A photodetection system having a secondary light scattering detector and its method of use.

Abstract

The present invention provides a system having an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream. [Solution] The system includes a light source 102 having two or more lasers, a photodetection system 100 having an unfiltered light scattering detector 104, and a processor having a memory, the memory being operably coupled to the processor, and the memory containing instructions, which, 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 data signals generated from the unfiltered light scattering detector.

Description

Background Art

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

[0002] One technique that uses light detection to characterize components within a sample is flow cytometry. Using data generated from the detected light, the characteristics of the components can be recorded and desired materials can be sorted.

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

Summary of the Invention

[0004] Aspects of the present disclosure include a system having an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream. A system according to a particular embodiment includes a light source having two or more lasers, a photodetection system having an unfiltered light scattering detector, and a processor having a memory, the memory operably coupled to the processor, and the memory includes instructions, which, 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 of data acquisition based on the data signals generated from the unfiltered light scattering detector. In some embodiments, one or more parameters of data acquisition include the timing of particle irradiation by each of the two or more lasers. In a particular case, the system includes a memory having instructions for adjusting one or more parameters of data acquisition based on the data signals generated from the unfiltered light scattering detector. For example, the duration of data acquisition may 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 flow stream in response to data signals generated from an unfiltered light scattering detector. In certain embodiments, the system includes a memory having instructions for generating one or more particle sorting parameters in response to data signals from an unfiltered light scattering detector. In some cases, the particle sorting parameter is the particle sorting timing.

[0005] In some embodiments, the system includes a photodetector having an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream irradiated by two or more lasers. In some embodiments, the light scattering detector is a side-scatter light detector. In other embodiments, the light scattering detector is a forward-scatter light detector. In other embodiments, the light scattering detector is a back-scatter light detector. The photodetector system of the subject may further include a filtered light scattering detector. In some cases, the filtered light scattering detector is configured to detect scattered light from one of the lasers of the light source by a sample in a flow stream. In certain cases, the filtered light scattering detector includes the light scattering detector and an optical tuning component (e.g., a bandpass filter, a dichroic mirror) configured to transmit light scattered by the sample from one laser to the light scattering detector. In the photodetector system, the optical tuning component may be positioned in the optical path between the filtered and unfiltered light scattering detectors, such that the optical tuning component (e.g., a beam splitter) is configured to transmit scattered light from the sample to the unfiltered and filtered light scattering detectors.

[0006] Aspects of the present disclosure also include methods for determining one or more parameters of data acquisition based on data signals generated from an unfiltered light scattering detector. A method according to a particular embodiment includes detecting light from a flow stream in a photodetector system comprising an unfiltered light scattering detector configured to detect scattered light from a sample in a flow 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 data signals generated from the unfiltered light scattering detector. In some embodiments, the parameter determined based on the data signals generated from the unfiltered light scattering detector is particle irradiation timing. In other embodiments, the parameter determined based on the data signals generated from the unfiltered light scattering detector is particle sorting parameters, such as particle sorting timing. In certain embodiments, the method further includes adjusting one or more parameters based on the data signals generated from the unfiltered light scattering detector, such as adjusting irradiation timing, data acquisition duration, or particle sorting timing.

[0007] Aspects of the present disclosure include kits, each comprising two or more light scattering detectors, optical filtering components, and optical tuning components for transmitting light to each of the light scattering detectors. The kits may further include other optical tuning components, such as an obscuration component comprising an optical aperture, a slit, an obscuration disk, and a scattering bar. [Brief explanation of the drawing]

[0008] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures.

[0009] [Figure 1]This figure shows the arrangement of components of a photodetection system according to a specific embodiment. [Figure 2] This figure shows how to detect light scattering by irradiating the distal end of a flow cell with a light system according to a specific embodiment. [Figure 3] This diagram shows a flowchart for determining and adjusting one or more parameters based on data signals generated from an unfiltered light scattering detector according to a specific embodiment. [Figure 4A] This diagram shows the functional block of a particle analysis system according to a specific embodiment. [Figure 4B] This figure shows a flow cytometer according to a specific embodiment. [Figure 5] This figure shows a functional block of one embodiment of a particle analyzer control system according to a specific embodiment. [Figure 6A] This is a schematic diagram of a particle sorting machine system according to a specific embodiment. [Figure 6B] This is a schematic diagram of a particle sorting machine system according to a specific embodiment. [Figure 7] This figure shows the configuration of a computing system according to a specific embodiment. [Figure 8A] The diagram illustrates how data acquisition is adjusted using data signals from an unfiltered light scattering detector according to a specific embodiment, and how the time-shifted laser pulses are shifted from the data acquisition window due to changes in particle flow rate according to a specific embodiment. [Figure 8B] The diagram illustrates adjusting data acquisition using data signals from an unfiltered light scattering detector according to a particular embodiment, and shows the rearrangement of the data acquisition window using an unfiltered light scattering detector according to a particular embodiment. [Modes for carrying out the invention]

[0010] A system is provided having an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream. The system according to a particular embodiment includes a light source having two or more lasers, a photodetection system having an unfiltered light scattering detector, and a processor having a memory, the memory being operably coupled to the processor, and the memory includes instructions, which, 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 data signals generated from the unfiltered light scattering detector. A method for determining one or more parameters for data acquisition using the system of the subject is also described.

[0011] Before the present invention is described in more detail, it should be understood that the present invention is not limited to the specific embodiments described and is therefore, of course, subject to change. Furthermore, since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to limit them.

[0012] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other described values ​​or intermediates within the range described herein are included in the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are likewise included in the invention, according to any specifically excluded limits in the described range. Where a described range includes one or both limits, ranges excluding either or both of those included limits are also likewise included in the invention.

[0013] Certain ranges are presented herein with numerical values ​​preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number it precedes, and for any number that is close to or approximates the preceding number. In determining whether a number is close to or approximates a specifically stated number, any unspecified number that is close to or approximates a specific number may, in the context in which it is presented, provide a substantial equivalent of the specifically stated number.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein may also be used in carrying out or testing the invention, but representative exemplary methods and materials are described below.

[0015] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials, and publications are cited in relation to those methods and / or materials. Any citation of a publication relates to its disclosure prior to the filing date, and the present invention should not be construed as acknowledging that such publication has no prior rights on the grounds of prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may need to be independently verified.

[0016] It should be noted that, as used herein and in the appended claims, the articles “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly indicated by the context. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to function as an antecedent for the use of exclusive terms such as “solely” and “only,” or for the use of “negative” limitation, in relation to the enumeration of claim elements.

[0017] As will be apparent to those skilled in the art upon reading this disclosure, each of the distinct embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with any of the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events, or in any other logically possible order.

[0018] Apparatus and methods have been, or will be, described with a functional description for grammatical fluidity, but unless explicitly stated under Section 112 of the United States Patent Act, the claims should not necessarily be interpreted as being limited by an interpretation of “means” or “steps,” but should be granted the full scope of the meaning and equivalents of the definitions provided by the claims under the doctrine of legal equivalents, and if the claims are explicitly stated under Section 112 of the United States Patent Act, they should be granted the full legal equivalents under Section 112 of the United States Patent Act.

[0019] As summarized above, a system is provided that has an unfiltered light scattering detector configured to detect scattered light from a sample within a frost stream. In a further description of embodiments of the present disclosure, first, an optical detection system including a light source having two or more lasers and an unfiltered light scattering detector will be described in more detail. Next, systems and methods for determining and adjusting one or more parameters of data acquisition based on a generated data signal from the unfiltered light scattering detector are described. A kit including one or more components of the optical detection system of the present subject matter is also provided.

[0020] Optical detection system Aspects of the present disclosure include an optical detection system having an unfiltered light scattering detector configured to detect scattered light from a sample within a flow stream irradiated by two or more lasers. The term "light scattering" is used herein in its conventional meaning to refer to the propagation of light energy from particles in a sample (e.g., flowing within a flow stream) that are deflected from the incident beam path, such as by reflection, refraction, or deflection of a light beam. In some embodiments, the scattered light is not luminescence from the components of the particles (e.g., fluorophores). In embodiments, the scattered light according to the present disclosure is not fluorescence or phosphorescence. In certain embodiments, the scattered light detected by the scattered light detector of the system of the present subject matter includes Mie scattering by particles within the flow stream. In other embodiments, the scattered light detected by the scattered light detector of the system of the present subject matter includes Rayleigh scattering by particles within the flow stream. In yet other embodiments, the scattered light detected by the scattered light detector of the system of the present subject matter includes Mie scattering and Rayleigh scattering by particles within the flow stream. The scattered light detector can be a side scattered light detector, a forward scattered light detector, a backward scattered light detector, and combinations thereof.

[0021] The photodetection system according to the embodiment includes an unfiltered light scattering detector. The term “unfiltered” is used herein to mean a light scattering detector that receives light from a sample that is not transmitted through optical components configured to regulate, reduce, or otherwise limit the propagation of one or more wavelengths of light from the sample to the active surface of the light scattering detector (e.g., the wavelengths of light from a laser used to irradiate the sample). For example, in some embodiments, the unfiltered light scattering detector of interest does not optically communicate with the sample via a bandpass filter. In other embodiments, the unfiltered light scattering detector of interest does not optically communicate with the sample via a dichroic mirror. In certain cases, scattered light from the sample is transmitted directly to the active surface of the unfiltered light scattering detector. In other cases, scattered light from the sample is transmitted to the active surface of the unfiltered light scattering detector through one or more light-propagating optical components, such as optical components that change the direction or focus of the light beam without reducing, regulating, or limiting the propagation of one or more wavelengths of light. In certain embodiments, scattered light from the sample is transmitted to the active surface of the unfiltered light scattering detector using one or more beam splitters, mirrors, lenses, or collimators.

[0022] As described above, the scattered light is detected by an unfiltered light scattering detector from a sample irradiated with a light source having two or more lasers, including, 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 fifty or more lasers. In an embodiment, for example, light scattered by the sample from three or more of the lasers, for example, from four or more of the lasers, for example, from five or more of the lasers, for example, from ten or more of the lasers, for example, from fifteen or more of the lasers, and from twenty-five or more of the lasers of the light source, is detected by an unfiltered light scattering detector. In a particular embodiment, the unfiltered light scattering detector is configured to detect light scattered by the sample from 50% or more (e.g., two of a total of four lasers) of the lasers of the light source, including, for example, 60% or more, for example, 70% or more, for example, 75% or more, for example, 80% or more, and 90% or more of the lasers of the light source. In a particular case, the unfiltered light scattering detector is configured to detect light scattered by the sample from all of the lasers of the light source.

[0023] In some embodiments, the photodetector system includes one or more filtered light scattering detectors. The term “filtered” is used herein to mean a light scattering detector that receives light from a sample transmitted through an optical component configured to regulate, reduce, or otherwise limit the propagation of at least one wavelength of light (e.g., one or more wavelengths of light from a laser used to irradiate the sample) from the sample to the active surface of the light scattering detector. The light transmitted to the light scattering detector may include an optical component that limits the propagation of one or more different wavelengths of light, including, for example, limiting the propagation of 5 or more different wavelengths, for example, 10 or more, for example, 25 or more, for example, 50 or more, for example, 100 or more, for example, 200 or more, for example, 300 or more and 500 or more different wavelengths of light. For example, in some embodiments, scattered light from a sample is transmitted to the active surface of a light scattering detector filtered through a bandpass filter. In other embodiments, scattered light from a sample is transmitted to the active surface of a light scattering detector filtered through a dichroic mirror.

[0024] Depending on the number of lasers used to irradiate the sample, in some embodiments the filtered light scattering detector is configured to detect light scattered by the sample from five or fewer lasers, including, for example, four or fewer lasers, three or fewer lasers, and two or fewer lasers. In a particular case, the filtered light scattering detector is configured to detect light scattered by the sample from one of the lasers of the light source. For example, the filtered light scattering detector may be configured to detect light scattered by 50% or less of the lasers of the light source (e.g., two of the four lasers in total), including, for example, 40% or less, 30% or less, 25% or less, 20% or less, and 10% or less of the lasers of the light source. In a particular case, the filtered light scattering detector is configured to detect light scattered by a single laser.

