A particle analyzer system having multiple optical fiber paths and an optical collection component with a single optical fiber output, and a method for using the same.

Abstract

A particle analyzer system is provided. The system of interest includes a light source having a plurality of lasers, each configured to illuminate a flow stream at its respective location; a photodetector system; and a single photodetector array having a plurality of photodetectors configured to detect light from the lasers. The photodetector system of interest includes a photocollection component having a single optical fiber output comprising a plurality of optical fiber paths, each arranged to collect light from the flow stream at one of its respective locations at its proximal end and transport the collected light to its distal end, and a combination of the distal ends of the plurality of optical fiber paths. The proximal ends of the plurality of optical paths in the present invention are spatially separated from each other. Methods and kits for carrying out the present invention are also provided.

Description

[Background technology]

[0001] The characterization of biological fluid analytes is a crucial part of biological research, medical diagnosis, and the assessment of a patient's overall health and wellness. Detecting biological fluid analytes, such as human blood or blood-derived products, can yield results that can play a role in determining treatment protocols for patients with various disease conditions.

[0002] Particle analysis (e.g., flow cytometry) is a technique used to characterize and often sort biological materials, such as cells in a blood sample or particles of interest in other types of biological or chemical samples. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir for containing sheath fluid. The flow cytometer transports particles (including cells) in the fluid sample into the flow cell as a flow of cells, while also directing the sheath fluid towards the flow cell. Light is irradiated into the flow stream to characterize its components. Variations in the material in the flow stream, such as morphology or the presence of fluorescent labels, can cause variations in the observed light, and these variations enable characterization and separation. To characterize the components in the flow stream, light must strike and collect the flow stream. The light source in the flow cytometer can vary and may include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0003] Conventional high-parameter flow cytometers typically use multiple excitation lasers focused via a final focusing lens (FFL) to irradiate particles with light of different wavelengths for a spot height in the core stream ranging from 10 to 20 μm. The lasers are typically spaced apart at various positions along the particle's migration path through the core stream, so that different lasers irradiate the same particle at different times, which is crucial for time discrimination of the signal. An objective lens collects light from the laser spot interacting with the particle and projects that light onto a set of optical fibers. Conventionally, each laser spot is projected onto its own optical fiber, i.e., one fiber per laser spot, and each optical fiber then carries the light from the laser spot to its respective detector array so that each excitation laser correlates with its respective detector array. Figure 1 shows a conventional flow cytometry system including individual optical fibers that collect light from each laser and carry that light to the corresponding photodetector array. As shown in Figure 1, individual fibers 102a, 102b, and 102c collect light 101a, 101b, and 101c, respectively, and transport that light to the corresponding detector arrays 103a, 103b, and 103c. In other words, each optical fiber transports a light spot from the objective lens to multiple detector arrays, such that there is one detector array for each light spot. [Overview of the project]

[0004] The inventors have recognized that conventional flow cytometry approaches, which include individual detector arrays for each laser, increase system complexity due to the large number of photodetector paths and components to support. Furthermore, the inventors have recognized that some conventional systems have limitations in the maximum time and interval that can be achieved between lasers without signal degradation. Also, while systems for photodetector array multiplexing, such as those described in U.S. Patent Application Publication No. 2021 / 0325292, which is incorporated entirely herein by reference, constitute an improvement in the field of photodetection, the inventors have recognized that further enhancements can be made. Therefore, systems and methods that improve the simplicity and efficiency of photocollection in flow cytometry are desirable. Embodiments of the present invention satisfy this need.

[0005] Aspects of the present disclosure include a system for analyzing particles in a flow stream. More specifically, the system of the present disclosure includes a light source having a plurality of lasers, each configured to illuminate a flow stream at a respective location, and a photodetector system having an optical collection component comprising a plurality of optical fiber paths, each arranged to collect light from the flow stream at one of the respective locations at a proximal end and transport the collected light to a distal end. The proximal ends of the plurality of optical paths in the system of interest are spatially separated from one another. The optical collection component also includes a single optical fiber output comprising a combination of the distal ends of the plurality of optical fiber paths. The system of interest also includes a single photodetector array comprising a plurality of photodetectors configured to detect light from the lasers transported through the single optical output of the optical collection component. In particular cases, the system of the present disclosure is a particle analyzer system.

[0006] In some embodiments, a single optical fiber output of an optical collection component comprises a fusion of the distal ends of multiple optical fiber paths. In other embodiments, the multiple optical fiber paths are joined via a binder. In certain cases, the binder is epoxy. In some embodiments, a single optical fiber output comprises a single housing containing multiple combined optical fiber paths. In some embodiments, the housing is circular or rectangular. In certain embodiments, the housing is configured to arrange the multiple optical fiber paths in tandem or horizontal rows. In certain embodiments, the number of optical fiber paths in the optical collection component ranges from 3 to 10. In some embodiments, the system of the disclosure comprises an equal number of lasers and optical fiber paths. In some embodiments, the proximal ends of the multiple optical fiber paths are separated by the same distance from the flow cell. In other embodiments, the proximal ends of the multiple optical fiber paths are separated by different distances from the flow cell. In further embodiments, the system of the disclosure comprises multiple optical collection components.

[0007] In some embodiments, the light source further comprises a first laser configured to illuminate the flowstream at a first position, and a plurality of lasers configured to illuminate the flowstream at each of the positions downstream of the first position. In some embodiments, the number of lasers in the light source is in the range of 3 to 10. In some cases, the lasers of the light source are configured to illuminate the flowstream at positions spaced 10 μm or less apart from each other.

[0008] In some embodiments, each photodetector in a photodetector array is configured to detect light at different times. In other embodiments, each photodetector in a photodetector array is configured to differentially detect light from one or more lasers. In yet another embodiment, each photodetector in a photodetector array is configured to detect one or more predetermined sets of light wavelengths. In some cases, each set of light wavelengths includes 50 or fewer different wavelengths. In another case, each photodetector in a photodetector array is a hybrid photodetector with a photocathode integrated with an avalanche diode. In yet another case, each photodetector in a photodetector array is optically connected to an optical tuning component configured to limit the wavelengths of one or more light detected by the photodetector. In some cases, the optical tuning component is a bandpass filter.

[0009] In some embodiments, the system of the Disclosure further comprises a processor, the processor comprising a memory operably coupled to the processor, which stores instructions, when executed by the processor, to cause the processor to assign each photodetector in a photodetector array to detect a given set of light wavelengths. In some embodiments, the system of the Disclosure further comprises a processor, the processor comprising a memory operably coupled to the processor, which stores instructions, when executed by the processor, to cause the processor to receive multiplexed data signals from the photodetector array. In some cases, the data signals are time-division multiplexed data signals. In other cases, the data signals are wavelength-division multiplexed data signals. In some embodiments, the system of the Disclosure further comprises a processor, the processor comprising a memory operably coupled to the processor, which stores instructions, when executed by the processor, to cause the processor to spectrally decompose the light detected by the photodetectors of the photodetector array. In some embodiments, the memory stores instructions for spectrally decomposing the light by calculating spectral separation matrices for each spectrum of the light detected by the photodetectors of the photodetector array. In some embodiments, the system of the Disclosure is incorporated into a flow cytometer. In certain cases, the flow cytometer is equipped with a particle sorter.

[0010] Aspects of the present disclosure also include methods for determining one or more parameters of particles in a flowstream. Methods according to particular embodiments include i) irradiating particles in a flowstream with a plurality of lasers, each configured to irradiate the flowstream at each location, and ii) detecting light from the particles using a photodetector system having a single optical fiber output comprising a plurality of optical fiber paths, each arranged to collect light from the flowstream at one of the respective locations at a proximal end and transport the collected light to a distal end, and a combination of the distal ends of the plurality of optical fiber paths, and a single photodetector array comprising a plurality of photodetectors configured to detect light from lasers transported through the single optical output of the photodetector component. The proximal ends of the optical fiber paths for use in the method in question are spatially separated from each other.

[0011] In some embodiments, the method of the Disclosure includes detecting one or more predetermined sets of light wavelengths using each photodetector in a photodetector array. In some embodiments, the method of the Disclosure includes generating a time-division multiplexed data signal. In other embodiments, the method of the Disclosure includes generating a wavelength-division multiplexed data signal. In some embodiments, the method of the Disclosure includes spectral decomposition of the light detected by the photodetectors in the photodetector array. In other embodiments, the method of the Disclosure further includes assigning each photodetector in the photodetector array to detect a predetermined set of light wavelengths. In some embodiments, the method of the Disclosure further includes identifying particles based on one or more determined parameters of the particles. In other embodiments, the method of the Disclosure further includes sorting particles based on one or more determined parameters of the particles.

[0012] A non-temporary computer-readable storage medium containing instructions for performing the method using the system in question is also provided. Some aspects of this disclosure further include a kit having one or more components of the system in question. [Brief explanation of the drawing]

[0013] This invention will be best understood by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures. [Figure 1] This shows light collection in a conventional flow cytometry system. [Figure 2] An optical collection component comprising multiple optical fiber paths, a single optical fiber output, and a single photodetector array is shown according to one embodiment of the present invention. [Figure 3A] This shows a configuration for combining optical fiber paths according to a specific embodiment. Figure 3A shows an example of a circular configuration for combining optical fiber paths. [Figure 3B] This shows a configuration for combining optical fiber paths according to a specific embodiment. Figure 3B shows a tandem configuration for combining optical fiber paths. [Figure 3C] A configuration for combining optical fiber paths according to a specific embodiment is shown. Figure 3C shows a horizontal configuration for combining optical fiber paths. [Figure 4] This shows an optical collection component comprising multiple optical fiber paths and a single optical fiber output, according to a specific embodiment. [Figure 5A] The irradiation of particles using the system of the present invention according to several embodiments is shown. Figure 5A shows a particle analyzer system according to a particular embodiment, which includes a light collection component for receiving light from particles in a flow stream. [Figure 5B] The irradiation of particles using the system of the present invention according to several embodiments is shown. Figure 5B shows the generated data signal from light detected by the photodetector of the photodetector array according to a particular embodiment. [Figure 6] This shows a multiplexing configuration of photodetectors within a photodetector array according to a specific embodiment. [Figure 7] A functional block diagram is shown for an example of a sorting control system according to a specific embodiment. [Figure 8]A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization, according to a specific embodiment, is shown. [Figure 9A] A schematic diagram of a particle sorting machine system according to a specific embodiment is shown. [Figure 9B] A schematic diagram of a particle sorting machine system according to a specific embodiment is shown. [Figure 10] A block diagram of a computing system according to a specific embodiment is shown. [Modes for carrying out the invention]

[0014] A particle analyzer system is provided. An embodiment of the system includes a light source having a plurality of lasers, each configured to illuminate a flowstream at its respective location; a photodetector system; and a single photodetector array comprising a plurality of photodetectors configured to detect light from the lasers. The photodetector system in question includes an optical collection component having a plurality of optical fiber paths, each arranged to collect light from the flowstream at one of its respective locations at its proximal end and transport the collected light to its distal end; and a single optical fiber output comprising a combination of the distal ends of the plurality of optical fiber paths. The proximal ends of the plurality of optical paths in this invention are spatially separated from one another. Methods and kits for carrying out this invention are also provided.

[0015] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described and can therefore vary. It should also be understood that the scope of the present invention is limited only by the appended claims, and that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0016] Where a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, unless the context clearly indicates otherwise, and any other listed or between values ​​within that range, are included in the invention. The upper and lower limits of these smaller ranges may independently be included within smaller ranges and are also included in the invention, subject to any specifically excluded limits within the listed ranges. Where a listed range includes one or both limits, the range excluding one or both of those limits is also included in the invention.

[0017] In this specification, certain ranges are presented with numbers preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number it precedes, as well as for any number that is close to or approximates the number preceded by the term. When determining whether a number is close to or approximates a specifically enumerated number, any close or approximate number that is not enumerated may be a number that, in the context in which the number is presented, presents a substantial equivalent of the specifically enumerated number.

[0018] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, but representative exemplary methods and materials are described below.

[0019] 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 the methods and / or materials cited in connection therewith. Any reference to a publication is for its disclosure prior to the filing date and should not be construed as an acknowledgment that the present invention has no prior rights to such publication by prior art. Furthermore, the publication dates presented may differ from the actual publication dates and may need to be independently verified.

[0020] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude optional elements. Therefore, this statement is intended to serve as a precedent for the use of exclusive terms such as “alone” and “only” in relation to the enumeration of elements of the claims or the use of “negative” limitations.

[0021] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any of the enumerated methods may be performed in the order of the enumerated events, or in any other logically possible order.

[0022] Apparatus and methods have grammatical fluidity with functional descriptions, or are described for that purpose, but claims should not necessarily be interpreted as being limited by constructing limitations of “means” or “steps” unless explicitly formulated under Section 112 of the United States Patent Act, and should be given the meaning of the definitions and the full scope of equivalents provided by the claims under the doctrine of equivalents, and should be clearly understood that if the claims are explicitly formulated under Section 112 of the United States Patent Act, a full set of statutory equivalents should be given under Section 112 of the United States Patent Act.

[0023] Particle analyzer system Aspects of this disclosure include a system for analyzing particles in a flow stream. The system of interest includes a light source having a plurality of lasers, each configured to illuminate the flow stream at its respective location, and a photodetector system. The system of interest provides a flow cytometer with improved cell sorting accuracy during cell sorting, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection. The photocollection component of the system of interest allows interrogation points to be separated by any distance, such as equal to, less than, or greater than a conventional (one or more) distance. In some aspects, the photocollection component allows the user to increase or decrease the distance and / or time between lasers without changing the input to the detector array. In other aspects, the use of the photocollection component of interest reduces light leakage compared to a system in which the component is not used. In some cases, the photocollection component reduces light leakage between laser intercepts by 10%, 20%, 30%, 50%, 80%, 90%, or 99% or more. In yet another embodiment, the optical collection component increases the amount of light that can be collected by the detector array without clipping the light at the edge of the optical fiber, and the number of lasers that can collect light simultaneously. In some cases, the optical collection component reduces the amount of uncollected light by more than 10%, 20%, 30%, 50%, 80%, 90%, or 99%. In other cases, the optical collection component improves the ability of the system to increase the number of lasers illuminating the flowstream by more than two lasers, more than three lasers, for example more than four lasers, for example more than five lasers, for example more than six lasers, for example more than twelve lasers (including more than sixteen lasers). Thus, the optical collection component improves the ability of the system to isolate low-intensity signals and minimize optical crosstalk from lasers and interceptors.

[0024] In a further embodiment, the system in question may not include an optical adjustment component (e.g., a reflective optical element) between the objective lens and the light-collecting component. In conventional flow cytometers, the reflective optical element increases the numerical aperture of the light from the objective lens before it reaches the optical fiber. In the system in question, the embodiment of the light-collecting component does not affect the numerical aperture of the light focused from the objective lens before it reaches the light-collecting component. Such an embodiment allows all light to be collected by the light-collecting component and detected by the photodetector array.

[0025] As described above, the optical collection component of the present invention includes a plurality of optical fiber paths. “Optical fiber path” means a plurality of optical fibers, each configured to collect light from a flow cell at a specific location and transport that light using its own path. The optical collection component of the system in question has an elongated structure having a plurality of proximal ends and a single distal end. The proximal end of the optical collection component is positioned adjacent to a flow cell configured to have a flow stream through which light is transported. As described above, the optical collection component includes a single optical fiber output comprising a combination of the distal ends of the plurality of optical fiber paths. “Combination” of distal ends means that, in contrast to the proximal ends of optical fiber paths that are spatially separated from each other, the single optical fiber output arranges the distal ends of the optical fiber paths so that they are not spatially separated from each other, i.e., they are physically connected and / or fused together to form a tightly packed whole. In certain embodiments, the distal ends of the optical fiber paths are indistinguishable from each other when combined into a single optical fiber output. In other words, of the plurality of optical fiber paths, each optical fiber path has a different proximal end than the proximal ends of the other optical fiber paths, but the optical fiber paths share the same distal end. In other words, the light collection component of the present invention is configured to collect light at different positions relative to the flow cell, but to output the collected light at a single (i.e., one) position.

