Optical detection system with auxiliary light scattering detector and its usage method

Abstract

A system is provided having an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream. The system according to a particular embodiment includes: a light source having two or more lasers; a light detection system having the unfiltered light scattering detector; and a processor having a memory operatively coupled to the processor, wherein the memory includes instructions that, when executed by the processor, cause the processor to generate one or more data signals in response to scattered light detected by the unfiltered light scattering detector from each of the two or more lasers; and to determine one or more parameters for data acquisition based on the generated data signals from the unfiltered light scattering detector. A method for determining one or more parameters for data acquisition using the subject system is also described.

Description

[0001] Cross-reference to related applications

[0002] This application relates to U.S. Provisional Patent Application Serial No. 62 / 981,932, filed February 26, 2020; the disclosure of which is incorporated herein by reference. Background Technology

[0003] For example, when samples are used in the diagnosis of diseases or medical conditions, optical detection is often employed to characterize the composition of the sample (e.g., a biological sample). When a sample is illuminated, light can be scattered by the sample, transmitted through the sample, and emitted by the sample (e.g., in a fluorescent manner). Variations in sample composition, such as morphology, absorbance, and the presence of fluorescent labels, can cause variations in the light scattered by the sample. To quantify these variations, the light is collected and directed onto the surface of a detector.

[0004] One technique that uses optical detection to characterize the components in a sample is flow cytometry. Using data generated based on the detected light, the properties of the components can be recorded, and desired substances can be sorted. A flow cytometer typically includes a sample reservoir for receiving fluid samples, such as blood samples, and a sheath reservoir containing sheath fluid. The flow cytometer delivers particles (including cells) from the fluid sample as a cell stream to a flow cell, while simultaneously directing the sheath fluid into the flow cell. Within the flow cell, a liquid sheath forms around the cell stream to impose a substantially uniform velocity on it. The flow cell hydrodynamically concentrates 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 light or by transmission spectroscopy, or it can be absorbed by one or more components in the sample and re-emitted as cold light. Summary of the Invention

[0005] This disclosure includes aspects of a system having an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream. A system according to a particular embodiment includes: a light source having two or more lasers; a light detection system having an unfiltered light scattering detector; and a processor having a memory operatively coupled to the processor, wherein the memory includes instructions that, when executed by the processor, cause the processor to generate one or more data signals in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector, and to determine one or more parameters for data acquisition based on the generated data signals from the unfiltered light scattering detector. In some embodiments, the one or more parameters for data acquisition include the timing of particle irradiation by each of the two or more lasers. In a particular instance, the system includes: a memory having instructions for adjusting one or more parameters for data acquisition based on the generated data signals from the unfiltered light scattering detector. For example, the duration of data acquisition can be adjusted (e.g., reducing the duration of data acquisition). In other embodiments, one or more parameters for data acquisition include parameters for identifying the position of particles in the fluid flow in response to the generated data signal from the unfiltered light scattering detector. In a particular embodiment, the system includes a memory having instructions for generating one or more particle sorting parameters in response to the data signal from the unfiltered light scattering detector. In some instances, the particle sorting parameters are particle sorting timing sequences.

[0006] In one embodiment, the system includes: a light detection system having an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream illuminated by the two or more lasers. In some embodiments, the light scattering detector is a side-scattering photodetector. In other embodiments, the light scattering detector is a front-scattering photodetector. In other embodiments, the light scattering detector is a back-scattering photodetector. The subject light detection system may also include a filtered light scattering detector. In some instances, the filtered light scattering detector is configured to detect light from one of the lasers scattered by the sample in the liquid stream. In a particular instance, the filtered light scattering detector includes: a light scattering detector; and a dimming component (e.g., a bandpass filter, a dichroic mirror) configured to transmit light from the one laser scattered by the sample to the light scattering detector. In the optical detection system, a dimming component may be disposed in the optical path between the filtered light scattering detector and the unfiltered light scattering detector, for example, wherein the dimming component (e.g., a beam splitter) is configured to transmit scattered light from the sample to the unfiltered light scattering detector and the filtered light scattering detector.

[0007] This disclosure also includes a method for determining one or more parameters of data acquisition based on the generated data signals from an unfiltered light scattering detector. A method according to a particular embodiment includes: detecting light from the liquid stream using a photodetector system comprising an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream irradiated by two or more lasers; generating one or more data signals in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector; and determining one or more parameters of data acquisition based on the generated data signals from the unfiltered light scattering detector. In some embodiments, the parameters determined based on the generated data signals from the unfiltered light scattering detector are the timing of particle irradiation. In other embodiments, the parameters determined based on the generated data signals from the unfiltered light scattering detector are particle sorting parameters, such as particle sorting timing. In a particular embodiment, the method further includes: adjusting one or more parameters based on the generated data signals from the unfiltered light scattering detector, such as adjusting the irradiation timing, data acquisition duration, or particle sorting timing.

[0008] This disclosure also includes a kit comprising: two or more light scattering detectors; a filter assembly; and a dimming assembly for transmitting light to each light scattering detector. The kit may also include other dimming assemblies, such as a masking assembly including an optical aperture, slits, a masking disk, and a scattering strip. Attached Figure Description

[0009] The invention can be best understood based on the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures:

[0010] Figure 1 The arrangement of components of a light detection system according to a particular embodiment is described.

[0011] Figure 2 A method for detecting light scattering by illuminating the far end of a flow cell using an optical system, according to a specific embodiment, is described.

[0012] Figure 3 A flowchart is depicted according to a specific embodiment for determining and adjusting one or more parameters based on a generated data signal from an unfiltered light scattering detector.

[0013] Figure 4A A functional block diagram of a particle analysis system according to a specific embodiment is depicted. Figure 4B A flow cytometer according to a specific embodiment is described.

[0014] Figure 5 A functional block diagram of an example of a particle analyzer control system according to a particular embodiment is depicted.

[0015] Figure 6A A schematic diagram of a particle sorter system according to a specific embodiment is depicted.

[0016] Figure 6B A schematic diagram of a particle sorter system according to a specific embodiment is depicted.

[0017] Figure 7 A block diagram of a computing system according to a particular embodiment is depicted.

[0018] Figure 8A and Figure 8B The method of adjusting data acquisition using data signals from an unfiltered light scattering detector according to a specific embodiment is illustrated. Figure 8A The illustration depicts a misalignment between a time-shifted laser pulse and a data acquisition window caused by a change in particle velocity, according to a specific embodiment. Figure 8B The diagram depicts the realignment of the data acquisition window using an unfiltered light scattering detector according to a specific embodiment. Detailed Implementation

[0019] A system is provided having an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream. The system, according to a particular embodiment, includes: a light source having two or more lasers; a light detection system having the unfiltered light scattering detector; and a processor having a memory operatively coupled to the processor, wherein the memory includes instructions that, when executed by the processor, cause the processor to generate one or more data signals in response to scattered light detected by the unfiltered light scattering detector from each of the two or more lasers, and to determine one or more parameters for data acquisition based on the generated data signals from the unfiltered light scattering detector. A method for determining one or more parameters for data acquisition using the subject system is also described.

[0020] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, and therefore variations are possible. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be defined only by the appended claims.

[0021] Where ranges of values ​​are provided, it should be understood that, unless the context clearly specifies otherwise, every intermediate value between the upper and lower limits of the range, accurate to one-tenth of the unit of the lower limit, and any other stated value or intermediate value within the stated range, is included in this invention. Smaller upper and lower limits may be independently included in these smaller ranges and are also included in this invention, subject to the explicit exclusion of any limit value from the stated range. Where a stated range includes one or both of the limit values, the range excluding any one or both of those included limit values ​​is also included in this invention.

[0022] Specific ranges are presented herein in numerical form as defined by the term “about”. The term “about” is used herein to provide an exact figure to which it is defined, as well as textual support for figures that are close to or approximate the figures defined by the term. In determining whether a figure is close to or approximates an explicitly listed figure, a close to or approximate unlisted figure may be a figure that is substantially equivalent to the explicitly listed figure in the context in which it is presented.

[0023] Unless otherwise defined, all technical and scientific data used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, representative illustrative methods and materials are described here.

[0024] All disclosures and patents referenced in this specification are incorporated herein by reference as if each individual disclosure or patent were expressly and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials in conjunction with the referenced disclosures. References to any disclosure are made prior to the date of this filing and should not be construed as an admission that the invention is not entitled to a prior art invention prior to that disclosure. Furthermore, the provided disclosure date may differ from the actual disclosure date, which may require separate confirmation.

[0025] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this description is intended as a basis for using exclusive terms such as “only,” “just,” etc., in conjunction with an enumeration of the elements of the claim, or for using a priori limiting term such as “negative.”

[0026] Those skilled in the art will understand upon reading this disclosure that each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any of the enumerated methods may be performed in the order of the enumerated events or in any other logically possible order.

[0027] Although the apparatus and method have been described or will be described for grammatical fluency and functional description, it is to be clearly understood that, unless expressly stated in accordance with 35 U.SC §112, the claims need not be construed as limiting by any means of constructing an “apparatus” or “step,” but should conform to the full scope of the meaning and equivalent meaning provided by the claims under the judicial interpretation of equivalent claims, and where the claims are expressly stated in accordance with 35 U.SC §112, they should conform to the full statutory equivalent meaning under 35 U.SC §112.

[0028] In summary, a system with an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream is provided. In embodiments further described in this disclosure, a system having a light source with two or more lasers and a light detection system including an unfiltered light scattering detector is first described in more detail. Next, a system and method for determining and adjusting one or more parameters of data acquisition based on generated data signals from the unfiltered light scattering detector are described. A kit having one or more components of a subject light detection system is also provided.

[0029] Optical detection system

[0030] Aspects of this disclosure include: a light detection system having an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream illuminated by two or more lasers. The term "light scattering" is used herein in its conventional sense to refer to the propagation of light energy from particles in a sample (e.g., flowing in a liquid stream), which are deflected from the incident light path, such as by reflection, refraction, or deflection of the light beam. In some embodiments, the scattered light is not cold light from a particle component (e.g., a fluorophore). In embodiments, the scattered light according to this disclosure is not fluorescence or phosphorescence. In a particular embodiment, the scattered light detected by the scattering photodetector of the subject system comprises Mie scattering through particles in the liquid stream. In other embodiments, the scattered light detected by the scattering photodetector of the subject system comprises Rayleigh scattering through particles in the liquid stream. In still other embodiments, the scattered light detected by the scattering photodetector of the subject system comprises both Mie scattering and Rayleigh scattering through particles in the liquid stream. The scattering photodetector may be a side-scattering photodetector, a front-scattering photodetector, a back-scattering photodetector, or a combination thereof.

[0031] The light detection system according to an embodiment includes an unfiltered light scattering detector. The term "unfiltered" is used herein to refer to a light scattering detector that receives light from a sample that has not been transmitted through an optical component configured to define, reduce, or otherwise restrict the propagation of one or more wavelengths of light from the sample (e.g., the wavelength of light from a laser used to illuminate the sample) to the effective surface of the light scattering detector. For example, in some embodiments, the unfiltered light scattering detector of interest is not optically connected to the sample via a bandpass filter. In other embodiments, the unfiltered light scattering detector of interest is not optically connected to the sample via a dichroic mirror. In a particular instance, scattered light from the sample is transmitted directly to the effective surface of the unfiltered light scattering detector. In other instances, scattered light from the sample is transmitted to the effective surface of the unfiltered light scattering detector via one or more light propagation optics, such as optics that change the direction or focus of the beam without reducing, defining, or restricting the propagation of one or more wavelengths of light. In a particular embodiment, one or more beam splitters, mirrors, lenses, or collimators are used to propagate scattered light from the sample to the effective surface of the unfiltered light scattering detector.

[0032] As described in more detail below, scattered light from a sample illuminated by a light source having a number of lasers is detected by an unfiltered light scattering detector, wherein the number is two or more lasers, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, for example, ten or more lasers, for example, fifteen or more lasers, for example, twenty-five or more lasers, and including fifty or more lasers. In an embodiment, the light scattered by the sample from two or more lasers of the light source is detected by the unfiltered light scattering detector, for example, from three or more lasers, for example, from four or more lasers, for example, from five or more lasers, for example, from ten or more lasers, for example, from fifteen or more lasers, and including light scattered by the sample from twenty-five or more lasers of the light source. In a particular embodiment, the unfiltered light scattering detector is configured to detect light scattered by the sample from 50% or more of the lasers in the light source (e.g., 2 out of a total of 4 lasers), such as 60% or more, 70% or more, 75% or more, 80% or more, and including 90% or more of the lasers in the light source. In a particular example, the unfiltered light scattering detector is configured to detect light scattered by the sample from all the lasers in the light source.

[0033] In some embodiments, the light detection system includes one or more filtered light scattering detectors. The term "filtered" is used herein to refer to a light scattering detector that receives light transmitted from a sample via optical components configured to define, reduce, or limit the propagation of at least one or more wavelengths of light from the sample (e.g., one or more wavelengths of light from a laser used to illuminate the sample) to the effective surface of the light scattering detector. The light transmitted to the light scattering photodetector may include optical components that limit the propagation of one or more different wavelengths of light, such as five or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, and including limiting the propagation of 500 or more different wavelengths of light. For example, in some embodiments, scattered light from the sample is transmitted to the effective surface of the filtered light scattering detector via a bandpass filter. In other embodiments, scattered light from the sample is transmitted to the effective surface of the filtered light scattering detector via a dichroic mirror.

