Method and system for modulating the intensity profile of a laser beam

Abstract

Aspects of the present disclosure include methods for modulating the intensity profile of a laser beam. The method, according to certain embodiments, includes illuminating an acousto-optic device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams, capturing an image of the output laser beam, determining from the captured image an intensity profile of the output laser beam along a horizontal axis, and adjusting one or more parameters of a waveform input to the acousto-optic device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile. Systems having a laser, an acousto-optic device, an imaging sensor, and a waveform generator, as well as a non-transitory computer-readable storage medium having instructions for practicing the subject methods, are also described.

Description

Background Art

[0001] Light detection is often used to characterize the components of a sample (e.g., a biological sample) when the sample is used for the diagnosis of a disease or condition. When the sample is irradiated, light is scattered by the sample, transmitted through the sample, and can also be emitted by the sample (e.g., by fluorescence). Variations in sample components such as morphology, absorption rate, and the presence of fluorescent labels can cause variations in the light scattered, transmitted, or emitted by the sample. To quantify these variations, light is collected and directed towards the surface of a detector.

[0002] One technique that utilizes light detection to characterize components within a sample is flow cytometry. Using the data generated from the detected light, the characteristics of the components can be recorded and desired materials can be sorted. A flow cytometer typically includes a sample reservoir for receiving a fluid sample such as a blood sample, and a sheath reservoir containing sheath fluid.

[0003] A flow cytometer transfers particles (including cells) in a fluid sample as a cell stream into a flow cell while directing the sheath fluid towards the flow cell. Inside the flow cell, a liquid sheath is formed around the cell stream to impart a substantially uniform velocity to the cell stream. The flow cell hydrodynamically constricts the cells in the stream to pass through the center of a light source within the flow cell. Light from the light source can be detected as scattered or by transmission spectroscopy, or absorbed by one or more components within the sample and re-emitted as luminescence.

Summary of the Invention

[0004] Aspects of this disclosure include methods for modulating the intensity profile of a laser beam. Methods according to particular embodiments include irradiating an acousto-optical device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams; capturing an image of the output laser beam; determining an intensity profile of the output laser beam along the horizontal axis from the captured image; and adjusting one or more parameters of a waveform input to the acousto-optical device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile. Systems having a laser, an acousto-optical device, an imaging sensor, and a waveform generator, as well as a non-temporary computer-readable storage medium having instructions for practicing the method of the subject, are also described.

[0005] In practicing the subject method, one or more parameters of the waveform input to the acousto-optical device are adjusted to modulate the intensity of the output laser beam. In some embodiments, the method includes adjusting one or more tones input to the acousto-optical device. In some cases, the amplitude of the tone is adjusted. In other cases, the frequency of the tone is adjusted. In certain cases, each of the tones input to the acousto-optical device is formed from a sum of sine waves, and the method includes adjusting one or more parameters of the sine wave of each input tone. For example, for each input tone, the frequency or amplitude of the sine wave may be adjusted.

[0006] In certain embodiments, the method includes determining the amplitude of each angularly deflected laser beam in the output laser beam and adjusting one or more parameters of the waveform input to the acousto-optical device for each of the angularly deflected laser beams. In some cases, the method includes determining the amplitude of each angularly deflected laser beam in the output laser beam and comparing each determined amplitude with a predetermined threshold intensity. In certain cases, the method includes adjusting the parameters of the waveform input to the acousto-optical device for each of the angularly deflected laser beams that are determined to be below a predetermined threshold intensity. If the intensity of an angularly deflected laser beam is determined to be below a predetermined threshold intensity, the method may include adjusting the frequency or amplitude of the tone of the waveform input to the acousto-optical device.

[0007] In some embodiments, the method includes determining the intensity profiles of multiple angularly deflected light beams across the horizontal axis of the output laser beam. In some embodiments, the intensity profiles are determined by a photodetector. In certain embodiments, an image of the output laser beam is captured by an imaging sensor or beam camera, etc. In some cases, an intensity profile plot is generated from the captured image of the output laser beam, etc. In certain cases, the method includes applying a predetermined threshold to the output laser beam intensity profile plot and identifying angularly deflected beams that fall below the applied predetermined threshold.

[0008] In some embodiments, the method includes generating an output laser beam having a substantially constant intensity profile along the horizontal axis. In some cases, the parameters of the waveform input to the acousto-optic device are tuned in a manner sufficient to generate an output laser beam having a constant intensity profile for one or more of the angularly deflected laser beams. In certain cases, one or more parameters of the input waveform are tuned to generate multiple angularly deflected laser beams having intensities that differ by 10% or less. In other cases, one or more parameters of the input waveform are tuned to generate multiple angularly deflected laser beams having intensities that differ by 5% or less. In embodiments, each of the angularly deflected laser beams along the horizontal axis is spatially isolated. In some cases, each angularly deflected laser beam at least partially overlaps with one other angularly deflected laser beam in the output laser beam.

[0009] Aspects of the present disclosure also include a system for modulating the intensity profile of a laser beam, the subject system (e.g., a particle analyzer) comprising a laser, an acousto-optical device (e.g., an acousto-optical deflector), a photodetector, a waveform generator configured to input a waveform to the acousto-optical device to produce an output laser beam having a plurality of angularly deflected laser beams, and a controller having a processor, the processor having a memory operably coupled to the processor having instructions, the instructions which, when executed by the processor, cause the processor to determine the intensity profile of the output laser beam along the horizontal axis at the photodetector, and to adjust one or more parameters of the waveform input to the acousto-optical device in response to the determined intensity profile to produce an output laser beam having a modulated intensity profile.

[0010] In some embodiments, the photodetector is an imaging sensor or camera, and the memory includes instructions for capturing an image of the output laser beam and adjusting one or more parameters of the waveform input to the acousto-optical device based on the captured image of the output laser beam. In some cases, the memory includes instructions for generating intensity profile plots of multiple angularly deflected light beams across the horizontal axis of the output laser beam. In certain cases, the memory includes instructions for generating intensity profile plots based on the captured image of the output laser beam. In some embodiments, the memory includes instructions for applying a predetermined threshold to the generated output laser beam intensity profile plot and identifying angularly deflected beams that fall below the applied predetermined threshold.

[0011] The subject system includes a waveform generator for inputting a waveform to an acousto-optical device so that, when irradiated by a laser, it generates multiple angle-deflected laser beams. In some embodiments, the waveform generator is an arbitrary waveform generator (AWG). In certain embodiments, the waveform generator is configured to input a waveform to an acousto-optical device so that it generates a local oscillator beam and multiple high-frequency comb beams. In some cases, the multiple high-frequency comb beams are spatially separated. In certain cases, each angle-deflected laser beam at least partially overlaps with one other angle-deflected laser beam in the output laser beam.

[0012] In some embodiments, one or more parameters of a waveform are adjusted by a waveform generator to modulate the intensity of the output laser beam. In some embodiments, the waveform generator is configured to adjust one or more tones of the waveform input to the acousto-optical device. In some cases, the waveform generator is configured to adjust the amplitude of the tones of the waveform input to the acousto-optical device. In other cases, the waveform generator is configured to adjust the frequency of the tones of the waveform input to the acousto-optical device.

[0013] In some embodiments, the waveform generator is configured to adjust one or more parameters of the input waveform for each angle-deflected laser beam generated by a laser-irradiated acousto-optic device. In certain embodiments, the system is configured to determine the amplitude of each angle-deflected laser beam in the output laser beam and to adjust one or more parameters of the waveform input to the acousto-optic device for each angle-deflected laser beam. In some cases, the system is configured to determine the amplitude of each angle-deflected laser beam in the output laser beam and to compare each determined amplitude with a predetermined threshold intensity. In certain cases, the waveform generator is configured to adjust the parameters of the waveform input to the acousto-optic device for each angle-deflected laser beam that is determined to be below a predetermined threshold intensity. If the intensity of an angle-deflected laser beam is determined to be below a predetermined threshold intensity, the waveform generator may be configured to adjust the frequency or amplitude of the tone of the waveform input to the acousto-optic device.

[0014] In certain embodiments, the system is configured to generate an output laser beam having a substantially constant intensity profile along the horizontal axis. In some cases, the waveform generator is configured to adjust the parameters of the waveform input to the acousto-optical device for one or more of the angularly deflected laser beams in a manner sufficient to generate an output laser beam having a constant intensity profile. In certain cases, the waveform generator adjusts one or more parameters of the input waveform to generate multiple angularly deflected laser beams having intensities that differ by less than 10%. In other cases, the waveform generator adjusts one or more parameters of the input waveform to generate multiple angularly deflected laser beams having intensities that differ by less than 5%. In some embodiments, the waveform generator adjusts one or more parameters of the input waveform to generate an output laser beam having a top-hat beam profile.

[0015] Aspects of the present disclosure also include a non-temporary computer-readable storage medium for modulating the output intensity profile of a laser in the subject system. The non-temporary computer-readable storage medium according to a particular embodiment includes instructions stored in the non-temporary computer-readable storage medium, comprising: an algorithm for irradiating an acousto-optical device with a laser to generate an output laser beam including a plurality of angularly deflected laser beams; an algorithm for determining the intensity profile of the output laser beam along the horizontal axis from a captured image; and an algorithm for adjusting one or more parameters of a waveform input to the acousto-optical device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile.

[0016] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for adjusting one or more tones of a waveform input to an acoustic-optical device. In some cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting the amplitude of the tones. In other cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting the frequency of the tones. In a specific case, the non-temporary computer-readable storage medium includes an algorithm for determining the amplitude of each angularly deflected laser beam in the output laser beam, and for each of the angularly deflected laser beams, an algorithm for adjusting one or more parameters of the waveform input to the acoustic-optical device.

[0017] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for determining the amplitude of each angularly deflected laser beam in the output laser beam, and an algorithm for comparing each determined amplitude with a predetermined threshold intensity. In some cases, the non-temporary computer-readable storage medium includes an algorithm for identifying angularly deflected laser beams having an intensity below a predetermined threshold intensity. In specific cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting the parameters of the waveform input to the acousto-optical device for each angularly deflected laser beam determined to be below a predetermined threshold intensity. For example, the non-temporary computer-readable storage medium may include an algorithm for adjusting the frequency or amplitude of the tone of the waveform input to the acousto-optical device for each angularly deflected laser beam.

[0018] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for determining the intensity profiles of multiple angularly deflected light beams across the horizontal axis of the output laser beam. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for determining the intensity profile based on data signals from a photodetector. In other embodiments, the non-temporary computer-readable storage medium includes an algorithm for capturing an image of the output laser beam using an imaging sensor or beam camera. In some cases, the non-temporary computer-readable storage medium includes an algorithm for generating a plot of the intensity profiles. In specific cases, the non-temporary computer-readable storage medium includes an algorithm for applying a predetermined threshold to the output laser beam intensity profile plot and an algorithm for identifying angularly deflected beams that fall below the applied predetermined threshold.

[0019] In certain embodiments, the non-temporary computer-readable storage medium includes an algorithm for generating an output laser beam having a substantially constant intensity profile along the horizontal axis. In some cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting the parameters of the waveform input to the acousto-optical device for one or more of the angularly deflected laser beams, and an algorithm for generating an output laser beam having a constant intensity profile. In certain cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting one or more parameters of the input waveform to generate multiple angularly deflected laser beams having intensities that differ by 10% or less. In other cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting one or more parameters of the input waveform to generate multiple angularly deflected laser beams having intensities that differ by 5% or less. [Brief explanation of the drawing]

[0020] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures.

[0021] [Figure 1A] This demonstrates the generation of an intensity profile plot of an output laser beam having multiple angularly deflected laser beams, according to a specific embodiment. [Figure 1B] This demonstrates how, by adjusting the intensity of one or more of the angularly deflected laser beams according to a specific embodiment, an output laser beam with a substantially constant intensity profile along the horizontal axis can be generated. [Figure 2] A flowchart is shown for modulating the intensity profile of an output laser beam having multiple angle-deflected laser beams according to a specific embodiment. [Figure 3A] This document describes a system for modulating an output laser beam having multiple angle-deflected laser beams, according to a specific embodiment. [Figure 3B]A system for modulating an output laser beam having a plurality of angularly deflected laser beams according to a particular embodiment is shown. [Figure 4A] A functional block diagram of a particle analysis system for computational-based sample analysis and particle characterization according to a particular embodiment is shown. [Figure 4B] A flow cytometer according to a particular embodiment is shown. [Figure 5] A functional block diagram for an example of a particle analyzer control system according to a particular embodiment is shown. [Figure 6] A block diagram of a computing system according to a particular embodiment is shown. **DETAILED DESCRIPTION**

[0022] Aspects of the present disclosure include a method for modulating the intensity profile of a laser beam. The method according to a particular embodiment includes irradiating an acousto-optic device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams, capturing an image of the output laser beam, determining from the captured image an intensity profile of the output laser beam along a horizontal axis, and adjusting one or more parameters of a waveform input to the acousto-optic device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile. A system having a laser, an acousto-optic device, an imaging sensor, and a waveform generator, as well as a non-transitory computer-readable storage medium having instructions for practicing the subject method, is also described.

[0023] Before the present invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, and thus, of course, may vary. 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] When a range of values is provided, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, as well as any other recited value or intermediate value within the recited range of this description, is understood to be encompassed by the present invention. The upper and lower limits of these smaller ranges may be individually included in the smaller ranges as well and are likewise encompassed by the present invention, subject to any specifically excluded limits in the recited range. When the recited range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present invention.

[0025] As used herein, the term "about" is used before a numerical value to present a specific range. As used herein, the term "about" is used to provide literal support for the exact number that it precedes, and for numbers that are close to, or approximate, the number that the term precedes. When determining whether a number is close to, or approximate to, a specifically recited number, the close or approximate unrecited number can be a number that provides a substantial equivalent of the specifically recited number in the context in which it is presented.

[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 invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, but representative and exemplary methods and materials are described below.

[0027] All publications and patents cited herein are incorporated herein by reference, and by being incorporated herein by reference, disclose and describe the relevant methods and / or materials from which those publications are cited, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Any citation of a publication relates to its disclosure prior to the filing date, and the present invention should not be construed as acknowledging that such publication has no prior rights on the grounds of prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may need to be individually verified.

[0028] It should be noted that, as used herein and in the appended claims, the articles “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly indicated by the context. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to function as an antecedent for the use of exclusive terms such as “solely” and “only,” or for the use of “negative” limitation, in relation to the enumeration of claim elements.

