acousto-optic device for a time of 0.001 microseconds or more, e.g., 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 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 ranging from 0.001 microseconds to 100 microseconds, e.g., from 0.01 microseconds to 75 microseconds, from 0.1 microseconds to 50 microseconds, from 1 microsecond to 25 microseconds, and including from 5 microseconds to 10 microseconds. In embodiments where two or more lasers are used to irradiate the acousto-optic device sequentially, the duration of irradiation of the acousto-optic device by each laser can be the same or different.

[0036] The time interval between irradiation with each laser can also vary as desired, with the independently spaced delays being 0.001 microseconds or more, for example, 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 15 microseconds or more, 30 microseconds or more, including 60 microseconds or more. For example, the time interval between irradiation with each light source can range from 0.001 microseconds to 60 microseconds, for example, 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microsecond to 25 microseconds, including 5 microseconds to 10 microseconds. In certain embodiments, the time interval between irradiation with each laser is 10 microseconds. In embodiments in which more than two (i.e., 3 or more) lasers are used to sequentially irradiate the acousto-optic device, the delay between irradiation by each laser can be the same or different. In certain embodiments, a single laser is used to irradiate the acousto-optic device, and a single laser is used to generate both the first set of angularly deflected laser beams and the second set of angularly deflected laser beams.

[0037] The acousto-optic device can be irradiated continuously or at discrete intervals. In some cases, the method includes continuously irradiating the acousto-optic device with the laser. In other cases, the acousto-optic device is irradiated with the laser 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, including every 1000 milliseconds or some other interval.

[0038] The acousto-optic device can be irradiated from varying distances, depending on the laser, for example, the distance being 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, including 50 mm or more. In addition, the angle of irradiation can also vary, with the angle of irradiation ranging from 10° to 90°, for example, 15° to 85°, 20° to 80°, 25° to 75°, including 30° to 60°, for example, 90°.

[0039] In some embodiments, the method includes irradiating the acousto-optic device with a laser and generating a local oscillation beam and a plurality of radio frequency comb beams, e.g., where the generated angularly deflected laser beam includes the local oscillation beam and two or more radio frequency comb beams, e.g., 3 or more radio frequency comb beams, 4 or more radio frequency comb beams, 5 or more radio frequency comb beams, 6 or more radio frequency comb beams, 7 or more radio frequency comb beams, 8 or more radio frequency comb beams, 9 or more radio frequency comb beams, 10 or more radio frequency comb beams, 12 or more radio frequency comb beams, 16 or more radio frequency comb beams, 24 or more radio frequency comb beams, including 48 or more radio frequency comb beams and the local oscillation beam.

[0040] In some embodiments, the acousto-optic device irradiated with the laser is an acousto-optic deflector (AOD). In other embodiments, the acousto-optic device is an acousto-optic frequency shifter (AOFS). In other embodiments, the acousto-optic device is an acousto-optic modulator (AOM).

[0041] In some embodiments, the method includes applying a radio frequency drive signal to the acousto-optic device while irradiating the acousto-optic device to generate the 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, e.g., 3 or more radio frequency drive signals, 4 or more radio frequency drive signals, 5 or more radio frequency drive signals, 6 or more radio frequency drive signals, 7 or more radio frequency drive signals, 8 or more radio frequency drive signals, 9 or more radio frequency drive signals, 10 or more radio frequency drive signals, 15 or more radio frequency drive signals, 25 or more radio frequency drive signals, 50 or more radio frequency drive signals, including 100 or more radio frequency drive signals.

[0042] In certain embodiments, the angularly deflected laser beams produced by the radio frequency drive signals each have an intensity based on an amplitude of the applied radio frequency drive signal. In embodiments, the method includes applying radio frequency drive signals having an amplitude sufficient to produce angularly deflected laser beams having a desired intensity. In some embodiments, each applied radio frequency drive signal independently has an amplitude of about 0.001 V to about 500 V, for example, about 0.005 V to about 400 V, about 0.01 V to about 300 V, about 0.05 V to about 200 V, about 0.1 V to about 100 V, about 0.5 V to about 75 V, about 1 V to 50 V, about 2 V to 40 V, 3 V to about 30 V, 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, about 0.01 MHz to about 300 MHz, about 0.05 MHz to about 200 MHz, about 0.1 MHz to about 100 MHz, about 0.5 MHz to about 90 MHz, about 1 MHz to about 75 MHz, about 2 MHz to about 70 MHz, about 3 MHz to about 65 MHz, about 4 MHz to about 60 MHz, including about 5 MHz to about 50 MHz.

[0043] In certain cases, the method includes generating the plurality of angularly deflected frequency shifted beams by irradiating an acousto-optic device such as described in Diebold et al., published in Nature Photonics, Vol. 7(10), pp. 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.

[0044] In implementing the subject methods, the first set of angularly deflected laser beams and the second set of angularly deflected laser beams are generated from an output laser beam having one local oscillator beam and a plurality of radio frequency comb beams. In some embodiments, generating the first set of angularly deflected laser beams and the second set of angularly deflected laser beams includes optically splitting the output laser beam from the acousto-optic device into two different sets of angularly deflected laser beams. The output laser beam from the acousto-optic device having one local oscillator beam and a plurality of radio frequency comb beams can be split using any convenient optical conditioning scheme, such as with a beamsplitter. The term "beamsplitter" is used herein in its conventional sense to refer to an optical component configured to cause a light beam to propagate along two or more different, and spatially separated, optical paths such that a predetermined portion of the light propagates along each optical path. Any convenient beam splitting scheme can be employed, such as a triangular prism, a silver mirror prism, a dichroic mirror prism, and other types of beamsplitters. The beamsplitter can be formed from any suitable material, so long as the beamsplitter is capable of causing the desired amount and wavelength of light to propagate along each optical path. For example, beamsplitters of interest can be made from glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glass), silica (e.g., fused silica), quartz, crystals (e.g., CaF2crystals), zinc selenide (ZnSe), F2, germanium (Ge) titanates (e.g., S-TIH11), borosilicates (e.g., BK7). In certain embodiments, the beamsplitter is formed from a polymeric material, such as, but not limited to, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics, such as PETG (polyethylene terephthalate glycol), as well as other polymeric plastic materials.In certain embodiments, the beam splitter is formed from a polyester, where the polyester of interest can include, but is not limited to, poly(alkylene terephthalate)s, such as poly(ethylene terephthalate) (PET), bottle grade PET (copolymer made based on monoethylene glycol, terephthalic acid, and other comonomers such as isophthalic acid, cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT), and poly(hexamethylene terephthalate); poly(alkylene adipate)s, such as poly(ethylene adipate), poly(l,4-butylene adipate), and poly(hexamethylene adipate); poly(alkylene suberate)s, such as poly(ethylene suberate); poly(alkylene sebacate)s, such as poly(ethylene sebacate); poly(e-caprolactone) and poly(beta-propiolactone); poly(alkylene isophthalate)s, such as poly(ethylene isophthalate); poly(2,6- naphthalene dicarboxylic acid alkylene ester), such as poly(ethylene 2,6-naphthalate); poly(alkylene sulfonyl-4,4'-dibenzoate), such as poly(ethylene sulfonyl-4,4'-dibenzoate); poly(p-phenylene alkylene dicarboxylate), such as poly(p-phenylene ethylene dicarboxylate); poly(trans-1,4-cyclohexane diyl alkylene dicarboxylate), such as poly(trans-1,4-cyclohexane diyl ethylene dicarboxylate); poly(l,4-cyclohexane-diyl alkylene dicarboxylate), such as poly(l,4-cyclohexane-diyl methylene ethylene dicarboxylate); poly([2.2.2]-bicyclooctane-l,4-alkylene dicarboxylate), such as poly([2.2.2]-bicyclooctane-l,4-ethylene dicarboxylate); lactic acid polymers and copolymers, such as (S)-polylactide, (R,S)-polylactide, poly(tetramethylglycolide), and poly(lactide-co-glycolide); polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A; polyamides, such as poly(paraphenylene terephthalamide); polyethylene terephthalate (e.g., Mylar™ polyethylene terephthalate); combinations thereof; and the like.

[0045] In certain embodiments, the optical conditioning component is a wedge beam splitter. In these embodiments, the beam splitter is a beam splitter having a wedge angle that produces non-collinear back reflections, such that the propagation of the collected light through the wedge beam splitter causes a small change in the angle of the light propagating to one or more of the light scatter detector and the brightfield photodetector. The wedge beam splitter according to embodiments of the present disclosure has a wedge angle in which the change in the angle of incidence of the collected light results in a deviation in the angle of the propagating light that is 0.001% or more, for example, 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 5% or more, including 10% or more. In some embodiments, the wedge angle of the wedge beam splitter is 5 arcminutes to 120 arcminutes, for example, 10 arcminutes to 115 arcminutes, 15 arcminutes to 110 arcminutes, 20 arcminutes to 105 arcminutes, 25 arcminutes to 100 arcminutes, 30 arcminutes to 105 arcminutes, 35 arcminutes to 100 arcminutes, 40 arcminutes to 95 arcminutes, including from 45 arcminutes to 90 arcminutes.

[0046] In some embodiments, the wedge beam splitter has a pass window of 150 nm to 5 pm, 180 nm to 8 pm, 185 nm to 2.1 pm, 200 nm to 6 pm, 200 nm to 11 pm, 250 nm to 1.6 pm, 350 nm to 2 pm, 600 nm to 16 pm, 1.2 pm to 8 pm, 2 pm to 16 pm, or some other wavelength range.

[0047] The target beam splitter is configured to split the angularly deflected laser beam output from the acousto-optic device into two different sets of angularly deflected laser beams. In some embodiments, the split beam ratio between the first set of angularly deflected laser beams and the second set of angularly deflected laser beams of the beam splitter is from 1:99 to 99:1, for example, 5:95 to 95:5, 10:90 to 90:10, 20:80 to 80:20, 25:75 to 75:25, including a split ratio of 50:50. In certain embodiments, the beam splitter is a 50:50 beam splitter in which the first set of angularly deflected laser beams is identical to the second set of angularly deflected laser beams (e.g., the first set of angularly deflected laser beams has the same amplitude and frequency as the second set of angularly deflected laser beams).

[0048] In some embodiments, each of the first and second sets of generated angularly deflected laser beams includes one local oscillation beam and a plurality of radio frequency comb beams. For example, each set of generated angularly deflected laser beams can include one local oscillation beam and two or more radio frequency comb beams, e.g., 3 or more radio frequency comb beams, 4 or more radio frequency comb beams, 5 or more radio frequency comb beams, 6 or more radio frequency comb beams, 7 or more radio frequency comb beams, 8 or more radio frequency comb beams, 9 or more radio frequency comb beams, 10 or more radio frequency comb beams, 12 or more radio frequency comb beams, 16 or more radio frequency comb beams, 24 or more radio frequency comb beams, can include one local oscillation beam and 48 or more radio frequency comb beams.

[0049] In embodiments, the radio frequency comb beams in each set of generated angularly deflected laser beams are spatially separated. Depending on the radio frequency drive signal applied to the acousto-optic device, the angularly deflected laser beams can be separated by 0.001 pm or more, e.g., 0.005 pm or more, 0.01 pm or more, 0.05 pm or more, 0.1 pm or more, 0.5 pm or more, 1 pm or more, 5 pm or more, 10 pm or more, 100 pm or more, 500 pm or more, 1000 pm or more, including 5000 pm or more. In some embodiments, one or more radio frequency comb beams overlap, e.g., overlap with an adjacent laser beam along a transverse axis of each set of angularly deflected laser beams. The overlap, e.g., the overlap of the spots, between adjacent angularly deflected laser beams can be an overlap of 0.001 pm or more, e.g., an overlap of 0.005 pm or more, an overlap of 0.01 pm or more, an overlap of 0.05 pm or more, an overlap of 0.1 pm or more, an overlap of 0.5 pm or more, an overlap of 1 pm or more, an overlap of 5 pm or more, an overlap of 10 pm or more, including an overlap of 100 pm or more.

[0050] In some embodiments, the local oscillation beams of each set of angularly deflected laser beams have a substantially constant intensity profile along the lateral axis. In some cases, the local oscillation beams of each set of angularly deflected laser beams have a beam profile with substantially constant intensity from each edge to the center, e.g., the intensity of the beam profile across the lateral axis differs by 10% or less, e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, including cases where the intensity of the beam profile across the lateral axis differs by 0.001% or less. In some embodiments, the local oscillation beams of each angularly deflected laser beam have a top-hat intensity profile along the lateral axis. In accordance with the conventional meaning, the term “top-hat” refers to an irradiation beam having a substantially uniform fluence (energy density) along one or more axes orthogonal to the optical axis of the irradiation beam. In embodiments, an output beam having a top-hat intensity profile exhibits little deviation in relative intensity from each edge to the center along the lateral axis, where along the lateral axis, the beam having the target top-hat intensity profile has an intensity at the center that is 95% to 99.9% of the intensity at the edges, e.g., 96% to 99.5%, including 98% to 99% of the intensity at the edges along the lateral axis.

[0051] In other embodiments, the local oscillation beams of each set of angularly deflected laser beams have a super-Gaussian intensity profile along the lateral axis. In accordance with the conventional meaning, the term “super-Gaussian” as used herein refers to an irradiation beam having an energy density along one or more axes orthogonal to the optical axis of the irradiation beam where the energy density is slightly higher at the center of the beam profile. In embodiments, a local oscillation beam having a super-Gaussian intensity profile exhibits a higher fluence at the edges of the beam along the lateral axis compared to a corresponding Gaussian intensity profile. In an example, the intensity of the beam having a super-Gaussian intensity profile at the edges along the lateral axis is 70%-90%, e.g., 75%-85%, of the intensity of the beam at the center, including where the intensity at the edges along the lateral axis is 80%-90% of the intensity of the beam at the center.

[0052] In some embodiments, two or more of the plurality of radio frequency comb beams in each set of angularly deflected laser beams have substantially the same intensity along the transverse axis. Depending on the number of radio frequency comb beams in each set of angularly deflected laser beams, two or more of the radio frequency comb beams can have the same intensity, for example, where 3 or more radio frequency comb beams have the same intensity, 4 or more radio frequency comb beams have the same intensity, 5 or more radio frequency comb beams have the same intensity, 6 or more radio frequency comb beams have the same intensity, 7 or more radio frequency comb beams have the same intensity, 8 or more radio frequency comb beams have the same intensity, 9 or more radio frequency comb beams have the same intensity, 10 or more radio frequency comb beams have the same intensity, 12 or more radio frequency comb beams have the same intensity, 16 or more radio frequency comb beams have the same intensity, 24 or more radio frequency comb beams have the same intensity, including cases where 48 or more radio frequency comb beams have the same intensity.

[0053] In other embodiments, the number of radio frequency comb beams in each set of angularly deflected laser beams that have different intensities can vary, for example, where 2 or more radio frequency comb beams have different intensities, 3 or more radio frequency comb beams have different intensities, 4 or more radio frequency comb beams have different intensities, 5 or more radio frequency comb beams have different intensities, 6 or more radio frequency comb beams have different intensities, 7 or more radio frequency comb beams have different intensities, 8 or more radio frequency comb beams have different intensities, 9 or more radio frequency comb beams have different intensities, 10 or more radio frequency comb beams have different intensities, 12 or more radio frequency comb beams have different intensities, 16 or more radio frequency comb beams have different intensities, 24 or more radio frequency comb beams have different intensities, including cases where 48 or more angularly deflected laser beams have different intensities.

[0054] In certain embodiments, the method further comprises determining the intensity profile of one or more of the radio frequency comb beams of the first and second sets of angularly deflected laser beams, for example, along the transverse axis or the longitudinal axis. The intensity profile can be measured using any convenient scheme, including but not limited to a scanning slit analyzer, a charge-coupled device (CCD, for example, an intensified charge-coupled device, ICCD), a position sensor, a power sensor (for example, a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, and other types of photodetectors. In some cases, to determine the intensity profile of a radio frequency comb beam, the relative intensity of each radio frequency comb beam is plotted as a function of the distance from the optical axis of the output beam (along the orthogonal transverse axis) to determine the intensity profile at the irradiance point. In certain embodiments, the relative intensity deviation at a predetermined distance from the optical axis is calculated to determine whether the beam profile of the output beam exhibits substantially constant intensity from each edge to the center along the transverse axis. In other embodiments, the relative intensity deviation of the beam profile of the output beam is calculated across the entire transverse axis to determine whether the output beam exhibits substantially constant intensity from each edge to the center.