[0026] The distal ends of optical fiber paths may be combined into a single optical fiber output in any convenient manner. In some embodiments, the single optical fiber output comprises a fusion of the distal ends of multiple optical fiber paths. In other words, the optical fiber paths are fused together at their distal ends (e.g., by fusion splicing). The fusion of optical fibers generally proceeds by applying heat to the optical fibers from a heat source. The heat source of choice includes, but is not limited to, lasers, gas flames, electric tungsten filaments, and electric arcs. In some embodiments, the multiple optical fiber paths are combined via a binder. The binders that can be used can vary and include, for example, optical adhesives. Any convenient optical adhesive can be used. For example, adhesives of choice may include epoxy (e.g., pure epoxy, polyester resins, and epoxy acrylates), photocurable acrylic resins, elastomers (e.g., silicones, silicone-free silanes), cyanoacrylates, and structural adhesives (e.g., those having resins and activators). In some embodiments, the adhesive is curable by exposure to light (e.g., UV light). In certain embodiments, the optical adhesive is transparent. The refractive index of the adhesive of choice may vary in some cases within a range including 1.9 to 2.3, e.g., 1.1 to 2.9, e.g., 1.2 to 2.8, e.g., 1.3 to 2.7, e.g., 1.4 to 2.6, e.g., 1.5 to 2.7, e.g., 1.6 to 2.6, e.g., 1.7 to 2.5, e.g., 1.8 to 2.4.

[0027] In certain cases, optical fiber paths are mechanically joined to each other at their distal ends to form a single optical fiber output. In such cases, the techniques used to mechanically join the optical fiber paths can vary. For example, optical fiber paths may be mechanically joined to each other using capillary tubes, V-grooves, elastomer splices, or swivel splices. In some embodiments, optical fiber paths are combined into a single optical fiber output via optical fiber connectors used to couple the optical fibers. Optical fiber connectors may be configured to couple to each other via any convenient mechanism. Couplings of interest include screw couplings, latch couplings, push-pull couplings, bayonet couplings, gendered couplings, snap-fit ​​couplings, and combinations thereof. In some embodiments, optical fiber paths are combined using ultra-small assembly (SMA) optical fiber connectors.

[0028] In some embodiments, a single optical fiber output comprises a single housing containing multiple optical fiber paths. The housing may be configured to arrange the optical fiber paths in any convenient manner. Exemplary housing shapes include, but are not limited to, circular, rectangular, tandem, horizontal, elliptical, triangular, square, kite, trapezoidal, parallelogram, rhombic, or different types of polygonal housings. In some embodiments, the housing is circular. In other embodiments, the housing is rectangular. In yet another embodiment, the housing is configured to arrange multiple optical fiber paths in tandem. In yet another embodiment, the housing is configured to arrange multiple optical fiber paths horizontally.

[0029] In some embodiments, each of the multiple optical fiber paths has an input diameter including 1050 μm to 1100 μm, in the range of 800 μm to 1000 μm, e.g., 700 μm to 1000 μm, e.g., 750 μm to 1000 μm, e.g., 800 μm to 1000 μm, e.g., 850 μm to 1000 μm, e.g., 900 μm to 1000 μm, e.g., 950 μm to 1000 μm, e.g., 700 μm to 1100 μm, e.g., 750 μm to 1100 μm, e.g., 800 μm to 1100 μm, e.g., 900 μm to 1100 μm, e.g., 950 μm to 1100 μm, e.g., 1000 μm to 1100 μm. In some cases, the input diameter of each of the multiple optical fiber paths is 800 μm. In other cases, the input diameter of each of the multiple optical fiber paths is 1000 μm.

[0030] In some embodiments, the range of laser illumination on the flowstream is less than or equal to the input diameter of each of the multiple optical fiber paths configured to collect light from the flowstream. For example, the laser in the system under consideration may be configured to illuminate positions on the flowstream ranging from 1000 μm or less, e.g., 800 μm or less, e.g., 600 μm or less, e.g., 400 μm or less, e.g., 200 μm or less, and each optical fiber input may have a diameter sufficient to collect light from each of the laser-illuminated positions, e.g., 200 μm or more, e.g., 400 μm or more, e.g., 600 μm or more, e.g., 800 μm or more, and if each optical fiber input is configured to collect light from the illuminated flowstream, it may have a diameter including 1000 μm or more. In some embodiments, the proximal end of each of the multiple optical fiber paths has a numerical aperture in the range of 0.10 to 0.15, e.g., 0.10, 0.11, 0.12, 0.13, 0.14, and 0.15. In some cases, the proximal end of each of the multiple optical fiber paths has a numerical aperture of 0.12.

[0031] In this embodiment, each of the multiple optical fiber paths is 300 μm to 1000 μm, for example 350 μm to 1000 μm, for example 400 μm to 1000 μm, for example 450 to 1000 μm, for example 500 μm to 1000 μm, for example 550 μm to 1000 μm, for example 600 μm to 1000 μm, for example 650 μm to 1000 μm, for example 700 μm to 1000 μm, for example 750 μm to 1 The output diameter includes 600μm to 800μm, within the range of 000μm, e.g., 800μm to 1000μm, e.g., 850μm to 1000μm, e.g., 900μm to 1000μm, e.g., 950μm to 1000μm, e.g., 300μm to 800μm, e.g., 350μm to 800μm, e.g., 400μm to 800μm, e.g., 450μm to 800μm, e.g., 500μm to 800μm. In some cases, the output diameter of each of the multiple optical fiber paths is 450μm. In other cases, the output diameter of each of the multiple optical fiber paths is 600μm. In yet another case, the output diameter of each of the multiple optical fiber paths is 800μm. In a specific case, the output diameter of each of the multiple optical fiber paths is 1000μm. In some embodiments, the distal end of each of the multiple optical fiber paths has a numerical aperture in the range of 0.20 to 0.25, for example, 0.20, 0.21, 0.22, 0.23, and 0.25. In a particular case, the distal end of each of the multiple optical fiber paths has a numerical aperture of 0.22.

[0032] In one embodiment, at least one of the multiple optical fiber paths tapers from the proximal end to the distal end. "Tapering" means that the diameter of one or more optical fiber paths gradually decreases from the proximal end to the distal end. In some cases, the input diameter of each of the multiple optical fiber paths is tapered, for example, from 1000 μm to 800 μm, from 1000 μm to 600 μm, from 1000 μm to 500 μm, from 1000 μm to 450 μm, from 1000 μm to 400 μm, from 1000 μm to 300 μm, from 800 μm to 600 μm, from 800 μm to 450 μm, from 800 μm to 400 μm, from 800 μm to 300 μm, from 600 μm to 450 μm, from 600 μm to 300 μm, and from 500 μm to 300 μm. In the embodiment, at least one of the multiple optical fiber paths does not taper from the proximal end to the distal end.

[0033] The number of optical fiber paths within an optical collection component can vary. In some embodiments, the optical collection component includes two or more optical fiber paths, e.g., three or more optical fiber paths, e.g., four or more optical fiber paths, e.g., five or more optical fiber paths, e.g., six or more optical fiber paths, e.g., twelve or more optical fiber paths, e.g., sixteen or more optical fiber paths, e.g., thirty-two or more optical fiber paths, and includes sixty-four or more optical fiber paths. In some embodiments, the number of optical fiber paths within an optical collection component is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 24, 32, 48, or 64. In certain embodiments, the number of optical fiber paths within an optical collection component is in the range of 3 to 10. In some embodiments, the systems of the present disclosure include an equal number of laser and optical fiber paths.

[0034] In some embodiments, the system of the present disclosure also includes an objective lens. Light from the flowstream can be collected by the objective lens. The objective lens may have any suitable magnification, such as 10x, 20x, 50x, and 100x. In some cases, the objective lens has a magnification of 20x.

[0035] The position of the proximal ends of the optical fiber paths relative to the flow cell can vary. In some embodiments, the proximal ends of multiple optical fiber paths are separated by the same distance from the flow cell. In some cases, where an objective lens is included, the proximal ends of multiple optical fiber paths are separated by the same distance from the objective lens. In some embodiments, the proximal ends of multiple optical fiber paths are separated by different distances from the flow cell. In some cases, the proximal ends of multiple optical fiber paths are separated by different distances from the objective lens. In other embodiments, the proximal ends of multiple optical fiber paths are in optical communication with the flow cell but are not in physical contact. For example, the proximal ends of multiple optical fiber paths may be positioned at a distance of 0.001 mm or more from the flow cell, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 10 mm or more, e.g., 25 mm or more, e.g., 50 mm or more, including a distance of 100 mm or more from the flow cell.

[0036] In some embodiments, the system of the present disclosure comprises a photodetection system comprising a plurality of light-collecting components. In some cases, the photodetection system comprises one or more light-collecting components, for example, two or more light-collecting components, three or more light-collecting components, four or more light-collecting components, five or more light-collecting components, six or more light-collecting components, ten or more light-collecting components, or twenty or more light-collecting components.

[0037] Figure 2 shows an optical collection component according to one embodiment of the present invention. As shown in Figure 2, the optical collection component 204 includes a plurality of optical fiber paths 202a, 202b, and 202c, each arranged to collect light 201a, 201b, and 201c from a flow stream (not shown) at its proximal end and to transport the collected light to its distal end. The proximal ends of the plurality of optical paths are spatially separated from each other. The optical collection component 204 includes a single optical fiber output 203 comprising a combination of the distal ends of the plurality of optical fiber paths. The single optical fiber output 203 transports light from the optical spots 201a, 201b, and 201c to a single detector array 205.

[0038] Figures 3A and 3B show different configurations for mechanically combining optical fiber paths with a single optical fiber output according to a particular embodiment. As shown in Figure 3A, fibers 301a, 301b, and 301c are arranged within a circular housing 302. As shown in Figure 3B, fibers 303a, 303b, and 303c are arranged within a housing 304 so that the fibers are in tandem. As shown in Figure 3C, fibers 305a, 305b, and 305c are arranged within a housing 306 so that the fibers are in horizontal rows.

[0039] Figure 4 shows an optical acquisition component comprising three optical fiber paths and a single optical fiber output according to a particular embodiment. In this example, the optical acquisition component consists of three tapering optical fiber paths. Each optical fiber path has an input core diameter of 800 μm with an numerical aperture (NA) of 0.12 and an output diameter of 450 μm with an NA of 0.22. The input optical fiber paths of the optical acquisition component can be moved independently, allowing the spacing between the input optical fiber paths to be changed to increase or decrease the spacing between laser intercepts without degrading data quality (sensitivity), adversely affecting the optical fiber path NA, or clipping the light from the objective lens. The single optical fiber output comprises a circular housing with the three optical fiber paths combined inside. The circular housing is an SMA housing. The SMA housing connects to a detector array and transmits light from all three optical fiber paths to a single detector array.

[0040] The system of interest may also include one or more optical adjustment components. As used herein, the term “optical adjustment” refers to any device that can change the spatial width of the irradiation, or any other characteristics of the irradiation from a light source, such as the direction of irradiation, wavelength, beam profile, beam width, beam intensity, focus, and pulse width. In some embodiments, the system may include optical adjustment components that adjust one or more of the irradiation direction, wavelength, beam profile, beam width, beam intensity, focus, and pulse width of the laser.

[0041] In other embodiments, the system may also include an optical tuning component that adjusts the light collected from the flowstream. In some cases, the optical tuning component adjusts the focus of the light from the flowstream from each of the lasers. In other cases, the optical tuning component adjusts the size of each beam spot from each of the lasers on an optical collection component (e.g., a single optical fiber). For example, the optical tuning component may be configured to reduce the size of each beam spot on the optical collection component by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.2 μm or more, e.g., 0.3 μm or more, e.g., 0.4 μm or more, e.g., 0.5 μm or more, e.g., 0.6 μm or more, e.g., 0.7 μm or more, e.g., 0.8 μm or more, e.g., 0.9 μm or more (including 1.0 μm or more). In some embodiments, the optical adjustment component is configured to reduce the size of each beam spot on the optical collection component by 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more (but not 95% or more). In specific cases, the optical adjustment component is configured to reduce the size of each beam spot on the optical collection component by 1.5 times or more, e.g., 2 times or more, e.g., 3 times or more, e.g., 4 times or more, e.g., 5 times or more (including 10 times or more).

[0042] In further cases, the optical adjustment component is configured to adjust the spacing between each beam spot from each laser on the optical collection component, for example, by reducing the spacing between each beam spot by 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 2 μm or more, e.g., 3 μm or more, e.g., 4 μm or more, e.g., 5 μm or more, e.g., 6 μm or more, e.g., 7 μm or more, e.g., 8 μm or more, e.g., 9 μm or more (including 10 μm or more). For example, the optical adjustment component may be configured to reduce the space between each beam spot by 5% or more, e.g., 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more (including 95% or more). In certain cases, the optical adjustment component is configured to reduce the space between each beam spot by 1.5 times or more, for example, 2 times or more, for example, 3 times or more, for example, 4 times or more, for example, 5 times or more (including 10 times or more).

[0043] In embodiments, optical adjustment components may include, but are not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In certain embodiments, the system of interest includes one or more focusing lenses. In one example, the focusing lens may be a reduction lens. In another example, the focusing lens may be a magnifying lens. In other embodiments, the system of interest includes one or more mirrors. However, in other embodiments, there are no optical adjustment components between the flow cell and the light collection component.

[0044] The system of the present disclosure includes a light source having a first laser configured to irradiate a flow stream at a first position, and one or more lasers configured to irradiate the flow stream at a position different from the first position, for example, a downstream position of the first position. In some embodiments, the light source includes two or more lasers configured to irradiate the flow stream at the downstream position, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, for example, six or more lasers, for example, twelve or more lasers, for example, sixteen or more lasers, for example, thirty-two or more lasers, and for example, sixteen

[0045] The type of laser may vary, and the laser of interest may 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. In other cases, the light source of interest may include dye lasers such as stilbene, coumarin, or rhodamine lasers. In yet other cases, the laser of interest may include metal vapor lasers such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof. In other cases, the light sources in question include solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, trim YAG lasers, ytterbium YAG lasers, ytterbium-2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0046] Each laser may be positioned at any suitable distance from the flow stream (for example, in a particle analyzer in a flow cytometer), including distances of 100 mm or more, such as 0.001 mm or more, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 25 mm or more from the flow stream. The laser may also be configured to irradiate the flow stream at any suitable angle (for example, with respect to the vertical axis of the flow stream), including angles of 30° to 60°, such as 90°, in the range of 10° to 90°, e.g., 15° to 85°, e.g., 20° to 80°, e.g., 25° to 75°.

[0047] Each laser may be independently configured to irradiate the flowstream continuously or at discrete intervals. In some cases, one or more of the lasers of the light source are configured to irradiate the flowstream continuously, such as continuous-wave lasers. In other cases, one or more of the lasers of the light source are configured to irradiate the flowstream at discrete intervals, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 1000 milliseconds, or at any other interval. When one or more of the lasers of the light source are configured to irradiate the sample at discrete intervals, the system may include one or more additional components to provide intermittent irradiation of the flowstream by the light source. For example, the systems in these embodiments may include one or more laser beam choppers, manual or computer-controlled beam stops for blocking the sample and exposing it to the light source.

[0048] The interval between irradiations by each laser may also vary, and delays of 60 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, may be independently separated as needed. For example, the interval between irradiations by each laser may be in the range of 5 microseconds to 10 microseconds, including 0.001 microseconds to 60 microseconds, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microseconds to 25 microseconds. In a particular embodiment, the interval between irradiations by each laser is 10 microseconds. In embodiments in which the sample is sequentially irradiated by more than two (i.e., three or more) lasers, the delays between irradiations by each light source may be the same or different.

[0049] In an embodiment, the light source includes a first laser configured to illuminate a flowstream at a first position, and one or more lasers configured to illuminate the flowstream at a position different from the first position, for example, a position downstream of the first position. For example, in one example, the light source includes a first laser configured to illuminate a flowstream at a first position, a second laser configured to illuminate a flowstream downstream of the first laser, a third laser configured to illuminate a flowstream downstream of the second laser, a fourth laser configured to illuminate a flowstream downstream of the third laser, and a fifth laser configured to illuminate a flowstream downstream of the fourth laser.

[0050] Depending on the flow rate of the flowstream and the distance between the irradiation positions of each laser, each laser is configured to independently irradiate the flowstream at a position 5 μm or more downstream from the irradiation position of the first laser, for example, 6 μm or more, for example, 7 μm or more, for example, 8 μm or more, for example, 9 μm or more, for example, 10 μm or more, for example, 15 μm or more, for example, 25 μm or more, for example, 50 μm or more, for example, 100 μm or more, for example, 250 μm or more, for example, 500 μm or more, including the case where each laser is independently configured to irradiate the flowstream at a position 1000 μm or more downstream from the irradiation position of the first laser. For example, the irradiation position of the flowstream by each laser may be located downstream from the irradiation position of the flowstream by the first laser by a distance of 5 μm to 5000 μm, e.g., 10 μm to 2500 μm, e.g., 25 μm to 1000 μm, e.g., 50 μm to 750 μm, e.g., 75 μm to 500 μm, including a distance of 100 μm to 250 μm. In some embodiments, each laser is configured to independently irradiate positions on the flowstream that are spaced 10 μm or less from each other, e.g., 9 μm or less, e.g., 8 μm or less, e.g., 7 μm or less, e.g., 6 μm or less (including positions on the flowstream that are spaced 5 μm or less).