[0029] As will be apparent to those skilled in the art upon reading this disclosure, each of the distinct embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events, or in any other logically possible order.

[0030] Apparatus and methods are described or will be described for grammatical fluidity with functional description, but claims should not necessarily be construed as being limited by constructing “means” or “step” limitations unless expressly formulated under Section 112 of the United States Patent Act, and should be granted the meaning of the definitions and the full scope of the equivalent provided by the claims under the statutory principle of equivalents, and should be explicitly understood that if the claims are expressly formulated under Section 112 of the United States Patent Act, a full statutory equivalent should be granted under Section 112 of the United States Patent Act.

[0031] As summarized above, this disclosure provides a method for modulating the intensity profile of a laser beam. Further description of embodiments of this disclosure first describes in more detail a method for determining the intensity profile of an output laser beam having multiple angularly deflected laser beams and modulating the intensity profile along the horizontal axis. Next, a system for practicing the subject method is also described, having a laser, an acousto-optical device, a laser beam detector, a waveform generator, and a controller for adjusting waveform parameters input to the acousto-optical device. A non-temporary computer-readable storage medium having instructions for practicing the subject method, and a kit having one or more components of the subject system are also provided.

[0032] Method for modulating an output laser beam having an angularly deflected laser beam Aspects of this disclosure include a method for modulating the intensity profile of an output laser beam having multiple angle-deflected laser beams (for example, for irradiating a sample in a flowstream). In practicing the method according to the embodiments, an acousto-optical device is irradiated with a laser to generate multiple angle-deflected laser beams, determines the intensity profile of the output laser beam along the horizontal axis, and adjusts one or more parameters of the waveform input to the acousto-optical device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile. Each angle-deflected laser beam is generated based on a waveform input to the acousto-optical device from a waveform generator, as described in more detail below. To modulate the intensity of each angle-deflected laser beam, one or more parameters of the input waveform can be adjusted as described herein. The term “angle-deflected laser beam” is used herein in its conventional sense to refer to a laser beam generated by an applied high-frequency drive signal through the interaction of acoustic waves within an acousto-optical device, where the optical beam from the laser generates one or more beamlets having optical frequency shifts and propagation angle deflections.

[0033] In embodiments, the method includes irradiating an acousto-optical device with a laser. The laser in question may include pulsed lasers or continuous-wave lasers. The type and number of lasers used in the subject method may vary and may be gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other cases, the method includes irradiating an acousto-optical device with a dye laser such as a stilbene, coumarin, or rhodamine laser. In further cases, the method includes irradiating an acousto-optic device with a metallic 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, or combinations thereof. In yet another case, the method includes irradiating an 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:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium-sapphire laser, a thulium-YAG laser, a ytterbium-YAG laser, a Y2O3 laser, or a cerium-doped laser, or combinations thereof. In other cases, the method includes the implementation of a semiconductor diode laser, an optically excited semiconductor laser (OPSL), or any of the aforementioned lasers at twice or three times their frequency.

[0034] Depending on the desired wavelength of light generated by the output laser beam (for example, for use when irradiating a sample in a flow stream), the laser may have specific wavelengths differing by 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. The acousto-optic device may be irradiated with one or more lasers, including, for example, two or more lasers, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, and ten or more lasers. The lasers may include any combination of several types of lasers. For example, in some embodiments, this method involves irradiating the acousto-optic device with a laser array, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0035] When two or more lasers are employed, the acousto-optical device may be irradiated by the lasers simultaneously, sequentially, or in combination thereof. For example, the acousto-optical device may be irradiated simultaneously by each of the lasers. In other embodiments, the acousto-optical device may be irradiated sequentially by each of the lasers. When two or more lasers are employed to sequentially irradiate the acousto-optical device, the time each laser irradiates the acousto-optical device may be 0.001 microseconds or more individually, including, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, and 60 microseconds or more. For example, the method may include irradiating the acousto-optical device with lasers for durations ranging from 0.001 microseconds to 100 microseconds, including, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In embodiments in which an acoustic-optical device is sequentially irradiated by two or more lasers, the duration for which the acoustic-optical device is irradiated by each laser may be the same or different.

[0036] The interval between irradiations by each laser may also vary, as desired, by individual delays of 0.001 microseconds or more, including, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, up to 10 microseconds or more, for example, up to 15 microseconds or more, for example, up to 30 microseconds or more, and 60 microseconds or more. For example, the interval between irradiations by each light source may range from 0.001 microseconds to 60 microseconds, including, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In a particular embodiment, the interval between irradiations by each laser is 10 microseconds. In embodiments in which the acousto-optical device is sequentially irradiated by more than two (i.e., three or more) lasers, the delay between irradiations by each laser may be the same or different.

[0037] Acousto-optical devices can be irradiated continuously or at discrete intervals. In some cases, the method involves continuously irradiating the acousto-optical device with a laser. In other cases, the acousto-optical device is irradiated with a laser at discrete intervals or at some other interval, including, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds.

[0038] Depending on the laser, the acousto-optical device may be illuminated from different distances, including, for example, 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, and 50 mm or more. The illumination angle may also vary, for example, from 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and for example, 90°, including 30° to 60°, within a range of 10° to 90°.

[0039] The method according to this disclosure includes inputting waveforms from a waveform generator (e.g., an arbitrary waveform generator) for each angle-deflected laser beam into an acousto-optical device to generate multiple angle-deflected laser beams of an output laser beam. In practicing the method of the subject, one or more waveforms may be generated and input to the acousto-optical device for each angle-deflected laser beam in the output beam, including, for example, inputting two or more, for example three or more, for example four or more, for example five or more, for example ten or more, for example fifteen or more, for example twenty-five or more, for example fifty or more, and one hundred or more waveforms into the acousto-optical device. In some embodiments, the input waveforms include one or more tones, for example two or more tones, for example three or more tones, for example four or more tones, for example five or more tones, and one or more tones. Each tone is a sum of different sine waves, such as the sum of two or more different sine waves, including in certain cases, for example, three or more, for example, four or more, for example, five or more, for example, ten or more, for example, twenty-five or more, for example, one hundred or more, and each tone is the sum of five hundred or more different sine waves.

[0040] In some embodiments, the angle-deflected laser beams generated by the input waveforms each have an intensity based on the amplitude of the waveform tone. In some embodiments, the waveform tones input to the acousto-optic device each have substantially the same amplitude individually, and the amplitudes of the waveform tones input to the acousto-optic device differ from each other by only 10% or less, including, for example, only 9% or less, for example 8% or less, for example 7% or less, for example 6% or less, for example 5% or less, for example 4% or less, for example 3% or less, for example 2% or less, for example 1% or less, for example 0.1% or less, and for example 0.01% or less. In some embodiments, the sine wave forming each tone has an amplitude that differs between tones by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less.

[0041] In practicing the subject method, the intensity profile of the output laser beam is determined along the horizontal axis. The intensity profile of the output laser beam can be measured by any convenient protocol, including but not limited to scanning slit profilers, charge-coupled devices (CCDs, e.g., ICCDs), positioning sensors, power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser photometers, and laser diode detectors, among other types of photodetectors. In some cases, to determine the intensity profile of the output light beam, the relative intensity of each output laser light beam is plotted as a function of the distance from the optical axis (along the orthogonal horizontal axis) of the output light beam to determine the intensity profile at the point of illumination. In certain embodiments, the deviation of relative intensity at a given distance from the optical axis is calculated to determine whether the beam profile of the output light beam exhibits substantially constant intensity from each edge to the center along the horizontal axis. In other embodiments, the deviation of relative intensity is calculated over the entire horizontal axis of the beam profile of the output light beam to determine whether the output light beam exhibits substantially constant intensity from the edge to the center.

[0042] In some embodiments, the intensity profile of the output laser beam is determined by capturing images of the output laser beam. For example, the method may include capturing two or more images of the output laser beam and determining the intensity profile of the output laser beam along the horizontal axis, including capturing three or more images of the output laser beam, for example, four or more images, for example, five or more images, for example, six or more images, for example, seven or more images, for example, eight or more images, for example, nine or more images, for example, ten or more images, for example, twenty-five or more images, for example, fifty or more images, and one hundred or more images of the output laser beam and determining the intensity profile of the output laser beam along the horizontal axis. If two or more images are captured, the multiple images may be automatically stitched together by a processor having a digital image processing algorithm.

[0043] The image of the output laser beam may be captured by any suitable device capable of capturing an optical image and converting it into an electronic data signal, including but not limited to charge-coupled devices, semiconductor charge-coupled devices (CCDs), active pixel sensors (APS), complementary metal-oxide-semiconductor (CMOS) image sensors, or N-type metal-oxide-semiconductor (NMOS) image sensors. In some embodiments, the imaging sensor is a CCD camera. For example, the camera may be a CCD (EMCCD) camera or an intensified CCD (ICCD) camera. In other embodiments, the imaging sensor is a CMOS camera.

[0044] Images of the output laser beam can be captured by one or more imaging sensors. In some embodiments, the intensity profile of the output laser beam is captured by a combination of imaging sensors, including, for example, two or more imaging sensors, for example, three or more imaging sensors, for example, four or more imaging sensors, and five or more imaging sensors. When two or more imaging sensors are employed, images of the output laser beam can be captured by the imaging sensors simultaneously, sequentially, or in combination thereof. For example, when images of the output laser beam are captured by two imaging sensors, the method of the subject may include simultaneously capturing images of the intensity profile of the output laser beam with both imaging sensors. In other embodiments, the method includes sequentially capturing images of the output laser beam with two imaging sensors. When images of the output laser beam are captured sequentially, the time between images captured by the imaging sensors can be, individually, 0.001 seconds or more, including, for example, 0.01 seconds or more, for example, 0.1 seconds or more, for example, 1 second or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 30 seconds or more, and 60 seconds or more. In an embodiment in which an image of the output laser beam is sequentially captured by two or more imaging sensors, the duration between each image capture may be the same or different.

[0045] Images of the output laser beam can be captured continuously or at discrete intervals. In some cases, the method involves capturing images continuously. In other cases, the method involves capturing images at discrete intervals, such as capturing images of a flow stream, including every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other interval.

[0046] In some embodiments, determining the intensity profile of an output laser beam involves generating an intensity profile plot of multiple angle-deflected laser beams along a horizontal axis. In generating the intensity profile plot, the method may include determining the intensity value from each angle-deflected laser beam and plotting each intensity value against the position of the angle-deflected laser beam along the horizontal axis. In some cases, the intensity value is determined based on the amplitude of the angle-deflected laser beam along the horizontal axis. In other cases, the intensity value is determined based on the brightness of the pixels produced by each angle-deflected laser beam in a captured image of the output laser beam. In some embodiments, each intensity value plotted in the intensity profile plot is based on a single measurement of the output laser beam. In other embodiments, each intensity value plotted in the intensity profile plot is based on the average intensity of each angle-deflected laser beam over a predetermined irradiation period. For example, the intensity value determined for each angle-deflected laser beam along the horizontal axis may be the average intensity of two or more measurements, including the average intensity determined from, for example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty-five or more, fifty or more, and one hundred or more measurements of the intensity of each angle-deflected laser beam.

[0047] Figure 1A shows the generation of an intensity profile plot of an output laser beam having multiple angle-deflected laser beams according to a specific embodiment. An image 100 of the output laser beam is captured by an imaging sensor, and the intensity of each angle-deflected laser beam is plotted based on its position along the horizontal axis to generate a profile plot 101. In image 100, the brightness of each pixel in the image at each position is used to determine the intensity of the angle-deflected laser beam on the output laser beam intensity profile plot 101.

[0048] In certain embodiments, the method may include evaluating the intensity profile of the output laser beam (e.g., by a human or computer-implemented protocol, as described in more detail below) and identifying one or more adjustments to generate an output laser beam having a desired intensity profile. Depending on the number of angular deflection laser beams in the output laser beam, the intensity of one or more of the angular deflection laser beams may be adjusted, including cases where the intensity of, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 25 or more, 50 or more, and 100 or more of the angular deflection laser beams is adjusted.

[0049] In some embodiments, the method includes adjusting the intensity of one or more of one or more angle-deflected laser beams in the output laser beam in such a way that two or more angle-deflected laser beams have the same intensity, including, for example, when three or more angle-deflected laser beams have the same intensity, for example, when four or more angle-deflected laser beams have the same intensity, for example, when five or more angle-deflected laser beams have the same intensity, for example, when six or more angle-deflected laser beams have the same intensity, for example, when seven or more angle-deflected laser beams have the same intensity, for example, when eight or more angle-deflected laser beams have the same intensity, for example, when nine or more angle-deflected laser beams have the same intensity, for example, when ten or more angle-deflected laser beams have the same intensity, for example, when 25 or more angle-deflected laser beams have the same intensity, for example, when 50 or more angle-deflected laser beams have the same intensity, and when 100 or more angle-deflected laser beams have the same intensity.

[0050] In some embodiments, the method includes adjusting the intensity of one or more of the angularly deflected laser beams of the output laser beam in such a way that the multiple angularly deflected laser beams of the output laser beam have intensities that differ from each other by only 10% or less, including, for example, 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, and 0.001% or less. In certain embodiments, the method includes adjusting the intensity of one or more of the angularly deflected laser beams in such a way that the output laser beam exhibits a top-hat beam profile along the horizontal axis. The term “top-hat” is used herein in its conventional sense to refer to an irradiated beam (e.g., light) having a substantially uniform flow rate (energy density) along one or more axes perpendicular to the optical axis of the irradiated beam. In the embodiment, the output light beam having a top-hat intensity profile exhibits little to no deviation in relative intensity from each edge to the center along the horizontal axis, and the light 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 along the horizontal axis, and for example, 96% to 99.5%, including 98% to 99% of the intensity at the edges along the horizontal axis.

[0051] In some cases, the method includes increasing the intensity of one or more of the angle-deflected laser beams to the intensity of a top-hat intensity profile, including increasing the intensity of, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more, for example, twenty-five or more, for example, fifty or more, and one hundred or more of the angle-deflected laser beams to the intensity of a top-hat intensity profile. In other cases, the method includes decreasing the intensity of one or more of the angle-deflected laser beams to the intensity of a top-hat intensity profile, including decreasing the intensity of, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more, for example, twenty-five or more, for example, fifty or more, and one hundred or more of the angle-deflected laser beams to the intensity of a top-hat intensity profile.

