Integrated optical fluid inspection module, flow sight meter having the same, and method for using the same

Abstract

To provide an optical module configured so that laser beams are aligned in a core stream in a flow cell.SOLUTION: An integrated optical fluid inspection module 100 includes: a flow cell 101 having an optical transmission flow passage for carrying particles in a flow stream 102; a linear fiber array 113 having a plurality of non-round core optical fibers integrated with the flow cell and configured to project excitation wavelength light to a corresponding focal point in the flow cell; and a plurality of optical fiber light propagation couplers 111a, 111b, and 111c for attaching a plurality of light sources to the linear fiber array in an operable manner.SELECTED DRAWING: Figure 1A

Description

[Background technology]

[0001] Flow cytometry is a technique used to characterize and sometimes separate biological material, such as cells in a blood sample or particles of interest in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer carries particles (including cells) in the fluid sample as a cell stream to a flow cell, while further directing the sheath fluid to the flow cell. To characterize the components of the flow stream, the flow stream is illuminated with light from a light source. Changes in the biological material in the flow stream, such as morphology or the presence of fluorescent labels, can change the light observed, and these changes allow for characterization and separation. Summary of the Invention [Problem to be solved by the invention]

[0002] In conventional flow cytometers, the flow cell must be properly aligned with the light source and light collection elements so that light illuminates the flow cell as intended. Assembling a flow cytometer is often a laborious process that requires highly skilled technicians to ensure proper alignment of the light source and light collection elements with the flow cell. Often, this alignment is accomplished by mounting the light source and / or flow cell to an xyz stage to position the flow cytometer components in three dimensions. In addition, the placement of mirrors and other optical components must be fine-tuned to properly direct the light emitted from the light source to the flow cell. These separate components require complex mechanical mountings that can be less robust and / or more costly.

[0003] The inventors have recognized that alignment of the laser beam with the flow core stream is critical to signal quality, and that conventional systems are time-consuming and costly to install and maintain because available alignment techniques require well-trained personnel and complex adjustable mechanical or electromechanical components. Thus, an optical module configured to align a laser beam with the core stream in a flow cell is desirable. The integrated opto-fluidic interrogation module, flow cytometer, methods, and kits described herein fulfill this need. [Means for solving the problem]

[0004] Aspects of the invention include an integrated opto-fluidic investigation module. The integrated opto-fluidic investigation module of interest includes a flow cell having an optically transparent flow path for carrying particles in a flow stream, a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each configured to project excitation wavelength light to a corresponding focal point within the flow cell, and a plurality of optical fiber light transmitting couplers for operably attaching a plurality of light sources to the linear fiber array. In some cases, the non-circular core optical fibers are rectangular core optical fibers, e.g., square optical fibers. In certain cases, the non-circular core optical fibers are configured to project excitation wavelength light having a top-hat profile to the focal points. The non-circular core fibers of the linear fiber array may be stacked vertically in some embodiments. In such embodiments, the cores of adjacent non-circular core fibers of the linear fiber array are separated from each other by a distance of 0.1 mm to 0.3 mm. In some cases, the linear fiber array is attached to an exterior surface of the flow cell. In some variations, the module includes focusing optics integrated with the flow cell and configured to relay the excitation wavelength light from the non-circular core optical fibers to the focal points. The focusing optics may be embedded in the flow cell in certain embodiments. In some variations, the focusing optics include a plurality of microlenses. In certain cases, the module includes a light detector (e.g., a forward scatter detector) that is integrated with the flow cell. In some cases, the module includes an electrical coupler for operably attaching an electrical line to the light detector. In further cases, the module includes an electrical line. In certain cases, the module includes an optical mask disposed between the focal point and the light detector. In some cases, the light detector is attached to an exterior surface of the flow cell. In some embodiments, the module includes a plurality of fiber optic focusing element couplers for operably attaching a plurality of fiber optic focusing elements to the flow cell, where the plurality of fiber optic focusing element couplers may be stacked (e.g., stacked vertically). The module according to some embodiments further includes a sample fluid input coupler for injecting a sample fluid into the flow cell.In some cases, the module includes a sheath fluid input coupler for injecting a sheath fluid into the flow cell and / or a fluid output coupler for receiving a flow stream from the flow cell. The flow cell may include, for example, a cuvette. In some variations, the module of the invention includes a collimating lens (e.g., a Fresnel lens) attached to an outer surface of the cuvette. In some embodiments, the collimating lens is an injection compression molded microlens. In some cases, the integrated optofluidic interrogation module includes N non-circular core optical fibers, N focal points, and N optical fiber light transmitting couplers, where N is an integer in the range of 2 to 10. In certain variations, the width of the flow cell is in the range of 75 mm to 125 mm.

[0005] Aspects of the invention further include flow cytometers. The subject flow cytometers include a plurality of light sources and an integrated opto-fluidic interrogation module of the invention (e.g., as described above). Aspects of the invention further include methods of analyzing samples. The methods of interest include introducing a particle sample into a flow cytometer of the invention and analyzing the particle sample by flow cytometry. Kits are also provided that include an integrated opto-fluidic interrogation module and components associated with the integrated opto-fluidic interrogation module.

[0006] Aspects of the invention further include methods of assembling a flow cytometer. Methods of interest include placing an integrated opto-fluidic interrogation module of the invention into a flow cytometer, operably attaching a plurality of light sources to the fiber optic light transfer coupler, and assembling the flow cytometer. Optionally, the method further includes operably attaching electrical lines to the electrical coupler of the module. In embodiments, the method includes operably attaching a plurality of fiber optic light collection elements to the plurality of fiber optic light collection element couplers of the module. Optionally, the method includes fluidly connecting a sample fluid line of the flow cytometer to the sample fluid input coupler of the module. In one variation, the method includes fluidly connecting a sheath fluid line of the flow cytometer to the sheath fluid input coupler of the module. Optionally, the method includes fluidly connecting a waste line of the flow cytometer to the fluid output coupler of the module. [Brief description of the drawings]

[0007] The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, in which:

[0008] [Figure 1A] FIG. 1 illustrates an integrated opto-fluidic investigation module according to an embodiment of the present invention. [Figure 1B] FIG. 1 illustrates an integrated opto-fluidic investigation module according to an embodiment of the present invention. [Figure 1C] FIG. 1 illustrates an integrated opto-fluidic investigation module according to an embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram illustrating a flow cytometer with an integrated opto-fluidic investigation module according to an embodiment of the present invention. [Diagram 3] FIG. 1 illustrates a control system according to an embodiment. [Figure 4A] FIG. 1 is a schematic diagram illustrating a particle sorter system according to an embodiment. [Figure 4B] FIG. 1 is a schematic diagram illustrating a particle sorter system according to an embodiment. [Diagram 5]FIG. 1 is a block diagram illustrating a computing system according to an embodiment. [Figure 6] 1 is a plot showing the illumination profile obtained from a linear fiber array of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] An integrated opto-fluidic interrogation module is provided that includes a flow cell having an optically transparent flow path for carrying particles in a flow stream, a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each configured to project excitation wavelength light at a corresponding focal point within the flow cell, and a plurality of fiber optic light transmissive couplers for operably attaching a plurality of light sources to the linear fiber array. Also provided are flow cytometers including the subject integrated opto-fluidic interrogation modules, and methods of use thereof.

[0010] Before the present invention is described in more detail, it should be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. The scope of the present invention will be limited only by the appended claims, and it should be further understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0011] When a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are further encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0012] In this specification, a range is provided with the term "about" before the numerical values. The term "about" is used herein to literally support the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately may be a number that, in the context in which the specifically stated number is presented, provides a substantial equivalent to the specifically stated number.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative and illustrative methods and materials are described.

[0014] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0015] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prelude to the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in relation to the recitation of claim elements.

[0016] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein comprises separate components and features which may be readily separated from, or combined with, any of the features of the other multiple embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0017] Although the systems and methods have been described or will be described for grammatical fluidity with functional descriptions, it should be clearly understood that the claims, unless expressly recited under 35 U.S.C. ...

[0018] Integrated optofluidic investigation module As mentioned above, aspects of the invention include an integrated opto-fluidic investigation module. The integrated opto-fluidic investigation module of interest comprises a flow cell having an optically transparent flow path for carrying particles in a flow stream, a linear fiber array having a plurality of non-circular core optical fibers each configured to project excitation wavelength light to a corresponding focal point in the flow cell, and a plurality of optical fiber light transmitting couplers for operably attaching a plurality of light sources to the linear fiber array. By "integrated" opto-fluidic investigation module, it is meant that at least the flow cell, the linear fiber array, and possibly the optical fiber light transmitting coupler are integrated together to form a combined integral structure, rather than existing as separate and distinct elements. The flow cell and the linear fiber array may be integrated to be mounted at a fixed position, the fixed position being selected to optically align the core stream of the flow cell with collection optics associated with the core stream. Thus, in some cases, the components of the integrated opto-fluidic investigation module (e.g., flow cell, linear fiber array, etc.) are in fixed positions relative to each other, e.g., to maintain proper optical alignment over time. In some cases, the components of the integrated opto-fluidic investigation module (e.g., flow cell, linear fiber array, etc.) cannot be easily separated without damaging one or more of the components. Thus, in some cases, the integrated opto-fluidic investigation module is adjustment-free, for example, because the components are already present in an optimal alignment. In some cases, the subject integrated opto-fluidic investigation modules reduce the need for manual adjustments to optically align, for example, because each component is in a fixed position relative to one another, thereby maintaining proper optical alignment. In some cases, the integrated optics of the integrated opto-fluidic investigation modules described herein may increase the efficiency of collecting fluorescent light emitted by particles in the flow cell, for example, by increasing the amount of fluorescent light collected relative to noise.

[0019] As described herein, a "flow cell" is described in the conventional sense to refer to a component, such as a cuvette, having a flow path for a liquid flow stream to carry particles within a sheath fluid. A cuvette of interest has a container with a passageway therethrough. The flow stream through which the flow path is configured may include a liquid sample injected from a sample tube. In some cases, the flow cell has a light-transmitting flow path. In such cases, the flow cell includes a transparent material (e.g., quartz) that allows the passage of light. In some cases, the flow cell is configured to be illuminated with light from a light source at one or more interrogation points. An "interrogation point" as described herein refers to an area within the flow cell where particles are illuminated by light from a light source, for example, for analysis. The size of the interrogation point may vary as desired. For example, if 0 μm represents the axis of the light emitted by the light source, the interrogation point may be within a range of -50 μm to 50 μm, such as -25 μm to 40 μm, such as -15 μm to 30 μm.

[0020] During use of the integrated optofluidic investigation module of the present invention, particles may be carried through the core stream of the flow stream. As used herein, "core stream" is referred to in the conventional sense to refer to a portion of the flow of sheath fluid (i.e., flow stream) in which particles are carried through the flow cell. Generally, particles are carried through the core stream in a single file arrangement. The size (e.g., diameter) of the core stream may vary as desired. In some cases, the diameter of the core stream may be approximately the same as the diameter of the particles being analyzed. Thus, in some cases, the diameter of the core stream is in the range of 5 μm to 25 μm, such as in the range of 10 μm to 20 μm. The diameter of the core stream may be adjusted in proportion to the pressure applied to the particles as they are injected into the sheath fluid flow. The flow rate of the sheath fluid remains constant. In this way, the particles are injected into the sheath fluid and are hydrodynamically focused, creating a laminar flow in which the particles travel at approximately the same speed along the same axis. Because the particles in the core stream travel along the same axis, the geometry of the core stream is defined by straight edges.

[0021] The linear fiber array of the integrated optofluidic investigation module comprises a plurality of non-circular core optical fibers. A linear fiber array as described herein is a fiber array formed at the end of a fiber bundle, with the constituent fibers arranged in a linear fashion. The optical fiber may be comprised of a cladding and a core. The core is the portion of the optical fiber made of a transparent material configured to transmit light, while the cladding confines the light primarily to the core. In other words, the core has a higher refractive index compared to the cladding. The cladding and core may be comprised of the same material or different materials. In some cases, the cladding and core are made of a silica-based glass (e.g., SiO 2 ). In other embodiments, the transparent material comprises a polymeric material. In such embodiments, the transparent material may include one or more materials such as, but not limited to, poly(methyl methacrylate) (PMMA), polystyrene, and poly(perfluorobutenyl vinyl ether) (CYTOP). In some cases, the optical fiber core has opposing edges that run parallel to the direction of the core stream, i.e., edges that run parallel to each other and are on opposite sides of the collection core of the optical fiber. In other words, the projection surface of the optical fiber core (i.e., the area of ​​the core from which light is emitted, see FIG. 1B) is defined by straight edges. By "extending parallel" to the core stream, it is meant that the opposing straight edges of the projection surface of the optical fiber core are positioned to run in a direction parallel to the axis along which particles are transported in the core stream. The opposing parallel edges may have any suitable length. In some cases, the length is in the range of 5 μm to 100 μm, such as 10 μm to 75 μm, such as 15 μm to 50 μm, such as 15 μm to 25 μm.

