Integrated optical fluid interrogation module, flow cytometer including integrated optical fluid interrogation module, and methods of use thereof

Abstract

An integrated optical fluid interrogation module is provided. An integrated optical fluid interrogation module of interest comprises: a flow cell comprising an optically accessible flow channel for transporting particles in a flow stream; a linear fiber array integrated with the flow cell and including a plurality of non-circular core fibers, each non-circular core fiber configured to project excitation wavelength light onto a corresponding focus within the flow cell; and a plurality of fiber optic light transmitter couplers for operably attaching the plurality of light sources to the linear fiber array. Flow cytometers including the subject integrated optical fluid interrogation module and methods of using the same are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application Serial No. 63 / 537,165, filed on September 7, 2023; the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Flow cytometry is a technique used to characterize biological materials and, in some instances, to sort biological materials, such as cells of 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 fluid reservoir containing a sheath fluid. A flow cytometer transmits particles, including cells, in a fluid sample to a flow cell as a cell stream while also directing a sheath fluid to the flow cell. To characterize the composition of the flow stream, the flow stream is illuminated with light from a light source. Changes in the material in the flow stream, such as morphology or the presence of a fluorescent label, can result in changes in the observed light, and these changes allow for characterization and separation.

[0004] In conventional flow cytometers, the flow cell must be properly aligned with the light source and light collecting elements so that the light illuminates the flow cell in the intended manner. The assembly of a flow cytometer is often a tedious process that requires a skilled technician to ensure that the light source and light collecting elements are properly aligned with the flow cell. Typically, this alignment is achieved by mounting the light source and / or flow cell on an xyz stage for three-dimensional positioning of the flow cytometer components. In addition, the positioning of mirrors and other optical components must be finely adjusted to properly direct the light emitted by the light source to the flow cell. Such separate components require complex mechanical mounting and may therefore lack robustness and / or be costly. Summary of the invention

[0005] The inventors have recognized that because alignment of the laser beam with the flow core stream is important for signal quality, and available alignment techniques require highly trained personnel, complex adjustable mechanical or electromechanical components, conventional systems are associated with long installation times, long service times, and high costs. Therefore, there is a need for an optical module configured to align a laser beam with the core stream in a flow cell. The integrated optofluidic interrogation module, flow cytometer, method, and kit described herein meet this need.

[0006] Aspects of the present invention include integrated optofluidic interrogation modules. The modules of interest include: a flow cell including an optically accessible flow channel for transporting particles in a flow stream; a linear fiber array integrated with the flow cell and including a plurality of non-circular core fibers, each non-circular core fiber being configured to project excitation wavelength light onto a corresponding focus within the flow cell; and a plurality of fiber optic light transmitter couplers for operably attaching a plurality of light sources to the linear fiber array. In some cases, the non-circular core fiber is a rectangular core fiber, such as a square core fiber. In selected instances, the non-circular core fiber is configured to project excitation wavelength light having a top hat profile onto a focus. In some embodiments, the non-circular core fibers in the linear fiber array can be stacked vertically. In some such embodiments, the cores of adjacent non-circular core fibers in the linear fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm. In some cases, the linear fiber array is attached to the outer surface of the flow cell. In some versions, the module includes a focusing optical system that is integrated with the flow cell and configured to relay excitation light from the non-circular core fiber to the focus. In selected embodiments, the focusing optical system can be embedded in the flow cell. In some versions, the focusing optical system includes a plurality of microlenses. In selected instances, the module includes a light detector (e.g., a forward scattering detector) integrated with the flow cell. In some cases, the module includes an electrical coupler for operably attaching an electrical wire to the light detector. In other cases, the module includes electrical wires. In selected cases, the module includes an optical mask located between the focus and the light detector. In some instances, the light detector is attached to the outer surface of the flow cell. In some embodiments, the module includes a plurality of fiber optic light collecting element couplers for operably attaching a plurality of fiber optic light collecting elements to the flow cell, and in some instances, these fiber optic light collecting element couplers can be stacked (e.g., vertically stacked). Modules according to some embodiments also include 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 certain versions, the module of the present invention includes a collimating lens (e.g., a Fresnel lens) attached to the 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 fiber optic transmitter couplers, where N is an integer from 2 to 10. In selected versions, the width of the flow cell ranges from 75 mm to 125 mm.

[0007] Aspects of the invention also include flow cytometers. The subject flow cytometer includes multiple light sources and an integrated optofluidic interrogation module of the invention (e.g., as described above). Aspects of the invention also include methods of analyzing samples. Methods of interest include introducing a particulate sample into a flow cytometer of the invention and then analyzing the particulate sample using flow cytometry. A kit including an integrated optofluidic interrogation module and its associated components is also provided.

[0008] Aspects of the present invention also include methods of assembling a flow cytometer. Methods of interest include positioning the integrated optofluidic interrogation module of the present invention into a flow cytometer and operably attaching a plurality of light sources to a fiber optic light transmitter coupler to assemble the flow cytometer. In some cases, the method also includes operably attaching electrical wires to an electrical coupler of the module. In an embodiment, the method includes operably attaching a plurality of fiber optic light collecting elements to a plurality of fiber optic light collecting element couplers of the module. In certain instances, the method includes fluidly connecting a sample fluid line of the flow cytometer to a sample fluid input coupler of the module. In certain versions, the method includes fluidly connecting a sheath fluid line of the flow cytometer to a sheath fluid input coupler of the module. In some cases, the method includes fluidly connecting a waste fluid line of the flow cytometer to a fluid output coupler of the module. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention is best understood from the following detailed description when read in conjunction with the accompanying drawings, which include the following:

[0010] Figures 1A to 1C An integrated optofluidic interrogation module according to some embodiments of the present invention is depicted.

[0011] Figure 2 Depicted is a schematic diagram of a flow cytometer including an integrated optofluidic interrogation module according to some embodiments of the present invention.

[0012] Figure 3 A control system according to certain embodiments is depicted.

[0013] FIG. 4A to FIG. 4B A schematic diagram of a particle sorter system is shown in accordance with certain embodiments.

[0014] Figure 5 A block diagram of a computing system is depicted in accordance with certain embodiments.

[0015] Figure 6 Depicted is the irradiance distribution obtained from a linear optical fiber array of the present invention. DETAILED DESCRIPTION

[0016] An integrated optofluidic interrogation module is provided. An integrated optofluidic interrogation module of interest includes: a flow cell including an optically accessible flow channel for transporting particles in a flow stream; a linear fiber array integrated with the flow cell and including a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light onto a corresponding focal point within the flow cell; and a plurality of fiber optic light transmitter couplers for operably attaching a plurality of light sources to the linear fiber array. A flow cytometer including the subject integrated optofluidic interrogation module and methods of using the same are also provided.

[0017] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as these embodiments may of course vary. It should also be understood that since the scope of the present invention will be limited only by the appended claims, the terminology used herein is only used for the purpose of describing specific embodiments and is not intended to be limiting.