[0052] Figure 1B shows a particular embodiment in which the intensity of one or more angle-deflected laser beams is adjusted to generate an output laser beam with a substantially constant intensity profile along the horizontal axis. An image 100 of the output laser beam is captured by an imaging sensor, and the intensity of each angle-deflected laser beam is plotted based on its position along the horizontal axis to generate a profile plot 101. Based on the generated intensity profile plot, the intensity of multiple angle-deflected laser beams in the output laser beam is increased (103). After adjusting the intensity of multiple angle-deflected laser beams, a second image 102 of the output laser beam is captured, and an intensity profile plot of the adjusted output laser beam 104 is generated. As described in more detail below, the intensity of each angle-deflected laser beam is adjusted by identifying the position of the angle-deflected laser beam in the output laser beam along the horizontal axis (either by the captured image or by the intensity profile plot), thereby adjusting the waveform input to the acousto-optical device.

[0053] In practicing the subject method, parameters of the waveform input to the acousto-optical device may be adjusted to adjust the intensity of one or more of the angularly deflected laser beams. In some embodiments, each angularly deflected laser beam whose intensity increase or decrease is desired is identified, and one or more parameters of the waveform input for each identified angularly deflected laser beam are adjusted, including, for example, adjusting two or more parameters, for example, three or more parameters, for example, four or more parameters, for example, five or more parameters, for example, six or more parameters, for example, seven or more parameters, for example, eight or more parameters, for example, nine or more parameters, for example, ten or more parameters, for example, twenty-five or more parameters, for example, fifty or more parameters, and one or more parameters of the waveform input for each identified angularly deflected laser beam.

[0054] In some cases, the method involves adjusting one or more tones of the input waveform for each identified angular deflection laser beam, including, for example, two or more tones, for example, three or more tones, for example, four or more tones, for example, five or more tones, and ten or more tones. In some cases, adjusting one or more tones of the input waveform for each identified angular deflection laser beam involves adjusting one or more distinct sine waves whose sum constitutes each tone, including, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, ten or more, for example, fifteen or more, for example, twenty-five or more, for example, fifty or more, and one hundred or more.

[0055] When the tone is adjusted, the method according to a particular embodiment includes adjusting the amplitude of the tone of the input waveform for each of the identified angle-deflected laser beams. In some cases, the amplitude of the tone of the input waveform can be increased by 5% or more, including increasing it by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more. In some cases, the amplitude of the tone of the input waveform can be decreased by 5% or more, including decreasing it by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more.

[0056] In other embodiments, the method includes adjusting the frequency of the input waveform tone for each of the identified angle-deflected laser beams. In some cases, the frequency of the input waveform tone may be increased by 5% or more, including increasing the frequency of the input waveform tone by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more. In other cases, the amplitude of the input waveform tone may be decreased by 5% or more, including decreasing the frequency of the input waveform tone by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more.

[0057] In certain embodiments, the method includes adjusting the tone of the waveform input to the acousto-optical device for each angle-deflected laser beam to have substantially the same amplitude, for example, the amplitudes of the tone of the waveform input to the acousto-optical device are adjusted to differ from each other by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less.

[0058] In some embodiments, the method includes adjusting the sine waves forming each tone to have substantially the same amplitude, for example, the amplitudes of the sine waves of each tone are adjusted to differ from each other by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less. In other embodiments, the method includes adjusting the sine waves forming each tone to have substantially the same frequency, for example, the frequencies of the sine waves of each tone are adjusted to differ from one another by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less.

[0059] In some embodiments, the adjustment of the input waveform may be repeated one or more times as desired, based, for example, on a determined output laser beam intensity profile (e.g., from a captured image of the output laser beam). In some embodiments, the method includes adjusting one or more parameters of the waveform input to the acousto-optic device for an angular deflection laser beam, and determining the output laser beam intensity profile generated by the adjusted input waveform. In these embodiments, the method may further include adjusting the input waveform one or more subsequent times, for example, two or more times, for example, three or more times, for example, four or more times, for example, five or more times, adjusting one or more parameters of the waveform input to the acousto-optic device for an angular deflection laser beam, and determining the output laser beam intensity profile generated by the adjusted input waveform ten or more times.

[0060] Figure 2 shows a flowchart for modulating the intensity profile of an output laser beam having multiple angle-deflected laser beams according to a specific embodiment. In step 201, an acousto-optical device (e.g., an acousto-optical modulator) is irradiated with a laser to generate an output laser beam having multiple angle-deflected laser beams. To generate multiple angle-deflected laser beams, the waveform of each angle-deflected laser beam is input from a waveform generator to the acousto-optical device. In step 202, an image of the beam profile of the output laser beam along the horizontal axis is captured. In some cases, the brightness of the pixels at each position in the captured image corresponds to the intensity of the angle-deflected laser beam in the intensity profile. In step 203, the intensity profile of the output laser beam is determined, for example, by generating intensity profile plots shown in Figures 1A and 1B. In step 204, one or more adjustments to the angle-deflected laser beam are determined based on one or more of the captured image generated in step 202 and the intensity profile plots generated in step 203. To adjust the intensity of the identified angle-deflected laser beams, the parameters of the waveform input to the acousto-optic device are adjusted for each angle-deflected laser beam. In some cases, one or more tones of the input waveform are adjusted by increasing or decreasing the amplitude of the tones of the input waveform. A second image of the output laser beam is captured in step 205 to generate an image, and the intensity profile of the modulated laser beam can be determined in step 206 (i.e., after adjustment of one or more input waveforms).

[0061] In some embodiments, the angular deflection laser beams in the output laser beam are spatially separated. Depending on the waveform input to the acousto-optic device and the desired irradiation profile of the output laser beam, the angular deflection laser beams can be separated by 0.001 μm or more, including, for example, only 0.005 μm or more, for example 0.01 μm or more, for example 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm or more, for example 1 μm or more, for example 5 μm or more, for example 10 μm or more, for example 100 μm or more, for example 500 μm or more, for example 1000 μm or more, for example 1000 μm or more, for example 500 μm or more, and for example 5000 μm or more. In some embodiments, the angular deflection laser beams overlap, for example, with adjacent angular deflection laser beams along the horizontal axis of the output laser beam. Overlap between adjacent angle-deflected laser beams (such as beam spot overlap) can be 0.001 μm or larger, including overlaps of 0.005 μm or larger, 0.01 μm or larger, 0.05 μm or larger, 0.1 μm or larger, 0.5 μm or larger, 1 μm or larger, 5 μm or larger, 10 μm or larger, and 100 μm or larger.

[0062] In certain embodiments, the method includes adjusting the spatial width of the output laser beam, such as adjusting the spatial width of the output laser beam along the horizontal axis of the output laser beam. Depending on the size of the output laser beam, the spatial width of the output laser beam can be increased by 0.001 μm or more, for example, by 0.005 μm or more, for example, by 0.01 μm or more, for example, by 0.05 μm or more, for example, by 0.1 μm or more, for example, by 0.5 μm or more, for example, by 1 μm or more, for example, by 5 μm or more, for example, by 10 μm or more, for example, by 100 μm or more, for example, by 500 μm or more, for example, by 1000 μm or more, for example, by 5000 μm or more, and for example, by 5000 μm or more. In other embodiments, the method includes reducing the spatial width of the output laser beam by 0.001 μm or more, for example, by 0.005 μm or more, for example, by 0.01 μm or more, for example, by 0.05 μm or more, for example, by 0.1 μm or more, for example, by 0.5 μm or more, for example, by 1 μm or more, for example, by 5 μm or more, for example, by 10 μm or more, for example, by 100 μm or more, for example, by 500 μm or more, for example, by 1000 μm or more, and for example, by 5000 μm or more.

[0063] In some embodiments, the method further includes irradiating a sample in a flow stream (e.g., in a flow cytometer) with an output laser beam having a substantially uniform intensity profile (e.g., a top-hat beam intensity profile). In some embodiments, the sample is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungus, or, in certain cases, a subset of the tissues, cells, or components of an animal that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to both a natural organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and urinary tract, tears, saliva, milk, blood cells, tumors, and organs. The biological sample can be any type of biological tissue, including both healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is blood or its derivatives, e.g., plasma, or other biological fluid samples, e.g., liquid samples such as tears, urine, or semen. In some cases, the sample is a blood sample, including whole blood, such as blood obtained by venipuncture or from a fingertip (the blood may or may not be combined with any reagents before the assay, such as preservatives or anticoagulants).

[0064] In certain embodiments, the sample source is a “mammal” or “animal of a mammal,” and these terms are broadly used to describe organisms within mammals, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is human. The method may be applied to samples obtained from human subjects of both sexes at any stage of development (i.e., neonates, infants, adolescents, and adults), and in certain embodiments, the human subject may be an adolescent, adolescent, or adult. While the present invention may be applied to samples from human subjects, it should be understood that it may also be applied to samples from other animal subjects (i.e., “non-human subjects”), such as birds, mice, rats, dogs, cats, livestock, and horses, but is not limited to the above.

[0065] In certain embodiments, the biological sample contains cells. Cells that may be present in the sample include eukaryotic cells (e.g., mammalian cells) and / or prokaryotic cells (e.g., bacterial or archaeal cells). The sample may be obtained from an in vitro source (e.g., a suspension of cells derived from cultured and grown laboratory cells) or an in vivo source (e.g., mammalian subjects, human subjects, etc.). In some embodiments, the cell sample is obtained from an in vitro source. The in vitro source includes, but is not limited to, prokaryotic (e.g., bacterial, archaeological) cell cultures, environmental samples containing prokaryotic and / or eukaryotic (e.g., mammalian, ostrich, fungal, etc.) cells, eukaryotic cell cultures (e.g., cultures of established cell lines, cultures of known or purchased cell lines, cultures of immortalized cell lines, cultures of primary cells, cultures of experimental yeast, etc.), tissue cultures, etc.

[0066] If the biological sample contains cells, the methods of this disclosure may include cell characterizing components, such as cell fragments, fragmented cell membranes, organelles, dead cells, or lysed cells. In some embodiments, the methods include characterizing extracellular vesicles of cells. Characterizing extracellular vesicles of cells may include identifying the type of extracellular vesicle within the cell or determining the size of the extracellular vesicle within the cell.

[0067] The sample in the flowstream may be irradiated continuously or at discrete intervals. In some cases, the method involves continuously irradiating the sample in the flowstream with the output laser beam. In other cases, the sample in the flowstream is irradiated with the output laser beam at discrete intervals or at some other interval, including, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds.

[0068] Samples in the flowstream may be irradiated with the output laser beam from different distances, including, for example, 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, and 50 mm or more. The irradiation angle may also vary, for example, from 15° to 85°, for example, from 20° to 80°, for example, from 25° to 75°, and for example, from 90°, including 30° to 60°, within a range of 10° to 90°.

[0069] The flow rate of the flowstream may vary depending on the light intensity, for example, and may be 1 μL / min or more, including 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 25 μL / min or more, 50 μL / min or more, 75 μL / min or more, 100 μL / min or more, 250 μL / min or more, 500 μL / min or more, 750 μL / min or more, and 1000 μL / min or more. In certain embodiments, the flow rate of the flow stream in the method of the subject is in the range of 1 μL / min to 500 μL / min, including, for example, 1 μL / min to 250 μL / min, for example, 1 μL / min to 100 μL / min, for example, 2 μL / min to 90 μL / min, for example, 3 μL / min to 80 μL / min, for example, 4 μL / min to 70 μL / min, for example, 5 μL / min to 60 μL / min, and 10 μL / min to 50 μL / min. In certain embodiments, the flow rate of the flow stream is 5 μL / min to 6 μL / min.

[0070] Methods according to specific embodiments also include detecting light from a sample in a flow stream. The detected light may be side-scattered light, forward-scattered light, emitted light, or a combination thereof. Preferred photodetection protocols include, but are not limited to, optical sensors or photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), sensitized charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In specific embodiments, light from a flow stream irradiated in the sample inspection area of ​​a particle sorting module is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In specific embodiments, light from an irradiated flow stream is measured by a charge-coupled device (CCD).

[0071] In some embodiments, light (e.g., forward scattered light, side scattered light, emitted light, etc.) is detected directly from the sample in the flowstream. In other embodiments, light from the sample in the flowstream is propagated to a detector having one or more optical adjustment components. "Optical adjustment" means that the light from the sample in the flowstream is modified as desired. For example, the beam path, direction, focus, or collimation of the light from the sample in the flowstream can be modified by the optical adjustment components. In some cases, the dimensions of the light collected from the sample in the flowstream are adjusted by, for example, increasing the dimensions by 10% or more, for example, 25% or more, for example, 50% or more, and 75% or more, and increasing the dimensions by 5% or more, or by, for example, decreasing the dimensions by 10% or more, for example, 25% or more, for example, 50% or more, and 75% or more, and focusing the light to reduce the dimensions of the light by 5% or more. In other cases, optical adjustment includes collimating the light. The term “collimate” is used in its conventional sense to refer to optically adjusting the collinearity of light propagation, or reducing divergence due to light from a common propagation axis. In some cases, collimation includes narrowing the spatial cross-section of a light beam. In certain embodiments, the optical adjustment component is a wavelength separator. The term “wavelength separator” is used herein in its conventional sense to refer to an optical protocol for separating polychromatic light into its constituent wavelengths. Wavelength separation in some embodiments may include selectively passing or blocking specific wavelengths or wavelength ranges of polychromatic light. Wavelength separation protocols in question include, but are not limited to, colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators, and combinations thereof, among other wavelength separation protocols. In some embodiments, the wavelength separator is an optical filter.For example, the optical filter may include a bandpass filter having a minimum bandwidth in the range of 2nm to 100nm, and a bandpass filter having a minimum bandwidth in the range of 3nm to 95nm, 5nm to 95nm, 10nm to 90nm, 12nm to 85nm, 15nm to 80nm, and 20nm to 50nm.