[0034] Depending on the number of lasers used to illuminate the sample, in some embodiments, the filtered light scattering detector is configured to detect light scattered by the sample from five or fewer lasers, such as four or fewer lasers, three or fewer lasers, and including two or fewer lasers. In a particular instance, the filtered light scattering detector is configured to detect light scattered by the sample from a single laser of the light source. For example, the filtered light scattering detector may be configured to detect light scattered by lasers comprising 50% or fewer of the light source's lasers (e.g., two out of a total of four lasers), such as 40% or fewer, such as 30% or fewer, such as 25% or fewer, such as 20% or fewer, and including 10% or fewer of the light source's lasers. In a particular instance, the filtered light scattering detector is configured to detect the scattered light by means of a single laser.

[0035] Scattered light can be detected by each photodetector at an angle relative to the incident illumination beam, for example, at angles of 1° or greater, 10° or greater, 15° or greater, 20° or greater, 25° or greater, 30° or greater, 45° or greater, 60° or greater, 75° or greater, 90° or greater, 135° or greater, or 150° or greater, and including a scattered light detector configured to detect light from particles in the sample at an angle of 180° or greater relative to the incident illumination beam. In a particular example, one or more of the filtered and unfiltered scattered light detectors are side-scattering photodetectors, for example, wherein the photodetectors are configured to detect scattered light propagating relative to the incident illumination beam from 30° to 120°, for example from 45° to 105°, and including from 60° to 90°. In a particular example, one or more of the filtered and unfiltered scattered light detectors are side-scattering photodetectors positioned at an angle of 90° relative to the incident illumination beam. In other examples, one or more of the filtered and unfiltered light scattering detectors are forward-scattering detectors, for example, detectors configured to detect scattered light propagating from 120° to 240° relative to the incident illumination beam, such as from 100° to 220°, such as from 120° to 200°, and including from 140° to 180°. In a particular example, one or more of the filtered and unfiltered light scattering detectors are forward-scattering photodetectors configured to detect scattered light propagating at an angle of 180° relative to the incident illumination beam. In still other examples, one or more of the filtered and unfiltered light scattering detectors are backscattering photodetectors configured to detect scattered light propagating from 1° to 30° relative to the incident illumination beam, such as from 5° to 25°, and including from 10° to 20°. In a particular instance, one or more of the filtered and unfiltered light scattering detectors are configured as backscattering photodetectors to detect scattered light propagating at an angle of 30° relative to the incident illumination beam.

[0036] Each light-scattering photodetector in the subject light detection system can be any suitable photodetector, such as an active pixel sensor (APS), avalanche photodiode, image sensor, charge-coupled device (CCD), enhancement charge-coupled device (ICCD), complementary metal-oxide-semiconductor (CMOS) image sensor or N-type metal-oxide-semiconductor (NMOS) image sensor, light-emitting diode, photon counter, calorimeter, thermoelectric detector, photoresistor, photovoltaic cell, photodiode, photomultiplier tube, phototransistor, quantum dot photoconductor, or combination thereof, and other types of photodetectors. In embodiments, a light-scattering photodetector may include one or more photodetectors, such as two or more, three or more, five or more, ten or more, and 25 or more photodetectors. In some instances, each light-scattering photodetector is a photodetector array. The term "photodetector array" is used in its conventional sense to refer to an arrangement or sequence of two or more photodetectors configured to detect light. In embodiments, the photodetector array may include two or more photodetectors, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, and even fifteen or more. In a particular embodiment, the photodetector array includes five photodetectors. The photodetectors can be arranged in any geometric configuration as needed, wherein arrangements of interest include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular shapes. The photodetectors in the light scattering photodetector array can be oriented at angles ranging from 10° to 180° relative to other photodetectors (with reference in the XZ plane), such as from 15° to 170°, from 20° to 160°, from 25° to 150°, from 30° to 120°, and including angles from 45° to 90°.

[0037] The light scattering photodetector disclosed herein is configured to measure light of one or more wavelengths, such as two or more wavelengths, five or more different wavelengths, ten or more different wavelengths, 25 or more different wavelengths, 50 or more different wavelengths, 100 or more different wavelengths, 200 or more different wavelengths, 300 or more different wavelengths, and including measuring light of 400 or more different wavelengths emitted by a sample in a liquid flow.

[0038] In some embodiments, a subject photodetector is configured to measure light over a wavelength range (e.g., 200 nm–1000 nm) acquired. In particular embodiments, a detector of interest is configured to acquire the spectrum of light over a wavelength range. For example, the system may include one or more detectors configured to acquire the spectrum of light over one or more wavelength ranges from 200 nm to 1000 nm. In still other embodiments, the detector of interest is configured to measure light of one or more specific wavelengths from a sample in a liquid stream. In embodiments, the light detection system is configured to measure light continuously or at discontinuous intervals. In some instances, the detector of interest is configured to continuously measure the acquired light. In other instances, the light detection system is configured to perform measurements at discontinuous intervals, such as every 0.001 ms, every 0.01 ms, every 0.1 ms, every 1 ms, every 10 ms, every 100 ms, and including every 1000 ms or some other interval.

[0039] In some embodiments, the light detection system includes: an unfiltered light scattering detector; a filtered light scattering detector; and a dimming component disposed in an optical path between the unfiltered and filtered light scattering detectors, the optical path being configured to transmit scattered light from the sample to both the unfiltered and filtered light scattering detectors. In a particular embodiment, dimming includes splitting the light beam such that a portion of the collected light (light scattered by the sample in the liquid stream) is transmitted to the unfiltered light scattering detector and another portion of the collected light is transmitted to the filtered light scattering detector.

[0040] In some embodiments, the dimming component is a beam splitter. The amount of light propagating through the dimming component to each light scattering photodetector can also vary, wherein in some embodiments, 50% or more of the collected light is transmitted to the light scattering photodetector, for example, 55% or more, 60% or more, 65% or more, 75% or more, 80% or more, 90% or more, and including 95% or more of the light collected by the subject light detection system transmitted through the dimming component to each light scattering photodetector. For example, the amount of light propagating through the dimming component to each light scattering photodetector can range from 25% to 99%, for example, from 30% to 95%, for example, from 35% to 90%, for example, from 40% to 85%, for example, from 45% to 80%, and including from 50% to 75%.

[0041] In some embodiments, 50% or less of the collected light is transmitted to the filtered light-scattering photodetector via a dimming component, for example, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, and including 5% or less of the light collected by the light detection system transmitted to the filtered light-scattering photodetector via the dimming component. For example, the amount of collected light propagated to the filtered light-scattering photodetector via the dimming component can range from 1% to 75%, for example, from 2% to 70%, from 3% to 65%, from 4% to 60%, and including 5% to 50%. In other embodiments, 50% or less of the collected light is transmitted to the unfiltered light-scattering photodetector via the dimming component, for example, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, and including 5% or less of the light collected by the light detection system transmitted to the unfiltered light-scattering photodetector via the dimming component. For example, the amount of collected light propagated to the unfiltered light-scattering photodetector via the dimming component can range from 1% to 75%, for example, from 2% to 70%, from 3% to 65%, from 4% to 60%, and including from 5% to 50%.

[0042] In some embodiments, the dimming component is a beam splitter. As used herein, the term "beam splitter" conventionally refers to an optical component configured to propagate light along two or more distinct optical paths, such that a predetermined portion of the light propagates along each path. Any convenient beam splitting protocol, such as utilizing triangular prisms, strip mirror prisms, dichroic mirror prisms, and other types of beam splitters, can be used. The beam splitter can be formed from any suitable material, as long as it can propagate the desired amount and wavelength of light to both the unfiltered and filtered light scattering detectors. For example, the beam splitter of interest can be formed from glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glass), silica (e.g., fused silica), quartz, crystals (e.g., CaF2 crystals), zinc selenide (ZnSe), F2, germanium (Ge), titanates (e.g., S-TIH11), or borosilicates (e.g., BK7). In certain embodiments, the bundle splitter is formed of a polymeric material, such as, but not limited to, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics, such as PETG (ethylene glycol-modified polyethylene terephthalate), and other polymeric plastic materials.In certain embodiments, the bundle splitter is formed of polyester, wherein the polyester of interest may include, but is not limited to: poly(alkyl terephthalate), such as poly(ethylene terephthalate) (PET), bottle-grade PET (a copolymer based on ethylene glycol, terephthalic acid and other comonomers such as isophthalic acid, cyclohexanediol, etc.), poly(butylene terephthalate) (PBT), and poly(hexamethylene terephthalate); poly(alkyl adipate), such as poly(ethylene adipate), poly(alkyl adipate 1), etc. ,4-Butanediol ester) and poly(hexamethylene adipate); poly(alkylene octanoate), such as poly(ethylene octanoate); poly(alkylene sebacate), such as poly(ethylene sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalate), such as poly(ethylene isophthalate); poly(alkylene 2,6-naphthalenedicarboxylate), such as poly(ethylene 2,6-naphthalenedicarboxylate); poly(alkylene sulfonyl-4,4'-dibenzoate), such as poly(alkylene sulfonyl-4,4'-dibenzoate). 4,4'-dibenzoic acid ethylene glycol ester); poly(alkylene terephthalate), for example poly(ethylene terephthalate); poly(trans-1,4-cyclohexanediyldicarboxylate), for example poly(trans-1,4-cyclohexanediyldicarboxylate); poly(1,4-cyclohexane-dimethylenedicarboxylate), for example poly(1,4-cyclohexane-dimethylenedicarboxylate); poly([2.2.2]-bicyclooctane-1,4-dimethylenedicarboxylate), for example poly([2 .2.2]-Bicyclooctane-1,4-dimethylenedicarboxylate (ethylene glycol); lactic acid polymers and copolymers, such as (S)-polylactide, (R,S)-polylactide, poly(tetramethylglycolic acid) and poly(lactide-co-glycolic acid); and bisphenol A polycarbonate, 3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A, 3,3′,5,5′-tetramethylbisphenol A; polyamides, such as poly(p-phenylene terephthalamide); polyethylene terephthalate (e.g., Mylar). TM Polyethylene terephthalate (PET); and combinations thereof.

[0043] In certain embodiments, the dimming component is a wedge beamsplitter. In these embodiments, the beamsplitter is a beamsplitter with a wedge angle that produces non-collinear back reflection, such that light acquired and propagated through the wedge beamsplitter results in a small change in the angle of light propagating to one or more of the unfiltered and filtered light scattering detectors. The wedge beamsplitter according to embodiments of this disclosure has a wedge angle, wherein the change in the incident angle of the acquired light results in an angular offset of 0.001% or more, such as 0.005% or more, such as 0.01% or more, such as 0.05% or more, such as 0.1% or more, such as 0.5% or more, such as 1% or more, such as 2% or more, such as 3% or more, such as 5% or more, and including 10% or more. In some embodiments, the wedge beamsplitter has a wedge angle ranging from 5 arcminutes to 120 arcminutes, such as from 10 arcminutes to 115 arcminutes, such as from 15 arcminutes to 110 arcminutes, such as from 20 arcminutes to 105 arcminutes, such as from 25 arcminutes to 100 arcminutes, such as from 30 arcminutes to 105 arcminutes, such as from 35 arcminutes to 100 arcminutes, such as from 40 arcminutes to 95 arcminutes, and including from 45 arcminutes to 90 arcminutes. In a particular embodiment, the wedge beamsplitter has a wedge angle sufficient to reduce or eliminate optical interference. In other embodiments, the wedge beamsplitter has a wedge angle sufficient to reduce or eliminate image artifacts from light measured by an unfiltered or filtered light scattering detector.

[0044] In some embodiments, the wedge beam splitter has a transparent window ranging from 150 nm to 5 μm, from 180 nm to 8 μm, from 185 nm to 2.1 μm, from 200 nm to 6 μm, from 200 nm to 11 μm, from 250 nm to 1.6 μm, from 350 nm to 2 μm, from 600 nm to 16 μm, from 1.2 μm to 8 μm, from 2 μm to 16 μm, or some other wavelength range.

[0045] A beam splitter of interest can be configured to divide the amount of light propagating to an unfiltered light scattering detector and a filtered light scattering detector as needed. In some embodiments, the beam splitter has a beam splitting ratio between the unfiltered and filtered light scattering detectors ranging from 1:99 to 99:1, such as from 5:95 to 95:5, such as from 10:90 to 90:10, such as from 20:80 to 80:20, and including beam splitting ratios from 25:75 to 75:25. In other embodiments, the beam splitter has a beam splitting ratio between the filtered and unfiltered light scattering detectors ranging from 1:99 to 99:1, such as from 5:95 to 95:5, such as from 10:90 to 90:10, such as from 20:80 to 80:20, and including beam splitting ratios from 25:75 to 75:25. In a particular embodiment,

[0046] In some embodiments, the spatial position of the beam splitter is adjustable, for example, by means of manual (hand) or by utilizing a motor-driven displacement device. For example, the angle of the beam splitter can be adjusted in the subject light detection system by 5° or greater, such as 10° or greater, 15° or greater, 20° or greater, 30° or greater, 45° or greater, 60° or greater, and including 75° or greater. In specific instances, the spatial position of the beam splitter can be adjusted in the light detection system by, for example, 1 mm or more, 5 mm or more, 10 mm or more, and including 25 mm or more. For example, any convenient motor-driven actuator can be used, such as a motor-driven displacement stage, a motor-driven lead screw assembly, a motor-operated gear actuator using a stepper motor, a servo motor, a brushless motor, a brushed DC motor, a micro stepper drive motor, a high-resolution stepper motor, and other types of motors. In one example, the horizontal or vertical position or orientation angle of the beam splitter can be adjusted using a motor-driven displacement device.