[0018] Where a range of values ​​is provided, it is to be understood that, unless the context clearly specifies otherwise, each intermediate value (to one tenth of the unit of the lower limit) between the upper and lower limits of the range and any other specified value or intermediate value in the range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present invention, subject to any specific exclusions in the range. Where the range includes one or two of the limits, the range excluding one or two of those included limits is also included in the present invention.

[0019] Certain ranges are given herein whose values ​​are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that, in the context in which it appears, provides a substantial equivalent to the specifically recited number.

[0020] 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 the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0021] All publications and patents cited in this specification are incorporated herein by reference, as if each individual publication or patent was expressly and individually indicated to be incorporated herein by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications. 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 priority over such publications due to prior invention. In addition, the publication dates provided may be different from the actual publication dates and may need to be independently confirmed.

[0022] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," "the," and the like include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only," and the like when reciting claim elements or using a "negative" limitation.

[0023] As will be apparent to those skilled in the art after reading this disclosure, each independent embodiment described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any enumerated method can be executed in the order of enumerated events or in any other logically possible order.

[0024] Although the system and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be expressly understood that unless expressly provided under 35 U.S.C. §112, the claims should in no way be interpreted as necessarily limited to "means" or "steps", but should be given the full scope of meaning and equivalents of the definitions provided in the claims in accordance with the doctrine of judicial equivalence, and if a claim is expressly provided under 35 U.S.C. §112, it should be given its full legal equivalents under 35 U.S.C. §112.

[0025] Integrated Optofluidic Interrogation Module

[0026] As described above, aspects of the present invention include integrated optofluidic interrogation modules. Modules of interest include: a flow cell including an optically accessible flow channel for transporting particles in a flow stream; a linear fiber array that is integrated with the flow cell and includes a plurality of non-circular core fibers, each of which is configured to project excitation wavelength light onto a corresponding focus within the flow cell; and a plurality of fiber optic transmitter couplers for operably attaching a plurality of light sources to the linear fiber array. An "integrated" optofluidic interrogation module means that at least the flow cell, the linear fiber array, and certain fiber optic transmitter couplers are combined with one another so that they form a coherent whole, rather than existing as separate and distinct elements. The flow cell and the linear fiber array can be integrated together so that they are attached to a fixed position, wherein the fixed position is selected to provide optical alignment with the core flow of the flow cell and the collection optical device associated with the core flow. Thus, in some instances, the components of the integrated optofluidic interrogation module (e.g., flow cell, linear fiber array, etc.) are in fixed positions relative to one another, for example, so that they maintain proper optical alignment over time. In some cases, the components of an integrated optofluidic interrogation module (e.g., a flow cell, a linear fiber array, etc.) cannot be easily separated into component parts without damaging one or more of the parts. Thus, in some cases, the integrated optofluidic interrogation module is non-adjustable, for example, because the components are already in an optimal alignment state. In some cases, the subject integrated optofluidic interrogation module reduces the need for manual adjustments to achieve optical alignment, for example, because each component is in a fixed position relative to each other, thereby maintaining proper optical alignment. In some instances, the integrated optical devices of the integrated optical interrogation modules described herein can improve the collection efficiency of fluorescence emitted by particles in the flow cell, for example, by increasing the amount of fluorescence collected relative to noise.

[0027] As described herein, a "flow cell" is described in its conventional sense and refers to a component (e.g., a cuvette) containing a flow channel for a flow stream for transmitting particles in a sheath fluid. The cuvette of interest includes a container having a channel extending therethrough. The flow stream for which the flow channel is configured may include a liquid sample injected from a sample tube. In some instances, the flow cell includes a light-accessible flow channel. In such an instance, the flow cell includes a transparent material (e.g., quartz) that allows light to pass therethrough. In some cases, the flow cell is configured to be irradiated with light from a light source at one or more interrogation points. The "interrogation point" discussed herein refers to an area within a flow cell in which particles are irradiated with light from a light source, such as for analysis. The size of the interrogation point can vary as desired. For example, where 0 μm represents the optical axis of the light emitted by the light source, the interrogation point may range from -50 μm to 50 μm, such as -25 μm to 40 μm, and includes -15 μm to 30 μm.

[0028] During the use of the integrated optical fluid interrogation module of the present invention, particles can be transmitted through the core flow of the flow stream. The "core flow" discussed herein refers to its conventional meaning to describe a portion of the sheath flow (i.e., the flow stream) in which the particles are transmitted through the flow cell. Typically, particles are transmitted through the core flow in a single-file manner. The size (e.g., diameter) of the core flow can vary as needed. In some instances, the diameter of the core flow may be approximately the same as the diameter of the particle being analyzed. Therefore, in some cases, the diameter of the core flow is 5 μm to 25 μm, including 10 μm to 20 μm. The core flow diameter can be adjusted in proportion to the pressure applied to the particles when the particles are injected into the sheath flow. The flow rate of the sheath liquid remains constant. In this way, the particles are injected into the sheath liquid and focused in a fluid dynamic manner so that a laminar flow is generated, and the particles travel along the same axis at approximately the same rate. Since the particles in the core flow travel along the same axis, the geometry of the core flow is defined by a straight edge.

[0029] The linear fiber array of the integrated optofluidic interrogation module includes a plurality of non-circular core optical fibers. The linear fiber array described herein is an array of optical fibers formed at the end of a fiber bundle, which arranges the constituent optical fibers linearly. The optical fiber can be composed of a cladding and a core. The core is a portion of the optical fiber composed of a transparent material configured to transport light, while the cladding confines the light primarily to the core. In other words, the core has a high refractive index relative to the cladding. The cladding and the core can be composed of the same or different materials. In some cases, the cladding and the core are composed of silicon-based glass (e.g., SiO2). In other embodiments, the transparent material comprises a polymer 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(perfluorobutylene vinyl ether) (CYTOP). In some cases, the core of the optical fiber has relative edges extending parallel to the direction of the core flow, i.e., edges extending parallel to each other and present on opposite sides of the collecting core of the optical fiber. In other words, the projection surface of the core of the optical fiber (i.e., the area of ​​the core through which light is emitted, see Figure 1B ) is defined by straight edges. "Parallel to" the core flow means that the opposite straight edges of the projection surface of the core of the optical fiber are arranged so that they extend in parallel directions relative to the axis along which particles are transported within the core flow. The opposite parallel edges can have any suitable length. In some cases, the length ranges from 5 μm to 100 μm, such as 10 μm to 75 μm, such as 15 μm to 50 μm, and includes 15 μm to 25 μm.

[0030] In addition to having relative edges extending parallel to the direction of the core flow, the optical fiber core of the linear optical fiber array can also have a projection surface having relative edges extending perpendicularly to the direction of the core flow. "Perpendicular to" the core flow means that the relative straight edges of the collection surface of the optical fiber light collector extend in a perpendicular direction relative to the following axis, along which particles are transmitted in the core flow. The relative vertical edges can have any suitable length. In some cases, the length ranges from 5μm to 150μm, such as 50μm to 100μm, such as 60μm to 90μm, and includes 70μm to 80μm. The core can have a plurality of suitable non-circular shapes. For example, in some cases, the non-circular core optical fiber is a rectangular core optical fiber. In some such cases, the non-circular core optical fiber is a square core optical fiber.