[0022] In addition to having opposing edges extending parallel to the direction of the core stream, the optical fiber core of the linear fiber array may have a projection surface having opposing edges extending perpendicular to the direction of the core stream. By "extending perpendicular" to the core stream, it is meant that the opposing linear edges of the collection surface of the optical fiber collector extend perpendicular to the axis along which the particles are carried in the core stream. The opposing perpendicular edges may have any suitable length. In some cases, the length is in the range of 5 μm to 150 μm, such as 50 μm to 100 μm, such as 60 μm to 90 μm, such as 70 μm to 80 μm. The core may have a number of suitable non-circular shapes. For example, in some cases, the non-circular core optical fiber is a rectangular core optical fiber. In such cases, the non-circular core optical fiber is a square core optical fiber.

[0023] In some cases, the non-circular core optical fiber is configured to project the excitation wavelength light having a top-hat profile to the focal point. In other words, in addition to directing the light without complex alignment procedures, the non-circular core optical fiber further shapes the beam such that the beam is emitted from the optical fiber with a flat-top beam profile. The term "top-hat" (sometimes also referred to as "flat-top") is used herein in the conventional sense to refer to an illumination beam (e.g., light) having a substantially uniform fluence (energy density) along one or more axes perpendicular to the optical axis of the illumination beam. In an embodiment, an output beam of light having a top-hat intensity profile has little or no deviation in relative intensity from each edge to the center along a horizontal axis, and a beam of light having a top-hat intensity profile of interest has an intensity at its center within a range of 95%-99.9% of the intensity at its edges along the horizontal axis, such as within a range of 96%-99.5%, e.g. 98%-99%, of the intensity at its edges along the horizontal axis. A non-circular core fiber as described herein can generate a flat-top profile by acting as a mode scrambler. In other words, the rectangular core geometry can stimulate coupled optical modes, which can result in a homogenized intensity distribution. The properties of non-circular core fibers are described, for example, in Hracek, B., & Bauerle, H. (2015). Optik & Photonik, 10(5), 16-18.

[0024] The non-circular core optical fibers are each configured to project excitation wavelength light to a corresponding focal point within the flow cell. A "focal point" refers to a particular area of ​​the interrogation zone of the flow cell that is illuminated by one or more lasers. In other words, the module is configured to focus the excitation wavelength light to a particular portion of the flow cell while preventing the light from irradiating other undesired locations of the flow cell. The distance between adjacent focal points is determined at least in part by the distance between the cores of adjacent optical fibers of the linear fiber array. In some embodiments, the non-circular core optical fibers are assembled in a linear array configuration such that the size and spacing of the optical fiber cores correspond to the desired dimensions to be emitted into the flow cell to excite particles in the core stream of the flow cell. The non-circular core optical fibers may be arranged in any suitable configuration. In some cases, the non-circular core fibers of the linear fiber array are vertically stacked. The distance between adjacent non-circular core fibers may vary as desired. In some cases, the fibers are vertically stacked and spaced apart by the thickness of the fiber cladding. In other cases, e.g. when it is desired to separate the foci by a larger distance, the claddings of adjacent optical fibers of the linear fiber array are separated from each other by a distance in the range of 0.01 mm to 1 mm, e.g. 0.01 mm to 0.5 mm, e.g. 0.01 mm to 0.25 mm, e.g. 0.01 mm to 0.1 mm, e.g. 0.01 mm to 0.05 mm, e.g. 0.01 mm to 0.025 mm. The distance between the cores of adjacent non-circular core fibers of the linear fiber array may also vary. In some cases, the cores of adjacent non-circular core fibers of the linear fiber array are separated from each other by a distance in the range of 0.01 mm to 1 mm, e.g. 0.05 mm to 0.5 mm, e.g. 0.1 mm to 0.25 mm, e.g. 0.1 mm to 0.15 mm (e.g. measured from the geometric center of the light projection surface of one core to the light projection surface of the adjacent core). In some cases, the cores of adjacent non-circular core fibers of the linear fiber array are separated from each other by a distance of 0.125 mm.

[0025] The particular placement of the optical fibers in the linear fiber array may be achieved in any convenient manner. In some cases, the linear fiber array has the optical fibers placed in V-shaped grooves in a surface sized and shaped to maintain a constant or no distance between the optical fibers. In other cases, the linear fiber array may use holes in the surface to position each fiber. When the fibers are placed through the appropriate holes, they will be properly aligned with the core stream. In some cases, the surface is a glass surface. In other cases, the surface is a polymer (e.g., plastic) surface. In still other cases, the surface is a metal surface.

[0026] The linear fiber array may be integrated with the flow cell in any convenient manner. In some cases, the linear fiber array is attached to the exterior surface of the flow cell. For example, in some embodiments, the linear fiber array is attached by adhesive. Any convenient optical adhesive may be used. For example, adhesives of interest include epoxies, photocurable acrylics, elastomers (e.g., silicones, silicone-free silanes), cyanoacrylates, and structural adhesives (e.g., adhesives including resins and activators). In some embodiments, the adhesive is curable by exposure to light (e.g., UV light). In some embodiments, the optical adhesive is transparent. In other cases, the components of the linear fiber array are embedded in the walls of the flow cell. In such cases, the focusing optics assembly may be integrated into the structure of the flow cell rather than being attached to the surface of the flow cell. In some cases, the linear fiber array is integrated with the flow cell by techniques such as press-fitting (i.e., interference fit). Press-fitting is a known technique for forming holes in a housing that are slightly smaller than the shape of the components (e.g., lenses) of the focusing optics assembly. The focusing optics assembly components and the housing may then be forcefully coupled together and then held together by friction.

[0027] As described above, the integrated optofluidic investigation module of the present invention includes a plurality of fiber optic optical couplers for operably attaching a plurality of light sources to a linear fiber array. The light emitting ends of the optical fibers are co-located in the linear fiber array, while the light receiving ends of the optical fibers are spatially separated and include a fiber optic optical coupler. Any suitable coupling mechanism configured to operably attach the light sources to the linear fiber array may be used with these fiber optic optical couplers. Couplings of interest include, but are not limited to, threaded couplings, latch couplings, push-pull couplings, bayonet couplings, male-female couplings, snap-fit ​​couplings, combinations thereof, and the like.

[0028] In some cases, the integrated optofluidic investigation module further comprises a plurality of fiber optic light transmitters, e.g., operably attached to a fiber optic light transmitting coupler associated with the linear fiber array. As described herein, a "fiber optic light transmitter" refers to an elongated structure having a proximal end and a distal end, the elongated structure being made of an optically transparent material configured to transmit light from a light source at the proximal end to a focusing optical assembly at the distal end. In some embodiments, the optically transparent material includes, but is not limited to, a glass material, such as silica (e.g., fused silica). In other embodiments, the optically transparent material includes a polymeric material. In such embodiments, the optically transparent material may include, but is not limited to, one or more materials, such as poly(methyl methacrylate) (PMMA), polystyrene, and poly(perfluorobutenyl vinyl ether) (CYTOP). In some embodiments, the fiber optic light collector is a single mode fiber. As understood in the art, a single mode fiber is an optical fiber that transmits one mode of light at a given time. In other cases, the fiber optic light collector is a multimode fiber. In contrast to single mode fibers, multimode fibers simultaneously propagate multiple modes of light. The diameter of the fiber optic light transmitter may vary as desired. In some embodiments, the diameter of the fiber optic light transmitter of interest is in the range of 0.1 mm to 3 mm, e.g., 0.2 mm to 2.5 mm, 0.3 mm to 2.2 mm, 0.4 mm to 2 mm, 0.5 mm to 1.5 mm, e.g., 0.6 mm to 1.2 mm. If a fiber optic light transmitting connector is provided, the fiber optic light transmitting connector itself may be connected to the laser in some embodiments.

[0029] In some cases, the integrated optofluidic investigation module of the present invention further comprises focusing optics integrated with the flow cell and configured to relay excitation wavelength light from the non-circular core optical fiber to a focal point. The focusing optics may, for example, comprise one or more lenses. In such cases, the focusing optics may comprise any convenient number of lenses, for example 1-10 lenses, 2-5 lenses, for example 2-4 lenses. In some embodiments, the lens or lenses in the focusing optics assembly are microlenses. In such embodiments, the microlenses of interest may have any convenient size and shape. In some cases, the focusing optics assembly comprises microlenses having diameters in the range of 10 μm to 5 mm, for example 50 μm to 2 mm, for example 100 μm to 1 mm. In some cases, the microlenses of interest are spherical. In some embodiments, the microlenses have a flat surface on one side and a spherical (for example convex) surface on the opposite side. In further embodiments, the microlenses are aspheric lenses, i.e. lenses that are not spherical or cylindrical in size. In embodiments of the focusing optics having multiple microlenses, each microlens of the focusing optics may be the same or different. For example, the microlenses may have various sizes and / or shapes as desired to focus the excitation wavelength light to a focal point in the flow cell. In some embodiments, the lenses are arranged in an array. The lenses of interest of the focusing optics may be made of glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glasses), silica (e.g., fused silica), quartz, crystal (e.g., CaF 2 Crystal), zinc selenide (ZnSe), F 2 The lenses may be formed from any suitable material, including, but not limited to, germanium titanate (Ge) (e.g., S-TIH11), borosilicate (e.g., BK7), or combinations thereof. The refractive index of the lenses of interest may vary within the range of 1 to 3, such as 1.1 to 2.9, such as 1.2 to 2.8, such as 1.3 to 2.7, such as 1.4 to 2.6, such as 1.5 to 2.7, such as 1.6 to 2.6, such as 1.7 to 2.5, such as 1.8 to 2.4, such as 1.9 to 2.3.

[0030] In some embodiments, a component of the focusing optics (e.g., a lens) may modify the beam profile of the light beam across one or more of the horizontal and vertical axes. As described herein, the horizontal and vertical axes refer to axes that are orthogonal to the optical axis (i.e., the beam path) of the light beam and, in embodiments, form the XY plane of the beam profile. The horizontal axis of the output beam of light is orthogonal to the longitudinal axis of the flow stream (e.g., the flow stream in a flow cytometer), and the vertical axis of the output beam of light is parallel to the longitudinal axis of the flow stream. In some embodiments, the focusing optics of interest modify the beam profile of the propagating light beam across the horizontal axis. In other embodiments, the lens of interest modify the beam profile of the propagating light beam across the vertical axis. In yet other embodiments, the lens of interest modify the beam profile of the propagating light beam across the horizontal and vertical axes. The focusing optics in these embodiments may include diffractive or refractive optics. In some embodiments, the focusing optics include a biconic lens. In other embodiments, the focusing optics include an achromatic lens.

[0031] The focusing optics may be integrated with the flow cell by any one of a variety of different means as desired. For example, the focusing optics may be stably associated with the outer surface of the flow cell. In some embodiments, the focusing optics may be attached by adhesive as described above. In some embodiments, the components of the focusing optics (e.g., one or more lenses) may be integrated (embedded) into the structure of the flow cell. In some cases, the components of the focusing optics are embedded in the walls of the flow cell. In such cases, the focusing optics may be integrated into the structure of the flow cell rather than being attached to the surface of the flow cell. In some cases, the lenses of the optical assembly may be integrated into the structure of the housing by a press fit (i.e., an interference fit). In further embodiments, the focusing optics is attached to the outer surface of the flow cell by optical contact bonding. As is known in the art, optical contact bonding involves joining two surfaces by utilizing intermolecular forces that exist between the two surfaces. Intermolecular forces associated with optical contact bonding include van der Waals forces, hydrogen bonding, and dipole-dipole interactions. In embodiments of the invention involving optical contact bonding, the surfaces of the focusing optics and the surfaces of the flow cell may be substantially conformal (i.e., flat and smooth) so that intermolecular forces may draw these elements together.

[0032] In some embodiments, the integrated opto-fluidic investigation module of the present invention comprises one or more photodetectors integrated with the flow cell. The number of photodetectors integrated with the flow cell may vary as desired. In some cases, the integrated opto-fluidic investigation module comprises one photodetector integrated with the flow cell. In other cases, the integrated opto-fluidic investigation module comprises multiple photodetectors integrated with the flow cell, such as 2 or more photodetectors, such as 3 or more photodetectors, such as 4 or more photodetectors, such as 5 or more photodetectors, such as 6 or more photodetectors, such as 7 or more photodetectors, such as 8 or more photodetectors, such as 9 or more photodetectors, such as 10 or more photodetectors. The one or more photodetectors may be integrated with the flow cell by any suitable technique (e.g., adhesive, press fit, etc.) as described above. In certain cases, the photodetector is attached to the outer surface of the flow cell.