[0055] In some embodiments, the intensity profile of the radio frequency comb beams of each set of angularly deflected laser beams is determined by capturing an image of the output laser beam. For example, the method can comprise capturing 2 or more images of the output laser beam to determine the intensity profile of the output laser beam along the transverse axis, for example, 3 or more images, 4 or more images, 5 or more images, 6 or more images, 7 or more images, 8 or more images, 9 or more images, 10 or more images, 25 or more images, 50 or more images, including capturing 100 or more images of the output laser beam to determine the intensity profile of the output laser beam along the transverse axis. Where more than one image is captured, the multiple images can be automatically stitched together by a processor having a digital image processing algorithm.

[0056] The image of the output laser beam from the radio frequency comb beams of each set of angularly deflected laser beams can be captured with any suitable device capable of capturing an optical image and converting it into an electronic data signal, including but not limited to a charge-coupled device, a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or a N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the imaging sensor is a CCD camera. For example, the camera can be an electron-multiplying CCD (EMCCD) camera or an intensified CCD (ICCD) camera. In other embodiments, the imaging sensor is a CMOS-type camera.

[0057] In certain embodiments, the method includes determining and adjusting the beam profile of the radio frequency comb beams of each set of angularly deflected laser beams, such as described in U.S. Provisional Patent Application No. 63 / 027,080, filed May 19, 2020, the disclosure of which is incorporated herein by reference.

[0058] In implementing the subject method, the first set of angularly deflected laser beams and the second set of angularly deflected laser beams are optically combined. In some embodiments, the first set of angularly deflected laser beams and the second set of angularly deflected laser beams are combined and propagate along a single optical path, such as where one or more beams from the first set of angularly deflected laser beams and the second set of angularly deflected laser beams overlap. For example, two or more of the angularly deflected laser beams (e.g., radio frequency comb beams) of the first and second sets of angularly deflected laser beams can overlap, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 24 or more, including 48 or more of the angularly deflected laser beams of the first and second sets of angularly deflected laser beams can overlap.

[0059] In other embodiments, the first set of angularly deflected laser beams and the second set of angularly deflected laser beams are combined and propagate along two parallel optical paths, such as where there is no overlap between the beams from the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In this embodiment, the optical path of the first set of angularly deflected laser beams is spatially separated from the second set of angularly deflected laser beams. In some cases, the optical path of the first set of angularly deflected laser beams is separated from the second set of angularly deflected laser beams by 0.00001 mm or more, such as 0.00005 mm or more, 0.0001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, including 2 mm or more. In certain embodiments, the first set of angularly deflected laser beams is combined and propagates along a different parallel optical plane than the second set of angularly deflected laser beams. In certain cases, the optical plane of the first set of angularly deflected laser beams is spatially separated from the optical plane of the second set of angularly deflected laser beams by 0.00001 mm or more, such as 0.00005 mm or more, 0.0001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, including 2 mm or more.

[0060] In embodiments, the first set of angularly deflected laser beams and the second set of angularly deflected laser beams are directed to irradiate the fluid stream. In some embodiments, the method comprises irradiating the same location on the fluid stream with the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In other embodiments, the method comprises irradiating a location on the fluid stream with the first set of angularly deflected laser beams that overlaps with a location on the fluid stream irradiated with the second set of angularly deflected laser beams. For example, the location on the fluid stream irradiated with the first set of angularly deflected laser beams can overlap the location on the fluid stream irradiated with the second set of angularly deflected laser beams by 0.00001 pm or more, for example, 0.00005 pm or more, 0.0001 pm or more, 0.005 pm or more, 0.01 pm or more, 0.05 pm or more, 0.1 pm or more, 0.5 pm or more, 1 pm or more, 10 pm or more, 50 pm or more, 100 pm or more, including 1000 pm or more.

[0061] In other embodiments, the method comprises irradiating a first location on the fluid stream with the first set of angularly deflected laser beams and a second location on the fluid stream with the second set of angularly deflected laser beams. For example, the method can comprise irradiating a different vertical location on the fluid stream with the first set of angularly deflected laser beams than the location irradiated with the second set of angularly deflected laser beams. Depending on the flow rate of the fluid stream, the method can comprise irradiating the fluid stream with the second set of angularly deflected laser beams at a location 0.001 pm or more downstream of the irradiation location of the first set of angularly deflected laser beams, for example, 0.005 pm or more, 0.01 pm or more, 0.05 pm or more, 0.1 pm or more, 0.5 pm or more, 1 pm or more, 5 pm or more, 10 pm or more, 100 pm or more, 250 pm or more, 500 pm or more, including 1000 pm or more downstream of the irradiation location of the first set of angularly deflected laser beams.

[0062] The methods of the present disclosure further comprise detecting light from the irradiated sample in the fluid stream. Suitable light detection schemes include, but are not limited to, optical sensors or 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, and combinations thereof, as well as other photodetectors.

[0063] The light signal from the fluid stream can be measured at one or more wavelengths, e.g., at 2 or more wavelengths, at 5 or more different wavelengths, at 10 or more different wavelengths, at 25 or more different wavelengths, at 50 or more different wavelengths, at 100 or more different wavelengths, at 200 or more different wavelengths, at 300 or more different wavelengths, including at 400 or more different wavelengths. In some embodiments, the method includes measuring light over a range of wavelengths (e.g., 200 nm - 1000 nm). For example, the method can include collecting a spectrum over one or more wavelength ranges in the 200 nm - 1000 nm wavelength range. In yet other embodiments, the method includes measuring light from the fluid stream at one or more specific wavelengths. For example, the light can be measured at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, the method includes measuring light at a wavelength corresponding to a fluorescence peak wavelength of certain fluorophores.

[0064] The light from the fluid stream can be measured continuously or at discrete intervals. In some cases, the method includes measuring the light continuously. In other cases, the light is measured at discrete intervals, e.g., every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds or some other interval. During the subject method, the light can be measured one or more times, e.g., 2 or more times, 3 or more times, 5 or more times, including 10 or more times. In certain embodiments, the light propagation is measured two or more times, in some cases, the data is averaged.

[0065] In some embodiments, the method includes detecting light from the sample illuminated by the first set of angularly deflected laser beams with a first set of photodetectors and detecting light from the sample illuminated by the second set of angularly deflected laser beams with a second set of photodetectors. In some cases, the first set of photodetectors includes a plurality of photomultiplier tubes, e.g., 2 or more photomultiplier tubes, 3 or more photomultiplier tubes, 4 or more photomultiplier tubes, 5 or more photomultiplier tubes, 6 or more photomultiplier tubes, 7 or more photomultiplier tubes, 8 or more photomultiplier tubes, 9 or more photomultiplier tubes, 10 or more photomultiplier tubes, 12 or more photomultiplier tubes, including 16 or more photomultiplier tubes. In other cases, the first set of photodetectors includes a plurality of photodiodes, e.g., 2 or more photodiodes, 3 or more photodiodes, 4 or more photodiodes, 5 or more photodiodes, 6 or more photodiodes, 7 or more photodiodes, 8 or more photodiodes, 9 or more photodiodes, 10 or more photodiodes, 12 or more photodiodes, including 16 or more photodiodes.

[0066] In embodiments, the method includes generating light signals with a first set of photodetectors and generating light signals with a second set of photodetectors. In some cases, the generated light signals include frequency-encoded fluorescence data from particles (e.g., cells) in the fluid stream. In certain cases, the frequency-encoded fluorescence data from the particles in the fluid stream is transformed to give spatial data for the particles. In some embodiments, the spatial data includes a horizontal dimension of the particles, a vertical dimension of the particles, a ratio of the particle dimensions along two different dimensions, a ratio of dimensions of components of the particles (e.g., a ratio of the horizontal dimension of the nucleus to the horizontal dimension of the cytoplasm).

[0067] In some embodiments, the frequency-encoded fluorescence data is transformed by a Fourier transform of the frequency-encoded fluorescence data. In some cases, the frequency-encoded fluorescence data is transformed by a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data. In other cases, the spatial data is computed by performing a short-time Fourier transform (STFT) on the frequency-encoded fluorescence data. In yet other cases, the spatial data is computed with a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data. In certain embodiments, the method further includes computing the spatial data by performing the transformation of the frequency-encoded fluorescence data with a phase correction component, e.g., as described in U.S. Patent Application No. 16 / 887,538, filed May 29, 2020, the disclosure of which is incorporated herein by reference.

[0068] In some embodiments, the method includes generating an image of the particles from the frequency-encoded fluorescence in the fluid stream. In some embodiments, the image of the particles can be generated from the frequency-encoded fluorescence in combination with detected light absorption, detected light scattering, or a combination thereof. In some cases, the image of the particles is generated from the frequency-encoded fluorescence alone. In other cases, the image of the object is generated from the frequency-encoded fluorescence and from light absorption detected from the sample (e.g., from a brightfield light detector). In yet other cases, the image of the particles is generated using the frequency-encoded fluorescence and light scattering detected from the sample (e.g., from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector). In still other cases, the image of the particles is generated from the frequency-encoded fluorescence in combination with detected light absorption, detected light scattering, and detected light emission. In still other cases, the image of the particles is generated from the frequency-encoded fluorescence and spectrally resolved light from a second set of photodetectors, as described in more detail below.

[0069] One or more images of the particles can be generated based on the light signals detected from one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In some embodiments, a single image of the particles is generated. In other embodiments, two or more images of the particles are generated, e.g., 3 or more, 4 or more, 5 or more, including 10 or more images generated based on the light signals detected from one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams.

[0070] In some embodiments, light from the sample illuminated by the second set of angularly deflected laser beams is detected by a second set of photodetectors. In some cases, the second set of photodetectors includes a plurality of photodiodes, e.g., a plurality of avalanche photodiodes. For example, the second set of photodetectors can include 2 or more photodiodes, e.g., 3 or more photodiodes, 4 or more photodiodes, 5 or more photodiodes, 6 or more photodiodes, 7 or more photodiodes, 8 or more photodiodes, 9 or more photodiodes, 10 or more photodiodes, 12 or more photodiodes, including 16 or more photodiodes. In other cases, the second set of photodetectors includes a plurality of photomultiplier tubes, e.g., 2 or more photomultiplier tubes, 3 or more photomultiplier tubes, 4 or more photomultiplier tubes, 5 or more photomultiplier tubes, 6 or more photomultiplier tubes, 7 or more photomultiplier tubes, 8 or more photomultiplier tubes, 9 or more photomultiplier tubes, 10 or more photomultiplier tubes, 12 or more photomultiplier tubes, including 16 or more photomultiplier tubes.

[0071] In some embodiments, the method includes detecting light from a sample in a fluid stream illuminated with one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams with a cluster wave division optical detection system having a wavelength separator for distinguishing light from the sample into a plurality of predetermined spectral ranges. In some cases, the method includes wavelength division multiplexing, in which light from the fluid stream illuminated with one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams is detected by a plurality of photodetectors (e.g., a plurality of avalanche photodiodes). For example, each photodetector in the set of photodetectors can be configured to detect one or more sets of predetermined wavelengths of light from the sample in the fluid stream. In these embodiments, data signals generated from the light of the predetermined wavelength sets from the plurality of photodetectors are multiplexed, and the wavelength division multiplexed data signals are output to the processor. For example, the wavelength division multiplexed data signals can include data signals generated from 2 or more different sets of predetermined wavelengths of light, e.g., 3 or more sets, 4 or more sets, 5 or more sets, 6 or more sets, 7 or more sets, 8 or more sets, 9 or more sets, 10 or more sets, 11 or more sets, including wavelength division multiplexed data signals including data signals generated from 12 or more different sets of predetermined wavelengths of light. In certain embodiments, the method includes generating wavelength division multiplexed data signals including data signals from 2 or more different spectra detected by the photodetectors, e.g., 3 or more, 4 or more, 5 or more, e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, including generating wavelength division multiplexed data signals including data signals from 12 or more different spectra detected by the photodetectors.

[0072] In some embodiments, the method includes spectrally resolving light detected from the sample in the fluid stream illuminated with one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In certain embodiments, the overlapping spectral components of the light are determined by computing a spectral unmixing matrix. In some embodiments, the spectrum of the light detected by each photodetector overlaps the spectrum of the light detected by at least one other detector of the plurality of photodetectors. In some cases, the spectrum of the light detected by one photodetector overlaps the spectrum of the light of at least one other detector by 5 nm or more, for example, 10 nm or more, 25 nm or more, including 50 nm or more. In certain cases, the spectrum of the light detected by one photodetector overlaps the spectrum of two or more other photodetectors, for example, where each overlap is 5 nm or more, for example, 10 nm or more, 25 nm or more, including 50 nm or more. In other embodiments, the spectrum of the light detected by a photodetector has a non-overlapping spectrum. In these embodiments, the spectrum of the light detected by each photodetector is adjacent to the spectrum of at least one other photodetector of the second set of photodetectors by 10 nm or less, 9 nm or less, 8 nm or less, 7 nm or less, 6 nm or less, 5 nm or less, 4 nm or less, 3 nm or less, 2 nm or less, including 1 nm or less.

[0073] In some embodiments, the method includes determining the overlap of the spectra of the light from the fluid stream and computing the contribution of each spectrum to the detected spectrum of the overlap. In some embodiments, spectrally resolving the light includes computing a spectral unmixing matrix. In certain embodiments, the method includes computing the spectral unmixing matrix to estimate the abundance of each contribution to the photodetector-detected light signal.

[0074] In some cases, computing the spectral unmixing matrix includes determining the abundance of a fluorophore associated with a particle in the fluid stream. The abundance of each fluorophore associated with a particle can be used to identify and classify the particle. In some cases, the identified or classified particles can be used to sort target particles (e.g., cells) in the sample. In certain embodiments, the computation of the spectral unmixing is performed such that the sorting is fast enough to sort the particles in real-time after detection by the light detection system.

[0075] In certain embodiments, the method includes spectrally resolving light detected by the plurality of photodetectors, for example as described in International Patent Application No. PCT / US2019 / 068395, filed December 23, 2019; U.S. Provisional Patent Application No. 62 / 971,840, filed February 7, 2020; and U.S. Provisional Patent Application No. 63 / 010,890, filed April 16, 2020, the disclosures of which are incorporated by reference herein in their entireties. For example, spectrally resolving light detected by the plurality of photodetectors can include solving a spectral unmixing matrix using one or more of: 1) a weighted least squares algorithm; 2) a Sherman-Morrison iterative inverse updater; 3) an LU matrix decomposition, for example, factoring a matrix into a product of a lower triangular (L) matrix and an upper triangular (U) matrix; 4) a modified Cholesky decomposition; 5) a weighted least squares algorithm computed by QR factorization; and 6) a weighted least squares algorithm computed by singular value decomposition.

[0076] Figure 1 A flowchart depicting irradiating a sample with a first set of angularly deflected laser beams and a second set of angularly deflected laser beams is depicted in accordance with certain embodiments. At step 101, an acousto-optic device (e.g., an acousto-optic deflector) is irradiated with a laser to generate an output laser beam having one local oscillation beam and a plurality of radio frequency comb beams. To generate the plurality of angularly deflected laser beams, a waveform of each angularly deflected laser beam is input from a waveform generator into the acousto-optic device. At step 102, the output laser beam is split (e.g., with a beam splitter) into a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, and each set of angularly deflected laser beams is caused to propagate along different optical paths at step 103. At step 104, the sets of laser beams are combined (e.g., with a mirror and a telescopic lens system) and directed to irradiate a fluid stream. The first set of angularly deflected laser beams is configured to irradiate different locations on the fluid stream (e.g., different vertical locations along a longitudinal axis of the fluid stream) than the second set of angularly deflected laser beams. At step 105, light from particles irradiated by the first set of angularly deflected laser beams is detected with a first set of photodetectors (e.g., a plurality of photomultiplier tubes). At step 105a, an image of the particles is generated based on data signals generated by the first set of photodetectors. At step 106, light from particles irradiated by the second set of angularly deflected laser beams is detected with a second set of photodetectors (e.g., a plurality of photodiodes), and at step 106a, the light from the particles is spectrally resolved based on data signals from the second set of photodetectors.

[0077] As described above, the methods include illuminating a sample in a fluid stream (e.g., in a particle analyzer of a flow cytometer) with a first set of angularly deflected laser beams and a second set of angularly deflected laser beams. In some embodiments, the sample is a biological sample. The term "biological sample" refers to a whole organism, a subset of plant, fungal, or animal tissue, a cell, or a constituent part, in some cases, that can be found in blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen, in accordance with its conventional meaning. Thus, a "biological sample" refers both to a natural organism or a subset of its tissue, as well as to a homogenate, lysate, or extract prepared from the organism or a subset of its tissue, including but not limited to: blood plasma; serum; spinal fluid; lymphatic fluid; skin, respiratory, gastrointestinal, cardiovascular, and genitourinary tract sections; tears; saliva; milk; blood cells; tumors; organs; and the like. The biological sample can be any type of biological tissue, including healthy tissue and pathological 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 derivative thereof (e.g., plasma), or other biological fluid sample, e.g., tears, urine, semen, etc., where in certain cases the sample is a blood sample, including a whole blood sample, e.g., blood taken by venipuncture or finger prick (where the blood can or can not be mixed with any reagents (e.g., preservatives, anticoagulants, etc.) prior to analysis).