[0072] In certain embodiments, the detector is positioned at a distance from the sample in the flowstream, and light from the sample in the flowstream is propagated to the detector through an optical relay system, such as an optical fiber or a free-space optical relay system. For example, the optical relay system may be an optical fiber relay bundle, and the light is transmitted to the detector through the optical fiber relay bundle. Any optical fiber relay system may be used to propagate light to the detector. In certain embodiments, suitable optical fiber relay systems for propagating light to the detector include, but are not limited to, optical fiber relay systems described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference. In other embodiments, the optical relay system is a free-space optical relay system. The term “free-space optical relay” is used herein in its conventional sense to refer to light propagation that directs light through free space to a detector using a configuration of one or more optical components. In certain embodiments, the free-space optical relay system includes a housing having a proximal and a distal edge, the proximal edge being coupled to the detector. A free-space optical relay system may include any combination of different optical tuning components, such as lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof. For example, in some embodiments, the free-space optical relay system in question includes one or more focusing lenses. In other embodiments, the free-space optical relay system in question includes one or more mirrors. In yet another embodiment, the free-space optical relay system includes a collimating lens. In certain embodiments, suitable free-space optical relay systems for propagating light to a detector are, but are not limited to, optical relay systems described, for example, U.S. Patents 7,643,142, 7,728,974, and 8,223,445, the disclosures of which are incorporated herein by reference.

[0073] A method according to a particular embodiment also includes measuring light from a sample in a flow stream at one or more wavelengths, including, for example, measuring light emitted by the sample in the flow stream at two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, and four hundred or more different wavelengths. In some embodiments, the method includes measuring light collected over a wavelength range (e.g., 200 nm to 1000 nm). For example, the method may include collecting the spectrum of light over one or more wavelength ranges from 200 nm to 1000 nm. In yet other embodiments, the method includes measuring light collected at one or more specific wavelengths. For example, the collected light may be measured at one or more of the following wavelengths: 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, 617nm, and any combination thereof. In certain embodiments, the method includes measuring the wavelength of light corresponding to the fluorescence peak wavelength of a particular fluorophore.

[0074] The collected light may be measured continuously or at discrete intervals. In some cases, the method involves continuously measuring the light. In other cases, the light may be measured at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other interval.

[0075] The collected light may be measured one or more times during the method of the subject, including, for example, two or more times, three or more times, five or more times, and ten or more times. In certain embodiments, light propagation is measured two or more times, and in certain cases, the data is averaged.

[0076] In certain cases, the flowstream is irradiated with multiple angle-deflected beams of frequency-shifted light, and cells in the flowstream are imaged by fluorescence imaging using radiofrequency tagged emission (FIRE), as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013), and U.S. Patents 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, and 10 Frequency-coded images are generated, such as those described in U.S. Patent Publications 451,538, 10,620,111, and U.S. Patent Publications 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, and these disclosures are incorporated herein by reference.

[0077] The method in certain embodiments also includes data acquisition, analysis, and recording by a computer or the like, with multiple data channels recording data from the sample as the sample passes through the detection area of ​​the system. In these embodiments, the analysis may include classifying and counting cells or components of cells (extracellular vesicles) such that each component exists as a set of digitized parameter values. The system of the subject may be configured to trigger on selected parameters to distinguish the particles of interest from the background and noise. A “trigger” refers to a pre-set threshold for detecting a parameter and may be used as a means to detect the passage of the components of interest through the detection area. Detection of an event exceeding the threshold of the selected parameter triggers data acquisition of the sample components. With respect to components in the medium being chemically analyzed, no data is acquired that would result in a response below the threshold.

[0078] In some embodiments, the method further includes sorting one or more particles (e.g., cells) from a sample. The term “sort” is used herein in its conventional sense and refers to separating components of a sample (e.g., non-cellular particles such as cells or biomolecules), and optionally delivering the separated components to one or more sample collection containers. For example, the method may include sorting a sample having two or more components, including sorting a sample having, for example, three or more components, for example, four or more components, for example, five or more components, for example, ten or more components, for example, fifteen or more components, and twenty-five or more components. One or more of the sample components, for example, two or more sample components, for example, three or more sample components, for example, four or more sample components, for example, five or more sample components, for example, ten or more sample components, may be separated from the sample and delivered to a sample collection container, and fifteen or more sample components may be separated from the sample and delivered to a sample collection container.

[0079] In some embodiments, methods for sorting components of a sample include sorting particles (e.g., cells in a biological sample) as described in U.S. Patents 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, etc., the disclosures of which are incorporated herein by reference. In some embodiments, the method includes sorting components of a sample using a particle sorting module such as those described in U.S. Patent Nos. 9,551,643 and 10,324,019, U.S. Patent Publication No. 2017 / 0299493, and International Patent Publication No. WO / 2017 / 040151, the disclosures of which are incorporated herein by reference. In certain embodiments, cells of a sample are sorted using a sorting determination module having multiple sorting determination units, such as those described in U.S. Patent Application No. 16 / 725,756 filed December 23, 2019, the disclosures of which are incorporated herein by reference.

[0080] System for modulating an output laser beam with an angle-bending laser beam As summarized above, aspects of the present disclosure include a system configured to generate an output laser beam having multiple angle-deflected laser beams (for example, for irradiating a sample in a flowstream). In embodiments, the system includes a laser, an acousto-optical device, a waveform generator configured to input a waveform to the acousto-optical device to generate an output laser beam having multiple angle-deflected laser beams, a photodetector configured to determine the intensity profiles of the multiple angle-deflected laser beams along a horizontal axis, and a controller having a processor, the processor having a memory operably coupled to the processor, the memory containing instructions stored in the memory, the instructions, when executed by the processor, causing the processor to determine the intensity profile of the output laser beam along a horizontal axis, and, in response to the determined intensity profile, to adjust one or more parameters of the waveform input to the acousto-optical device to generate an output laser beam having a modulated intensity profile. In certain cases, as will be described in more detail below, the system of the subject is configured to adjust one or more parameters of the waveform input to the acousto-optical device to generate an output beam having multiple angle-deflected laser beams having substantially the same intensity (for example, having a top-hat intensity profile).

[0081] In embodiments, the subject system includes one or more lasers. The lasers in question may include pulsed lasers or continuous-wave lasers. The types and number of lasers used in the subject method may vary and may be gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other cases, the method includes irradiating an acousto-optic device with a dye laser such as a stilbene, coumarin, or rhodamine laser. In further cases, the method includes irradiating an acousto-optic device with a metallic 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, or combinations thereof. In yet another case, the method includes irradiating an 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:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium-sapphire laser, a thulium-YAG laser, a ytterbium-YAG laser, a Y2O3 laser, or a cerium-doped laser, or combinations thereof. In other cases, the method includes the implementation of a semiconductor diode laser, an optically excited semiconductor laser (OPSL), or any of the aforementioned lasers at twice or three times their frequency.

[0082] Depending on the desired wavelength of light produced by the output laser beam (for example, for use when irradiating a sample in a flow stream), the laser may have specific wavelengths differing by 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. The system may include one or more lasers, for example, two or more lasers, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, and ten or more lasers. The lasers may include any combination of several types of lasers. For example, in some embodiments, the system of the subject includes 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.

[0083] When two or more lasers are employed, each laser may be configured to irradiate simultaneously, sequentially, or in combination thereof. For example, lasers may be configured to irradiate an acousto-optical device simultaneously. In other embodiments, lasers are configured to irradiate an acousto-optical device sequentially. When the system includes two or more lasers for sequentially irradiating an acousto-optical device, the time each laser is configured for irradiation may be 0.001 microseconds or more individually, including, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, and 60 microseconds or more. For example, each laser may be configured to irradiate for a duration in the range of 0.001 microseconds to 100 microseconds, including, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In embodiments in which an acoustic-optical device is sequentially irradiated by two or more lasers, the duration for which the acoustic-optical device is irradiated by each laser may be the same or different.

[0084] Each laser may be configured to irradiate with individual separation by a delay of 0.001 microseconds or more, including, for example, periods of 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, only 10 microseconds or more, for example, only 15 microseconds or more, for example, only 30 microseconds or more, and only 60 microseconds or more. For example, the period between irradiations from each light source may be in the range of 0.001 microseconds to 60 microseconds, including, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In a particular embodiment, the period between irradiations from each laser is 10 microseconds. In embodiments in which the acousto-optical device is sequentially irradiated by more than two (i.e., three or more) lasers, the delay between irradiations from each laser may be the same or different.

[0085] Each laser can be configured to emit light continuously or at discrete intervals. In some cases, each laser is configured to operate continuously. In other cases, each laser is configured to operate at discrete intervals or some other interval, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds.

[0086] The laser can be movably positioned from the acousto-optical device at different distances, including, for example, 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, and 50 mm or more. The laser can also be movably positioned at an angle of 90°, for example, at an angle of 90°, within an illumination angle range of 10° to 90°, including, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and 30° to 60°.

[0087] The acousto-optic device can be any simple acousto-optic protocol configured to frequency shift laser light using applied acoustic waves. 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 another embodiment, the acousto-optic device is an acousto-optic modulator. The acousto-optic device in the system of the subject is configured to generate an angle-deflected laser beam from light from a laser and from a waveform generator.

[0088] In some embodiments, the system includes a waveform generator configured to generate a waveform for each angularly deflected laser beam to an acousto-optical device and input the waveforms to the acousto-optical device in order to generate multiple angularly deflected laser beams of the output laser beam. The waveform generator may include, but is not limited to, a direct digital synthesizer, an electronic pulse generator, and an arbitrary waveform generator. The waveform generator may be configured to generate one or more waveforms for each angularly deflected laser beam in the output beam, including, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, ten or more, for example, fifteen or more, for example, twenty-five or more, for example, fifty or more, and one hundred or more waveforms. In some embodiments, the input waveform includes one or more tones, for example, two or more tones, for example, three or more tones, for example, four or more tones, for example, five or more tones, and ten or more tones. Each tone is a sum of different sine waves, such as the sum of two or more different sine waves, including in certain cases where each tone is the sum of three or more different sine waves, four or more different sine waves, five or more different sine waves, ten or more different sine waves, twenty-five or more different sine waves, one hundred or more different sine waves, and one hundred or more different sine waves.

[0089] In some embodiments, the waveform generator is configured to generate a waveform, and the angle-deflected laser beam generated by the input waveform each has an intensity based on the amplitude of the tone of the waveform. In some embodiments, the waveform generator is configured to generate a waveform having a tone and input the waveform to an acousto-optical device, where the tones each have substantially the same amplitude, such that the amplitudes of the tones of the waveforms input to the acousto-optical device differ from each other by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less. In some embodiments, the sine wave forming each tone has an amplitude that differs between tones by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less.

[0090] A system according to a particular embodiment includes a photodetector for determining the intensity profile of the output laser beam along the horizontal axis. The photodetector for determining the intensity profile of the output laser beam may include, but is not limited to, any convenient photodetector protocol, including, scanning slit profilers, charge-coupled devices (CCDs, e.g., ICCDs), positioning sensors, power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser photometers, and laser diode detectors.

[0091] As described above, in some cases, the intensity profile of the output laser beam is determined by capturing an image of the output laser beam. In these embodiments, the system may include, but is not limited to, any suitable device capable of capturing an optical image and converting it into an electronic data signal, including a charge-coupled device, a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the imaging sensor is a CCD camera. For example, the camera may be a CCD (EMCCD) camera or an intensified CCD (ICCD) camera. In other embodiments, the imaging sensor is a CMOS camera.

[0092] The system may include one or more imaging sensors. In some embodiments, the system includes combinations of imaging sensors, such as two or more imaging sensors, three or more imaging sensors, four or more imaging sensors, and five or more imaging sensors. When the system includes two or more imaging sensors, the imaging sensors may be configured to operate simultaneously, sequentially, or in combination thereof. In some embodiments, the imaging sensors are configured to simultaneously capture images of the intensity profile of the output laser beam. In other embodiments, the imaging sensors are configured to sequentially capture images of the intensity profile of the output laser beam. When images of the output laser beam are captured sequentially, the time between images captured by the imaging sensors may be 0.001 seconds or more, individually, including, for example, 0.01 seconds or more, for example, 0.1 seconds or more, for example, 1 second or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 30 seconds or more, and 60 seconds or more.

[0093] The imaging sensor may be configured to capture images of the output laser beam continuously or at discrete intervals. In some cases, the imaging sensor is configured to capture images continuously. In other cases, the imaging sensor is configured to capture images at discrete intervals or some other interval, including capturing images of a flow stream every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds.

[0094] Figure 3A shows a system for modulating an output laser beam having multiple angle-deflected laser beams according to a particular embodiment. System 300 includes a laser 301 configured to illuminate an acousto-optical device (e.g., an acousto-optical deflector) 302, which communicates with a waveform generator 305. Parameters (e.g., a tone formed from a sum of sinusoids) are input to the waveform generator 305 (e.g., an arbitrary waveform generator) to generate multiple waveforms, which are then transmitted to the acousto-optical device 302 to generate multiple angle-deflected laser beams. The multiple angle-deflected laser beams include a local oscillator beam (LO beam) and multiple high-frequency shift comb beams (combs), separated within System 300 by pick-off mirrors. 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 in a beam splitter (BS1). Images of the beam profiles of multiple angle-deflected laser beams are captured by the imaging sensor 303, and an intensity profile plot may be generated based on the captured images. One or more parameters of the waveform used to generate the multiple angle-deflected laser beams may be adjusted to modulate the beam profile of the output laser beam and re-input to the waveform generator 305. In certain cases, a modulated output laser beam with an intensity profile that exhibits little to no variation along the horizontal axis may be used to irradiate a sample in a flow stream propagated through the flow cell 304.

[0095] Figure 3B shows a system for modulating an output laser beam having multiple angle-deflected laser beams according to a particular embodiment. System 310 includes a laser 301a configured to illuminate an acousto-optical device 302a that communicates with a waveform generator 305a. The waveform from the waveform generator 305a is transmitted to the acousto-optical device 302a to generate multiple angle-deflected laser beams. At a first pick-off mirror (pick-off mirror 1), the local oscillator beam (LO beam) and multiple high-frequency shift comb beams (combs) are separated. The local oscillator beam passes through a top-hat lens and is recombined with the comb beams at a first beam splitter (BS1). The first set of beams from beam splitter BS1 is propagated to a second pick-off mirror (pick-off mirror 2), which transmits a portion of the first set of beams to a flow cell 304a. The second set of beams from the beam splitter BS1 is recombined with light from the second pick-off mirror (pick-off mirror 2) and transmitted to the imaging sensor 303a. Images of the beam profiles of the multiple angle-deflected laser beams are captured by the imaging sensor 303a, and an intensity profile plot may be generated based on the captured images. One or more parameters of the waveform used to generate the multiple angle-deflected laser beams may be adjusted to modulate the beam profile of the output laser beam and re-input to the waveform generator 305a.