[0033] The light detector may be any convenient particle-modulated light detector. "Particle-modulated light" refers to light received from a particle in a flow stream after the particle is illuminated with light from a light source. In some cases, the particle-modulated light is side-scattered light. As described herein, side-scattered light refers to light that is refracted and reflected off the surface and internal structures of a particle. In further embodiments, the particle-modulated light includes forward-scattered light (i.e., light that travels primarily through or around the particle in a forward direction). In still other cases, the particle-modulated light includes fluorescent light (i.e., light emitted from a fluorescent dye after illumination with excitation wavelength light).

[0034] In some embodiments, the one or more particle modulation light detectors include one or more forward scattered light detectors configured to detect forward scattered light. For example, a module of interest may include one forward scattered light detector or multiple forward scattered light detectors, such as two or more, such as three or more, such as four or more, such as five or more forward scattered light detectors. In some embodiments, the module includes one forward scattered light detector. In other embodiments, the module includes two forward scattered light detectors.

[0035] Any convenient detector for detecting collected light may be used with the forward scattered light detector described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other detectors. In some embodiments, the collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10cm 2 , e.g. 0.05 cm 2 ~9 cm 2 , e.g. 0.1 cm 2 ~8 cm 2 , e.g. 0.5 cm 2 ~7 cm 2 , e.g. 1 cm 2 ~5 cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .

[0036] In embodiments, the forward scattered light detector is configured to measure light continuously or at discrete intervals. In some cases, the detector of interest is configured to measure the collected light continuously. In other cases, the detector of interest is configured to measure at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, such as every 1000 milliseconds, or other intervals.

[0037] In some embodiments, the module comprises one or more wavelength separators disposed between the flow cell and the one or more particle modulation light detectors. The term "wavelength separator" is used herein in its conventional sense to refer to an optical component configured to separate light collected from a sample into predetermined spectral regions. In some embodiments, the module comprises one wavelength separator. In other embodiments, the module comprises multiple wavelength separators, e.g., 2 or more wavelength separators, e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 6 or more, e.g., 7 or more, e.g., 8 or more, e.g., 9 or more, e.g., 10 or more, e.g., 15 or more, e.g., 25 or more, e.g., 50 or more, e.g., 75 or more, e.g., 100 or more wavelength separators. In some embodiments, the wavelength separator is configured to separate light collected from a sample into predetermined spectral regions by passing light having a predetermined spectral region and reflecting light in one or more remaining spectral regions. In other embodiments, the wavelength separator is configured to separate light collected from a sample into predetermined spectral regions by passing light having a predetermined spectral region and absorbing light in one or more remaining spectral regions. In yet other embodiments, the wavelength separator is configured to spatially diffract light collected from the sample into a predetermined spectral region. Each wavelength separator may be any convenient light separation protocol, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In certain embodiments, the wavelength separator of the subject light detection system is a dichroic mirror.

[0038] In some cases, the one or more photodetectors incorporated in the flow cell include an electrical coupler for operably attaching electrical wires to the one or more photodetectors. Any element configured to couple a photodetector (such as the photodetectors described above) to electrical wires for transmitting a signal generated by the photodetector may be used, such as a thread, a bayonet, a fastener, a blind-mate connection, or the like. In some cases, the module further includes electrical wires. The electrical wires may be constructed of any suitable insulated conductive material (e.g., copper).

[0039] In some cases, the integrated optofluidic investigation module comprises one or more optical conditioning elements configured to modulate the light received by the one or more photodetectors. For example, in some cases, the module comprises an optical filter disposed between the focal point and the photodetector. The optical filter is configured to restrict, reduce or limit the propagation of at least one or more wavelengths of light from the sample (e.g., one or more of the wavelengths of light of a laser used to illuminate the sample) to the active surface of the photodetector. The optical filter may limit the propagation of one or more different wavelengths of light, for example, 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, such as 100 or more, such as 200 or more, such as 300 or more, such as 500 or more different wavelengths of light. For example, in some embodiments, the optical filter is a bandpass filter. In other embodiments, the optical filter is a dichroic mirror. Additionally or alternatively, the module of the present invention may comprise an optical mask disposed between the focal point and the photodetector. In some cases, the optical mask is configured to prevent a certain portion of light (e.g., an incident beam from a laser) from being detected.

[0040] In addition to or in lieu of a photodetector integrated with the flow cell, the integrated optical fluidic investigation module may comprise one or more fiber optic light collection element couplers for operably attaching one or more fiber optic light collection elements to the flow cell. In such embodiments, the fiber optic light collection element is configured to collect particle modulated light emitted by particles conveyed through the one or more focal points after such particles are illuminated with excitation wavelength light. The fiber optic light collection element coupler may operably attach the one or more fiber optic light collection elements to the flow cell by any convenient mechanism. In some embodiments, the fiber optic light collection element coupler may operably attach the fiber optic light collection element to the flow cell such that the fiber optic light collection element requires little, if any, optical adjustment to ensure proper optical alignment with the flow cell. In some embodiments, the fiber optic light collection element coupler may physically couple the fiber optic light collection element to the flow cell, for example, with an adhesive. In other embodiments, the fiber optic light collection element coupler is configured to releasably attach the fiber optic light collection element. Suitable coupling mechanisms include, for example, screws, latches, push-pull, bayonet, snap, male-female, etc.

[0041] The number of fiber optic focusing couplers may vary. In some cases, the integrated optofluidic investigation module comprises one fiber optic focusing coupler. In other cases, the integrated optofluidic investigation module comprises a plurality of fiber optic focusing couplers, e.g., two or more fiber optic focusing couplers, e.g., three or more fiber optic focusing couplers, e.g., four or more fiber optic focusing couplers, e.g., five or more fiber optic focusing couplers, e.g., six or more fiber optic focusing couplers, e.g., seven or more fiber optic focusing couplers, e.g., eight or more fiber optic focusing couplers, e.g., nine or more fiber optic focusing couplers, e.g., ten or more fiber optic focusing couplers. When the module comprises a plurality of fiber optic focusing couplers, the plurality of fiber optic focusing couplers may be arranged in any suitable manner relative to each other. In some embodiments, the fiber optic focusing elements are stacked vertically. In such embodiments, each fiber optic focusing element may be configured to collect light from a particular focal point.

[0042] In embodiments of the method including both a fiber array and one or more fiber optic focusing couplers, the linear fiber array and the one or more fiber optic focusing couplers may occupy any convenient location relative to one another relative to the flow cell. In some embodiments, the fiber optic focusing coupler is attached to the flow cell on the opposite side of the module from the linear fiber array. In other embodiments, the fiber optic focusing coupler is attached to the flow cell at an orthogonal or near orthogonal location relative to the linear fiber array. The one or more fiber optic focusing couplers may be positioned within the module such that the fiber optic focusing elements connected to the fiber optic focusing coupler are in a suitable position to collect one or more types of particle modulated light (e.g., forward scattered light, side scattered light, fluorescent light, or a combination thereof).

[0043] In some embodiments, the module further comprises one or more fiber optic focusing elements operably attached to the plurality of fiber optic focusing elements. In certain embodiments, the fiber optic focusing elements are configured to collect fluorescence emitted by particles conveyed through the focal point after such particles are illuminated with the excitation wavelength light. In some embodiments, the fiber optic focusing elements comprise a transparent material, such as a glass material, including but not limited to silica (e.g., fused silica). In other embodiments, the transparent material comprises a polymeric material. In such embodiments, the transparent material may comprise one or more materials, such as, but not limited to, poly(methyl methacrylate) (PMMA), polystyrene, and poly(perfluorobutenyl vinyl ether) (CYTOP). The diameter of the fiber optic light transmitter may vary as desired. In some embodiments, the diameter of the fiber optic light transmitter of interest is in the range of 0.1 mm to 3 mm, e.g., 0.2 mm to 2.5 mm, 0.3 mm to 2.2 mm, 0.4 mm to 2 mm, 0.5 mm to 1.5 mm, e.g., 0.6 mm to 1.2 mm. In some embodiments, the subject modules include one fiber optic focusing element. In other embodiments, the modules include multiple fiber optic focusing elements. For example, a flow cytometer of interest may have a fiber optic focusing element per focal point and / or per fiber optic focusing element coupler. Thus, a flow cytometer may have one or more fiber optic focusing elements, e.g., 2 or more, 3 or more, 4 or more, 5 or more, e.g., 6 or more fiber optic focusing elements. In some embodiments, the one or more fiber optic focusing elements have a focusing surface that is optically aligned with the core stream and has opposing edges that run parallel to the direction of the core stream. Exemplary fiber optic focusing elements are described in U.S. Patent Application Publication No. 2022 / 0236165, the disclosure of which is incorporated herein by reference in its entirety.

[0044] In embodiments, the integrated opto-fluidic interrogation module includes collimating optics disposed between the flow stream and the photodetector and / or fiber optic focusing coupler. For example, in some cases, the module includes a collimating lens. In some embodiments where the flow cell includes a cuvette, the collimating lens is attached to an outer surface of the cuvette. The collimating lens may be attached to the outer surface of the cuvette by any suitable means, such as an optical adhesive. In certain cases, the collimating lens is an injection compression molded microlens. In some cases, the collimating lens is a Fresnel lens.

[0045] As mentioned above, the number of non-circular core optical fibers, focal points and optical fiber light transmitting couplers may vary within embodiments of the invention. In some cases, the modules of the invention comprise the same number of non-circular core optical fibers, focal points and optical fiber light transmitting couplers. In other words, the integrated optofluidic investigation module comprises N non-circular core optical fibers, N focal points and N optical fiber light transmitting couplers, where N is any suitable integer. In some cases, N is in the range of 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 10, such as 2 to 5, such as 2 to 4. For example, N may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 in some cases. If these elements are provided, the module may further comprise N optical fiber focusing elements and / or N optical fiber light transmitters.

[0046] Any convenient flow cell that delivers a fluid sample to a sample interrogation region may be used in the subject modules, hi some embodiments, the flow cell is a cylindrical flow cell, a frusto-conical flow cell, or a flow cell having a proximal cylindrical portion defining a longitudinal axis and a distal frusto-conical portion terminating in a flat surface having an orifice perpendicular to the longitudinal axis.

[0047] In some cases, the module includes a fluid output coupler for receiving the flow stream from the flow cell. The fluid output coupler may be any component configured to collect and convey the flow stream from the flow cell. In some cases, the fluid output coupler has a connector at a distal end configured to allow a waste line (e.g., tubing) to be connected. Any suitable connector may be included. Exemplary connectors include, for example, quick disconnect connectors, threaded connectors, luer connectors, multi-port connectors, tri-clamp fittings, and puncture seal sterile fittings. Suitable quick disconnect connectors include, but are not limited to, snap-on (ball latch) connectors, bayonet connectors, threaded connectors, non-latching connectors, single shut-off connectors, double shut-off connectors, non-shut-off connectors, dry break connectors, roller lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors. The waste line may convey liquid to another location, such as a waste reservoir. In some embodiments where the module is configured for use with vacuum-driven fluids, the fluid output coupler is configured with an outlet fitting. An outlet fitting of interest has an elongated structure and an opening at a proximal end of the elongated structure for receiving a flow stream from a distal end of the flow cell. In some cases, the outlet fitting is configured to reduce bubble formation at an interface between the outlet fitting and the flow cell (e.g., a cuvette of the flow cell). Suitable outlet fittings are described in U.S. Patent Application Publication No. 2023 / 0046207, the disclosure of which is incorporated herein by reference.

[0048] In an embodiment, the module further comprises a sample fluid input coupler for injecting a sample fluid into the flow cell. In some cases, for example, the sample fluid input coupler may have an orifice that interfaces with the orifice of the flow cell so that the sample fluid can be delivered to the orifice of the flow cell. Additionally, in some embodiments, the sample fluid input coupler has a connector configured to fluidly couple the sample fluid input coupler to a fluid line (e.g., tubing) that is connected to a sample fluid source. Any suitable connector may be included. Exemplary connectors include, for example, quick disconnect connectors, threaded connectors, luer connectors, multi-port connectors, tri-clamp fittings, and puncture seal sterile fittings. Suitable quick disconnect connectors include, but are not limited to, snap-on (ball latch) connectors, bayonet connectors, threaded connectors, non-latching connectors, single shut-off connectors, double shut-off connectors, non-shut-off connectors, dry break connectors, roller lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors. In some cases, the sample fluid input coupler is or includes a sample injection needle. In such cases, the sample injection needle has a passage therethrough for delivering sample fluid from a sample injection line at a proximal end to a flow cell at a distal end to generate a core stream. In some cases, the sample injection needle is constructed and arranged to maintain an intact core stream under constant or varying flow conditions. Sample injection needles that may be adapted for use are described in U.S. Patent Application Serial No. 18 / 215,402, the entire disclosure of which is incorporated herein by reference.