[0047] Figure 1 An arrangement of components of a light detection system according to a specific embodiment is depicted. The light detection system 100 includes: a flow cell 101, illuminated by a light source 102 having lasers 102a, 102b, 102c, and 102d. A beam splitter 105 transmits light scattered by particles in a sample from the lasers 102a, 102b, 102c, and 102d to an unfiltered light scattering detector 103 (front light scattering detector) and a filtered light scattering detector 104. Light from the beam splitter 105 is transmitted to the filtered light scattering detector 104 via a bandpass filter 104a, which is configured to limit the propagation of light from the lasers 102a, 102b, and 102c and to transmit only the scattered light from the laser 102d to the light scattering detector 104.

[0048] In some embodiments, light from the dimming component is propagated through a shielding component to one or more of an unfiltered light scattering detector and a filtered light scattering detector. In these embodiments, the shielding component is configured to reduce the amount of light transmitted to the detector, for example, by reducing the amount of transmitted light by 1% or more, such as 5% or more, such as 10% or more, such as 25% or more, such as 40% or more, and including reducing the amount of transmitted light by 50% or more. Any convenient shielding scheme can be used, including but not limited to optical openings (e.g., pinholes) or slits. The size of the optical aperture can be varied as needed, wherein the range of the opening of interest is from 0.001 mm to 10 mm, such as from 0.005 mm to 9.5 mm, such as from 0.01 mm to 9 mm, such as from 0.05 mm to 8.5 mm, such as from 0.1 mm to 8 mm, such as from 0.5 mm to 7.5 mm, and including from 1 mm to 5 mm. The shielding slit of interest can also be varied, wherein the width of the slit ranges from 0.001 mm to 10 mm, for example from 0.005 mm to 9.5 mm, for example from 0.01 mm to 9 mm, for example from 0.05 mm to 8.5 mm, for example from 0.1 mm to 8 mm, for example from 0.5 mm to 7.5 mm, and including from 1 mm to 5 mm. The length of the shielding slit can vary according to the width of the light propagating to the light scattering detector, and can be in the range of 1 mm to 50 mm, for example from 2 mm to 45 mm, for example from 3 mm to 40 mm, for example from 4 mm to 35 mm, and including from 5 mm to 25 mm.

[0049] The shielding component used to reduce the amount of light transmitted to the light scattering detector can be of any convenient shape, wherein the cross-sectional shape of interest includes, but is not limited to: cross-sectional shapes composed of straight lines, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; cross-sectional shapes composed of curves, such as circles, ellipses; and irregular shapes, such as parabolic bottoms coupled to the top of a plane. In some embodiments, the shielding component is circular. In other embodiments, the shielding component is elliptical. In still other embodiments, the shielding component is polygonal, such as square or rectangular. The width of the shielding component can vary, ranging from 1 mm to 25 mm in some instances, such as from 2 mm to 22 mm, such as from 3 mm to 20 mm, such as from 4 mm to 17 mm, and including from 5 mm to 15 mm. The length of each shielding component ranges from 1 mm to 50 mm, such as from 2 mm to 45 mm, such as from 3 mm to 40 mm, such as from 4 mm to 35 mm, such as from 5 mm to 30 mm, and including from 10 mm to 20 mm.

[0050] In some embodiments, the light received by the subject light detection system can be transmitted by a light acquisition system. The light acquisition system can be any suitable light acquisition scheme for collecting and directing the light. In some embodiments, the light acquisition system includes optical fibers, such as fiber optic repeater bundles. In other embodiments, the light acquisition system is a free-space optical repeater system.

[0051] In embodiments, the light acquisition system can be physically coupled to the light detection system, for example, by using an adhesive, co-molding together, or integrating into the light detection system. In certain embodiments, the light acquisition system and the light detection system are integrated into a single unit. In some instances, the light acquisition system is coupled to the light detection system using a connector that fastens the light acquisition system to the light detection system, such as using hook and loop fasteners, magnets, latches, slots, countersunk holes, counterbores, grooves, pins, tethers, hinges, Velcro, non-permanent adhesives, or combinations thereof.

[0052] In other embodiments, the optical detection system and the optical acquisition system are optically connected but not physically in contact. In these embodiments, the optical acquisition system may be positioned 0.001 mm or more away from the optical detection system, for example, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 10 mm or more, 25 mm or more, 50 mm or more, and up to 100 mm or more away.

[0053] In a particular embodiment, the optical harvesting system includes optical fiber. For example, the optical harvesting system may be a fiber optic repeater bundle, and light is transmitted to the optical detection system via the fiber optic repeater bundle. Any fiber optic repeater system can be used to propagate light to the optical detection system. In a particular embodiment, suitable fiber optic repeater systems for propagating light to the optical detection system include, but are not limited to, those described in, for example, U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0054] In other embodiments, the light acquisition system is a free-space optical relay system. The phrase "free-space optical relay" as used herein is, in its conventional sense, used to refer to the configuration of one or more optical components for directing light through free space to a light detection system. In a particular embodiment, the free-space optical relay system includes a housing having a near end and a far end, the near end being coupled to the light detection system. The free-space relay system may include any combination of various dimming components, such as lenses, mirrors, slits, pinholes, wavelength splitters, or combinations thereof. For example, in some embodiments, the free-space optical relay system of interest includes one or more focusing lenses. In other embodiments, the subject free-space optical relay system includes one or more mirrors. In still other embodiments, the free-space optical relay system includes a collimating lens. In a particular embodiment, suitable free-space optical relay systems for propagating light to the light detection system are, but are not limited to, those described in, for example, U.S. Patent Nos. 7,643,142, 7,728,974, and 8,223,445, the disclosure of which is incorporated herein by reference.

[0055] As summarized above, aspects of this disclosure also include a system for measuring scattered light from a sample. A system according to a particular embodiment includes: a light source having two or more lasers; a light detection system including an unfiltered light scattering detector as described above; and a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate one or more data signals in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector, and to determine one or more parameters for data acquisition based on the generated data signals from the unfiltered light scattering detector.

[0056] In embodiments, the light source includes two or more lasers, such as three or more lasers, four or more lasers, five or more lasers, ten or more lasers, fifteen or more lasers, twenty-five or more lasers, and even fifty or more lasers. Depending on the composition of the sample (e.g., cells, beads, non-cellular particles, etc.), the lasers can emit light with wavelengths ranging from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, and including from 400 nm to 800 nm. Each laser can be either a pulsed laser or a continuous-wave laser. For example, the laser can be: a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, or a neon-copper (N) laser. eCu ​​lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or second or third harmonics of any of the above lasers.

[0057] In certain embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some instances, the beam generator includes: a laser; and a radio frequency generator configured to apply a radio frequency drive signal to the acousto-optic device to generate two or more angle-shifted laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser in the beam generator of interest can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, or a helium-selenium (HSe) laser. HeSe lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titania-sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers and combinations thereof.

[0058] The acousto-optic device can be any convenient acousto-optic scheme configured to frequency-shift a laser using applied acoustic waves. In a particular embodiment, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angle-shifted laser beam based on light from a laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0059] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angularly offset laser beams in the output laser beam, for example, to apply 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and includes being configured to apply 100 or more radio frequency drive signals.

[0060] In some instances, in order to generate an intensity distribution of an angularly offset laser beam in the output laser beam, the controller is configured to apply an RF drive signal with amplitudes varying from approximately 0.001V to approximately 500V, for example from approximately 0.005V to approximately 400V, for example from approximately 0.01V to approximately 300V, for example from approximately 0.05V to approximately 200V, for example from approximately 0.1V to approximately 100V, for example from approximately 0.5V to approximately 75V, for example from approximately 1V to approximately 50V, for example from approximately 2V to approximately 40V, for example from approximately 3V to approximately 30V, and including from approximately 5V to approximately 25V. In some embodiments, each applied radio frequency drive signal has a frequency from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and includes frequencies from about 5 MHz to about 50 MHz.

[0061] In a particular embodiment, the controller includes a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with angle-shifted laser beams having a desired intensity distribution. For example, the memory may include instructions for generating two or more angle-shifted laser beams of equal intensity, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more; and the memory may also include instructions for generating 100 or more angle-shifted laser beams of equal intensity. In other embodiments, instructions may be included for generating two or more angle-shifted laser beams of different intensities, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more; and the memory may also include instructions for generating 100 or more angle-shifted laser beams of different intensities.

[0062] In a particular embodiment, the controller has: a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having an intensity that gradually increases from the edge of the output laser beam along the horizontal axis toward the center. In these examples, the intensity of the angularly offset laser beam at the center of the output beam along the horizontal axis can be in the range of 0.1% to about 99% of the intensity of the angularly offset laser beam at the edge of the output laser beam, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, and includes about 10% to about 50% of the intensity of the angularly offset laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller includes a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge of the output laser beam along the horizontal axis toward the center. In these examples, the intensity of the angularly offset laser beam at the edge of the output beam along the horizontal axis can be in the range of 0.1% to about 99% of the intensity of the angularly offset laser beam at the center of the output laser beam, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, and includes about 10% to about 50% of the intensity of the angularly offset laser beam at the center of the output laser beam along the horizontal axis. In some other embodiments, the controller includes a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having a Gaussian intensity distribution along a horizontal axis. In still other embodiments, the controller includes a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having a top hat intensity distribution along a horizontal axis.

[0063] In embodiments, the beam generator of interest can be configured to generate spatially separated angularly offset laser beams within the output laser beam. Depending on the applied RF drive signal and the desired illumination distribution of the output laser beam, the angularly offset laser beams can be separated by 0.001 μm or more, for example, 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, and including 5000 μm or more. In some embodiments, the system is configured to generate overlapping angularly offset laser beams within the output laser beam, for example, overlapping with adjacent angularly offset laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angular offset laser beams (e.g., the overlap of beam points) can be 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.05 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, and including 100 μm or more.

[0064] In a particular instance, a beam generator configured to generate two or more frequency-shifted beams includes a laser excitation module, the disclosure of which is incorporated herein by reference to U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publications 2017 / 0133857 and 2017 / 0350803.

[0065] In one embodiment, light scattered from a light source by particles in the sample is detected by an unfiltered light scattering detector. In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate one or more data signals in response to scattered light from each of two or more lasers detected by the unfiltered light scattering detector, and to determine one or more parameters for data acquisition based on the generated data signals from the unfiltered light scattering detector.

[0066] In some embodiments, one or more parameters of data acquisition determined based on generated data signals from the unfiltered light scattering detector include the timing of data acquisition from one or more photodetectors in the subject system. For example, the timing of data acquisition from one or more other light scattering detectors, emission photodetectors, transmission photodetectors, or flow imaging sensors can be determined using generated data signals from the unfiltered light scattering detector. In some embodiments, one or more parameters of data acquisition determined based on generated data signals from the unfiltered light scattering detector include the identification of the position of particles in the flow. In other embodiments, one or more parameters of data acquisition determined based on generated data signals from the unfiltered light scattering detector include the duration between light scattered by particles in the sample from each laser. In other embodiments, one or more parameters of data acquisition determined based on generated data signals from the unfiltered light scattering detector include the velocity variation of particles in the sample. In a particular instance, the system includes a memory having instructions for generating one or more particle sorting parameters in response to data signals from the unfiltered light scattering detector. In some instances, the particle sorting parameters are particle sorting timing sequences, such as timing sequences for charging droplets containing particles.

[0067] In certain instances, the system includes a memory having instructions for adjusting one or more parameters of data acquisition based on a generated data signal from the unfiltered light scattering detector. In some embodiments, the system includes a memory having instructions for changing the duration of data acquisition (i.e., the data acquisition window). In some instances, the memory includes instructions for reducing the duration of data acquisition by 5% or more, such as 10% or more, such as 25% or more, and including reducing the duration of data acquisition by 50% or more. For example, the duration of data acquisition may be reduced by 0.0001 μs or more, such as 0.0005 μs or more, such as 0.001 μs or more, such as 0.005 μs or more, such as 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.5 μs or more, such as 1 μs or more, and including 5 μs or more.

[0068] In other embodiments, the system includes a memory having instructions for changing the timing of data acquisition. In some instances, the memory includes instructions for adjusting the timing of data acquisition by 5% or more, such as 10% or more, such as 25% or more, and including adjusting the timing of data acquisition by 50% or more. For example, the timing of data acquisition may be adjusted by 0.0001 μs or more, such as 0.0005 μs or more, such as 0.001 μs or more, such as 0.005 μs or more, such as 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.5 μs or more, such as 1 μs or more, and including 5 μs or more.