[0025] Scattered light can be detected by each photodetector at angles to the incident beam of light irradiation, including, for example, 1° or more, 10° or more, 15° or more, 20° or more, 25° or more, 30° or more, 45° or more, 60° or more, 75° or more, 90° or more, 135° or more, 150° or more, and when the scattering light detector is configured to detect light from particles in the sample at angles of 180° or more to the incident beam of light irradiation. In certain cases, one or more of the filtered and unfiltered light scattering detectors are, for example, side scattering light detectors, where the photodetector is positioned to detect scattered light propagating at 30° to 120° to the incident beam of light irradiation, including, for example, 45° to 105° and 60° to 90°. In certain cases, one or more of the filtered and unfiltered light scattering detectors are side-scattering light detectors positioned at a 90° angle to the incident beam of light irradiation. In other cases, one or more of the filtered and unfiltered light scattering detectors are forward-scattering light detectors, for example, positioned to detect scattered light propagating from 120° to 240° relative to the incident beam of light irradiation, including, for example, 100° to 220°, for example, 120° to 200°, and 140° to 180° relative to the incident beam of light irradiation. In certain cases, one or more of the filtered and unfiltered light scattering detectors are forward-scattering light detectors positioned to detect scattered light propagating at a 180° angle to the incident beam of light irradiation. In further examples, one or more of the filtered and unfiltered light scattering detectors are backscattered light detectors positioned to detect scattered light propagating from 1° to 30° relative to the incident light beam, including 5° to 25° and 10° to 20° relative to the incident light beam.In certain cases, one or more of the filtered and unfiltered light scattering detectors are backscatter detectors positioned to detect scattered light propagating at a 30° angle to the incident beam of light irradiation.

[0026] Each light scattering photodetector in the photodetection system of this subject may be any suitable photosensor, among other types of photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhanced charge-coupled devices (ICCDs), complementary metal-oxide-semiconductor (CMOS) image sensors or N-type metal-oxide-semiconductor (NMOS) image sensors, light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In embodiments, the light scattering photodetector may include one or more photosensors, for example, two or more, for example, three or more, for example, five or more, for example, ten or more, and twenty-five or more photodetectors. In some cases, each light scattering photodetector is a photodetector array. The term “photodetector array” is used in the conventional sense 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, for example, three or more photodetectors, for example, four or more photodetectors, for example five or more photodetectors, for example six or more photodetectors, for example seven or more photodetectors, for example eight or more photodetectors, for example nine or more photodetectors, for example ten or more photodetectors, for example twelve or more photodetectors, and fifteen or more photodetectors. In a particular embodiment, the photodetector array includes five photodetectors. The photodetectors may be arranged in any geometric configuration as needed, and configurations of interest include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, non-rectangular, decagonal, dodecagonal, circular, elliptical, and irregularly shaped configurations. The photodetectors in a light scattering photodetector array may be oriented at angles ranging from 10° to 180° relative to the other (as referenced in the XZ plane), including, for example, 15° to 170°, for example, 20° to 160°, for example, 25° to 150°, for example, 30° to 120°, and 45° to 90°.

[0027] The light scattering photodetector of the present disclosure is configured to measure light collected at one or more wavelengths, including, for example, measuring light emitted by a sample in a flow stream at two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, and four hundred or more different wavelengths.

[0028] In some embodiments, the photodetector of the subject is configured to measure light collected over a wavelength range (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect the spectrum of light over a range of wavelengths. For example, the system may include one or more detectors configured to collect the spectrum of light over one or more wavelengths within the 200 nm to 1000 nm wavelength range. In yet another embodiment, the detector of interest is configured to measure light from a sample in a flow stream at one or more specific wavelengths. In embodiments, the photodetector system is configured to measure light continuously or at distinct intervals. In some cases, the detector of interest is configured to measure the collected light continuously. In other cases, the photodetector system is configured to measure at distinct intervals, such as measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at several other intervals.

[0029] In some embodiments, the photodetector system includes an unfiltered light scattering detector, a filtered light scattering detector, an unfiltered light scattering detector, and an optical tuning component positioned in the optical path between the filtered light scattering detector and the unfiltered light scattering detector, configured to transmit scattered light from a sample to the unfiltered and filtered light scattering detectors. In certain embodiments, the optical tuning includes splitting the beam of light such that one portion of the collected light (light scattered by the sample in the flow stream) is transmitted to the unfiltered light scattering detector and another portion of the collected light is transmitted to the filtered light scattering detector.

[0030] In some embodiments, the optical tuning component is a beam splitter. The amount of light propagated to each light scattering detector through the optical tuning component can also vary in some embodiments, for example, if more than 50% of the collected light is transmitted to each light scattering detector through the optical tuning component, including more than 55%, more than 60%, more than 65%, more than 75%, more than 80%, more than 90%, and more than 95% of the light collected by the photodetection system of this subject. For example, the amount of light propagated to each light scattering detector through the optical tuning component may be in the range of 25% to 99%, including, for example, 30% to 95%, 35% to 90%, 40% to 85%, 45% to 80%, and 50% to 75%.

[0031] In some embodiments, up to 50% of the collected light, including, 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 up to 5% of the light collected by the photodetector system, is transmitted through the optical tuning component to the filtered light scattering photodetector. For example, the amount of collected light transmitted through the optical tuning component to the filtered light scattering photodetector may be in the range of 1% to 75%, including, for example, 2% to 70%, 3% to 65%, 4% to 60%, and 5% to 50%. In other embodiments, for example, 50% or less of the collected light, including 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 5% or less of the light collected by the photodetector system, is transmitted through the optical tuning component to an unfiltered light scattering photodetector. For example, the amount of collected light transmitted through the optical tuning component to the unfiltered light scattering photodetector may be in the range of 1% to 75%, including 2% to 70%, 3% to 65%, 4% to 60%, and 5% to 50%.

[0032] In other embodiments, the optical adjustment component is a beam splitter. The term “beam splitter” is used herein in the conventional sense to refer to an optical component configured to propagate light along two or more different optical paths such that a given portion of light propagates along each optical path. Any convenient optical beam splitting protocol can be employed, among other types of beam splitters, including triangular prisms, elongated mirror prisms, dichroic mirror prisms, and the like. The beam splitter can be formed from any suitable material, insofar as it can propagate a desired amount and wavelength of light to unfiltered and filtered light scattering detectors. For example, the beam splitter of interest may be formed from glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glass), silica (e.g., condensed silica), quartz, crystal (e.g., CaF2 crystal), zinc selenide (ZnSe), F2, germanium (Ge) titanate (e.g., S-TIH11), or borosilicate (e.g., BK7). In certain embodiments, the beam splitter may be formed from polymer materials such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or other polymeric plastic materials, in particular copolymers of these thermoplastics such as PETG (glycol-modified polyethylene terephthalate), but is not limited to the following. In certain embodiments, the beam splitter is formed from polyester, and the polyester of interest is not limited to, but includes, poly(ethylene terephthalate) (PET), bottle-grade PET (copolymers made based on monoethylene glycol, terephthalic acid, and other comonomers such as isophthalic acid, cyclohexanedimethanol), poly(butylene terephthalate) (PBT), and poly(hexamethylene terephthalate), poly(ethylene adipate), poly(1,Poly(alkylene adipates) such as 4-butylene adipate and poly(hexamethylene adipate), poly(alkylenesberates) such as poly(ethylenesberate), poly(alkylenesebate) such as poly(ethylenesebacate), poly(ε-caprolactone) and poly(β-propiolactone), poly(alkylene isophthalates) such as poly(ethylene isophthalate), poly(ethylene 2,6-naphthalene-dicarboxylate) Poly(alkylene 2,6-naphthalene-dicarboxylate), poly(ethylenesulfonyl-4,4'-dibenzoate), poly(p-phenylene ethylenedicarboxylate), and poly(trans-1,4-cyclohexanediylethylenedicarboxylate). Poly(1,4-cyclohexane-dimethylene ethylenedicarboxylate), poly(1,4-cyclohexane-dimethylene ethylenedicarboxylate), poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylenedicarboxylate), poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylenedicarboxylate), (S)-polylactide, (R,S)-polylactide, poly(tetramethyl glycolide), and poly Examples include lactic acid polymers and copolymers such as (lactide-co-glycolide), as well as polycarbonates of bisphenol A, 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® polyethylene terephthalate), and combinations thereof.

[0033] In other embodiments, the optical adjustment component is a wedge-shaped beam splitter. In these embodiments, the beam splitter is a beam splitter having a wedge angle, which generates non-collinear back reflection such that the propagation of collected light through the wedge-shaped beam splitter results in a small change in the angle of light propagated to one or more of the unfiltered and filtered light scattering detectors. The wedge-shaped beam splitter according to embodiments of the present disclosure has a wedge angle such that the change in the incident angle of collected light results in a deviation of 0.001% or more in the propagated light angle, including, for example, 0.005% or more, for example, 0.01%, for example, 0.05% or more, for example, 0.1% or more, for example, 0.5% or more, for example, 1% or more, for example, 2% or more, for example, 3% or more, for example, 5% or more, and 10% or more. In some embodiments, the wedge beam splitter has a wedge angle of 5 to 120 arcs, including, for example, 10 to 115 arcs, 15 to 110 arcs, 20 to 105 arcs, 25 to 100 arcs, 30 to 105 arcs, 35 to 100 arcs, 40 to 95 arcs, and 45 to 90 arcs. In certain embodiments, the wedge beam splitter has a wedge angle sufficient to reduce or eliminate optical interference. In other embodiments, the wedge beam splitter has a wedge angle sufficient to reduce or eliminate image artifacts from light measured by an unfiltered or filtered light scattering detector.

[0034] In some embodiments, the wedge-shaped beam splitter has a transparent window in the following wavelength ranges: 150 nm to 5 μm, 180 nm to 8 μm, 185 nm to 2.1 μm, 200 nm to 6 μm, 200 nm to 11 μm, 250 nm to 1.6 μm, 350 nm to 2 μm, 600 nm to 16 μm, 1.2 μm to 8 μm, 2 μm to 16 μm, or several other wavelength ranges.

[0035] The beam splitter of interest may be configured to split the amount of light propagated to an unfiltered and filtered light scattering detector, as needed. In some embodiments, the beam splitter has a beam splitting ratio of 1:99 to 99:1 between an unfiltered and filtered light scattering detector, including, for example, 5:95 to 95:5, for example, 10:90 to 90:10, for example, 20:80 to 80:20, and 25:75 to 75:25. In other embodiments, the beam splitter has a beam splitting ratio of 1:99 to 99:1 between a filtered and unfiltered light scattering detector, including, for example, 5:95 to 95:5, for example, 10:90 to 90:10, for example, 20:80 to 80:20, and 25:75 to 75:25.

[0036] In some embodiments, the spatial position of the beam splitter can be adjusted manually (by human hands) or by a motor-driven displacement device. For example, the angle of the beam splitter can be adjusted by 5° or more in the photodetection system of this subject, including, for example, 10° or more, 15° or more, 20° or more, 30° or more, 45° or more, 60° or more, and 75° or more. In specific cases, the spatial position of the beam splitter can be adjusted within the photodetection system, including, for example, 1 mm or more, 5 mm or more, 10 mm or more, and 25 mm or more. For example, any convenient motor-driven actuator can be used, such as a motor differential displacement step, a motor-driven lead screw assembly, or, among other types of motors, a stepping motor, a servo motor, a brushless electric motor, a brushed DC motor, a microstepped motor, or a motor-operated geared actuator using a high-resolution stepping motor. In one embodiment, the horizontal or vertical position or orientation angle of the beam splitter can be adjusted by a motor-driven displacement device.

[0037] Figure 1 shows the arrangement of components of a photodetector system according to a particular embodiment. The photodetector system 100 includes a flow cell 101 illuminated by a light source 102 having lasers 102a, 102b, 102c, and 102d. Light scattered by particles in the sample from lasers 102a, 102b, 102c, and 102d is transmitted to an unfiltered light scattering detector 103 (forward light scattering detector) and a filtered light scattering detector 104 using a beam splitter 105. Light from the beam splitter 105 is transmitted to the filtered light scattering detector 104 through a bandpass filter 104a, which is configured to restrict the propagation of light from lasers 102a, 102b, and 102c and to transmit only scattered light from laser 102d to the light scattering detector 104.