[0049] In an embodiment, the module further comprises a sheath fluid input coupler for injecting sheath fluid into the flow cell. For example, the sheath fluid input coupler may have an orifice that interfaces with the orifice of the flow cell so that sheath fluid can be delivered to the orifice of the flow cell. In addition, the sheath fluid input coupler has a connector configured to fluidly couple the sheath fluid input coupler to a fluid line (e.g., tubing) that is connected to a sheath fluid source. Any suitable connector may be included. Exemplary connectors include, for example, quick disconnect connectors, threaded connectors, luer connectors, multi-port connectors, tri-clamp fittings, and puncture seal sterile fittings. Suitable quick disconnect connectors include, but are not limited to, snap-on (ball latch) connectors, bayonet connectors, threaded connectors, non-latching connectors, single shut-off connectors, double shut-off connectors, non-shut-off connectors, dry break connectors, roller lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors.

[0050] 1A-1C show an integrated opto-fluidic investigation module according to an embodiment of the present invention. As shown in FIG. 1A, the integrated opto-fluidic investigation module 100 comprises a flow cell 101 configured to carry particles within a flow stream 102 (i.e., a core stream of the flow stream 102). The integrated opto-fluidic investigation module 100 comprises a plurality of fiber optic light transmitting couplers 111a, 111b, 111c. Each fiber optic light transmitting coupler may be configured to be operatively attached to a fiber optic light transmitter, which may itself be coupled to a laser. In an alternative embodiment, the fiber optic light transmitting couplers 111a-111c may be directly coupled to a laser (i.e., without an intermediate optical fiber). Also shown is a linear fiber array 113 integrated with the flow cell 101, the linear fiber array having a plurality of non-circular core optical fibers, each configured to project excitation wavelength light to a corresponding focal point within the flow stream 102 of the flow cell 101. The light propagates through optical fibers 112a, 112b, 112c via optical fiber light transmitting couplers 111a, 111b, 111c to a linear fiber array 113. In the embodiment of Figure 1A, the module 100 further comprises focusing optics, shown in the form of a lens 114 integrated with the flow cell 101, configured to relay excitation wavelength light from the non-circular core optical fiber to a focal point. Alternatively, the focusing optics may comprise multiple micro lenses.

[0051] 1B is a diagram showing the light emitting surface of the linear fiber array 113. As shown in FIG. 1B, the linear fiber array 113 has a vertical stack 115 of a non-circular core optical fiber 116a, a non-circular core optical fiber 116b, and a non-circular core optical fiber 116c. The non-circular core optical fiber 116a is continuous with the optical fiber 112a and the optical fiber coupler 111a such that the laser light received at the optical fiber coupler 111a propagates along the optical fiber 112a and is emitted at the non-circular core optical fiber 116a. The non-circular core optical fiber 116b is continuous with the optical fiber 112b and the optical fiber coupler 111b such that the laser light received at the optical fiber coupler 111b propagates along the optical fiber 112b and is emitted at the non-circular core optical fiber 116b. The non-circular core optical fiber 116c is continuous with the optical fiber 112c and the optical fiber optical transmission coupler 111c such that the laser light received at the optical fiber optical transmission coupler 111c propagates along the optical fiber 112c and is emitted at the non-circular core optical fiber 116c. The non-circular core optical fiber 116a, the non-circular core optical fiber 116b and the non-circular core optical fiber 116c have a core 117a, a core 117b and a core 117c, respectively. In the embodiment of Figure 1B, the cores 117a, 117b, 117c are rectangular and configured to project excitation wavelength light having a top hat profile to a focal point. The cores of adjacent non-circular core optical fibers are separated by a distance d, which in this case is determined by the cladding thickness of each of the non-circular core optical fibers 116a, 116b, 116c (i.e., the portion of the non-circular core optical fibers 116a, 116b, 116c that surrounds the cores 117a, 117b, 117c, respectively). In this arrangement, the fiber outputs are mechanically and optically matched to the desired core stream positions of the respective laser pump beams.

[0052] FIG. 1C illustrates an embodiment of a flow cell 101. A linear fiber array 113 projects excitation wavelength light as described above with respect to FIGS. 1A and 1B. The light passes through a lens 114. A non-circular core optical fiber 116a projects the excitation wavelength light to a focal point 126a in the flow stream 102 of the flow cell 101. A non-circular core optical fiber 116b projects the excitation wavelength light to a focal point 126b in the flow stream 102 of the flow cell 101. A non-circular core optical fiber 116c projects the excitation wavelength light to a focal point 126c in the flow stream 102 of the flow cell 101. Illumination of particles within focal points 126a-126c produces forward scattered light that propagates primarily in the forward direction. A forward scatter detector 123 is integrated with the flow cell 101 and configured to detect this forward scattered light. Also shown are an optical mask 121 and an optical filter 122 integral with the flow cell 101. Attached to the forward scatter detector 123 is an electrical coupler 124 for operably attaching electrical lines 125 to the forward scatter detector 123. In addition to forward scattered light, side scattered light and / or fluorescent light is produced by illuminating particles within focal points 126a-126c. A vertical stack 119 of fiber optic collector couplers 119a, 119b, 119c is provided for operably attaching the fiber optic collectors 120a, 120b, 120c, respectively, to the flow cell 101. Fiber optic collectors 120a, 120b, 120c, operably attached to fiber optic collector couplers 119a, 119b, 119c, respectively, may be configured to transmit fluorescent and / or side-scattered light from focal points 126a, 126b, 126c, which may be transmitted to one or more photodetectors as desired.

[0053] Flow cytometer Aspects of the invention include a flow cytometer. The flow cytometer of interest includes a plurality of light sources and an integrated optofluidic interrogation module of the invention (e.g., as described above). The number of light sources in the flow cytometer may vary. In some cases, the number of light sources is in the range of 2-10, such as 2-5, such as 2-4. Any convenient light source may be used as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near infrared diode laser. In other embodiments, the laser may be a helium neon (HeNe) laser. In some cases, the laser may be a gas laser, such as a helium neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, or a fluorine laser. 2 Lasers of interest include 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, flow cytometers of interest include dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers. In still other cases, lasers of interest 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 still other cases, flow cytometers of interest include solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO 4 Laser, Nd:YCa 4 O(BO 3 ) 3 Laser, Nd:YCOB laser, Ti:sapphire laser, Thulium YAG laser, Ytterbium YAG laser, Yb 2 O 3laser, or cerium doped laser, and combinations thereof.

[0054] The laser light source according to an embodiment may further include one or more optical adjustment components. In an embodiment, the optical adjustment components are disposed between the light source and the flow cell and may include any device capable of changing the spatial width of the illumination from the light source or other characteristics of the illumination, such as the illumination direction, wavelength, beam width, beam intensity, and focus. The optical adjustment protocol may include any convenient device that adjusts one or more characteristics of the light source, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In an embodiment, the flow cytometer of interest includes one or more focusing lenses. The focusing lens may be, in one example, a reduction lens. In yet another embodiment, the flow cytometer of interest includes optical fibers.

[0055] The light source may be positioned at any suitable distance from the flow cell, for example the light source and the flow cell are separated by 0.005 mm or more, such as 0.01 mm or more, for example 0.05 mm or more, such as 0.1 mm or more, for example 0.5 mm or more, such as 1 mm or more, for example 5 mm or more, such as 10 mm or more, for example 25 mm or more, such as 100 mm or more. In addition, the light source may be positioned at any suitable angle relative to the flow cell, for example at an angle in the range of 10 degrees to 90 degrees, such as 15 degrees to 85 degrees, for example 20 degrees to 80 degrees, such as 25 degrees to 75 degrees, for example 30 degrees to 60 degrees, for example at an angle of 90 degrees.

[0056] In some embodiments, the light source of interest may include multiple lasers, e.g., 2 or more lasers, e.g., 3 or more lasers, e.g., 4 or more lasers, e.g., 5 or more lasers, e.g., 10 or more lasers, e.g., 15 or more lasers, configured to provide laser light for separate application to the flow stream. Depending on the desired wavelength of light for irradiating the flow stream, each laser may have a different specific wavelength within the range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. In some embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0057] The fiber optic light transmitting coupler of the linear fiber array of the integrated opto-fluidic interrogation module may be configured to interface directly with the laser such that light output by the laser is directed to the appropriate optical fiber of the linear fiber array. In an embodiment, the flow cytometer of the present invention includes a fiber optic light collector operably attached to the fiber optic light collector coupler of the integrated opto-fluidic interrogation module. Details regarding the fiber optic light collector are described above in connection with the integrated opto-fluidic interrogation module of the present invention.

[0058] The flow cytometer of the present invention may include a photodetector operably attached to the fiber optic collection element. The photodetector is configured to detect the particle modulated light transmitted by the fiber optic collection element and generate a signal based on a characteristic (e.g., intensity) of the light. For example, the particle modulated light detector or detectors may include one or more side scatter light detectors for detecting side scatter wavelengths of light (i.e., light refracted and reflected by the surface and internal structure of the particle). In some embodiments, the module includes one side scatter light detector. In other embodiments, the module includes multiple side scatter light detectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more side scatter light detectors.

[0059] Any convenient detector for detecting collected light may be used in the side scatter light detector described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof, among other detectors. In some embodiments, the collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10cm 2 , e.g. 0.05 cm 2 ~9 cm 2 , e.g. 0.1 cm 2 ~8 cm 2 , e.g. 0.5 cm 2 ~7 cm 2 , e.g. 1 cm 2 ~5 cm 2The photomultiplier tube has an active detection surface area of ​​each region within the range of .

[0060] In embodiments, the subject modules further comprise a fluorescence light detector configured to detect light at one or more fluorescence wavelengths, hi other embodiments, the modules comprise a plurality of fluorescence light detectors, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, 5 or more, 10 or more, 15 or more, e.g., 20 or more fluorescence light detectors.

[0061] Any convenient detector for detecting collected light may be used with the fluorescence photodetectors described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof, among other detectors. In some embodiments, the collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10 cm 2 , e.g. 0.05 cm 2 ~9 cm 2 , e.g. 0.1 cm 2 ~8 cm 2 , e.g. 0.5 cm 2 ~7 cm 2 , e.g. 1 cm 2 ~5 cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .

[0062] When a module of interest has multiple fluorescent light detectors, each fluorescent light detector may be identical, or the collection of fluorescent light detectors may be a combination of different types of detectors. For example, when a module of interest has two fluorescent light detectors, in some embodiments, the first fluorescent light detector is a CCD type device and the second fluorescent light detector (or image sensor) is a CMOS type device. In other embodiments, both the first fluorescent light detector and the second fluorescent light detector are CCD type devices. In yet other embodiments, both the first fluorescent light detector and the second fluorescent light detector are CMOS type devices. In yet other embodiments, the first fluorescent light detector is a CCD type device and the second fluorescent light detector is a photomultiplier tube (PMT). In yet other embodiments, the first fluorescent light detector is a CMOS type device and the second fluorescent light detector is a photomultiplier tube. In yet other embodiments, both the first fluorescent light detector and the second fluorescent light detector are photomultiplier tubes.

[0063] In embodiments of the present disclosure, the fluorescence light detector of interest is configured to measure collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., twenty-five or more different wavelengths, e.g., fifty or more different wavelengths, e.g., one or more than one hundred different wavelengths, e.g., two or more than one hundred different wavelengths, e.g., two or more than one hundred different wavelengths, e.g., three or more than one hundred different wavelengths, e.g., four ...

[0064] In some embodiments, the fluorescence detector of interest is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect a spectrum of light over a range of wavelengths. For example, a module may have one or more detectors configured to collect a spectrum of light over one or more of the wavelength ranges of 200 nm to 1000 nm. In yet other embodiments, the detector of interest is configured to measure light emitted from a sample in the flow stream at one or more particular wavelengths. For example, a module may have one or more detectors configured to measure light at one or more of: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In an embodiment, the one or more detectors may be configured to pair with a particular fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0065] As described above, embodiments of the integrated opto-fluidic interrogation module of the present invention include a sample fluid input coupler for injecting a flow stream into a flow cell, a sheath fluid input coupler for injecting a sheath fluid into the flow cell, and / or a fluid output coupler for receiving a flow stream from the flow cell. The flow cytometer may include any suitable mechanism or mechanisms for providing sheath fluid and sample fluid to the sample fluid input coupler and the sheath fluid input coupler. For example, the sample fluid input coupler may be fluidly connected to a sample fluid line (e.g., tubing) that is fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidly connected to a sheath fluid line that is fluidly connected to a sheath fluid reservoir. Similarly, the flow cytometer may include any suitable mechanism or mechanisms for managing waste from the flow stream. The fluid output coupler may be fluidly connected to a waste line that is fluidly connected to a waste reservoir. A fluid management system that may be adapted for use in the subject flow cytometer is described in U.S. Patent Application Publication No. 2022 / 0341838, the entire disclosure of which is incorporated herein by reference.