[0031] In some instances, the non-circular core fiber is configured to project excitation wavelength light having a top hat profile onto a focal point. In other words, in addition to directing the light without the need for a complex alignment process, the non-circular core fiber can also shape the beam so that it emits a beam having a flat top beam profile from the fiber. The term "top hat" (sometimes also referred to as "flat top") is used herein in its conventional sense to refer to an illumination beam (e.g., light) having a nearly uniform flux (energy density) along one or more axes orthogonal to the optical axis line of the illumination beam. In an embodiment, the output beam of light having a top hat intensity distribution exhibits little or no deviation in relative intensity from each edge to the center along the horizontal axis, wherein the intensity of the beam having the top hat intensity distribution of interest at the center is 95% to 99.9%, such as 96% to 99.5%, of the intensity at the edge along the horizontal axis, and includes 98% to 99% of the intensity at the edge along the horizontal axis. Non-circular core fibers, such as those described herein, are capable of producing a flat top distribution by assuming the function of a mode scrambler. In other words, the rectangular core geometry is able to stimulate coupled optical modes, thereby providing a uniform intensity distribution. This property of non-circular core fibers is discussed in, for example, Hracek, B. and H. (2015). Optik & Photonik, 10(5), 16-18.

[0032] Each non-circular core fiber is configured to project the excitation wavelength light onto a corresponding focus within the flow cell. "Focus" means a specific area of ​​the interrogation zone of the flow cell that is irradiated by one or more lasers. In other words, the module is configured to focus the excitation wavelength light onto a specific part of the flow cell while preventing the light from irradiating the flow cell at another undesired position. The distance between adjacent focal points depends at least in part on the distance between the cores of adjacent optical fibers in the linear fiber array. In some embodiments, the non-circular core optical fibers are assembled into a linear array structure so that the size and spacing of the optical fiber cores correspond to the desired size of particles to be placed in the flow cell to excite the core flow of the flow cell. The non-circular core optical fiber can have any suitable arrangement. In some cases, the non-circular core optical fibers in the linear fiber array are stacked vertically. The distance between adjacent non-circular core optical fibers can vary as needed. In some cases, the optical fibers are in a vertically stacked arrangement, separated only by the thickness of their cladding. In other cases, such as when the focal points are expected to be spaced a larger distance apart, the distances between the claddings of adjacent optical fibers in a linear optical fiber array are spaced from each other by a range of 0.01 mm to 1 mm, such as 0.01 mm to 0.5 mm, such as 0.01 mm to 0.25 mm, such as 0.01 mm to 0.1 mm μm, such as 0.01 mm to 0.05 mm, and including 0.01 mm to 0.025 mm. The distance between the cores of adjacent non-circular core optical fibers in a linear optical fiber array may also vary. In some cases, the cores of adjacent non-circular core optical fibers in a linear optical fiber array are spaced from each other by a distance (e.g., measured from the geometric center of the light projection surface of one core to the light projection surface of an adjacent core), the distance being in the range of 0.01 mm to 1 mm, such as 0.05 mm to 0.5 mm, such as 0.1 mm to 0.25 mm, and including 0.1 mm to 0.15 mm. In some cases, the cores of adjacent non-circular core optical fibers in a linear optical fiber array are spaced from each other by a distance of 0.125 mm.

[0033] The particular arrangement of optical fibers within a linear optical fiber array can be achieved in any convenient manner. In some cases, the linear optical fiber array includes optical fibers placed in V-grooves in a surface, and the size and shape of the V-grooves can be set to keep a certain distance between the optical fibers or not keep a certain distance between them. In other cases, the linear optical fiber array can use holes in the surface to position each optical fiber. When the optical fiber passes through the appropriate holes, proper alignment of the optical fiber with the core flow is achieved. In some cases, the surface is a glass surface. In other cases, the surface is a polymer (e.g., plastic) surface. In some other cases, the surface is a metal surface.

[0034] The linear fiber array can be integrated with the flow cell in any convenient manner. In some cases, the linear fiber array is attached to the outer surface of the flow cell. For example, in some embodiments, the linear fiber array is attached via an adhesive. Any convenient optical adhesive can be used. For example, the adhesive of interest may include epoxy resins, light-cured acrylic resins, elastomers (e.g., silicone, silicone-free silane), cyanoacrylates, and structural adhesives (e.g., adhesives with resins and activators). In some embodiments, the adhesive can be cured 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 wall of the flow cell. In this case, the focusing optical assembly can be incorporated into the structure of the flow cell instead of being attached to its surface. In some cases, the linear fiber array is integrated with the flow cell via a technique such as press fit (i.e., interference fit). Press fit is a known technique, i.e., a hole slightly smaller than the shape of the focusing optical assembly (e.g., lens) is produced in the housing. After the focusing optical assembly and the housing are forced together, the focusing optical assembly and the housing can then be held together by friction.

[0035] As described above, the integrated optofluidic interrogation module of the present invention includes a plurality of fiber optic transmitter couplers for operably attaching a plurality of light sources to a linear fiber optic array. While the light emitting ends of the optical fibers are co-located in the linear fiber optic array, the light receiving ends of the optical fibers are spatially separated and include fiber optic transmitter couplers. These couplers may employ any suitable coupling mechanism configured to operably attach a light source to a linear fiber optic array. Couplings of interest include, but are not limited to, screw couplings, latch couplings, push-pull couplings, bayonet couplings, gendered couplings, snap couplings, combinations thereof, and the like.

[0036] In some cases, the integrated optical fluid interrogation module also includes a plurality of fiber optic transmitters, for example, operably attached to a fiber optic transmitter coupler associated with a linear optical fiber array. As described herein, a "fiber optic transmitter" refers to an elongated structure having a proximal end and a distal end, wherein the elongated structure is made of a light transparent material that is 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 transparent material includes a glass material, such as but not limited to silica (e.g., fused silica). In other embodiments, the transparent material includes a polymer 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(perfluorobutylene vinyl ether) (CYTOP). In some embodiments, the fiber optic light collector is a single-mode optical fiber. As understood in the art, a single-mode optical fiber is an optical fiber that transmits light having one mode at a given time. In other instances, the fiber optic light collector is a multimode optical fiber. Compared to a single-mode optical fiber, a multimode optical fiber simultaneously transmits light having multiple modes. The diameter of the fiber optic light transmitter can vary as desired. In some embodiments, the diameter of the subject fiber optic transmitter ranges from 0.1 mm to 3 mm, such as 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, and including 0.6 mm to 1.2 mm. In some embodiments, the fiber optic transmitter connector (if included) itself can be connected to the laser.