[0051] In certain embodiments, the light source is a light beam generator configured to generate two or more frequency-shifted light beams. In some cases, the light beam generator includes a laser and a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous-wave laser. The acousto-optic device may be any convenient acousto-optic protocol configured to frequency-shift the laser light using the applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the system of interest is configured to generate an angle-deflected laser beam from light from the laser and the applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital combiner (DDS), arbitrary waveform generator (AWG), or electric pulse generator.

[0052] In one embodiment, the controller is configured to apply high-frequency drive signals to an acousto-optical device to generate a desired number of angularly deflected laser beams within the output laser beam, and includes being configured to apply, for example, three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals, and being configured to apply one hundred or more high-frequency drive signals.

[0053] 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 high-frequency drive signals with varying amplitudes, including approximately 5V to 25V, for example, approximately 0.001V to 500V, approximately 0.005V to 400V, approximately 0.01V to 300V, approximately 0.05V to 200V, approximately 0.1V to 100V, approximately 0.5V to 75V, approximately 1V to 50V, approximately 2V to 40V, and approximately 3V to 30V. In some embodiments, each applied high-frequency drive signal has a frequency range of approximately 5 MHz to approximately 50 MHz, such as approximately 0.001 MHz to approximately 500 MHz, for example 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.

[0054] In certain embodiments, the controller has a processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam having an angle-deflected laser beam having a desired intensity profile. For example, the memory may include instructions for generating two or more angle-deflected laser beams having the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may include instructions for generating 100 or more angle-deflected laser beams having the same intensity. In other embodiments, it may include instructions for generating two or more angle-deflected laser beams having different intensities, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may include instructions for generating 100 or more angle-deflected laser beams having different intensities.

[0055] In certain embodiments, the controller has a processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge toward 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 be in the range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., In other embodiments, the controller has a processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge toward 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 be in the range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., In yet another embodiment, the controller has a processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause 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 processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along the horizontal axis.

[0056] In some embodiments, the target light beam generator may be configured to generate spatially separated, angle-deflected laser beams within the output laser beam. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams may be separated by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more (including 5000 μm or more). In some embodiments, the system is configured to generate angle-deflected laser beams within the output laser beam that overlap, for example, with 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) may be 0.001 μm or larger, e.g., 0.005 μm or larger, e.g., 0.01 μm or larger, e.g., 0.05 μm or larger, e.g., 0.1 μm or larger, e.g., 0.5 μm or larger, e.g., 1 μm or larger, e.g., 5 μm or larger, e.g., 10 μm or larger (including overlap of 100 μm or larger).

[0057] In certain cases, a light beam generator configured to generate two or more frequency-shifted light beams includes the laser excitation modules described in U.S. Patent Nos. 9,423,353, 9,784,661 and 10,006,852, and U.S. Patent Application Publications 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0058] The system of the present disclosure further comprises a single photodetector array including a plurality of photodetectors configured to detect light from a laser transported through a single optical output of an optical collecting component. In some embodiments, the optical collecting component may be physically bonded to the single photodetector array using an adhesive or the like, co-molded together, or integrated with the photodetector array. In certain embodiments, the optical collecting component and the photodetector array are integrated into a single unit. In other embodiments, the optical collecting component is bonded to the photodetector array using connectors, for example, hook-and-loop fasteners, magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, non-permanent adhesives, or a combination thereof.

[0059] In other embodiments, the photodetector array and the light collection component are in optical communication but not in physical contact. For example, the light collection component may be positioned at a distance of 0.001 mm or more from the photodetector array, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 10 mm or more, e.g., 25 mm or more, e.g., 50 mm or more (including 100 mm or more from the photodetector array).

[0060] The photodetector system includes a single photodetector array having multiple photodetectors configured to detect light from a laser transported through a single optical output of an optical collection component. “Single” photodetector array means that the system includes only one photodetector array per photodetector component. The term “photodetector array” is used in its conventional sense to refer to an array or series of two or more photodetectors configured to detect light. In certain embodiments, the photodetector array includes four or more photodetectors, e.g., five or more photodetectors, e.g., ten or more photodetectors, e.g., twenty-five or more photodetectors, e.g., fifty or more photodetectors.

[0061] The photodetectors in the system in question may be any convenient photodetection protocol, including, but are not limited to, photodetectors such as active pixel sensors (APS), four-quadrant photodiodes, image sensors, charge-coupled devices (CCDs), sensitized charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In some embodiments, one or more of the photodetectors in the photodetector array are hybrid photodetectors including a photocathode integrated with an avalanche diode. In other embodiments, each photodetector in the photodetector array is a hybrid photodetector including a photocathode integrated with an avalanche diode. In some cases, the photocathode of the hybrid photodetector is a GaAs / GaAsP photocathode.

[0062] The photodetectors may be arranged in a photodetector array in any geometric configuration as needed, and the desired arrangements may include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The photodiodes in the photodiode array may be oriented at angles including 45° to 90°, in the range of 10° to 180°, e.g., 15° to 170°, e.g., 20° to 160°, e.g., 25° to 150°, e.g., 30° to 120°, relative to other photodiodes (referenced in the XZ plane). The photodiode array may be any suitable shape, including shapes consisting of straight lines, e.g., squares, rectangles, trapezoids, triangles, hexagons, etc., shapes consisting of curves, e.g., circles, ellipses, and irregular shapes, e.g., a parabolic bottom coupled to a flat top. In certain embodiments, the photodiode array has a rectangular active surface.

[0063] Each photodetector in the array may have an active surface having a width in the range of 5 μm to 250 μm, such as 10 μm to 225 μm, such as 15 μm to 200 μm, such as 20 μm to 175 μm, such as 25 μm to 150 μm, such as 30 μm to 125 μm, including 50 μm to 100 μm, and a length in the range of 5 μm to 250 μm, such as 10 μm to 225 μm, such as 15 μm to 200 μm, such as 20 μm to 175 μm, such as 25 μm to 150 μm, such as 30 μm to 125 μm, including 50 μm to 100 μm. The surface area of each photodiode in the array is 25 μm 2 ~10000 μm 2 For example, 50 μm 2 ~9000 μm 2 For example, 75 μm 2 ~8000 μm 2 For example, 100 μm 2 ~7000 μm 2 For example, 150 μm 2 ~6000 μm 2 in the range of, including 200 μm 2 ~5000 μm 2 and including.

[0064] The size of the photodetector array may vary according to the amount and intensity of light, the number of photodiodes, and the desired sensitivity, and may have a length in the range of 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm, including 5 mm to 25 mm. The width of the photodiode array may also vary in the range of 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm, including 5 mm to 25 mm. Therefore, the active surface of the photodiode array is 0.1 mm 2 ~10000 mm 2 For example, 0.5 mm 2 ~5000 mm 2 For example, 1 mm 2 ~1000 mm2 , for example 5mm 2 ~500mm 2 10mm 2 ~100mm 2 It may also include the range that is included.

[0065] In an embodiment, the photodetector of choice is configured to measure collected light with wavelengths including 5000 or more different wavelengths, such as one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths of light, e.g., 15 or more, e.g., 25 or more, e.g., 50 or more, e.g., 100 or more, e.g., 200 or more, e.g., 300 or more, e.g., 400 or more, e.g., 500 or more, e.g., 1000 or more, e.g., 1500 or more, e.g., 2500 or more. In a particular embodiment, the photodetector is configured to measure the spectrum of light, for example, the spectrum of light includes wavelengths including 1500 nm or more, extending to e.g., 50 nm or more, e.g., 100 nm or more, e.g., 1000 nm or more. For example, a photodetector is configured to measure light in the range of 200 nm to 1500 nm, for example, 400 nm to 1100 nm.

[0066] In some embodiments, the photodetector system is configured to measure light continuously or at discrete intervals. In some cases, the photodetectors in a photodetector array are configured to take measurements of the collected light continuously. In other cases, the detectors in a photodetector array are configured to take measurements at discrete intervals, for example, measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds (including every 1000 milliseconds), or at some other interval.

[0067] In some embodiments, the photodetector array is configured to propagate light between each photodetector in the array using mirrors, beam splitters, or lenses, etc. In some embodiments, each photodetector in the photodetector array is optically in communication with an optical tuning component configured to limit the wavelength of one or more light detected by the photodetector. In some embodiments, the optical tuning component is a bandpass filter. In other embodiments, the optical tuning component is a dichroic mirror. In a particular embodiment, the photodetector array of choice includes dichroic mirrors adjacent to one or more photodetectors in the array, e.g., dichroic mirrors adjacent to two or more photodetectors in the array, e.g., dichroic mirrors adjacent to three or more photodetectors in the array, e.g., dichroic mirrors adjacent to four or more photodetectors in the array, e.g., dichroic mirrors adjacent to five or more photodetectors in the array, e.g., dichroic mirrors adjacent to six or more photodetectors in the array, e.g., dichroic mirrors adjacent to seven or more photodetectors in the array, and includes dichroic mirrors adjacent to eight or more photodetectors in the array. In certain cases, the photodetector array includes a dichroic mirror adjacent to each photodetector in the array. In other embodiments, the photodetector array includes one or more beam splitters for propagating light to each photodetector. For example, the photodetector array may include two or more beam splitters, e.g., three or more beam splitters, e.g., four or more beam splitters, e.g., five or more beam splitters, e.g., six or more beam splitters, e.g., seven or more beam splitters, e.g., eight or more beam splitters, e.g., nine or more beam splitters, or even ten or more beam splitters.

[0068] In some embodiments, the photodetectors in a photodetector array are configured to detect light at different times. In some cases, light propagates sequentially across each of the photodetectors in the array, for example, light at a first time (t N1 ) to the first photodetector (detector) N1 ) is detected by the second photodetector (detector)N2 It is propagated in the second time (t N2 ) is detected by a further photodetector (detector) N+x Light propagates through each of the ) and time (t N+x ) are detected. In these embodiments, the light detection by the photodetectors in the photodetector array is temporally separated. In some embodiments, the light detection by the photodetectors in the photodetector array is temporally separated for 0.01 ps or more, e.g., 0.05 ps or more, e.g., 0.1 ps or more, e.g., 0.5 ps or more, e.g., 1.0 ps or more, e.g., 2 ps or more, e.g., 3 ps or more, e.g., 4 ps or more, e.g., 5 ps or more, e.g., 10 ps or more, e.g., 25 ps or more, e.g., 50 ps or more, e.g., 75 ps or more, e.g., 100 ps or more (including 500 ps or more). As will be described in more detail below, the generated light signals from each of the photodetectors may be temporally separated data signals. In certain cases, the temporally separated light detection in some embodiments of the present disclosure provides a reduction in simultaneous photon measurement in the photodetectors in the photodetector array, including cases where there is no simultaneous photon measurement in any of the photodetectors in the photodetector array.

[0069] In some embodiments, each photodetector in the photodetector array is configured to differentially detect light from a flow stream. In some embodiments, the photodetector array is configured to detect two or more predetermined sets of light wavelengths, for example, the photodetector array detects three or more different sets of light wavelengths, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., eleven or more (including twelve or more different sets of light wavelengths). Each predetermined set of light wavelengths may include five or more different light wavelengths, e.g., ten or more, e.g., fifteen or more, e.g., 25 or more, e.g., fifteen

[0070] In a particular embodiment, each photodetector detects a predetermined spectral range X of light.s It is configured to detect (nanometers, in nm units). The predetermined spectral range may vary, and in a particular embodiment, the photodetector of the target has a spectral range of light including 150 nm to 200 nm, ranging from 50 nm to 300 nm, for example 75 nm to 275 nm, for example 100 nm to 250 nm, for example 125 nm to 225 nm (X s ) are configured to detect. In certain embodiments, each photodetector is configured to detect a spectral range of light extending to 100 nm (i.e., X s (=100nm).

[0071] Figure 5A shows a system for irradiating particles in a flowstream according to a specific embodiment. Lasers 501, 502, 503, 504, and 505 irradiate the flowstream at positions 501a, 502a, 503a, 504a, and 505a, respectively. The light collected from the flowstream is collected by a focusing lens 506 and then collected at one of the positions 507a, 507b, 507c, 507d, and 507e at the proximal ends of the optical fiber path and transported through a single optical fiber output 508. In some embodiments, the focusing lens 506 is absent. The proximal ends 507a-e of the optical fiber path are spatially separated from each other. The single optical fiber output 508 comprises a combination of the distal ends of the optical fiber path. Light collected from the flowstream is transported through an optical fiber path to the photodetector array 510, where the photodetector array processes the light into photodetectors d1, d2, d3, d4, d5, d6, d7, d8, d9, d 10 d 11 d n It propagates to each of them.

[0072] Figure 5B shows the data signals from light detected by a photodetector in a single photodetector array according to a specific embodiment. As shown in Figure 5B, each of the photodetectors (shown in Figure 5A) is d1, d2, d3, d4, d5, d6, d7, d8, d9, d 10 d 11 d nThe light transported from the light collection component 509 is detected differentially. Detectors d1, d2, d3, d4, d5, d6, d7, d8, d9, d 10 d 11 d n It is configured to generate data signals for all wavelengths of light carried from laser 501 and laser 502. Detectors d3, d4, d5, d6, d7, d8, d9, d 10 d 11 d n It is configured to generate data signals of light detected from laser 503. Detectors d5, d6, d7, d8, d9, d 10 d 11 d n It is configured to generate data signals of light detected from laser 504. Detectors d6, d7, d8, d9, d 10 d 11 d n It is configured to generate a data signal of the light detected from the laser 505.

[0073] In certain embodiments, the photodetectors in the photodetector array are configured to detect light from one or more specific lasers in a light source. In some cases, each laser is assigned to one or more photodetectors such that the light from the laser (e.g., as scattered light from a flow stream, or as fluorescence from fluorophores irradiated by the laser in a flow stream) is detected by one or more assigned photodetectors in the photodetector array. Depending on the number of lasers in the light source (as described above) and the number of photodetectors in the photodetector array, the light from each laser may be assigned to 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, eight or more photodetectors, for example, ten or more photodetectors (including twelve or more photodetectors). In certain embodiments, the light from the lasers in the light source may be detected by all photodetectors in the photodetector array. In other embodiments, light from lasers in a light source may be detected by 90% or less (9 out of 10 photodetectors), for example 80% or less (16 out of 20 photodetectors), for example 75% or less (9 out of 12 photodetectors) (including 50% or less (8 out of 16 photodetectors) in the photodetector array). In some embodiments, the system of the present disclosure also includes a processor having memory operably coupled to the processor, which stores instructions that, when executed by the processor, cause the processor to assign each photodetector in the photodetector array to detect one or more predetermined sets of wavelengths of light, such as light from one or more lasers in a light source.

[0074] Figure 6 shows a multiplexed configuration of photodetectors in a photodetector array according to a specific embodiment. As shown in Figure 6, light from lasers 601, 602, 603, 604, and 605 is directed to photodetectors d1, d2, d3, d4, d5, d6, d7, d8, d9, and d 10 d 11 d 12 d 13 d nIt is detected by [this method]. The generated data signal output from each photodetector is assigned a specified data channel (e.g., V13, SSC, UV12). The assignment is also determined by a bandpass filter optically coupled to the photodetector (e.g., FC1~FC n This also includes data on the type and mapping location (e.g., number 1 to number 37).

[0075] In some embodiments, each photodetector in a photodetector array is configured to generate one or more data signals in response to detected light. In some embodiments, the data signals generated by the photodetector array are multiplexed data signals. In some embodiments, the photodetector system is configured for time-division multiplexing, where time discrimination is used to separate different photons carried to the photodetector array by a single light-collecting component. As described above, each photodetector may be configured to detect light from a flow stream at different times, and the output data signals from multiple photodetectors may be multiplexed. In these embodiments, the time-division multiplexed data signals may be output to a processor. For example, the time-division multiplexed data signals may include generated data signals from light detected at two or more different times, for example, four or more different times, for example, eight or more different times, for example, six or more different times, for example, thirty-two or more different times, for example, sixty-four or more different times, for example, one-two or more different times (including two-two or more different times).