[0038] In some embodiments, light from an optical adjustment component is propagated through an obfuscation component to one or more of an unfiltered and filtered light scattering detector. In these embodiments, the obfuscation component is configured to reduce the amount of light transmitted to the detector, for example, by reducing the amount of light transmitted by 5% or more, 10% or more, 25% or more, 40% or more, and 50% or more, and by reducing the amount of light transmitted by 1% or more. Any convenient obfuscation protocol may be employed, including but not limited to optical apertures (e.g., pinholes) or slits. The size of the optical aperture may vary as desired, and apertures of interest range from 0.001 mm to 10 mm, including, for example, 0.005 mm to 9.5 mm, 0.01 mm to 9 mm, 0.05 mm to 8.5 mm, 0.1 mm to 8 mm, 0.5 mm to 7.5 mm, and 1 mm to 5 mm. The obscuration slit can also vary in width from 0.001 mm to 10 mm, including, for example, 0.005 mm to 9.5 mm, 0.01 mm to 9 mm, 0.05 mm to 8.5 mm, 0.1 mm to 8 mm, 0.5 mm to 7.5 mm, and 1 mm to 5 mm. The length of the obscuration slit can vary depending on the width of the light propagating to the light scattering detector and may be in the range of 1 mm to 50 mm, including, for example, 2 mm to 45 mm, 3 mm to 40 mm, 4 mm to 35 mm, and 5 mm to 25 mm.

[0039] The obscuring element employed to reduce the amount of light transmitted to the light scattering detector can be any convenient shape, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons; curved cross-sectional shapes such as circles and ellipses; and irregular shapes such as a parabolic base joined to a planar top. In some embodiments, the obscuring element is circular. In other embodiments, the optical adjustment element is elliptical. In yet another embodiment, the obscuring element is polygonal, such as a square or rectangle. The width of the obscuring element may vary in some cases from 1 mm to 25 mm, including, for example, 2 mm to 22 mm, for example, 3 mm to 20 mm, for example, 4 mm to 17 mm, and 5 mm to 15 mm. The length of each obscuring component is in the range of 1 mm to 50 mm, including, for example, 2 mm to 45 mm, 3 mm to 40 mm, 4 mm to 35 mm, 5 mm to 30 mm, and 10 mm to 20 mm.

[0040] In some embodiments, the light received by the photodetector system of this subject may be transmitted by an optical collection system. The optical collection system may be any suitable optical collection protocol that collects and directs the light. In some embodiments, the optical collection system includes optical fibers, such as optical fiber relay bundles. In other embodiments, the optical collection system is a free-space optical relay system.

[0041] In embodiments, the optical collection system may be physically coupled to one or more photodetectors, for example, with an adhesive, molded together, or integrated into a photodetector. In certain embodiments, the optical collection system and the photodetector are integrated into a single unit. In some cases, the optical collection system is coupled to the photodetector by connectors that secure the optical collection system to the photodetector, for example, hook and loop fasteners, magnets, latches, notches, countersinks, counterbores, grooves, pins, tethers, hinges, Velcro®, non-permanent adhesives, or a combination thereof.

[0042] In other embodiments, the photodetector and the optical collection system communicate optically but do not have physical contact. In embodiments, the optical collection system may be positioned at a distance of 0.001 mm or more from the photodetector, including, for example, 0.005 mm or more, 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, 10 mm or more, for example, 25 mm or more, for example, 50 mm or more, and 100 mm or more.

[0043] In certain embodiments, the optical collection system includes optical fibers. For example, the optical collection system may be an optical fiber relay bundle, through which light is transmitted to a photodetector. Any optical fiber relay system may be used to propagate light to the photodetector. In certain embodiments, suitable optical fiber relay systems for propagating light to the photodetector include, but are not limited to, optical fiber relay systems described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0044] In other embodiments, the optical collection system is a free-space optical relay system. The term “free-space optical relay” is used herein in its conventional sense to refer to optical propagation employing a configuration of one or more optical components to direct light through free space to a photodetector. In certain embodiments, the free-space optical relay system includes a housing having a proximal end and a distal end, the proximal end being coupled to the photodetector. The free-space relay system may include any combination of different optical tuning components, such as one or more lenses, mirrors, slits, pinholes, wavelength separators, 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 free-space optical relay system of this subject includes one or more mirrors. In yet another embodiment, the free-space optical relay system includes a collimating lens. In certain embodiments, suitable free-space optical relay systems for propagating light to an optical detection system include, but are not limited to, optical relay systems described in U.S. Patents No. 7,643,142, No. 7,728,974 and No. 8,223,445, the disclosures of which are incorporated herein by reference.

[0045] As summarized above, aspects of the present disclosure also include systems for measuring scattered light from a sample. A system according to a particular embodiment includes a light source having two or more lasers, a photodetection system including an unfiltered light scattering detector as described above herein, and a processor having a memory operably coupled to the processor, the memory containing instructions stored in the memory, and when the instructions are executed by the processor, the processor causes 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 data signals generated from the unfiltered light scattering detector.

[0046] In the embodiment, the sample includes 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 fifty or more lasers. Depending on the components in the sample (e.g., cells, beads, non-cellular particles, etc.), the lasers emit wavelengths of light that vary in the range of 200 nm to 1500 nm, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. Each laser may independently be a pulsed laser or a continuous wave laser. For example, lasers include gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof; dye lasers such as stilbene, coumarin, or rhodamine lasers; helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, and neon-copper lasers. Solid-state lasers such as metal vapor lasers including (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers; photo-excited semiconductor lasers (OPSLs); or implementations with frequencies two or three times the frequency of any of the above lasers.

[0047] In certain embodiments, the light source is a light beam generator configured to produce two or more frequency-shifted beams of light. In some cases, the light beam generator includes a laser and a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optical device to produce two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous wave laser. For example, the laser in the light beam generator of interest may be a gas laser such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, or xenon-fluorine (XeF) excimer laser, or a combination thereof; a dye laser such as a stilbene, coumarin, or rhodamine laser; a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, or a helium-selenium (HeS) laser. e) Solid-state lasers may include metal vapor lasers such as helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof; ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, thulium-YAG lasers, ytterbium-YAG lasers, Y2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0048] The acousto-optic device can be any convenient acousto-optic protocol configured to detect frequency-shifted laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the system of this subject is configured to generate an angularly deflected laser beam from light from a laser and an applied radio frequency drive signal. The radio frequency drive signal can be applied to the acousto-optic device equipped with any suitable radio frequency drive signal source, such as a direct digital synthesizer (DDS), any waveform generator (AWG), or an electrical pulse generator.

[0049] In embodiments, the controller is configured to apply radio frequency drive signals to an acoustic-optical device to generate a desired number of angularly deflected laser beams, including, for example, three or more radio frequency drive signals, for example, four or more radio frequency drive signals, for example, five or more radio frequency drive signals, for example, six or more radio frequency drive signals, for example, seven or more radio frequency drive signals, for example, eight or more radio frequency drive signals, for example, nine or more radio frequency drive signals, for example, ten or more radio frequency drive signals, for example, fifteen or more radio frequency drive signals, for example, twenty-five or more radio frequency drive signals, for example, fifty or more radio frequency drive signals, and for example, one hundred or more radio frequency drive signals.

[0050] In some cases, to generate an intensity profile of the angularly deflected laser beam within the output laser beam, the controller is configured to apply a radio frequency drive signal with a varying amplitude, including, for example, approximately 0.001V to approximately 500V, approximately 0.005V to approximately 400V, approximately 0.01V to approximately 300V, approximately 0.05V to approximately 200V, approximately 0.1V to approximately 100V, approximately 0.5V to approximately 75V, approximately 1V to approximately 50V, approximately 2V to approximately 40V, approximately 3V to approximately 30V, and approximately 5V to approximately 25V. Each applied radio frequency drive signal has frequencies ranging from approximately 0.001 MHz to approximately 500 MHz, including, in some embodiments, frequencies from approximately 0.005 MHz to approximately 400 MHz, for example, approximately 0.01 MHz to approximately 300 MHz, for example, approximately 0.05 MHz to approximately 200 MHz, for example, approximately 0.1 MHz to approximately 100 MHz, for example, approximately 0.5 MHz to approximately 90 MHz, for example, approximately 1 MHz to approximately 75 MHz, for example, approximately 2 MHz to approximately 70 MHz, for example, approximately 3 MHz to approximately 65 MHz, for example, approximately 4 MHz to approximately 60 MHz, and approximately 5 MHz to approximately 50 MHz.

[0051] In certain embodiments, the controller has a memory operably coupled to a processor, the memory containing instructions stored in the memory, and when an instruction is executed by the processor, it causes the processor to generate an output laser beam having an angular deflection laser beam with a desired intensity profile. For example, the memory may contain instructions to generate two or more angular deflection laser beams having the same intensity, for example, 3 or more, for example, 4 or more, for example, 5 or more, for example, 10 or more, for example, 25 or more, for example, 50 or more, and the memory may contain instructions to generate 100 or more angular deflection laser beams having the same intensity. In other embodiments, the memory may contain instructions to generate two or more angular deflection laser beams having different intensities, for example, 3 or more, for example, 4 or more, for example, 5 or more, for example, 10 or more, for example, 25 or more, for example, 50 or more, and the memory may contain instructions to generate 100 or more angular deflection laser beams having different intensities.

[0052] In certain embodiments, the controller has memory operably coupled to a processor, the memory containing instructions stored in the memory, and when an instruction is executed by the processor, the processor causes the processor to generate an output laser beam having increasing intensity from the edge to the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may range from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, for example, 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%, and about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has memory operably coupled to the processor, the memory containing internally stored instructions, and when the instructions are executed by the processor, the processor causes the processor to generate an output laser beam having increasing intensity from the edge to the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may range from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, for example, 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%, and about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller has a memory operably coupled to the processor, the memory containing instructions stored internally, and when an instruction is executed by the processor, the processor causes the processor to generate an output laser beam having an intensity profile having a Gaussian distribution along the horizontal axis.In yet another embodiment, the controller has a memory operably coupled to a processor, the memory containing instructions stored in the memory, and when an instruction is executed by the processor, the processor causes the processor to generate an output laser beam having a top-hat intensity profile along the horizontal axis.

[0053] In some embodiments, the light beam generator of interest may be configured to generate an angularly deflected laser beam within a spatially separated output laser beam. Depending on the applied radio frequency drive signal and the desired irradiation profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, including, for example, 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, 100 μm or more, 500 μm or more, 1000 μm or more, and 5000 μm or more. In some embodiments, the system is configured to generate an angularly deflected laser beam within an output laser beam that overlaps with an adjacent angularly deflected laser beam along the horizontal axis of the output laser beam, etc. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 0.001 μm or larger, including overlaps of 0.005 μm or larger, e.g., overlaps of 0.01 μm or larger, e.g., overlaps of 0.05 μm or larger, e.g., overlaps of 0.1 μm or larger, e.g., overlaps of 0.5 μm or larger, e.g., overlaps of 1 μm or larger, e.g., overlaps of 5 μm or larger, e.g., overlaps of 10 μm or larger, and overlaps of 100 μm or larger.

[0054] In certain cases, a light beam generator configured to produce two or more beams of frequency-shifted light includes laser excitation modules such as 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.

[0055] In some embodiments, light scattered by particles in a sample from a light source is detected by an unfiltered light scattering detector. In some embodiments, the system includes a processor having memory operably coupled to the processor, the memory containing internally stored instructions, which, 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 an 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 some embodiments, one or more parameters of data acquisition determined based on data signals generated from an unfiltered light scattering detector include the timing of data acquisition by one or more photodetectors in the system of this subject. For example, the timing for data acquisition by one or more other light scattering detectors, synchrotron radiation detectors, transmission photodetectors, and flowstream imaging sensors may be determined using data signals generated from an unfiltered light scattering detector. In some embodiments, one or more parameters of data acquisition determined based on data signals generated from an unfiltered light scattering detector include identifying the location of particles in the flowstream. In other embodiments, one or more parameters of data acquisition determined based on data signals generated from an unfiltered light scattering detector include the duration of light scattering by particles in the sample from each laser. In other embodiments, one or more parameters of data acquisition determined based on data signals generated from an unfiltered light scattering detector include changes in flow velocity due to particles in the sample. In certain cases, the system includes a memory having instructions for generating one or more particle sorting parameters in response to data signals from an unfiltered light scattering detector. In some cases, the particle sorting parameters are particle sorting timings, such as the timing for charging particle-containing droplets.