[0037] In some cases, the integrated optofluidic interrogation module of the present invention also includes a focusing optical system integrated with the flow cell, which is configured to relay the excitation light from the non-circular core optical fiber to the focus. The focusing optical system may include, for example, one or more lenses. In this case, the focusing optical system may include any convenient number of lenses, such as 1 to 10 lenses, 2 to 5 lenses, and including 2 to 4 lenses. In some embodiments, one or more lenses in the focusing optical assembly are microlenses. In such embodiments, the subject microlenses may have any convenient size and shape. In some instances, the focusing optical assembly includes microlenses with diameters ranging from 10μ to 5mm, such as 50μm to 2mm and including 100μm to 1mm. In some cases, the subject microlenses are spherical in shape. In an embodiment, the microlens has a flat surface on one side and a spherical (e.g., convex) surface on the other side. In another embodiment, the microlens is an aspherical lens, i.e., a lens having an aspherical or cylindrical size. In an embodiment of a focusing optical system comprising a plurality of microlenses, each microlens in the focusing optical system may be the same or different. For example, the microlenses can have different sizes and / or shapes as needed to focus the excitation wavelength light to the focal point of the flow cell. In some embodiments, the lenses are arranged in an array. The lenses of interest in the focusing optical system can be formed of any suitable material, including but not limited to glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21 or N-LAF35 glass), silica (e.g., fused silica), quartz, crystals (e.g., CaF2 crystals), zinc selenide (ZnSe), F2, germanium titanate (Ge) (e.g., S-TIH11), borosilicates (e.g., BK7), or combinations thereof. The refractive index of the lens of interest may range from 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 and including 1.9 to 2.3.

[0038] In some embodiments, a component (e.g., a lens) of the focusing optical system can modify the beam profile of the light beam on one or more of the horizontal axis and the vertical axis. As described herein, the horizontal axis and the vertical axis refer to axes orthogonal to the optical axis (i.e., the beam path) of the light beam, and in embodiments, form an XY plane of the beam profile. The horizontal axis of the output light beam 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 light beam is parallel to the longitudinal axis of the flow stream. In some embodiments, the focusing optical system of interest modifies the beam profile of the propagating light beam on the horizontal axis. In other embodiments, the lens of interest modifies the beam profile of the propagating light beam on the vertical axis. In other embodiments, the lens of interest modifies the beam profile of the propagating light beam on the horizontal axis and the vertical axis. The focusing optical system in these embodiments may include a diffractive optical device or a refractive optical device. In some embodiments, the focusing optical system includes a biconical lens; in other embodiments, the focusing optical system includes an achromatic lens.

[0039] The focusing optical system can be integrated with the flow cell by any of a variety of different means as needed. For example, the focusing optical system can be stably associated with the outer surface of the flow cell. In some embodiments, the focusing optical system can be attached via an adhesive attachment (e.g., the above-mentioned adhesive). In some embodiments, the components of the focusing optical system (e.g., one or more lenses) can be incorporated (i.e., embedded) into the structure of the flow cell. In some cases, the components of the focusing optical system are embedded in the wall of the flow cell. In this case, the focusing optical system can be incorporated into the structure of the flow cell instead of being attached to its surface. In some cases, the lens of the optical component can be incorporated into the structure of the housing via a press fit (i.e., an interference fit). In other embodiments, the focusing optical system is attached to the outer surface of the flow cell via optical contact bonding. As is known in the art, optical contact bonding involves joining two surfaces by utilizing the intermolecular forces existing between the surfaces. The intermolecular forces associated with optical contact bonding include van der Waals forces, hydrogen bonds, and dipole-dipole interactions. In embodiments of the invention involving optical contact bonding, the surfaces of the focusing optics and flow cell may be substantially conformal (ie, flat and smooth) such that intermolecular forces can pull the elements together.

[0040] In some embodiments, the integrated optical fluid interrogation module of the present invention includes one or more photodetectors integrated with the flow cell. The number of photodetectors integrated with the flow cell can vary as needed. In some cases, the integrated optical fluid interrogation module includes a single photodetector integrated with the flow cell. In other cases, the integrated optical fluid interrogation module includes 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, and including 10 or more photodetectors. One or more photodetectors can be integrated with the flow cell via any suitable technology, such as the above-mentioned technology (e.g., adhesive, press fit, etc.). In selected cases, the photodetector is attached to the outer surface of the flow cell.

[0041] The light detector can be any convenient particle modulated light detector. "Particle modulated light" means light received from particles after light from a light source illuminates the particles in the flow cell. In some cases, particle modulated light is side scattered light. As described herein, side scattered light refers to light refracted and reflected from the surface and internal structure of the particles. In other embodiments, particle modulated light includes forward scattered light (i.e., light that passes through or travels around the particles primarily in the forward direction). In yet other cases, particle modulated light includes fluorescence (i.e., light emitted by a fluorescent dye after the fluorescent dye is illuminated with light of an excitation wavelength).

[0042] In some embodiments, one or more particle modulated light detectors include one or more forward scattered light detectors configured to detect forward scattered light. For example, the subject module may include 1 forward scattered light detector or multiple forward scattered light detectors, such as 2 or more, such as 3 or more, such as 4 or more and including 5 or more forward scattered light detectors. In some embodiments, the module includes 1 forward scattered light detector. In other embodiments, the module includes 2 forward scattered light detectors.

[0043] Any convenient detector for detecting the collected light can be used in the forward scattered light detector described herein. Detectors of interest may include, but are not limited to, optical sensors or detectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In some embodiments, the collected light is measured using 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, such as a photomultiplier tube having an effective detection surface area of ​​0.01 cm per region. 2 Up to 10cm 2 , for example 0.05cm 2 Up to 9cm 2 , for example 0.1cm 2 Up to 8cm 2 , for example 0.5cm 2 Up to 7cm 2 And including 1cm 2 Up to 5cm 2 Photomultiplier tube.

[0044] In an embodiment, the forward scattered light detector is configured to measure light continuously or at discrete intervals. In some instances, the detector of interest is configured to measure the collected light continuously. In other instances, 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, and including every 1000 milliseconds or at some other intervals.

[0045] In some embodiments, the module includes one or more wavelength separators between the flow cell and one or more particle modulated light detectors. The term "wavelength separator" is used in its conventional sense herein and refers to an optical component configured to separate the light collected from the sample into a predetermined spectral range. In some embodiments, the module includes a single wavelength separator. In other embodiments, the module includes a plurality of wavelength separators, such as 2 or more wavelength separators, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 15 or more, such as 25 or more, such as 50 or more, such as 75 or more and including 100 or more wavelength separators. In some embodiments, the wavelength separator is configured to separate the light collected from the sample into a predetermined spectral range by passing the light with a predetermined spectral range and reflecting the light of one or more remaining spectral ranges. In other embodiments, the wavelength separator is configured to separate the light collected from the sample into a predetermined spectral range by passing the light with a predetermined spectral range and absorbing the light of one or more remaining spectral ranges. In yet other embodiments, the wavelength separator is configured to spatially diffract the light collected from the sample into a predetermined spectral range. Each wavelength separator can 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 in the subject light detection system is a dichroic mirror.