[0076] In other embodiments, the photodetector system is configured for wavelength division multiplexing, in which light of different wavelengths from a flow stream is transported through a single photocollection component and detected by multiple photodetectors in a photodetector array. As described above, each photodetector may be configured to detect one or more predetermined sets of light wavelengths. In these embodiments, the output data signals generated by the predetermined sets of light wavelengths from the multiple photodetectors are multiplexed, and the wavelength division multiplexed data signal is output to a processor. For example, the wavelength division multiplexed data signal may include generated data signals from two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., eleven or more different predetermined sets of light wavelengths, or it may be a wavelength division multiplexed data signal including generated data signals from twelve or more predetermined sets of light wavelengths. In a particular embodiment, the wavelength division multiplexed data signal includes a wavelength division multiplexed data signal that includes generated data signals from two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., eleven or more different spectral light detected by a photodetector, and includes a wavelength division multiplexed data signal that includes generated data signals from twelve or more different spectral light detected by a photodetector.

[0077] In some embodiments, the system of the present disclosure includes a memory that, when executed by a processor, stores instructions causing the processor to spectrally decompose the light detected by the photodetectors of the photodetector array. The term “spectrally decompose” is used herein in its conventional sense to mean spectrally distinguishing the light from a flow stream by assigning or attributing overlapping wavelengths of light to each contributing component (e.g., fluorophores in the flow stream, scattered light from each laser). In certain embodiments, overlapping spectral components of light are determined by calculating a spectral separation matrix (as described in more detail below). In some embodiments, the spectrum of light detected by each photodetector overlaps with the spectrum of light detected by at least one other detector in the photodetector array. In some cases, the spectrum of light detected by the photodetectors in the photodetector array overlaps with the spectrum of light from at least one other detector by 5 nm or more, e.g., 10 nm or more, e.g., 25 nm or more (including 50 nm or more). In certain cases, the light spectrum detected by a photodetector overlaps with the spectra of two or more other photodetectors in the photodetector array, for example, each overlap being 5 nm or more, e.g., 10 nm or more, e.g., 25 nm or more, and including 50 nm or more. In other embodiments, the light spectra detected by photodetectors in the photodetector array have non-overlapping spectra. In these embodiments, the light spectrum detected by each photodetector is adjacent to the spectrum of at least one other photodetector within 10 nm, e.g., 9 nm or less, e.g., 8 nm or less, e.g., 7 nm or less, e.g., 6 nm or less, e.g., 5 nm or less, e.g., 4 nm or less, e.g., 3 nm or less, e.g., 2 nm or less (including 1 nm or less).

[0078] In some embodiments, the system of the present disclosure includes a processor having memory operably coupled to the processor, which stores instructions that, when executed by the processor, cause the processor to spectrally decompose the light from each photodetector in a photodetector array. In some embodiments, the overlap of the light spectra from the flow stream is determined, and the respective contributions to the overlapping detected light spectra are calculated. In some embodiments, spectral decomposition of light includes calculating a spectral separation matrix. In certain embodiments, calculating the spectral separation matrix may be used to estimate the abundance of each contribution to the light signal detected by the photodetectors in the photodetector array.

[0079] In some cases, calculating the spectral separation matrix involves determining the abundance of fluorophores associated with target particles in the flow stream. The abundance of each fluorophore associated with the target particles can be used to identify and classify the particles. In some cases, the identified or classified particles can be used to sort target particles (e.g., cells) in a sample. In certain embodiments, calculating the spectral separation is done so that the sorting is fast enough to sort particles in real time after detection by a photodetector system.

[0080] In certain embodiments, the system is configured to spectrally decompose light detected by multiple photodetectors in a photodetector array, for example, as described in International Patent Application PCT / US2019 / 068395, filed on 23 December 2019, whose entire disclosure is incorporated herein by reference. For example, light detected by multiple photodetectors in a photodetector array may be spectrally decomposed by solving a spectral separation matrix using one or more of the following: 1) a weighted least squares algorithm, 2) a Sherman-Morrison iterative inverse updater, 3) LU matrix decomposition, e.g., where the matrix is ​​decomposed into the product of a lower triangular (L) matrix and an upper triangular (U) matrix, 4) a modified Cholesky decomposition, 5) a QR factorization, or 6) a singular value decomposition to compute a weighted least squares algorithm.

[0081] In certain embodiments, the system of the Disclosure is part of or located within a flow cytometry system. Appropriate flow cytometry systems include, but are not limited to, those disclosed herein by reference: 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 (pt 1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222 (4): 335-344; and Herbig, et al. (2007) Crit Rev Ther This may include those described in Drug Carrier Syst. 24(3):203-255.In certain cases, the target flow cytometry system is the BD Biosciences FACSCanto® flow cytometer, BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Accuri® C6 Plus 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 LSRFortessa® X-20 flow cytometer, BD Biosciences FACSPresto® flow cytometer, BD Biosciences FACSVia® flow cytometer, and BD Biosciences FACSCalibur® cell sorter, BD Biosciences FACSCount® cell sorter, BD Biosciences This includes FACSLyric® cell sorters, BD Biosciences Via® cell sorters, BD Biosciences Influx® cell sorters, BD Biosciences Jazz® cell sorters, BD Biosciences Aria® cell sorters, BD Biosciences FACSAria® II cell sorters, BD Biosciences FACSAria® III cell sorters, BD Biosciences FACSAria® Fusion cell sorters, and BD Biosciences FACSMelody® cell sorters, BD Biosciences FACSymphony® S6 cell sorters, etc.

[0082] In some embodiments, the system in question is U.S. Patent Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, and 10,302,5 No. 45, No. 10,145,793, No. 10,113,967, No. 10,006,852, No. 9,952,076, No. 9,933,341, No. No. 9,726,527, No. 9,453,789, No. 9,200,334, No. 9,097,640, No. 9,095,494, No. 9,092,03 No. 4, No. 8,975,595, No. 8,753,573, No. 8,233,146, No. 8,140,300, No. 7,544,326, No. 7,2 01,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, Flow cytometry systems such as those described in Patent Nos. 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766 are incorporated herein by reference.

[0083] In certain embodiments, the system in question is configured to sort one or more particles (e.g., cells) from a sample. The term “sorting” is used herein in its conventional sense to mean separating components of a sample (e.g., non-cellular particles such as cells or biomolecules) and, in some cases, delivering the separated components to one or more sample collection containers. For example, the system in question may be configured to sort a sample having two or more components, e.g., three or more components, e.g., four or more components, e.g., five or more components, e.g., ten or more components, e.g., fifteen or more components, or to sort a sample having twenty-five or more components. One or more of the sample components may be separated from the sample and delivered to a sample collection container, e.g., two or more sample components, e.g., three or more sample components, e.g., four or more sample components, e.g., five or more sample components, e.g., ten or more sample components (including fifteen or more sample components) may be separated from the sample and delivered to a sample collection container.

[0084] In some embodiments, the particle sorting system of interest is configured to sort particles using a sealed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, particles of a sample (e.g., cells) are sorted using a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application No. 16 / 725,756, filed December 23, 2019, whose disclosure is incorporated herein by reference. In some embodiments, a method for sorting components of a sample includes sorting particles (e.g., cells in a biological sample) using a particle sorting module having deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference.

[0085] Figure 7 shows a functional block diagram of an example of a control system, such as an analysis controller 700, for analyzing and displaying biological events. The analysis controller 700 can be configured to implement various processes for controlling the graphical display of biological events.

[0086] The particle analyzer or sorting system 702 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 702 can be configured to provide biological event data to the analysis controller 700. A data communication channel can be included between the particle analyzer 702 and the analysis controller 700. The biological event data can be provided to the analysis controller 700 via the data communication channel.

[0087] The analysis controller 700 can be configured to receive biological event data from the particle analyzer 702. The biological event data received from the particle analyzer 702 may include flow cytometry event data. The analysis controller 700 can be configured to provide a graphical display to the display device 706, including a first plot of the biological event data. The analysis controller 700 can be further configured to render a region of interest as a gate around the collection of biological event data shown by the display device 706, for example, overlaid on the first plot. 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 show particle parameters or saturation detector data.

[0088] The analysis controller 700 can be further configured to display the biological event data within the gate on the display device 706 in a way that distinguishes it from other events in the biological event data outside the gate. For example, the analysis controller 700 can be configured to render the colors of the biological event data contained within the gate in a way that distinguishes them from the colors of the biological event data outside the gate. The display device 706 can be implemented as a monitor, a tablet computer, a smartphone, or any other electronic device configured to present a graphical interface.

[0089] The analysis controller 700 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 710. The mouse 710 can initiate gate selection signals to the analysis controller 700 that identify gates to be displayed on the display device 706 or operated via the display device (for example, by clicking gates when the cursor is positioned on or inside a desired gate). In some implementations, the first device can be implemented as a keyboard 708, or as other means of providing input signals to the analysis controller 700, 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 7, the mouse 710 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0090] A trigger event can cause the analysis controller 700 to change how the data is displayed, which parts of the data are actually displayed on the display device 706, and / or provide input for further processing, such as selecting a target population for particle sorting.

[0091] In some embodiments, the analysis controller 700 can be configured to detect when gate selection is initiated by the mouse 710. The analysis controller 700 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a specific distribution of biological event data received by the analysis controller 700.

[0092] The analysis controller 700 can be connected to a storage device 704. The storage device 704 can be configured to receive and store biological event data from the analysis controller 700. The storage device 704 can also be configured to receive and store flow cytometry event data from the analysis controller 700. The storage device 704 can be further configured to allow the analysis controller 700 to retrieve biological event data, such as flow cytometry event data.

[0093] The display device 706 can be configured to receive display data from the analysis controller 700. The display data may include plots of biological event data and gates that outline sections of the plots. The display device 706 can be further configured to change the information presented according to the input received from the analysis controller 700, in conjunction with input from the particle analyzer 702, the memory device 704, the keyboard 708, and / or the mouse 710.

[0094] In some implementations, the analysis controller 700 can generate a user interface for receiving exemplary events for selection. For example, the user interface may include controls for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates may be provided before the collection of event data for the sample, or based on an initial set of events for a portion of the sample.

[0095] As described above with respect to Figure 7, element 702 may be a particle analyzer or a sorting system. Thus, the system of the present invention may include a particle analyzer that can be used to analyze and characterize particles, whether or not the particles are physically sorted and placed into a collection container. For example, Figure 8 shows a functional block diagram of a particle analysis system for computationally based sample analysis and particle characterization. In some embodiments, the particle analysis system 800 is a flow system. The particle analysis system 800 includes a fluid system 802. The fluid system 802 may include or be combined with a sample tube 810 and a moving fluid column in the sample tube through which sample particles 830 (e.g., cells) move along a common sample path 820.

[0096] The particle analysis system 800 includes a detection system 804 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 808 generally refers to a monitoring area 840 of the common sample path. Detection may, in some implementations, include detecting light or one or more other properties of a particle 830 as it passes through the monitoring area 840. Figure 8 shows one detection station 808 with one monitoring area 840. Some implementations of the particle analysis system 800 may include multiple detection stations. Furthermore, some detection stations may monitor two or more areas. In the present invention, at least one detection station includes the light collection component and a single detector array described herein.

[0097] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be called event data. The data points can be multidimensional data points containing values ​​for each characteristic measured for the particle. The detection system 804 is configured to collect a series of such data points in a first time interval.

[0098] The particle analysis system 800 may also include a control system 806. The control system 806 may include one or more processors, an amplitude control circuit 926 and / or a frequency control circuit 924 as shown in Figure 9A. The control system 806 shown in Figure 8 can be operably associated with the fluid system 802. The control system 806 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 804 during a first time interval. The control system 806 may be further configured to generate an experimental signal frequency based on the number of data points in that portion of the first time interval. The control system 806 may further compare the experimental signal frequency with that of a calculated signal frequency or a predetermined signal frequency.

[0099] Figure 9A is a schematic diagram of a particle sorting system 900 according to one embodiment presented herein (for example, an embodiment in which 702 in Figure 7 is the particle sorter). In some embodiments, the particle sorting system 900 is a cell sorting system. As shown in Figure 9A, a droplet-forming transducer 902 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 901, which may be coupled to a nozzle 903, may include a nozzle 903, or may be a nozzle 903. Within the fluid conduit 901, a sheath fluid 904 hydrodynamically focuses a sample fluid 906 containing particles 909 into a moving fluid column 908 (e.g., a stream). Within the moving fluid column 908, the particles 909 (e.g., cells) are aligned in a vertical line across a monitoring area 911 (e.g., where laser streams intersect) and are irradiated by an irradiation source 912 (e.g., a laser). The vibration of the droplet-forming transducer 902 causes the moving fluid column 908 to decompose into multiple droplets 910, some of which contain particles 909.

[0100] During operation, a detection station 914 (e.g., an event detector) identifies a target particle (or target cell) as it crosses the monitoring area 911. The detection station 914 supplies input to a timing circuit 928, which then supplies input to a flash charge circuit 930. At a droplet separation point indicated by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 908 so that the target droplet becomes charged. The target droplet 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) to deflect the droplet into a container such as a collection tube or a multi-well or microwell sample plate, where the wells or microwells can be associated with specific target droplets. As shown in Figure 9A, the droplets can be collected in a drain receptacle 938.

[0101] The detection system 916 (e.g., a droplet boundary detector) helps to automatically determine the phase of the droplet drive signal as the target particle passes through the monitoring area 911. 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 916 enables the instrument to accurately calculate the location of each detected particle in the droplet. The detection system 916 can be input to amplitude signals 920 and / or phase signals 918, which are then input to amplitude control circuits 926 and / or frequency control circuits 924 (via amplifier 922). The amplitude control circuits 926 and / or frequency control circuits 924 then control the droplet formation transducer 902. The amplitude control circuits 926 and / or frequency control circuits 924 may be included in a control system.

[0102] In some implementations, the sorting electronics (e.g., detection system 916, detection station 914, and processor 940) can be coupled with a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the particle event data. In some implementations, the detection system 916 and the detection station 914 can be implemented as a single detection unit, or they can be communicatively coupled so that either the detection system 916 or the detection station 914 can collect event measurements and provide them to non-collecting elements.

[0103] Figure 9B is a schematic diagram of a particle sorting system according to one embodiment presented herein. The particle sorting system 900 shown in Figure 9B includes deflection plates 952, 954. An electric charge can be applied via stream-charging wires in barbs. This creates a stream of droplets 910 containing particles 909 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The particle information is analyzed by sorting electronic equipment or other detection systems (not shown in Figure 9B). The deflection plates 952, 954 can be independently controlled to attract or repel charged droplets, guiding the droplets toward a desired collection container (e.g., one of 972, 974, 976, or 978). As shown in Figure 9B, the deflection plates 952, 954 can be controlled to guide the particles toward container 974 along a first path 962, or toward container 978 along a second path 968. If the particles are not of interest (for example, they 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 964. Such uncharged droplets may enter the waste receptacle via the aspirator 970 or the like.

[0104] Sorting electronics may be included to initiate measurement data collection, receive fluorescence signals from particles, and determine how to adjust the deflection plates to sort the particles. An exemplary implementation of the embodiment shown in Figure 9B includes the BD FACSAria® line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0105] Computer control system Aspects of the present disclosure further include a computer-controlled system, the system including one or more computers for fully automated or partially automated systems, as described in Section 1. More specifically, the system of the present disclosure includes a computer having a computer-readable storage medium in which a computer program is stored, the computer program when loaded into the computer including instructions for irradiating a plurality of lasers onto particles in a flow stream, instructions for detecting light carried from an optical collection component having a single photodetector array having a plurality of photodetectors, instructions for generating a multiplexed data signal from the photodetector array, and instructions for determining one or more parameters of a particle based on the multiplexed data signal. In some embodiments, the computer program includes instructions for generating a time-division multiplexed data signal. In other embodiments, the computer program includes instructions for generating a wavelength-division multiplexed data signal.