[0057] In certain cases, the system includes memory containing instructions for adjusting one or more parameters of data acquisition based on data signals generated from an unfiltered light scattering detector. In some embodiments, the system includes memory containing instructions for changing the duration of data acquisition (i.e., the data acquisition window). In some cases, the memory includes instructions for reducing the data acquisition duration by, for example, 10% or more, 25% or more, and 50% or more, and by 5% or more. For example, the data acquisition duration may be reduced by 0.0001 μs or more, including, for example, 0.0005 μs or more, 0.001 μs or more, 0.005 μs or more, 0.01 μs or more, 0.05 μs or more, 0.1 μs or more, 0.5 μs or more, 1 μs or more, and 5 μs or more.

[0058] In other embodiments, the system includes memory having instructions for changing the timing of data acquisition. In some cases, the memory includes instructions for adjusting the timing of data acquisition by 5% or more, including, for example, 10% or more, for example, 25% or more, and 50% or more. For example, the timing of data acquisition can be adjusted by 0.0001 μs or more, including, for example, 0.0005 μs or more, for example, 0.001 μs or more, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, and 5 μs or more.

[0059] In certain embodiments, the system includes a memory having instructions for adjusting one or more particle sorting parameters in response to data signals from an unfiltered light scattering detector. In some cases, the memory includes instructions for adjusting particle sorting timing, such as the timing for charging particle-containing droplets. In certain cases, the memory includes instructions for adjusting the timing for charging particle-containing droplets by 5% or more, for example, 10% or more, for example, 25% or more, and 50% or more. For example, the timing for charging particle-containing droplets can be adjusted by 0.0001 μs or more, for example, 0.0005 μs or more, for example, 0.001 μs or more, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, and 5 μs or more.

[0060] In yet another embodiment, the memory includes instructions to adjust the drop drive frequency in response to data signals generated from an unfiltered light scattering detector. In some cases, the drop drive frequency includes, for example, 0.01 Hz or higher, for example 0.05 Hz or higher, for example 0.1 Hz or higher, for example 0.25 Hz or higher, for example 0.5 Hz or higher, for example 1 Hz or higher, for example 2.5 Hz or higher, for example 5 Hz or higher, for example 10 Hz or higher, and 25 Hz or higher, and increases. For example, the drop drive frequency may increase by 1% or more, including, for example 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, and increasing the drop drive frequency by 90% or more. In other cases, the drop drive frequency is reduced to, for example, 0.01 Hz or higher, 0.05 Hz or higher, 0.1 Hz or higher, 0.25 Hz or higher, 0.5 Hz or higher, 1 Hz or higher, 2.5 Hz or higher, 5 Hz or higher, 10 Hz or higher, and 25 Hz or higher. For example, the drop drive frequency may be reduced by 1% or more, including, for example, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and reducing the drop frequency by 90% or more.

[0061] In yet another embodiment, the memory includes instructions to adjust the drop delay in response to the data signal generated from an unfiltered light scattering detector. In some cases, the drop delay may be 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 by increasing the drop delay by 10 μs or more. For example, the drop delay may be increased by, for example, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and by increasing the drop frequency by 90% or more. In other cases, the drop frequency may be reduced by, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.3 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2.5 μs or more, for example, 5 μs or more, for example, 7.5 μs or more, and by reducing the drop delay by 10 μs or more. For example, the drop delay may be reduced by, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, and by reducing the drop delay by 90% or more, and by 1% or more.

[0062] In certain embodiments, the system further includes a flow cell configured to propagate a sample within a flow stream. In some cases, an unfiltered light scattering detector is configured to detect light scattered by the sample from one or more lasers configured to irradiate the distal end of the flow cell or near it. In certain cases, the lasers are configured to irradiate from the distal end of the flow cell at positions of 0.0001 μm to 10 μm, including, for example, 0.0005 μm to 9.5 μm, for example, 0.001 μm to 9 μm, for example, 0.005 μm to 8.5 μm, for example, 0.01 μm to 8 μm, for example, 0.05 μm to 7.5 μm, for example, 0.1 μm to 7 μm, and from the distal end of the flow cell at positions of 0.0001 μm to 10 μm, including 0.5 μm to 5 μm. In some embodiments, the system is configured to adjust one or more parameters as described above using data signals from the light scattering detector that detect scattered light from the sample irradiated at the distal end of the flow cell. In certain cases, the data signal from this light scattering detector is used to adjust one or more particle sorting parameters, such as particle sorting timing. In certain embodiments, using the data signal from a light scattering detector that detects scattered light from a sample irradiated at the distal end of the flow cell is sufficient to offset the velocity gradient across the flow stream, such as when particles flowing in the center of the flow stream or near the flow stream travel faster than particles flowing at the outer edge of the flow stream or near the flow stream.

[0063] Figure 2 illustrates the detection of light scattering by irradiating a flow cell at its distal end according to a specific embodiment. The flow cell 201, containing particles 201a flowing through it, is detected by the light scattering detector 204 and then irradiated by a laser 203 that generates pulse 203a. The flow cell 201 is then irradiated at its distal end by a laser 202 that generates pulse 202a. In certain cases, the pulse 202a detected by the light scattering detector can be used to adjust the timing of particle sorting (e.g., the timing of charging of particle-containing droplets).

[0064] Figure 3 shows a flowchart for determining and adjusting one or more parameters based on data signals generated from an unfiltered light scattering detector having a photodetector system according to a particular embodiment. In step 301, light from particles in the flow stream is detected by an unfiltered light scattering detector. One or more data signals from the unfiltered light scattering detector are generated in step 302. In step 303, one or more parameters of the photodetector system are determined based on the unfiltered light scattering detector data signals. For example, particle positions may be identified, the flow velocity of particles in the flow stream, the timing of particles passing through each laser beam, and the duration between the times a particle passes through each laser beam may be determined in step 303. In step 304, one or more parameters for data acquisition or particle sorting may be adjusted.

[0065] Any convenient flow cell may be used to propagate the fluid sample to the sample inspection area. In some embodiments, the flow cell includes a proximal cylindrical portion defining the longitudinal axis and a distal frustoconical portion terminating at a flat surface with an orifice, which is perpendicular to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) can vary in the range of 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, and 4 mm to 8 mm. Similarly, the length of the distal frustoconical portion (measured along the longitudinal axis) can vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, e.g., 3 mm to 8 mm, and 4 mm to 7 mm. In some embodiments, the diameter of the flow cell nozzle chamber can vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, e.g., 3 mm to 8 mm, and 4 mm to 7 mm.

[0066] In certain cases, the flow cell does not include a cylindrical portion, and the entire internal chamber of the flow cell is formed in a frustoconical shape. In these embodiments, the length of the frustoconical internal chamber (measured along the longitudinal axis lateral to the nozzle orifice) may range from 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, and 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical internal chamber may range from 1 mm to 10 mm, including, for example, 2 mm to 9 mm, e.g., 3 mm to 8 mm, and 4 mm to 7 mm.

[0067] In the embodiment, the sample flowstream originates from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flowstream, the flow cell orifice can be any suitable shape, and the cross-sectional shape of interest can be, but is not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic bottom joined to a planar top. In a particular embodiment, the flow cell of interest has a circular orifice. The nozzle orifice size can vary in some embodiments from 1 μm to 20000 μm, including, for example, 2 μm to 17500 μm, 5 μm to 15000 μm, 10 μm to 12500 μm, 15 μm to 10000 μm, 25 μm to 7500 μm, 50 μm to 5000 μm, 75 μm to 1000 μm, 100 μm to 750 μm, and 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0068] In some embodiments, the flow cell includes a sample injection port configured to supply the sample to the flow cell nozzle. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the internal chamber of the flow cell. Depending on the desired characteristics of the flowstream, the rate of the sample transmitted to the flow cell chamber by the sample injection port may be 1 μL / min or more, including, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 15 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, and 100 μL / min or more. In some cases, the rate of the sample transmitted to the flow cell chamber by the sample injection port may be 1 μL / min or more, including, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 15 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, and 100 μL / min or more.

[0069] The sample injection port may be an orifice positioned within the wall of the internal chamber, or a conduit positioned at the proximal end of the internal chamber. If the sample injection port is an orifice positioned within the wall of the internal chamber, the sample injection port orifice may be any preferred shape, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic bottom joined to a planar top. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0070] In certain cases, the sample injection port is a conduit positioned at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a conduit positioned to have an orifice aligned with the flow cell orifice. When the sample injection port is a conduit positioned in line with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any preferred shape, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons; curved cross-sectional shapes such as circles and ellipses; and irregular shapes such as a parabolic bottom joined to a planar top. The orifice of the conduit may vary depending on its shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, and 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 orifice of the sample injection port may include a slanted tip with a bevel angle in the range of 1° to 10°, including, for example, bevel angles of 2° to 9°, 3° to 8°, 4° to 7°, and 5°.

[0071] In some embodiments, the flow cell also includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid to the internal chamber of the flow cell, for example, together with the sample, to create a layered flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid transmitted to the flow cell chamber may be 25 μL / s or more, including, for example, 50 μL / s or more, 75 μL / s or more, 100 μL / s or more, 250 μL / s or more, 500 μL / s or more, 750 μL / s or more, 1000 μL / s or more, and 2500 μL / s or more.

[0072] In some embodiments, the sheath fluid injection port is an orifice positioned within the wall of the internal chamber. The sheath fluid injection port orifice can be any preferred shape, and the cross-sectional shape of interest is not limited to, but includes, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic bottom coupled to a planar top. The size of the sample injection port orifice can vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0073] In some embodiments, the system further includes a pump that fluid-communicates with the flow cell to propagate the flow stream through the flow cell. Any convenient fluid pump protocol may be employed to control the flow of the flow stream through the flow cell. In certain cases, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the system of this subject is configured to transmit fluid through the flow cell at a suitable rate for detecting light from a sample in the flow stream. In some cases, the sample flow rate in the flow cell is 1 μL / min (microliters per minute) or more, including, for example, 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 25 μL / min or more, 50 μL / min or more, 75 μL / min or more, 100 μL / min or more, 250 μL / min or more, 500 μL / min or more, 750 μL / min or more, and 1000 μL / min or more. For example, the system may include a pump configured to pass the sample through the flow cell at a rate in the range of 1 μL / min to 500 μL / min, including, for example, 1 μL / min to 250 μL / min, for example, 1 μL / min to 100 μL / min, for example, 2 μL / min to 90 μL / min, for example, 3 μL / min to 80 μL / min, for example, 4 μL / min to 70 μL / min, for example, 5 μL / min to 60 μL / min, and 10 μL / min to 50 μL / min. In a particular embodiment, the flow rate of the flow stream is 5 μL / min to 6 μL / min.

[0074] In a particular embodiment, the system of this subject is a flow cytometry system using the photodetection system described above. Flow cytometry systems are described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.) Flow Cytometry Protocols, Methods in molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49 (pt1): 17-28; Linden, et al. Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222 (4): 335-344; and Herbig, et al. (2007) Crit RevTher Drug Carrier This disclosure, including but not limited to Syst.24(3):203-255, is incorporated herein by reference.In specific cases, the flow cytometry systems of interest include: BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortess® X-20 flow cytometer, and BD Biosciences FACSCalibur® flow cytometer, BD Biosciences FACSCount® cell sorter, BD Biosciences FACSLyric® cell sorter, and BD Biosciences Via® cell sorter, BD Biosciences Influx® cell sorter, BD Biosciences Jazz® cell sorter, and BD Biosciences Examples include the Aria® cell sorting machine and the BD Biosciences FACSMelody® cell sorting machine.

[0075] In some embodiments, the particle sorting system of this subject matter is referred to 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, and 8,140,300. Flow cytometry systems such as those described in Patent Nos. 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, and 5,602,039 are incorporated herein by reference.