[0066] Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997);Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997);Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995);Virgo, et al. (2012) Ann Clin Biochem. Jan;49(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 Drug Carrier Syst. 24(3):203-255.In some cases, flow cytometry systems of interest include BD Biosciences FACSCanto™ flow cytometers, BD Biosciences FACSCanto™ II flow cytometers, BD Accuri™ flow cytometers, BD Accuri™ C6 Plus flow cytometers, BD Biosciences FACSCelesta™ flow cytometers, BD Biosciences FACSLyric™ flow cytometers, BD Biosciences FACSVerse™ flow cytometers, BD Biosciences FACSymphony™ flow cytometers, BD Biosciences LSRFortessa™ flow cytometers, BD Biosciences LSRFortessa™ X-20 flow cytometers, BD Biosciences FACSPresto™ flow cytometers, BD Biosciences FACSVia™ flow cytometers, and BD Biosciences FACSCalibur™ cell sorters, BD Biosciences FACSCount™ cell sorters, BD Biosciences FACSLyric™ cell sorters, BD These include BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, and BD Biosciences FACSDiscover™ cell sorter.

[0067] In some embodiments, the subject system may be configured as described in U.S. Pat. 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, 10,302,545, 10,145,793, 10,113,967, 10,006,852, 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,097,641, 9,100,972, 9,200,334, 9,300,972 ...0, 9,100,972, 9,100,972, 9,200,972, 9,300,972, 9,453,789, 9,200,334, 9,097,640, 9,100,972, 9,100,972, 9,200,972, 9,3 Nos. 5494, 9092034, 8975595, 8753573, 8233146, 8140300, 7544326, 7201875, 7129505, 6821740, 6813017, 6809804, 6372506, 5700692, 5643796, 5627040, 5620842, 5602039, 4987086, and 4498766. In some embodiments in which the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter, as described in U.S. Provisional Patent Application No. 63 / 431,803 and U.S. Provisional Patent Application No. 63 / 465,057, the entire disclosures of which are incorporated herein by reference.

[0068] Figure 2 is a schematic diagram of a flow cytometer with an integrated opto-fluidic investigation module 220 according to one embodiment of the present invention. The integrated opto-fluidic investigation module 220 includes a flow cell 205, a linear fiber array 204, a focusing optics 206 having a plurality of microlenses, a cuvette 207, a collimating lens 208, fiber optic focusing element couplers 209a-209c, an optical mask 210, an optical filter 211, a forward scatter detector 212, a sheath fluid input coupler 213, a sample fluid input coupler 214, and a fluid output coupler 215. The flow cytometer shown in Figure 2 includes lasers 201a, 201b, and 201c operably attached to optical fibers 203a, 203b, and 203c, respectively, via fiber optic light transmitting connectors 202a, 202b, and 202c. The optical fibers 203a-c terminate in a linear fiber array 204 that is integrated with a flow cell 205 and includes a plurality of non-circular core optical fibers each configured to project excitation wavelength light to a corresponding focal point within a cuvette 207 of the flow cell 205 (e.g., as described above in connection with FIG. 1B). In the embodiment of FIG. 2, the optofluidic interrogation module 220 includes a collimating lens 208 that is attached to the outer surface of the cuvette 207. The collimating lens 208 is configured to collimate the particle-modulated light emitted from the cuvette 207. The forward scattered light from the cuvette 207 passes through an optical mask 210 and an optical filter 211 before proceeding to a forward scatter detector 212. The side scattered light and / or fluorescent light may be collected by a fiber optic collector (not shown) that is operably attached to the fiber optic collector couplers 209a-c. Sample fluid may be supplied to the flow cell 205 via a sample fluid input coupler 214, which itself may be fluidly connected to a sample fluid line (e.g., tubing) that is fluidly connected to a sample fluid reservoir. Sheath fluid may be supplied to the flow cell 205 via a sheath fluid input coupler 213, which itself may be fluidly connected to a sheath fluid line that is fluidly connected to a sheath fluid reservoir.Sheath fluid (e.g., waste fluid) emitted from the flow cell may be removed via fluid output coupler 215. A waste line may be fluidly connected to fluid output coupler 215, which may deposit the waste fluid in a waste reservoir.

[0069] During operation, the operation of the flow cytometer is controlled by the controller / processor 290, and measurement data from the detectors may be stored in memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detectors to receive output signals from the detectors, and may be further coupled to electrical and electromechanical components of the flow cytometer to control the lasers 201a-201c, fluid flow parameters, and the like. Input / output (I / O) functionality 297 may also be provided in the system. The memory 295, controller / processor 290, and I / O functionality 297 may be provided entirely as an integral part of the flow cytometer. In such an embodiment, a display may also form part of the I / O functionality 297 to present experimental data to a user of the flow cytometer. Alternatively, some or all of the memory 295 and the controller / processor 290 and I / O functionality 297 may be part of one or more external devices, such as a general purpose computer. In some embodiments, some or all of the memory 295 and controller / processor 290 may be in wireless or wired communication with the flow cytometer. The controller / processor 290 in conjunction with the memory 295 and I / O functionality 297 may be configured to perform a variety of functions associated with the preparation and analysis of flow cytometer experiments.

[0070] The various fluorescent molecules of the fluorochrome panel used in a flow cytometer experiment emit light in their own characteristic wavelength bands. The particular fluorescent labels used in the experiment and the associated fluorescent emission bands of the fluorescent labels may be selected to generally match the filter windows of the detector. The I / O functionality 297 may be configured to receive data regarding a flow cytometer experiment having a panel of fluorescent labels and a plurality of cell populations having a plurality of markers, each cell population having a subset of the plurality of markers. The I / O functionality 297 may be further configured to receive biological data assigning one or more markers to one or more cell populations, marker concentration data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, may further be stored in memory 295. The controller / processor 290 may be configured to evaluate one or more assignments of labels to markers.

[0071] In some embodiments, the subject flow cytometer is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having a plurality of sorting determination units, such as described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module having deflection plates, such as described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0072] 3 is a functional block diagram of an example of a control system for analyzing and displaying biological events, such as a processor 300. The processor 300 may be configured to perform various processes for controlling the graphical display of biological events.

[0073] A flow cytometer or sorting system 302 may be configured to acquire bio-event data. For example, a flow cytometer may generate flow cytometry event data (e.g., particle-modulated light data). The flow cytometer 302 may be configured to provide the bio-event data to the processor 300. A data communication channel may be provided between the flow cytometer 302 and the processor 300. The bio-event data may be provided to the processor 300 via the data communication channel.

[0074] The processor 300 may be configured to receive bio-event data from the flow cytometer 302. The bio-event data received from the flow cytometer 302 may include flow cytometry event data. The processor 300 may be configured to provide a graphical representation including a first plot of the bio-event data to the display device 306. The processor 300 may be further configured to render a region of interest as a gate (e.g., a first gate) around a population of the bio-event data displayed by the display device 306, e.g., overlaid on the first plot. In some embodiments, the gate may be a logical combination of one or more illustrated regions of interest plotted on a histogram or bivariate plot of a parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0075] The processor 300 may further be configured to display the bioevent data on the display device 306 within the gate differently than other events within the bioevent data outside the gate. For example, the processor 300 may be configured to render the color of the bioevent data included within the gate differently than the color of the bioevent data outside the gate. The display device 306 may be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to provide a graphical interface.

[0076] The processor 300 may be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device may be implemented as a mouse 310. The mouse 310 may transmit a gate selection signal identifying a gate to be displayed on or manipulated via the display device 306 (e.g., by clicking on or within the desired gate when the cursor is on the desired gate) to the processor 300. In some implementations, the first device may be implemented as a keyboard 308 or other means for providing input signals to the processor 300, such as a touch screen, a pen, a photodetector, or a voice recognition system. Some input devices may include multiple input functions. In such implementations, each input function may be considered an input device. For example, as shown in FIG. 3, the mouse 310 may include a right mouse button and a left mouse button, and the right mouse button and the left mouse button may each generate a trigger event.

[0077] A trigger event can cause the processor 300 to change how the data is displayed, what portions of the data are actually displayed on the display device 306, and / or provide input for further processing, such as selection of a population of interest for particle sorting.

[0078] In some embodiments, the processor 300 may be configured to detect when a gate selection is initiated by the mouse 310. The processor 300 may further be configured to automatically modify the visualization of the plot to facilitate gating. This modification may be based on a particular distribution of the bio-event data received by the processor 300. In some embodiments, the processor 300 extends the first gate such that a second gate is generated (e.g., as described above).

[0079] The processor 300 may be connected to a storage device 304. The storage device 304 may be configured to receive and store bio-event data from the processor 300. The storage device 304 may be further configured to receive and store flow cytometry event data from the processor 300. The storage device 304 may be further configured to enable retrieval of bio-event data, such as flow cytometry event data, by the processor 300.

[0080] The display device 306 may be configured to receive display data from the processor 300. The display data may include plots of the biological event data and gates outlining sections of the plots. The display device 306 may be further configured to modify the information displayed in response to input received from the processor 300 in conjunction with input from the flow cytometer 302, the storage device 304, the keyboard 308, and / or the mouse 310.

[0081] In some embodiments, the processor 300 can generate a user interface for receiving exemplary events for screening. For example, the user interface can include a mechanism for receiving exemplary events or exemplary images. The exemplary events or images, or exemplary gates, can be provided prior to collection of event data for a sample, or based on an initial set of events for a portion of a sample.

[0082] FIG. 4A is a schematic diagram illustrating a particle sorting system 400 (e.g., flow cytometer 302) according to one embodiment presented herein. In some embodiments, the particle sorting system 400 is a cell sorting system. As shown in FIG. 4A, a droplet forming transducer 402 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 401, which may be coupled to, include, or be a nozzle 403. Within the fluid conduit 401, a sheath fluid 404 hydrodynamically focuses a sample fluid 406 containing particles 409 into a moving fluid column 408 (e.g., a stream). Within the moving fluid column 408, the particles 409 (e.g., cells) are aligned in a single file and traverse a monitoring region 411 (e.g., intersecting a laser stream) that is illuminated by an illumination source 412 (e.g., a laser). Vibration of the droplet forming transducer 402 causes the moving fluid column 408 to break up into a number of droplets 410 , some of which contain particles 409 .

[0083] In operation, a detection station 414 (e.g., an event detector) identifies when a particle of interest (or cell of interest) crosses the monitoring region 411. The detection station 414 feeds a timing circuit 428, which in turn feeds a flash charge circuit 430. At a droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 408 such that the droplet of interest carries a charge. The droplet of interest can contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the charged droplets into a receptacle, such as a collection tube or a multi-well or microwell sample plate, where a well or microwell can be specifically associated with the droplet of interest. As shown in FIG. 4A, the droplets can be collected in a drain receptacle 438.

[0084] The detection system 416 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal when a particle of interest passes through the monitoring region 411. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 416 enables the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 416 may provide an amplitude signal 420 and / or a phase signal 418, which are then provided (via an amplifier 422) to an amplitude control circuit 426 and / or a frequency control circuit 424. The amplitude control circuit 426 and / or the frequency control circuit 424 then control the droplet forming transducer 402. The amplitude control circuit 426 and / or the frequency control circuit 424 may be provided within the control system.

[0085] In some embodiments, the sorting electronics (e.g., detection system 416, detection station 414, and processor 440) may be coupled to a memory configured to store detected events and sorting decisions based on the detected events. The sorting decisions may be included in the event data for the particles. In some embodiments, detection system 416 and detection station 414 may be implemented as a single detection unit or may be communicatively coupled such that event measurements may be collected by either detection system 416 or detection station 414 and provided to a non-collection element.

[0086] FIG. 4B is a schematic diagram showing a particle sorting system according to one embodiment presented herein. The particle sorting system 400 shown in FIG. 4B includes deflection plates 452 and 454. An electric charge can be applied via a stream charging wire in the barb, thus generating a stream of droplets 410 containing the particles 409 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The information about the particles is analyzed, such as by sorting electronics (not shown in FIG. 4B) or other detection systems. Deflection plates 452 and 454 can be independently controlled to attract or repel the charged droplets and direct them toward a desired collection vessel (e.g., one of 472, 474, 476, or 478). 4B, deflector 452 and deflector 454 can be controlled to direct particles along a first path 462 toward a container 474 or along a second path 468 toward a container 478. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector may allow the particle to continue along flow path 464. Such uncharged droplets may be channeled into a waste container, such as via aspirator 470.