[0106] In some embodiments, the system of the present disclosure includes a computer having a computer-readable storage medium in which a computer program is stored, and the computer program, when loaded into the computer, includes instructions for irradiating a flow stream with a first laser at a first position, and for irradiating a plurality of lasers at positions different from the first position, for example, downstream of the first position. In some cases, the computer program includes instructions for irradiating each of the plurality of lasers at a position 5 μm or more downstream from the position of irradiation by the first laser, for example 6 μm or more, for example 7 μm or more, for example 8 μm or more, for example 9 μm or more, for example 10 μm or more, for example 15 μm or more, for example 25 μm or more, for example 50 μm or more, for example 100 μm or more, for example 250 μm or more, for example 500 μm or more, and the computer program includes instructions for irradiating each laser independently at a position 1000 μm or more downstream from the position of irradiation by the first laser. For example, the computer program may include instructions for irradiating the flowstream at a distance including 100 μm to 250 μm, such as 5 μm to 5000 μm, e.g., 10 μm to 2500 μm, e.g., 25 μm to 1000 μm, e.g., 50 μm to 750 μm, e.g., 75 μm to 500 μm, from the irradiation position of the flowstream by the first laser. In some embodiments, the computer program includes instructions for independently irradiating each laser at positions on the flowstream that are spaced 10 μm or less from each other, e.g., 9 μm or less, e.g., 8 μm or less, e.g., 7 μm or less, e.g., 6 μm or less (including positions on the flowstream that are spaced 5 μm or less from each other).

[0107] In a specific case, the computer program includes instructions to irradiate the flowstream with a first laser at a first location in the flowstream, instructions to irradiate the flowstream with a second laser at a second location downstream of the first location in the flowstream, instructions to irradiate the flowstream with a third laser at a third location downstream of the second location in the flowstream, instructions to irradiate the flowstream with a fourth laser at a fourth location downstream of the third location in the flowstream, and instructions to irradiate the flowstream with a fifth laser at a fifth location downstream of the fourth location in the flowstream.

[0108] In some embodiments, the computer program includes instructions for detecting light using each photodetector in the photodetector array at different times. In some cases, the light propagates sequentially across each of the photodetectors in the array, and the computer program gives instructions for a first time (t N1 ) to the first photodetector (detector) N1 ) detects light, and the second time (t N2 ) has a second photodetector (detector) N2 ) detects light and time (t N+x ) and further photodetectors (detectors) N+x The program includes instructions for detecting light using each of the following: In certain cases, the computer program includes instructions for generating time-separated data signals, such as data signals generated by the detection of degraded photons through simultaneous photon measurement in photodetectors within a photodetector array.

[0109] In some embodiments, the system includes a computer having a computer-readable storage medium in which a computer program is stored, and the computer program, when loaded into the computer, includes instructions for differentially detecting light from a flow stream. In some embodiments, the computer program includes instructions for using a photodetector array to detect two or more predetermined sets of light wavelengths, for example, three or more, for example four or more, for example five or more, for example six or more, for example seven or more, for example eight or more, for example nine or more, for example ten or more, for example eleven or more different sets of light wavelengths (including twelve or more different sets of light wavelengths).

[0110] In some embodiments, the computer program includes instructions for detecting light from one or more specific lasers of a light source. In some cases, the computer program includes instructions for assigning each laser to one or more photodetectors. For example, the computer program may include instructions for assigning lasers to 90% or less of the photodetectors in a photodetector array (e.g., 9 out of 10 photodetectors), 80% or less (e.g., 16 out of 20 photodetectors), 75% or less (e.g., 9 out of 12 photodetectors) (including 50% or less of the photodetectors in a photodetector array (e.g., 8 out of 16 photodetectors)). In certain embodiments, the computer program includes instructions for assigning lasers to all photodetectors in the photodetector array.

[0111] In some embodiments, the computer program includes instructions for spectrally decomposing light detected by photodetectors in a photodetector array, such as by calculating a spectral separation matrix for each spectrum of detected light. In some embodiments, the computer program includes instructions for determining the overlap of each spectrum of light detected from the flow stream and calculating each contribution to the overlapping light spectra. In some cases, the computer program includes instructions for estimating the abundance of each contribution to the light signal detected by photodetectors in the photodetector array. In specific cases, the computer program includes instructions for spectrally decomposing light by solving the spectral separation matrix using one or more of the following: 1) a weighted least squares algorithm, 2) a Sherman-Morrison iterative inverse updater, 3) LU matrix decomposition, e.g., where the matrix is ​​decomposed into the product of a lower triangular (L) matrix and an upper triangular (U) matrix, 4) a modified Cholesky decomposition, 5) a QR factorization, or 6) calculating a weighted least squares algorithm by singular value decomposition.

[0112] In some embodiments, the computer program includes instructions for determining one or more parameters of irradiated particles in a flow stream from generated data signals from a photodetector array. In some embodiments, the computer program includes instructions for identifying particles based on one or more determined parameters of the particles. In other embodiments, the computer program includes instructions for sorting particles based on one or more determined parameters of the particles.

[0113] In an embodiment, the system includes an input module, a processing module, and an output module. The system in question may include both hardware and software components, and the hardware components may take the form of one or more platforms, for example, in the form of servers, such that functional elements, i.e., elements of the system that perform specific tasks of the system (e.g., managing information input and output, processing information, etc.), can be executed by running software applications across one or more computer platforms that represent the system.

[0114] 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 can access memory in which instructions for performing steps of the method in question are stored. 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 several other processors that are available or will be available. The processor runs the operating system, which interfaces with firmware and hardware in a well-known way and facilitates the processor coordinating and executing the functions of various computer programs that can be written in various programming languages ​​such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically works with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, according to all known technologies. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow the user to manually align the light source with the flow stream based on first and second optical signals. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0115] System memory may be any of the various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read-and-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices may be any of the various known or future devices, including compact disk drives, tape drives, or floppy disk drives. Such types of memory storage devices typically read from and / or write to program storage media (not shown), such as compact disks. Any of these program storage media, or others currently in use or to be developed in the future, may be considered computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0116] In some embodiments, a computer program product is described that comprises a computer-usable medium having stored control logic (a computer software program including program code). When the control logic is executed by the computer's processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of a hardware state machine for performing the functions described herein will be obvious to those skilled in the art.

[0117] Memory can be any suitable device on which a 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 fixed or portable device). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium holding the required program code. Programming can be provided to the processor remotely via a communication channel, or it can be pre-stored in a computer program product, such as memory or any other portable or fixed computer-readable storage medium, using one of those memory-related devices. For example, magnetic or optical disks can carry programs and can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of computer program products, and algorithms for use in carrying out the method as described above. Programming according to the present invention can be recorded on a computer-readable medium, e.g., any medium that a computer can directly read and access. Such mediums include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tapes, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROMs, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0118] The processor can also access communication channels to communicate with users in remote locations. Remote locations mean that the user is not in direct contact with the system, but rather relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0119] 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 is 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 cellular communication such as code division multiple access (CDMA) and global system for mobile communications (GSM).

[0120] In one embodiment, the communication interface is configured to include one or more physical ports or interfaces, such as a USB port, an RS-232 port, or any other suitable electrical connection port that enables data communication between the system in question and other external devices, such as a computer terminal configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0121] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol that enables the target system to communicate with computer terminals and / or networks, other devices such as communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with it.

[0122] In one embodiment, the communication interface is configured to provide a connection for data transfer using Internet Protocol (IP), Short Message Service (SMS), wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or WiFi connection to the Internet via a WiFi hotspot.

[0123] In one embodiment, the system in question is configured to communicate wirelessly with a server device via a communication interface using common standards such as 802.11, 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 it may be a larger device such as a desktop computer or consumer electronics 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.

[0124] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network device or server device and data stored in a target system, such as an optional data storage unit, using one or more of the communication protocols and / or mechanisms described above.

[0125] The output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other types of remote communication. The output manager may also provide information generated by the processing module to a user in 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 implemented according to various known techniques. As some examples, the data may include SQL, HTML or XML documents, email or other files, or data in other formats. The data may also include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. One or more platforms present in the system under consideration may be any kind of known computer platform or a kind to be developed in the future, but they are typically computers of a class commonly referred to as servers. However, they may also be mainframe computers, workstations, or other types of computers. They may be connected via any known or future type of cabling or other communication systems, including wireless systems, or they may be networked or otherwise connected. They may be located in the same place or they may be physically separated.Various operating systems can be used on any computer platform, depending on the type and / or manufacturer of the selected computer platform. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBM i®, Android®, SGI IRIX®, Oracle Solaris®, and others.

[0126] Figure 10 shows a general architecture of an exemplary computing device 1000 according to a particular embodiment. The general architecture of the computing device 1000 shown in Figure 10 includes the configuration of computer hardware and software components. However, it is not necessary to show all of these generally conventional elements in order to provide a practicable disclosure. As shown, the computing device 1000 includes a processing unit 1010, a network interface 1020, a computer-readable media drive 1030, an input / output device interface 1040, a display 1050, and an input device 1060, all of which can communicate with each other via a communication bus. The network interface 1020 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 1010 may receive information and instructions from other computing systems or services via the network. The processing unit 1010 may also communicate with memory 1070 and further provide output information to an optional display 1050 via the input / output device interface 1040. For example, analysis software (such as data analysis software or programs like FlowJo®) stored as executable instructions in the non-temporary memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 1040 may also accept input from an optional input device 1060, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, speech recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0127] Memory 1070 may contain computer program instructions (grouped as modules or components in some embodiments) that the processing unit 1010 executes to implement one or more embodiments. Memory 1070 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 1070 may store an operating system 1072 that provides computer program instructions for use by the processing unit 1010 in the general management and operation of the computing device 1000. Data may be stored in a data storage device 1090. Memory 1070 may further include computer program instructions and other information for implementing embodiments of the present disclosure.

[0128] A method for analyzing particles in a flow stream using the target system. Aspects of the present disclosure also include a method for determining one or more parameters of a particle in a flowstream. The method of interest includes i) irradiating a particle in a flowstream with a plurality of lasers, each configured to irradiate the flowstream at each location, and ii) detecting light from the particle using a photodetector system having an optical collection component comprising a plurality of optical fiber paths, each arranged to collect light from the flowstream at one of the respective locations at the proximal end and transport the collected light to the distal end. The proximal ends of the plurality of optical paths are spatially separated from each other.

[0129] In carrying out the method in question, particles in a flowstream are irradiated by a first laser at a first position in the flowstream, and then irradiated by one or more lasers at a different position in the flowstream, for example, downstream of the first position. In some embodiments, the particles are irradiated in the flowstream by two or more lasers, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, for example, six or more lasers, for example, twelve or more lasers, for example, sixteen or more lasers, for example, thirty-two or more lasers, and the irradiation of particles in the flowstream by sixty-four or more lasers is included. In some embodiments, each laser used to irradiate the flowstream may vary and emit wavelengths including 400 nm to 800 nm, such as 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.

[0130] In some embodiments, the type of laser used to irradiate the flowstream may vary and may 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. In other cases, the light source in question may include dye lasers such as stilbene, coumarin, or rhodamine lasers. In yet another case, the laser in question may include metal vapor lasers such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof. In other cases, the light sources in question include solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, trim YAG lasers, ytterbium YAG lasers, ytterbium-2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0131] The method of interest involves irradiating particles in a flowstream with multiple lasers. In some embodiments, the flowstream may be irradiated by each laser from any suitable distance, for example, at distances of 100 mm or more, including distances of 0.001 mm or more, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 25 mm or more from the flowstream. The flowstream may also be irradiated by each laser at any suitable angle (for example, with respect to the vertical axis of the flowstream), for example, at angles including 30° to 60°, including angles of 90°, including angles in the range of 10° to 90°, e.g., 15° to 85°, e.g., 20° to 80°, e.g., 25° to 75°.

[0132] In some embodiments, the method of interest involves irradiating particles in a flowstream continuously or at discrete intervals. In some cases, the flowstream is irradiated continuously with one or more lasers, such as a continuous-wave laser that irradiates the flowstream continuously. In other cases, the flowstream is irradiated at discrete intervals using one or more lasers, such as irradiating the flowstream at discrete intervals including, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at any other interval.

[0133] The interval between irradiations by each laser may also vary, and delays of 60 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, may be independently separated as needed. For example, the interval between irradiations by each laser may be in the range of 5 microseconds to 10 microseconds, including 0.001 microseconds to 60 microseconds, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microseconds to 25 microseconds. In a particular embodiment, the interval between irradiations by each laser is 10 microseconds. In embodiments in which the sample is sequentially irradiated by more than two (i.e., three or more) lasers, the delays between irradiations by each light source may be the same or different.

[0134] In embodiments, the method of interest includes irradiating particles in a flowstream at a first position in the flowstream, and irradiating the particles with one or more lasers at a different flowstream position, for example, a position downstream of the first position. For example, in one example, the method includes irradiating the flowstream with a first laser at a first position in the flowstream, irradiating the flowstream with a second laser at a second position in the flowstream downstream of the first position, irradiating the flowstream with a third laser at a third position in the flowstream downstream of the second position, irradiating the flowstream with a fourth laser at a fourth position in the flowstream downstream of the third position, and irradiating the flowstream with a fifth laser at a fifth position in the flowstream downstream of the fourth position.

[0135] Depending on the flow rate of the flowstream and the distance between the irradiation positions of each laser, the flowstream may be independently irradiated by each laser at a position 5 μm or more downstream from the irradiation position of the first laser, for example, 6 μm or more, for example, 7 μm or more, for example, 8 μm or more, for example, 9 μm or more, for example, 10 μm or more, for example, 15 μm or more, for example, 25 μm or more, for example, 50 μm or more, for example, 100 μm or more, for example, 250 μm or more, for example, 500 μm or more, or the flowstream may be independently irradiated by each laser at a position 1000 μm or more downstream from the irradiation position of the first laser. For example, the flowstream may be irradiated by each laser at a distance including 100 μm to 250 μm, such as 5 μm to 5000 μm, e.g., 10 μm to 2500 μm, e.g., 25 μm to 1000 μm, e.g., 50 μm to 750 μm, e.g., 75 μm to 500 μm, from the position of the flowstream irradiated by the first laser, which is 5 μm to 500 μm downstream. In some embodiments, the flowstream is irradiated independently by each laser at positions on the flowstream that are spaced 10 μm or less from each other, e.g., 9 μm or less, e.g., 8 μm or less, e.g., 7 μm or less, e.g., 6 μm or less (including positions on the flowstream that are spaced 5 μm or less from each other).

[0136] In certain embodiments, the method of interest involves irradiating a sample with two or more frequency-shifted light beams. As described above, a light beam generator component having a laser and an acousto-optical device for frequency-shifting the laser light may be used. In these embodiments, the method involves irradiating the acousto-optical device with a laser. Depending on the wavelength of the desired light produced 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, e.g., 250 nm and 1250 nm, e.g., 300 nm and 1000 nm, e.g., 350 nm and 900 nm (including 400 nm and 800 nm). The acousto-optical device may be irradiated with one or more lasers, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers (including ten or more lasers). The lasers may include any combination of lasers. For example, in some embodiments, the method involves 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.

[0137] When two or more lasers are used, 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 by each of the lasers simultaneously. In other embodiments, the acousto-optical device is irradiated by each of the lasers sequentially. When two or more lasers are used to sequentially irradiate the acousto-optical device, the time each laser irradiates the acousto-optical device can independently be 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 30 microseconds or more, and e.g., 60 microseconds or more. For example, the method may include irradiating the acousto-optical device with lasers for durations in the range of 5 microseconds to 10 microseconds, e.g., 0.01 microseconds to 75 microseconds, e.g., 0.1 microseconds to 50 microseconds, e.g., 1 microsecond to 25 microseconds. In embodiments in which an acoustic-optical device is sequentially irradiated with two or more lasers, the duration for which the acoustic-optical device is irradiated by each laser may be the same or different.

[0138] Acousto-optical devices can be irradiated continuously or at discrete intervals. In some cases, the method involves continuously irradiating the acousto-optical device with a laser. In other cases, the acousto-optical device is irradiated with a laser at discrete intervals, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds (including every 1000 milliseconds), or at any other interval.

[0139] Depending on the laser, the acousto-optical device may be illuminated from various distances, including 50 mm and above, such as 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 2.5 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 15 mm or more, e.g., 25 mm or more. The angle or illumination may also vary in an angle of, for example, 90°, within a range of 30° to 60°, such as 10° to 90°, e.g., 15° to 85°, e.g., 20° to 80°, e.g., 25° to 75°.

[0140] In one embodiment, the method includes applying high-frequency drive signals to an acousto-optical device to generate an angularly deflected laser beam. Two or more high-frequency drive signals, for example, three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals (including one hundred or more high-frequency drive signals) may be applied to the acousto-optical device to generate an output laser beam having a desired number of angularly deflected laser beams.