[0069] In a particular embodiment, the system includes a memory having instructions for adjusting one or more particle sorting parameters in response to data signals from the unfiltered light scattering detector. In some instances, the memory includes instructions for adjusting the particle sorting timing, such as the timing for charging a droplet containing particles. In a particular instance, the memory includes instructions for adjusting the timing for charging the droplet containing particles by 5% or more, such as 10% or more, such as 25% or more, and including adjusting the timing for charging the droplet containing particles by 50% or more. For example, the timing for charging the droplet containing particles can be adjusted by 0.0001 μs or more, such as 0.0005 μs or more, such as 0.001 μs or more, such as 0.005 μs or more, such as 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.5 μs or more, such as 1 μs or more, and including 5 μs or more.

[0070] In other embodiments, the memory includes instructions for adjusting the drop drive frequency in response to a generated data signal from the unfiltered light scattering detector. In some instances, the drop drive frequency is increased by, for example, 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more, and including 25 Hz or more. For example, the drop drive frequency may be increased by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, and including increasing the drop drive frequency by 90% or more. In other instances, the drip drive frequency is reduced by, for example, 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more, and including 25 Hz or more. For example, the drip drive frequency may be reduced by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, and including reducing the drip frequency by 90% or more.

[0071] In other embodiments, the memory includes instructions for adjusting the dripping delay in response to a generated data signal from the unfiltered light scattering detector. In some instances, the dripping delay is increased by, for example, 0.01 μs or more, 0.05 μs or more, 0.1 μs or more, 0.3 μs or more, 0.5 μs or more, 1 μs or more, 2.5 μs or more, 5 μs or more, 7.5 μs or more, and includes increasing the dripping delay by 10 μs or more. For example, the dripping delay may be increased by 1% or more, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and includes increasing the dripping delay by 90% or more. In other instances, the dripping frequency is reduced by, for example, 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.3 μs or more, such as 0.5 μs or more, such as 1 μs or more, such as 2.5 μs or more, such as 5 μs or more, such as 7.5 μs or more, and includes reducing the dripping delay by 10 μs or more. For example, the dripping delay can be reduced by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, and includes reducing the dripping delay by 90% or more.

[0072] In certain embodiments, the system further includes a flow cell configured to deliver a sample in the flow stream. In some instances, the unfiltered light scattering detector is configured to detect light scattered by the sample from one or more lasers configured to illuminate at or near the distal end of the flow cell. In certain instances, the lasers are configured to illuminate at locations from 0.0001 μm to 10 μm from the distal end of the flow cell, for example, 0.0005 μm to 9.5 μm, 0.001 μm to 9 μm, 0.005 μm to 8.5 μm, 0.01 μm to 8 μm, 0.05 μm to 7.5 μm, 0.1 μm to 7 μm, and including locations from 0.5 μm to 5 μm from the distal end of the flow cell. In some embodiments, the system is configured to use data signals from the light scattering detector detecting scattered light from a sample illuminated at the distal end of the flow cell to adjust one or more parameters as described above. In a particular instance, the data signal from this light scattering detector is used to adjust one or more particle sorting parameters, such as particle sorting timing. In a particular embodiment, the data signal from the light scattering detector, which detects scattered light from a sample illuminated at the far end of the flow cell, is sufficient to offset velocity gradients across the flow, such as particles flowing at or near the center of the flow traveling faster than particles flowing at or near the outer edge of the flow.

[0073] Figure 2 A method for detecting light scattering based on distal illumination of a flow cell is described according to a specific embodiment. A flow cell 201 having particles 201a flowing through it is illuminated by a laser 203, and a pulse 203a is generated when a particle is detected by a light scattering detector 204. A laser 202 distally illuminates the flow cell 201 to generate a pulse 202a. In a specific example, the pulse 202a detected by the light scattering detector can be used to adjust the timing for particle sorting (e.g., the timing for charging a droplet containing particles).

[0074] Figure 3 A flowchart is depicted illustrating the use of an optical detection system to determine and adjust one or more parameters based on a generated data signal from an unfiltered light scattering detector, according to a specific embodiment. At step 301, light from particles in a liquid flow is detected using the unfiltered light scattering detector. At step 302, one or more data signals are generated by the unfiltered light scattering detector. At step 303, one or more parameters of the optical detection system are determined based on the data signals from the unfiltered light scattering detector. For example, at step 303, particle positions can be identified, and the flow velocity of particles in the liquid flow, the timing of particles passing through each laser beam, and the duration between the times particles pass through each laser beam can be determined. At step 304, one or more parameters for data acquisition or particle sorting can be adjusted.

[0075] Any convenient flow cell for delivering fluid samples to a sample exploration area can be used. In some embodiments, the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical 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) can vary from 1 mm to 15 mm, for example from 1.5 mm to 12.5 mm, for example from 2 mm to 10 mm, for example from 3 mm to 9 mm, and including 4 mm to 8 mm. The length of the distal truncated conical portion (measured along the longitudinal axis) can also vary from 1 mm to 10 mm, for example from 2 mm to 9 mm, for example from 3 mm to 8 mm, and including 4 mm to 7 mm. In some embodiments, the diameter of the nozzle chamber of the flow cell can vary from 1 mm to 10 mm, for example from 2 mm to 9 mm, for example from 3 mm to 8 mm, and including 4 mm to 7 mm.

[0076] In certain instances, the flow cell does not include a cylindrical portion, and the entire inner cavity of the flow cell is truncated conical. In these embodiments, the length of the truncated conical cavity (measured along the longitudinal axis transverse to the nozzle orifice) can range from 1 mm to 15 mm, for example from 1.5 mm to 12.5 mm, for example from 2 mm to 10 mm, for example from 3 mm to 9 mm, and including from 4 mm to 8 mm. The diameter of the proximal end of the truncated conical cavity can range from 1 mm to 10 mm, for example from 2 mm to 9 mm, for example from 3 mm to 8 mm, and including from 4 mm to 7 mm.

[0077] In some embodiments, the sample fluid flows out from an orifice at the distal end of the flow cell. Depending on the desired flow characteristics, the flow cell orifice can be of any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to: cross-sectional shapes composed of straight lines, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; cross-sectional shapes composed of curves, such as circles, ellipses; and irregular shapes, such as parabolic bottoms coupled to flat tops. In a particular embodiment, the flow cell of interest has a circular orifice. In some embodiments, the size of the nozzle orifice can vary from 1 μm to 20000 μm, for example from 2 μm to 17500 μm, for example from 5 μm to 15000 μm, for example from 10 μm to 12500 μm, for example from 15 μm to 10000 μm, for example from 25 μm to 7500 μm, for example from 50 μm to 5000 μm, for example from 75 μm to 1000 μm, for example from 100 μm to 750 μm, and including 150 μm to 500 μm. In a particular embodiment, the nozzle orifice is 100 μm.

[0078] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell. In another embodiment, the sample injection system is configured to provide a suitable sample flow into the flow cell cavity. Depending on the desired flow characteristics, the rate at which the sample is transferred to the flow cell cavity through the sample injection port can be 1 μL / min or faster, such as 2 μL / min or faster, such as 3 μL / min or faster, such as 5 μL / min or faster, such as 10 μL / min or faster, such as 15 μL / min or faster, such as 25 μL / min or faster, such as 50 μL / min or faster, and including 100 μL / min or faster. In some instances, the rate at which the sample is transferred to the flow cell cavity through the sample injection port is 1 μL / sec or faster, such as 2 μL / sec or faster, such as 3 μL / sec or faster, such as 5 μL / sec or faster, such as 10 μL / sec or faster, such as 15 μL / sec or faster, such as 25 μL / sec or faster, such as 50 μL / sec or faster, and including 100 μL / sec or faster.

[0079] The sample injection port can be a hole disposed in the inner wall of the cavity or a conduit disposed at the proximal end of the cavity. When the sample injection port is a hole disposed in the inner wall of the cavity, the sample injection port hole can be any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: cross-sectional shapes composed of straight lines, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; cross-sectional shapes composed of curves, such as circles, ellipses, etc.; and irregular shapes, such as parabolic bottom coupled to flat top. In a particular embodiment, the sample injection port has a circular hole. The size of the sample injection port hole can vary according to the shape, and in a particular example, it has an opening ranging from 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as from 0.75 mm to 2.25 mm, such as from 1 mm to 2 mm, and including from 1.25 mm to 1.75 mm, such as 1.5 mm.

[0080] In a specific instance, the sample injection port is a conduit located proximally within the flow cell lumen. For example, the sample injection port may be a conduit configured with a sample injection port orifice aligned with the flow cell orifice. When the sample injection port is a conduit aligned with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including but not limited to: cross-sectional shapes composed of straight lines, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; cross-sectional shapes composed of curves, such as circles, ellipses; and irregular shapes, such as parabolic bottoms coupled to flat tops. The orifice of the conduit can vary in shape, and in a specific instance, has an opening ranging from 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, from 0.75 mm to 2.25 mm, for example, from 1 mm to 2 mm, and including from 1.25 mm to 1.75 mm, 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 sample injection port orifice may include a beveled tip with a bevel angle ranging from 1° to 10°, such as from 2° to 9°, such as from 3° to 8°, such as from 4° to 7°, and including a bevel angle of 5°.

[0081] In some embodiments, the flow cell further includes a sheath fluid injection port configured to provide sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to provide a flow of sheath fluid into the flow cell cavity, for example, to generate a stratified flow of sheath fluid around the sample fluid flow together with the sample. Depending on the desired flow characteristics, the rate at which the sheath fluid is transported to the flow cell cavity can be 25 μL / sec or faster, for example 50 μL / sec or faster, for example 75 μL / sec or faster, for example 100 μL / sec or faster, for example 250 μL / sec or faster, for example 500 μL / sec or faster, for example 750 μL / sec or faster, for example 1000 μL / sec or faster, and includes 2500 μL / sec or faster.

[0082] In some embodiments, the sheath fluid injection port is a hole disposed in the inner wall of the cavity. The sheath fluid injection port hole can be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to: cross-sectional shapes composed of straight lines, such as square, rectangular, trapezoidal, triangular, hexagonal, etc.; cross-sectional shapes composed of curves, such as circular, elliptical; and irregular shapes, such as parabolic bottom coupled to flat top. The size of the sample injection port hole can vary according to the shape, and in certain instances, it has an opening ranging from 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as from 0.75 mm to 2.25 mm, such as from 1 mm to 2 mm, and including from 1.25 mm to 1.75 mm, such as 1.5 mm.

[0083] In some embodiments, the system further includes a pump in fluid communication with the flow cell to deliver a fluid flow through the flow cell. Any convenient fluid pump scheme can be used to control the flow of fluid through the flow cell. In a particular instance, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the subject system is configured to deliver fluid through the flow cell at a rate suitable for detecting light from a sample in the fluid flow. In some instances, the sample flow rate in the flow cell is 1 μL / min or faster, such as 2 μL / min or faster, such as 3 μL / min or faster, such as 5 μL / min or faster, such as 10 μL / min or faster, such as 25 μL / min or faster, such as 50 μL / min or faster, such as 75 μL / min or faster, such as 100 μL / min or faster, such as 250 μL / min or faster, such as 500 μL / min or faster, such as 750 μL / min or faster, and including 1000 μL / min or faster. For example, the system may include a pump configured to flow the sample through the flow cell at rates ranging from 1 μL / min to 500 μL / min, such as from 1 μL / min to 250 μL / min, such as from 1 μL / min to 100 μL / min, such as from 2 μL / min to 90 μL / min, such as from 3 μL / min to 80 μL / min, such as from 4 μL / min to 70 μL / min, such as from 5 μL / min to 60 μL / min, and including rates from 10 μL / min to 50 μL / min. In a particular embodiment, the flow rate is from 5 μL / min to 6 μL / min.

[0084] In a particular embodiment, the subject system is a flow cytometry system using the optical detection system described above. Suitable flow cytometry systems may include, but are not limited to, those described below: Ormerod (ed.), FlowCytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (ed.), Flow Cytometry Protocols, Methods in Molecular Biology, Vol. 91, Humana Press (1997); Practical FlowCytometry, 3rd Edition, Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem, Jan., 49 (Part 1): 17-28; Linden et al., Semin Throm Hemost, Oct. 2004, 30(5): 502-11; Alison et al., J Pathol, December 2010, 222(4):335-344; and Herbig et al. (2007) Crit RevTher Drug Carrier Syst, 24(3):203-255; the contents of which are incorporated herein by reference. In particular, flow cytometry systems of interest include BD Biosciences FACSCanto TM II flow cytometer, BDAccuri TM Flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSL Fortes TM X-20 flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BDBiosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TMCell sorter, and BDBiosciencesVia TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter, and BD Biosciences FACSMelody TM Cell sorters, etc.

[0085] In some embodiments, the subject particle sorting system is a flow cytometry cell counting system, such as those described in: U.S. Patent Nos. 10,006,852; 9,952,076; 9,933,341; 9,784,661; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753. 573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; The entire contents of these publications are incorporated herein by reference.