[0087] Sorting electronics can be included to initiate measurement collection, receive fluorescent signals for the particles, and determine how to adjust the deflection plates to sort the particles. Exemplary implementations of the embodiment shown in Figure 4B include the BD FACSAria™ line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0088] FIG. 5 illustrates a general configuration of an exemplary computing device 500 according to an embodiment. The general configuration of the computing device 500 illustrated in FIG. 5 includes an arrangement of computer hardware and software components. However, not all of these general conventional elements need to be illustrated to provide a useful disclosure. As illustrated, the computing device 500 includes a processing unit 510, a network interface 520, a computer-readable medium drive 530, an input / output device interface 540, a display 550, and an input device 560, all of which may communicate with each other via a communication bus. The network interface 520 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 510 may receive information and instructions from other computing systems or services via the network. The processing unit 510 may further communicate with a memory 570 and may further provide output information for an optional display 550 via the input / output device interface 540. For example, analysis software (e.g., data analysis software or programs, such as FlowJo®), stored as executable instructions in non-transitory memory of the analysis system, can display flow cytometry event data to a user. Input / output device interface 540 may further accept input from any input device 560, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device.

[0089] The memory 570 may include computer program instructions (grouped in some embodiments as modules or components) that the processing unit 510 executes to implement one or more embodiments. The memory 570 typically includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. The memory 570 may store an operating system 572 that provides computer program instructions for use by the processing unit 510 in the general management and operation of the computing device 500. Data may be stored in the data storage device 590. The memory 570 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0090] How to analyze the sample As noted above, aspects of the invention include methods of analyzing a sample, including (a) introducing a particle sample into a flow cytometer comprising a plurality of light sources and an integrated optofluidic interrogation module of the invention, and (b) analyzing the particle sample by flow cytometry.

[0091] The sample to be analyzed can be any sample of interest to the user. In certain embodiments, the sample contains biological components or is a biological sample. The term "biological sample" is used in the conventional sense to refer to a sample that is derived from or contains a subset of tissues, cells or components of a whole organism, such as a prokaryotic cell, eukaryotic cell, plant, fungus, or animal, where the subset of tissues, cells or components may be found in blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, or semen. Thus, a "biological sample" refers to both a naturally occurring organism or a subset of its tissues, as well as homogenates; isolated, purified or enriched biological particles (e.g., DNA, RNA, proteins, subcellular organelles, etc.); and lysates or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymphatic fluid, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, genitourinary tract, tears, saliva, milk, blood cells, tumors, organs. A biological sample may be tissue from any type of organism, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, such as plasma, tears, urine, semen, etc., and in some cases, the sample is a blood sample, including whole blood, such as blood obtained from a venipuncture or finger prick (blood may or may not be combined with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0092] In some embodiments, the sample source is a "mammal" or "mammalian," which terms are used broadly to describe organisms belonging to the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult), and in some embodiments, the human subject is a juvenile, adolescent, or adult. Although embodiments of the present disclosure may be applied to samples from human subjects, it should be understood that the methods may also be performed on samples from other animal subjects (i.e., "non-human subjects"), including, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0093] Cells of interest may be subject to characterization according to various parameters, such as phenotypic characteristics identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analysis droplets determined to contain target cells. A variety of cells may be characterized using the subject methods. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and red blood cells. Target cells of interest include cells bearing convenient cell surface markers or antigens that may be taken up or labeled by convenient affinity agents or conjugates thereof. For example, the target cell may comprise cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0094] The featured methods may further use the particles in research, clinical trials, or therapy. In some embodiments, the subject methods obtain individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods obtain cells from a fluid or tissue sample used as a research or diagnostic specimen for diseases such as cancer. Similarly, the subject methods obtain cells from a fluid or tissue sample used for therapy. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissue, may be prepared and introduced into a subject as a therapeutic treatment. Conditions that may be treated by administration of flow cytometrically sorted samples include, but are not limited to, blood disorders, immune system disorders, organ damage, and the like.

[0095] A typical cell therapy protocol may include the steps of sample collection, cell isolation, genetic modification, culture, in vitro expansion, cell harvesting, sample volume reduction, sample washing, biopreservation, storage, and introduction of cells into a subject. The protocol may begin by collecting viable cells and tissue from a tissue source of the subject to generate a cell and / or tissue sample. The sample may be collected by any suitable procedure, including, for example, administering a cell mobilizing agent to the subject, drawing blood from the subject, removing bone marrow from the subject, etc. After the sample is collected, cell enrichment may be performed by multiple methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells may be genetically modified by any convenient method, for example, nuclease-mediated gene editing. The genetically modified cells may be cultured, activated, and expanded in vitro. In some cases, the cells are preserved, for example, cryopreserved, and stored for future use. When in use, the cells may be thawed and then administered to the patient, for example, the cells may be infused into the patient.

[0096] How to Assemble a Flow Cytometer Aspects of the invention further include a method of assembling a flow cytometer, the method including (a) disposing an integrated opto-fluidic interrogation module in a flow cytometer including a plurality of light sources, and (b) operably attaching a plurality of fiber optic light transmissive couplers to the plurality of light sources to assemble the flow cytometer. As described in detail above, the integrated opto-fluidic interrogation module of the present disclosure includes a flow cell having an optically transparent flow path for carrying particles in a flow stream, a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each configured to project excitation wavelength light to a corresponding focal point within the flow cell, and a plurality of fiber optic light transmissive couplers for operably attaching the plurality of light sources to the linear fiber array.

[0097] As described above, embodiments of the integrated opto-fluidic interrogation module include a photodetector integrated with the flow cell and an electrical coupler for operably attaching electrical wires to the photodetector. In some cases, the electrical wires are operably attached to the electrical coupler when the flow cytometer is assembled. Additionally, embodiments of the integrated opto-fluidic interrogation module include a plurality of fiber optic focusing element couplers for operably attaching a plurality of fiber optic focusing elements to the flow cell. In some cases, the plurality of fiber optic focusing elements are operably attached to the plurality of fiber optic focusing element couplers when the flow cytometer is assembled. If the module includes a sample fluid input coupler for injecting a flow stream into the flow cell, some embodiments of the disclosed method include fluidically connecting a sample fluid line of the flow cytometer to the sample fluid input coupler. If the module includes a sheath fluid input coupler for injecting a sheath fluid into the flow cell, some embodiments of the disclosed method include fluidically connecting a sheath fluid line of the flow cytometer to the sheath fluid input coupler. If the module has a fluid output coupler for receiving the flow stream from the flow cell, some embodiments of the disclosed methods fluidly connect a waste line of the flow cytometer to the fluid output coupler.

[0098] However, the methods of assembling a flow cytometer disclosed herein do not include mechanical mounting and alignment. In other words, once the integrated opto-fluidic interrogation module is placed into the flow cytometer and connected to the associated components (e.g., laser, fiber optic focusing elements, electrical lines, etc.) due to the integrated nature of the integrated opto-fluidic interrogation module, the optical components are already in precise optical alignment. In some cases, the methods of the present invention reduce assembly and / or alignment time by 20% or less, such as 30% or less, such as 50% or less, such as 60% or less than conventional methods that do not include integrated optics.

[0099] kit As discussed above, aspects of the invention further include kits. Kits of interest include an integrated opto-fluidic investigation module (e.g., as described above). In embodiments of the subject kits, one integrated opto-fluidic investigation module is included. In some cases, the kits include multiple integrated opto-fluidic investigation modules. In such cases, the number of integrated opto-fluidic investigation modules in the subject kits is in the range of 2-20, such as 2-10, e.g., 2-5.

[0100] The kit may further comprise one or more other components useful for assembling a flow cytometer. In some cases, the kit comprises one or more electrical lines for transmitting a signal generated by a photodetector integrated with the flow cell. In some cases, the kit comprises a plurality of electrical lines, e.g., two or more electrical lines, e.g., three or more electrical lines, e.g., four or more electrical lines, e.g., five or more electrical lines. The kit according to some embodiments comprises a plurality of fiber optic light collecting elements. For example, the number of fiber optic light collecting elements may be in the range of 2-20, e.g., in the range of 2-10. In some cases, the kit comprises fluidic components, such as sample fluid lines, sheath fluid lines and / or waste lines, which may be operably attached to the module.

[0101] The kits may further comprise instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be provided is information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, a package insert, etc. Yet another form in which these instructions may be present is a computer readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Yet another form in which these instructions may be present is a website address that may be used via the internet to access the information at a remote location.

[0102] usefulness The subject integrated opto-fluidic interrogation modules, flow cytometers, and methods are used in a variety of applications where it is desirable to improve resolution and accuracy in determining parameters of analytes (e.g., cells, particles) in biological samples. For example, the present invention may be used to facilitate assembly of a flow cytometer, i.e., to eliminate the need for laborious assembly and adjustment. Thus, the subject integrated opto-fluidic interrogation modules, flow cytometers, and methods are used where it is desirable to simplify the assembly of complex flow cytometers, which typically involve laborious mechanical mounting and alignment. As a result, the subject integrated opto-fluidic interrogation modules, flow cytometers, and methods may be used to reduce the assembly costs of flow cytometers and to increase the robustness of results obtained from flow cytometry. In some embodiments, the present invention may be used where it is desirable to have components of a flow cytometer that are modular, i.e., such that components can be swapped for different modules as needed to achieve the functionality of interest. In further cases, the present invention may be used where it is desirable to increase the collection efficiency of a flow cytometer.

[0103] The present disclosure can be used to characterize many types of analytes, particularly those relevant to protocols for medical diagnosis or patient care, including, but not limited to, proteins (including both unbound proteins and proteins bound to the surfaces of structures such as cells), nucleic acids, viral particles, etc. Furthermore, samples can be obtained from in vitro or in vivo sources, and the sample can be a diagnostic sample.

[0104] The following are offered by way of example and not by way of limitation.

[0105] experiment An illumination profile was obtained for the light emitted by a linear fiber array arranged as shown in Figure 1B. The resulting illumination profile is shown in Figure 6. As shown in Figure 6, the illumination is uniform and regularly spaced, indicating that linear fiber arrays are suitable for use in illuminating core streams.

[0106] Regardless of the scope of the appended claims, the present disclosure is further defined by the following caveats.

[0107] Addendum 1. A flow cell having an optically transparent flow path for carrying particles in a flow stream; a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each configured to project excitation wavelength light to a corresponding focal point within the flow cell; a plurality of optical fiber light transmitting couplers for operably attaching a plurality of optical sources to the linear fiber array; an integrated opto-fluidic investigation module comprising:

[0108] Clause 2. The integrated opto-fluidic investigation module of clause 1, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0109] Clause 3. The integrated opto-fluidic investigation module of clause 2, wherein the non-circular core optical fiber is a square core optical fiber.

[0110] Clause 4. The integrated opto-fluidic investigation module of any one of clauses 1-3, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile to a focal point.

[0111] Clause 5. The integrated optofluidic investigation module of any one of clauses 1 to 4, wherein the non-circular core fibers of the linear fiber array are stacked vertically.

[0112] Clause 6. The integrated optofluidic investigation module of clause 5, wherein the cores of adjacent non-circular core fibers of the linear fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm.

[0113] Clause 7. The integrated opto-fluidic investigation module of any one of clauses 1-6, wherein the linear fiber array is attached to an outer surface of the flow cell.

[0114] Clause 8. The integrated optofluidic investigation module of any one of clauses 1-7, further comprising focusing optics integrated with the flow cell and configured to relay excitation wavelength light from the non-circular core optical fiber to a focal point.

[0115] Clause 9. The integrated opto-fluidic interrogation module of clause 8, wherein the focusing optics are embedded in the flow cell.

[0116] Clause 10. The integrated opto-fluidic investigation module of clause 8 or 9, wherein the focusing optics comprises a plurality of microlenses.

[0117] Clause 11. The integrated opto-fluidic investigation module of any one of clauses 1 to 10, further comprising an optical detector integrated with the flow cell.

[0118] Clause 12. The integrated opto-fluidic investigation module of clause 11, further comprising an electrical coupler for operably attaching electrical lines to the optical detector.

[0119] Clause 13. The integrated opto-fluidic investigation module of clause 12, further comprising an electrical line.

[0120] Addendum 14. The integrated opto-fluidic investigation module of any one of Addendums 11 to 13, further comprising an optical filter disposed between the focal point and the photodetector.