[0078] In certain embodiments, the sample is derived from a "mammal," a term that is used broadly herein to describe organisms that belong to the class Mammalia, including, without limitation, humans, other primates, dogs, cats, mice, rats, and the like. In certain cases, the subject is a human. The methods can be applied to samples taken from male and female subjects at any stage of development (i.e., neonates, infants, juveniles, adolescents, adults), where in certain embodiments the human subject is a juvenile, adolescent, or adult. While embodiments of the disclosure can be applied to samples taken from human subjects, it will be appreciated that the methods can also be applied to samples taken from other animal subjects (i.e., in "non-human subjects"), including, without limitation, birds, mice, rats, dogs, cats, livestock, and horses.

[0079] In certain embodiments, the biological sample contains cells. Cells that can be present in the sample include eukaryotic cells (e.g., mammalian cells) and / or prokaryotic cells (e.g., bacterial cells or archaeal cells). The sample can be obtained from an ex vivo source (e.g., a cell suspension derived from laboratory cells grown in culture) or an in vivo source (e.g., a mammalian subject, a human subject, etc.). In some embodiments, the cell sample is obtained from an ex vivo source. Ex vivo sources include, but are not limited to, prokaryotic (e.g., bacterial, archaeal) cell cultures, environmental samples containing prokaryotic and / or eukaryotic (e.g., mammalian, protist, fungal, etc.) cells, eukaryotic cell cultures (e.g., established cell line cultures, known or purchased cell line cultures, immortalized cell line cultures, primary cell cultures, laboratory yeast cultures, etc.), tissue cultures, etc.

[0080] Where the biological sample includes cells, the methods of the application can include characterizing components of the cells, e.g., cell debris, disrupted cell membranes, organelles, dead or lysed cells. In some embodiments, the methods include characterizing extracellular vesicles of the cells. Characterizing extracellular vesicles of the cells can include identifying a type of extracellular vesicle in the cells, or determining a size of extracellular vesicles in the cells.

[0081] The sample in the fluid stream can be illuminated with one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams either continuously or at discrete intervals. In some cases, the methods include illuminating the sample in the fluid stream with one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams continuously. In other cases, the sample in the fluid stream is illuminated with one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams at discrete intervals, e.g., every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds, or some other interval.

[0082] The sample in the fluid stream can be illuminated with the first set of angularly deflected laser beams and the second set of angularly deflected laser beams from varying distances, e.g., the distances are 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, 1 mm or greater, 2.5 mm or greater, 5 mm or greater, 10 mm or greater, 15 mm or greater, 25 mm or greater, including 50 mm or greater. In addition, the angle of illumination can also vary, ranging from 10° to 90°, 15° to 85°, 20° to 80°, 25° to 75°, including 30° to 60°, e.g., at a 90° angle.

[0083] The flow rate of the fluid stream can vary, e.g., depending on the intensity of the light and can be 1 pL / min or more, e.g., 2 pL / min or more, 3 pL / min or more, 5 pL / min or more, 10 pL / min or more, 25 pL / min or more, 50 pL / min or more, 75 pL / min or more, 100 pL / min or more, 250 pL / min or more, 500 pL / min or more, 750 pL / min or more, including 1000 pL / min or more. In certain embodiments, the flow rate of the fluid stream in the subject methods ranges from 1 pL / min to 500 pL / min, e.g., from 1 pL / min to 250 pL / min, from 1 pL / min to 100 pL / min, from 2 pL / min to 90 pL / min, from 3 pL / min to 80 pL / min, from 4 pL / min to 70 pL / min, from 5 pL / min to 60 pL / min, including from 10 pL / min to 50 pL / min. In certain embodiments, the flow rate of the fluid stream is from 5 pL / min to 6 pL / min.

[0084] The methods in certain embodiments further include data acquisition, analysis, and recording, such as using a computer, wherein multiple data channels record data generated as the sample passes through the detection region of the system. In these embodiments, analysis can include classification and counting of cells or cellular components (extracellular vesicles) such that each component exists as a set of digitalized parameter values. The subject system can be set to trigger at selected parameters in order to distinguish particles of interest from background and noise. “Trigger” refers to a preset threshold for a detection parameter and can be used as a means to detect passage of a component of interest through the detection region. Detection of an event that exceeds the selected parameter threshold triggers acquisition of data for the sample component. No data acquisition is performed for components in the medium being analyzed that elicit a response below the threshold.

[0085] In some embodiments, the method further comprises sorting one or more particles (e.g., cells) of the sample. The term “sorting” is used herein in its conventional sense to refer to separating components (e.g., cells, non-cellular particles such as biological macromolecules) of a sample and, in some cases, delivering the separated components to one or more sample collection vessels. For example, the method can comprise sorting a sample having 2 or more components, e.g., 3 or more components, 4 or more components, 5 or more components, 10 or more components, 15 or more components, including sorting a sample having 25 or more components. One or more of the sample components can be separated from the sample and delivered to a sample collection vessel, e.g., 2 or more sample components, 3 or more sample components, 4 or more sample components, 5 or more sample components, 10 or more sample components, including 15 or more sample components can be separated from the sample and delivered to a sample collection vessel.

[0086] In some embodiments, methods for sorting sample components include sorting particles (e.g., cells in a biological sample), e.g., as described in U.S. Pat. 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 method comprises sorting components of a sample using a particle sorting module, e.g., those described in U.S. Pat. Nos. 9,551,643 and 10,324,019, U.S. Pat. Pub. No. 2017 / 0299493, and International Pat. Pub. No. WO / 2017 / 040151, the disclosures of which are incorporated herein by reference. In certain embodiments, cells of a sample are sorted using a sorting decision module having a plurality of sorting decision units, e.g., those described in U.S. Pat. App. No. 16 / 725,756, filed December 23, 2019, the disclosure of which is incorporated herein by reference.

[0087] System for irradiating a fluid stream with an angularly deflected laser beam

[0088] As described above, aspects of the present disclosure include systems for generating angularly deflected laser beams to illuminate a sample in a fluid stream. A system according to certain embodiments includes a beam generator having a laser, an acousto-optic device configured to generate an output laser beam having a plurality of angularly deflected laser beams in response to irradiation of the laser, a first optical conditioning assembly configured to generate a first set of angularly deflected laser beams and a second set of angularly deflected laser beams from the output laser beam, and a second optical conditioning assembly configured to direct the first set of angularly deflected laser beams and the second set of angularly deflected laser beams onto a sample in a fluid stream. As described above, the angularly deflected laser beams generated by the systems described herein are laser beams that are produced via the interaction of an acoustic wave produced in an acousto-optic device by an applied radio frequency drive signal with a light beam from a laser, thereby producing one or more sub-beams having a light frequency shift and a propagation angular deflection. Each set of angularly deflected laser beams includes a plurality of laser sub-beams, e.g., 3 or more sub-beams, 4 or more sub-beams, 5 or more sub-beams, 6 or more sub-beams, 7 or more sub-beams, 8 or more sub-beams, 9 or more sub-beams, 10 or more sub-beams, 12 or more sub-beams, 16 or more sub-beams, 24 or more sub-beams, including 48 or more sub-beams.

[0089] In embodiments, the beam generator comprises one or more lasers. In certain embodiments, the target beam generator comprises a single laser, and the first set of angularly deflected laser beams and the second set of angularly deflected laser beams are generated by the same laser. The target laser can comprise a pulsed laser or a continuous wave laser. The type and number of lasers used in the subject methods 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 beam generator comprises a dye laser, such as a diphenylstilbene, coumarin, or rhodamine laser. In yet other cases, the beam generator comprises 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 still other cases, the beam generator comprises 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 YAG thulium laser, a YAG ytterbium laser, a diytterbium oxide laser, or a cerium-doped laser, and combinations thereof. In still other cases, the beam generator comprises a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a frequency-doubled or frequency-tripled implementation of any of the above lasers.

[0090] The lasers can have different specific wavelengths depending on the desired wavelength of light produced in the output laser beam (e.g., for irradiating a sample in a fluid stream), which can range from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, including from 400 nm to 800 nm. The beam generator can comprise one or more lasers, such as 2 or more lasers, 3 or more lasers, 4 or more lasers, 5 or more lasers, including 10 or more lasers. The lasers can comprise any combination of laser types. For example, in some embodiments, the beam generator can comprise 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.

[0091] The lasers can be configured to irradiate the acousto-optic device simultaneously or sequentially or in combination. For example, the lasers can be configured to irradiate the acousto-optic device simultaneously. In other embodiments, the lasers are configured for sequential irradiation. Where the beam generator includes more than one laser configured to sequentially irradiate the acousto-optic device, the time at which each laser irradiates the acousto-optic device can independently be 0.001 microseconds or more, for example, 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 30 microseconds or more, including 60 microseconds or more. For example, the method can include irradiating the acousto-optic device with a laser for a duration ranging from 0.001 microseconds to 100 microseconds, for example, 0.01 microseconds to 75 microseconds, 0.1 microseconds to 50 microseconds, 1 microsecond to 25 microseconds, including 5 microseconds to 10 microseconds.

[0092] The time interval between irradiation with each laser can also vary as desired, with the independently spaced delay being 0.001 microseconds or more, for example, 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 15 microseconds or more, 30 microseconds or more, including 60 microseconds or more. For example, the time interval between irradiation with each light source can range from 0.001 microseconds to 60 microseconds, for example, 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microsecond to 25 microseconds, including 5 microseconds to 10 microseconds. In certain embodiments, the time interval between irradiation with each laser is 10 microseconds. In embodiments where more than two (i.e., 3 or more) lasers sequentially irradiate the acousto-optic device, the delay between irradiation by each laser can be the same or different.

[0093] In some embodiments, the lasers of the beam generator are configured to irradiate the acousto-optic device continuously or at discrete intervals. In some cases, the lasers are configured for continuous irradiation of the acousto-optic device. In other cases, the lasers are configured to irradiate the acousto-optic device 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, including every 1000 milliseconds, or some other interval.

[0094] The laser can be operatively positioned at a distance from the acousto-optic device, which distance can vary, e.g., 0.01 mm or more, e.g., 0.05 mm or more, 0.1 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, including 50 mm or more. In addition, the laser can be operatively positioned at an irradiation angle ranging from 10° to 90°, e.g., 15° to 85°, 20° to 80°, 25° to 75°, including 30° to 60°, e.g., 90°.

[0095] In embodiments, the beam generator is configured to generate a plurality of angularly deflected laser beams by irradiating the acousto-optic device with the laser. In some embodiments, the beam generator is configured to generate an output laser beam having one local oscillator beam and a plurality of radio frequency comb beams, e.g., where the generated angularly deflected laser beams include one local oscillator beam and two or more radio frequency comb beams, e.g., 3 or more radio frequency comb beams, 4 or more radio frequency comb beams, 5 or more radio frequency comb beams, 6 or more radio frequency comb beams, 7 or more radio frequency comb beams, 8 or more radio frequency comb beams, 9 or more radio frequency comb beams, 10 or more radio frequency comb beams, 12 or more radio frequency comb beams, 16 or more radio frequency comb beams, 24 or more radio frequency comb beams, including one local oscillator beam and 48 or more radio frequency comb beams.

[0096] The acousto-optic device can be any convenient acousto-optic scheme configured to cause laser frequency shifting using an applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. In other embodiments, the acousto-optic device is an acousto-optic frequency shifter. In yet other embodiments, the acousto-optic device is an acousto-optic modulator. The acousto-optic device in the subject system is configured to generate angularly deflected laser beams from light from the laser and waveforms from the waveform generator.

[0097] In some embodiments, the acousto-optic device in the subject system is configured to generate angularly deflected laser beams 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 with any suitable radio frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator. 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, such as configured to apply 3 or more radio frequency drive signals, such as, for example, 4 or more radio frequency drive signals, 5 or more radio frequency drive signals, 6 or more radio frequency drive signals, 7 or more radio frequency drive signals, 8 or more radio frequency drive signals, 9 or more radio frequency drive signals, 10 or more radio frequency drive signals, 15 or more radio frequency drive signals, 25 or more radio frequency drive signals, 50 or more radio frequency drive signals, including configured to apply 100 or more radio frequency drive signals.

[0098] In certain embodiments, the radio frequency drive signal is generated with a waveform generator configured to generate and input into the acousto-optic device a waveform for each angularly deflected laser beam that reaches the acousto-optic device. The waveform generator can be configured to generate one or more waveforms for each angularly deflected laser beam in the output laser beam, such as, for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, 50 or more, including 100 or more waveforms. In some embodiments, the input waveform includes one or more tones, such as, for example, 2 or more tones, 3 or more tones, 4 or more tones, 5 or more tones, including 10 or more tones. In certain cases, each tone is a sum of 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 25 or more, 100 or more different sinusoidal waves, including cases where each tone is a sum of 500 or more different sinusoidal waves.

[0099] To produce an intensity profile of the angularly deflected laser beams in the output laser beam, the controller is configured to apply radio frequency drive signals having varying amplitudes, e.g., from about 0.001 V to about 500 V, from about 0.005 V to about 400 V, from about 0.01 V to about 300 V, from about 0.05 V to about 200 V, from about 0.1 V to about 100 V, from about 0.5 V to about 75 V, from about 1 V to 50 V, from about 2 V to 40 V, from 3 V to about 30 V, including from about 5 V to about 25 V. In some embodiments, each applied radio frequency drive signal has a frequency from about 0.001 MHz to about 500 MHz, e.g., from about 0.005 MHz to about 400 MHz, from about 0.01 MHz to about 300 MHz, from about 0.05 MHz to about 200 MHz, from about 0.1 MHz to about 100 MHz, from about 0.5 MHz to about 90 MHz, from about 1 MHz to about 75 MHz, from about 2 MHz to about 70 MHz, from about 3 MHz to about 65 MHz, from about 4 MHz to about 60 MHz, including from about 5 MHz to about 50 MHz.

[0100] 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 produce the output laser beam having the angularly deflected laser beams with a desired intensity profile. For example, the memory can include instructions to produce two or more angularly deflected laser beams having the same intensity, e.g., 3 or more, 4 or more, 5 or more, 10 or more, 25 or more, 50 or more, including the memory can include instructions to produce 100 or more angularly deflected laser beams having the same intensity. In other embodiments, the memory can include instructions to produce two or more angularly deflected laser beams having different intensities, e.g., 3 or more, 4 or more, 5 or more, 10 or more, 25 or more, 50 or more, including the memory can include instructions to produce 100 or more angularly deflected laser beams having different intensities.

[0101] In certain embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to produce an output laser beam having an increasing intensity along the lateral 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 in a range from 0.1% to about 99%, e.g., 0.5% to about 95%, 1% to about 90%, about 2% to about 85%, about 3% to about 80%, about 4% to about 75%, about 5% to about 70%, about 6% to about 65%, about 7% to about 60%, about 8% to about 55%, and including a range 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 lateral axis. In other embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to produce an output laser beam having an increasing intensity along the lateral 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 edge of the output beam can be in a range from 0.1% to about 99%, e.g., 0.5% to about 95%, 1% to about 90%, about 2% to about 85%, about 3% to about 80%, about 4% to about 75%, about 5% to about 70%, about 6% to about 65%, about 7% to about 60%, about 8% to about 55%, and including a range 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 lateral axis. In yet other embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to produce an output laser beam having a Gaussian distribution of intensity along the lateral axis. In still other embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to produce an output laser beam having a top-hat intensity distribution along the lateral axis.

[0102] In embodiments, the system is configured to produce spatially separated angularly deflected laser beams in the output laser beam. The angularly deflected laser beams can be separated by 0.001 pm or more, for example, 0.005 pm or more, for example 0.01 pm or more, 0.05 pm or more, 0.1 pm or more, 0.5 pm or more, 1 pm or more, 5 pm or more, 10 pm or more, 100 pm or more, 500 pm or more, 1000 pm or more, 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 system is configured to produce angularly deflected laser beams in the output laser beam that overlap, for example, with angularly deflected laser beams adjacent along a transverse axis of the output laser beam. The overlap (e.g., the overlap of the spots) between adjacent angularly deflected laser beams can be an overlap of 0.001 pm or more, for example, an overlap of 0.005 pm or more, an overlap of 0.01 pm or more, an overlap of 0.05 pm or more, an overlap of 0.1 pm or more, an overlap of 0.5 pm or more, an overlap of 1 pm or more, an overlap of 5 pm or more, an overlap of 10 pm or more, including an overlap of 100 pm or more.