[0086] In certain instances, the subject system is a flow cytometry system configured to image particles in a fluid stream using fluorescence imaging with radio frequency labeled emission (FIRE), such as those described in Diebold et al., Nature Photonics, Vol. 7(10), 806-810 (2013); and those described in U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852 and U.S. Patent Publications 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0087] In some embodiments, the system of interest includes a particle analysis system that can be used to analyze and characterize particles by physically sorting them into a collection container, with or without the use of physical sorting. Figure 4A A functional block diagram illustrating an example particle analysis system is shown. In some embodiments, particle analysis system 401 is a flow system. Figure 4AThe particle analysis system 401 shown can be configured to perform the methods described herein, either fully or partially. The particle analysis system 401 includes a fluid system 402. The fluid system 402 may include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, wherein particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0088] Particle analysis system 401 includes a detection system 404 configured to acquire a signal from each particle as it passes one or more detection stations along a common sample path. Detection station 408 typically refers to a monitoring area 407 along the common sample path. In some implementations, detection may include detecting light or one or more of their other properties as particles 403 pass through monitoring area 407. Figure 4A The image shows a detection station 408 with a monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations can monitor more than one area.

[0089] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be referred to as event data. The data point can be a multidimensional data point that includes values ​​of various properties measured for the particle. The detection system 404 is configured to acquire a sequence of such data points in a first time interval.

[0090] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on a Poisson distribution and the number of data points acquired by the detection system 404 during the first time interval. The control system 406 may also be configured to generate an experimental signal frequency based on the number of data points in that portion of the first time interval. The control system 406 may also compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0091] Figure 4BA system 400 for flow cytometry is shown according to an illustrative embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scattering detector 430, a side scattering detector 435, a fluorescence acquisition 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.

[0092] The 115a-c laser is excited to emit light in the form of a laser beam. Figure 4B In the example system, the laser beams emitted from excitation lasers 415a-415c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first directed through one or more of beam splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits UV light (light with wavelengths in the range of 10 to 400 nm) and reflects both 488 nm and 633 nm light.

[0093] The laser beam is then directed to focusing lens 420, which focuses the beam onto the portion of the fluid flow containing the sample particles within flow chamber 425. A flow chamber is a component of a fluid system that directs particles (typically one at a time) in the flow toward the focused laser beam used for probing. A flow chamber may include the flow cell of a benchtop cytometer or the nozzle tip of an airflow cytometer.

[0094] Depending on the characteristics of the particles (e.g., their size, internal structure) and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles, light from a laser beam interacts with the particles in the sample through diffraction, refraction, reflection, scattering, absorption, and re-emission at various wavelengths. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be routed through one or more of the following: beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence acquisition lens 440 to one or more of the following: front scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a-460f.

[0095] The fluorescence collecting lens 440 collects light emitted due to particle-laser beam interactions and routes this light to one or more beamsplitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number indicates the center of the spectral band. The second number provides the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit light with wavelengths equal to or shorter than a specific wavelength. Long-pass filters, such as long-pass filters 455a-455b, transmit light with wavelengths equal to or longer than a specific wavelength. For example, long-pass filter 455a, a 670 nm long-pass filter, transmits light with wavelengths equal to or longer than 670 nm. Filters are typically selected to optimize the detector's characteristics for a specific fluorescent dye. The filter 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.

[0096] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be classified as, for example, short-pass and long-pass based on the properties of the filters. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits light of 620 nm or shorter wavelengths and reflects light of longer wavelengths than 620 nm in different directions. In one embodiment, beam splitters 445a-445g may include optical mirrors, such as dichroic mirrors.

[0097] A forward-scattering detector 430 is positioned slightly off-center from the beam orientation along the axis of the flow cell and is configured to detect diffracted light, the excitation light traveling primarily in the forward direction through or near the particle. The intensity of the light detected by the forward-scattering detector depends on the overall size of the particle. The forward-scattering detector may include a photodiode. A side-scattering detector 435 is configured to detect light based on refraction and reflection from the particle's surface and internal structure, and tends to increase with increasing particle structural complexity. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side-scattering detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected at the forward-scattering detector 430, the side-scattering detector 435, and the fluorescence detector can be converted into electrical signals (voltages) by the detectors. This data can provide information about the sample.

[0098] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present invention is not limited to Figure 4B The flow cytometer described can include any flow cytometer known in the art. For example, a flow cytometer can have any number of lasers, beam splitters, filters, and detectors employing various wavelengths and different configurations.

[0099] During operation, the cytometer is controlled by a controller / processor 490, and measurement data from the detector can be stored in a memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is 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 the laser, fluid flow parameters, etc. An input / output (I / O) function 497 can also be provided in the system. The memory 495, controller / processor 490, and I / O 497 can all be provided as integrated components of the flow cytometer 410. In such embodiments, a display can also form part of the I / O function 497 for presenting experimental data to a user of the cytometer 400. Alternatively, some or all of the memory 495, controller / processor 490, and I / O function can be components of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 495 and controller / processor 490 can communicate wirelessly or wiredly with the cytometer 410. The controller / processor 490, which combines memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0100] Based on the definition of the configuration of filters and / or beam splitters in the optical path from the flow cell 425 to each detector. Figure 4BThe system shown includes six different detectors (which may be referred to herein as “filter windows” for specifying detectors) for detecting fluorescence in six different wavelength bands. Different fluorescent molecules used in flow cytometry experiments will emit light in their own characteristic wavelength bands. Specific fluorescent labels used for the experiment and its associated fluorescence emission bands can be selected to generally coincide with the filter windows of the detectors. However, due to the availability of more detectors and the use of more labels, a perfect correspondence between filter windows and fluorescence emission spectra is impossible. In reality, while the peaks of the emission spectrum of a particular fluorescent molecule may lie within the filter window of a particular detector, some of the emission spectrum of that label will also overlap with the filter windows of one or more other detectors. This can be referred to as spillover. I / O 497 can be configured to receive data on flow cytometry experiments having groups of fluorescent labels and multiple cell populations with multiple labels, each cell population having a subset of multiple labels. I / O 497 can also be configured to receive biological data assigning one or more labels to one or more cell populations, label density data, emission spectrum data, data on label assignment to one or more labels, and cytometry configuration data. Flow cytometry experimental data, such as label profile characteristics and flow cytometry configuration data, can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more designated labels to flags.

[0101] Figure 5 A functional block diagram illustrates an example of a particle analyzer control system (e.g., analyzer controller 500) for analyzing and displaying biological events. The analyzer controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0102] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry cell counting event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0103] Analysis controller 500 may be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry counting event data. Analysis controller 500 may be configured to provide a graphical display of a first graph including the biological event data to display device 506. For example, analysis controller 500 may also be configured to render a region of interest as a gate surrounding the cluster of biological event data shown by display device 506, overlapping with the first graph. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest drawn based on a single-parameter histogram or a bivariate graph. In some embodiments, the display may be used to display particle parameters or saturated detector data.

[0104] The analysis controller 500 can also be configured to display bio-event data on the display device 506 that differs from other events in the bio-event data outside the door. For example, the analysis controller 500 can be configured to render the colors of the bio-event data contained within the door differently from the colors of the bio-event data outside the door. The display device 506 can be implemented as a monitor, tablet computer, smartphone, or other electronic device with a graphical interface.

[0105] The analysis controller 500 can be configured to receive a door selection signal identifying a door from a first input device. For example, the first input device can be implemented as a mouse 510 (e.g., by clicking when the cursor is over or in the desired door). The mouse 510 can send a door selection signal to the analysis controller 500 identifying a door to be displayed on or manipulated via the display device 506. In some implementations, the first device can be implemented as a keyboard 508 or other tool, such as a touchscreen, stylus, light detector, or voice recognition system, for providing input signals to the analysis controller 500. Some input devices may include multiple input functions. In such implementations, all input functions can be considered as input devices. For example, such as... Figure 5 As shown, mouse 510 may include a right mouse button and a left mouse button, both of which can generate trigger events.

[0106] Triggering events can cause the analysis controller 500 to change the way 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 group of interest for particle sorting.

[0107] In some embodiments, the analysis controller 500 may be configured to detect when a gating selection is initiated via a mouse 510. The analysis controller 500 may also be configured to automatically modify the visualization plot to facilitate the gating process. The modification may be based on a specific distribution of the biological event data received by the analysis controller 500.

[0108] The analysis controller 500 can be connected to the storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can also be configured to allow retrieval of biological event data, such as flow cytometry event data, via the analysis controller 500.

[0109] Display device 506 can be configured to receive display data from analysis controller 500. The display data may include graphs of biological event data and cross-sectional views of delineated plots. Display device 506 can also be configured to change the presented information based on a combination of input received from analysis controller 500 and input from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0110] In some implementations, the analysis controller 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include controls for receiving sample events or sample images. Sample events, images, or sample gates may be provided before acquiring event data for the samples or based on an initial set of events from a portion of the samples.

[0111] In some embodiments, the system of interest includes a particle sorter system. Figure 6A This is a schematic diagram of a particle sorter system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. Figure 6A As shown, a drop-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, include, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically concentrates a sample fluid 606, including particles 609, into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, particles 609 (e.g., cells) align in a single file across a monitoring area 611 (e.g., a laser-stream intersection) irradiated by an irradiation source 612 (e.g., a laser). Vibration of the drop-forming transducer 602 causes the moving fluid column 608 to split into multiple droplets 610, some of which contain particles 609.

[0112] In operation, when a particle (or cell) of interest crosses the monitoring area 611, a detection stage 614 (e.g., an event detector) identifies it. The detection stage 614 inputs to a timing circuit 628, which in turn inputs to a flash charge circuit 630. At a drop interruption point notified by a timed drop delay (Δt), a flash charge can be applied to the moving fluid column 608 to charge the droplet of interest. The droplet of interest may include one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect the droplet into a container (e.g., a collection tube or a porous or microporous sample tray, where the pores or micropores can be associated with the droplet of particular interest). Figure 6A As shown, the droplets can be collected in the discharge container 638.

[0113] A detection system 616 (e.g., a drop boundary detector) is used to automatically determine the phase of the drop drive signal as a particle of interest passes through the monitoring region 611. An exemplary drop boundary detector is described in U.S. Patent No. 7,679,039, the entirety of which is incorporated herein by reference. The detection system 616 allows the instrument to accurately calculate the position of each particle detected in the droplet. The detection system 616 may input an amplitude signal 620 and / or a phase signal 618, which are then (via amplifier 622) input to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the drop forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0114] In some implementations, sorting electronics (e.g., detection system 616, detection stage 614, and processor 640) may be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions may be included in the event data of the particles. In some implementations, detection system 616 and detection stage 614 may be implemented as a single detection unit or communicatively coupled so that event measurement results can be collected by one of detection system 616 or detection stage 614 and provided to a non-collecting element.

[0115] Figure 6B This is a schematic diagram of a particle sorter system according to an embodiment presented herein. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via current-charged wires in the barbs. This produces a series of droplets 610 containing particles 610 to be analyzed. The particles can be illuminated using one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. This information is then processed by, for example, sorting electronics or other detection systems. Figure 6B(Not shown in the image) Analyze particle information. Deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets to guide them to the target collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflectors 652 and 654 can be controlled to direct particles toward container 674 along a first path 662 or orient them toward container 678 along a second path 668. If the particles are of no interest (e.g., do not exhibit scattering or illuminance information within a specific sorting range), the deflectors can allow the particles to continue away along the flow path 664. Such uncharged droplets can, for example, enter the waste container via a suction device 670.

[0116] It may include sorting electronics to initiate the collection of measurement results, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to result in the sorting of the particles. Figure 6B The example implementation shown includes a commercially available BD FACSAria system provided by Becton, Dickinson (Franklin Lakes, New Jersey). TM A series of flow cytometers.

[0117] Computer control system

[0118] This disclosure also includes a computer control system, wherein the system further includes one or more computers for full or partial automation. In some embodiments, the system includes: a computer having a computer-readable storage medium storing a computer program, wherein, when loaded onto the computer, the computer program includes: instructions for irradiating a liquid stream with a light source having two or more lasers; an algorithm for detecting light scattered from the irradiated liquid stream using an unfiltered light scattering detector; and, in a particular instance, an algorithm for generating a data signal from the unfiltered light scattering detector and determining one or more parameters for data acquisition based on the generated data signal from the unfiltered light scattering detector. In some embodiments, the memory includes an algorithm for determining the timing of data acquisition by one or more photodetectors in the subject system. In other embodiments, the memory includes an algorithm for identifying the position of particles in the liquid stream. In still other embodiments, the memory includes an algorithm for determining the duration between light scattered by particles in the sample from each laser. In yet other embodiments, the memory includes an algorithm for generating one or more particle sorting parameters in response to data signals from the unfiltered light scattering detector.

[0119] In certain instances, the system includes: a memory having an algorithm for adjusting one or more parameters of data acquisition based on generated data signals from the unfiltered light scattering detector. In some instances, the system includes: a memory having an algorithm for changing the duration of data acquisition (i.e., the data acquisition window). In other instances, the system includes: a memory having an algorithm for adjusting the timing of data acquisition. In still other instances, the system includes: a memory having an algorithm for adjusting one or more particle sorting parameters in response to data signals from the unfiltered light scattering detector, such as timing for charging a droplet containing particles. In still other instances, the system includes: a memory having an algorithm for adjusting the droplet drive frequency in response to generated data signals from the unfiltered light scattering detector. In still other instances, the system includes: a memory having an algorithm for adjusting the droplet delay in response to generated data signals from the unfiltered light scattering detector.