[0096] In some embodiments, the system includes a controller having a processor, the processor having a memory operablely coupled to the processor, the memory containing instructions stored in the memory, the instructions causing the processor to determine the intensity profile of the output laser beam by generating an intensity profile plot of multiple angle-deflected laser beams along a horizontal axis when executed by the processor. In some cases, the memory contains instructions for determining the intensity value from each angle-deflected laser beam and instructions for plotting each intensity value against the position of the angle-deflected laser beam along the horizontal axis. In some cases, the memory contains instructions for determining the intensity value based on the amplitude of the angle-deflected laser beam along the horizontal axis. In other cases, the memory contains instructions for determining the intensity value based on the brightness of the pixels generated by each angle-deflected laser beam in a captured image of the output laser beam.

[0097] In some embodiments, the system includes a controller having a processor, the processor having a memory operably coupled to the processor, the memory containing instructions stored in the memory, the instructions causing the processor to plot each intensity value based on a single measurement of the output laser beam when executed by the processor. In other embodiments, the memory contains instructions for plotting each intensity based on the average intensity of each angular deflection laser beam over a predetermined irradiation period. For example, the intensity value determined for each angular deflection laser beam along the horizontal axis may be the average intensity of two or more measurements of intensity for each angular deflection laser beam, including the average intensity determined from, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more, for example, 25 or more, for example, 50 or more, and 100 or more measurements of the intensity of each angular deflection laser beam.

[0098] In certain embodiments, the system includes a controller having a processor, the processor having a memory operablely coupled to the processor, the memory containing instructions stored in the memory, the instructions causing the processor to evaluate the intensity profile of the output laser beam and determine one or more adjustments to produce an output laser beam having a desired intensity profile when executed by the processor. In some cases, the memory contains instructions for identifying one or more of the angular deflection laser beams of the output laser beam for adjustment. Depending on the number of angular deflection laser beams in the output laser beam, the intensity of one or more of the angular deflection laser beams may be adjusted, including cases where the intensity of, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 25 or more, 50 or more, and 100 or more of the angular deflection laser beams is adjusted.

[0099] In some embodiments, the system includes a controller having a processor, the processor having memory operably coupled to the processor, the memory including instructions stored in the memory, the instructions causing the processor to adjust the intensity of one or more of the angle-deflected laser beams in an output laser beam such that two or more angle-deflected laser beams have the same intensity, including, for example, three or more angle-deflected laser beams have the same intensity, for example, four or more angle-deflected laser beams have the same intensity, for example, five or more angle-deflected laser beams have the same intensity, for example, six or more angle-deflected laser beams have the same intensity, for example, seven or more angle-deflected laser beams have the same intensity, for example, eight or more angle-deflected laser beams have the same intensity, for example, nine or more angle-deflected laser beams have the same intensity, for example, ten or more angle-deflected laser beams have the same intensity, for example, 25 or more angle-deflected laser beams have the same intensity, for example, 50 or more angle-deflected laser beams have the same intensity, and 100 or more angle-deflected laser beams have the same intensity.

[0100] In some embodiments, the memory includes instructions for adjusting the intensity of one or more of the angular deflection laser beams of the output laser beam in such a way that the multiple angular deflection laser beams of the output laser beam have intensities that differ from each other by only 10% or less, including, for example, 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, and 0.001% or less. In certain embodiments, the memory includes instructions for adjusting the intensity of one or more of the angular deflection laser beams in such a way that the output laser beam exhibits a top-hat beam profile along the horizontal axis.

[0101] In some cases, the system includes a controller having a processor, the processor having memory operably coupled to the processor, the memory containing instructions stored in the memory, the instructions, when executed by the processor, to increase the intensity of one or more of the angularly deflected laser beams to the intensity of a top-hat intensity profile, including increasing the intensity of, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more, for example, twenty-five or more, for example, fifty or more, and one hundred or more of the angularly deflected laser beams to the intensity of a top-hat intensity profile. In other cases, the memory includes instructions to reduce the intensity of one or more of the angular deflection laser beams to the intensity of a top-hat intensity profile, including reducing the intensity of, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more, for example, twenty-five or more, for example, fifty or more, and one hundred or more of the angular deflection laser beams to the intensity of a top-hat intensity profile.

[0102] In certain embodiments, the system includes a controller having a processor, the processor having a memory operably coupled to the processor, the memory containing instructions stored in the memory, the instructions causing the processor to adjust the intensity of one or more of the angular deflection laser beams by adjusting the parameters of the waveforms input to the acousto-optical device when executed by the processor. In some embodiments, the memory identifies each angular deflection laser beam whose intensity needs or decreases or is desired, and for each identified angular deflection laser beam, contains instructions for adjusting one or more parameters of the waveform, for example, the memory contains instructions for adjusting two or more parameters of the input waveform for each identified angular deflection laser beam, including adjusting, for example, three or more parameters, for example, four or more parameters, for example, five or more parameters, for example, six or more parameters, for example, seven or more parameters, for example, eight or more parameters, for example, nine or more parameters, for example, ten or more parameters, for example, twenty-five or more parameters, for example, fifty or more parameters, and one hundred or more parameters.

[0103] In some cases, the memory includes instructions for adjusting one or more tones of the input waveform for each identified angular deflection laser beam, including, for example, two or more tones, for example, three or more tones, for example, four or more tones, for example, five or more tones, and ten or more tones. In some cases, the memory includes instructions for adjusting one or more distinct sine waves whose sum constitutes each tone, including adjusting, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, ten or more, for example, fifteen or more, for example, twenty-five or more, for example, fifty or more, and one hundred or more of the sine waves of each tone of the input waveform for each identified angular deflection laser beam.

[0104] In certain embodiments, the memory includes instructions for adjusting the amplitude of the input waveform tone for each of the identified angle-deflected laser beams. In some cases, the memory includes instructions for increasing the amplitude of the input waveform tone by 5% or more, including increasing the amplitude of the input waveform tone by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more. In other cases, the memory includes instructions for decreasing the amplitude of the input waveform tone by 5% or more, including decreasing the amplitude of the input waveform tone by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more.

[0105] In other embodiments, the memory includes instructions for adjusting the frequency of the input waveform tone for each of the identified angle-deflected laser beams. In some cases, the memory includes instructions for increasing the frequency of the input waveform tone by 5% or more, including increasing the frequency of the input waveform tone by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more. In other cases, the memory includes instructions for decreasing the frequency of the input waveform tone by 5% or more, including decreasing the frequency of the input waveform tone by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, and 99% or more.

[0106] In certain embodiments, the system includes a controller having a processor, the processor having memory operably coupled to the processor, the memory including instructions stored in the memory, the instructions which, when executed by the processor, cause the processor to adjust the tone of the waveform input to the acousto-optical device for each angle-deflected laser beam to have substantially the same amplitude, for example, the amplitudes of the tone of the waveform input to the acousto-optical device are adjusted to differ from each other by only 10% or less, including, for example, only 9% or less, for example 8% or less, for example 7% or less, for example 6% or less, for example 5% or less, for example 4% or less, for example 3% or less, for example 2% or less, for example 1% or less, for example 0.1% or less, and 0.01% or less. In some embodiments, the memory includes instructions for adjusting the sine waves forming each tone to have substantially the same amplitude, for example, the amplitudes of the sine waves of each tone are adjusted to differ from each other by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less. In other embodiments, the memory includes instructions for adjusting the sine waves forming each tone to have substantially the same frequency, for example, the frequencies of the sine waves of each tone are adjusted to differ from each other by only 10% or less, including, for example, 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, 0.1% or less, and 0.01% or less.

[0107] In some embodiments, the system includes a flow cell configured to propagate a sample in a flow stream. Any convenient flow cell for propagating a fluid sample to a sample inspection area may be employed, and in some embodiments, the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating at a flat surface having an orifice transverse to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) may vary in the range of 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, and 4 mm to 8 mm. The length of the distal frustoconical portion (measured along the longitudinal axis) may also vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, e.g., 3 mm to 8 mm, and 4 mm to 7 mm. The diameter of the flow cell nozzle chamber may vary in some embodiments within a range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, 3 mm to 8 mm, and 4 mm to 7 mm.

[0108] In certain cases, the flow cell does not include a cylindrical portion, and the entire inner chamber of the flow cell is frustoconical. In these embodiments, the length of the frustoconical inner chamber (measured along the longitudinal axis transverse to the nozzle orifice) may range from 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, and 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical inner chamber may range from 1 mm to 10 mm, including, for example, 2 mm to 9 mm, e.g., 3 mm to 8 mm, and 4 mm to 7 mm.

[0109] In the embodiment, the sample flowstream is emitted from an orifice at the distal edge of the flow cell. Depending on the desired characteristics of the flowstream, the flow cell orifice may be any suitable shape, and the cross-sectional shape of the subject is not limited to these, but includes, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic base joined to the top of a plane. In a particular embodiment, the flow cell of the subject has a circular orifice. The size of the nozzle orifice may vary in some embodiments within a range of 1 μm to 20000 μm, including, for example, 2 μm to 17500 μm, 5 μm to 15000 μm, 10 μm to 12500 μm, 15 μm to 10000 μm, 25 μm to 7500 μm, 50 μm to 5000 μm, 75 μm to 1000 μm, 100 μm to 750 μm, and 150 μm to 500 μm. In a particular embodiment, the nozzle orifice is 100 μm.

[0110] In some embodiments, the flow cell includes a sample injection port configured to supply a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the internal chamber of the flow cell. Depending on the desired characteristics of the flowstream, the rate of the sample transmitted to the flow cell chamber by the sample injection port may be 1 μL / min or more, including, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 15 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, and 100 μL / min or more. In some cases, the rate of the sample transmitted to the flow cell chamber by the sample injection port is 1 μL / second or more, including, for example, 2 μL / second or more, for example, 3 μL / second or more, for example, 5 μL / second or more, for example, 10 μL / second or more, for example, 15 μL / second or more, for example, 25 μL / second or more, for example, 50 μL / second or more, and 100 μL / second or more.

[0111] The sample injection port may be an orifice positioned in the wall of the internal chamber, or a conduit positioned at the proximal end of the internal chamber. If the sample injection port is an orifice positioned in the wall of the internal chamber, the sample injection port orifice may be any preferred shape, and the cross-sectional shape of the subject is not limited, but includes, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal, curved cross-sectional shapes such as circular and elliptical, and irregular shapes such as a parabolic base joined to the top of a plane. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, including 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, and 1.5 mm, including 1.25 mm to 1.75 mm.

[0112] In certain cases, the sample injection port is a conduit positioned at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a conduit positioned to have an orifice of the sample injection port along the flow cell orifice. If the sample injection port is a conduit positioned along the flow cell orifice, the cross-sectional shape of the sample injection tube may be any preferred shape, and is not limited to these, but includes, for example, straight cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic base joined to a plane top. The orifice of the conduit may vary depending on the shape, and in some cases, it may have an opening in the range of 0.1 mm to 5.0 mm, including 1.25 mm to 1.75 mm, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and for example, 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip having a bevel angle in the range of 1° to 10°, including bevel angles of, for example, 2° to 9°, 3° to 8°, 4° to 7°, and 5°.

[0113] In some embodiments, the flow cell also includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid to the internal chamber of the flow cell, for example, together with the sample, to create a layered flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid transmitted to the flow cell chamber may be 25 μL / s or more, including, 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, and 2500 μL / s or more.

[0114] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the internal chamber. The sheath fluid injection port orifice may have any preferred shape, and the cross-sectional shape of the subject is not limited to these, but includes, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic base coupled to a plane top. The size of the sample injection port orifice may vary depending on the shape, and in some cases, it has an opening in the range of 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and for example 1.5 mm, including 1.25 mm to 1.75 mm.

[0115] In some embodiments, the system further includes a pump that fluid-communicates with the flow cell to propagate a flow stream through the flow cell. Any convenient fluid pump protocol may be used to control the flow of the flow stream through the flow cell. In certain cases, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the system of the subject is configured to transport fluid through the flow cell at a suitable rate for detecting light from a sample in the flow stream. In some cases, the sample flow rate in the flow cell is 1 μL / min (microliters per minute) or more, including, for example, 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 25 μL / min or more, 50 μL / min or more, 75 μL / min or more, 100 μL / min or more, 250 μL / min or more, 500 μL / min or more, 750 μL / min or more, and 1000 μL / min or more. For example, the system may include a pump configured to flow the sample through a flow cell at a rate in the range of 1 μL / min to 500 μL / min, including, for example, 1 μL / min to 250 μL / min, for example, 1 μL / min to 100 μL / min, for example, 2 μL / min to 90 μL / min, for example, 3 μL / min to 80 μL / min, for example, 4 μL / min to 70 μL / min, for example, 5 μL / min to 60 μL / min, and 10 μL / min to 50 μL / min. In a particular embodiment, the flow rate of the flow stream is 5 μL / min to 6 μL / min.

[0116] The system also includes one or more detectors for detecting light from a sample in a flow stream (e.g., in a flow cytometer). The detectors may be configured to detect side-scattered light, forward-scattered light, emitted light, or a combination thereof. Preferred photodetection protocols include, but are not limited to, optical sensors or photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, light from a flow stream irradiated in the sample inspection area of ​​a particle sorting module is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the light is measured by a charge-coupled device (CCD).

[0117] The system may also include one or more optical tuning components. For example, the system may include wavelength separators such as lenses, mirrors, collimators, and tinted glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, and monochromators. In certain embodiments, the system includes a beam splitter and, for example, an optical inverter component for inverting the output laser beam along the horizontal axis, as described above. In certain embodiments, the detector is positioned spaced apart from the sample in the flowstream, and light from the sample in the flowstream is propagated to the detector through an optical relay system, such as an optical fiber or a free-space optical relay system. For example, the optical relay system may be an optical fiber optical relay bundle, and the light is transmitted to the detector through the optical fiber optical relay bundle. Any optical fiber optical relay system may be used to propagate light to the detector. In certain embodiments, suitable optical fiber optical relay systems for propagating light to the detector include, but are not limited to, optical fiber optical relay systems such as those described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference. In other embodiments, the optical relay system is a free-space optical relay system. The term “free-space optical relay” is used herein, in its conventional sense, to refer to light propagation that directs light through free space to a detector using a configuration of one or more optical components. In certain embodiments, the free-space optical relay system includes a housing having a proximal and distal edge, the proximal edge being coupled to the detector. The free-space relay system may include any combination of different optical tuning components, such as one or more of lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof. For example, in some embodiments, the free-space optical relay system in question includes one or more focusing lenses. In other embodiments, the free-space optical relay system in question includes one or more mirrors. In yet another embodiment, the free-space optical relay system includes a collimating lens.In certain embodiments, suitable free-space optical relay systems for propagating light to a detector include, but are not limited to, optical relay systems described in, for example, U.S. Patents No. 7,643,142, No. 7,728,974, and No. 8,223,445, the disclosures of which are incorporated herein by reference.