[0141] An angle-deflected laser beam generated by a high-frequency drive signal has an intensity based on the amplitude of the applied high-frequency drive signal. In some embodiments, the method includes applying a high-frequency drive signal having an amplitude sufficient to generate an angle-deflected laser beam having a desired intensity. In some cases, each applied high-frequency drive signal independently has an amplitude including about 5V to about 25V, such as about 0.001V to about 500V, e.g., about 0.005V to about 400V, e.g., about 0.01V to about 300V, e.g., about 0.05V to about 200V, e.g., about 0.1V to about 100V, e.g., about 0.5V to about 75V, e.g., about 1V to 50V, e.g., about 2V to 40V, e.g., 3V to about 30V. In some embodiments, each applied high-frequency drive signal has a frequency range of approximately 5 MHz to approximately 50 MHz, such as approximately 0.001 MHz to approximately 500 MHz, for example 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.

[0142] In these embodiments, the angle-deflected laser beams within the output laser beam are spatially separated. Depending on the applied high-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, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more (including 5000 μm or more). In some embodiments, the angle-deflected laser beams overlap with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam, for example. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) may be 0.001 μm or larger, e.g., 0.005 μm or larger, e.g., 0.01 μm or larger, e.g., 0.05 μm or larger, e.g., 0.1 μm or larger, e.g., 0.5 μm or larger, e.g., 1 μm or larger, e.g., 5 μm or larger, e.g., 10 μm or larger (including overlap of 100 μm or larger).

[0143] In certain cases, a flowstream is irradiated with multiple beams of frequency-shifted light, and cells in the flowstream are imaged by fluorescence imaging, which uses high-frequency tagged radiation to generate frequency-coded images, as described, for example, in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013), and in U.S. Patent Nos. 9,423,353, 9,784,661, 10,006,852, and U.S. Patent Application Publications 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0144] In some embodiments, a flowstream may be illuminated with one or more lasers via an optical adjustment component. In some cases, the optical adjustment component is configured to change the spatial width of the illumination from one or more lasers or some other characteristic of the illumination, such as the illumination direction, wavelength, beam profile, beam width, beam intensity, focus, and pulse width. In some embodiments, the method includes adjusting one or more of the illumination direction, wavelength, beam profile, beam width, beam intensity, focus, and pulse width of the lasers using optical adjustment.

[0145] Light from a flowstream can be transported to a photodetector array through an optical collection component. In some embodiments, the light from the flowstream is transported directly to the proximal end of the optical collection component (i.e., there is no component in between). In other embodiments, the light from the flowstream is sent to the proximal end of the optical collection component through an optical adjustment component. In some cases, the optical adjustment component adjusts the focus of the light from the flowstream from each of the lasers. In other cases, the optical adjustment component adjusts the size of each beam spot from each of the lasers on the optical collection component (e.g., a single optical fiber). For example, the optical adjustment component may be configured to reduce the size of each beam spot on the light collection component by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.2 μm or more, e.g., 0.3 μm or more, e.g., 0.4 μm or more, e.g., 0.5 μm or more, e.g., 0.6 μm or more, e.g., 0.7 μm or more, e.g., 0.8 μm or more, e.g., 0.9 μm or more (including 1.0 μm or more). In some embodiments, the optical adjustment component may be configured to reduce the size of each beam spot on the light collection component by 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more (including 95% or more). In certain cases, the optical adjustment component is configured to reduce the size of each beam spot on the light collection component by 1.5 times or more, for example, 2 times or more, for example, 3 times or more, for example, 4 times or more, for example, 5 times or more (including 10 times or more).

[0146] In other cases, the method of interest may include using an optical adjustment component to adjust the spacing between each beam spot from each laser on the optical collection component. For example, the spacing between each beam spot may be reduced by 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 2 μm or more, e.g., 3 μm or more, e.g., 4 μm or more, e.g., 5 μm or more, e.g., 6 μm or more, e.g., 7 μm or more, e.g., 8 μm or more, e.g., 9 μm or more (including 10 μm or more). For example, the spacing between each beam spot may be reduced by 5% or more, e.g., 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more (including 95% or more) using an optical adjustment component. In certain cases, the spacing between beam spots is reduced by 1.5 times or more, for example, 2 times or more, for example, 3 times or more, for example, 4 times or more, for example, 5 times or more (including 10 times or more), using optical adjustment components.

[0147] The method of interest involves detecting light from a particle using a photodetection system having an optical collection component and a single photodetector array. When carrying out the method of interest, the method of interest includes collecting light by using the optical collection component. The optical collection component comprises multiple optical fiber paths and a single optical fiber output for using photon collection from the objective lens of a flow cytometer to a single detector array. The multiple optical fiber paths are each arranged to collect light from the flow stream at one of their respective positions at their proximal ends and to transport the collected light to their distal ends. The proximal ends of the multiple optical paths are spatially separated from each other. The single optical fiber output comprises a combination of the distal ends of the multiple optical fiber paths.

[0148] The distal ends of optical fiber paths may be combined into a single optical fiber output in any convenient manner. In some embodiments, the single optical fiber output comprises a fusion of the distal ends of multiple optical fiber paths. In other words, the optical fiber paths are fused together at their distal ends (e.g., by fusion splicing). The fusion of optical fibers generally proceeds by applying heat to the optical fibers from a heat source. The heat source of choice includes, but is not limited to, lasers, gas flames, electric tungsten filaments, and electric arcs. In some embodiments, the multiple optical fiber paths are combined via a binder. The binders that can be used can vary and include, for example, optical adhesives. Any convenient optical adhesive can be used. For example, adhesives of choice may include epoxy (e.g., pure epoxy, polyester resins, and epoxy acrylates), photocurable acrylic resins, elastomers (e.g., silicones, silicone-free silanes), cyanoacrylates, and structural adhesives (e.g., those having resins and activators). In some embodiments, the adhesive is curable by exposure to light (e.g., UV light). In certain embodiments, the optical adhesive is transparent. The refractive index of the adhesive of choice may vary in some cases within a range including 1.9 to 2.3, e.g., 1.1 to 2.9, e.g., 1.2 to 2.8, e.g., 1.3 to 2.7, e.g., 1.4 to 2.6, e.g., 1.5 to 2.7, e.g., 1.6 to 2.6, e.g., 1.7 to 2.5, e.g., 1.8 to 2.4.

[0149] In certain cases, optical fiber paths are mechanically joined to each other at their distal ends to form a single optical fiber output. In such cases, the techniques used to mechanically join the optical fiber paths can vary. For example, optical fiber paths may be mechanically joined to each other using capillary tubes, V-grooves, elastomer splices, or swivel splices. In some embodiments, optical fiber paths are combined into a single optical fiber output via optical fiber connectors used to couple the optical fibers. Optical fiber connectors may be configured to couple to each other via any convenient mechanism. Couplings of interest include screw couplings, latch couplings, push-pull couplings, bayonet couplings, gendered couplings, snap-fit ​​couplings, and combinations thereof. In some embodiments, optical fiber paths are combined using ultra-small assembly (SMA) optical fiber connectors.

[0150] In some embodiments, a single optical fiber output comprises a single housing containing multiple optical fiber paths. The housing may be configured to arrange the optical fiber paths in any convenient manner. Exemplary housing shapes include, but are not limited to, circular, rectangular, tandem, horizontal, elliptical, triangular, square, kite, trapezoidal, parallelogram, rhombic, or different types of polygonal housings. In some embodiments, the housing is circular. In other embodiments, the housing is rectangular. In yet another embodiment, the housing is configured to arrange multiple optical fiber paths in tandem. In yet another embodiment, the housing is configured to arrange multiple optical fiber paths horizontally.

[0151] In embodiments, the method of interest may include transporting light from a flow stream to a photodetector array using multiple optical collection components. In some cases, the method includes transporting light from a flow stream to a photodetector array using one or more optical collection components, for example, two or more optical collection components, three or more optical collection components, four or more optical collection components, five or more optical collection components, six or more optical collection components, or ten or more optical collection components. Further details regarding the optical collection components of the present invention can be found above.

[0152] The method of the objective includes detecting light transported from a light collection component using a photodetector array having multiple photodetectors. In certain embodiments, the method of the objective includes detecting light using a photodetector array having four or more photodetectors, e.g., five or more photodetectors, e.g., ten or more photodetectors, e.g., twenty or more photodetectors, and e.g., fifty or more photodetectors. In some embodiments, the photodetectors may include, but are not limited to, active pixel sensors (APS), four-quadrant photodiodes, image sensors, charge-coupled devices (CCDs), sensitized charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the light is detected using one or more hybrid photodetectors, including a photocathode integrated with an avalanche diode. In some cases, the photocathode of the hybrid photodetector is a GaAs / GaAsP photocathode.

[0153] The photodetectors may be arranged in a photodetector array in any geometric configuration as needed, and the desired arrangements may include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The photodiodes in the photodiode array may be oriented at angles ranging from 45° to 90° relative to other photodiodes (referenced in the XZ plane), such as 10° to 180°, e.g., 15° to 170°, e.g., 20° to 160°, e.g., 25° to 150°, e.g., 30° to 120°. The photodiode array may be any suitable shape, such as a shape consisting of straight lines, e.g., square, rectangle, trapezoid, triangle, hexagon, etc., a shape consisting of curves, e.g., circular, elliptical, and irregular shapes, e.g., a parabolic bottom coupled to a flat top. In certain embodiments, the photodiode array has a rectangular active surface.

[0154] Each photodetector in the array may have an active surface having a width in the range of 50 μm to 100 μm, such as 5 μm to 250 μm, e.g., 10 μm to 225 μm, e.g., 15 μm to 200 μm, e.g., 20 μm to 175 μm, e.g., 25 μm to 150 μm, e.g., 30 μm to 125 μm, and a length in the range of 50 μm to 100 μm, such as 5 μm to 250 μm, e.g., 10 μm to 225 μm, e.g., 15 μm to 200 μm, e.g., 20 μm to 175 μm, e.g., 25 μm to 150 μm, e.g., 30 μm to 125 μm, and the surface area of ​​each photodiode in the array is 25 μm. 2 ~10000μm 2 For example, 50 μm 2 ~9000μm 2 For example, 75 μm 2 ~8000μm 2 For example, 100 μm 2 ~7000μm 2 For example, 150 μm 2 ~6000μm 2 200 μm 2 ~5000μm 2 This is the range that includes it.

[0155] The size of the photodetector array may vary depending on the amount and intensity of light, the number of photodiodes and the desired sensitivity, and may have a length including 5 mm to 25 mm, in the range of 0.01 mm to 100 mm, e.g., 0.05 mm to 90 mm, e.g., 0.1 mm to 80 mm, e.g., 0.5 mm to 70 mm, e.g., 1 mm to 60 mm, e.g., 2 mm to 50 mm, e.g., 3 mm to 40 mm, e.g., 4 mm to 30 mm. The width of the photodiode array may also vary in the range of 0.01 mm to 100 mm, e.g., 0.05 mm to 90 mm, e.g., 0.1 mm to 80 mm, e.g., 0.5 mm to 70 mm, e.g., 1 mm to 60 mm, e.g., 2 mm to 50 mm, e.g., 3 mm to 40 mm, e.g., 4 mm to 30 mm, e.g., 5 mm to 25 mm. Therefore, the active surface of the photodiode array is 0.1 mm 2 ~10000mm 2 For example, 0.5mm 2 ~5000mm 2 For example, 1 mm 2 ~1000mm 2 , for example 5mm 2 ~500mm 2 10mm 2 ~100mm 2 It may also include the range that is included.

[0156] In an embodiment, the collected light is measured at wavelengths including 5000 or more different wavelengths, such as one or more wavelengths, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths of light, for example, 15 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, for example, 400 or more, for example, 500 or more, for example, 1000 or more, for example, 1500 or more, for example, 2500 or more. In a particular embodiment, the method includes measuring the spectrum of light using a photodetector, for example, the spectrum of light includes wavelengths including 1500 nm or more, ranging from 50 nm or more, for example, 100 nm or more, for example, 200 nm or more, for example, 300 nm or more, for example, 400 nm or more, for example, 500 or more, for example, 600 nm or more, for example, 700 nm or more, for example, 800 nm or more, for example, 900 nm or more, for example, 1000 nm or more. For example, the method may include measuring light in the range of 200 nm to 1500 nm, for example, 400 nm to 1100 nm.

[0157] In embodiments, the method of the object of interest includes measuring light continuously or at discrete intervals. In some cases, the light measurements are taken continuously. In other cases, the light measurements are taken at discrete intervals, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds (including every 1000 milliseconds), or at any other interval.

[0158] In some embodiments, the method of the objective involves detecting light using each of the photodetectors in the photodetector array at different times. In some cases, the light propagates sequentially across each of the photodetectors in the array, for example, the light at a first time (t N1 ) to the first photodetector (detector) N1 ) is detected by the second photodetector (detector) N2 It is propagated in the second time (t N2 ) is detected by a further photodetector (detector) N+x Light propagates through each of the ) and time (t N+x) is detected. In these embodiments, the method includes temporally separated optical detections. In some embodiments, the detection of light by detectors in the detector array is temporally separated by 0.01 ps or more, such as 0.05 ps or more, such as 0.1 ps or more, such as 0.5 ps or more, such as 1.0 ps or more, such as 2 ps or more, such as 3 ps or more, such as 4 ps or more, such as 5 ps or more, such as 10 ps or more, such as 25 ps or more, such as 50 ps or more, such as 75 ps or more, such as 100 ps or more (including 500 ps or more). In some embodiments, the method includes generating data signals temporally separated from each of the detectors.

[0159] In some embodiments, the method of interest includes differentially detecting light from the flow stream using each detector in the detector array. In some embodiments, two or more predetermined sets of light wavelengths are detected using the detector array, for example, the detector array detects three or more different sets of light wavelengths, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as eleven or more, including twelve or more different sets of light wavelengths. Each predetermined set of light wavelengths may include five or more different light wavelengths, such as ten or more, such as fifteen or more, such as twenty-five or more, such as fifty or more, such as one hundred or more, such as two hundred or more, such as three hundred or more, such as four hundred or more, such as five hundred or more, such as one thousand or more, such as one thousand five hundred or more, such as two thousand five hundred or more different light wavelengths, and may include five thousand or more different light wavelengths.

[0160] In certain embodiments, the method includes detecting a predetermined spectral range X of light s (in nanometers, nm units) using each detector. The predetermined spectral range may vary, and in certain embodiments, the spectral range of light (X s) extends from 50nm to 300nm, for example, 75nm to 275nm, for example, 100nm to 250nm, for example, 125nm to 225nm, and includes 150nm to 200nm. In certain embodiments, the spectral range of light detected by each photodetector extends to 100nm (i.e., X s (=100nm).

[0161] In certain embodiments, the method of interest involves detecting light from one or more specific lasers in a light source using photodetectors in a photodetector array. In some cases, the method involves assigning each laser to one or more photodetectors such that the light from the lasers (e.g., as scattered light from a flow stream, or as fluorescence from fluorophores irradiated by the lasers in a flow stream) is detected by one or more assigned photodetectors in the photodetector array. Depending on the number of lasers in the light source (as described above) and the number of photodetectors in the photodetector array, the light from each laser may be assigned to 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, eight or more photodetectors, for example, ten or more photodetectors (including twelve or more photodetectors). In certain embodiments, the light from the lasers in the light source may be detected by all photodetectors in the photodetector array. In other embodiments, light from lasers in a light source may be detected by 90% (9 out of 10 photodetectors), for example 80% (16 out of 20 photodetectors), for example 75% (9 out of 12 photodetectors), for example 50% (8 out of 16 photodetectors) in a photodetector array. In some embodiments, the method includes assigning each photodetector in a photodetector array to detect one or more predetermined sets of wavelengths of light, such as light from one or more lasers in a light source.

[0162] In some embodiments, the method of interest includes determining one or more parameters of particles in a flow stream. In embodiments, each photodetector in a photodetector array is configured to generate one or more data signals in response to detected light. In some cases, the data signals generated by the photodetector array are multiplexed data signals. In certain embodiments, the method includes time-division multiplexing, in which time discrimination is used to separate different photons carried to the photodetector array (e.g., a single optical fiber) by a single optical collection component. As described above, each photodetector may be configured to detect light from the flow stream at different times, and the output data signals from multiple photodetectors may be multiplexed. In these embodiments, the time-division multiplexed data signals may be output to a processor. For example, the time-division multiplexed data signals may include generated data signals from light detected at two or more different times, e.g., four or more different times, e.g., eight or more different times, e.g., sixteen or more different times, e.g., 32 or more different times, e.g., sixteen or more different times, e.g., 128 or more different times (including 256 or more different times).