[0076] In certain cases, the system described in this subject is described in Diebold, et al. Nature. Flow cytometry systems configured to image particles in a flow stream by fluorescence imaging using radio frequency tagging emission (FIRE), such as those described in Photonics Vol. 7(10); 806-810 (2013), and U.S. Patents No. 9,423,353, 9,784,661, 10,006,852, and U.S. Patent Publications 2017 / 0133857 and 2017 / 0350803, which are incorporated herein by reference.

[0077] In some embodiments, the system of interest includes a particle analysis system that can be used to analyze and characterize particles, with or without physically separating the particles into a collection container. Figure 4A shows a functional block diagram of an embodiment of the particle analysis system. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 shown in Figure 4A can be configured to carry out, for example, the methods described herein, either entirely or partially. The particle analysis system 401 includes a fluid engineering system 402. The fluid engineering system 402 includes, or can be combined with, a sample tube 405 and a moving fluid column within the sample tube through which sample particles 403 (e.g., cells) move along a common sample pathway 409.

[0078] The particle analysis system 401 detects each particle along a common sample path to one or more detection stations. The system includes a detection system 404 configured to collect a signal from each particle as it passes through the common sample path. The detection station 408 generally points to a monitoring area 407 of the common sample path. In some implementations, detection may include detecting light or one or more other properties of a particle 403 as it passes through the monitoring area 407. Figure 4A shows one detection station 408 with one monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor two or more areas.

[0079] Each signal is assigned a signal value to form a data point for each particle. As described above, this data may be referred to as event data. The data points may be multidimensional data points containing the values ​​of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0080] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuits, and / or frequency control circuits. The indicated control system can be operably associated with the fluid engineering system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of a first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 during a first time interval. The control system 406 may be further configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may additionally compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0081] Figure 4B shows a system 400 for flow cytometry described in an exemplary embodiment of the present invention. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long-pass filters ("LP") 455a-455b, and one or more fluorescence detectors 460a-460f.

[0082] The excitation lasers 115a-415c emit light in the form of laser beams. In the system of the embodiment shown in Figure 4B, the wavelengths of the laser beams emitted from the excitation lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first directed through one or more of the beam splitters 445a and 445b. Beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. Beam splitter 445b transmits UV light (light with wavelengths in the range of 10-400 nm) and reflects light at 488 nm and 633 nm.

[0083] The laser beam is then directed to a focusing lens 420, which focuses the beam onto the portion of the fluid stream where the sample particles in the flow chamber 425 are located. The flow chamber is part of a hydrodynamic system that directs the particles in the stream (typically one at a time) to the focused laser beam for inspection. The flow chamber may consist of a flow cell in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0084] Light from a laser beam can determine the size, internal structure, and attachment of particles. Depending on the characteristics of the particles, such as the presence of one or more naturally occurring fluorescent molecules on or within the particles, the particles in the sample interact with the fluorescent light through diffraction, refraction, reflection, scattering, and absorption using re-emission at various different wavelengths. The fluorescent light, as well as the diffracted, refracted, reflected, and scattered light, can be routed through one or more of the beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collection lenses 440 to one or more of the forward scattering detector 430, side scattering detector 435, and one or more fluorescence detectors 460a-460f.

[0085] The fluorescence collecting lens 440 collects light emitted from the particle-laser beam interaction and routes the light toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or 500 nm to 520 nm. Short-pass filters transmit wavelengths of light below a specified wavelength. Long-pass filters, such as long-pass filters 455a-455b, transmit wavelengths of light above a specified wavelength. For example, long-pass filter 455a, a 670 nm long-pass filter, transmits light above 670 nm. Filters are often selected to optimize the detector's specificity for a particular fluorescent dye. The filter can be configured so that the spectral bands of the light transmitted to the detector are close to the emission peaks of the fluorescent dye.

[0086] A beam splitter directs light of different wavelengths in different directions. Beam splitters can be characterized by their filtering characteristics, such as short-pass and long-pass filtering. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits light with wavelengths below 620 nm and reflects light with wavelengths above 620 nm in different directions. In one embodiment, beam splitters 445a to 445g may be equipped with optical mirrors such as dichroic mirrors.

[0087] The forward scatter detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, excitation light traveling almost forward through or around the particle. The intensity of the light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the surface and internal structure of the particle and tends to increase as the particle complexity of the structure increases. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detectors may include photomultiplier tubes. The signals detected by the forward scatter detector 430, the side scatter detector 435, and the fluorescence detectors can be converted into electronic signals (voltages) by the detectors. This data can provide information about the sample.

[0088] Those skilled in the art will recognize that the flow cytometer described in one embodiment of the present invention is not limited to the flow cytometer shown in Figure 4B, but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in various different configurations.

[0089] During operation, the cytometer operation is controlled by the controller / processor 490, and measurement data from the detector is stored in memory 495 and can be processed by the controller / processor 490. Although not explicitly shown, the controller / processor 190 may be coupled to the detector to receive output signals from there, and may also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, etc. Input / output (I / O) capabilities 497 may also be provided within the system. Memory 495, controller / processor 490, and I / O 497 may be provided entirely as an integral part of the flow cytometer 410. In such embodiments, a display may also form part of the I / O capabilities 497 for presenting experimental data to the user of the cytometer 400. Alternatively, some or all of the memory 495 and controller / processor 490 and I / O capabilities may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 495 and controller / processor 490 may communicate wirelessly or wired with the cytometer 410. The controller / processor 490, along with memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0090] The system shown in Figure 4B includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as “filter windows” for a given detector), as defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in flow cytometry experiments emit light in their own characteristic wavelength bands. Specific fluorescent labels used in experiments and their associated fluorescence emission bands may be selected to roughly match the filter windows of the detectors. However, as more detectors are provided and more labels are utilized, a perfect correspondence between filter windows and fluorescence emission spectra is impossible. It is generally true that while the emission spectrum peaks of a particular fluorescent molecule may lie within the filter window of one particular detector, some of the emission spectra of that label will also overlap with the filter windows of one or more other detectors. This may be referred to as spillover. The I / O497 can be configured to receive data on flow cytometry experiments with panels of fluorescent labels and multiple cell populations having multiple markers, each cell population having a subset of multiple markers. I / O497 can also be configured to receive biological data for assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data for assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 495. The controller / processor 490 can be configured to evaluate the assignment of one or more labels to the markers.

[0091] Figure 5 shows a functional block diagram of one embodiment of a particle analyzer control system, such as an analysis controller 500, for analyzing and displaying biological events. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0092] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel can 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.

[0093] The analysis controller or sorting system 500 can be configured to receive biological event data from the particle analyzer 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry event data. The analysis controller 500 can be configured to provide a graphical display to the display device 506, including a first plot of the biological event data. The analysis controller 500 can be further configured to render, for example, a region of interest, overlaid on the first plot as a gate around the population of biological event data shown by the display device 506. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest drawn on a single-parameter histogram or bivariate plot. In some embodiments, a display may be used to display particle parameters or saturation detector data.

[0094] The analysis controller 500 can be further configured to display biological event data within the gate on the display device 506 differently from other events in the biological event data outside the gate. For example, the analysis controller 500 can be configured to render the colors of the biological event data contained within the gate in a way that is distinct from the colors of the biological event data outside the gate. The display device 506 can be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0095] The analysis controller 500 can be configured to receive gate selection signals from a first input device that identify gates. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate gate selection signals to the analysis controller 500 that identify gates displayed on or operated via the display device 506 (for example, by clicking on or within a desired gate when the cursor is positioned there). In some implementations, the first device can be implemented as a keyboard 508, or other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, optical detector, or speech recognition system. Some input devices can include multiple input functions. In such implementations, each input function can be considered an input device. For example, as shown in Figure 5, the mouse 510 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0096] The trigger event can cause the analysis controller 500 to change the format in which 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 a population of interest for particle sorting.

[0097] In some embodiments, the analysis controller 500 can be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically correct the plot visualization to facilitate the gating process. The correction can be based on a specific distribution of biological event data received by the analysis controller 500.

[0098] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can be further configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0099] The display device 506 can be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that show the contours of areas in the plots. The display device 506 can be further configured to modify the information presented according to the input received from the analysis controller 500, in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0100] In some implementations, the analysis controller 500 can generate a user interface for receiving example events for sorting. For example, the user interface may include controls for receiving example events or example images. Example events, example images, or example gates may be provided before the collection of event data for the sample, or based on an initial set of events for a portion of the sample.

[0101] In some embodiments, the system of interest includes a particle sorting system. Figure 6A is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) described in one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in Figure 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to a nozzle 603, or may include a nozzle 603, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically concentrates a sample fluid 606 into a moving fluid column 608 (e.g., a stream) containing particles 609. Within the moving fluid column 608, the particles 609 (e.g., cells) are arranged in a single column and traverse a monitoring area 611 (e.g., a laser stream intersection) and are irradiated by an irradiation source 612 (e.g., a laser). The vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to split into multiple droplets 610, some of which contain particles 609.

[0102] During operation, detection station 614 (e.g., event detector) identifies when a particle (or cell of interest) crosses the monitoring area 611. Detection station 614 supplies input to timing circuit 628, which then supplies input to flash charge circuit 630. At droplet splitting points, a flash charge may be applied to the moving fluid column 608, notified by a timed droplet delay (Δt), so that the droplet of interest carries the charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown), which can deflect the droplet into a collection tube or a container such as a multi-well or microwell sample plate, where wells or microwells can be associated with droplets of particular interest. However, as shown in Figure 6A, the droplet can be collected in a drain container 638.

[0103] The detection system 616 (e.g., a droplet boundary detector) plays a role in automatically determining the phase of the droplet driving signal as particles of interest pass through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 enables the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 supplies input to amplitude signals 620 and / or phase signals 618, which can then supply input to amplitude control circuits 626 and / or frequency control circuits 624 (via amplifier 622). The amplitude control circuits 626 and / or frequency control circuits 624 then control the droplet formation transducer 602. The amplitude control circuits 626 and / or frequency control circuits 624 may be included within a control system.

[0104] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store detected events and sorting decisions based thereon. The sorting decision can be included in the event data for the particles. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit, or they can be communicatively coupled so that event measurements can be collected by either the detection system 616 or the detection station 614 and provided to non-collecting elements.

[0105] Figure 6B is a schematic diagram of a particle sorting system described in one embodiment presented herein. The particle sorting system 600 shown in Figure 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream charging wire in a barb. This creates a stream of droplets 610 containing particles 610 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed by sorting electronics or other detection systems (not shown in Figure 6B), etc. The deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets and guide the droplets toward a desired collection container (e.g., one of 672, 674, 676, or 678). As shown in Figure 6B, the deflection plates 652 and 654 can be controlled to guide the particles toward container 674 along a first path 662 or toward container 678 along a second path 668. If the particles are not of interest (e.g., do not exhibit scattering or illumination information within the specified sorting range), the deflection plate may allow the particles to continue along the flow path 664. Such uncharged droplets may then be transferred into the waste container, for example, via the aspirator 670.

[0106] Sorting electronics may be included to initiate the collection of measurement data, receive fluorescence signals related to particles, and determine how to adjust the deflection plates to induce particle sorting. An exemplary implementation of the embodiment shown in Figure 6B is the BD FACSAria® flow cytometer, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0107] Computer control system Aspects of the present disclosure further include a computer-controlled system, the system further including one or more computers for full or partial automation. In some embodiments, the system includes a computer having a computer-readable storage medium in which a computer program is stored, the computer program, when loaded into the computer, includes instructions to irradiate a flow stream with a light source having two or more lasers; an algorithm for detecting scattered light from the irradiated flow stream with an unfiltered light scattering detector; in certain cases, an algorithm for generating a data signal from the unfiltered light scattering detector; and an algorithm for 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 system of the subject. In other embodiments, the memory includes an algorithm for identifying the location of particles in the flow stream. In yet another embodiment, the memory includes an algorithm for determining the duration between light scattering by particles in the sample from each laser. In yet another embodiment, the memory includes an algorithm for generating one or more particle sorting parameters in response to the data signal from the unfiltered light scattering detector.