[0103] In certain instances, the purpose beam generator generates a plurality of angularly deflected frequency shifted beams by irradiating an acousto-optic device including, but not limited to, those described in Diebold et al., Nature Photonics, Vol. 7(10); pp. 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; and U.S. Provisional Patent Application No. 63 / 027,080, filed May 19, 2020, the disclosures of which are incorporated herein by reference.

[0104] In embodiments, the system is configured to generate a first set of angularly deflected laser beams and a second set of angularly deflected laser beams from an output beam from the acousto-optic device. In some embodiments, the system includes an optical conditioning assembly configured to split the output laser beam from the acousto-optic device into two different sets of angularly deflected laser beams. In certain embodiments, the optical conditioning assembly is a beamsplitter. As described above, the beamsplitter can be any optical assembly configured to propagate a light beam along two or more different and spatially separated optical paths such that predetermined portions of the light propagate along each optical path. The beamsplitter can be any convenient beamsplitting scheme, such as a triangular prism, a silver mirror prism, a dichroic mirror prism, and other types of beamsplitters. The beamsplitter can be formed of any suitable material so long as the beamsplitter is capable of propagating the desired amount and wavelength of light along each optical path. For example, the target beamsplitter can be formed of glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glass), silica (e.g., fused silica), quartz, a crystal (e.g., a CaF2crystal), zinc selenide (ZnSe), F2, germanium (Ge) titanate (e.g., S-TIH11), borosilicate (e.g., BK7). In certain embodiments, the beamsplitter is formed of a polymeric material, such as, but not limited to, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics, such as PETG (polyethylene terephthalate glycol-modified), as well as other polymeric plastic materials.In certain embodiments, the beam splitter is formed from a polyester, where the target polyester can include, but is not limited to, poly(alkylene terephthalate)s, such as poly(ethylene terephthalate) (PET), bottle grade PET (copolymer made based on monoethylene glycol, terephthalic acid, and other comonomers such as isophthalic acid, cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT), and poly(hexamethylene terephthalate); poly(alkylene adipate)s, such as poly(ethylene adipate), poly(l,4-butylene adipate), and poly(hexamethylene adipate); poly(alkylene suberate)s, such as poly(ethylene suberate); poly(alkylene sebacate)s, such as poly(ethylene sebacate); poly(e-caprolactone) and poly(beta-propiolactone); poly(alkylene isophthalate)s, such as poly(ethylene isophthalate); poly(2,6- naphthalene dicarboxylic acid alkylene ester), such as poly(ethylene 2,6-naphthalate); poly(alkylene sulfonyl-4,4'-dicarboxybenzoate), such as poly(ethylene sulfonyl-4,4'-dicarboxybenzoate); poly(p-phenylene alkylene dicarboxylate), such as poly(p-phenylene ethylene dicarboxylate); poly(trans-1,4-cyclohexane diyl alkylene dicarboxylate), such as poly(trans-1,4-cyclohexane diyl ethylene dicarboxylate); poly(l,4-cyclohexane-diyl alkylene dicarboxylate), such as poly(l,4-cyclohexane-diyl methylene ethylene dicarboxylate); poly([2.2.2]-bicyclooctane-l,4-alkylene dicarboxylate), such as poly([2.2.2]-bicyclooctane-l,4-ethylene dicarboxylate); lactic acid polymers and copolymers, such as (S)-polylactide, (R,S)-polylactide, poly(tetramethylglycolide), and poly(lactide-co-glycolide); polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A; polyamides, such as poly(paraphenylene terephthalamide); polyethylene terephthalate (e.g., Mylar™ polyethylene terephthalate); combinations thereof; and the like.

[0105] In certain embodiments, the optical conditioning component is a wedge beam splitter. In these embodiments, the beam splitter is a beam splitter having a wedge angle that produces non-collinear back reflections, such that the propagation of light collected by the wedge beam splitter causes a small change in the angle of light propagating to one or more of the light scatter detector and the brightfield photodetector. The wedge beam splitter according to embodiments of the present disclosure has a wedge angle in which a change in the angle of incidence of the collected light results in a deviation in the angle of the propagating light that is 0.001% or more, for example, 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 5% or more, including 10% or more. In some embodiments, the wedge angle of the wedge beam splitter is 5 arcminutes to 120 arcminutes, for example, 10 arcminutes to 115 arcminutes, 15 arcminutes to 110 arcminutes, 20 arcminutes to 105 arcminutes, 25 arcminutes to 100 arcminutes, 30 arcminutes to 105 arcminutes, 35 arcminutes to 100 arcminutes, 40 arcminutes to 95 arcminutes, including from 45 arcminutes to 90 arcminutes.

[0106] In some embodiments, the wedge beam splitter has a clear aperture of 150 nm to 5 pm, 180 nm to 8 pm, 185 nm to 2.1 pm, 200 nm to 6 pm, 200 nm to 11 pm, 250 nm to 1.6 pm, 350 nm to 2 pm, 600 nm to 16 pm, 1.2 pm to 8 pm, 2 pm to 16 pm, or some other range of wavelengths.

[0107] The target beam splitter is configured to split the angularly deflected laser beams output from the acousto-optic device into two distinct sets of angularly deflected laser beams. In some embodiments, the split beam ratio between the first set of angularly deflected laser beams and the second set of angularly deflected laser beams of the beam splitter is 1 :99 to 99: 1, for example, 5:95 to 95:5, 10:90 to 90:10, 20:80 to 80:20, 25:75 to 75:25, including a 50:50 split ratio. In certain embodiments, the beam splitter is a 50:50 beam splitter in which the first set of angularly deflected laser beams is identical to the second set of angularly deflected laser beams (e.g., the first set of angularly deflected laser beams has the same amplitude and frequency as the second set of angularly deflected laser beams).

[0108] In some embodiments, the beam generator is configured to generate a first set and a second set of angularly deflected laser beams including one local oscillator beam and a plurality of radio frequency comb beams. In some cases, the beam generator is configured to generate a plurality of sets of angularly deflected laser beams including one local oscillator beam and two or more radio frequency comb beams, for example, 3 or more radio frequency comb beams, 4 or more radio frequency comb beams, 5 or more radio frequency comb beams, 6 or more radio frequency comb beams, 7 or more radio frequency comb beams, 8 or more radio frequency comb beams, 9 or more radio frequency comb beams, 10 or more radio frequency comb beams, 12 or more radio frequency comb beams, 16 or more radio frequency comb beams, 24 or more radio frequency comb beams, including one local oscillator beam and 48 or more radio frequency comb beams.

[0109] In embodiments, the radio frequency comb beams in each set of generated angularly deflected laser beams are spatially separated. The angularly deflected laser beams can be separated by 0.001 pm or more, for example, 0.005 pm or more, 0.01 pm or more, 0.05 pm or more, 0.1 pm or more, 0.5 pm or more, 1 pm or more, 5 pm or more, 10 pm or more, 100 pm or more, 500 pm or more, 1000 pm or more, including 5000 pm or more, depending on the radio frequency drive signal applied to the acousto-optic device. In some embodiments, one or more of the radio frequency comb beams overlap, for example, along a transverse axis of each set of angularly deflected laser beams, with an adjacent laser beam. The overlap, for example, the overlap of the spots, between adjacent angularly deflected laser beams can be an overlap of 0.001 pm or more, for example, an overlap of 0.005 pm or more, an overlap of 0.005 pm or more, an overlap of 0.01 pm or more, an overlap of 0.05 pm or more, an overlap of 0.1 pm or more, an overlap of 0.5 pm or more, an overlap of 1 pm or more, an overlap of 5 pm or more, an overlap of 10 pm or more, including an overlap of 100 pm or more.

[0110] In some embodiments, the beam generator is configured to generate a local oscillator beam in each set of angularly deflected laser beams having a substantially constant intensity profile along a lateral axis. In some cases, the local oscillator beam of each set of angularly deflected laser beams has a beam profile with substantially constant intensity from each edge to the center, e.g., an intensity along a lateral axis of the beam profile differs by 10% or less, e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, including cases where the intensity along a lateral axis of the beam profile differs by 0.001% or less. In some embodiments, the beam generator is configured to generate a local oscillator beam in each set of angularly deflected laser beams having a top hat intensity profile along a lateral axis. In embodiments, the local oscillator beam in each set of angularly deflected laser beams having a top hat intensity profile exhibits little deviation in relative intensity along a lateral axis from each edge to the center, where a beam having a target top hat intensity profile has an intensity at the center that is 95% to 99.9%, e.g., 96% to 99.5%, of the intensity at the edges along a lateral axis, including 98% to 99% of the intensity at the edges along a lateral axis.

[0111] A beam generator according to certain embodiments includes a beam shaping assembly for generating a local oscillator beam having a desired intensity profile along a lateral axis. The beam shaping assembly in these embodiments can include diffractive optics, refractive optics, or an array of lenses, e.g., an array of cylindrical lenses. In some embodiments, the beam shaping assembly is an aspheric cylindrical lens having a cylindrical axis oriented at a right angle, e.g., a laser linear generator lens (e.g., a Powell lens). Examples of laser linear generator lenses include, but are not limited to, the lenses described in U.S. Patent Nos. 4,826,299; 5,283,694; 7,400,457; and 7,329,860, the disclosures of which are incorporated herein by reference.

[0112] The objective beam shaping assembly (e.g., a cylindrical lens, a laser line generator lens, a Powell lens) can be made of any suitable material, including but not limited to glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or NLAF35 glass), silica (e.g., fused silica), quartz, crystal (e.g., CaF2 crystal), zinc selenide (ZnSe), F2, germanium titanate (e.g., S-TIH11), borosilicate (e.g., BK7). In some embodiments, the objective beam shaping assembly has a transmittance window with a wavelength range of 150 nm to 5 pm, 180 nm to 8 pm, 185 nm to 2.1 pm, 200 nm to 6 pm, 200 nm to 11 pm, 250 nm to 1.6 pm, 350 nm to 2 pm, 600 nm to 1.6 pm, 1.2 pm to 8 pm, 2 pm to 16 pm, or other wavelength ranges. The refractive index of the objective beam shaping assembly can vary, with a range of 1 to 3, e.g., 1.1 to 2.9, 1.2 to 2.8, 1.3 to 2.7, 1.4 to 2.6, 1.5 to 2.7, 1.6 to 2.6, 1.7 to 2.5, 1.8 to 2.4, including 1.9 to 2.3.

[0113] In some cases, the beam shaping assembly is a Powell lens positioned to receive two or more different beams of light at different angles of incidence at substantially the same location and generate an output beam having a beam intensity profile in which the intensity at the center of the beam is 75% to 99.9% of the intensity at the edge along the lateral axis. Depending on the laser in the subject system, the Powell lens can have a different diameter, with a range of 2 mm to 15 mm, e.g., 2.5 mm to 14.5 mm, 3 mm to 14 mm, 3.5 mm to 13.5 mm, 4 mm to 13 mm, 4.5 mm to 12.5 mm, 5 mm to 12 mm, 5.5 mm to 11.5 mm, 6 mm to 11 mm, including 7 mm to 10 mm. The Powell lens can also vary in sector angle, with a range of 0.1° to 90°, e.g., 0.5° to 85°, 1° to 80°, 5° to 75°, 10° to 70°, 15° to 65°, including a Powell lens with a sector angle of 20° to 60°. In certain embodiments, the subject beam generator includes only a single beam shaping optical assembly (e.g., a single Powell lens) and is configured to receive two or more different beams of light at different angles of incidence at substantially the same location and generate an output beam having a predetermined beam intensity profile along the lateral axis (e.g., a top-hat beam intensity profile).

[0114] In embodiments, the output beam preserves the power density of the light from each laser received by the beam shaping assembly such that the power from each laser is reduced by 10% or less, e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, including a reduction of 0.0001% or less. The output power produced can be determined by any suitable scheme, including but not limited to employing power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser luminometers, laser diode detectors, CCD or CMOS photosensors, and other types of photodetectors to measure incident and output power. To determine the change in power of the generated output beam, the power of the incident light impinging on the beam shaping assembly can be measured with one or more of the foregoing power meters (e.g., with a hand-held optical power meter or thermopile power meter) and compared to the laser power propagating through the beam shaping assembly (i.e., the power of the generated output beam).

[0115] In some embodiments, the beam generator is configured such that two or more of the plurality of radio frequency comb beams in each set of angularly deflected laser beams have substantially the same intensity along the transverse axis. For example, the beam generator can be configured to generate a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, where two or more of the radio frequency comb beams can independently have the same intensity, e.g., where 3 or more of the radio frequency comb beams have the same intensity, 4 or more of the radio frequency comb beams have the same intensity, 5 or more of the radio frequency comb beams have the same intensity, 6 or more of the radio frequency comb beams have the same intensity, 7 or more of the radio frequency comb beams have the same intensity, 8 or more of the radio frequency comb beams have the same intensity, 9 or more of the radio frequency comb beams have the same intensity, 10 or more of the radio frequency comb beams have the same intensity, 12 or more of the radio frequency comb beams have the same intensity, 16 or more of the radio frequency comb beams have the same intensity, 24 or more of the radio frequency comb beams have the same intensity, including where 48 or more of the radio frequency comb beams have the same intensity.

[0116] In other embodiments, the optical beam generator is configured to generate a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, where two or more of the radio frequency comb of beams in each set of angularly deflected laser beams independently have different intensities, e.g., where 3 or more of the radio frequency comb of beams have different intensities, 4 or more of the radio frequency comb of beams have different intensities, 5 or more of the radio frequency comb of beams have different intensities, 6 or more of the radio frequency comb of beams have different intensities, 7 or more of the radio frequency comb of beams have different intensities, 8 or more of the radio frequency comb of beams have different intensities, 9 or more of the radio frequency comb of beams have different intensities, 10 or more of the radio frequency comb of beams have different intensities, 12 or more of the radio frequency comb of beams have different intensities, 16 or more of the radio frequency comb of beams have different intensities, 24 or more of the radio frequency comb of beams have different intensities, including cases where 48 or more of the angularly deflected laser beams have different intensities.

[0117] A system according to certain embodiments includes a photodetector for determining the intensity profile of the laser beams of one or more of the first set of angularly deflected laser beams and the second set of angularly deflected laser beams along the transverse axis. The photodetector for determining the intensity profile of the sets of angularly deflected laser beams can be any convenient photodetector scheme, including but not limited to a scanning slit profiler, a charge-coupled device (CCD, e.g., an intensified charge-coupled device, ICCD), a position sensor, a power sensor (e.g., a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, and other types of photodetectors.

[0118] As noted above, in some cases the intensity profile of each set of angularly deflected laser beams is determined by capturing an image. In these embodiments, the system can include any suitable device capable of capturing an optical image and converting it into an electronic data signal, including but not limited to a charge-coupled device, a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or a N-type metal-oxide semiconductor (NMOS) image sensor. In some embodiments, the imaging sensor is a CCD camera. For example, the camera can be an electron multiplying CCD (EMCCD) camera or an intensified CCD (ICCD) camera. In other embodiments, the imaging sensor is a CMOS-type camera.

[0119] In certain embodiments, the system includes one or more photodetectors and a processor with memory having stored thereon instructions for determining and adjusting the beam profile of the angularly deflected laser beams, such as those described in U.S. Provisional Patent Application No. 63 / 027,080, filed May 19, 2020, the disclosure of which is incorporated herein by reference.

[0120] In embodiments, the beam generator includes an optical conditioning assembly for optically combining the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. The optical conditioning assembly can be any convenient optical conditioning scheme suitable for combining multiple beams and can include mirrors, lenses and other types of free space optical relay devices, as well as fiber optic light combining assemblies. In certain embodiments, the beam generator includes a mirror to combine the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In other embodiments, the beam generator includes a lens system, such as a telescopic lens system, to combine the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In still other embodiments, the beam generator includes a mirror and a lens system to combine the first set of angularly deflected laser beams and the second set of angularly deflected laser beams.

[0121] In some embodiments, the optical conditioning assembly is configured to combine the first set of angularly deflected laser beams and the second set of angularly deflected laser beams and cause them to propagate along a single optical path, e.g., where one or more beams from the first set of angularly deflected laser beams and the second set of angularly deflected laser beams overlap. For example, two or more of the angularly deflected laser beams (e.g., radio frequency comb beams) of the first and second sets of angularly deflected laser beams can overlap, e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 24 or more, including 48 or more of the angularly deflected laser beams of the first and second sets of angularly deflected laser beams can overlap.