[0163] In other embodiments, the method of interest involves wavelength division multiplexing, in which light of different wavelengths from a flow stream is transported through a single optical collection component and detected by multiple photodetectors in a photodetector array. In these embodiments, each photodetector may be configured to detect one or more predetermined sets of light wavelengths. In these embodiments, the data signals generated by the predetermined sets of light wavelengths from the multiple photodetectors are multiplexed, and the wavelength division multiplexed data signal is output to a processor. For example, the wavelength division multiplexed data signal may include generated data signals from two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., eleven or more different predetermined sets of light wavelengths, and may include a wavelength division multiplexed data signal including generated data signals from twelve or more predetermined sets of light wavelengths. In a particular embodiment, the method includes generating a wavelength division multiplexed data signal that includes data signals from two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., eleven or more different spectral light detected by a photodetector, and also includes generating a wavelength division multiplexed data signal that includes data signals from twelve or more different spectral light detected by a photodetector.

[0164] In some embodiments, the method of the objective includes spectral decomposition of light detected by photodetectors in a photodetector array. In certain embodiments, overlapping spectral components of light are determined by calculating a spectral separation matrix. In some embodiments, the spectrum of light detected by each photodetector overlaps with the spectrum of light detected by at least one other detector in the photodetector array. In some cases, the spectrum of light detected by photodetectors in a photodetector array overlaps with the spectrum of light from at least one other detector by 5 nm or more, e.g., 10 nm or more, e.g., 25 nm or more (including 50 nm or more). In certain cases, the spectrum of light detected by a photodetector overlaps with the spectra of two or more other photodetectors in the photodetector array, e.g., each overlap being 5 nm or more, e.g., 10 nm or more, e.g., 25 nm or more, including 50 nm or more. In other embodiments, the spectrum of light detected by photodetectors in a photodetector array has non-overlapping spectra. In these embodiments, the light spectrum detected by each photodetector is adjacent to the spectrum of at least one other photodetector within 10 nm, for example, within 9 nm, for example, within 8 nm, for example, within 7 nm, for example, within 6 nm, for example, within 5 nm, for example, within 4 nm, for example, within 3 nm, for example, within 2 nm (including less than 1 nm).

[0165] In some embodiments, the method of interest includes determining the overlap of light spectra from a flow stream and calculating the respective contributions to the overlapping detected light spectra. In some embodiments, spectral decomposition of light includes calculating a spectral separation matrix. In certain embodiments, the method includes calculating a spectral separation matrix to estimate the abundance of each contribution to the light signal detected by photodetectors in a photodetector array.

[0166] In some cases, calculating the spectral separation matrix involves determining the abundance of fluorophores associated with target particles in the flow stream. The abundance of each fluorophore associated with the target particles can be used to identify and classify the particles. In some cases, the identified or classified particles can be used to sort target particles (e.g., cells) in a sample. In certain embodiments, calculating the spectral separation is done so that the sorting is fast enough to sort particles in real time after detection by a photodetector system.

[0167] In certain embodiments, the method of interest includes spectrally decomposing light detected by multiple photodetectors in a photodetector array, as described, for example, in International Patent Application PCT / US2019 / 068395, filed on 23 December 2019, the entire disclosure of which is incorporated herein by reference. For example, spectrally decomposing light detected by multiple photodetectors in a photodetector array may include solving the spectral separation matrix using one or more of the following: 1) a weighted least squares algorithm, 2) a Sherman-Morrison iterative inverse updater, 3) LU matrix decomposition, for example, where the matrix is ​​decomposed into the product of a lower triangular (L) matrix and an upper triangular (U) matrix, 4) a modified Cholesky decomposition, 5) by QR factorization, or 6) by singular value decomposition.

[0168] In certain embodiments, the method of interest comprises sorting one or more particles (e.g., cells) of a sample in a flowstream. For example, the method may comprise sorting two or more components of a sample, e.g., three or more components, e.g., four or more components, e.g., five or more components, e.g., ten or more components, e.g., fifteen or more components, or even twenty-five or more components of a sample. In certain embodiments, the sample is a biological sample. The term “biological sample” is used in its conventional sense to refer to an entire organism, an entire plant, an entire fungus, or, in certain cases, a subset of animal tissue, cells, or components that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Therefore, “biological sample” refers to both a subset of an organism or its tissues in their natural state and homogenates, lysates, or extracts prepared from a subset of the organism or its tissues, including but not limited to plasma, serum, cerebrospinal fluid, lymph, skin, respiratory, gastrointestinal, cardiovascular, and urogenital tract sections, tears, saliva, milk, blood cells, tumors, and organs. A biological sample can be any type of living tissue, including both healthy tissue and affected tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, a biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, and in some cases, a blood sample including whole blood, such as blood obtained from a venipuncture or fingertip puncture (blood may or may not be combined with any reagents such as preservatives and anticoagulants before assay).

[0169] In certain embodiments, the source of the sample is “mammal” or “mammalian,” and these terms are broadly used to describe organisms belonging to the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys). In some cases, the subject is human. The method can be applied to samples obtained from human subjects of both sexes and any developmental stage (i.e., neonatal, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is juvenile, adolescent, or adult. While the present invention may be applied to samples derived from human subjects, it should be understood that the method can also be carried out on samples derived from other animal subjects such as birds, mice, rats, dogs, cats, livestock and horses (i.e., in “non-human subjects”), but is not limited to these.

[0170] When sorting particles, the method includes data acquisition, analysis, and recording by a computer or the like, with multiple data channels recording data from each detector used. In these embodiments, the analysis may include spectrally decomposed light (e.g., by calculating a spectral separation matrix) as described above. The results of this analysis can be transported to a sorting system configured to generate a set of digitized parameters based on particle classification.

[0171] In some embodiments, a method for sorting components of a sample includes sorting particles (e.g., cells in a biological sample) using a particle sorting module having deflection plates, such as the one described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, cells in a sample are sorted using a sorting decision module having multiple sorting decision units, such as the one described in U.S. Patent Application No. 16 / 725,756, filed December 23, 2019, whose disclosure is incorporated herein by reference.

[0172] Non-temporary computer-readable storage medium for analyzing particles in a flowstream Aspects of this disclosure further include a non-temporary computer-readable storage medium having instructions for carrying out the methods covered herein. The computer-readable storage medium may be used in one or more computers for the full or partial automation of a system for carrying out the methods described herein. In certain embodiments, instructions by the methods described herein may be encoded on the computer-readable medium in the form of “programming,” and as used herein, the term “computer-readable medium” refers to any non-temporary storage medium involved in providing instructions and data to a computer for execution and processing. Suitable examples of non-temporary storage media include hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state disks, and network-attached storage (NAS), whether such devices are inside or outside a computer. A file containing information may be “stored” on the computer-readable medium, and “stored” means recording the information so that it is accessible and retrievable by a computer at a later date. The computer implementations described herein may be carried out using programming, which may be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Python, Java (Sun Microsystems, Inc., Santa Clara, CA), JavaScript, Visual Basic (Microsoft Corp., Redmond, WA), C, C#, C++ (AT&T Corp., Bedminster, NJ), Go, R, Swift, PHP, and many other languages.

[0173] In some embodiments, the target computer-readable storage medium includes a stored computer program, which, when loaded into a computer, includes instructions having an algorithm for irradiating particles in a flowstream with a plurality of lasers, each configured to irradiate the flowstream at its respective location; an algorithm for detecting light carried from the particles using a photodetection system having the aforementioned light collection component and a single photodetector array having a plurality of photodetectors; an algorithm for generating a multiplexed data signal from the photodetector array; and an algorithm for determining one or more parameters of the particles based on the multiplexed data signal. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for generating a time-division multiplexed data signal. In other embodiments, the non-temporary computer-readable storage medium includes an algorithm for generating a wavelength-division multiplexed data signal.

[0174] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for irradiating the flow stream with a first laser at a first position and for irradiating the flow stream with one or more lasers at a different position from the first position, for example, a position downstream of the first position. In some cases, the non-temporary computer-readable storage medium includes an algorithm for irradiating the flow stream with each of a plurality of lasers at a position 5 μm or more downstream from the position of irradiation by the first laser, for example 6 μm or more, for example 7 μm or more, for example 8 μm or more, for example 9 μm or more, for example 10 μm or more, for example 15 μm or more, for example 25 μm or more, for example 50 μm or more, for example 100 μm or more, for example 250 μm or more, for example 25 μm or more, for example 500 μm or more, for example 500 μm or more, and the non-temporary computer-readable storage medium includes an algorithm for independently irradiating the flow stream with each laser at a position 1000 μm or more downstream from the position of irradiation by the first laser. For example, a non-temporary computer-readable storage medium includes an algorithm for irradiating a flowstream at a distance including 100 μm to 250 μm, from the irradiation position of the flowstream by the first laser, at a distance of 5 μm to 5000 μm, e.g., 10 μm to 2500 μm, e.g., 25 μm to 1000 μm, e.g., 50 μm to 750 μm, e.g., 75 μm to 500 μm. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for independently irradiating each laser at positions on the flowstream that are spaced 10 μm or less from each other, e.g., 9 μm or less, e.g., 8 μm or less, e.g., 7 μm or less, e.g., 6 μm or less (including positions on the flowstream that are spaced 5 μm or less from each other).

[0175] In certain cases, the non - transitory computer - readable storage medium includes an algorithm for irradiating a first laser on a flow stream at a first position of the flow stream, an algorithm for irradiating a second laser on the flow stream at a second position of the flow stream downstream of the first position, an algorithm for irradiating a third laser on the flow stream at a third position of the flow stream downstream of the second position, an algorithm for irradiating a fourth laser on the flow stream at a fourth position of the flow stream downstream of the third position, and an algorithm for irradiating a fifth laser on the flow stream at a fifth position of the flow stream downstream of the fourth position.

[0176] In some embodiments, the non - transitory computer - readable storage medium includes an algorithm for detecting light using each photodetector in a photodetector array at different times. In some cases, the non - transitory computer - readable storage medium includes an algorithm for detecting light by a first photodetector (detector N1 ) at a first time (t N1 ), detecting light by a second photodetector (detector N2 ) at a second time (t N2 ), and an algorithm for detecting light using each of additional photodetectors (detectors N+x ) at a time (t N+x ). In certain cases, the non - transitory computer - readable storage medium includes an algorithm for generating temporally separated data signals, such as data signals generated by detecting photons with reduced photon coincidence measurements in the photodetectors within the photodetector array.

[0177] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for differentially detecting light from a flow stream. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for detecting two or more predetermined sets of light wavelengths, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., eleven or more different sets of light wavelengths (including twelve or more different sets of light wavelengths), using a photodetector array.

[0178] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for detecting light from one or more specific lasers of a light source. In some cases, the non-temporary computer-readable storage medium includes an algorithm for assigning each laser to one or more photodetectors. The non-temporary computer-readable storage medium may include an algorithm for assigning lasers to 90% (9 out of 10 photodetectors) or less of the photodetectors in a photodetector array, for example, 80% (16 out of 20 photodetectors) or less, for example, 75% (9 out of 12 photodetectors) or less (including 50% (8 out of 16 photodetectors) or less of the photodetectors in a photodetector array). In certain embodiments, the non-temporary computer-readable storage medium includes an algorithm for assigning lasers to all photodetectors in the photodetector array.

[0179] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for spectrally decomposing light detected by photodetectors in a photodetector array, such as by calculating a spectral separation matrix for each spectrum of detected light. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for determining the overlap of each spectrum of light detected from a flow stream and calculating each contribution to the overlapping light spectra. In some cases, the non-temporary computer-readable storage medium includes an algorithm for estimating the abundance of each contribution to the optical signal detected by photodetectors in a photodetector array. In specific cases, the non-temporary computer-readable storage medium includes an algorithm for spectrally decomposing light by solving a spectral separation matrix using one or more of the following: 1) a weighted least squares algorithm, 2) a Sherman-Morrison iterative inverse updater, 3) LU matrix decomposition, e.g., where the matrix is ​​decomposed into the product of a lower triangular (L) matrix and an upper triangular (U) matrix, 4) modified Cholesky decomposition, 5) by QR factorization, or 6) calculating a weighted least squares algorithm by singular value decomposition.

[0180] In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for determining one or more parameters of irradiated particles in a flow stream from generated data signals from a photodetector array. In some embodiments, the non-temporary computer-readable storage medium includes an algorithm for identifying particles based on one or more determined parameters of the particles. In other embodiments, the non-temporary computer-readable storage medium includes an algorithm for sorting particles based on one or more determined parameters of the particles.

[0181] Non-temporary computer-readable storage media may be used in one or more computer systems having a display and operator input devices. Operator input devices may be, for example, a keyboard, a mouse, etc. A processing module includes a processor that can access memory in which instructions for performing steps of the method in question are stored. A processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of several other processors that are available or will be available. The processor runs an operating system, which interfaces with firmware and hardware in well-known ways and facilitates the processor coordinating and executing the functions of various computer programs that can be written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically works 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, all according to known technologies.

[0182] kit This disclosure further provides a kit comprising the plurality of lasers described herein, an optical collection component, and a photodetector array. The kit of interest includes an optical collection component comprising a plurality of optical fiber paths and a single optical fiber output, wherein each of the plurality of optical fiber paths is configured to collect light from a flow stream at its proximal end and to transport the collected light to its distal end, the proximal ends of the plurality of optical paths are spatially separated from each other, and the single optical fiber output comprises a combination of the distal ends of the plurality of optical fiber paths described herein.

[0183] The various components of the kit may reside 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 each optical fiber, photodetector, or laser, may reside in a sealed pouch, such as a sterile foil pouch or envelope.

[0184] In addition to the components described above, the kit may further include instructions for carrying out the method in question (in a particular embodiment). These instructions may be present in the kit in various forms, and one or more of these forms may be present in the kit. One possible form of these instructions is printed information on a suitable medium or substrate, e.g., one or more sheets of paper on which the information is printed, the kit's packaging, or accompanying documents. Yet another form of these instructions is a computer-readable medium on which the information is recorded, e.g., a diskette, a compact disc (CD), or a portable flash drive. Yet another possible form of these instructions is a website address that can be used via the Internet to access the information at the removed site.

[0185] usefulness The systems, methods, computer systems, and kits described herein are used in a variety of applications where it is desirable to analyze and sort particulate components in samples in fluid media, such as biological samples. The disclosure is also used in flow cytometry where it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In some embodiments, the optical collection component of the systems described herein offers the advantage of increasing or decreasing the distance and / or time between lasers without changing the input to the detector array. In other embodiments, the optical collection component of the systems described herein offers the advantage of increasing the distance and / or time between lasers so that a single optical output fiber bundle reduces light leakage between laser intercepts. In yet another embodiment, the optical collection component of the systems described herein offers the advantage of increasing the distance and / or time between lasers so that a single optical output fiber bundle increases the amount of light that the detector array can collect and the number of lasers that can be collected simultaneously without clipping light at the edges of the optical fiber. In certain embodiments, the system in question provides a fully automated protocol that requires little to no human input for adjusting the flow cytometer in use.

[0186] This disclosure also relates to applications in which cells prepared from biological samples may be desired for use in research, laboratory testing, or therapy. In some embodiments, the methods and devices of this disclosure may facilitate the acquisition of individual cells prepared from biological samples of target fluids or tissues. For example, the methods and systems of this disclosure facilitate the acquisition of cells from fluid or tissue samples used as research or diagnostic samples for diseases such as cancer. Similarly, the methods and systems of this disclosure 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, fluids) with higher efficiency and lower cost compared to conventional flow cytometry systems.