[0108] In certain cases, the system includes memory containing an algorithm for adjusting one or more parameters of data acquisition based on data signals generated from an unfiltered light scattering detector. In some cases, the system includes memory containing an algorithm for changing the duration of data acquisition (i.e., the data acquisition window). In other cases, the system includes memory containing an algorithm for adjusting the timing of data acquisition. In yet another case, the system includes memory containing an algorithm for adjusting one or more particle sorting parameters in response to data signals from an unfiltered light scattering detector, such as the timing for charging particle-containing droplets. In yet another case, the system includes memory containing an algorithm for adjusting the drop drive frequency in response to data signals generated from an unfiltered light scattering detector. In yet another case, the system includes memory containing an algorithm for adjusting the drop delay in response to data signals generated from an unfiltered light scattering detector.

[0109] In the embodiment, the system includes an input module, a processing module, and an output module. The system of this subject may include both hardware and software components, and the hardware components may take the form of one or more platforms, for example, a server, so that functional elements, i.e., those elements of the system that perform specific tasks of the system (such as managing the input and output of information, processing information, etc.), can be executed by the execution of software applications on and across one or more computer platforms represented in the system.

[0110] 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 that accesses memory having instructions stored to perform the steps of the method of this subject. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or one of other processors that are available or will become available. The processor runs the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor's coordination and execution of the functions of various computer programs that can be written in various programming languages ​​such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system usually works in coordination with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, according to all known technologies. The processor may be any preferred analog or digital system. In some embodiments, the processor includes analog electronics that allow the user to manually align the light source with the flow stream based on first and second optical signals. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0111] System memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including compact disk drives, tape drives, removable hard disk drives, or disk drives. Such types of memory storage devices typically read from and / or write to program storage media (not shown), such as compact disks, magnetic tapes, removable hard disks, or floppy disks, respectively. Any of these program storage media, or others currently in use or to be developed in the future, can be considered computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0112] In some embodiments, the computer program product is described in terms of a computer-usable medium having control logic (a computer software program including program code) stored therein. When the control logic is executed by a processor, it causes the computer and the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of hardware state machines for performing the functions described herein will be apparent to those skilled in the art.

[0113] Memory can be any suitable device from which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic or optical disks, or tapes, or RAM, or any other suitable device, whether fixed or portable). The processor may include a general-purpose digital microprocessor preferably programmed from a computer-readable medium carrying the required program code. The programming can be provided to the processor remotely via a communication channel, or it can be pre-stored in memory or any other portable or fixed computer-readable storage medium, using one of those devices together with the memory. For example, a magnetic disk or optical disk can carry programming that can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product, and algorithms for use in carrying out the above methods. The programming according to the present invention can be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, floppy disks, hard disk storage media, magnetic storage media such as magnetic tape, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROMs, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0114] The processor may also have access to a communication channel for communicating with the user at a remote location. A remote location means that the user does not have direct contact with the system, but rather relays input information to the input manager from an external device, such as a computer connected to any other suitable communication channel, including a wide area network ("WAN"), telephone network, satellite network, or mobile phone (i.e., smartphone).

[0115] In some embodiments, the systems according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and code division multiple access (CDMA) or global system for mobile communications (GSM).

[0116] In one embodiment, the communication interface is configured to include one or more communication ports, such as a physical port or interface, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the system of the subject and other external devices, such as computer terminals (e.g., in a clinic or hospital environment), configured for similar complementary data communication.

[0117] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the System of the Subject Matter to communicate with other devices, such as computer terminals and / or networks, communicable mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0118] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet Protocol (IP) via a mobile phone network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0119] In one embodiment, the system of this subject is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11 or Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or appliance. In some embodiments, the server device has a display such as a liquid crystal display (LCD), as well as input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0120] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network or server device and data stored within the system of the subject, for example, in an optional data storage unit, using one or more of the communication protocols and / or mechanisms described above.

[0121] An output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. A graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. Functional elements of a computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. An output manager may also provide information generated by a processing module to a user at a remote location, for example, via the internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be carried out according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the system of this subject are typically of the class of computers commonly referred to as servers, but may be any type of known or future computer platform. They may also be mainframe computers, workstations, or other computer types. They may be connected via other communication systems, including any known or future type of cabling or wireless systems, whether networked or not. They may be located in the same location or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems may be adopted on any of the computer platforms. Suitable operating systems include Windows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux®, OS / 400, Compaq Tru64 Unix, SGI IRIX, and Siemens Reliant Unix.

[0122] Figure 7 shows a general architecture of an exemplary computing device 700 described in a particular embodiment. The general architecture of the computing device 700 shown in Figure 7 includes the arrangement of computer hardware and software components. The computing device 700 may include more (or fewer) elements than those shown in Figure 7. However, it is not necessary to show all of these generally conventional elements in order to provide a valid disclosure. As illustrated, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which may communicate with each other via a communication bus. The network interface 720 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via the network. The processing unit 710 may also communicate with and from memory 770, and further, provide output information to an optional display 750 via the input / output device interface 740. The input / output device interface 740 can also accept input from optional input devices 760, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input devices.

[0123] Memory 770 may include computer program instructions (grouped as modules or components in some embodiments) executed by the processing unit 710 to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. Memory 770 may further include computer program instructions and other information for implementing embodiments of the present disclosure.

[0124] method Aspects of the present disclosure also include methods for determining one or more parameters for data acquisition based on data signals generated from an unfiltered light scattering detector. Methods of a particular embodiment include detecting light from a flow stream, in a photodetector system comprising an unfiltered light scattering detector configured to detect scattered light from a sample in a flow 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 for data acquisition based on the data signals generated from the unfiltered light scattering detector.

[0125] Scattered light can be detected by each photodetector at angles to the incident beam of light irradiation, including, for example, 1° or more, 10° or more, 15° or more, 20° or more, 25° or more, 30° or more, 45° or more, 60° or more, 75° or more, 90° or more, 135° or more, 150° or more, and when the scattering light detector is configured to detect light from particles in the sample at angles of 180° or more to the incident beam of light irradiation. In certain cases, for example, when the photodetector is positioned to detect scattered light propagating at 30° to 120° to the incident beam of light irradiation, including, for example, 45° to 105° and 60° to 90°, one or more of the filtered and unfiltered light scattering detectors are side scattering light detectors. In certain cases, one or more of the filtered and unfiltered light scattering detectors are side-scattering light detectors positioned at a 90° angle to the incident beam of light irradiation. In other cases, one or more of the filtered and unfiltered light scattering detectors are forward-scattering light detectors, for example, positioned to detect scattered light propagating from 120° to 240° relative to the incident beam of light irradiation, including, for example, 100° to 220°, for example, 120° to 200°, and 140° to 180° relative to the incident beam of light irradiation. In certain cases, one or more of the filtered and unfiltered light scattering detectors are forward-scattering light detectors positioned to detect scattered light propagating at a 180° angle to the incident beam of light irradiation. In further examples, one or more of the filtered and unfiltered light scattering detectors are backscattered light detectors positioned to detect scattered light propagating from 1° to 30° relative to the incident light beam, including 5° to 25° and 10° to 20° relative to the incident light beam.In certain cases, one or more of the filtered and unfiltered light scattering detectors are backscatter detectors positioned to detect scattered light propagating at a 30° angle to the incident beam of light irradiation.

[0126] Each light scattering photodetector in the photodetection system of this subject may be any suitable photosensor, among other types of photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhanced charge-coupled devices (ICCDs), complementary metal-oxide-semiconductor (CMOS) image sensors or N-type metal-oxide-semiconductor (NMOS) image sensors, light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In embodiments, the light scattering photodetector may include one or more photosensors, for example, two or more, for example, three or more, for example, five or more, for example, ten or more, and twenty-five or more photodetectors. In some cases, each light scattering photodetector is a photodetector array. The term “photodetector array” is used in the conventional sense 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, for example, three or more photodetectors, for example, four or more photodetectors, for example five or more photodetectors, for example six or more photodetectors, for example seven or more photodetectors, for example eight or more photodetectors, for example nine or more photodetectors, for example ten or more photodetectors, for example twelve or more photodetectors, and fifteen or more photodetectors. In a particular embodiment, the photodetector array includes five photodetectors. The photodetectors may be arranged in any geometric configuration as needed, and configurations of interest include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, non-rectangular, decagonal, dodecagonal, circular, elliptical, and irregularly shaped configurations. The photodetectors in a light scattering photodetector array may be oriented at angles ranging from 10° to 180° relative to the other (as referenced in the XZ plane), including, for example, 15° to 170°, for example, 20° to 160°, for example, 25° to 150°, for example, 30° to 120°, and 45° to 90°.

[0127] In embodiments of the present disclosure, the detector of interest is configured to measure light collected at one or more wavelengths, including, for example, measuring light emitted by a sample in a flow stream at two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, and four hundred or more different wavelengths.

[0128] In some embodiments, the photodetector of the subject is configured to measure light collected over a wavelength range (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect the spectrum of light over a range of wavelengths. For example, the system may include one or more detectors configured to collect the spectrum of light over one or more wavelengths within the 200 nm to 1000 nm wavelength range. In yet another embodiment, the detector of interest is configured to measure light from a sample in a flow stream at one or more specific wavelengths. In embodiments, the method includes measuring light continuously or at distinct intervals. In some cases, the detector of interest is configured to measure the collected light continuously. In other cases, the photodetector system is configured to measure at distinct intervals, such as measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at several other intervals.

[0129] 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 by one or more photodetectors in the system of the subject based on the generated data signals from the unfiltered light scattering detector. For example, the timing for data acquisition by one or more other light scattering detectors, synchrotron radiation detectors, transmission photodetectors, and flowstream imaging sensors may be determined using the generated data signals from the unfiltered light scattering detector. In some embodiments, the method includes identifying the location of particles in the flowstream based on the generated data signals from the unfiltered light scattering detector. In other embodiments, the method includes determining the duration of light scattering by particles in the sample from each laser based on the generated data signals from the unfiltered light scattering detector. In other embodiments, the method includes determining changes in flow rate due to particles in the sample based on the generated data signals from the unfiltered light scattering detector. In specific cases, the method includes determining sorting parameters in response to data signals from the unfiltered light scattering detector. In some cases, the particle sorting parameter is the timing of particle sorting, such as the timing for charging the particle-containing droplets.

[0130] In certain cases, the method includes adjusting one or more parameters of data acquisition based on the data signal generated from an unfiltered light scattering detector. In some embodiments, the method includes changing the duration of data acquisition (i.e., the data acquisition window). In some cases, changing the duration of data acquisition includes reducing the duration of data acquisition by, for example, 10% or more, for example, 25% or more, and 50% or more, and reducing it by 5% or more. For example, the duration of data acquisition may be reduced by 0.0001 μs or more, including, for example, 0.0005 μs or more, for example, 0.001 μs or more, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, and 5 μs or more.

[0131] In other embodiments, the method includes changing the timing of data acquisition. In some cases, the method includes instructions to adjust the timing of data acquisition by 5% or more, including, for example, 10% or more, for example, 25% or more, and 50% or more. For example, the timing of data acquisition can be adjusted by 0.0001 μs or more, including, for example, 0.0005 μs or more, for example, 0.001 μs or more, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, and 5 μs or more.

[0132] In certain embodiments, the method includes adjusting one or more particle sorting parameters in response to data signals from an unfiltered light scattering detector. In some cases, the method includes adjusting particle sorting timing, such as the timing for charging particle-containing droplets. In certain cases, the timing for charging particle-containing droplets is adjusted by 5% or more, including, for example, 10% or more, for example, 25% or more, and 50% or more. For example, the timing for charging particle-containing droplets can be adjusted by 0.0001 μs or more, including, for example, 0.0005 μs or more, for example, 0.001 μs or more, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, and 5 μs or more.