[0120] In one embodiment, the system includes an input module, a processing module, and an output module. The system may include both hardware and software components, wherein the hardware components may take the form of one or more platforms, such as servers, so that functional elements—those that perform specific tasks of the system (e.g., managing the input and output of information, processing information, etc.)—can be executed by running software applications distributed on one or more computer platforms representing the system.

[0121] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor having access to memory having instructions stored thereon for executing steps of a subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, buffer memory, data backup units, and many other devices. The processor may be a commercially available processor or may be one of other processors that are available or will become available. The processor executes the operating system, and the operating system is connected to firmware and hardware in a well-known manner and facilitates the processor in coordinating and executing the functions of various computer programs that can be written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system, which typically works with the processor, coordinates and executes 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 entirely according to known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes: analog electronics that allow a user to manually align a light source with a liquid flow based on a first optical signal and a second optical signal. In some embodiments, for example, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0122] System memory can be any of a variety of known or future memory storage devices. Examples include any commercially available random access memory (RAM), magnetic media such as resident hard disks or magnetic tapes, optical media such as optical discs, flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. These types of memory storage devices typically read from and / or write to program storage media (not shown), such as optical discs, magnetic tapes, removable hard disks, or floppy disks, respectively. Any of these or other program storage media currently in use or to be developed in the future can be considered a computer program product. It will be understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0123] In some embodiments, a computer program product is described as comprising a computer-usable medium having a computer software program storing control logic (including program code). When executed by a processor / computer, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementing a hardware state machine to perform the functions described herein will be apparent to those skilled in the art.

[0124] The memory can be any suitable device on which the processor can store and receive data, such as magnetic, optical, or solid-state storage devices (including magnetic disks or optical discs or magnetic tapes or RAM or any other suitable device, either fixed or portable). The processor can include a general-purpose digital microprocessor appropriately programmed based on a computer-readable medium carrying the necessary program code. The program can be provided to the processor remotely via a communication channel or pre-stored in a computer program product, such as memory, or some other portable or fixed computer-readable storage medium using any of those devices combined with memory. For example, a magnetic disk or optical disc can carry the program and can be read by a disk writer / reader. The system of the present invention also includes programs, such as computer program products or algorithms used to practice the methods described above. The program according to the invention can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROMs; portable flash drives; and mixtures of these types of devices, such as magnetic / optical storage media.

[0125] The processor may also have access to a communication channel to communicate with a user at a remote location. A remote location indicates that the user is not in direct contact with the system and that input information from external devices is relayed to the input manager, such as computers connected to a wide area network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones).

[0126] In some embodiments, the system 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., RFID, Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB)). Communication protocols and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0127] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as USB ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject system and other external devices, such as (e.g., in a doctor's office or in a hospital environment) computer terminals configured for similar complementary data communication.

[0128] In one embodiment, the communication interface is configured for infrared communication. The system may use a communication or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user may use in combination.

[0129] In one embodiment, the communication interface is configured to provide data transmission via the following connections: Internet Protocol (IP) over a mobile network, Short Message Service (SMS), a wireless connection to a personal computer (PC) connected to a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0130] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, for example using common standards such as 802.11 or... The protocol is either RF or IrDA infrared. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop computer; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has: a display, such as a liquid crystal display (LCD); and input devices, such as buttons, keyboard, mouse, or touchscreen.

[0131] In some embodiments, the communication interface is configured to automatically or semi-automatically transmit data stored in the subject system (e.g., in an optional data storage unit) to a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0132] The output controller may include any of a variety of known display devices for presenting information to a user, regardless of whether the user is human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of picture elements. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. The functional elements of the computer may communicate with each other via a system bus. In alternative embodiments, some of these communications may be implemented using a network or other types of remote communication. According to known techniques, the output manager may also provide information generated by the processing module to a user at a remote location (e.g., via the Internet, telephone, or satellite network). Presenting data through the output manager may be implemented according to a variety of known techniques. According to 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 URLs to allow the user to retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or a type to be developed in the future, but it will typically be a category commonly referred to as a server in computers. However, it can also be a mainframe computer, workstation, or other computer type. It can be connected via any known or future type of cable or other communication system, including wireless systems, whether networked or otherwise. It can be co-located or physically separated. Depending on the type and / or brand of the chosen computer platform, various operating systems can be used on any of these 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, etc.

[0133] Figure 7 The overall architecture of an example computing device 700 according to a particular embodiment is described. Figure 7 The depicted computing device 700's overall architecture includes the arrangement of computer hardware and software components. The computing device 700 may include... Figure 7The diagram shows more (or fewer) of those components. However, it is not necessary to show all of these generally conventional components to provide a achievable disclosure. As shown, computing device 700 includes processing unit 710, network interface 720, computer-readable media drive 730, input / output device interface 740, display 750, and input device 760, all of which can communicate with each other via a communication bus. Network interface 720 can provide connectivity to one or more networks or computing systems. Processing unit 710 can therefore receive information and instructions from other computing systems or services via the network. Processing unit 710 can also communicate bidirectionally with memory 770 and also provide output information to optional display 750 via input / output device interface 740. Input / output device interface 740 can also receive input from optional input device 760, such as keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input device.

[0134] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments), which processing unit 710 executes to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store operating system 772, which provides computer program instructions for use by processing unit 710 in the general management and operation of computing device 700. Memory 770 may also include computer program instructions and other information for implementing aspects of this disclosure.

[0135] method

[0136] This disclosure also includes a method for determining one or more parameters of data acquisition based on generated data signals from an unfiltered light scattering detector. A method according to a particular embodiment includes: detecting light from the liquid stream using a light detection system comprising an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream irradiated by two or more lasers; generating one or more data signals in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector; and determining one or more parameters of data acquisition based on the generated data signals from the unfiltered light scattering detector.

[0137] Scattered light can be detected by each photodetector at an angle relative to the incident illumination beam, such as 1° or greater, 10° or greater, 15° or greater, 20° or greater, 25° or greater, 30° or greater, 45° or greater, 60° or greater, 75° or greater, 90° or greater, 135° or greater, or 150° or greater, and includes a scattered light detector configured to detect light from particles in the sample at an angle of 180° or greater relative to the incident illumination beam. In a particular example, one or more of the filtered and unfiltered scattered light detectors are side-scattering photodetectors, for example, wherein the photodetectors are configured to detect scattered light propagating relative to the incident illumination beam from 30° to 120°, such as from 45° to 105° and including from 60° to 90°. In a particular example, one or more of the filtered and unfiltered scattered light detectors are side-scattering photodetectors positioned at an angle of 90° relative to the incident illumination beam. In other examples, one or more of the filtered and unfiltered light scattering detectors are forward-scattering detectors, for example, detectors configured to detect propagated scattered light from 120° to 240° relative to the incident illumination beam, such as from 100° to 220°, such as from 120° to 200°, and including from 140° to 180° relative to the incident illumination beam. In a particular example, one or more of the filtered and unfiltered light scattering detectors are forward-scattering photodetectors configured to detect propagated scattered light at an angle of 180° relative to the incident illumination beam. In still other examples, one or more of the filtered and unfiltered light scattering detectors are backscattering photodetectors configured to detect propagated scattered light from 1° to 30° relative to the incident illumination beam, such as from 5° to 25°, and including from 10° to 20° relative to the incident illumination beam. In a particular instance, one or more of the filtered and unfiltered light scattering detectors are configured as backscattering photodetectors to detect scattered light propagating at an angle of 30° relative to the incident illumination beam.

[0138] Each light-scattering photodetector in the subject light detection system can be any suitable photodetector, such as an active pixel sensor (APS), avalanche photodiode, image sensor, charge-coupled device (CCD), enhancement charge-coupled device (ICCD), complementary metal-oxide-semiconductor (CMOS) image sensor or N-type metal-oxide-semiconductor (NMOS) image sensor, light-emitting diode, photon counter, calorimeter, thermoelectric detector, photoresistor, photovoltaic cell, photodiode, photomultiplier tube, phototransistor, quantum dot photoconductor, or combination thereof, and other types of photodetectors. In embodiments, a light-scattering photodetector may include one or more photodetectors, such as two or more, three or more, five or more, ten or more, and 25 or more photodetectors. In some instances, each light-scattering photodetector is a photodetector array. In its conventional sense, the term "photodetector array" is used to refer to an arrangement or series of two or more photodetectors configured to detect light. In embodiments, the photodetector array may include two or more photodetectors, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, and even fifteen or more. In a particular embodiment, the photodetector array includes five photodetectors. The photodetectors can be arranged in any geometric configuration as needed, wherein arrangements of interest include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular shapes. The photodetectors in the light scattering photodetector array can be oriented relative to others (with reference in the XZ plane) at angles ranging from 10° to 180°, such as from 15° to 170°, from 20° to 160°, from 25° to 150°, from 30° to 120°, and including from 45° to 90°.

[0139] The light scattering detector disclosed herein is configured to measure light of one or more wavelengths, such as two or more wavelengths, such as five or more different wavelengths, such as ten or more different wavelengths, such as 25 or more different wavelengths, such as 50 or more different wavelengths, such as 100 or more different wavelengths, such as 200 or more different wavelengths, such as 300 or more different wavelengths, and including measuring light of 400 or more different wavelengths emitted by a sample in a liquid stream.

[0140] In some embodiments, a subject photodetector is configured to measure light over a certain wavelength range (e.g., 200 nm–1000 nm). In a particular embodiment, the detector of interest is configured to acquire the spectrum of light over a wavelength range. For example, the system may include one or more detectors configured to acquire the spectrum of light over one or more wavelengths in the range of 200 nm–1000 nm. In still other embodiments, the detector of interest is configured to measure light over one or more specific wavelengths from a sample in a liquid stream. In embodiments, the method includes measuring light continuously or at discontinuous intervals. In some instances, the detector of interest is configured to continuously measure the acquired light. In other instances, the light detection system is configured to perform measurements at discontinuous intervals, such as every 0.001 ms, every 0.01 ms, every 0.1 ms, every 1 ms, every 10 ms, every 100 ms, and including every 1000 ms or some other interval.

[0141] In some embodiments, the method includes generating one or more data signals from an unfiltered light scattering detector and determining one or more parameters for data acquisition based on the generated data signals from the unfiltered light scattering detector. In some embodiments, the method includes determining the timing of data acquisition from one or more photodetectors in a subject system based on the generated data signals from the unfiltered light scattering detector. For example, the timing of data acquisition from one or more other light scattering detectors, emission photodetectors, transmission photodetectors, or flow imaging sensors can be determined using the generated data signals from the unfiltered light scattering detector. In some embodiments, the method includes identifying the position of particles in the flow based on the generated data signals from the unfiltered light scattering detector. In other embodiments, the method includes determining the duration of light scattered from each laser by particles in the sample based on the generated data signals from the unfiltered light scattering detector. In other embodiments, the method includes determining changes in the flow velocity of particles in the sample based on the generated data signals from the unfiltered light scattering detector. In a particular instance, the method includes determining sorting parameters in response to the data signals from the unfiltered light scattering detector. In some instances, the particle sorting parameters are particle sorting timing sequences, such as timing sequences for charging droplets containing particles.

[0142] In certain instances, the method includes adjusting one or more parameters of data acquisition based on the generated data signal from the unfiltered light scattering detector. In some embodiments, the method includes changing the duration of data acquisition (i.e., the data acquisition window). In some instances, changing the duration of data acquisition includes reducing the duration of data acquisition by 5% or more, such as 10% or more, such as 25% or more, and including reducing the duration of data acquisition by 50% or more. For example, the duration of data acquisition may be reduced by 0.0001 μs or more, such as 0.0005 μs or more, such as 0.001 μs or more, such as 0.005 μs or more, such as 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.5 μs or more, such as 1 μs or more, and including 5 μs or more.

[0143] In other embodiments, the method includes changing the timing of data acquisition. In some instances, the method includes instructions to adjust the timing of data acquisition by 5% or more, such as 10% or more, such as 25% or more, and includes adjusting the timing of data acquisition by 50% or more. For example, the timing of data acquisition may be adjusted by 0.0001 μs or more, such as 0.0005 μs or more, such as 0.001 μs or more, such as 0.005 μs or more, such as 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.5 μs or more, such as 1 μs or more, and including 5 μs or more.

[0144] In certain embodiments, the method includes adjusting one or more particle sorting parameters in response to data signals from the unfiltered light scattering detector. In some instances, the method includes adjusting the particle sorting timing, such as the timing for charging a droplet containing particles. In certain instances, the timing for charging the droplet containing particles is adjusted by 5% or more, such as 10% or more, such as 25% or more, and may include adjusting the timing for charging the droplet containing particles by 50% or more. For example, the timing for charging the droplet containing particles may be adjusted by 0.0001 μs or more, such as 0.0005 μs or more, such as 0.001 μs or more, such as 0.005 μs or more, such as 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.5 μs or more, such as 1 μs or more, and may include 5 μs or more.

[0145] In other embodiments, the method includes adjusting the drop drive frequency in response to a generated data signal from the unfiltered light scattering detector. In some instances, the drop drive frequency is increased by, for example, 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more, and including 25 Hz or more. For example, the drop drive frequency may be increased by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, and including increasing the drop drive frequency by 90% or more. In other instances, the drop drive frequency is decreased by, for example, 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more, and including 25 Hz or more. For example, the drip drive frequency can be reduced by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, and includes reducing the drip frequency by 90% or more.