[0118] In certain embodiments, the subject system is a flow cytometric system that uses the above-described photodetector system for detecting light emitted by a sample in a flow stream. Suitable flow cytometry systems are not limited to these, but include: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30(5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222(4): 335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier. This may include the information contained in Syst.24(3):203-255, which are incorporated herein by reference.In certain cases, the flow cytometry systems covered include: BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortess® X-20 flow cytometer, and BD Biosciences FACSCalibur® cell sorter, a BD Biosciences FACSCount® cell sorter, BD Biosciences FACSLyric® cell sorter, and BD Biosciences Via® cell sorter, BD Biosciences Influx® cell sorter, BD Biosciences Jazz® cell sorter, and BD Biosciences This includes Aria® cell sorting machines and BD Biosciences FACSMelody® cell sorting machines, among others.

[0119] In certain cases, the subject system is as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013), as well as U.S. Patents 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. Patents. Flow cytometry systems configured to image particles in a flow stream by fluorescence imaging using radio frequency tagged emission (FIRE), such as those described in Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, which are incorporated herein by reference.

[0120] 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 and refers to separating components of a sample (e.g., non-cellular particles such as cells and biomolecules), and optionally delivering the separated components to one or more sample collection containers. For example, the subject system may be configured to sort a sample having two or more components, including sorting a sample having, for example, three or more components, for example, four or more components, for example, five or more components, for example, ten or more components, for example, fifteen or more components, and twenty-five or more components. One or more of the sample components, for example, two or more sample components, for example, three or more sample components, for example, four or more sample components, for example, five or more sample components, for example, ten or more sample components may be separated from the sample and delivered to a sample collection container, and fifteen or more sample components may be separated from the sample and delivered to a sample collection container.

[0121] In some embodiments, the particle sorting system is configured to sort particles using a sealed particle sorting module, such as that 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 of a sample (e.g., cells) are sorted using a sorting determination module having multiple sorting determination units, such as that 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, for example, U.S. Patent Nos. 10,006,852, 9,952,076, 9,933,341, 9,784,661, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, and Flow cytometric systems, such as those described in Patent Nos. 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, and 5,602,039, are incorporated herein by reference in their entirety.

[0122] In some embodiments, the system is a particle analyzer that can analyze and characterize particles with or without physically separating them into a collection container using 401 (Figure 4A). Figure 4A shows a functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 shown in Figure 4A may be configured to perform the methods described herein, either entirely or in part. The particle analysis system 401 includes a fluid engineering system 402. The fluid system 402 may include, or may be coupled with, a sample tube 405 and a moving fluid column in the sample tube in which sample particles 403 (e.g., cells) move along a common sample path 409.

[0123] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection stations 408 generally refer to a monitoring area 407 of the common sample path. In some embodiments, detection may include detecting light or one or more other properties of particles 403 as they pass through the monitoring area 407. Figure 4A shows one detection station 408 with one monitoring area 407. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor two or more areas.

[0124] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data may be referred to as event data. The data points may be multidimensional data points containing the values ​​of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0125] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuits, and / or frequency control circuits. The indicated control system may be operably associated with the fluid engineering system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of a first period, based on a Poisson distribution and the number of data points collected by the detection system 404 during the first period. The control system 406 may be further configured to generate an experimental signal frequency based on the number of data points in a portion of the first period. The control system 406 may additionally compare the experimental signal frequency with that of a calculated signal frequency or a predetermined signal frequency.

[0126] Figure 4B shows a system 400 for flow cytometry according to an exemplary embodiment of the present invention. This system 400 includes a flow cytometer 410, a controller / processor 490, and memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence focusing lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long-pass ("LP") filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0127] The excitation lasers 415a–415c emit light in the form of laser beams. The wavelengths of the laser beams emitted from excitation lasers 415a–415c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system shown in Figure 4B. The laser beams are initially guided through one or more of the beam splitters 445a and 445b. Beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. Beam splitter 445b transmits UV light (light with wavelengths in the range of 10–400 nm) and reflects light at 488 nm and 633 nm.

[0128] The laser beam is then guided to a focusing lens 420, which focuses the beam onto the portion of the fluid stream where the sample particles are located within the flow chamber 425. The flow chamber is part of a fluid engineering system that guides particles in the stream to the focused laser beam, typically one at a time, for investigation. The flow chamber may contain a flow cell within a benchtop flow cytometer or a nozzle tip within a stream-in-air cytometer.

[0129] Light from the laser beam interacts with particles in the sample by diffraction, refraction, reflection, scattering, and absorption, resulting in re-emission at various different wavelengths, depending on the particle's characteristics, such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally occurring on or within the particle. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be routed through one or more of the beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence focusing lenses 440 to one or more of the forward scattering detector 430, side scattering detector 435, and one or more fluorescence detectors 460a-460f.

[0130] The fluorescence focusing lens 440 collects light emitted from the interaction between particle laser beams and routes that light toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a to 450e, allow a narrow wavelength range to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first digit represents the center of the spectral band. The second digit provides the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm, or 500 nm to 520 nm, on each side of the center of the spectral band. Short-pass filters transmit wavelengths of light below a specified wavelength. Long-pass filters, such as long-pass filters 455a to 455b, transmit wavelengths of light above a specified wavelength. For example, long-pass filter 455a, a 670 nm long-pass filter, transmits wavelengths of light above 670 nm. Filters are often selected to optimize the specificity of the detector for a particular fluorescent dye. These filters can be configured so that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0131] A beam splitter directs light of different wavelengths in different directions. Beam splitters can be characterized by filtering characteristics such as short-pass and long-pass. For example, beam splitter 445g is a 620SP beam splitter, meaning that this beam splitter 445g transmits light with wavelengths shorter than 620nm and reflects light with wavelengths longer than 620nm in different directions. In one embodiment, beam splitters 445a to 445g may be equipped with optical mirrors such as dichroic mirrors.

[0132] The forward scatter detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light and excitation light that travels almost forward through or around the particle. The intensity of the light detected by the forward scatter detector depends on the overall particle size. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect diffracted and reflected light from the surface and internal structure of the particle and tends to increase as the particle structure becomes more complex. 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 may include photomultiplier tubes. The signals detected by the forward scatter detector 430, the side scatter detector 435, and the fluorescence detectors can be converted into electronic signals (voltages) by the detectors. This data can provide information about the sample.

[0133] Those skilled in the art will recognize that the flow cytometer according to one embodiment of the present invention is not limited to the flow cytometer shown in Figure 4B, but may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in various different configurations.

[0134] During operation, the flow cytometer's operation is controlled by the controller / processor 490, and measurement data from the detector can be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 190 can be coupled to the detector to receive output signals from it, and can also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, etc. An input / output (I / O) function unit 497 may also be provided within the system. The memory 495, controller / processor 490, and I / O 497 may be provided collectively as an integral part of the flow cytometer 410. In such embodiments, a display may also form part of the I / O function unit 497 for presenting experimental data to the user of the flow cytometer 400. Alternatively, the memory 495, controller / processor 490, and some or all of the I / O function unit may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 495 and the controller / processor 490 can communicate with the flow cytometer 410 wirelessly or via a wired connection. Together with the memory 495 and I / O 497, the controller / processor 490 can be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0135] The system illustrated in Figure 4B includes six different detectors that detect fluorescence within six different wavelength bands (which may be referred to herein as “filter windows” for a given detector), as defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in flow cytometry experiments emit light in their own characteristic wavelength bands. Specific fluorescent labels used in experiments, and their associated fluorescence emission bands, may be selected to roughly coincide with the filter windows of the detectors. However, as more detectors are provided and more labels are utilized, a perfect correspondence between filter windows and fluorescence emission spectra is impossible. It is generally true that the emission spectrum peak of a particular fluorescent molecule may lie within the filter window of one particular detector, while a portion of the emission spectrum of that label may also overlap with the filter windows of one or more other detectors. This may be referred to as spill signaling. I / O497 may be configured to receive data relating to flow cytometry experiments with panels of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O497 may also be configured to receive biodata for assigning one or more markers to one or more cell populations, marker concentration data, emission spectral data, data for assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as label spectral characteristics and flow cytometer configuration data, may also be stored in memory 495. Controller / processor 490 may be configured to evaluate the assignment of one or more labels to the markers.

[0136] Figure 5 shows a functional block diagram of one embodiment of a particle analyzer control system, including an analysis controller 500, for analyzing and displaying biological events. The analysis controller 500 may be configured to perform various processes for controlling the graphical display of biological events.

[0137] The particle analyzer 502 may be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometric event data. The particle analyzer 502 may be configured to provide biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0138] The analysis controller 500 may be configured to receive bioevent data from the particle analyzer 502. The bioevent data received from the particle analyzer 502 may include flow cytometric event data. The analysis controller 500 may be configured to provide a graphical display to the display device 506, including a first plot of the bioevent data. The analysis controller 500 may be further configured to render the region of interest as a gate around the population of bioevent data shown by the display device 506, for example, superimposed on the first plot. In some embodiments, the gate may be a logical combination of one or more image regions of interest drawn on a single-parameter histogram or bivariate plot. In some embodiments, the display may be used to display particle parameters or saturated detector data.

[0139] The analysis controller 500 may be further configured to display bio-event data on the display device 506 within the gate separately from other events in the bio-event data outside the gate. For example, the analysis controller 500 may be configured to render the colors of the bio-event data contained within the gate in a way that distinguishes them from the colors of the bio-event data outside the gate. The display device 506 may be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0140] The analysis controller 500 may be configured to receive gate selection signals from a first input device that identify gates. For example, the first input device may be implemented as a mouse 510. This mouse 510 can initiate gate selection signals to the analysis controller 500, which identifies gates displayed or manipulated via the display device 506 (for example, by clicking on a desired gate when positioning a cursor there). In some embodiments, the first device may be implemented as a keyboard 508, or other means for providing input signals to the analysis controller 500, such as a touchscreen, input pen, photodetector, or voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, as shown in Figure 5, the mouse 510 may include a right mouse button and a left mouse button, each of which may generate an activation event.

[0141] This activation event may cause the analysis controller 500 to change how the data is displayed, which parts of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting the population to be sorted.

[0142] In some embodiments, the analysis controller 500 may be configured to detect when gate selection was initiated by the mouse 510. The analysis controller 500 may be further configured to automatically correct the plot visualization to facilitate the gate control process. This correction may be based on a specific distribution of biological event data received by the analysis controller 500.

[0143] The analysis controller 500 may be connected to a storage device 504. This storage device 504 may be configured to receive and store biometric data from the analysis controller 500. The storage device 504 may also be configured to receive and store flow cytometric event data from the analysis controller 500. The storage device 504 may be further configured by the analysis controller 500 to allow retrieval of biometric data, such as flow cytometric event data.

[0144] The display device 506 may be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that outline the plots. The display device 506 may be further configured to modify the information presented in accordance with the input received from the analysis controller 500, in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0145] In some embodiments, the analysis controller 500 can generate a user interface to receive exemplary events for sorting. For example, this user interface may include controls for receiving exemplary events or exemplary images. Exemplary events or images, or exemplary gates, may be provided before the collection of event data for the sample, or based on an initial set of events for a portion of the sample.

[0146] Computer control system Aspects of the present disclosure further include a computer-controlled system for practicing the subject method, the system further including one or more computers for full or partial automation of the system for practicing the method described herein. In some embodiments, the system includes a computer having a computer-readable storage medium in which a computer program is stored, the computer program, when loaded onto the computer, includes instructions for irradiating an acousto-optical device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams; instructions for determining an intensity profile of the output laser beam along a horizontal axis; and instructions for adjusting one or more parameters of a waveform input to the acousto-optical device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile. In certain embodiments, the computer program includes instructions for capturing an image of the intensity profile of the output laser beam along a horizontal axis; and instructions for determining the intensity profile based on the captured image.

[0147] In some embodiments, the system includes an input module, a processing module, and an output module. In some embodiments, the system of the subject may include an input module for inputting parameters or information relating to an acousto-optical device (e.g., an acousto-optical deflector), a laser, a waveform generator, the sample of the applied light source, its intensity, and wavelength (discrete or range), the flow cell diameter, the number of optical channels, the number of detection areas, the duration of irradiation by the light source, the number of different light sources, the distance from the light source to the flow channel, the focal length of any optical tuning component, the refractive index of the flow channel medium (e.g., a sheath fluid), the presence of any wavelength separator, the bandpass width, opacity, characteristics of the wavelength separator including the grating spacing, and the characteristics and sensitivity of the photodetector.

[0148] After the processing module has performed one or more steps of the subject method, the output module communicates the results to the user, for example, by displaying them on a monitor or by printing a report.

[0149] The subject system may include both hardware and software components, and the hardware components may take the form of one or more platforms, for example, in the form of servers, and as a result, the functional elements of the system, i.e., those elements of the system that perform specific tasks (such as managing input / output of information, processing information, etc.), may be executed by the execution of software applications on and across one or more computer platforms represented by the system.

[0150] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard or a mouse. The processing module includes a processor that accesses a memory storing instructions for performing steps of the method of the subject, such as irradiating an acousto-optical device with a laser to generate an output laser beam having multiple angularly deflected laser beams, capturing an image of the intensity profile of the output laser beam, and adjusting the tone of the waveform input to the acousto-optical device for each angularly deflected laser beam to have substantially the same amplitude.