[0046] In some cases, one or more photodetectors combined with the flow cell include an electrical coupler for operably attaching wires to the one or more photodetectors. Any element configured to couple a photodetector (such as those described above) to a wire to convey a signal generated by the photodetector can be used, such as a thread, a bayonet, a bracket, a blind-mate connection, etc. In some cases, the module also includes a wire. The wire can be made of any suitable insulating conductive material (such as copper).

[0047] In some instances, the integrated optical fluid interrogation module includes one or more optical adjustment elements, and the one or more optical adjustment elements are configured to modulate the light received by one or more photodetectors. For example, in some instances, the module includes an optical filter located between the focus and the photodetector. The optical filter is configured to constrain, reduce or limit the propagation of at least one or more wavelengths of light (e.g., one or more wavelengths of light of a laser used to illuminate the sample) from the sample to the effective surface of the photodetector. The optical filter can limit the propagation of one or more different wavelengths of light, such as 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 different wavelengths of light and include limiting the propagation of 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. In addition or alternatively, the module of the present invention may include an optical mask located between the focus and the photodetector. In some cases, the optical mask is configured to block certain portions of light from being detected (eg, an incident beam from a laser).

[0048] In addition to or in lieu of a light detector integrated with the flow cell, the integrated optical fluid interrogation module may include one or more fiber optic light collecting element couplers for operably attaching one or more fiber optic light collecting elements to the flow cell. In such an embodiment, the fiber optic light collecting element is configured to collect particle modulated light emitted by particles transmitted through one or more focal points after being irradiated with excitation wavelength light. The fiber optic light collecting element coupler may operably attach one or more fiber optic light collecting elements to the flow cell via any convenient mechanism. In some embodiments, the fiber optic light collecting element coupler may operably attach the fiber optic light collecting element to the flow cell so that the fiber optic light collecting element requires very little optical adjustment (if any) to ensure proper optical alignment with the flow cell. In some embodiments, the fiber optic light collecting element coupler may physically couple the fiber optic light collecting element to the flow cell, for example using an adhesive. In other embodiments, the fiber optic light collecting element coupler is configured to releasably attach the fiber optic light collecting element. Suitable coupling mechanisms include, for example, screws, latches, push-pull, bayonet, snap, male and female connectors, and the like.

[0049] The number of fiber optic light collecting element couplers can vary. In some cases, the integrated optofluidic interrogation module includes a single fiber optic light collecting element coupler. In other cases, the module includes multiple fiber optic light collecting element couplers, such as 2 or more couplers, such as 3 or more couplers, such as 4 or more couplers, such as 5 or more couplers, such as 6 or more couplers, such as 7 or more couplers, such as 8 or more couplers, such as 9 or more couplers, and including 10 or more couplers. When the module includes multiple fiber optic light collecting element couplers, the couplers in the multiple couplers can be arranged relative to each other in any suitable manner. In some embodiments, the fiber optic light collecting element couplers are stacked vertically. In such embodiments, each fiber optic light collecting element can be configured to collect light from a specific focus.

[0050] In embodiments of the method including an optical fiber array and one or more fiber optic light collecting element couplers, the linear optical fiber array and the one or more fiber optic light collecting element couplers can occupy any convenient position relative to each other relative to the flow cell. In some embodiments, the fiber optic light collecting element coupler is attached to the flow cell on the opposite side of the module relative to the optical fiber linear optical fiber array. In other embodiments, the fiber optic light collecting element coupler is attached to the flow cell at an orthogonal or nearly orthogonal position relative to the linear optical fiber array. One or more fiber optic light collecting element couplers can be positioned within the module so that the fiber optic light collecting element connected thereto is in a suitable position to collect one or more types of particle modulated light (e.g., forward scatter, side scatter, fluorescence, or a combination thereof).

[0051] In some embodiments, the module also includes one or more fiber optic light collecting elements operably attached to a plurality of fiber optic light collecting elements. In certain embodiments, the fiber optic light collecting element is configured to collect fluorescence emitted by particles transmitted through the focus after being irradiated with excitation wavelength light. In some embodiments, the fiber optic light collecting element includes a transparent material, such as a glass material, including but not limited to silica (e.g., fused silica). In other embodiments, the transparent material includes a polymer 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(perfluorobutylene vinyl ether) (CYTOP). The diameter of the fiber optic light transmitter may vary as desired. In some embodiments, the diameter of the subject fiber optic light transmitter ranges from 0.1 mm to 3 mm, such as 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, and includes 0.6 mm to 1.2 mm. In some embodiments, the subject module includes a single fiber optic light collecting element. In other embodiments, the module includes a plurality of fiber optic light collecting elements. For example, a flow cytometer of interest may include a fiber optic light collection element and / or a fiber optic light collection element coupler for each focal point. Thus, a flow cytometer may include one or more fiber optic light collection elements, such as two or more, three or more, four or more, five or more, and including six or more fiber optic light collection elements. In some embodiments, one or more fiber optic light collection elements are optically aligned with the core flow and include a collection surface having relative edges extending parallel to the direction of the core flow. Exemplary fiber optic light collection elements are described in U.S. Patent Application Publication No. 2022 / 0236165, the disclosure of which is incorporated herein by reference in its entirety.

[0052] In an embodiment, the integrated optofluidic interrogation module includes collimating optics located between the flow stream and the photodetector and / or fiber optic light collection element 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 the outer surface of the cuvette. The collimating lens can be attached to the outer surface of the cuvette via any suitable means (e.g., an optical adhesive). In selected cases, the collimating lens is an injection compression molded microlens. In some cases, the collimating lens is a Fresnel lens.

[0053] As described above, the number of non-circular core fibers, focal points, and fiber optic transmitter couplers may be different in embodiments of the present invention. In some cases, the modules of the present invention include an equal number of non-circular core fibers, focal points, and fiber optic transmitter couplers. In other words, the integrated optofluidic interrogation module includes N non-circular core fibers, N focal points, and N fiber optic transmitter couplers, where N is any suitable integer. In some cases, N ranges from 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, and includes 2 to 4. For example, N can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in some cases. In the case of including these elements, the module can also include N fiber optic light collection elements and / or N fiber optic transmitters.

[0054] Any convenient flow cell that propagates a fluid sample to a sample interrogation region may be employed in the subject module. In some embodiments, the flow cell is a cylindrical flow cell, a frustoconical flow cell, or a flow cell comprising a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating in a flat surface having an orifice transverse to the longitudinal axis.