[0187] Notwithstanding the attached claims, this disclosure is also defined by the following clauses: 1. A light source comprising multiple lasers, each configured to irradiate a flow stream at its respective position, Light detection system and The light detection system is equipped with, A plurality of optical fiber paths, each arranged to collect light from a flowstream at one of its respective positions at the proximal end and to transport the collected light to the distal end, wherein the proximal ends of the plurality of optical fiber paths are spatially separated from each other, and A single optical fiber output comprising a combination of the distal ends of multiple optical fiber paths An optical collection component equipped with, A single photodetector array comprising multiple photodetectors configured to detect light from a laser transported through a single optical output of an optical collection component, and A particle analyzer equipped with the following features. 2. The particle analyzer described in Clause 1, wherein a single optical fiber output comprises a fusion section of the distal ends of multiple optical fiber paths. 3. A particle analyzer as described in Clause 1, in which multiple optical fiber paths are combined via a binder. 4. The binder is epoxy, as described in Clause 3 of the particle analyzer. 5. A single optical fiber output is provided in a single housing with multiple optical fiber paths combined inside, as described in Clause 1 of the particle analyzer. 6. The housing is a circular housing, as described in Clause 5. 7. The housing is a rectangular housing, as specified in Clause 5. 8. The particle analyzer described in Clause 7, wherein the housing is configured to have multiple optical fiber paths arranged in a tandem row. 9. The particle analyzer described in Clause 7, wherein the housing is configured to have multiple optical fiber paths arranged in a row. 10. A particle analyzer according to any one of the preceding clauses, wherein each of the multiple optical fiber paths has an input diameter in the range of 800 μm to 1000 μm. 11. A particle analyzer according to any one of the preceding clauses, wherein the proximal end of each of the multiple optical fiber paths has an numerical aperture in the range of 0.10 to 0.15. 12. A particle analyzer according to any one of the preceding clauses, wherein each of the multiple optical fiber paths has an output diameter in the range of 400 μm to 1000 μm. 13. A particle analyzer according to any one of the preceding clauses, wherein the distal end of each of the multiple optical fiber paths has an numerical aperture in the range of 0.20 to 0.25. 14. A particle analyzer according to any one of the preceding clauses, wherein at least one of the multiple optical fiber paths tapers from the proximal end to the distal end. 15. A particle analyzer according to any one of the multiple optical fiber paths, wherein at least one of the optical fiber paths does not taper from the proximal end to the distal end. 16. A particle analyzer as described in any one of the preceding clauses, wherein the number of lasers in the light source is in the range of 3 to 10. 17. The light source is, A first laser configured to irradiate a flowstream at a first position, Multiple lasers configured to illuminate the flowstream at a position downstream of the first position and A particle analyzer as described in Clause 16, comprising the following features. 18. A particle analyzer according to any one of the preceding clauses, wherein the lasers in the plurality of lasers are configured to irradiate flow streams at positions separated from each other by no more than 10 μm. 19. A particle analyzer as described in any one of the preceding clauses, wherein the number of optical fiber paths in the optical collection component is in the range of 3 to 10. 20. A particle analyzer comprising an equal number of laser and optical fiber paths, as described in any one of clauses 16 to 19. 21. A particle analyzer according to any one of the preceding clauses, wherein the proximal ends of multiple optical fiber paths are separated by the same distance from the flow cell. 22. A particle analyzer according to any one of the preceding clauses, wherein the proximal ends of multiple optical fiber paths are separated from the flow cell by different distances. 23. The particle analyzer is the particle analyzer described in any one of the preceding clauses, comprising a plurality of optical collection components. 24. A particle analyzer according to any one of the preceding clauses, wherein the photodetectors in the photodetector array are configured to detect light at different times. 25. A particle analyzer according to any one of the preceding clauses, wherein each photodetector in the photodetector array is configured to differentially detect light from one or more lasers. 26. A particle analyzer according to any one of the preceding clauses, wherein each photodetector in the photodetector array is configured to detect one or more predetermined sets of light wavelengths. 27. Each set of light wavelengths includes 50 or fewer different wavelengths, as described in Clause 26 of the particle analyzer. 28. A particle analyzer according to any one of the preceding clauses, wherein each photodetector in the photodetector array is a hybrid photodetector comprising a photocathode integrated with an avalanche diode. 29. A particle analyzer according to any one of the preceding clauses, wherein each photodetector in the photodetector array is in optical communication with an optical tuning component configured to limit the wavelength of one or more light detected by the photodetector. 30. The optical adjustment component is a bandpass filter, as described in Clause 29 of the particle analyzer. 31. The particle analyzer according to any one of the clauses 26 to 30, further comprising a processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to assign each photodetector in a photodetector array to detect a given set of wavelengths of light. 32. The particle analyzer according to any one of the preceding clauses, further comprising a processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to receive multiplexed data signals from a photodetector array. 33. The data signal is a time-division multiplexed data signal, as described in Clause 32 of the particle analyzer. 34. The data signal is a wavelength division multiplexed data signal, as described in Clause 32 of the particle analyzer. 35. The particle analyzer according to any one of the preceding clauses, further comprising a processor having memory operably coupled to the processor, the memory storing instructions that, when executed by the processor, cause the processor to spectrally decompose light detected by the photodetectors of a photodetector array. 36. The particle analyzer described in Clause 35, wherein the memory stores instructions for spectrally decomposing light by calculating a spectral separation matrix for each spectrum of light detected by the photodetectors of the photodetector array. 37. A particle analyzer is a particle analyzer as described in any one of the preceding clauses, which is incorporated into a flow cytometer. 38. A flow cytometer is a particle analyzer as described in Clause 37, which is equipped with a particle sorter. 39. A method for determining one or more parameters of particles in a flow stream, the method being: Multiple lasers, each configured to illuminate the flowstream at a different location, are used to irradiate particles in the flowstream, This involves detecting light from particles using a photodetection system, where the photodetection system is A plurality of optical fiber paths, each arranged to collect light from a flowstream at one of its respective positions at the proximal end and to transport the collected light to the distal end, wherein the proximal ends of the plurality of optical fiber paths are spatially separated from each other, and A single optical fiber output with a combination of the distal ends of multiple optical fiber paths. An optical collection component equipped with, A single photodetector array comprising multiple photodetectors configured to detect light from a laser transported through a single optical output of an optical collection component, and It has the ability to detect, Generating multiplexed data signals from a photodetector array, Determining one or more parameters of a particle based on multiplexed data signals and Methods that include... 40. The method according to Clause 39, wherein a single optical fiber output comprises a fusion section of the distal ends of multiple optical fiber paths. 41. The method according to Clause 39, wherein multiple optical fiber paths are joined together via a binder. 42. The method according to Clause 39, wherein a single optical fiber output comprises a single housing with multiple optical fiber paths combined inside. 43. The housing is a circular housing, as described in Clause 42. 44. The housing is a rectangular housing, as described in Clause 42. 45. The method according to Clause 44, wherein the housing is configured to arrange multiple optical fiber paths in a cascaded manner. 46. ​​The method according to clause 44, wherein the housing is configured to arrange multiple optical fiber paths in a row. 47. The method according to any one of the claims 39 to 46, wherein each of the multiple optical fiber paths has an input diameter in the range of 800 μm to 1000 μm. 48. The method according to any one of the claims 39 to 47, wherein each of the multiple optical fiber paths has an output diameter in the range of 400 μm to 1000 μm. 49. The method according to any one of the multiple optical fiber paths, wherein at least one of the optical fiber paths tapers from the proximal end to the distal end. 50. The method according to any one of the multiple optical fiber paths, wherein at least one optical fiber path does not taper from the proximal end to the distal end. 51. The number of lasers in the light source is in the range of 3 to 10, as described in any one of the clauses 39 to 50. 52. The number of optical fiber paths in the optical collection component is in the range of 3 to 10, as described in any one of the clauses 39 to 51. 53. The method according to clause 51 or 52, wherein the number of lasers in the multiple lasers and the number of optical fiber paths in the optical collection component are equal. 54. The method according to any one of the claims 39 to 53, wherein multiple optical fiber paths are configured to irradiate flow streams at positions separated from each other by no more than 10 μm. 55. The method according to any one of the clauses 39 to 54, wherein the proximal ends of multiple optical fiber paths are separated by the same distance from the flow cell. 56. The method according to any one of the clauses 39 to 55, wherein the proximal ends of multiple optical fiber paths are separated from the flow cell by different distances. 57. The method according to any one of the clauses 39 to 56, wherein the generated data signal is a time-division multiplexed data signal. 58. The method according to any one of the clauses 39 to 56, wherein the generated data signal is a wavelength division multiplexed data signal. 59. The method according to any one of the claims 39 to 58, comprising detecting one or more predetermined sets of light wavelengths using each photodetector in a photodetector array. 60. The method according to clause 59, wherein the photodetectors in the photodetector array are configured to detect light at different times. 61. Each set of light wavelengths includes 50 or fewer different wavelengths, as described in Clause 59. 62. The method according to Clause 59, wherein each set of light wavelengths includes 25 or fewer different wavelengths. 63. The method according to any one of the clauses 39 to 62, comprising spectrally decomposing light detected by a photodetector in a photodetector array. 64. The method according to any one of the clauses 39 to 63, further comprising assigning each photodetector in a photodetector array to detect a predetermined set of light wavelengths. 65. The method according to any one of the clauses 39 to 64, further comprising identifying a particle based on one or more determined parameters of the particle. 66. The method according to any one of the clauses 39 to 65, further comprising sorting particles based on one or more determined parameters of the particles. 67. The particle is a cell, as described in any one of the clauses 39 to 66. 68. A plurality of optical fiber paths, each arranged to collect light from a flowstream at its proximal end and transport the collected light to its distal end, wherein the proximal ends of the plurality of optical fiber paths are spatially separated from each other. A single optical fiber output comprising a combination of the distal ends of multiple optical fiber paths Light collection component equipped with A kit that includes the following: 69. A single optical fiber output comprising a fusion section of the distal ends of multiple optical fiber paths, as described in Clause 68. 70. The kit described in Clause 68, in which multiple optical fiber paths are joined together via a binder. 71. The binder is epoxy, as described in Clause 70 of the kit. 72. A single optical fiber output is a kit as described in Clause 68, comprising a single housing with multiple optical fiber paths combined inside. 73. The housing is a circular housing, as described in Clause 72 of the kit. 74. The housing is a rectangular housing, as described in Clause 72 of the kit. 75. The housing is configured to arrange multiple optical fiber paths in a cascaded manner, as described in Clause 74. 76. The housing is configured to arrange multiple optical fiber paths in a row, as described in Clause 74. 77. A kit according to any one of the clauses 68 to 76, wherein each of the multiple optical fiber paths has an input diameter in the range of 800 μm to 1000 μm. 78. The kit according to any one of clauses 68 to 77, wherein the proximal end of each of the multiple optical fiber paths has an numerical aperture in the range of 0.10 to 0.15. 79. A kit according to any one of clauses 68 to 78, wherein each of the multiple optical fiber paths has an output diameter in the range of 400 μm to 1000 μm. 80. The kit according to any one of clauses 68 to 79, wherein the distal end of each of the multiple optical fiber paths has an numerical aperture in the range of 0.20 to 0.25. 81. A kit according to any one of the multiple optical fiber paths, wherein at least one of the optical fiber paths tapers from the proximal end to the distal end. 82. A kit according to any one of the multiple optical fiber paths, wherein at least one of the optical fiber paths does not taper from the proximal end to the distal end. 83. A kit according to any one of clauses 68 to 82, further comprising a light source having multiple lasers, each configured to illuminate a flowstream at its respective position. 84. The number of lasers in the light source is in the range of 3 to 10, as described in Clause 83. 85. The light source is, A first laser configured to irradiate a flowstream at a first position, Multiple lasers configured to illuminate the flowstream at a position downstream of the first position and A kit as described in Clause 83 or 84, comprising: 86. The kit according to any one of clauses 68 to 85, further comprising a photodetector array having multiple photodetectors configured to detect light from a laser transported through a single optical output of an optical collection component. 87. The kit described in Clause 86, wherein the photodetectors in the photodetector array are configured to detect light at different times. 88. The kit described in Clause 86 or 87, wherein each photodetector in the photodetector array is configured to differentially detect light from one or more lasers. 89. The kit according to clause 86 or 87, wherein each photodetector in the photodetector array is configured to detect one or more predetermined sets of light wavelengths. 90. Each set of light wavelengths includes 50 or fewer different wavelengths, as described in Clause 89. 91. Each set of light wavelengths includes 25 or fewer different wavelengths, as described in the kit in Clause 90. 92. Each photodetector in the photodetector array is a hybrid photodetector comprising a photocathode integrated with an avalanche diode, as described in any one of the kits in Clauses 86 to 91. 93. The hybrid photodetector is the kit described in Clause 92, comprising a GaAs / GaAsP photocathode. 94. The kit according to any one of clauses 86 to 93, wherein each photodetector in the photodetector array is in optical communication with an optical tuning component configured to limit the wavelength of one or more light detected by the photodetector. 95. The optical adjustment component is a bandpass filter, as described in the kit in Clause 94.

[0188] While the aforementioned inventions are described in some detail as examples and illustrations for clarity, 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 the present invention without departing from the spirit or scope of the appended claims.

[0189] Therefore, the above merely illustrates the principles of the present invention. Those skilled in the art will understand that various configurations embodying the principles of the present invention and falling within its spirit and scope can be devised, although not expressly described or illustrated herein. Furthermore, all examples and conditional statements enumerated herein are intended primarily to help the reader understand the principles of the present invention and the concepts to which the inventors have contributed to advancing the art, and should be construed as not being limited to such specifically enumerated examples and conditions. Moreover, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any developed elements that perform the same function regardless of their structure. Furthermore, nothing disclosed herein is intended to be made available to the public, whether such disclosure is expressly described in the claims or not.

[0190] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments illustrated and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims. In the claims, § 112(f) or § 112(6) of the U.S. Patent Act is explicitly defined as being invoked for limitation in the claims only if the exact phrase “means” or the exact phrase “step” is placed at the beginning of such limitation in the claims. If such exact phrase is not used in limitation of the claims, § 112(f) or § 112(6) of the U.S. Patent Act is not invoked.

[0191] Cross-reference of related applications In accordance with Section 119(e) of the United States Patent Act, this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 450,443, filed on 7 March 2023, and the entire disclosure of said application is incorporated herein by reference.

Claims

1. A light source comprising multiple lasers, each configured to illuminate a flowstream at its respective position, Light detection system and Equipped with, The aforementioned light detection system is A plurality of optical fiber paths, each arranged to collect light from the flow stream at one of the respective positions at its proximal end and to transport the collected light to its distal end, wherein the proximal ends of the plurality of optical fiber paths are spatially separated from each other, and A single optical fiber output comprising the combination of the distal ends of the plurality of optical fiber paths. An optical collection component equipped with, A single photodetector array comprising multiple photodetectors configured to detect light from the laser transported through the single optical output of the optical collection component, A particle analyzer equipped with the following features.

2. The particle analyzer according to claim 1, wherein the single optical fiber output comprises a fusion portion of the distal ends of the plurality of optical fiber paths.

3. The particle analyzer according to claim 1, wherein the plurality of optical fiber paths are combined via a binder.

4. The particle analyzer according to claim 1, wherein the single optical fiber output comprises a single housing in which the plurality of optical fiber paths are combined internally.

5. The particle analyzer according to any one of claims 1 to 4, wherein each of the plurality of optical fiber paths has an input diameter in the range of 800 μm to 1000 μm.

6. The particle analyzer according to any one of claims 1 to 5, wherein the proximal end of each of the plurality of optical fiber paths has an numerical aperture in the range of 0.10 to 0.

15.

7. The particle analyzer according to any one of claims 1 to 6, wherein each of the plurality of optical fiber paths has an output diameter in the range of 400 μm to 1000 μm.

8. The particle analyzer according to any one of claims 1 to 7, wherein the distal end of each of the plurality of optical fiber paths has an numerical aperture in the range of 0.20 to 0.

25.

9. The particle analyzer according to any one of claims 1 to 8, wherein the number of lasers in the light source is in the range of 3 to 10.

10. The aforementioned light source is A first laser configured to irradiate a flowstream at a first position, A plurality of lasers configured to irradiate the flow stream at a position downstream of the first position and A particle analyzer according to claim 9, comprising:

11. The particle analyzer according to any one of claims 1 to 10, wherein the lasers in the plurality of lasers are configured to irradiate the flow stream at positions separated from each other by 10 μm or less.

12. A particle analyzer according to any one of claims 1 to 11, comprising a plurality of light collection components.

13. A particle analyzer according to any one of claims 1 to 12, which is incorporated into a flow cytometer.

14. The particle analyzer according to claim 13, wherein the flow cytometer is equipped with a particle sorter.

15. A method for determining one or more parameters of particles in a flowstream, Multiple lasers, each configured to illuminate the flowstream at a different location, are used to irradiate particles in the flowstream, The method involves detecting light from the particles using a photodetection system, wherein the photodetection system is A plurality of optical fiber paths, each arranged to collect light from the flow stream at one of the respective positions at its proximal end and to transport the collected light to its distal end, wherein the proximal ends of the plurality of optical fiber paths are spatially separated from each other, and A single optical fiber output comprising the combination of the distal ends of the plurality of optical fiber paths. An optical collection component equipped with, A single photodetector array comprising multiple photodetectors configured to detect light from the laser transported through the single optical output of the optical collection component, It has the ability to detect, The process involves generating multiplexed data signals from the aforementioned photodetector array, Determining one or more parameters of the particle based on the multiplexed data signal. Methods that include...

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