[0122] In other embodiments, the optical conditioning assembly is configured to combine and propagate the first set of angularly deflected laser beams and the second set of angularly deflected laser beams along two parallel optical paths, e.g., with no overlap between the beams from the first set of angularly deflected beams and the second set of angularly deflected laser beams. In this embodiment, the optical paths of the first set of angularly deflected laser beams are spatially separated from the second set of angularly deflected laser beams. In some cases, the optical paths of the first set of angularly deflected laser beams are separated from the second set of angularly deflected laser beams by 0.00001 mm or more, e.g., 0.00005 mm or more, 0.0001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, including 2 mm or more. In certain embodiments, the first set of angularly deflected laser beams are combined and propagated along a different parallel optical plane than the second set of angularly deflected laser beams. In certain cases, the optical plane of the first set of angularly deflected laser beams is spatially separated from the optical plane of the second set of angularly deflected laser beams by 0.00001 mm or more, e.g., 0.00005 mm or more, 0.0001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, including 2 mm or more.

[0123] In some embodiments, the optical conditioning assembly is configured to propagate the first set of angularly deflected laser beams and the second set of angularly deflected laser beams onto the fluid stream, e.g., to illuminate particles of a sample in the fluid stream. In some embodiments, the optical conditioning assembly is configured to propagate the first set of angularly deflected laser beams and the second set of angularly deflected laser beams onto the fluid stream at the same location. In other embodiments, the optical conditioning assembly is configured to propagate the first set of angularly deflected laser beams onto the fluid stream at a location that overlaps with a location on the fluid stream that is illuminated with the second set of angularly deflected laser beams. For example, the location on the fluid stream that is illuminated with the first set of angularly deflected laser beams can overlap with the location on the fluid stream that is illuminated with the second set of angularly deflected laser beams by 0.00001 pm or more, e.g., 0.00005 pm or more, 0.0001 pm or more, 0.005 pm or more, 0.01 pm or more, 0.05 pm or more, 0.1 pm or more, 0.5 pm or more, 1 pm or more, 10 pm or more, 50 pm or more, 100 pm or more, including 1000 pm or more.

[0124] In other embodiments, the optical conditioning assembly is configured to propagate the first set of angularly deflected laser beams to a first location on the fluid stream and to propagate the second set of angularly deflected laser beams to a second location on the fluid stream. For example, the optical conditioning assembly can be configured to propagate the first set of angularly deflected laser beams to a different vertical position on the fluid stream than the second set of angularly deflected laser beams. Depending on the flow rate of the fluid stream, the optical conditioning assembly can be configured to propagate the second set of angularly deflected laser beams to a location downstream of the illuminated location of the first set of angularly deflected laser beams by 0.001 pm or more, e.g., 0.005 pm or more, 0.01 pm or more, 0.05 pm or more, 0.1 pm or more, 0.5 pm or more, 1 pm or more, 5 pm or more, 10 pm or more, 100 pm or more, 250 pm or more, 500 pm or more, including a location downstream of the illuminated location of the first set of angularly deflected laser beams by 1000 pm or more.

[0125] The system also includes one or more detectors for detecting light from the sample in the fluid stream (e.g., in a flow cytometer). Suitable light detection schemes include, but are not limited to, optical sensors or 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, and combinations thereof, as well as other photodetectors.

[0126] In some embodiments, the system includes a first set of photodetectors for detecting light from the sample illuminated by the first set of angularly deflected laser beams, and a second set of photodetectors for detecting light from the sample illuminated by the second set of angularly deflected laser beams. In some cases, the first set of photodetectors includes a plurality of photomultiplier tubes, e.g., 2 or more photomultiplier tubes, 3 or more photomultiplier tubes, 4 or more photomultiplier tubes, 5 or more photomultiplier tubes, 6 or more photomultiplier tubes, 7 or more photomultiplier tubes, 8 or more photomultiplier tubes, 9 or more photomultiplier tubes, 10 or more photomultiplier tubes, 12 or more photomultiplier tubes, including 16 or more photomultiplier tubes. In other cases, the first set of photodetectors includes a plurality of photodiodes, e.g., 2 or more photodiodes, 3 or more photodiodes, 4 or more photodiodes, 5 or more photodiodes, 6 or more photodiodes, 7 or more photodiodes, 8 or more photodiodes, 9 or more photodiodes, 10 or more photodiodes, 12 or more photodiodes, including 16 or more photodiodes.

[0127] In some embodiments, each set of photodetectors is configured to generate a light signal in response to light from particles in the fluid stream illuminated by the respective set of angularly deflected laser beams. In some cases, the generated light signal includes frequency-encoded fluorescence data from the particles. In certain cases, the system includes a controller with 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 transform the frequency-encoded fluorescence data from the particles to give spatial data for the particles. In some embodiments, the spatial data includes a horizontal dimension of the particles, a vertical dimension of the particles, a ratio of the particle dimensions along two different dimensions, a ratio of the dimensions of a component of the particles.

[0128] In some embodiments, the system includes a controller with 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 convert the frequency-encoded fluorescent data to spatial data by a Fourier transform. In some cases, the memory includes instructions for converting the frequency-encoded fluorescent data by a discrete Fourier transform (DFT). In other cases, the memory includes instructions for computing the spatial data by performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescent data. In yet other cases, the memory includes instructions for computing the spatial data using a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescent data. In certain embodiments, the system includes a controller with a processor having a memory for computing spatial data, such as described in U.S. Patent Application No. 16 / 887,538, filed May 29, 2020, the disclosure of which is incorporated herein by reference.

[0129] In some embodiments, the system includes a controller with 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 an image of a particle in the fluid stream from the frequency-encoded fluorescent light. In some embodiments, the memory includes instructions for generating the particle image from the frequency-encoded fluorescent light in combination with detected light absorption, detected light scattering, or a combination thereof. In certain cases, the memory includes instructions for generating the particle image from the frequency-encoded fluorescent light only. In other cases, the memory includes instructions for generating the particle image from the frequency-encoded fluorescent light and detected light absorption from the sample (e.g., from a brightfield light detector). In yet other cases, the memory includes instructions for generating the particle image from the frequency-encoded fluorescent light using detected light scattering from the sample (e.g., from a side scatter detector, a forward scatter detector, or a combination of a side scatter detector and a forward scatter detector). In still other cases, the memory includes instructions for generating the particle image from the frequency-encoded fluorescent light and a combination of detected light absorption, detected light scattering, and detected light emission. In still other cases, the memory includes instructions for generating the particle image from the frequency-encoded fluorescent light and spectrally resolved light from a second set of photodetectors, as described in more detail below.

[0130] In some embodiments, the memory includes instructions for generating a single image of the particle. In other embodiments, the memory includes instructions for generating two or more images of the particle, e.g., 3 or more, 4 or more, 5 or more, including 10 or more images.

[0131] In some embodiments, the system includes a second set of photodetectors configured to detect light from the sample illuminated by the second set of angularly deflected laser beams. In some cases, the second set of photodetectors includes a plurality of photodiodes, e.g., a plurality of avalanche photodiodes. For example, the second set of photodetectors can include 2 or more photodiodes, e.g., 3 or more photodiodes, 4 or more photodiodes, 5 or more photodiodes, 6 or more photodiodes, 7 or more photodiodes, 8 or more photodiodes, 9 or more photodiodes, 10 or more photodiodes, 12 or more photodiodes, including 16 or more photodiodes. In other cases, the second set of photodetectors includes a plurality of photomultiplier tubes, e.g., 2 or more photomultiplier tubes, 3 or more photomultiplier tubes, 4 or more photomultiplier tubes, 5 or more photomultiplier tubes, 6 or more photomultiplier tubes, 7 or more photomultiplier tubes, 8 or more photomultiplier tubes, 9 or more photomultiplier tubes, 10 or more photomultiplier tubes, 12 or more photomultiplier tubes, including 16 or more photomultiplier tubes.

[0132] In some embodiments, the system includes a cluster wave division optical detection system having a wavelength separator for separating light from the sample into a plurality of predetermined spectral ranges. In some cases, one or more of the first set of photodetectors and the second set of photodetectors are configured for wavelength division multiplexing, in which different wavelengths of light come from the fluid stream. For example, each photodetector in the set of photodetectors can be configured to detect one or more sets of predetermined wavelengths of light from the sample in the fluid stream. In these embodiments, data signals generated from the light of the predetermined wavelength sets from the plurality of photodetectors are multiplexed, and the wavelength division multiplexed data signals are output to the processor. For example, the wavelength division multiplexed data signals can include data signals generated from 2 or more different sets of predetermined wavelengths of light, e.g., 3 or more sets, 4 or more sets, 5 or more sets, 6 or more sets, 7 or more sets, 8 or more sets, 9 or more sets, 10 or more sets, 11 or more sets, including wavelength division multiplexed data signals including data signals generated from 12 or more different sets of predetermined wavelengths of light. In certain embodiments, the plurality of photodetectors are configured to generate wavelength division multiplexed data signals including data signals from 2 or more different optical spectra detected by the photodetectors, e.g., 3 or more, 4 or more, 5 or more, e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, including wavelength division multiplexed data signals including data signals from 12 or more different optical spectra detected by the photodetectors.

[0133] In some embodiments, the system includes a controller with 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 spectrally resolve light detected from irradiation of a sample in a fluid stream. In certain embodiments, the memory includes instructions for determining overlapping spectral components of the light by computing a spectral unmixing matrix. In some embodiments, the spectrum of light detected by each photodetector overlaps the spectrum of light detected by at least one other detector in the set of photodetectors. In some cases, the spectrum of light detected by one photodetector overlaps the spectrum of light of at least one other detector in the set of photodetectors by 5 nm or more, for example, 10 nm or more, 25 nm or more, including 50 nm or more. In certain cases, the spectrum of light detected by one photodetector overlaps the spectrum of light of two or more other photodetectors in the set of photodetectors, for example, where each overlap is 5 nm or more, for example, 10 nm or more, 25 nm or more, including 50 nm or more. In other embodiments, the spectrum of light detected by one photodetector in the set of photodetectors has a non-overlapping spectrum. In these embodiments, the spectrum of light detected by each photodetector is adjacent to the spectrum of at least one other photodetector in the set of photodetectors by 10 nm or less, 9 nm or less, 8 nm or less, 7 nm or less, 6 nm or less, 5 nm or less, 4 nm or less, 3 nm or less, 2 nm or less, including 1 nm or less.

[0134] In some embodiments, the system includes a controller with 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 determine overlap of spectra of light from a fluid stream irradiated with a set of angularly deflected laser beams and compute a contribution of each laser beam to the detected overlapping spectra. In some embodiments, the memory includes instructions for spectrally resolving the light by computing a spectral unmixing matrix. In certain embodiments, the memory includes instructions for computing the spectral unmixing matrix to estimate an abundance of each contribution to a signal of light detected by a photodetector in the set of photodetectors.

[0135] In some cases, the memory includes instructions for computing the spectral unmixing matrix by determining an abundance of a fluorophore associated with a particle in the fluid stream. The memory can include instructions for identifying an abundance of each fluorophore associated with a particle. The memory can further include instructions for classifying the particle. In some cases, the identified or classified particle can be used to sort a target particle (e.g., a cell) in the sample.

[0136] In certain embodiments, a system can include an optical detection system for spectrally resolving light, such as described in International Patent Application No. PCT / US2019 / 068395, filed December 23, 2019, U.S. Provisional Patent Application No. 62 / 971,840, filed February 7, 2020, and U.S. Provisional Patent Application No. 63 / 010,890, filed April 16, 2020, the disclosures of which are incorporated by reference herein in their entireties. The system includes a controller with 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 solve a spectral unmixing matrix using one or more of: 1) a weighted least squares algorithm; 2) a Sherman-Morrison iterative inverse updater; 3) an LU matrix decomposition, such as a decomposition of a matrix into a product of a lower triangular (L) matrix and an upper triangular (U) matrix; 4) a modified Cholesky decomposition; 5) a weighted least squares algorithm by QR factorization; and 6) a weighted least squares algorithm by singular value decomposition.

[0137] Figure 2 A system for illuminating a sample in a fluid stream with a plurality of angularly deflected laser beams is depicted in accordance with certain embodiments. The system 200 includes a laser 201 configured to illuminate an acousto-optic device (e.g., an acousto-optic deflector) 202. The plurality of angularly deflected laser beams includes one local oscillator beam (LO beam) and a plurality of radio frequency shifted comb beams, which are separated in the system 200 with a pick-off mirror (pick-off mirror 1). The local oscillator beam passes through a top hat lens (e.g., a Powell lens) to generate a constant beam profile and is recombined with the comb beams using a beam splitter (BS). A first set of angularly deflected laser beams 203 propagates from the beam splitter along a first optical path, and a second set of angularly deflected laser beams 204 propagates from the beam splitter along a second optical path. In certain embodiments, the second set of angularly deflected laser beams 204 can pass through beam shaping optics 208 (e.g., a prism pair) that can be used to change the vertical beam size. The sets of angularly deflected laser beams 203 and 204 are combined and directed to a flow cell 205. In this embodiment, pick-off mirror 2 is placed near the focal planes of lenses (L4) and (L7), where the two focused beams are separated in a direction perpendicular to the base plane. To avoid laser damage to the coating of pick-off mirror 2 due to the small size of the focused beams, pick-off mirror 2 is slightly offset from the focal planes (SI) of lenses (L4) and (L7) by an amount delta. In some cases, delta is a trade-off between the separation of the two beams and the power density on the pick-off mirror. Light from the irradiated sample is collected by a collection lens 206 and detected by an optical detection system 207, which can include a first set of photodetectors (e.g., a plurality of photomultiplier tubes) and a second set of photodetectors (e.g., a plurality of photodiodes).

[0138] Figure 3 Illumination of a fluid stream in a flow cell with two sets of angularly deflected laser beams is depicted in accordance with certain embodiments. As Figure 2 As depicted in FIG. 3, the first set of angularly deflected laser beams 301 combine with the second set of angularly deflected laser beams 302 and propagate to the flow cell for illuminating a sample in the fluid stream. The first set of angularly deflected laser beams propagate through lens L4 and combine with the second set of angularly deflected laser beams from pick-off mirror 2. The combined set of angularly deflected laser beams propagate along different optical planes, through a telescopic lens system combined with L5 and L6, and are focused at two different locations along the longitudinal axis of the flow cell (i.e., vertically separated). In this embodiment, the focal planes S1 and S2 are paired with each other, and the magnification of the telescopic lens (the ratio of f2 / f1) can be determined by the spacing ratio of the beams at S2 and S1.

[0139] In certain embodiments, one or more of the first set of photodetectors and the second set of photodetectors are spatially positioned apart from the fluid stream, and light from the illuminated fluid stream propagates to the photodetectors through an optical relay system, such as through an optical fiber or a free-space optical relay system. For example, the optical relay system can be an optical fiber relay bundle and the light propagates through the optical fiber relay bundle to the photodetectors. Any optical fiber relay system can be employed to propagate light from the fluid stream to the photodetectors. In certain embodiments, suitable optical fiber relay systems for propagating light to the detectors include, but are not limited to, those described in, for example, U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0140] In other embodiments, the optical relay system comprises a free-space optical relay system. The term "free-space optical relay" is used herein in its conventional sense to refer to the propagation of light using a configuration of one or more optical components to direct light from the illuminated fluid stream through free space to the photodetectors. In certain embodiments, the free-space optical relay system comprises a housing having a proximal end and a distal end. The free-space relay system can comprise any combination of different optical conditioning components, such as one or more of lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof. For example, in some embodiments, the target free-space optical relay system comprises one or more focusing lenses. In other embodiments, the target free-space optical relay system comprises one or more mirrors. In yet other embodiments, the free-space optical relay system comprises a collimating lens. In certain embodiments, the free-space optical relay system includes, but is not limited to, those described in, for example, U.S. Patent Nos. 7,643,142; 7,728,974; and 8,223,445, the disclosures of which are incorporated herein by reference.

[0141] In some embodiments, the system includes a flow cell configured to convey a sample in a fluid stream. Any suitable flow cell that conveys a fluid sample to a sample interrogation region can be employed, where in some embodiments the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating in a planar surface having an orifice transverse to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) can vary, ranging from 1 mm to 15 mm, for example, 1.5 mm to 12.5 mm, 2 mm to 10 mm, 3 mm to 9 mm, including 4 mm to 8 mm. The length of the distal frustoconical portion (measured along the longitudinal axis) can also vary, ranging from 1 mm to 10 mm, 2 mm to 9 mm, 3 mm to 8 mm, including 4 mm to 7 mm. In some embodiments, the diameter of the flow cell nozzle chamber can vary, ranging from 1 mm to 10 mm, for example, 2 mm to 9 mm, 3 mm to 8 mm, including 4 mm to 7 mm.