[0055] In some cases, the module includes a fluid output coupler for receiving a flow stream from a flow cell. The fluid output coupler can be any component configured to collect flow streams from a flow cell and convey flow streams. In some cases, the fluid output coupler includes a connector at a distal end, which is configured so that a waste liquid line (e.g., a pipeline) can be connected to the fluid output coupler. Any suitable connector can be included. Exemplary connectors include, for example, quick disconnect connectors, threaded connectors, Luer (luer) connectors, multi-port connectors, tri clamp fittings, and puncture and seal sterile connectors. Suitable quick disconnect connectors include, but are not limited to, snap-on (ball-latch) connectors, bayonet connectors, threaded connectors, non-latch connectors, single-close connectors, double-close connectors, non-close connectors, dry disconnect connectors, roller lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors. The waste liquid line can deliver liquid to another location, such as a waste liquid reservoir. In some embodiments where the module is configured for vacuum-driven fluids, the fluid output coupler is composed of an outlet connector. The outlet connector of interest has an elongated structure and an opening at the proximal end of the elongated structure for receiving a flow stream from the distal end of the flow cell. In some cases, the outlet connector is configured to reduce the formation of bubbles at the interface between the outlet connector and the flow cell (e.g., a cuvette of the flow cell). Suitable outlet connectors are described in U.S. Patent Application Publication No. 2023 / 0046207, which description is incorporated herein by reference.

[0056] In an embodiment, the module also includes a sample fluid input coupler for injecting sample fluid into the flow cell. In some cases, for example, the sample fluid input coupler can include an orifice with the orifice interface of the flow cell so that the sample fluid can be provided to the flow cell. In addition, in some embodiments, the sample fluid input coupler includes a connector, which is configured to connect the sample fluid input coupler fluid to the fluid line (for example, pipeline) connected to the sample fluid source. Any suitable connector can be included. Exemplary connectors include, for example, quick disconnect connectors, threaded connectors, Luer connectors, multi-port connectors, three-clamp connectors, and puncture and seal aseptic connectors. Suitable quick disconnect connectors include, but are not limited to, snap-on (ball-latch) connectors, bayonet connectors, threaded connectors, non-latch connectors, single shut-off connectors, double shut-off connectors, non-shut-off connectors, dry disconnect connectors, roller lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors. In some cases, the sample fluid input coupler is a sample injection needle or includes a sample injection needle. In some such cases, the sample injection needle has a channel therethrough for delivering the sample fluid from the sample injection line at the proximal end to the flow cell at the distal end to produce a core flow. In some instances, the sample injection needle is configured and positioned to maintain a complete core flow under flow conditions that differ by one or more orders of magnitude. Sample injection needles that may be suitable for use are described in U.S. Patent Application No. 18 / 215,402; the disclosure of which is incorporated herein by reference in its entirety.

[0057] In an embodiment, the module also includes a sheath liquid input coupler for injecting sheath liquid into the flow cell. For example, the sheath liquid input coupler may include an orifice interfaced with the orifice of the flow cell so that sheath liquid can be provided to the flow cell. In addition, the sheath liquid input coupler includes a connector configured to connect the sheath liquid input coupler fluid to a fluid line (e.g., a pipeline) connected to a sheath liquid source. Any suitable connector may be included. Exemplary connectors include, for example, quick disconnect connectors, threaded connectors, Luer connectors, multi-port connectors, tri-clamp connectors, and puncture and seal sterile connectors. Suitable quick disconnect connectors include, but are not limited to, snap-on (ball-latch) connectors, bayonet connectors, threaded connectors, non-latch connectors, single-close connectors, double-close connectors, non-close connectors, dry disconnect connectors, roller wheel lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors.

[0058] Figures 1A to 1C An integrated optofluidic interrogation module according to some embodiments of the present invention is depicted. Figure 1AAs shown, the integrated optofluidic interrogation module 100 includes a flow cell 101, which is configured to transmit particles in a flow stream 102 (i.e., the core stream of the flow stream 102). The integrated optofluidic interrogation module 100 has a plurality of fiber optic transmitter couplers 111a, 111b, and 111c. Each fiber optic transmitter coupler can be configured to be operably attached to a fiber optic transmitter, which itself can be coupled to a laser. In an alternative embodiment, the fiber optic transmitter couplers 111a-111c can be directly coupled to the laser (i.e., without an intermediate optical fiber). A linear optical fiber array 113 is also shown that is integrated with the flow cell 101 and includes a plurality of non-circular core optical fibers, each of which is configured to project excitation wavelength light onto a corresponding focus within the flow stream 102 in the flow cell 101. The light propagates through optical fibers 112a, 112b, and 112c via fiber optic transmitter couplers 111a, 111b, and 111c, respectively, and propagates to the linear optical fiber array 113. Figure 1A In an embodiment of the present invention, the module 100 further comprises a focusing optical system, shown in the form of a lens 114, which is integrated with the flow cell 101 and configured to relay the excitation light from the non-circular core fiber to a focal point. Alternatively, the focusing optical system may comprise a plurality of micro lenses.

[0059] Figure 1B 1 shows a view of the light emitting surface of the linear optical fiber array 113. Figure 1B As shown, the linear optical fiber array 113 includes a vertical stack 115 of non-circular core optical fibers 116a, 116b, and 116c. The non-circular core optical fiber 116a is continuous with the optical fiber 112a and the optical fiber optical transmitter coupler 111a, so that the laser light received at the optical fiber optical transmitter 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 optical transmitter coupler 111b, so that the laser light received at the optical fiber optical transmitter 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 transmitter coupler 111c, so that the laser light received at the optical fiber optical transmitter coupler 111c propagates along the optical fiber 112c and is emitted at the non-circular core optical fiber 116c. The non-circular core optical fibers 116a, 116b, and 116c include cores 117a, 117b, and 117c, respectively. Figure 1BIn an embodiment of the present invention, cores 117a, 117b, and 117c are rectangular in shape and are configured to project excitation wavelength light having a top-hat profile to a focal point. The cores of adjacent non-circular core fibers are separated by a core spacing distance d, which in this case is determined by the cladding thickness of each of the non-circular core fibers 116a, 116b, and 116c (i.e., the portion of the non-circular core fibers 116a, 116b, and 116c surrounding the cores 117a, 117b, and 117c, respectively). In this arrangement, the fiber output is mechanically and optically matched to the desired core flow position of the corresponding laser excitation beam.

[0060] Figure 1C An embodiment of a flow cell 101 is shown. A linear fiber array 113 projects excitation wavelength light, as described above with respect to Figure 1A and Figure 1B As described. The light passes through the lens 114. The non-circular core fiber 116a projects the excitation wavelength light onto a focus 126a within the flow stream 102 of the flow cell 101. The non-circular core fiber 116b projects the excitation wavelength light onto a focus 126b within the flow stream 102 of the flow cell 101. The non-circular core fiber 116c projects the excitation wavelength light onto a focus 126c within the flow stream 102 of the flow cell 101. Irradiating the particles in the focus 126a-126c will produce forward scattered light that propagates primarily in the forward direction. The forward scattering detector 123 is integrated with the flow cell 101 and is configured to detect the forward scattered light. An optical mask 121 and an optical filter 122 integrated with the flow cell 101 are also shown. An electrical coupler 124 for operably attaching an electrical wire 125 to the detector 123 is attached to the forward scattering detector 123. In addition to forward scattered light, irradiating particles in the focal points 126a-126c can generate side scattered light and / or fluorescence. A vertical stack 119 comprising fiber optic light collecting element couplers 119a, 119b, and 119c is used to operably attach fiber optic light collecting elements 120a, 120b, and 120c to the flow cell 101, respectively. Fiber optic light collecting elements 120a, 120b, and 120c operably attached to the fiber optic light collecting element couplers 119a, 119b, and 119c can be configured to deliver fluorescence and / or side scattered light from the focal points 126a, 126b, and 126c, respectively. The light can be delivered to one or more light detectors as needed.