[0146] In other embodiments, the method includes adjusting the droplet delay in response to a generated data signal from the unfiltered light scattering detector. In some instances, the droplet delay is increased by, for example, 0.01 μs or more, 0.05 μs or more, 0.1 μs or more, 0.3 μs or more, 0.5 μs or more, 1 μs or more, 2.5 μs or more, 5 μs or more, 7.5 μs or more, and includes increasing the droplet delay by 10 μs or more. For example, the droplet delay may be increased by 1% or more, for example, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and includes increasing the droplet delay by 90% or more. In other instances, the dripping frequency is reduced by, for example, 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.3 μs or more, such as 0.5 μs or more, such as 1 μs or more, such as 2.5 μs or more, such as 5 μs or more, such as 7.5 μs or more, and includes reducing the dripping delay by 10 μs or more. For example, the dripping delay can be reduced by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, and includes reducing the dripping delay by 90% or more.

[0147] Figure 8A and Figure 8B This illustrates adjusting data acquisition using data signals from an unfiltered light scattering detector according to a specific embodiment. Figure 8A Data acquisition windows (801a, 802a, 803a, 804a) for particles irradiated by four different lasers 801, 802, 803, and 804 are depicted. Figure 8A As shown, due to the change in the flow velocity of the particles in the flow cell, the time-shifted pulse of the laser becomes misaligned with the data acquisition windows 801a, 802a, 803a, and 804a. Figure 8B The diagram illustrates how using an unfiltered light scattering detector that detects scattered light from each of lasers 801, 802, 803, and 804 allows the data acquisition window to be realigned with the particle illumination passing through each laser (i.e., the timing of data acquisition is adjusted). In addition to adjusting the timing of data acquisition, the duration of data acquisition can be reduced (i.e., the width of the data acquisition windows 801a, 802a, 803a, and 804a), as... Figure 8B As shown.

[0148] In embodiments, the particles irradiated in the fluid stream can be cells, for example, where the sample in the fluid stream is a biological sample. In its conventional sense, the term "biological sample" is used to refer to any subset, cell, or component of an organism, plant, fungus, or animal tissue that can be found in certain conditions in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sheep cord blood, urine, vaginal fluid, and semen. Therefore, "biological sample" refers to both native organisms and subsets of their tissues, and to homogenates, lysates, or extracts prepared from subsets of organisms or their tissues, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tracts, tears, saliva, milk, blood cells, tumors, and organs. Biological samples can be any type of organic tissue, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some instances, the sample is a blood sample, including all blood, such as blood obtained by venipuncture or finger prick (wherein the blood may or may not be mixed with any reagents such as preservatives, anticoagulants, etc. before the test).

[0149] In certain embodiments, the sample source is "mammal" (or "mammalian"), a term used broadly to describe organisms in the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subject is human. The method can be applied to samples obtained from human subjects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, young adults, and adults), wherein in certain embodiments, the human subject is an adolescent, young adult, or adult. While the invention can be applied to samples from human subjects, it should be understood that the method can also be performed on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0150] When practicing the subject-matter method, a sample containing particles (e.g., in a flow cytometer) is illuminated using a light source having two or more lasers, such as three or more lasers, four or more lasers, five or more lasers, ten or more lasers, fifteen or more lasers, twenty-five or more lasers, and including fifty or more lasers. Depending on the composition of the sample (e.g., cells, beads, non-cellular particles, etc.), the lasers can emit light with wavelengths ranging from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, and including from 400 nm to 800 nm. Each laser can be either a pulsed laser or a continuous-wave laser. For example, the laser can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, or a strontium laser. Neon-copper (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or second or third harmonics of any of the above lasers.

[0151] The sample can be illuminated continuously or at discontinuous intervals. In some instances, the method involves illuminating the sample continuously with a light source. In other instances, the sample is illuminated at discontinuous intervals with a light source, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or at some other interval.

[0152] Depending on the light source, the sample can be illuminated from the following varying distances: for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2.5 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 15 mm or more, for example, 25 mm or more, and including 50 mm or more. Furthermore, the illumination angle can vary from 10° to 90°, for example, from 15° to 85°, for example, from 20° to 80°, for example, from 25° to 75°, and including a range from 30° to 60°, for example, a 90° angle.

[0153] In certain embodiments, the method includes illuminating a sample with two or more frequency-shifted beams. As described above, a beam generator assembly having a laser and an acousto-optic device for frequency shifting the laser can be used. In these embodiments, the method includes illuminating the acousto-optic device with a laser. Depending on the desired wavelength of light generated in the output laser beam (e.g., when illuminating a sample in a liquid stream), the laser can have a specific wavelength varying from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm, and including from 400 nm to 800 nm. One or more lasers can be used to illuminate the acousto-optic device, such as two or more lasers, such as three or more lasers, such as four or more lasers, such as five or more lasers, and including ten or more lasers. The lasers can include any combination of various types of lasers. For example, in some embodiments, the method includes irradiating an acousto-optic device with a laser array, such as an array of one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0154] When using more than one laser, the lasers can be used to illuminate the acousto-optic device simultaneously, sequentially, or in a combination of both. For example, each of the lasers can be used to illuminate the acousto-optic device simultaneously. In other embodiments, each of the lasers is used to illuminate the acousto-optic device sequentially. When more than one laser is used to illuminate the acousto-optic device sequentially, the duration for which each laser illuminates the acousto-optic device can be 0.001 microseconds or longer, for example, 0.01 microseconds or longer, for example, 0.1 microseconds or longer, for example, 1 microsecond or longer, for example, 5 microseconds or longer, for example, 10 microseconds or longer, for example, 30 microseconds or longer, and including 60 microseconds or longer. For example, the method may include illuminating the acousto-optic device with lasers for durations ranging from 0.001 microseconds to 100 microseconds, for example, from 0.01 microseconds to 75 microseconds, for example, from 0.1 microseconds to 50 microseconds, for example, from 1 microsecond to 25 microseconds, and including from 5 microseconds to 10 microseconds. In an embodiment, when two or more lasers are used to sequentially irradiate the acousto-optic device, the duration of irradiation by each laser on the acousto-optic device may be the same or different.

[0155] The time interval between the irradiation of each laser can also be varied as needed, with separate delays of 0.001 microseconds or longer, such as 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 15 microseconds or longer, 30 microseconds or longer, and including 60 microseconds or longer. For example, the range of the time interval between the irradiation of each light source can be from 0.001 microseconds to 60 microseconds, such as from 0.01 microseconds to 50 microseconds, such as from 0.1 microseconds to 35 microseconds, such as from 1 microsecond to 25 microseconds, and including from 5 microseconds to 10 microseconds. In a particular embodiment, the time interval between the irradiation of each laser is 10 microseconds. In embodiments, when the acousto-optic device is sequentially irradiated by more than two (i.e., three or more) lasers, the delay between the irradiation of each laser can be the same or different.

[0156] The acousto-optic device can be illuminated continuously or at discontinuous intervals. In some instances, the method includes illuminating the acousto-optic device continuously using a laser. In other instances, the acousto-optic device is illuminated at discontinuous intervals using a laser, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or at some other interval.

[0157] Depending on the laser, the acousto-optic device can be irradiated from the following varying distances: for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2.5 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 15 mm or more, for example, 25 mm or more, and including 50 mm or more. Furthermore, the irradiation angle can also vary from 10° to 90°, for example, from 15° to 85°, for example, from 20° to 80°, for example, from 25° to 75°, and including a range from 30° to 60°, for example, a 90° angle.

[0158] In an embodiment, the method includes applying radio frequency (RF) drive signals to an acousto-optic device to generate an angle-shifted laser beam. Two or more RF drive signals may be applied to the acousto-optic device to generate an output laser beam having a desired number of angle-shifted laser beams, such as three or more RF drive signals, four or more RF drive signals, five or more RF drive signals, six or more RF drive signals, seven or more RF drive signals, eight or more RF drive signals, nine or more RF drive signals, ten or more RF drive signals, fifteen or more RF drive signals, 25 or more RF drive signals, 50 or more RF drive signals, and including 100 or more RF drive signals.

[0159] The angle-shifted laser beams generated by the radio frequency (RF) drive signal each have an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal having an amplitude sufficient to generate an angle-shifted laser beam with the desired intensity. In some instances, each applied RF drive signal has an amplitude ranging from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to 50V, for example from about 2V to 40V, for example from 3V to about 30V, and including from about 5V to about 25V. In some embodiments, each applied radio frequency drive signal has a frequency from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and including from about 5 MHz to about 50 MHz.

[0160] In these embodiments, the angularly offset laser beams in the output laser beam are spatially separated. Depending on the applied RF drive signal and the desired illumination distribution of the output laser beam, the angularly offset laser beams can be separated by 0.001 μm or more, for example, 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, and including 5000 μm or more. In some embodiments, the angularly offset laser beams overlap, for example, by overlapping adjacent angularly offset laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angular offset laser beams (e.g., the overlap of beam points) can be 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, and including 100 μm or more.

[0161] kit

[0162] This disclosure also includes a kit comprising: two or more light scattering detectors; a filtering assembly; and a dimming assembly for transmitting light to each light scattering detector. The kit may also include other dimming assemblies as described herein, such as a shielding assembly including an optical aperture, slit, and shielding disk, and a scattering strip. According to a particular embodiment, the kit also includes: optical components for transmitting light, such as collimating lenses, mirrors, wavelength splitters, pinholes, etc. The kit may also include: light harvesting components, such as optical fibers (e.g., fiber optic repeater bundles) or components for free-space repeater systems. In some instances, the kit also includes: one or more photodetectors, such as photomultiplier tubes (e.g., metal-encapsulated photomultiplier tubes). In a particular embodiment, the kit includes: one or more components of a beam generator, such as a direct digital synthesizer, an acousto-optic deflector, a beam combiner lens, and a Powell lens.

[0163] The various assay components of the kit may be present in separate containers, or some or all of them may be pre-assembled. For example, in some instances, one or more components of the kit, such as two or more light scattering detectors, are contained in a sealed bag, such as a sterile foil bag or envelope.

[0164] In addition to the components described above, the theme kit may also include (in a particular embodiment) instructions for practicing the theme method. These instructions may exist in various forms within the theme kit, with one or more of them present in the kit. One form of these instructions may be printed information located on a suitable medium or substrate (e.g., one or more sheets of paper with printed information), in the kit's packaging, in a packaging insert, etc. Another form of these instructions is a computer-readable medium on which information is recorded, such as a floppy disk, optical disc (CD), portable flash drive, etc. Yet another possible form of these instructions is a website address, which can be used via the Internet to access information at a deleted site.

[0165] practicality

[0166] Subject methods and optical detection systems are used for characterizing samples by optical properties, particularly for identifying and distinguishing cells within the samples. In some embodiments, the systems and methods described herein are used for flow cytometry characterization of biological samples. In a particular instance, this disclosure is used to enhance the measurement of light on an illuminated sample collected from a flow cytometer. Embodiments of this disclosure are used, for example, in research and high-throughput laboratory testing when it is desirable to improve the efficiency of flow cytometry measurements. This disclosure is intended to provide flow cytometers that offer better cell sorting accuracy, better particle acquisition, less energy consumption, particle charging efficiency, more precise particle charging, and better particle deflection during cell sorting.

[0167] This disclosure can also be used in applications where it may be desirable to use cells prepared from biological samples for research, laboratory testing, or therapeutic purposes. In some embodiments, the subject methods and apparatus can facilitate the acquisition of unique cells prepared from a target fluid or tissue biological sample. For example, the subject methods and systems facilitate the acquisition of cells from fluid or tissue samples used as research or diagnostic samples for a disease (e.g., cancer). Similarly, the subject methods and systems facilitate the acquisition of cells from fluid or tissue samples intended for use in therapy. Compared to conventional flow cytometry systems, the methods and apparatus of this disclosure allow for the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with greater efficiency and lower cost.

[0168] The aspects of the subject matter described herein, including embodiments, may be beneficial individually or in combination with one or more other aspects or embodiments. Without limiting the specification, specific, non-limiting aspects numbered 1-70 are provided hereinafter in this disclosure. It will be apparent to those skilled in the art, upon reading this disclosure, that each of the individually numbered aspects can be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects explicitly provided below:

[0169] 1. A system comprising:

[0170] Light source, including two or more lasers;

[0171] The optical detection system includes an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream irradiated by the two or more lasers; and

[0172] A processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to operate as follows:

[0173] One or more data signals are generated in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector; and

[0174] One or more parameters for data acquisition are determined based on the generated data signal from the unfiltered light scattering detector.

[0175] 2. The system according to claim 1, wherein the one or more parameters of the data acquisition include the timing of particle irradiation through each of the two or more lasers.

[0176] 3. The system according to any one of 1-2, wherein the memory further includes instructions that, when executed by the processor, cause the processor to adjust one or more parameters of the data acquisition based on the generated data signal from the unfiltered light scattering detector.

[0177] 4. The system according to 3, wherein adjusting the one or more parameters of data acquisition includes adjusting the data acquisition duration.

[0178] 5. The system according to 4, wherein adjusting the data acquisition duration includes reducing the data acquisition duration.