[0151] The processing module includes a processor that accesses memory containing stored instructions for performing steps of the method of the subject. The processing module may also include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or one of other processors that are available or will become available. The processor runs the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor's coordination and execution of various computer programs that can be written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is well known in the art. The operating system typically works in conjunction with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, according to all known technologies. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow the user to manually align the light source with the flow stream based on a first optical signal and a second optical signal. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0152] System memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read-and-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including compact disk drives, tape drives, removable hard disk drives, or disk drives. Such types of memory storage devices typically read from and / or write to program storage media (not shown), such as compact disks, magnetic tapes, removable hard disks, or magnetic disks, respectively. Any of these program storage media, or others currently in use or to be developed in the future, can be considered computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0153] In some embodiments, a computer program product is described that comprises a computer-usable medium having internally stored control logic (a computer software program including program code). When the control logic is executed by a computer processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of hardware state machines for performing the functions described herein will be apparent to those skilled in the art.

[0154] Memory may be any suitable device on which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic or optical disks, tapes or RAM, or any other suitable device, whether fixed or portable). The processor may include a general-purpose digital microprocessor preferably programmed from a computer-readable medium that carries the required program code. The programming may be provided to the processor remotely via a communication channel, or it may be pre-stored in a computer program product such as memory or some other portable or fixed computer-readable storage medium using any of those devices in relation to memory. For example, a magnetic disk or optical disk may carry programming that can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product, and algorithms for use in carrying out the above method. The programming according to the present invention may be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as magnetic disks, hard disk storage media, and magnetic tapes; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROMs; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0155] The processor may also have access to a communication channel for communicating with the user at a remote location. A remote location means that the user does not have direct contact with the system, but relays input information to the input manager from an external device, such as a computer connected to any other suitable communication channel, including a wide area network ("WAN"), telephone network, satellite network, or mobile phone (i.e., smartphone).

[0156] In some embodiments, the systems according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and code division multiple access (CDMA) or global system for mobile communications (GSM).

[0157] In one embodiment, the communication interface is configured to include one or more communication ports, such as a physical port or interface, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment), configured for similar complementary data communication.

[0158] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, thereby enabling the subject system to communicate with other devices such as computer terminals and / or networks, communicable mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0159] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet Protocol (IP) via a mobile phone network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0160] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11 or Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or appliance. In some embodiments, the server device has a display such as a liquid crystal display (LCD), and input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0161] In some embodiments, the communication interface is configured to automatically or semi-automatically transmit data stored within the subject system, for example, in an optional data storage unit, to a network or server device using one or more of the above-described communication protocols and / or mechanisms.

[0162] The output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may be logically and / or physically organized, typically as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other types of remote communication. The output manager may also provide information generated by processing modules to a user at a remote location, for example, via the internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be carried out according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. One or more platforms present within the subject system are typically of the class of computers commonly referred to as servers, but may be any known or future type of computer platform. They may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cabling, or other communication systems, including wireless systems, whether networked or not. They may be located in the same place or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems may be adopted on any of the computer platforms. 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, and Zorin OS.

[0163] Figure 6 shows a general architecture of an exemplary computing device 600 according to a particular embodiment. The general architecture of the computing device 600 shown in Figure 6 includes the arrangement of computer hardware and software components. The computing device 600 may include more (or fewer) elements than those shown in Figure 6. However, not all of these generally traditional elements are necessarily shown in order to provide a valid disclosure. As illustrated, 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 each other via a communication bus. The network interface 620 can provide connectivity to one or more networks or computing systems. Thus, the processing unit 610 can receive information and instructions from other computing systems or services via the network. The processing unit 610 can also communicate with memory 670 and can further provide output information for the optional display 650 via the input / output device interface 640. The input / output device interface 840 can also receive input from an optional input device 660, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0164] Memory 670 may include computer program instructions (which in some embodiments may be grouped as modules or components) that the processing unit 610 executes sequentially to implement one or more embodiments. Memory 670 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 670 may 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. Memory 670 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0165] Non-temporary computer-readable storage medium Aspects of this disclosure further include a non-temporary computer-readable storage medium having instructions for practicing the subject matter. The computer-readable storage medium may be used on one or more computers for the full or partial automation of a system for practicing the method described herein. In certain embodiments, instructions by the method described herein may be coded into a computer-readable medium in the form of “programming,” in which the term “computer-readable medium” means, as used herein, any non-temporary storage medium involved in providing instructions and data to a computer for execution and processing. Examples of preferred non-temporary storage media include magnetic disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state disks, and network-attached storage devices (NAS), whether such devices are located inside or outside a computer. A file containing information may be “stored” on a computer-readable medium, where “stored” means recording the information so that it can be accessed and retrieved by a computer at a later date. The computer implementations described herein may be executed using programming 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), as well as many other languages.

[0166] In some embodiments, the computer-readable storage medium includes a computer program stored in the computer-readable storage medium, which, when loaded into a computer, includes instructions for an algorithm for irradiating an acousto-optical device with a laser to generate an output laser beam having multiple angularly deflected laser beams; an algorithm for detecting scattered light from particles with a photodetector; an algorithm for determining the intensity profile of the output laser beam along the horizontal axis; and an algorithm for adjusting one or more parameters of a waveform input to the acousto-optical device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for capturing an image of the intensity profile of the output laser beam along the horizontal axis, and instructions for determining the intensity profile based on the captured image.

[0167] In some embodiments, a non-temporary computer-readable storage medium includes an algorithm for determining the intensity profile of an output laser beam, and includes generating an intensity profile plot of multiple angle-deflected laser beams along a horizontal axis. In generating the intensity profile plot, the method may include determining an intensity value from each angle-deflected laser beam and plotting each intensity value against the position of the angle-deflected laser beam along the horizontal axis. In some cases, the non-temporary computer-readable storage medium includes an algorithm for determining the intensity value based on the amplitude of the angle-deflected laser beams along the horizontal axis. In other cases, the non-temporary computer-readable storage medium includes an algorithm for determining the intensity value based on the brightness of pixels produced by each angle-deflected laser beam in a captured image of the output laser beam. In some embodiments, each intensity value plotted in the intensity profile plot is based on a single measurement of the output laser beam. In other embodiments, the non-temporary computer-readable storage medium includes an algorithm for plotting each intensity value based on the average intensity of each angle-deflected laser beam over a predetermined irradiation period.

[0168] In certain embodiments, the non-temporary computer-readable storage medium includes an algorithm for evaluating the intensity profile of the output laser beam and determining whether one or more adjustments are necessary to produce an output laser beam having a desired intensity profile. In some cases, the non-temporary computer-readable storage medium includes an algorithm for identifying one or more angular deflection laser beams of the output laser beam for adjustment.

[0169] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for adjusting the intensity of one or more angular-deflected laser beams in such a way that two or more angular-deflected laser beams in the output laser beam have the same intensity. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for adjusting the intensity of one or more of the angular-deflected laser beams in such a way that the multiple angular-deflected laser beams of the output laser beam have intensities that differ from each other by only 10% or less, including, for example, 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, and 0.001% or less. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for adjusting the intensity of one or more of the angular-deflected laser beams in such a way that the output laser beam exhibits a top-hat beam profile along the horizontal axis.

[0170] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for adjusting the parameters of the waveform input to the acousto-optical device. In some cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting one or more tones of the waveform input for each of the identified angular-bent laser beams. In certain cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting the amplitude of the tone of the waveform input for each of the identified angular-bent laser beams. In other cases, the non-temporary computer-readable storage medium includes an algorithm for adjusting the frequency of the tone of the waveform input for each of the identified angular-bent laser beams. In certain embodiments, the non-temporary computer-readable storage medium includes an algorithm for adjusting the tones of the waveform input to the acousto-optical device for each angular-bent laser beam to have substantially the same amplitude. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for adjusting the sine waves forming each tone to have substantially the same frequency.

[0171] Non-temporary computer-readable storage media may be used on one or more computer systems having a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. A processing module includes a processor that accesses memory having stored instructions to perform steps of the method of the subject. A processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or one of other processors that are available or will become available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system usually works in cooperation with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, according to all known technologies.

[0172] kit Aspects of the present invention further include a kit comprising one or more lasers, an acousto-optical device (e.g., an acousto-optical deflector), a waveform generator, and an imaging sensor. In some embodiments, the imaging sensor is a camera. In some cases, the kit includes any waveform generator. The kit may further include one or more optical adjustment components, such as beam splitters, lens systems, or beam inverters as described herein. The various assay components of the kit may be in separate containers, or some or all of them may be pre-assembled. For example, in some cases, one or more components of the kit, e.g., each detector, may be in a sealed pouch, such as a sterile foil pouch or envelope.

[0173] In addition to the components described above, the subject kit may further include (in certain embodiments) instructions for carrying out the subject method. These instructions may be present in the kit in various forms, and one or more of them may be present in the kit. One possible form in which these instructions may be present is as information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, the kit packaging, accompanying documentation, etc. Yet another form of these instructions may be a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), portable flash drive, etc. Yet another possible form of these instructions may be a website address that can be used over the internet to access the information at a remote site.

[0174] Utility The subject system, method, and computer system finds use in a variety of applications where it is desirable to analyze and sort particulate components in a sample in a fluid medium, such as biological samples. The disclosure also finds use in flow cytometry, where it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In embodiments, the disclosure reduces the need for user input or manual adjustment during sample analysis by flow cytometry. In certain embodiments, the subject system provides a fully automated protocol, resulting in little to no adjustment to the flow cytometer in use, even with any human input.

[0175] This disclosure also finds applications in which cells prepared from biological samples may be desired for use in research, laboratory testing, or therapy. In some embodiments, the methods and devices of this disclosure can facilitate the acquisition of individual cells prepared from a biological fluid or tissue sample of interest. For example, the methods and systems of this disclosure facilitate the acquisition of cells from a fluid or tissue sample used as a specimen for research or diagnosis of diseases such as cancer. Similarly, the methods and systems of this disclosure facilitate the acquisition of cells from a fluid or tissue sample used in therapy. The methods and devices of this disclosure enable the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, body fluids) with improved efficiency and lower cost compared to conventional flow cytometry systems.

[0176] Notwithstanding the attached claims, this disclosure is also defined by the following appendix:

[0177] 1. A method for modulating the intensity profile of a laser in a particle analyzer, The process involves irradiating an acoustic-optical device with a laser to generate an output laser beam containing multiple angle-deflected laser beams, and To capture an image of the output laser beam, From the captured image, the intensity profile of the output laser beam along the horizontal axis is determined, In response to the determined intensity profile, one or more parameters of the waveform input to the acousto-optical device are adjusted to generate an output laser beam having a modulated intensity profile. Methods that include... 2. The method is the method described in Appendix 1, which includes adjusting one or more tones of the input waveform. 3. Each tone of the input waveform contains one or more sine waves, as described in Appendix 2. 4. The method according to Appendix 2 or 3, which includes adjusting the amplitude of one or more tones of the input waveform. 5. The method is, Determining the amplitude of each angularly deflected laser beam in the output laser beam, For each angle-deflected laser beam, adjust one or more parameters of the waveform input to the acousto-optical device. The method described in any one of the appendices 1 to 4, further including the method described in any one of the appendices 1 to 4.

[0178] 6. The method according to Appendix 5, which includes adjusting the amplitude of the tone of the input waveform for each angle-deflected laser beam. 7. The method according to Appendix 5, which includes adjusting the frequency of the tone of the input waveform for each angle-deflected laser beam. 8. The method described in any one of the appendices 1 to 5, wherein one or more parameters of the input waveform are adjusted to produce an output laser beam having a substantially constant intensity profile along the horizontal axis. 9. The method according to Appendix 8, wherein one or more parameters of the input waveform are adjusted to generate multiple angularly deflected laser beams having intensities that differ by no more than 10%. 10. The method according to Appendix 8, wherein one or more parameters of the input waveform are adjusted to generate multiple angularly deflected laser beams having intensities that differ by no more than 5%.

[0179] 11. The method according to any one of the appendices 1 to 10, further comprising generating an intensity profile plot from a captured image of the output laser beam. 12. The acousto-optic device is an acousto-optic deflector, as described in any one of the methods described in Appendix 1 to 11. 13. Each waveform input to the acousto-optical device is generated by an arbitrary waveform generator (AWG) using one of the methods described in Appendix 1 to 12. 14. A plurality of angle-deflected laser beams, comprising a local oscillator (LO) beam and a plurality of high-frequency comb beams, as described in any one of Appendices 1 to 13. 15. Multiple angle-deflected laser beams are spatially separated, according to one of the methods described in Appendices 1 to 14.

[0180] 16. The method according to Appendix 15, wherein each angle-deflected laser beam at least partially overlaps with one other angle-deflected laser beam in the output laser beam. 17. The particle analyzer is part of a flow cytometer, as described in any one of the methods described in Appendix 1 to 16.

[0181] 18. Irradiating an acoustic-optical device with a laser to generate an output laser beam containing multiple angle-deflected laser beams, Determining the intensity profile of the output laser beam along the horizontal axis, In response to the determined intensity profile, one or more parameters of the waveform input to the acousto-optical device are adjusted to generate an output laser beam having a modulated intensity profile. Methods that include... 19. The intensity profile of the output laser beam is determined from a captured image of the output laser beam, as described in Appendix 18. 20. The image of the output laser beam is captured by a camera as described in Appendix 19. 21. The image of the output laser beam is captured by an imaging light sensor as described in Appendix 19. 22. The method according to any one of the appendices 19-21, further comprising generating an intensity profile plot from captured images of the output laser beam.

[0182] 23. The method is the method described in any one of the appendices 18 to 22, which includes adjusting one or more tones of the input waveform. 24. Each tone of the input waveform contains one or more sine waves, as described in Appendix 23. 25. The method according to Appendix 23 or 24, which includes adjusting the amplitude of one or more tones of an input waveform. 26. The method is, Determining the amplitude of each angularly deflected laser beam in the output laser beam, For each angle-deflected laser beam, adjust one or more parameters of the waveform input to the acousto-optical device. The method described in any one of the appendices 18 to 25, further including the method described in any one of the appendices 18 to 25. 27. The method according to Appendix 26, comprising adjusting the amplitude of the tone of the input waveform for each angle-deflected laser beam.

[0183] 28. The method according to Appendix 26, comprising adjusting the frequency of the tone of the input waveform for each angle-deflected laser beam. 29. The method according to any one of the appendices 18 to 28, wherein one or more parameters of the input waveform are adjusted to produce an output laser beam having a substantially constant intensity profile along the horizontal axis. 30. The method according to Appendix 29, wherein one or more parameters of the input waveform are adjusted to generate multiple angularly deflected laser beams having intensities that differ by no more than 10%. 31. The method according to Appendix 29, wherein one or more parameters of the input waveform are adjusted to generate multiple angularly deflected laser beams having intensities that differ by no more than 5%. 32. The acousto-optical device is an acousto-optical deflector, as described in any one of the methods described in Appendix 18 to 31.