[0142] In certain cases, the flow cell does not include a cylindrical portion and the entire flow cell interior is frustoconical. In these embodiments, the length of the frustoconical interior (measured along the longitudinal axis transverse to the nozzle orifice) can range from 1 mm to 15 mm, for example, 1.5 mm to 12.5 mm, 2 mm to 10 mm, 3 mm to 9 mm, including 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical interior can range from 1 mm to 10 mm, for example, 2 mm to 9 mm, 3 mm to 8 mm, including 4 mm to 7 mm.

[0143] In some embodiments, the sample fluid stream exits the flow cell 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 sample fluid stream, where the orifice cross-sectional shape includes, but is not limited to, straight-sided cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curved-sided cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., parabolic base joined to a planar top). In certain embodiments, the flow cell has a circular orifice. The size of the nozzle orifice can vary, ranging in some embodiments from 1 μιη to 20,000 μιη, for example, 2 μιη to 17,500 μιη, 5 μιη to 15,000 μιη, 10 μιη to 12,500 μιη, 15 μιη to 10,000 μιη, 25 μιη to 7,500 μιη, 50 μιη to 5,000 μιη, 75 μιη to 1,000 μιη, 100 μιη to 750 μιη, including 150 μιη to 500 μιη. In certain embodiments, the nozzle orifice is 100 μιη.

[0144] In some embodiments, the flow cell comprises a sample inlet configured to provide a sample to the flow cell. In embodiments, the sample inlet system is configured to provide a suitable sample flow to the flow cell lumen. The rate of delivery of sample to the flow cell chamber through the sample inlet can be 1 pL / min or more, for example, 2 pL / min or more, 3 pL / min or more, 5 pL / min or more, 10 pL / min or more, 15 pL / min or more, 25 pL / min or more, 50 pL / min or more, including 100 pL / min or more, depending on the desired characteristics of the fluid flow. In some cases, the rate of delivery of sample to the flow cell chamber through the sample inlet can be 1 pL / s or more, for example, 2 pL / s or more, 3 pL / s or more, 5 pL / s or more, 10 pL / s or more, 15 pL / s or more, 25 pL / s or more, 50 pL / s or more, including 100 pL / s or more.

[0145] The sample inlet can be an orifice in the wall of the lumen or a conduit at the proximal end of the lumen. In cases where the sample inlet is an orifice in the wall of the lumen, the sample inlet orifice can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: straight-sided cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curved-sided cross-sectional shapes (e.g., circular, elliptical, etc.), and irregular shapes (e.g., parabolic bottom connected to a planar top). In some embodiments, the sample inlet has a circular orifice. The size of the sample inlet orifice can vary depending on the shape, and in some cases, the opening ranges from 0.1 mm to 5.0 mm, for example, 0.2 to 3.0 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0146] In some cases, the sample inlet is a conduit located proximal to the interior of the flow cell. For example, the sample inlet can be a conduit positioned such that the sample inlet orifice is in-line with the flow cell orifice. In cases where the sample inlet is a conduit that is in-line with the flow cell orifice, the cross-sectional shape of the sample inlet tube can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: straight-sided cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curved-sided cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., parabolic bottom connected to a planar top). The orifice of the conduit can vary in shape, in some cases ranging from 0.1 mm to 5.0 mm, for example, 0.2 to 3.0 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, including 1.25 mm to 1.75 mm, for example 1.5 mm. The tip shape of the sample inlet can be the same as the cross-sectional shape of the sample inlet tube, or it can be different. For example, the sample inlet orifice can include a beveled tip with a bevel angle ranging from 1° to 10°, for example, 2° to 9°, 3° to 8°, 4° to 7°, including a bevel angle of 5°.

[0147] In some embodiments, the flow cell also includes a sheath inlet configured to provide sheath fluid to the flow cell. In embodiments, the sheath inlet system is configured to provide a flow of sheath fluid to the interior of the flow cell, for example, to combine with the sample to form a layered sheath fluid flow around the sample fluid flow. The rate at which the sheath fluid is delivered to the flow cell chamber can be 25 μL / s or more, for example, 50 μL / s or more, 75 μL / s or more, 100 μL / s or more, 250 μL / s or more, 500 μL / s or more, 750 μL / s or more, 1000 μL / s or more, including 2500 μL / s or more, depending on the desired properties of the fluid flow.

[0148] In some embodiments, the sheath inlet is an orifice in the wall of the interior. The sheath inlet orifice can be any suitable shape, where cross-sectional shapes of interest include, but are not limited to: straight-sided cross-sectional shapes (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curved-sided cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., parabolic bottom connected to a planar top). The size of the sheath inlet orifice can vary in shape, in some cases ranging from 0.1 mm to 5.0 mm, for example, 0.2 to 3.0 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0149] In some embodiments, the system further comprises a pump in fluid communication with the flow cell to convey the fluid stream through the flow cell. Any suitable fluid pump scheme can be employed to control the flow rate of the fluid stream through the flow cell. In some cases, the system comprises a peristaltic pump, e.g., 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 fluid stream. In some cases, the flow rate of the sample stream in the flow cell is 1 pL / min (picoliter / minute) or more, e.g., 2 pL / min or more, 3 pL / min or more, 5 pL / min or more, 10 pL / min or more, 25 pL / min or more, 50 pL / min or more, 75 pL / min or more, 100 pL / min or more, 250 pL / min or more, 500 pL / min or more, 750 pL / min or more, including 1000 pL / min or more. For example, the system can comprise a pump configured to flow a sample through the flow cell at a flow rate ranging from 1 pL / min to 500 pL / min, e.g., from 1 pL / min to 250 pL / min, from 1 pL / min to 100 pL / min, from 2 pL / min to 90 pL / min, from 3 pL / min to 80 pL / min, from 4 pL / min to 70 pL / min, from 5 pL / min to 60 pL / min, including from 10 pL / min to 50 pL / min. In certain embodiments, the flow rate of the fluid stream is from 5 pL / min to 6 pL / min.

[0150] In certain embodiments, the subject system is a particle analyzer employing the above-described light beam generator and light detection system. In certain embodiments, the subject system is a flow cytometer system. Suitable flow cytometer systems and methods include, but are not limited to, those described in the following publications: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University 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., Thromb Haemost. 2004 Oct;30(5):502-11; Alison et al., J Pathol. 2010 Dec;222(4):335-344; and Herbig et al. (2007), Therapeutic Drug Carrier Systems Review, 24(3):203-255; the contents of which are incorporated herein by reference. In certain cases, the target flow cytometer system includes a BD Biosciences FACSCanto TM Flow Cytometer, BD Biosciences FACSCanto TM II Flow Cytometer, BD Accuri TM Flow Cytometer, BD Accuri TM Flow Cytometer, BD Biosciences FACSCelesta TM Flow Cytometer, BD Biosciences FACSLyric TM Flow Cytometer, BD Biosciences FACSVerse TM Flow Cytometer, BD Biosciences FACSymphony TM Flow Cytometer, BD Biosciences LSRFortessa TM Flow Cytometer, BD Biosciences LSRFortessa TM Flow Cytometer, BD Biosciences FACSPresto TM Flow Cytometer, BD Biosciences FACSVia TM Flow Cytometer, and BD Biosciences FACSCalibur TMCell Sorter, BD Biosciences FACSCount TM Cell Sorter, BD Biosciences FACSLyric TM Cell Sorter, BD Biosciences Via TM Cell Sorter, BD Biosciences Influx™ Cell Sorter, BD Biosciences Jazz TM Cell Sorter, BD Biosciences Aria TM Cell Sorter, BD Biosciences FACSAria TM II Cell Sorter, BD Biosciences FACSAria TM III Cell Sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell Sorter, BD Biosciences FACSymphony TM S6 Cell Sorter, etc.

[0151] In certain embodiments, the subject system is configured to sort one or more 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 such as biological macromolecules) of a sample and, in some cases, transferring the separated components to one or more sample collection vessels. For example, the subject system can be configured for sorting a sample having 2 or more components, e.g., 3 or more components, 4 or more components, 5 or more components, 10 or more components, 15 or more components, including sorting a sample having 25 or more components. One or more sample components can be separated from the sample and transferred to a sample collection vessel, e.g., 2 or more sample components, 3 or more sample components, 4 or more sample components, 5 or more sample components sample components, 10 or more sample components, including 15 or more sample components can be separated from the sample and transferred to a sample collection vessel.

[0152] In some embodiments, the target particle sorting system is configured to sort particles using, for example, those enclosed particle sorting modules described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, the particles (e.g., cells) of a sample are sorted using, for example, those sorting decision modules having multiple sorting decision units described in U.S. Patent Application No. 16 / 725,756, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, the subject particle sorting system is a flow cytometer system, such as those described in U.S. Patents Nos. 10,663,47; 610,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 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; 4,987,086; 4,498,766; the disclosures of which are incorporated herein by reference in their entireties.

[0153] In some embodiments, the system is a particle analysis instrument, wherein the particle analysis system 401 (e.g., a flow cytometer system) can be used to analyze and characterize particles with or without sorting the particles into collection vessels. Figure 4A ) can be used to analyze and characterize particles with or without sorting the particles into collection vessels. Figure 4A A functional block diagram of a particle analysis system for computing sample analysis and particle characterization is shown. In some embodiments, the particle analysis system 401 is a flow system. For example, Figure 4A The particle analysis system 401 shown in FIG. 610,620,111 can be configured to perform, in whole or in part, the methods described herein. The particle analysis system 401 includes a fluidics system 402. The fluidics system 402 can include or be coupled to a sample tube 405, and include or be coupled to a moving column of fluid within the sample tube, in which particles 403 (e.g., cells) of a sample move along a common sample path 409.

[0154] Particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as each particle passes one or more detection sites along a common sample path. A detection site 408 generally refers to a monitored region 407 of the common sample path. In some embodiments, detection can include detecting light or one or more other characteristics of a particle 403 as the particle 403 passes the monitored region 407. In Figure 4A In the illustrated embodiment, one detection site 408 is shown having one monitored region 407. Some embodiments of particle analysis system 401 can include multiple detection sites. Further, some detection sites can monitor multiple regions.

[0155] Each signal is assigned a signal value to form a data point for each particle. As noted above, this data can be referred to as event data. The data points can be multi-dimensional data points including values for respective characteristics measured for the particle. Detection system 404 is configured to collect a series of such data points over a first time interval.

[0156] Particle analysis system 401 can also include a control system 406. Control system 406 can include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system can be operatively associated with flow control system 402. Control system can be configured to generate a calculated signal frequency for at least a portion of the first time interval based on a Poisson distribution and a number of data points collected by detection system 404 during the first time interval. Control system 406 can be further configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. Control system 406 can additionally compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0157] Figure 4B A system 400 for a flow cytometer is shown in accordance with one illustrative embodiment of the present application. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. 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.

[0158] Excitation lasers 115a-c emit light in the form of laser beams. In Figure 4BIn the example system of FIG. 4, the wavelengths of the laser beams emitted from the excitation lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively. 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 in the range of 10 to 400 nm) and reflects light at 488 nm and 633 nm.

[0159] The laser beams are then directed to the focusing lens 420, which focuses the beams onto a portion of the fluid stream within the flow chamber 425 where the sample particles are located. The flow chamber is part of a flow cytometry system that directs the particles in the medium, typically one at a time, to the focused laser beams for review. The flow chamber can include a flow cell in a benchtop cytometer or a nozzle tip in an air flow cytometer.

[0160] Light from the laser beams interacts with the particles in the sample by diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various different wavelengths depending on the characteristics of the particles, such as their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally occurring on or in the particles. The fluorescent emissions, as well as the diffracted, refracted, reflected, and scattered light, can be delivered to one or more of the forward scatter detector 430, the side scatter detector 435, and the one or more fluorescence detectors 460a-460f through one or more of the beam splitters 445a-445g, the bandpass filters 450a-450e, the longpass filters 455a-455b, and the fluorescence collection lens 440.

[0161] The fluorescence collection lens 440 collects light emitted from the particle-laser beam interaction and delivers the light toward one or more beam splitters and filters. Bandpass filters, such as the bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, the 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, the 510 / 20 filter extends 10 nm on either side of the center of the spectral band, or the range is 500 nm to 520 nm. Shortpass filters transmit light at or shorter than a specified wavelength. Longpass filters, such as the longpass filters 455a-455b, transmit light at or longer than a specified wavelength. For example, the longpass filter 455a, which is a 670 nm longpass filter, transmits light at or longer than 670 nm. The filters are typically selected to optimize the specificity of the detector for a particular fluorescent dye. The filters can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0162] The beam splitters direct different wavelengths of light in different directions. The beam splitters are characterized by filter properties, such as short pass and long pass. For example, beam splitter 445g is a 620 SP beam splitter, meaning that beam splitter 445g transmits light having a wavelength of 620 nm or shorter and reflects light having a wavelength longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g can include optical mirrors, such as dichroic mirrors.

[0163] Forward scatter detector 430 is positioned slightly off axis from the direct beam of light passing through the flow cell and is configured to detect diffracted light, i.e., excitation light that travels through or around the particle primarily in a forward direction. The intensity of light detected by the forward scatter detector is dependent on the overall size of the particle. The forward scatter detector can include a photodiode. 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. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detectors can include photomultiplier tubes. The signals detected at the forward scatter detector 430, side scatter detector 435, and fluorescence detectors can be converted to electronic signals (voltage) by the detectors. This data can provide sample-related information.

[0164] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present application is not limited to the flow cytometer depicted in FIG. 4, but can include any flow cytometer known in the art. For example, the flow cytometer can have any number of lasers, beam splitters, filters, and detectors in various wavelengths and various different configurations. Figure 4B

[0165] ​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. While not explicitly shown, controller / processor 190 is coupled to the detectors to receive output signals therefrom, and 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) functionality 497 can also be provided in the system. Memory 495, controller / processor 490, and I / O 497 can be provided entirely as an integral part of flow cytometer 410. In such embodiments, a display can also form part of I / O functionality 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 functionality 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, along with memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0166] Figure 4B The illustrated system includes six different detectors that detect fluorescence in six different wavelength bands (which can be referred to herein as "filter windows" for a given detector) as defined by the configuration of filters and / or beam splitters in the beam path from flow cell 425 to each detector. Different fluorescent molecules used in a flow cytometer experiment will emit light in their own characteristic wavelength band. The particular fluorescent tags used in an experiment and their associated fluorescence emission bands can be chosen to generally coincide with the filter windows of the detectors. However, as more detectors are provided and more tags are utilized, perfect correspondence between filter windows and fluorescence emission spectra is not possible. Often, while the peak of the emission spectrum of a particular fluorescent molecule can lie within the filter window of one particular detector, some of the emission spectrum of that tag will also overlap with the filter windows of one or more other detectors. This can be referred to as spillover. I / O 497 can be configured to receive data regarding a flow cytometer experiment having a set of fluorescent tags and a plurality of cell populations having a plurality of markers, each cell population having a subset of the plurality of tags. 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 a tag to one or more markers, and cytometer configuration data. Flow cytometer experiment data such as tag spectral characteristics and flow cytometer configuration data can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more tag assignments of markers.

[0167] Figure 5 A functional block diagram of one example of a particle analyzer control system, such as an analysis controller 500, for analyzing and displaying biological events is shown. The analysis controller 500 can be configured to implement various processes for controlling graphical displays of biological events.

[0168] The particle analyzer 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometer 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 via the data communication channel.

[0169] 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 cytometer event data. The analysis controller 500 can be configured to provide a graphical display including a first graph of the biological event data to a display device 506. The analysis controller 500 can be further configured to display a target region by the display device 506 as a gate around the biological event data population overlaid on the first graph. In some embodiments, the gate can be a logical combination of one or more target graphical regions 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.

[0170] The analysis controller 500 can be further configured to display the biological event data within the gate differently from other events of the biological event data outside the gate on the display device 506. For example, the analysis controller 500 can be configured such that the color of the biological event data included within the gate is distinguished from the 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 present a graphical interface.

[0171] The analysis controller 500 can be configured to receive a gate selection signal identifying a gate from a first input device. 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 or manipulated via the display device 506 (e.g., by clicking on or in it when the cursor is located in the desired gate). In some embodiments, the first device can be implemented as a keyboard 508 or other device 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 embodiments, each of the input functions can be considered an input device. For example, as Figure 5As shown, the mouse 510 can include a mouse right button and a mouse left button, each of which can generate a trigger event.

[0172] 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, such as selecting a target population for particle sorting.

[0173] In some embodiments, the analysis controller 500 can be configured to detect when the mouse 510 initiates a gate selection. The analysis controller 500 can be further configured to automatically modify the graphical 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.

[0174] 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 the analysis controller 500 to retrieve biological event data, such as flow cytometer event data.

[0175] The display device 506 can be configured to receive display data from the analysis controller 500. The display data can include graphs of the biological event data and gates outlining portions of the graphs. The display device 506 can be further configured to change the 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.