[0061] Flow cytometry

[0062] Aspects of the present invention include flow cytometers. The flow cytometer of interest includes multiple light sources and an integrated optofluidic interrogation module of the present invention (e.g., as described above). The number of light sources in the flow cytometer can vary. In some instances, the number of light sources ranges from 2 to 10, such as 2 to 5, and includes 2 to 4. Any convenient light source can be used as the light source described herein. In some embodiments, the light source is a laser. In an embodiment, the laser can be any convenient laser, such as a continuous wave laser. For example, the laser can 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 can be a helium-neon (HeNe) laser. In some instances, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser or a combination thereof. In other examples, the subject flow cytometer includes a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In other examples, the laser of interest includes a metal-vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In other examples, the subject flow cytometer includes a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, a Yb2O3 laser or a cerium-doped laser and combinations thereof.

[0063] The laser light source according to some embodiments may also include one or more optical adjustment components. In some embodiments, the optical adjustment component is located 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 some other characteristics of the illumination (e.g., illumination direction, wavelength, beam width, beam intensity, and focus). The optical adjustment protocol may include any convenient device for adjusting one or more characteristics of the light source, including but not limited to lenses, reflectors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In some embodiments, the flow cytometer of interest includes one or more focusing lenses. In one example, the focusing lens may be a de-magnifying lens. In other embodiments, the flow cytometer of interest includes an optical fiber.

[0064] The light source can be positioned at any suitable distance from the flow cell, for example, the light source is spaced 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 5 mm or more, for example 10 mm or more, for example 25 mm or more and including a distance of 100 mm or more from the flow cell. In addition, the light source can be positioned at any suitable angle relative to the flow cell, for example, at an angle of 10 to 90 degrees, for example 15 to 85 degrees, for example 20 to 80 degrees, for example 25 to 75 degrees and including an angle of 30 to 60 degrees, for example, at an angle of 90 degrees.

[0065] In some embodiments, the light source of interest includes a plurality of lasers configured to provide laser light for discretely illuminating the flow stream, such as 2 or more lasers configured to provide laser light for discretely illuminating the flow stream, such as 3 or more lasers, such as 4 or more lasers, such as 5 or more lasers, such as 10 or more lasers and including 15 or more lasers. Depending on the desired wavelength of light for illuminating the flow stream, each laser can have a specific wavelength of 200nm to 1500nm, such as 250nm to 1250nm, such as 300nm to 1000nm, such as 350nm to 900nm and including 400nm to 800nm. In some embodiments, the laser of interest can include one or more of a 405nm laser, a 488nm laser, a 561nm laser, and a 635nm laser.

[0066] The fiber optic light transmitter coupler of the linear fiber array of the integrated optofluidic interrogation module can be configured to directly interface with the laser so that light output by the laser propagates to the appropriate optical fiber in the linear fiber array. In an embodiment, the flow cytometer of the present invention includes a fiber optic light collection element operably attached to the fiber optic light collection element coupler of the integrated optofluidic interrogation module. Detailed information about the fiber optic light collection element is provided above with reference to the integrated optofluidic interrogation module of the present invention.

[0067] The flow cytometer of the present invention may include a light detector operably attached to a fiber optic light collecting element. The light detector is configured to detect particle modulated light transmitted by the fiber optic light collecting element and generate a signal based on the characteristics (e.g., intensity) of the particle modulated light. For example, one or more particle modulated light detectors may include one or more side scattering light detectors for detecting side scattering wavelengths of light (i.e., light refracted and reflected from the surface and internal structure of the particles). In some embodiments, the module includes a single side scattering light detector. In other embodiments, the module includes multiple side scattering light detectors, such as 2 or more, such as 3 or more, such as 4 or more, and including 5 or more.

[0068] Any convenient detector for detecting the collected light can be used in the side scattered light detector described herein. Detectors of interest may include, but are not limited to, optical sensors or detectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, the collected light is measured using 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 certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area of ​​0.01 cm per region. 2 Up to 10cm 2 , for example 0.05cm 2 Up to 9cm 2 , for example 0.1cm 2 Up to 8cm 2 , for example 0.5cm 2 Up to 7cm 2 And including 1cm 2 Up to 5cm 2 Photomultiplier tube.

[0069] In an embodiment, the subject module also includes a fluorescence detector configured to detect light of one or more fluorescence wavelengths. In other embodiments, the module includes multiple fluorescence detectors, such as 2 or more, such as 3 or more, such as 4 or more, 5 or more, 10 or more, 15 or more, and including 20 or more fluorescence detectors.

[0070] Any convenient detector for detecting the collected light may be used in the fluorescence detector described herein. Detectors of interest may include, but are not limited to, optical sensors or detectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, the collected light is measured using 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 certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area of ​​0.01 cm per region. 2 Up to 10cm 2 , for example 0.05cm 2 Up to 9cm 2 , for example 0.1cm 2 Up to 8cm 2 , for example 0.5cm 2 Up to 7cm 2 And including 1cm 2 Up to 5cm 2 Photomultiplier tube.

[0071] When the subject module includes multiple fluorescence detectors, each fluorescence detector can be the same, or the collection of fluorescence detectors can be a combination of different types of detectors. For example, in the case where the subject module includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector (or imaging sensor) is a CMOS type device. In other embodiments, the first fluorescence detector and the second fluorescence detector are both CCD type devices. In some other embodiments, the first fluorescence detector and the second fluorescence detector are both CMOS type devices. In other embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector is a photomultiplier tube (PMT). In some other embodiments, the first fluorescence detector is a CMOS type device and the second fluorescence detector is a photomultiplier tube. In some other embodiments, the first fluorescence detector and the second fluorescence detector are both photomultiplier tubes.

[0072] In embodiments of the present disclosure, the fluorescence detector of interest is configured to measure light collected at one or more wavelengths, such as at 2 or more wavelengths, such as at 5 or more different wavelengths, such as at 10 or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and including measuring light emitted by a sample in a flow stream at 400 or more different wavelengths. In some embodiments, 2 or more detectors in a module as described herein are configured to measure collected light of the same or overlapping wavelengths.

[0073] In some embodiments, the fluorescence detector of interest is configured to measure the light collected in a certain wavelength range (e.g., 200nm to 1000nm). In certain embodiments, the detector of interest is configured to collect spectrum in a certain wavelength range. For example, a module may include one or more detectors, and the one or more detectors are configured to collect spectrum in one or more wavelength ranges in the wavelength range of 200nm to 1000nm. In some other embodiments, the detector of interest is configured to measure the light emitted by the sample in the flow stream at one or more specific wavelengths. For example, a module may include one or more detectors, and the one or more detectors are configured to measure the light at one or more wavelengths in 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, 617nm and any combination thereof. In certain embodiments, one or more detectors may be configured to pair with specific fluorophores, such as those used with the sample in a fluorescence assay.