[0133] In yet another embodiment, the method includes adjusting the drop drive frequency in response to a data signal generated from an unfiltered light scattering detector. In some cases, the drop drive frequency includes, for example, 0.01 Hz or higher, for example 0.05 Hz or higher, for example 0.1 Hz or higher, for example 0.25 Hz or higher, for example 0.5 Hz or higher, for example 1 Hz or higher, for example 2.5 Hz or higher, for example 5 Hz or higher, for example 10 Hz or higher, and 25 Hz or higher, and increases. For example, the drop drive frequency may increase by 1% or more, including, for example 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, and increasing the drop drive frequency by 90% or more. In other cases, the drop drive frequency is reduced to, for example, 0.01 Hz or higher, 0.05 Hz or higher, 0.1 Hz or higher, 0.25 Hz or higher, 0.5 Hz or higher, 1 Hz or higher, 2.5 Hz or higher, 5 Hz or higher, 10 Hz or higher, and 25 Hz or higher. For example, the drop drive frequency may be reduced by 1% or more, including, for example, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and reducing the drop frequency by 90% or more.

[0134] In yet another embodiment, the method includes adjusting the drop delay in response to a data signal generated from an unfiltered light scattering detector. In some cases, the drop delay may be 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 by increasing the drop delay by 10 μs or more. For example, the drop delay may be increased by, for example, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and by increasing the drop frequency by 90% or more. In other cases, the drop frequency may be reduced by, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.3 μs or more, for example, 0.5 μs or more, for example, 1 μs or more, for example, 2.5 μs or more, for example, 5 μs or more, for example, 7.5 μs or more, and by reducing the drop delay by 10 μs or more. For example, the drop delay may be reduced by, for example, 5% or more, for example, 10% or more, for example, 15% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, and by reducing the drop delay by 90% or more, and by 1% or more.

[0135] Figures 8A and 8B illustrate the adjustment of data acquisition using data signals from an unfiltered light scattering detector described in a particular embodiment. Figure 8A shows the data acquisition windows (801a, 802a, 803a, 804a) for particles irradiated by four different lasers 801, 802, 803, and 804. As shown in Figure 8A, time-shifted pulses from the lasers resulting from changes in the particle flow rate in the flow cell are misaligned with the data acquisition windows 801a, 802a, 803a, and 804a. Figure 8B shows that the data acquisition windows can be realigned (i.e., the timing of data acquisition can be adjusted) for particle irradiation by each laser using an unfiltered light scattering detector that detects scattered light from each of the lasers 801, 802, 803, and 804. In addition to adjusting the timing of data acquisition, the duration of data acquisition (i.e., the width of the data acquisition windows 801a, 802a, 803a, and 804a) can be reduced as shown in Figure 8B.

[0136] In embodiments, the particles irradiated within the flowstream may be cells, for example, when the sample within the flowstream is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungus, or, in specific cases, a subset of animal tissues, cells, or components that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to both a natural organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and urinary tract, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be any type of biological tissue, including both healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, and in some cases, the sample is a blood sample including whole blood, such as blood obtained from a venipuncture or finger stick (the blood may or may not be combined with any reagents such as preservatives and anticoagulants before the assay).

[0137] In certain embodiments, the source of the sample is “mammal” or “mammalian,” terms widely used to refer to organisms within 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 cases, the subject is human. The method may be applied to samples obtained from human subjects of both sexes at any stage of development (i.e., neonates, infants, adolescents, and adults), and in certain embodiments, the human subject may be adolescent, adolescent, or adult. While the present invention may be applied to samples from human subjects, it should be understood that it may also be applied to samples from other animal subjects (i.e., “non-human subjects”), such as birds, mice, rats, dogs, cats, livestock, and horses, but is not limited to the present invention.

[0138] When implementing the method described in this subject, samples containing particles (for example, flow cytometer flow) The sample (within the stream) is irradiated with a light source having two or more lasers, including, for example, 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 fifty or more lasers. Depending on the components in the sample (e.g., cells, beads, non-cellular particles, etc.), the lasers emit wavelengths of light that vary in the range of 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. Each laser can independently be a pulsed laser or a continuous wave laser. For example, lasers include gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof; dye lasers such as stilbene, coumarin, or rhodamine lasers; helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, and neon-copper lasers. Solid-state lasers such as metal vapor lasers including (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers; photo-excited semiconductor lasers (OPSLs); or implementations with frequencies two or three times the frequency of any of the above lasers.

[0139] The sample may be irradiated continuously or at separate intervals. In some cases, the method involves continuously irradiating the sample within the sample with a light source. In other cases, the sample is irradiated with a light source at separate intervals, including every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or several other intervals.

[0140] Depending on the light source, the sample may be irradiated from varying distances, including, for example, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and 50 mm or more. The angle or irradiation can also vary, for example, at an angle of 90°, in the range of 10° to 90°, including, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and 30° to 60°.

[0141] In certain embodiments, the method includes irradiating a sample with two or more beams of frequency-shifted light. As described above, a light beam generator component having an acousto-optical device for frequency-shifting lasers may be employed. In these embodiments, the method includes irradiating the acousto-optical device with lasers. Depending on the desired wavelength of light generated in the output laser beam (for example, for use when irradiating a sample in a flow stream), the laser may have a specific wavelength that varies between 200 nm and 1500 nm, including, for example, 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, and 400 nm to 800 nm. The acousto-optical device may be irradiated with one or more lasers, including, for example, two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, and ten or more lasers. The lasers may include any combination of several types of lasers. For example, in some embodiments, the method includes irradiating the acousto-optical device with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0142] When two or more lasers are employed, the acousto-optical device may be irradiated by the lasers simultaneously, sequentially, or in combination thereof. For example, the acousto-optical device may be irradiated simultaneously by each of the lasers. In other embodiments, the acousto-optical device is irradiated sequentially by each of the lasers. When two or more lasers are employed to sequentially irradiate the acousto-optical device, the time each laser irradiates the acousto-optical device may be 0.001 microseconds or more, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, and 60 microseconds or more, for each individual laser may be 0.001 microseconds or more. For example, the method may include irradiating the acousto-optical device with lasers for durations ranging from 0.001 microseconds to 100 microseconds, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In embodiments in which an acoustic-optical device is sequentially irradiated by two or more lasers, the duration for which the acoustic-optical device is irradiated by each laser may be the same or different.

[0143] The interval between irradiations by each laser may also be individually separated and varied by delays of 0.001 microseconds or more, including, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 15 microseconds or more, for example, 30 microseconds or more, and 60 microseconds or more, as needed. For example, the interval between irradiations by each light source may be in the range of 0.001 microseconds to 60 microseconds, including, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In a particular embodiment, the interval between irradiations by each laser is 10 microseconds. In embodiments in which the acousto-optical device is sequentially irradiated by more than two (i.e., three or more) lasers, the delays between irradiations by each laser may be the same or different.

[0144] Acousto-optical devices may be irradiated continuously or at separate intervals. In some cases, the method involves continuously irradiating the acousto-optical device with a laser. In other cases, the acousto-optical device is irradiated with a laser at separate intervals, including every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or several other intervals.

[0145] Depending on the laser, the acousto-optic device may be illuminated from varying distances, including, for example, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and 50 mm or more. The angle or illumination can also vary, for example, at an angle of 90°, in the range of 10° to 90°, including, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and 30° to 60°.

[0146] In embodiments, the method includes applying radio frequency drive signals to an acousto-optical device to generate an angularly deflected laser beam. Applying two or more radio frequency drive signals to the acousto-optical device may generate an output laser beam having a desired number of angularly deflected laser beams, such as three or more radio frequency drive signals, four or more radio frequency drive signals, five or more radio frequency drive signals, six or more radio frequency drive signals, seven or more radio frequency drive signals, eight or more radio frequency drive signals, nine or more radio frequency drive signals, ten or more radio frequency drive signals, fifteen or more radio frequency drive signals, twenty-five or more radio frequency drive signals, fifty or more radio frequency drive signals, and one hundred or more radio frequency drive signals.

[0147] The angle-deflected laser beams generated by the radio frequency drive signals each have an intensity based on the amplitude of the applied radio frequency drive signal. In some embodiments, the method includes applying a radio frequency drive signal having an amplitude sufficient to generate an angle-deflected laser beam at a desired intensity. In some cases, each applied radio frequency drive signal independently has an amplitude of about 0.001V to about 500V, including, for example, about 0.005V to about 400V, for example, about 0.01V to about 300V, for example, about 0.05V to about 200V, for example, about 0.1V to about 100V, for example, about 0.5V to about 75V, for example, about 1V to about 50V, for example, about 2V to about 40V, for example, 3V to about 30V, and about 5V to about 25V. Each applied radio frequency drive signal has frequencies ranging from approximately 0.001 MHz to approximately 500 MHz, including, in some embodiments, frequencies from approximately 0.005 MHz to approximately 400 MHz, for example, approximately 0.01 MHz to approximately 300 MHz, for example, approximately 0.05 MHz to approximately 200 MHz, for example, approximately 0.1 MHz to approximately 100 MHz, for example, approximately 0.5 MHz to approximately 90 MHz, for example, approximately 1 MHz to approximately 75 MHz, for example, approximately 2 MHz to approximately 70 MHz, for example, approximately 3 MHz to approximately 65 MHz, for example, approximately 4 MHz to approximately 60 MHz, and approximately 5 MHz to approximately 50 MHz.

[0148] In these embodiments, the angle-deflected laser beams within the output laser beam are spatially separated. Depending on the applied radio frequency drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams can be separated by 0.001 μm or more, including, for example, 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, 100 μm or more, 500 μm or more, 1000 μm or more, and 5000 μm or more. In some embodiments, the angle-deflected laser beams overlap with, for example, adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 0.001 μm or larger, including overlaps of 0.005 μm or larger, e.g., overlaps of 0.01 μm or larger, e.g., overlaps of 0.05 μm or larger, e.g., overlaps of 0.1 μm or larger, e.g., overlaps of 0.5 μm or larger, e.g., overlaps of 1 μm or larger, e.g., overlaps of 5 μm or larger, e.g., overlaps of 10 μm or larger, and overlaps of 100 μm or larger.

[0149] kit Aspects of the present disclosure further include a kit comprising two or more light scattering detectors, an optical filtering component, and an optical tuning component for transmitting light to each of the light scattering detectors. The kit may further include other optical tuning components as described herein, such as an obscuration component including an optical aperture, slit, and obscuration disk, and a scattering bar. The kit described in a particular embodiment also includes an optical component for transmitting light, such as a collimating lens, mirror, wavelength separator, and pinhole. The kit may also include an optical collection component, such as an optical fiber (e.g., an optical fiber relay bundle) or a component for a free-space relay system. In some cases, the kit further includes one or more photodetectors, such as a photomultiplier tube (e.g., a metal-packaged photomultiplier tube). In a particular embodiment, the kit includes one or more components of a light beam generator, such as a direct digital synthesizer, an acousto-optic deflector, a beam combination lens, and a Powell lens.

[0150] The various assay components of the kit may be located in separate containers, or some or all of them may be pre-assembled. For example, in some cases, one or more components of the kit, such as two or more light scattering detectors, may be located in a sealed pouch, such as a sterile foil pouch or envelope.

[0151] In addition to the components described above, the kit of this subject may further include (in certain embodiments) instructions for carrying out the methods of this subject. These instructions may be present in the kit of this subject in various forms, and one or more of these forms may be present in the kit. One form in which these instructions may be present is, for example, as printed information in the kit packaging, such as on a suitable medium or substrate such as one or more sheets of paper on which the information is printed, in the kit packaging, or in accompanying documents. Yet another form of these instructions may be a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), or portable flash drive. Yet another form in which these instructions may be present is a website address that can be used via the internet to access the information at a remote site.

[0152] Utility The methods and photodetection systems of this subject matter find applications where the characterization of a sample by its optical properties, particularly the identification and differentiation of cells in the sample, is desired. In some embodiments, the systems and methods described herein find applications in the flow cytometry characterization of biological samples. In specific cases, the disclosure finds applications to enhance the measurement of light collected from an irradiated sample in a flow stream within a flow cytometer. Embodiments of the disclosure find applications where enhanced effectiveness of measurements in flow cytometry is desired, such as in research and high-throughput laboratory testing. The disclosure also finds applications where it is desirable to provide a flow cytometer having improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting.