[0184] 33. Each waveform input to the acousto-optical device is generated by an arbitrary waveform generator (AWG) using one of the methods described in Appendix 18 to 32. 34. The method according to any one of the appendices 18 to 33, wherein the multiple angle-deflected laser beams include a local oscillator (LO) beam and multiple high-frequency comb beams. 35. Multiple angle-deflected laser beams are spatially separated, according to any one of the methods described in Appendices 18-34. 36. The method according to Appendix 35, wherein each angle-deflected laser beam at least partially overlaps with one other angle-deflected laser beam in the output laser beam.

[0185] 37. A particle analyzer, Laser and, Acoustic-optical devices and, A waveform generator configured to input a waveform to an acoustic-optical device and generate an output laser beam containing multiple angle-deflected laser beams, Image sensor and A controller equipped with a processor It is equipped with, The processor has memory operably coupled to the processor, the memory contains instructions stored in the memory, and when an instruction is executed by the processor, the processor... The process involves capturing an image of the output laser beam with an imaging light sensor, From the captured image, the intensity profile of the output laser beam along the horizontal axis is determined, In response to the determined intensity profile, one or more parameters of the waveform input to the acousto-optical device are adjusted to generate an output laser beam having a modulated intensity profile. A particle analyzer that performs this operation. 38. Acousto-optical devices are acousto-optical deflectors, as described in Appendix 37, for particle analyzers. 39. The imaging light sensor is a camera, as described in Appendix 37 or 38, for the particle analyzer. 40. A particle analyzer as described in any one of the appendices 37-39, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor causes the processor to adjust one or more tones of an input waveform. 41. A particle analyzer as described in Appendix 40, wherein each tone of the input waveform contains one or more sine waves.

[0186] 42. A particle analyzer as described in Appendix 40 or 41, wherein the memory includes instructions stored in the memory, and when the instructions are executed by the processor, the processor causes the processor to adjust the amplitude of one or more tones of an input waveform. 43. Memory contains instructions stored in memory, and when an instruction is executed by the processor, the processor... Determining the amplitude of each angularly deflected laser beam in the output laser beam, For each angle-deflected laser beam, adjust one or more parameters of the waveform input to the acousto-optical device. A particle analyzer described in any one of the appendices 37 to 42, which performs the following action. 44. The particle analyzer described in Appendix 43, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts the amplitude of the input waveform tone for each angle-deflected laser beam. 45. The particle analyzer described in Appendix 43, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts the frequency of the tone of the input waveform for each angle-deflected laser beam. 46. ​​A particle analyzer as described in Appendix 43, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts one or more parameters of an input waveform to generate multiple angularly deflected laser beams having intensities that differ by no more than 10%.

[0187] 47. A particle analyzer as described in Appendix 43, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts one or more parameters of an input waveform to generate multiple angularly deflected laser beams having intensities that differ by no more than 5%. 48. The particle analyzer described in Appendix 43, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor causes the processor to generate an intensity profile plot from a captured image of the output laser beam. 49. The waveform generator is an arbitrary waveform generator (AWG), as described in any one of the appendices 37-48. 50. A particle analyzer as described in any one of the appendices 37 to 49, comprising multiple angle-deflected laser beams, including a local oscillator (LO) beam and multiple radio frequency comb beams. 51. A particle analyzer described in any one of the appendices 37-50, in which multiple angle-deflected laser beams are spatially separated.

[0188] 52. A particle analyzer as described in Appendix 51, wherein each angle-deflected laser beam at least partially overlaps with one other angle-deflected laser beam in the output laser beam. 53. The particle analyzer is a particle analyzer that is part of a flow cytometer, as described in any one of the appendices 37 to 52.

[0189] 54. It is a system, Laser and, Acoustic-optical devices and, A waveform generator configured to input a waveform to an acoustic-optical device and generate an output laser beam containing multiple angle-deflected laser beams, Photodetector and, A controller equipped with a processor It is equipped with, The processor has memory operably coupled to the processor, the memory contains instructions stored in the memory, and when an instruction is executed by the processor, the processor... The photodetector determines the intensity profile of the output laser beam along the horizontal axis, and In response to the determined intensity profile, one or more parameters of the waveform input to the acousto-optical device are adjusted to generate an output laser beam having a modulated intensity profile. A system that enables this to happen. 55. The photodetector is an imaging sensor, as described in Appendix 54 of the system. 56. The imaging sensor is a camera, as described in Appendix 54 of the system. 57. The memory contains instructions stored in the memory, and when the instructions are executed by the processor, the system contains instructions that cause the processor to generate an intensity profile plot of the output laser beam, as described in any one of the appendices 54 to 56. 58. The system described in any one of appendices 55 to 57, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor causes the processor to generate an intensity profile plot from a captured image of the output laser beam.

[0190] 59. The acousto-optical device is an acousto-optical deflector, as described in any one of the appendices 54-58. 60. A system described in any one of the appendices 54-59, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor causes the processor to adjust one or more tones of an input waveform. 61. The system described in Appendix 60, in which each tone of the input waveform contains one or more sine waves. 62. The system as described in Appendix 60 or 61, wherein the memory includes instructions stored in the memory, and when the instructions are executed by the processor, the processor causes the processor to adjust the amplitude of one or more tones of an incoming waveform. 63. Memory contains instructions stored in memory, and when an instruction is executed by the processor, the processor... Determining the amplitude of each angularly deflected laser beam in the output laser beam, For each angle-deflected laser beam, adjust one or more parameters of the waveform input to the acousto-optical device. A system described in any one of the appendices 54-62 that performs the following action.

[0191] 64. The system as described in Appendix 63, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts the amplitude of the input waveform tone for each angle-deflected laser beam. 65. The system as described in Appendix 64, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts the frequency of the input waveform tone for each angle-deflected laser beam. 66. The system as described in Appendix 65, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts one or more parameters of an input waveform to generate multiple angularly deflected laser beams having intensities that differ by no more than 10%. 67. The system as described in Appendix 65, wherein the memory contains instructions stored in the memory, and when the instructions are executed by the processor, the processor adjusts one or more parameters of an input waveform to generate multiple angularly deflected laser beams having intensities that differ by no more than 5%. 68. The waveform generator is an arbitrary waveform generator (AWG), as described in any one of the systems listed in Appendix 54-67.

[0192] 69. A system of multiple angle-deflected laser beams, comprising a local oscillator (LO) beam and multiple high-frequency comb beams, as described in any one of the appendices 54 to 68. 70. Multiple angle-deflected laser beams are spatially separated in a system as described in any one of the appendices 54-69. 71. The system as described in Appendix 70, wherein each angle-deflected laser beam at least partially overlaps with one other angle-deflected laser beam in the output laser beam. 72. The system is a flow cytometer, as described in any one of the appendices 54 to 71.

[0193] 73. A non-temporary computer-readable storage medium, The instruction includes a command stored in a non-temporary computer-readable storage medium for modulating the intensity profile of the laser in the particle analyzer, and the command is: An algorithm for generating an output laser beam containing multiple angle-deflected laser beams by irradiating an acoustic-optical device with a laser, An algorithm for capturing an image of the output laser beam, An algorithm for determining the intensity profile of the output laser beam along the horizontal axis from captured images, The algorithm includes, in response to the determined intensity profile, adjusting one or more parameters of the waveform input to the acousto-optical device to generate an output laser beam having a modulated intensity profile, Non-temporary computer-readable storage medium. 74. A non-temporary computer-readable storage medium as described in Appendix 73, comprising an algorithm for adjusting one or more tones of an input waveform. 75. A non-temporary computer-readable storage medium as described in Appendix 74, in which each tone of the input waveform contains one or more sine waves. 76. A non-temporary computer-readable storage medium as described in Appendix 74 or 75, comprising an algorithm for adjusting the amplitude of one or more tones of an input waveform. 77. Non-temporary computer-readable storage media are, An algorithm for determining the amplitude of each angularly deflected laser beam in the output laser beam, For each angle-deflected laser beam, an algorithm is used to adjust one or more parameters of the waveform input to the acoustic-optical device. A non-temporary computer-readable storage medium, including any one of the items described in appendices 73 to 76.

[0194] 78. A non-temporary computer-readable storage medium as described in Appendix 77, comprising an algorithm for adjusting the amplitude of the input waveform tone for each angle-deflected laser beam. 79. A non-temporary computer-readable storage medium as described in Appendix 77, comprising an algorithm for adjusting the frequency of the tone of the input waveform for each angle-deflected laser beam. 80. A non-temporary computer-readable storage medium according to any one of the appendices 73 to 79, comprising an algorithm for adjusting one or more parameters of an input waveform to produce an output laser beam having a substantially constant intensity profile along the horizontal axis. 81. A non-temporary computer-readable storage medium as described in Appendix 80, comprising an algorithm for generating multiple angularly deflected laser beams having intensities that differ by no more than 10% by adjusting one or more parameters of an input waveform. 82. A non-temporary computer-readable storage medium as described in Appendix 80, comprising an algorithm for generating multiple angularly deflected laser beams having intensities that differ by no more than 5% by adjusting one or more parameters of an input waveform. 83. A non-temporary computer-readable storage medium is a non-temporary computer-readable storage medium as described in any one of Appendices 73 to 82, which includes an algorithm for generating an intensity profile plot from captured images of an output laser beam.

[0195] 84. It is a kit, Laser and, Acoustic-optical devices and, Waveform generator, Image sensor and A kit that includes the following: 85. The imaging sensor is a camera, as described in Appendix 84 of the kit. 86. The acousto-optical device is an acousto-optical deflector, as described in Appendix 84 or 85. 87. The waveform generator is an arbitrary waveform generator (AWG), as specified in one of the kits listed in Appendix 84-86. 88. A kit as described in any one of the appendices 84 to 87, further comprising one or more optical adjustment components. 89. The optical adjustment component is the kit described in Appendix 88, which includes a top-hat beam shaper. 90. The top hat beam shaper is the kit described in Appendix 89, equipped with a Powell lens.

[0196] Although the above inventions have been described in some detail by examples and illustrations for the sake of clear understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made to these inventions in light of the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0197] Therefore, the above merely illustrates the principles of the present invention. Those skilled in the art will understand that various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised, although not expressly described or shown herein. Furthermore, all examples and conditional language enumerated herein are intended primarily to help the reader understand the principles of the present invention and the concepts to which the inventors contribute to further the art, and should be construed as not being limited to such specifically enumerated examples and conditions. Furthermore, all descriptions herein describing the principles, aspects and embodiments of the present invention, as well as their specific examples, are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any elements developed to perform the same function, regardless of their structure. Furthermore, nothing disclosed herein is intended to be publicly dedicated, whether such disclosure is expressly stated in the claims or not.

[0198] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims. In the claims, Section 112(f) or Section 112(6) of the U.S. Patent Act is expressly defined as being referenced for the limitation in the claims only when the exact phrase “means for” or the exact phrase “steps for” is enumerated at the commencement of such limitation in the claims, and if such exact phrase is not used in the limitation in the claims, Section 112(f) or Section 112(6) of the U.S. Patent Act is not referenced.

[0199] cross reference In accordance with Section 119(e) of the United States Patent Act, this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 027,080, filed on 19 May 2020, the entire disclosure of that application is incorporated herein by reference.

Claims

1. A method for modulating the intensity profile of a laser in a particle analyzer, The process involves irradiating an acoustic-optical device with a laser to generate an output laser beam containing multiple angle-deflected laser beams, and The process involves capturing an image of the output laser beam, From the captured image, the intensity profile of the output laser beam along the horizontal axis is determined, In response to the determined intensity profile, one or more parameters of the waveform input to the acousto-optical device are adjusted to generate an output laser beam having a specified intensity profile for exciting and analyzing particles. Methods that include...

2. The method according to claim 1, comprising adjusting one or more tones of the input waveform.

3. The method according to claim 2, wherein each tone of the input waveform includes one or more sine waves.

4. The method according to claim 2 or 3, comprising adjusting the amplitude of one or more tones of the input waveform.

5. Determining the amplitude of each angularly deflected laser beam in the output laser beam, For each angle-deflected laser beam, one or more parameters of the waveform input to the acousto-optical device are adjusted. The method according to any one of claims 1 to 4, further comprising:

6. The method according to claim 5, comprising adjusting the amplitude of the tone of the input waveform for each angle-deflected laser beam, and / or adjusting the frequency of the tone of the input waveform for each angle-deflected laser beam.

7. The method according to any one of claims 1 to 6, wherein one or more parameters of the input waveform are adjusted to produce an output laser beam having a substantially constant intensity profile along the horizontal axis.

8. The method according to any one of claims 1 to 7, further comprising generating an intensity profile plot from a captured image of the output laser beam.

9. The method according to any one of claims 1 to 8, wherein the acousto-optical device is an acousto-optical deflector.

10. The method according to any one of claims 1 to 9, wherein each waveform input to the acousto-optical device is generated by an arbitrary waveform generator (AWG).

11. The method according to any one of claims 1 to 10, wherein the plurality of angle-deflected laser beams include a local oscillator (LO) beam and a plurality of high-frequency comb beams.

12. The method according to any one of claims 1 to 11, wherein the plurality of angle-deflected laser beams are spatially separated.

13. The method according to claim 12, wherein each of the angle-deflected laser beams at least partially overlaps with one other angle-deflected laser beam in the output laser beam.

14. The method according to any one of claims 1 to 13, wherein the particle analyzer is part of a flow cytometer.

15. It is a particle analyzer, Laser and, Acoustic-optical devices and, A waveform generator configured to input a waveform to the aforementioned acoustic-optical device and generate an output laser beam including multiple angle-deflected laser beams, Image sensor and A controller equipped with a processor It is equipped with, The processor has a memory operablely coupled to the processor, the memory includes instructions stored in the memory, and when an instruction is executed by the processor, the processor The imaging light sensor captures an image of the output laser beam, From the captured image, the intensity profile of the output laser beam along the horizontal axis is determined, In response to the determined intensity profile, one or more parameters of the waveform input to the acousto-optical device are adjusted to generate an output laser beam having a specified intensity profile for exciting and analyzing particles. A particle analyzer that performs this operation.

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