[0176] In some embodiments, the analysis controller 500 can generate a user interface to receive exemplary events for sorting. For example, the user interface can include a control for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates can be provided prior to collection of event data for a sample, or based on an initial set of events for a portion of a sample.

[0177] Computer control system

[0178] Aspects of the present disclosure further include a computer-controlled system for implementing the subject methods, wherein the system further comprises one or more computers to implement full automation or partial automation of the systems for implementing the methods described herein. In some embodiments, the system comprises a computer having a computer-readable storage medium having stored thereon a computer program, wherein the computer program, when loaded onto the computer, contains instructions for irradiating an acousto-optic device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams, thereby generating a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, and instructions for detecting light from a sample irradiated with the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In embodiments, the system comprises an input module, a processing module, and an output module. In some embodiments, the subject system can comprise an input module for inputting parameters or information about the acousto-optic device (e.g., acousto-optic deflector), the laser, the radio frequency drive source (e.g., waveform generator), the sample, the intensity and wavelength (discrete or range) of the applied light sources, the flow cell diameter, the number of light channels, the number of detection regions, the duration of light source irradiation, the number of different light sources, the distance of light sources to the flow channel, the focal length of any optical conditioning components, the refractive index of the flow channel medium (e.g., sheath fluid), the presence of any wavelength separators, the characteristics of the wavelength separators (including bandpass width, opacity, grating pitch), and the characteristics and sensitivity of the photodetectors.

[0179] In some embodiments, the subject computer-readable storage medium comprises a computer program stored thereon, wherein the computer program, when loaded onto the computer, contains instructions for irradiating an acousto-optic device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams, thereby generating a first set of angularly deflected laser beams and a second set of angularly deflected laser beams, and instructions for detecting light from a sample irradiated with the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In some embodiments, the computer-readable storage medium contains instructions for detecting light from a sample irradiated with the first set of angularly deflected laser beams with a first set of photodetectors and detecting light from a sample irradiated with the second set of angularly deflected laser beams with a second set of photodetectors.

[0180] In some embodiments, the computer program includes instructions for generating optical signals from the first set of photodetectors. In some cases, the generated optical signals include frequency-encoded fluorescence data from particles (e.g., cells) in the fluid stream. In certain cases, the computer program includes instructions for transforming the frequency-encoded fluorescence data from the particles to give spatial data for the particles. In some embodiments, the computer program includes instructions for transforming the frequency-encoded fluorescence data by a Fourier transform of the frequency-encoded fluorescence data. In some cases, the computer program includes instructions for transforming the frequency-encoded fluorescence data by a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data. In other cases, the computer program includes instructions for computing the spatial data by performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data. In yet other cases, the computer program includes instructions for computing the spatial data using a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data.

[0181] In certain embodiments, the computer program includes instructions for computing the spatial data by performing a transform of the frequency-encoded fluorescence data using a phase correction component, such as described in U.S. Patent Application No. 16 / 887,538, filed May 29, 2020, the disclosure of which is incorporated by reference herein.

[0182] In some embodiments, the computer program includes instructions for generating an image of the particles in the fluid stream from the frequency-encoded fluorescence. In some cases, the computer program includes instructions for generating the image from the frequency-encoded fluorescence in combination with detected optical absorption, detected optical scattering, or a combination thereof. The computer program can include instructions for generating one or more images of the particles based on the detected optical signals from the first set of angularly deflected laser beams. In some embodiments, a single image of the particles is generated. In other embodiments, two or more images of the particles are generated, such as 3 or more, 4 or more, 5 or more, including 10 or more images generated based on the detected optical signals from the sample illuminated by the first set of angularly deflected laser beams.

[0183] In other embodiments, the computer program includes instructions for generating optical signals from the second set of photodetectors. In some cases, the computer program includes instructions for wavelength division multiplexing the different wavelengths of light from the fluid stream illuminated with the second set of angularly deflected laser beams. In some embodiments, the computer program includes instructions for spectrally resolving the light detected from the sample in the fluid stream illuminated with the second set of angularly deflected laser beams. In certain embodiments, the computer program includes instructions for determining the overlapping spectral components of the light by computing a spectral unmixing matrix. In some embodiments, the computer program includes instructions for determining the overlap of the spectra of the light from the fluid stream illuminated with the second set of angularly deflected laser beams and an algorithm for computing the contribution of each laser beam to the detected overlapping spectrum. In some embodiments, the computer program includes instructions for spectrally resolving the light by computing a spectral unmixing matrix. In certain embodiments, the computer program includes instructions for computing a spectral unmixing matrix to estimate the abundance of each contribution of a photodetector in the second set of photodetectors to a detected optical signal.

[0184] In some cases, the computer program includes instructions for computing a spectral unmixing matrix to determine the abundance of a fluorophore associated with a particle in the fluid stream. In certain cases, the computer program includes instructions for identifying and classifying the particle based on the abundance of each fluorophore associated with the particle. In certain embodiments, the computer program includes instructions for spectrally resolving the light detected by the plurality of photodetectors in the second set of photodetectors by solving the spectral unmixing matrix using one or more of: 1) a weighted least squares algorithm; 2) a Sherman-Morrison iterative inverse updater; 3) LU matrix decomposition, e.g., factoring a matrix into the product of a lower triangular (L) matrix and an upper triangular (U) matrix; 4) a modified Cholesky decomposition; 5) a weighted least squares algorithm computed by QR factorization; and 6) a weighted least squares algorithm computed by singular value decomposition.

[0185] After the processing module performs one or more steps of the subject method, the output module communicates the results to a user, e.g., by displaying on a monitor or by printing a report.

[0186] The subject system can include hardware and software components, where the hardware components can take the form of one or more platforms, e.g., in the form of servers, such that the functional elements of the system, i.e., the elements of the system that perform particular tasks (e.g., managing the input and output of information, processing information, etc.) can function by executing software applications on the one or more computer platforms with which the system is equipped.

[0187] The system may include a display and an operator input device. For example, the operator input device may be a keyboard, a mouse, etc. The processing module includes a processor that can access memory having instructions stored thereon for performing standard method steps, such as generating a first set of angle-deflected laser beams and a second set of angle-deflected laser beams, and directing the combined laser beam group onto a sample in a fluid flow and detecting the light from the sample irradiated by the first set of angle-deflected laser beams and the second set of angle-deflected laser beams.

[0188] The processing module includes a processor that can access memory storing instructions for performing steps of a target method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor can be a commercially available processor or any other processor that is already available or will be available in the future. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and helps the processor coordinate and execute the functions of various computer programs written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system typically works with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, communication control, and related services according to known technologies. The processor can be any suitable analog or digital system.

[0189] System memory can be any of a variety of known or future memory storage devices. Examples include any generally available random access memory (RAM), magnetic media (e.g., resident hard disks or magnetic tapes), optical media (e.g., optical discs), flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. Memory storage devices of this type typically read content from and / or write content to program storage media (not shown), including optical discs, magnetic tapes, removable hard disks, or floppy disks. Any of these program storage media, or other media currently in use or that may be developed in the future, can be considered a computer program product. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0190] In some embodiments, a computer program product is described that includes a computer usable medium having control logic stored therein that, when executing a computer processor, enables the processor to perform functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine enabled to perform the functions described herein is clearly within the scope of the relevant art.

[0191] The memory can be any suitable device in which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic disk, optical disk, or tape or RAM, or any other suitable fixed or portable device). The processor can include a general purpose digital microprocessor that has been suitably programmed based on a computer readable medium carrying the necessary program code. The program code can be provided remotely to the processor over a communication channel or pre-stored in a computer program product using any device connected to the memory, such as a memory or some other portable or fixed computer readable storage medium. For example, a magnetic or optical disk can carry the program code and can be read using a disk writer / reader. The system of the present invention also includes program code, such as a computer program product, algorithm, for implementing the methods described above. The program code according to the present invention can be recorded in a computer readable medium, such as any medium that can be directly read and accessed by a computer. The medium includes, but is 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; mixtures of these categories, such as magnetic / optical storage media.

[0192] The processor can also access a communication channel to communicate with a user located at a remote location. The remote location means that the user has no direct contact with the system and instead forwards input information from an external device (e.g., 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.

[0193] In some embodiments, the system according to the present disclosure can be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface can be configured for wired or wireless communication, including but not limited to: radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication scheme, WiFi, infrared communication, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication scheme, and cellular communication, such as code division multiple access (CDMA) or global system for mobile communication (GSM).

[0194] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces (e.g., USB ports, RS-232 ports) or any other suitable electrical connection ports, to enable data communication between the subject system and any external device, e.g., a computer terminal configured to enable similar complementary data communication (e.g., in a physician's office or hospital environment).

[0195] In one embodiment, the communication interface is configured for infrared communication, Bluetooth communication, or any other suitable wireless communication scheme to enable the subject system to communicate with other devices, e.g., a computer terminal and / or network, a Bluetooth-enabled mobile phone, a personal digital assistant, or any other communication device that can be used in conjunction by the user.

[0196] In one embodiment, the communication interface is configured to provide data transfer connection through a mobile phone network, a short message service (SMS), using an internet protocol (IP); to provide wireless connection to a personal computer (PC) within a local area network (LAN) connected to the internet; or to provide WiFi connection at a WiFi hotspot to connect to the internet.

[0197] In one embodiment, the subject system is configured to wirelessly communicate with a server device through the communication interface, e.g., using a general standard such as 802.11 or Bluetooth RF scheme or IrDA infrared scheme. The server device can be another portable device, e.g., a smartphone, a personal digital assistant (PDA), or a laptop; or a larger device, e.g., a desktop computer, a piece of equipment, etc. In some embodiments, the server device has a display, e.g., a liquid crystal display (LCD), and an input device (e.g., a button, a keyboard, a mouse, or a touch screen).

[0198] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the subject system (e.g., data stored in the optional data storage unit) with a network or a server device using one or more of the above communication schemes and / or mechanisms.

[0199] The output controller can include a controller for any of a variety of known display devices to provide information to a local or remote user, whether human or machine. If any of the display devices provide visual information, the information can typically be logically and / or physically composed of an array of image elements. A graphical user interface (GUI) controller can include any of a variety of known or later-developed software programs to provide a graphical input and output interface between the system and a user, as well as to process user input. The functional elements of the computer can communicate with each other by way of the system bus. Some of this communication can be accomplished with 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 at a remote location according to known techniques, such as through the Internet, a telephone, or a satellite network, for example. The output manager can implement data display according to a variety of known techniques. In 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 Internet URL addresses so that the user can retrieve other SQL, HTML, XML, or other documents or data from a remote source. One or more of the platforms in the subject system can be any type of known computer platform or one later-developed, although it is typically of a type of computer commonly referred to as a server. However, the platforms can also be mainframe computers, workstations, or other types of computers. They can be connected by any known or later-developed cable or other communication system, including wireless systems connected by networking or other means. They can be located in the same location or physically separated. A variety of operating systems can be employed on any of the computer platforms, depending on the type and / or brand of computer platform selected. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, Ubuntu, Zorin OS, and the like.

[0200] Figure 6 A general architecture of an example computing device 600 is depicted in accordance with certain embodiments. Figure 6 The general architecture of the computing device 600 depicted in the middle includes an arrangement of computer hardware and software components. The computing device 600 can include more or fewer components, and the components depicted are not necessarily all required components. In some embodiments, the computing device 600 can not include all of the components depicted in the middle. Figure 6Those of ordinary skill in the art will recognize that many of the elements shown in FIG. 6 are optional and that the computing device 600 can include more, or fewer, elements than those shown. However, it is not necessary that all of these generally conventional elements be shown in order to provide an enabling disclosure. 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 via the network. The processing unit 610 can also communicate with a memory 670 and further provide output information to an optional display 650 via the input / output device interface 640. The input / output device interface 640 can also receive input from an optional input device 660, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input device.

[0201] 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 general management and operation of the computing device 600. The memory 670 can further include computer program instructions and other information for implementing various aspects of the present disclosure.

[0202] Non-transitory computer-readable storage medium

[0203] 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 employed on one or more computers to achieve complete automation or partial automation of a system to implement the methods described herein. In certain embodiments, instructions in accordance with the methods described herein can be encoded onto a computer-readable medium in "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 such 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 the computer can access and retrieve the information at a later time. The computer-implemented methods described herein can be performed using programs that can be written in one or more of any number of computer programming languages. Such languages include, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), among any number of others.

[0204] In some embodiments, the target computer-readable storage medium includes a computer program stored thereon, where the computer program, when loaded onto a computer, contains instructions for: having an algorithm that irradiates an acousto-optic device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams, thereby generating a first set of angularly deflected laser beams and a second set of angularly deflected laser beams; and for detecting light from a sample irradiated with the first set of angularly deflected laser beams and the second set of angularly deflected laser beams. In some embodiments, the non-transitory computer-readable storage medium contains an algorithm that detects light from a sample irradiated with the first set of angularly deflected laser beams with a first set of photodetectors and detects light from a sample irradiated with the second set of angularly deflected laser beams with a second set of photodetectors.

[0205] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating an optical signal from the first set of photodetectors. In some cases, the generated optical signal includes frequency-encoded fluorescence data from particles (e.g., cells) in the fluid stream. In certain cases, the non-transitory computer-readable storage medium includes an algorithm for transforming the frequency-encoded fluorescence data from the particles to give spatial data for the particles. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for transforming the frequency-encoded fluorescence data by a Fourier transform of the frequency-encoded fluorescence data. In some cases, the non-transitory computer-readable storage medium includes an algorithm for transforming the frequency-encoded fluorescence data by a discrete Fourier transform (DFT) of the frequency-encoded fluorescence data. In other cases, the non-transitory computer-readable storage medium includes an algorithm for computing the spatial data by performing a short-time Fourier transform (STFT) of the frequency-encoded fluorescence data. In yet other cases, the non-transitory computer-readable storage medium includes an algorithm for computing the spatial data using a digital lock-in amplifier to heterodyne and demultiplex the frequency-encoded fluorescence data.

[0206] In certain embodiments, the non-transitory computer-readable storage medium further includes an algorithm for computing the spatial data by performing a transform of the frequency-encoded fluorescence data using a phase correction component, as described, for example, in U.S. Patent Application No. 16 / 887,538, filed May 29, 2020, the disclosure of which is incorporated herein by reference.

[0207] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for generating an image of a particle in the fluid stream from the frequency-encoded fluorescence. In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating the image from the frequency-encoded fluorescence in combination with detected optical absorption, detected optical scattering, or a combination thereof. The non-transitory computer-readable storage medium can include an algorithm for generating one or more images of the particle based on the detected optical signal from the first set of angularly deflected laser beams. In some embodiments, a single image of the particle is generated. In other embodiments, two or more images of the particle are generated, for example, 3 or more, 4 or more, 5 or more, including 10 or more images generated based on the detected optical signal from the sample illuminated by the first set of angularly deflected laser beams.

[0208] In other embodiments, the non-transitory computer-readable storage medium contains algorithms for generating optical signals from the second set of photodetectors. In some cases, the non-transitory computer-readable storage medium contains algorithms for wavelength division multiplexing light of different wavelengths from the fluid stream illuminated with the second set of angularly deflected laser beams. In some embodiments, the non-transitory computer-readable storage medium contains algorithms for spectrally resolving light detected from a sample in the fluid stream illuminated with the second set of angularly deflected laser beams. In certain embodiments, the non-transitory computer-readable storage medium contains algorithms for determining overlapping spectral components of light by computing a spectral unmixing matrix. In some embodiments, the non-transitory computer-readable storage medium contains instructions for determining an overlap of spectra of light from the fluid stream illuminated with the second set of angularly deflected laser beams and algorithms for computing a contribution of each laser beam to the detected overlapping spectrum. In some embodiments, the non-transitory computer-readable storage medium contains algorithms for spectrally resolving light by computing a spectral unmixing matrix. In certain embodiments, the non-transitory computer-readable storage medium contains algorithms for computing a spectral unmixing matrix to estimate an abundance of each contribution of a photodetector in the second set of photodetectors to a detected optical signal.

[0209] In some cases, the non-transitory computer-readable storage medium contains algorithms for computing a spectral unmixing matrix to determine an abundance of a fluorophore associated with a particle in the fluid stream. In certain cases, the non-transitory computer-readable storage medium contains algorithms for identifying and classifying the particle based on the abundance of each fluorophore associated with the particle. In certain embodiments, the non-transitory computer-readable storage medium contains algorithms for spectrally resolving light detected by a plurality of photodetectors in the second set of photodetectors by solving a spectral unmixing matrix using one or more of: 1) a weighted least squares algorithm; 2) a Sherman-Morrison iterative inverse updater; 3) LU matrix decomposition, e.g., factoring a matrix into a product of a lower triangular (L) matrix and an upper triangular (U) matrix; 4) a modified Cholesky decomposition; 5) a weighted least squares algorithm computed by QR factorization; and 6) a weighted least squares algorithm computed by singular value decomposition.