[0153] This disclosure also finds applications in which cells prepared from biological samples may be desired for use in research, laboratory testing, or therapy. In some embodiments, the methods and devices of this subject matter may facilitate the acquisition of individual cells prepared from a fluid or tissue biological sample of interest. For example, the methods and systems of this subject matter facilitate the acquisition of cells from fluid or tissue samples used as specimens for the study or diagnosis of diseases such as cancer. Similarly, the methods and systems of this subject matter facilitate the acquisition of cells from fluid or tissue samples used in therapy. The methods and devices of this disclosure enable the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, body fluids) with improved efficiency and lower cost compared to conventional flow cytometry systems.

[0154] The aspects (including embodiments) of the subject matter described herein may be useful on their own or in combination with one or more other aspects or embodiments. Without limiting the description, certain non-limiting aspects of the disclosure are provided as appendices numbered 1 to 70. As will be apparent to those skilled in the art upon reading the disclosure, each of the individually numbered aspects (appendices) may be used or combined with any of the aforementioned aspects or subsequent individually numbered aspects (appendices). This is intended to provide support for all such combinations of aspects (appendices), and is not limited to the combinations of aspects (appendices) expressly provided below.

[0155] 1. A system, A light source equipped with two or more lasers, A photodetection system comprising an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream irradiated by two or more lasers, A processor comprising memory operablely coupled to the processor, When memory contains instructions stored in memory, and an instruction is executed by the processor, the processor, The process involves generating one or more data signals in response to scattered light from each of two or more lasers detected by an unfiltered light scattering detector, Based on the data signal generated from an unfiltered light scattering detector, determine one or more parameters for data acquisition. The processor and A system equipped with these features. 2. The system described in Appendix 1, wherein one or more parameters for data acquisition include the timing of particle irradiation by each of two or more lasers. 3. The system as described in Appendix 1 or 2, wherein the memory further includes instructions, and when the instructions are executed by the processor, the processor adjusts one or more parameters of data acquisition based on the data signals generated from an unfiltered light scattering detector. 4. The system described in Appendix 3, in which adjusting one or more parameters of data acquisition includes adjusting the data acquisition duration. 5. The system described in Appendix 4, which includes adjusting the data acquisition period to reduce the duration of data acquisition.

[0156] 6. The system described in any one of Appendix 1 to 5, wherein the memory further contains instructions, and when the instructions are executed by the processor, the processor causes the processor to identify the position of a particle in the flow stream in response to a data signal generated from an unfiltered light scattering detector. 7. The system as described in Appendix 6, wherein the memory further contains instructions, and when the instructions are executed by the processor, the processor causes the processor to generate one or more particle sorting parameters in response to data signals from an unfiltered light scattering detector. 8. A system as described in Appendix 7, in which one or more particle sorting parameters, including particle sorting timing, are used. 9. A system according to any one of the appendices 1 to 8, further comprising a flow cell for propagating a sample within a flowstream. 10. The system as described in Appendix 9, wherein the flow cell includes a proximal end and a distal end, and the light source is configured to irradiate a sample in the flow stream with one of the lasers at the distal end of the flow cell.

[0157] 11. The system as described in Appendix 9, wherein the memory contains instructions, and when the instructions are executed by the processor, the processor causes the processor to generate one or more particle sorting parameters in response to data signals generated by an unfiltered light scattering detector in response to light scattered by laser irradiation at the distal end of a flow cell. 12. The system described in Appendix 11, wherein one or more particle sorting parameters include particle sorting timing. 13. A system described in any one of the appendices 1 to 12, wherein the light source comprises four or more lasers. 14. A system described in any one of the appendices 1 to 13, wherein an unfiltered light scattering detector is configured to detect forward scattered light from a sample. 15. The system according to any one of the appendices 1 to 14, further comprising a filtered light scattering detector configured to detect light scattered by a sample from one of the laser light sources.

[0158] 16. The filtered light scattering detector Light scattering detector and, An optical tuning component configured to transmit light scattered by the sample from one laser to a light scattering detector, and The system described in Appendix 15, comprising the above. 17. The system described in Appendix 16, wherein the optical adjustment component comprises a bandpass filter. 18. The system according to any one of the appendices 15 to 17, wherein the photodetection system comprises an optical tuning component configured to transmit scattered light from a sample to an unfiltered light scattering detector and a filtered light scattering detector. 19. The system described in Appendix 18, wherein the optical adjustment component includes a beam splitter. 20. The system described in Appendix 19, wherein the optical adjustment component comprises a wedge-shaped beam splitter.

[0159] 21. The system described in Appendix 20, wherein the wedge-shaped beam splitter has a wedge angle of 5 to 120 arcs. 22. The system described in Appendix 20, wherein the wedge-shaped beam splitter has a wedge angle of 10 to 60 arcs. 23. The system according to any one of the appendices 19 to 22, wherein the beam splitter is configured to transmit a first predetermined amount of scattered light from the sample to an unfiltered light scattering detector and a second predetermined amount of scattered light from the sample to a filtered light scattering detector.

[0160] 24. A photodetection system including an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream irradiated by two or more lasers, for detecting light from a flow stream, The process involves generating one or more data signals in response to scattered light from each of two or more lasers detected by an unfiltered light scattering detector, Based on the data signal generated from an unfiltered light scattering detector, determine one or more parameters for data acquisition. Methods that include... 25. The method according to Appendix 24, wherein one or more parameters for data acquisition include the timing of particle irradiation by each of two or more lasers. 26. The method according to Appendix 24 or 25, further comprising adjusting one or more parameters of data acquisition based on the data signal generated from an unfiltered light scattering detector. 27. The method described in Appendix 26, including adjusting the data acquisition duration. 28. The method described in Appendix 27, including reducing the data acquisition duration.

[0161] 29. The method according to any one of the appendices 24-28, comprising identifying the position of a particle in a flow stream in response to a data signal generated from an unfiltered light scattering detector. 30. The method according to Appendix 29, further comprising generating one or more particle sorting parameters in response to a data signal from an unfiltered light scattering detector. 31. The method according to Appendix 30, wherein one or more particle sorting parameters include particle sorting timing. 32. The method according to any one of the appendices 24 to 31, wherein the flowstream is propagated through a flow cell. 33. The method according to Appendix 32, wherein the flow cell includes a proximal end and a distal end, and the sample is irradiated with one of the lasers in the flowstream at the distal end of the flow cell.

[0162] 34. The method according to Appendix 33, further comprising generating one or more particle sorting parameters in response to a data signal generated by an unfiltered light scattering detector in response to light scattered by laser irradiation at the distal end of a flow cell. 35. The method described in Appendix 34, wherein one or more particle sorting parameters include particle sorting timing. 36. The method according to Appendix 24, further comprising irradiating a sample in a flow stream within an inspection field with a light source comprising two or more lasers. 37. The method according to any one of the appendices 24 to 36, wherein each laser has an irradiation wavelength of 200 nm to 800 nm. 38. The method according to any one of the appendices 24-37, wherein the flowstream is irradiated with four or more lasers.

[0163] 39. The method according to any one of the appendices 24 to 38, comprising detecting forward light scattering with an unfiltered light scattering detector. 40. The method according to Appendix 39, further comprising detecting scattered light from a sample with a filtered light scattering detector. 41. The method according to Appendix 40, wherein a filtered light scattering detector is configured to detect light scattered by a sample from one of two or more lasers. 42. The filtered light scattering detector Light scattering detector and, An optical tuning component configured to transmit light scattered by the sample from one laser to a light scattering detector, and The method described in Appendix 41, comprising: 43. The method according to Appendix 42, wherein the optical adjustment component comprises a bandpass filter.

[0164] 44. The method according to any one of the appendices 40 to 43, wherein the photodetection system comprises an optical tuning component configured to transmit scattered light from a sample to an unfiltered light scattering detector and a filtered light scattering detector. 45. The method according to Appendix 44, wherein the optical adjustment component comprises a beam splitter. 46. ​​The method according to Appendix 45, wherein the optical adjustment component comprises a wedge-shaped beam splitter. 47. The method according to Appendix 46, wherein the wedge-shaped beam splitter has a wedge angle of 5 to 120 arcs. 48. The method according to Appendix 46, wherein the wedge-shaped beam splitter has a wedge angle of 10 to 115 arcs.

[0165] 49. The method according to Appendix 46, wherein the wedge-shaped beam splitter has a wedge angle of 10 to 60 arcs. 50. The method according to any one of the appendices 45 to 48, wherein the beam splitter is configured to transmit a first predetermined amount of scattered light from the sample to an unfiltered light scattering detector and a second predetermined amount of scattered light from the sample to a filtered light scattering detector.

[0166] 51. A photodetection system comprising an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream irradiated by two or more lasers. 52. The photodetection system described in Appendix 51, wherein an unfiltered light scattering detector is configured to detect forward scattered light from a sample. 53. The photodetection system according to Appendix 51 or 52, wherein an unfiltered light scattering detector is configured to detect forward scattered light from a sample in a flow stream irradiated by four or more lasers. 54. The photodetection system according to any one of the appendices 51 to 53, further comprising a filtered light scattering detector configured to detect light scattered by a sample from one of two or more lasers. 55. The filtered light scattering detector, Light scattering detector and, An optical tuning component configured to transmit light scattered by the sample from one laser to a light scattering detector, The light detection system described in Appendix 54, comprising:

[0167] 56. The photodetection system according to Appendix 55, wherein the optical adjustment component comprises a bandpass filter. 57. The photodetection system according to any one of the appendices 54 to 56, comprising an optical tuning component configured to transmit scattered light from a sample to an unfiltered light scattering detector and a filtered light scattering detector. 58. The optical detection system described in Appendix 57, wherein the optical adjustment component comprises a beam splitter. 59. The photodetection system according to Appendix 58, wherein the optical adjustment component comprises a wedge-shaped beam splitter. 60. The photodetection system described in Appendix 59, wherein the wedge-shaped beam splitter has a wedge angle of 5 to 120 arcs.

[0168] 61. The photodetection system described in Appendix 60, wherein the wedge-shaped beam splitter has a wedge angle of 10 to 60 arcs. 62. A photodetection system according to any one of the appendices 57 to 61, wherein the beam splitter is configured to transmit a first predetermined amount of scattered light from the sample to an unfiltered light scattering detector and a second predetermined amount of scattered light from the sample to a filtered light scattering detector. 63. A photodetection system according to any one of the appendices 51 to 62, wherein an unfiltered light scattering detector is configured to generate one or more data signals in response to scattered light from each of two or more lasers.

[0169] 64. It is a kit, Two light scattering detectors, Bandpass filter and, Beam splitter and A kit that includes the following: 65. The kit described in Appendix 64, further comprising a light source including two or more lasers. 66. A kit as described in Appendix 64 or 65, wherein the light source includes four or more lasers. 67. A kit that includes a wedge-shaped beam splitter, as described in any one of the appendices 64-66. 68. The kit described in Appendix 67, wherein the wedge-shaped beam splitter has a wedge angle of 5 to 120 arcs.

[0170] 69. The kit described in Appendix 68, wherein the wedge-shaped beam splitter has a wedge angle of 10 to 60 arcs. 70. A kit that includes an additional mount, as described in any one of the appendices 64-69.

[0171] Although the above inventions have been described in some detail by examples and illustrations for the sake of clear understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of this disclosure without departing from the spirit or scope of the appended claims.

[0172] Therefore, the above merely illustrates the principles of the present invention. Those skilled in the art will understand that various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised, although not expressly described or shown herein. Furthermore, all examples and conditional terms listed herein are not limited to such specifically listed examples and conditions, but are primarily intended to help the reader understand the principles of the present invention. Moreover, all descriptions herein describing the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and those to be developed in the future, i.e., any elements developed to perform the same function, regardless of their structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

[0173] Cross-reference of related applications This application relates to U.S. Provisional Patent Application No. 62 / 981,932, filed on 26 February 2020, the disclosure of which is incorporated herein by reference.

Claims

[Claim 1] A light source comprising two or more lasers, A photodetection system comprising an unfiltered light scattering detector configured to detect scattered light from a sample in a flow stream irradiated by two or more lasers, A processor comprising memory operably coupled to the processor, The memory includes instructions stored in the memory, and when an instruction is executed by the processor, 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, Based on the data signal generated from the unfiltered light scattering detector, one or more parameters for data acquisition are determined. Processor and A system equipped with these features.

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