[0179] 6. The system according to any one of 1-5, wherein the memory further includes instructions that, when executed by the processor, cause the processor to identify the position of a particle in the liquid flow in response to the generated data signal from the unfiltered light scattering detector.

[0180] 7. The system according to 6, wherein the memory further includes instructions that, when executed by the processor, cause the processor to generate one or more particle sorting parameters in response to the data signal from the unfiltered scattering detector.

[0181] 8. The system according to 7, wherein the one or more particle sorting parameters include particle sorting timing.

[0182] 9. The system according to any one of 1-8 further includes: a flow cell for conveying the sample in the liquid flow.

[0183] 10. The system of claim 9, wherein the flow cell includes a proximal end and a distal end, and the light source is configured to irradiate the sample in the flow with a laser at the distal end of the flow cell.

[0184] 11. The system of claim 9, wherein the memory includes instructions that, when executed by the processor, cause the processor to generate one or more particle sorting parameters in response to a data signal generated by irradiating the scattered light at the far end of the flow cell using a laser, via the unfiltered light scattering detector.

[0185] 12. The system according to 11, wherein the one or more particle sorting parameters include particle sorting timing.

[0186] 13. The system according to any one of 1-12, wherein the light source comprises four or more lasers.

[0187] 14. The system according to any one of 1-13, wherein the unfiltered light scattering detector is configured to detect forward-scattered light from the sample.

[0188] 15. The system according to any one of 1-14, further comprising: a filtered light scattering detector configured to detect light from a laser source scattered by the sample.

[0189] 16. The system of claim 15, wherein the filtered light scattering detector comprises:

[0190] Light scattering detector; and

[0191] A dimming component is configured to transmit light scattered by the sample from one of the lasers to the light scattering detector.

[0192] 17. The system of claim 16, wherein the dimming component includes a bandpass filter.

[0193] 18. The system according to any one of 15-17, wherein the light detection system includes: a dimming component configured to transmit scattered light from the sample to the unfiltered light scattering detector and the filtered light scattering detector.

[0194] 19. The system of claim 18, wherein the dimming assembly includes a beam splitter.

[0195] 20. The system of claim 19, wherein the dimming assembly includes a wedge beam splitter.

[0196] 21. The system of claim 20, wherein the wedge beam splitter includes a wedge angle ranging from 5 arcminutes to 120 arcminutes.

[0197] 22. The system of claim 20, wherein the wedge beam splitter includes a wedge angle ranging from 10 arcminutes to 60 arcminutes.

[0198] 23. The system according to any one of 19-22, wherein the beam splitter is configured to: transmit a first predetermined amount of scattered light from the sample to the unfiltered light scattering detector and transmit a second predetermined amount of scattered light from the sample to the filtered light scattering detector.

[0199] 24. A method comprising:

[0200] A light detection system is used to detect light from a liquid flow, including an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid flow illuminated by two or more lasers.

[0201] One or more data signals are generated in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector; and

[0202] The one or more parameters for data acquisition are determined based on the generated data signal from the unfiltered light scattering detector.

[0203] 25. The method of 24, wherein the one or more parameters of the data acquisition include the timing of particle irradiation through each of the two or more lasers.

[0204] 26. The method according to any one of 24-25, further comprising: adjusting the one or more parameters of data acquisition based on the generated data signal from the unfiltered light scattering detector.

[0205] 27. The method according to 26, wherein the method includes adjusting the data acquisition duration.

[0206] 28. The method according to 27, wherein the method includes reducing the data acquisition duration.

[0207] 29. The method according to any one of 24-28, wherein the method includes identifying the position of a particle in the liquid flow in response to the generated data signal from the unfiltered light scattering detector.

[0208] 30. The method of 29, wherein the method further comprises generating one or more particle sorting parameters in response to the data signal from the unfiltered light scattering detector.

[0209] 31. The method according to 30, wherein the one or more particle sorting parameters include particle sorting timing.

[0210] 32. The method according to any one of 24-31, wherein the liquid flow is conveyed through a flow-through pool.

[0211] 33. The method according to 32, wherein the flow cell includes a proximal end and a distal end and a laser is used to irradiate the sample in the flow at the distal end of the flow cell.

[0212] 34. The method of 33, wherein the method further comprises: generating one or more particle sorting parameters in response to a data signal generated by irradiating the scattered light at the far end of the flow cell using a laser, via the unfiltered light scattering detector.

[0213] 35. The method according to 34, wherein the one or more particle sorting parameters include particle sorting timing.

[0214] 36. The method of claim 24 further comprises: illuminating the sample in the fluid stream in the probe domain with a light source comprising two or more lasers.

[0215] 37. The method according to any one of 24-36, wherein each laser has an illumination wavelength from 200 nm to 800 nm.

[0216] 38. The method according to any one of 24-37, wherein the liquid flow is irradiated by four or more lasers.

[0217] 39. The method according to any one of 24-38, wherein the method comprises: detecting forward light scattering using the unfiltered light scattering detector.

[0218] 40. The method according to 39, wherein the method further comprises: detecting scattered light from the sample using a filtered light scattering detector.

[0219] 41. The method of 40, wherein the filtered light scattering detector is configured to detect light scattered by the sample from one of the two or more lasers.

[0220] 42. The method according to 41, wherein the filtered light scattering detector comprises:

[0221] Light scattering detector; and

[0222] A dimming component is configured to transmit light scattered by the sample from a laser to the light scattering detector.

[0223] 43. The method according to 42, wherein the dimming component includes a bandpass filter.

[0224] 44. The method according to any one of 40-43, wherein the light detection system comprises: a dimming component configured to transmit scattered light from the sample to the unfiltered light scattering detector and the filtered light scattering detector.

[0225] 45. The method according to 44, wherein the dimming component includes a beam splitter.

[0226] 46. ​​The method according to 45, wherein the dimming assembly includes a wedge beam splitter.

[0227] 47. The method according to 46, wherein the wedge beam splitter includes a wedge angle ranging from 5 arcminutes to 120 arcminutes.

[0228] 48. The method according to 46, wherein the wedge beam splitter includes a wedge angle ranging from 10 arcminutes to 115 arcminutes.

[0229] 49. The method according to 46, wherein the wedge beam splitter includes a wedge angle ranging from 10 arcminutes to 60 arcminutes.

[0230] 50. The method according to any one of 45-48, wherein the beam splitter is configured to: transmit a first predetermined amount of scattered light from the sample to the unfiltered light scattering detector and transmit a second predetermined amount of scattered light from the sample to the filtered light scattering detector.

[0231] 51. A light detection system comprising: an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream irradiated by two or more lasers.

[0232] 52. The light detection system of claim 51, wherein the unfiltered light scattering detector is configured to detect forward-scattered light from the sample.

[0233] 53. The optical detection system according to any one of 51-52, wherein the unfiltered light scattering detector is configured to detect forward-scattered light from a sample in a liquid stream irradiated by four or more lasers.

[0234] 54. The light detection system according to any one of 51-53 further includes: a filtered light scattering detector configured to detect light scattered by the sample from one of the two or more lasers.

[0235] 55. The optical detection system according to 54, wherein the filtered light scattering detector comprises:

[0236] Light scattering detector; and

[0237] A dimming component is configured to transmit light scattered by the sample from one of the lasers to the light scattering detector.

[0238] 56. The light detection system according to 55, wherein the dimming component includes a bandpass filter.

[0239] 57. The light detection system according to any one of 54-56, wherein the light detection system comprises: a dimming component configured to transmit scattered light from the sample to the unfiltered light scattering detector and the filtered light scattering detector.

[0240] 58. The light detection system according to 57, wherein the dimming component includes a beam splitter.

[0241] 59. The light detection system according to 58, wherein the dimming component includes a wedge beam splitter.

[0242] 60. The optical detection system according to claim 59, wherein the wedge beam splitter includes a wedge angle ranging from 5 arcminutes to 120 arcminutes.

[0243] 61. The optical detection system according to 60, wherein the wedge beam splitter includes a wedge angle ranging from 10 arcminutes to 60 arcminutes.

[0244] 62. The optical detection system according to any one of 57-61, wherein the beam splitter is configured to: transmit a first predetermined amount of scattered light from the sample to the unfiltered light scattering detector and transmit a second predetermined amount of scattered light from the sample to the filtered light scattering detector.

[0245] 63. The optical detection system according to any one of 51-62, wherein the unfiltered light scattering detector is configured to generate one or more data signals in response to scattered light from each of the two or more lasers.

[0246] 64. A kit comprising:

[0247] Two light scattering detectors;

[0248] Bandpass filters; and

[0249] Beam splitter.

[0250] 65. The kit according to 64 further includes: a light source, comprising two or more lasers.

[0251] 66. The kit according to any one of 64-65, wherein the light source comprises four or more lasers.

[0252] 67. The kit according to any one of 64-66, wherein the beam splitter includes a wedge beam splitter.

[0253] 68. The kit according to 67, wherein the wedge beam splitter includes a wedge angle ranging from 5 arcminutes to 120 arcminutes.

[0254] 69. The kit according to 68, wherein the wedge beam splitter includes a wedge angle ranging from 10 arcminutes to 60 arcminutes.

[0255] 70. The kit according to any one of 64-69 further includes a support.

[0256] Although the invention has been described in detail by way of illustration and example for the purpose of clear understanding, it will be apparent to those skilled in the art, based on the teachings of this disclosure, that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

[0257] Therefore, the foregoing only illustrates the principles of the invention. It will be understood that those skilled in the art will be able to design various arrangements embodying the principles of the invention and included within its spirit and scope but not explicitly described or shown herein. Furthermore, all examples and conditional descriptions listed herein are primarily intended to help the reader understand the principles of the invention, and are not intended to limit the scope to these explicitly listed examples and conditions. Moreover, all statements herein listing the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to include both structural and functional equivalents. Furthermore, these equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any element developed that performs the same function (regardless of its structure). Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied by the appended claims.

Claims

1. A system comprising: Light source, including two or more lasers; The optical detection system includes an unfiltered light scattering detector configured to detect scattered light from a sample in a liquid stream irradiated by the two or more lasers. as well as A processor includes memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following operations: One or more data signals are generated in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector; and One or more parameters for data acquisition are determined based on the generated data signal from the unfiltered light scattering detector, wherein the one or more parameters for data acquisition include the timing of particle irradiation through each of the two or more lasers.

2. The system according to claim 1, wherein, The memory also includes instructions that, when executed by the processor, cause the processor to adjust one or more parameters of the data acquisition based on the generated data signal from the unfiltered light scattering detector.

3. The system according to claim 2, wherein, Adjusting one or more parameters of data acquisition includes reducing the duration of data acquisition.

4. The system according to claim 1, wherein, The memory also includes instructions that, when executed by the processor, cause the processor to generate one or more particle sorting parameters in response to the data signal from the unfiltered scattering detector.

5. The system according to claim 4, wherein, The one or more particle sorting parameters include particle sorting timing.

6. The system according to claim 1, further comprising: A flow cell for conveying the sample in the liquid stream. The flow cell includes a proximal end and a distal end, and the light source is configured to irradiate the sample in the flow with a laser at the distal end of the flow cell.

7. The system according to claim 6, wherein, The memory includes instructions that, when executed by the processor, cause the processor to generate one or more particle sorting parameters in response to a data signal generated by irradiating the scattered light at the far end of the flow cell using a laser, via the unfiltered light scattering detector.

8. The system according to claim 1, further comprising: A filtered light scattering detector is configured to detect light from one of the lasers of the light source scattered by the sample, wherein the filtered light scattering detector comprises: Light scattering detector; and A dimming component is configured to transmit light scattered by the sample from one of the lasers to the light scattering detector.

9. The system according to claim 8, wherein, The optical detection system includes: a dimming component configured to transmit scattered light from the sample to the unfiltered light scattering detector and the filtered light scattering detector, and The dimming component is configured to transmit a first predetermined amount of scattered light from the sample to the unfiltered light scattering detector and a second predetermined amount of scattered light from the sample to the filtered light scattering detector.

10. A method comprising: The light from a liquid stream is detected using an optical detection system, the optical detection system including an unfiltered light scattering detector configured to detect scattered light from a sample in the liquid stream irradiated by two or more lasers; One or more data signals are generated in response to scattered light from each of the two or more lasers detected by the unfiltered light scattering detector; as well as One or more parameters for data acquisition are determined based on the generated data signal from the unfiltered light scattering detector, wherein the one or more parameters for data acquisition include the timing of particle irradiation through each of the two or more lasers.

11. The method of claim 10, further comprising: One or more parameters of the data acquisition are adjusted based on the generated data signal from the unfiltered light scattering detector.

12. The method according to claim 11, wherein, The one or more particle sorting parameters include particle sorting timing.

13. The method according to claim 10, wherein, The method further includes detecting the scattered light from the sample using a filtered light scattering detector.

14. The method according to claim 13, wherein, The light detection system includes a dimming component configured to transmit scattered light from the sample to the unfiltered light scattering detector and the filtered light scattering detector.

15. The method according to claim 14, wherein, The dimming component is configured to transmit a first predetermined amount of scattered light from the sample to the unfiltered scattering detector, and to transmit a second predetermined amount of scattered light from the sample to the filtered scattering detector.

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