[0210] The non-transitory computer readable storage medium can be used on one or more computer systems having 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 method. The processing module can include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input-output controllers, cache memory, data backup units, and many other devices. The processor can be a commercially available processor, or any of the other processors already available or that can become available. The processor executes the operating system, which, in known fashion, interfaces with the firmware and hardware and assists the processor in coordinating and executing the functions of the various computer programs that can be written in a variety of programming languages such as, for example, Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system typically cooperates with the processor to manage and prioritize data, input and output (I / O) requests, and other functions of the computer system. The operating system also provides scheduling, input-output control, file and data management, memory management, communication control, and related services, according to known techniques.

[0211] Kit

[0212] Aspects of the present invention also include kits, where the kit includes a laser, an acousto-optic device (e.g., an acousto-optic deflector), a beam splitter, and a telescopic lens system. In some embodiments, the laser is a continuous wave laser. In some cases, the beam splitter is a 50:50 beam splitter. The kit can further include one or more additional optical conditioning components, such as one or more mirrors, beam shapers, such as Powell lenses described herein.

[0213] The various assay components of the kit can be present in separate containers, or some or all of them can be pre-assembled. For example, in some cases, one or more components of the kit, such as the acousto-optic device, the beam splitter, and the telescopic lens system components, are present in separate sealed bags (e.g., sterile foil bags or envelopes).

[0214] In addition to the components described above, the subject kits can further include (in certain embodiments) instructions for carrying out 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, attached to the kit, e.g., as paper, foil, or other commonly used materials. Such instructions can also be present in the form of the computer readable medium, e.g., a floppy disc, CD, portable flash drive, or other medium. Alternatively, the instructions can be present in the subject kits in the form of a website address which leads to a website which has information pertaining to use of the kit components. Another form of instructions which can be present is a downloadable electronic file, e.g., in PDF format, which can be downloaded to a computer and used in a standard manner with the aid of the computer.

[0215] Utility

[0216] The subject systems, methods, and computer systems can be used in a variety of applications that require analysis and sorting of particle components in a sample (e.g., a biological sample) within a fluid stream. The present invention can also be used in flow cytometers where there is a need to provide a flow cytometer that has improved cell sorting accuracy, enhanced particle collection capability, reduced energy consumption, improved particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting. In embodiments, the present invention reduces the need for user input or manual adjustments during sample analysis with a flow cytometer. In certain embodiments, the subject systems provide a fully automated solution such that there is little to no need for manual input to adjust the flow cytometer during use.

[0217] The present invention can also be used 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 apparatus facilitate obtaining single 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 test sample 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 apparatus of the present invention are capable of isolating and collecting cells from a biological sample (e.g., an organ, a tissue, a tissue fragment, a fluid) with higher efficiency and lower cost.

[0218] The present disclosure is also defined by the following clauses, despite the appended claims:

[0219] 1. A method of illuminating a sample in a fluid stream, the method comprising:

[0220] generating a first set of angularly deflected laser beams and a second set of angularly deflected laser beams;

[0221] causing the first set of angularly deflected laser beams to propagate along a different optical path than the second set of angularly deflected laser beams; and

[0222] combining the first set of angularly deflected laser beams with the second set of angularly deflected laser beams and directing the combined sets of laser beams onto a sample in a fluid stream; and

[0223] detecting light from the sample.

[0224] 2. The method of clause 1, wherein the first set of angularly deflected laser beams and the second set of angularly deflected laser beams each comprise one local oscillator (LO) beam and a plurality of radio frequency comb beams.

[0225] 3. The method of clause 2, wherein the method comprises irradiating an acousto-optic device with a laser to generate the local-oscillation light beam and the plurality of radio-frequency comb light beams.

[0226] 4. The method of any one of clauses 2-3, wherein the local-oscillation light beam comprises a substantially constant intensity profile along a transverse axis.

[0227] 5. The method of clause 4, wherein the local-oscillation light beam has a Gaussian intensity profile along a longitudinal axis.

[0228] 6. The method of any one of clauses 2-5, wherein the plurality of radio-frequency comb light beams each have substantially the same intensity.

[0229] 7. The method of any one of clauses 1-6, wherein the first set of angularly- deflected laser beams and the second set of angularly-deflected laser beams are generated by a single laser.

[0230] 8. The method of clause 7, wherein the laser is a continuous-wave laser.

[0231] 9. The method of any one of clauses 1-8, wherein the first set of angularly- deflected laser beams propagate to the fluid stream along an optical plane that is parallel to an optical plane of the second set of angularly-deflected laser beams.

[0232] 10. The method of clause 9, wherein the optical plane of the first set of angularly- deflected laser beams is spatially separated from the optical plane of the second set of angularly-deflected laser beams by a predetermined distance.

[0233] 11. The method of any one of clauses 1-10, wherein the method comprises directing the first set of angularly-deflected laser beams to a different spatial location on the fluid stream than the second set of angularly-deflected laser beams.

[0234] 12. The method of clause 11, wherein the first set of angularly-deflected laser beams are directed to a different vertical position on the fluid stream than the second set of angularly-deflected laser beams.

[0235] 13. The method of any one of clauses 1-12, wherein the method further comprises:

[0236] generating a first set of optical signals in response to irradiating the sample with the first set of angularly-deflected laser beams; and

[0237] generating a second set of optical signals in response to irradiating the sample with the second set of angularly-deflected laser beams.

[0238] 14. The method of clause 13, wherein the first set of optical signals comprises time-domain fluorescence emission signals.

[0239] 15. The method of clause 14, further comprising generating an image of particles in the sample based on a plurality of time-domain fluorescence emission signals.

[0240] 16. The method of clause 15, wherein generating the image comprises frequency de-multiplexing each fluorescence emission signal.

[0241] 17. The method of clause 16, wherein frequency de-multiplexing comprises obtaining a Fourier transform of the fluorescence signal.

[0242] 18. The method of any one of clauses 13-17, wherein the method comprises determining spectral abundances from particles in the sample based on a first set of optical signals from the sample.

[0243] 19. The method of any one of clauses 13-18, wherein the second set of optical signals comprises time-domain fluorescence emission signals.

[0244] 20. The method of clause 19, further comprising generating an image of particles in the sample based on a plurality of time-domain fluorescence emission signals.

[0245] 21. The method of clause 20, wherein generating the image comprises frequency de-multiplexing each fluorescence emission signal.

[0246] 22. The method of clause 21, wherein frequency de-multiplexing comprises obtaining a Fourier transform of the fluorescence signal.

[0247] 23. The method of any one of clauses 13-22, wherein the method comprises determining spectral abundances from particles in the sample based on a second set of optical signals from the sample.

[0248] 24. The method of any one of clauses 13-23, wherein the method comprises:

[0249] generating the first set of optical signals with a first set of photodetectors; and

[0250] generating the second set of optical signals with a second set of photodetectors.

[0251] 25. The method of clause 24, wherein:

[0252] the first set of photodetectors comprises a plurality of photomultiplier tubes and the second set of photodetectors comprises a plurality of photodiodes; or

[0253] The first set of photodetectors includes a plurality of photodiodes, and the second set of photodetectors includes a plurality of photomultiplier tubes.

[0254] 26. The method of clause 25, wherein the photodiodes are avalanche photodiodes.

[0255] 27. A system for illuminating a sample in a fluid stream, the system comprising:

[0256] a beam generator comprising:

[0257] a laser;

[0258] an acousto-optic device configured to generate, in response to irradiation by the laser, an output laser beam comprising a plurality of angularly deflected laser beams;

[0259] a first optical conditioning component configured to generate, from the output laser beam, a first set of angularly deflected laser beams and a second set of angularly deflected laser beams; and

[0260] a second optical conditioning component configured to direct the first set of angularly deflected laser beams and the second set of angularly deflected laser beams onto a sample in the fluid stream; and

[0261] a light detection component for detecting light from the sample.

[0262] 28. The system of clause 27, wherein the acousto-optic device comprises an acousto-optic deflector.

[0263] 29. The system of any one of clauses 27-28, wherein the beam generator comprises a single laser.

[0264] 30. The system of any one of clauses 27-29, wherein the first optical conditioning component comprises a beam splitter.

[0265] 31. The system of clause 30, wherein the beam splitter is a 50:50 beam splitter.

[0266] 32. The system of any one of clauses 27-31, wherein the second optical conditioning component is configured to cause the first set of angularly deflected laser beams to propagate along a different optical plane than the second set of angularly deflected laser beams.

[0267] 33. The system of clause 32, wherein the optical plane of the first set of angularly deflected laser beams is spatially separated from the optical plane of the second set of angularly deflected laser beams by a predetermined distance.

[0268] 34. The system of any of Clauses 32-33, wherein the second optical conditioning assembly is configured to direct the first set of angularly deflected laser beams to different spatial locations on the fluid stream than the second set of angularly deflected laser beams.

[0269] 35. The system of Clause 34, wherein the second optical conditioning assembly is configured to direct the first set of angularly deflected laser beams to different vertical locations on the fluid stream than the second set of angularly deflected laser beams.

[0270] 36. The system of any of Clauses 27-35, wherein the second optical conditioning assembly comprises a telescopic lens system.

[0271] 37. The system of any of Clauses 27-36, wherein the light detection assembly comprises:

[0272] a first set of photodetectors configured to detect light from a sample illuminated by the first set of angularly deflected laser beams; and

[0273] a second set of photodetectors configured to detect light from a sample illuminated by the second set of angularly deflected laser beams.

[0274] 38. The system of Clause 37, wherein the first set of photodetectors comprises a plurality of photomultiplier tubes.

[0275] 39. The system of Clause 37, wherein the first set of photodetectors is configured to generate time-domain fluorescence emission signals in response to light from particles in a sample illuminated by the first set of angularly deflected laser beams.

[0276] 40. The system of Clause 39, further comprising a controller comprising a processor having 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 an image of particles in the sample based on a plurality of time-domain fluorescence emission signals.

[0277] 41. The system of any of Clauses 37-40, wherein the first set of photodetectors is configured to determine a spectral abundance of particles in a sample illuminated by the first set of angularly deflected laser beams.

[0278] 42. The system of any of Clauses 37-41, wherein the second set of photodetectors comprises a plurality of photodiodes.

[0279] 43. The system of Clause 35, wherein the photodiodes are avalanche photodiodes.

[0280] 44. The system of any of clauses 37-43, wherein the second set of photodetectors is configured to generate time-domain fluorescence emission signals in response to light from particles in a sample illuminated by the second set of angularly deflected laser beams.

[0281] 45. The system of clause 44, further comprising a controller comprising a processor having 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 an image of particles in the sample based on a plurality of time-domain fluorescence emission signals.

[0282] 46. The system of any of clauses 37-45, wherein the second set of photodetectors is configured to determine a spectral abundance of particles in a sample illuminated by the second set of angularly deflected laser beams.

[0283] 47. The system of any of clauses 27-46, wherein the output laser beam comprises a local oscillator (LO) beam and a plurality of radio frequency comb beams.

[0284] 48. The system of any of clauses 27-47, wherein the first set of angularly deflected laser beams and the second set of angularly deflected laser beams each comprise a local oscillator beam and a plurality of radio frequency comb beams.

[0285] 49. The system of any of clauses 47-48, wherein the local oscillator beam comprises a substantially constant intensity profile along a transverse axis.

[0286] 50. The system of clause 49, wherein the local oscillator beam has a Gaussian intensity profile along a longitudinal axis.

[0287] 51. The system of any of clauses 47-50, wherein the plurality of radio frequency comb beams each have substantially the same intensity.

[0288] 52. The system of any of clauses 27-51, wherein the system is a particle analyzer.

[0289] 53. The system of clause 52, wherein the particle analyzer is part of a flow cytometer.

[0290] 54. A kit comprising:

[0291] a laser;

[0292] an acousto-optic device;

[0293] a beam splitter; and

[0294] Telescopic lens system.

[0295] 55. The kit of clause 54, wherein the laser is a continuous wave laser.

[0296] 56. The kit of any one of clauses 54-55, wherein the acousto-optic device is an acousto-optic deflector.

[0297] 57. The kit of any one of clauses 54-56, wherein the beam splitter is a 50:50 beam splitter.

[0298] 58. The kit of any one of clauses 54-57, further comprising a top hat beam shaper.

[0299] 59. The kit of clause 58, wherein the top hat beam shaper comprises a Powell lens.

[0300] 60. The kit of any one of clauses 54-59, further comprising a light detection system comprising:

[0301] a plurality of photomultiplier tubes; and

[0302] a plurality of photodiodes.

[0303] 61. The kit of clause 60, wherein the photodiodes are avalanche photodiodes.

[0304] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be appreciated that certain changes and modifications can be practiced within the scope of the appended claims, which have been described with specificity in order to satisfy the legal requirements of a patent specification, but which might include other similar techniques or components known or available to those skilled in the art.

[0305] Therefore, the foregoing merely illustrates the principles of the application. It will 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 thus 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 any means described herein as well as any functional equivalents thereof are within the scope of the application. Furthermore, the described subject matter is intended to cover adaptations and modifications in the form, substance, and arrangement of the elements disclosed herein. It is to be understood that the above description is intended to be illustrative, and not restrictive. Many other examples will be apparent to those of skill in the art upon reading the above description. The scope of the application should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with their full scope of equivalents.

[0306] Therefore, the scope of the application is not limited to the exemplary embodiments shown and described herein. Rather, the scope of the application is defined by the appended claims and their equivalents.

Claims

1. A system for irradiating a sample in a fluid flow, the system comprising: A beam generator comprising: Laser; An acousto-optic device configured to generate an output laser beam comprising a plurality of angle-deflected laser beams in response to irradiation by the laser; A first optical adjustment assembly, configured to generate a first set of angle-deflected laser beams and a second set of angle-deflected laser beams from the output laser beam; and A second optical adjustment assembly configured to guide the first set of angle-deflected laser beams and the second set of angle-deflected laser beams onto the sample in the fluid flow, wherein the first set of angle-deflected laser beams and the second set of angle-deflected laser beams are focused at different positions along the longitudinal axis of the fluid flow; and a light detection assembly for detecting light from the sample.

2. The system according to claim 1, wherein, The acousto-optic device includes an acousto-optic deflector.

3. The system according to any one of claims 1-2, wherein, The beam generator comprises a single laser.

4. The system according to claim 1, wherein, The first optical adjustment component includes a beam splitter.

5. The system according to claim 1, wherein, The second optical adjustment component is configured to cause the first set of angle-deflected laser beams to propagate along an optical plane different from that of the second set of angle-deflected laser beams.

6. The system according to claim 1, wherein, The second optical adjustment assembly includes a telescopic lens system.

7. The system according to claim 1, wherein, The optical detection component includes: A first set of photodetectors, configured to detect light from a sample irradiated by the first set of angle-deflected laser beams; and The second set of photodetectors is configured to detect light from the sample irradiated by the second set of angle-deflected laser beams.

8. The system according to claim 7, wherein, The first set of photodetectors is configured to generate a time-domain fluorescence emission signal in response to light from particles in the sample irradiated by the first set of angle-deflected laser beams.

9. The system of claim 8, further comprising a controller including 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 an image of particles in the sample based on a plurality of time-domain fluorescence emission signals.

10. The system according to any one of claims 7-9, wherein, The second set of photodetectors is configured to determine the spectral abundance of particles in the sample irradiated by the first set of angle-deflected laser beams.

11. The system according to claim 10, wherein, The system is a particle analyzer.

12. The system according to claim 11, wherein, The particle analyzer is part of a flow cytometer.

13. A method for irradiating a sample in a fluid flow, the method comprising: Generate the first set of angle-deflecting laser beams and the second set of angle-deflecting laser beams; The first set of angle-deflected laser beams propagates along an optical path different from that of the second set of angle-deflected laser beams; as well as The first set of angle-deflected laser beams and the second set of angle-deflected laser beams are combined and the combined laser beams are guided onto the sample in the fluid flow, wherein the first set of angle-deflected laser beams and the second set of angle-deflected laser beams are focused at different positions along the longitudinal axis of the fluid flow; as well as Detect the light from the sample.

14. The method according to claim 13, wherein, The first set of angle-deflecting laser beams and the second set of angle-deflecting laser beams each include a local oscillation beam and multiple radio frequency comb beams.

15. The method according to claim 14, wherein, The method includes irradiating an acousto-optic device with a laser to generate the local oscillation beam and the plurality of radio frequency comb beams.

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