[0121] Addendum 15. The integrated opto-fluidic investigation module of any one of Addendums 11 to 14, further comprising an optical mask disposed between the focal point and the photodetector.

[0122] Appendix 16. An integrated opto-fluidic investigation module according to any one of appendices 11 to 15, wherein the optical detector is a forward scatter detector.

[0123] Addendum 17. The integrated opto-fluidic investigation module of any one of Addendums 11 to 16, wherein the optical detector is attached to an outer surface of the flow cell.

[0124] Clause 18. The integrated opto-fluidic investigation module of any one of clauses 1 to 17, further comprising a plurality of fiber optic focusing element couplers for operably mounting the plurality of fiber optic focusing elements to the flow cell.

[0125] Clause 19. The integrated opto-fluidic investigation module of clause 18, wherein the fiber optic concentrator couplers are vertically stacked.

[0126] Clause 20. The integrated opto-fluidic investigation module of clause 18 or 19, further comprising a plurality of optical fiber focusing elements.

[0127] Clause 21. The integrated opto-fluidic investigation module of any one of clauses 1 to 20, further comprising a sample fluid input coupler for injecting sample fluid into the flow cell.

[0128] Clause 22. The integrated opto-fluidic investigation module of any one of clauses 1 to 21, further comprising a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0129] Clause 23. The integrated opto-fluidic investigation module of any one of clauses 1 to 22, further comprising a fluid output coupler for receiving the flow stream from the flow cell.

[0130] Clause 24. The integrated optofluidic investigation module of any one of clauses 1 to 23, wherein the flow cell comprises a cuvette.

[0131] Clause 25. The integrated opto-fluidic investigation module of clause 24, further comprising a collimating lens attached to an outer surface of the cuvette.

[0132] Clause 26. The integrated optofluidic investigation module of clause 25, wherein the collimating lens is an injection compression molded microlens.

[0133] Clause 27. The integrated optofluidic investigation module of clause 25 or 26, wherein the collimating lens is a Fresnel lens.

[0134] Appendix 28. N noncircular core optical fibers; N foci, and N fiber optic optical transmission couplers It is equipped with 28. The integrated opto-fluidic investigation module of any one of claims 1-27, wherein N is an integer in the range of 2-10.

[0135] Addendum 29. The integrated opto-fluidic investigation module of any one of Addendums 1 to 28, wherein the width of the flow cell is in the range of 75 mm to 125 mm.

[0136] Appendix 30. Multiple light sources, and Integrated optofluidic investigation module It is equipped with The integrated optofluidic investigation module: a flow cell having an optically transparent flow passage for carrying particles in a flow stream; a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each configured to project excitation wavelength light to a corresponding focal point within the flow cell; a plurality of optical fiber light transmitting couplers for operably attaching a plurality of optical sources to the linear fiber array; The flow cytometer has

[0137] Clause 31. The flow cytometer of clause 30, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0138] Clause 32. The flow cytometer of clause 31, wherein the non-circular core optical fiber is a square core optical fiber.

[0139] Addendum 33. A flow cytometer according to any one of Addendums 30 to 32, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top hat profile to a focal point.

[0140] Addendum 34. A flow cytometer according to any one of Addendums 30 to 33, wherein the non-circular core fibers of the linear fiber array are stacked vertically.

[0141] Clause 35. The flow cytometer of clause 34, wherein the cores of adjacent non-circular core fibers of the linear fiber array are separated from each other by a distance of 0.1 mm to 0.3 mm.

[0142] Addendum 36. A flow cytometer according to any one of Addendums 30 to 35, wherein the linear fiber array is attached to an outer surface of the flow cell.

[0143] Addendum 37. A flow cytometer according to any one of Addendums 30 to 36, wherein the integrated opto-fluidic investigation module has focusing optics integrated with the flow cell and configured to relay excitation wavelength light from the non-circular core optical fiber to a focal point.

[0144] 38. The flow cytometer of claim 37, wherein the focusing optics are embedded in the flow cell.

[0145] Addendum 39. A flow cytometer according to any one of Addendums 37 and 38, wherein the focusing optics comprises a plurality of microlenses.

[0146] Addendum 40. The flow cytometer of any one of Addendums 30 to 39, wherein the integrated opto-fluidic interrogation module has an optical detector integrated with the flow cell.

[0147] Clause 41. The flow cytometer of clause 40, wherein the integrated opto-fluidic interrogation module has an electrical coupler for operably attaching electrical lines to the optical detector.

[0148] Addendum 42. The flow cytometer of Addendum 41, further comprising an electrical line.

[0149] Addendum 43. A flow cytometer according to any one of Addendums 40 to 42, wherein the integrated opto-fluidic investigation module has an optical filter disposed between the focal point and the photodetector.

[0150] Addendum 44. A flow cytometer according to any one of Addendums 40 to 43, wherein the integrated opto-fluidic investigation module has an optical mask disposed between the focal point and the photodetector.

[0151] Appendix 45. A flow cytometer according to any one of appendices 40 to 44, wherein the light detector is a forward scatter detector.

[0152] Addendum 46. A flow cytometer according to any one of Addendums 40 to 45, wherein the optical detector is attached to the outer surface of the flow cell.

[0153] Addendum 47. The flow cytometer of any one of Addendums 30-46, wherein the integrated optical fluidic investigation module has a plurality of fiber optic focusing element couplers for operably mounting the plurality of fiber optic focusing elements to the flow cell.

[0154] 48. The flow cytometer of claim 47, wherein the fiber optic focusing couplers are vertically stacked.

[0155] Addendum 49. The flow cytometer of any one of Addendums 47 and 48, further comprising a plurality of optical fiber focusing elements.

[0156] Clause 50. The flow cytometer of clause 49, further comprising a plurality of optical detectors operably attached to the plurality of optical fiber focusing elements, respectively.

[0157] Addendum 51. The flow cytometer of any one of Addendums 30 to 50, wherein the integrated opto-fluidic investigation module has a sample fluid input coupler for injecting sample fluid into the flow cell.

[0158] Addendum 52. A flow cytometer according to any one of Addendums 30 to 51, wherein the integrated opto-fluidic investigation module has a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0159] Addendum 53. The flow cytometer of any one of Addendums 30-52, wherein the integrated opto-fluidic interrogation module has a fluid output coupler for receiving the flow stream from the flow cell.

[0160] Addendum 54. A flow cytometer according to any one of Addendums 30 to 53, wherein the flow cell has a cuvette.

[0161] Clause 55. The flow cytometer of clause 54, wherein the integrated opto-fluidic interrogation module has a collimating lens attached to an outer surface of the cuvette.

[0162] Item 56. The flow cytometer of item 55, wherein the collimating lens is an injection compression molded microlens.

[0163] Addendum 57. A flow cytometer according to Addendum 55 or 56, wherein the collimating lens is a Fresnel lens.

[0164] Addendum 58. The integrated opto-fluidic investigation module is N noncircular core optical fibers; N foci, and N fiber optic optical transmission couplers 58. The flow cytometer according to any one of Appendices 30 to 57, wherein N is an integer in the range of 2 to 10.

[0165] Addendum 59. A flow cytometer according to any one of Addendums 30 to 58, wherein the width of the flow cell is within the range of 75 mm to 125 mm.

[0166] Addendum 60. A method for analyzing a sample, comprising: (a) introducing a particle sample into a flow cytometer having a plurality of light sources and an integrated opto-fluidic interrogation module, the integrated opto-fluidic interrogation module having a flow cell having an optically transparent flow path for carrying particles in a flow stream, a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each optical fiber being configured to project excitation wavelength light at a corresponding focal point within the flow cell, and a plurality of optical fiber light transmissive couplers for operably attaching the plurality of light sources to the linear fiber array; (b) Analyzing the particle sample by flow cytometry.

[0167] Clause 61. The method of clause 60, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0168] Clause 62. The method of clause 61, wherein the non-circular core optical fiber is a square core optical fiber.

[0169] Addendum 63. The method of any one of Addendums 60-62, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top hat profile to a focal point.

[0170] Addendum 64. The method of any one of Addendums 60-63, wherein the non-circular core fibers of the linear fiber array are stacked vertically.

[0171] Clause 65. The method of clause 64, wherein the cores of adjacent non-circular core fibers of the linear fiber array are separated from each other by a distance of 0.1 mm to 0.3 mm.

[0172] Addendum 66. The method of any one of Addendums 60 to 65, wherein the linear fiber array is attached to an outer surface of the flow cell.

[0173] Addendum 67. The method of any one of Addendums 60-66, wherein the integrated opto-fluidic investigation module has focusing optics integrated with the flow cell and configured to relay excitation wavelength light from the non-circular core optical fiber to a focal point.

[0174] Item 68. The method of item 67, wherein the focusing optics is embedded in the flow cell.

[0175] Addendum 69. The method of any one of Addendums 67 to 68, wherein the focusing optics comprises a plurality of microlenses.

[0176] Addendum 70. The method of any one of Addendums 60-69, wherein the integrated opto-fluidic interrogation module has an optical detector integrated with the flow cell.

[0177] Addendum 71. The method of Addendum 70, wherein the integrated opto-fluidic interrogation module has an electrical coupler for operably attaching an electrical line to the optical detector.

[0178] Addendum 72. The method of Addendum 71, wherein the flow cytometer further comprises an electrical wire.

[0179] Addendum 73. The method of any one of Addendums 60-72, wherein the integrated opto-fluidic investigation module has an optical filter disposed between the focal point and the photodetector.

[0180] Addendum 74. The method of any one of Addendums 70-73, wherein the integrated opto-fluidic investigation module has an optical mask disposed between the focal point and the photodetector.

[0181] Addendum 75. The method of any one of Addendums 70-74, wherein the light detector is a forward scatter detector.

[0182] Addendum 76. The method of any one of Addendums 70 to 75, wherein the optical detector is attached to the outer surface of the flow cell.

[0183] Addendum 77. The method of any one of Addendums 60-76, wherein the integrated optical fluidic investigation module has a plurality of fiber optic focusing element couplers for operably mounting the plurality of fiber optic focusing elements to the flow cell.

[0184] Addendum 78. The method of Addendum 77, wherein the fiber optic concentrator couplers are stacked vertically.

[0185] Addendum 79. The method of any one of Addendums 77 and 78, wherein the flow cytometer further comprises a plurality of optical fiber focusing elements.

[0186] Addendum 80. The method of Addendum 79, wherein the flow cytometer further comprises a plurality of optical detectors operably attached to the plurality of optical fiber focusing elements, respectively.

[0187] Addendum 81. The method of any one of Addendums 60-80, wherein the integrated opto-fluidic interrogation module has a sample fluid input coupler for injecting sample fluid into the flow cell.

[0188] Addendum 82. The method of any one of Addendums 60-81, wherein the integrated opto-fluidic investigation module has a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0189] Addendum 83. The method of any one of Addendums 60-82, wherein the integrated opto-fluidic interrogation module has a fluid output coupler for receiving the flow stream from the flow cell.

[0190] Addendum 84. The method of any one of Addendums 60 to 83, wherein the flow cell has a cuvette.

[0191] Addendum 85. The method of Addendum 84, wherein the integrated optofluidic investigation module has a collimating lens attached to an outer surface of the cuvette.

[0192] Addendum 86. The method of Addendum 85, wherein the collimating lens is an injection compression molded microlens.

[0193] Addendum 87. The method of any one of Addendums 85 to 86, wherein the collimating lens is a Fresnel lens.

[0194] Addendum 88. The integrated opto-fluidic investigation module is N noncircular core optical fibers; N foci, and N fiber optic optical transmission couplers and N is an integer in the range of 2 to 10.

[0195] Addendum 89. The method of any one of Addendums 60 to 88, wherein the width of the flow cell is in the range of 75 mm to 125 mm.

[0196] Addendum 90. A method for assembling a flow cytometer, comprising: (a) placing an integrated opto-fluidic interrogation module, the integrated opto-fluidic interrogation module having a flow cell having an optically transparent flow path for carrying particles in a flow stream, a linear fiber array having a plurality of non-circular core optical fibers integral with the flow cell, each optical fiber being configured to project excitation wavelength light at a corresponding focal point within the flow cell, and a plurality of optical fiber light transmissive couplers for operatively attaching the plurality of light sources to the linear fiber array, in a flow cytometer having a plurality of light sources; (b) operatively attaching a plurality of fiber optic light transmitting couplers to a plurality of light sources to assemble a flow cytometer.

[0197] Clause 91. The method of clause 90, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0198] Clause 92. The method of clause 91, wherein the non-circular core optical fiber is a square core optical fiber.

[0199] Addendum 93. The method of any one of Addendums 90-92, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile to a focal point.

[0200] Addendum 94. The method of any one of Addendums 90-93, wherein the non-circular core fibers of the linear fiber array are stacked vertically.

[0201] Clause 95. The method of clause 94, wherein the cores of adjacent non-circular core fibers of the linear fiber array are separated from each other by a distance of 0.1 mm to 0.3 mm.

[0202] Addendum 96. The method of any one of Addendums 90 to 95, wherein the linear fiber array is attached to an outer surface of the flow cell.

[0203] Addendum 97. The method of any one of Addendums 90-96, wherein the integrated opto-fluidic investigation module has focusing optics integrated with the flow cell and configured to relay excitation wavelength light from the non-circular core optical fiber to a focal point.

[0204] 98. The method of claim 97, wherein the focusing optics is embedded in the flow cell.

[0205] Addendum 99. The method of any one of Addendums 97 to 98, wherein the focusing optical system comprises a plurality of microlenses.

[0206] Addendum 100. The method of any one of Addendums 90-99, wherein the integrated opto-fluidic interrogation module further comprises an optical detector integrated with the flow cell.

[0207] Addendum 101. The method of Addendum 100, wherein the integrated opto-fluidic interrogation module has an electrical coupler for operably attaching electrical lines to the optical detector.

[0208] Clause 102. The method of clause 101, operably attaching the electrical line to an electrical coupler.

[0209] Addendum 103. The method of any one of Addendums 100-102, wherein the integrated opto-fluidic investigation module has an optical filter disposed between the focal point and the photodetector.

[0210] Addendum 104. The method of any one of Addendums 100 to 103, wherein the integrated opto-fluidic investigation module has an optical mask positioned between the focal point and the photodetector.

[0211] Addendum 105. The method of any one of Addendums 100 to 104, wherein the light detector is a forward scatter detector.

[0212] Addendum 106. The method of any one of Addendums 100 to 105, wherein the optical detector is attached to an outer surface of the flow cell.

[0213] Addendum 107. The method of any one of Addendums 90-106, wherein the integrated optical fluidic investigation module has a plurality of fiber optic focusing element couplers for operably attaching the plurality of fiber optic focusing elements to the flow cell.

[0214] Addendum 108. The method of Addendum 107, wherein the fiber optic concentrator couplers are stacked vertically.

[0215] Addendum 109. The method of any one of Addendums 107 to 108, further comprising operably attaching a plurality of fiber optic concentrating elements to a plurality of fiber optic concentrating element couplers.

[0216] Addendum 110. The method of any one of Addendums 90-109, wherein the integrated opto-fluidic investigation module has a sample fluid input coupler for injecting sample fluid into the flow cell.

[0217] Addendum 111. The method of Addendum 110, further comprising fluidly connecting a sample fluid line of a flow cytometer to a sample fluid input coupler.

[0218] Addendum 112. The method of any one of Addendums 90-111, wherein the integrated opto-fluidic investigation module has a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0219] Addendum 113. The method of Addendum 112, fluidly connecting a sheath fluid line of a flow cytometer to a sheath fluid input coupler.

[0220] Addendum 114. The method of any one of Addendums 90-113, wherein the integrated optical-fluidic investigation module has a fluid output coupler for receiving the flow stream from the flow cell.

[0221] Clause 115. The method of clause 114, further comprising fluidly connecting a waste line of the flow cytometer to the fluid output coupler.

[0222] Addendum 116. The method of any one of Addendums 90 to 115, wherein the flow cell has a cuvette.

[0223] Addendum 117. The method of Addendum 116, wherein the integrated opto-fluidic investigation module has a collimating lens attached to an outer surface of the cuvette.

[0224] Addendum 118. The method of Addendum 117, wherein the collimating lens is an injection compression molded microlens.

[0225] Addendum 119. The method of any one of Addendums 117 and 118, wherein the collimating lens is a Fresnel lens.

[0226] Addendum 120. The integrated opto-fluidic investigation module is N noncircular core optical fibers; N foci, and N fiber optic optical transmission couplers and N is an integer in the range of 2 to 10.

[0227] Addendum 121. The method of any one of Addendums 90 to 120, wherein the width of the flow cell is in the range of 75 mm to 125 mm.

[0228] Addendum 122. Equipped with an integrated opto-fluidic investigation module, The integrated optofluidic investigation module: a flow cell having an optically transparent flow passage for carrying particles in a flow stream; a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each configured to project excitation wavelength light to a corresponding focal point within the flow cell; a plurality of optical fiber light transmitting couplers for operably attaching a plurality of optical sources to the linear fiber array; The kit has:

[0229] Addendum 123. The kit of Addendum 122, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0230] Clause 124. The kit of clause 123, wherein the non-circular core optical fiber is a square core optical fiber.

[0231] Addendum 125. The kit of any one of Addendums 122-124, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top hat profile to a focal point.

[0232] Addendum 126. The kit of any one of Addendums 122-125, wherein the non-circular core fibers of the linear fiber array are stacked vertically.

[0233] Addendum 127. The kit of Addendum 126, wherein the cores of adjacent non-circular core fibers of the linear fiber array are separated from each other by a distance of 0.1 mm to 0.3 mm.

[0234] Addendum 128. A kit according to any one of Addendums 122 to 127, wherein the linear fiber array is attached to an outer surface of the flow cell.

[0235] Addendum 129. A kit according to any one of Addendums 122 to 128, wherein the integrated optofluidic investigation module has focusing optics integrated with the flow cell and configured to relay excitation wavelength light from the non-circular core optical fiber to a focal point.

[0236] Addendum 130. The kit of Addendum 129, wherein the focusing optics is embedded in the flow cell.

[0237] Addendum 131. The kit of any one of Addendums 129 and 130, wherein the focusing optical system comprises a plurality of microlenses.

[0238] Addendum 132. The kit of any one of Addendums 122 to 131, wherein the integrated optofluidic investigation module further comprises an optical detector integrated with the flow cell.

[0239] Addendum 133. The kit of Addendum 132, wherein the integrated opto-fluidic interrogation module further comprises an electrical coupler for operably attaching electrical lines to the optical detector.

[0240] Item 134. The kit of item 133, further comprising an electrical wire.

[0241] Addendum 135. A kit according to any one of Addendums 132 to 134, wherein the integrated optofluidic investigation module has an optical filter disposed between the focal point and the photodetector.

[0242] Addendum 136. A kit according to any one of Addendums 132 to 135, wherein the integrated optofluidic investigation module has an optical mask disposed between the focal point and the photodetector.

[0243] Addendum 137. The kit of any one of Addendums 132 to 136, wherein the light detector is a forward scatter detector.

[0244] Addendum 138. A kit according to any one of Addendums 132 to 137, wherein the optical detector is attached to the outer surface of the flow cell.

[0245] Addendum 139. A kit according to any one of Addendums 122 to 138, wherein the integrated optical fluidic investigation module has a plurality of fiber optic focusing element couplers for operably attaching a plurality of fiber optic focusing elements to the flow cell.

[0246] Addendum 140. The kit of Addendum 139, wherein the fiber optic concentrator couplers are stacked vertically.

[0247] Addendum 141. The kit of any one of Addendums 139 and 140, further comprising a plurality of optical fiber focusing elements.

[0248] Addendum 142. The kit of any one of Addendums 122-141, wherein the integrated opto-fluidic investigation module has a sample fluid input coupler for injecting sample fluid into the flow cell.

[0249] Addendum 143. A kit according to any one of Addendums 122 to 142, wherein the integrated opto-fluidic investigation module has a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0250] Addendum 144. A kit according to any one of Addendums 122-143, wherein the integrated opto-fluidic investigation module has a fluid output coupler for receiving the flow stream from the flow cell.

[0251] Addendum 145. A kit according to any one of Addendums 122 to 144, wherein the flow cell has a cuvette.

[0252] Addendum 146. The kit of Addendum 145, wherein the integrated optofluidic investigation module has a collimating lens attached to an outer surface of the cuvette.

[0253] Addendum 147. The kit of Addendum 146, wherein the collimating lens is an injection compression molded microlens.

[0254] Addendum 148. The kit of Addendum 146 or 147, wherein the collimating lens is a Fresnel lens.

[0255] Addendum 149. The integrated opto-fluidic investigation module is N noncircular core optical fibers; N foci, and N fiber optic optical transmission couplers wherein N is an integer in the range of 2 to 10.

[0256] Addendum 150. A kit according to any one of Addendums 122 to 149, wherein the width of the flow cell is within the range of 75 mm to 125 mm.

[0257] Addendum 151. The kit of any one of Addendums 122-150, comprising a plurality of integrated optofluidic investigation modules.

[0258] Although the foregoing invention has been described in some detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art, in view of the teachings of the invention, that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

[0259] Thus, the foregoing merely illustrates the nature of the invention. It is clear that those skilled in the art may devise various configurations that embody the nature of the invention and are within the spirit and scope of the invention, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended essentially to aid the reader in understanding the nature of the invention and the concepts given by the inventors to advance the art, and should not be construed as being limited to the examples and conditions specifically described as such. Furthermore, all descriptions herein that describe the nature, aspects, and embodiments of the invention, as well as specific examples of the invention, are intended to include both structural and functional equivalents of the invention. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., all elements developed that perform the same function, regardless of structure. Furthermore, the descriptions disclosed herein are not intended to be publicly available, regardless of whether such disclosure is expressly set forth in the claims.

[0260] Therefore, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied in the appended claims. With respect to claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly provided to be invoked with respect to a limitation in a claim only when the precise phrase "means for" or "step for" is recited at the beginning of such limitation in the claim, and if such precise phrase is not used in the limitation of the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0261] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Application No. 63 / 537,165, filed September 7, 2023, the entire disclosure of which is incorporated herein by reference.

Claims

1. a flow cell having an optically transparent flow passage for carrying particles in a flow stream; a linear fiber array having a plurality of non-circular core optical fibers integral with the flow cell, each optical fiber being configured to project excitation wavelength light to a corresponding focal point within the flow cell; a plurality of fiber optic light transmitting couplers for operatively attaching a plurality of light sources to said linear fiber array; an integrated opto-fluidic investigation module comprising:

2. The integrated opto-fluidic investigation module of claim 1 , wherein the non-circular core optical fiber is a rectangular core optical fiber.

3. The integrated opto-fluidic investigation module of claim 2 , wherein the non-circular core optical fiber is a square core optical fiber.

4. The integrated opto-fluidic investigation module of any one of claims 1 to 3, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile to the focal point.

5. The integrated opto-fluidic investigation module of any one of claims 1 to 4, wherein the non-circular core optical fibers of the linear fiber array are vertically stacked.

6. 6. The integrated opto-fluidic investigation module of claim 5, wherein the cores of adjacent non-circular core optical fibers of the linear fiber array are separated from each other by a distance of 0.1 mm to 0.3 mm.

7. The integrated opto-fluidic investigation module of any one of claims 1 to 6, wherein the linear fiber array is attached to an outer surface of the flow cell.

8. The integrated opto-fluidic investigation module of any one of claims 1 to 7, further comprising focusing optics integrated with the flow cell and configured to relay excitation wavelength light from the non-circular core optical fiber to the focal point.

9. The integrated opto-fluidic investigation module of claim 8 , wherein the focusing optics are embedded in the flow cell.

10. The integrated opto-fluidic investigation module of claim 8 or 9, wherein the focusing optics comprises a plurality of microlenses.

11. The integrated opto-fluidic investigation module of any one of claims 1 to 10, further comprising an optical detector integrated with the flow cell.

12. The integrated opto-fluidic investigation module of claim 11 , further comprising an optical filter disposed between the focal point and the photodetector.

13. 13. The integrated opto-fluidic investigation module of claim 11 or 12, wherein the optical detector is attached to an outer surface of the flow cell.

14. A plurality of light sources; and Integrated optofluidic investigation module It is equipped with The integrated opto-fluidic investigation module comprises: a flow cell having an optically transparent flow passage for carrying particles in a flow stream; a linear fiber array having a plurality of non-circular core optical fibers integral with the flow cell, each optical fiber being configured to project excitation wavelength light to a corresponding focal point within the flow cell; a plurality of fiber optic light transmitting couplers for operably attaching said plurality of light sources to said linear fiber array; The flow cytometer has

15. 1. A method for analyzing a sample, comprising the steps of: (a) introducing a particle sample into a flow cytometer comprising a plurality of light sources and an integrated opto-fluidic interrogation module, the integrated opto-fluidic interrogation module having a flow cell having an optically transparent flow path for carrying particles in a flow stream, a linear fiber array having a plurality of non-circular core optical fibers integrated with the flow cell, each optical fiber being configured to project excitation wavelength light at a corresponding focal point within the flow cell, and a plurality of optical fiber light transmissive couplers for operably attaching the plurality of light sources to the linear fiber array; (b) analyzing the particle sample by flow cytometry.

Images