[0074] As described above, embodiments of the integrated optical fluid 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 a flow cell, and / or a fluid output coupler for receiving a flow stream from a flow cell. The flow cytometer may include any suitable one or more 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 fluidically connected to a sample fluid line (e.g., a pipe) that is fluidically connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidically connected to a sheath fluid line that is fluidically connected to a sheath fluid reservoir. Similarly, the flow cytometer may include any suitable one or more mechanisms for managing waste fluid from the flow stream. The fluid output coupler may be fluidically connected to a waste liquid line that is fluidically connected to a waste liquid storage device. A fluid management system that may be applicable to the subject flow cytometer is provided in U.S. Patent Application Publication No. 2022 / 0341838, the entire disclosure of which is incorporated herein by reference.

[0075] Suitable flow cytometry systems may include, but are not limited to, 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 edition, Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49(pt1): 17-28; Linden et al., Semin Throm Hemost. 2004 Oct; 30(5): 502-11; Alison et al. J Pathol, 2010 Dec; 222(4): 335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosure of which is incorporated herein by reference. In certain embodiments, the flow cytometry system of interest includes a BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TMFlow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter, BD Biosciences FACSAria TM II cell sorter, BD Biosciences FACSAria TM III cell sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorter, BD Biosciences FACSymphony TM S6 cell sorter, BD Biosciences FACSDiscover TM Cell sorter, etc.

[0076] In some embodiments, the subject system is a flow cytometry system, such as 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, 9,0 95,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, 4,498,766, the entire disclosures of which are incorporated herein by reference. In some embodiments where the flow cytometer is a particle sorter, the particle sorter is an image-activated particle sorter. Image-activated particle sorters are described in U.S. Provisional Patent Application Nos. 63 / 431,803 and 63 / 465,057; the entire disclosures of which are incorporated herein by reference.

[0077] Figure 2 A schematic diagram of a flow cytometer including an integrated optofluidic interrogation module 220 according to some embodiments of the present invention is shown. The integrated optofluidic interrogation module 220 includes a flow cell 205, a linear optical fiber array 204, a focusing optical system 206 including a plurality of microlenses, a cuvette 207, a collimating lens 208, a fiber optic light collection element coupler 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. Figure 2 The flow cytometer shown includes lasers 201a, 201b, and 201c, which are operably attached to optical fibers 203a, 203b, and 203c via fiber optic light delivery connectors 202a, 202b, and 202c, respectively. The optical fibers 203a-203c 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 (e.g., as described above with respect to Figure 1B Each non-circular core optical fiber is configured to project the excitation wavelength light onto a corresponding focus within the cuvette 207 of the flow cell 205. Figure 2In an embodiment of the invention, the optical fluid interrogation module 220 includes a collimating lens 208 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 proceeds to the forward scattering detector 212 after passing through the optical mask 210 and the optical filter 211. The side scattered light and / or fluorescence can be collected by a fiber optic light collection element (not shown) operably attached to the fiber optic light collection element connector 209a-209c. The sample fluid can be supplied to the flow cell 205 via a sample fluid input coupler 214, which itself can be fluidically connected to a sample fluid line (e.g., a pipe) connected to a sample fluid reservoir fluid. The sheath fluid can be supplied to the flow cell 205 via a sheath fluid input coupler 213, which itself can be fluidically connected to a sheath fluid line connected to a sheath fluid reservoir fluid. Sheath fluid (eg, waste) flowing out of the flow cell can be removed via fluid output coupler 215. A waste line can be fluidly connected to fluid output coupler 215, which can deposit waste fluid in a waste reservoir.

[0078] In operation, the cytometer operation is controlled by the controller / processor 290, and the measurement data from the detector can be stored in the memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detector to receive the output signal from the detector, and can also be coupled to the electrical and electromechanical components of the flow cytometer to control the lasers 201a-201c, fluid flow parameters, etc. Input / output (I / O) capabilities 297 can also be provided in the system. The memory 295, the controller / processor 290 and the I / O 297 can be completely set as an integrated part of the flow cytometer. In such an embodiment, the display can also form a part of the I / O capability 297 for presenting experimental data to the user of the cytometer 210. Alternatively, the memory 295 and the controller / processor 290 and some or all of the I / O capabilities can be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, some or all of the memory 295 and the controller / processor 290 can communicate wirelessly or wired with the cytometer 210. Controller / processor 290 along with memory 295 and I / O 297 can be configured to perform various functions related to the preparation and analysis of flow cytometric experiments.

[0079] Different fluorescent molecules in a group of fluorescent dyes for flow cytometer experiments will emit light in their own characteristic wavelength bands. Specific fluorescent labels for experiments and their associated fluorescence emission bands can be selected to be substantially consistent with the filter window of the detector. I / O 297 can be configured to receive data about flow cytometer experiments, which have a group of fluorescent labels and multiple cell colonies with multiple marks, and each cell colony has a subset of the multiple marks. I / O 297 can also be configured to receive biological data, label density data, emission spectrum data, data and cytometer configuration data that one or more marks are assigned to one or more cell colonies by one or more marks. Flow cytometer experimental data (such as label spectrum characteristics and flow cytometer configuration data) can also be stored in the memory 295. Controller / processor 290 can be configured to evaluate one or more distributions of labels assigned to marks.

[0080] In some embodiments, the subject flow cytometer is a particle sorting system configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed on 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 decision module having multiple sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781, filed on December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting sample components includes a particle sorting module having a deflection plate, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed on March 28, 2017, the disclosure of which is incorporated herein by reference.

[0081] Figure 3 A functional block diagram of one example of a control system (eg, processor 300) for analyzing and displaying biological events is shown. Processor 300 can be configured to implement various processes for controlling the graphical display of biological events.

[0082] The flow cytometer or sorting system 302 can be configured to acquire biological event data. For example, the flow cytometer can generate flow cytometric event data (e.g., particle modulated light data). The flow cytometer 302 can be configured to provide the biological event data to the processor 300. A data communication channel can be included between the flow cytometer 302 and the processor 300. The biological event data can be provided to the processor 300 via the data communication channel.

[0083] Processor 300 can be configured to receive biological event data from flow cytometer 302. The biological event data received from flow cytometer 302 can include flow cytometry event data. Processor 300 can be configured to provide a graphical display of a first drawing including biological event data to display device 306. Processor 300 can also be configured to present a region of interest as, for example, a gate (e.g., a first gate) overlaid on the first drawing, surrounding a population of biological event data shown by display device 306. In some embodiments, a gate can be a logical combination of one or more graphical regions of interest drawn on a single parameter histogram or a bivariate graph. In some embodiments, a display can be used to display particle parameters or saturated detector data.

[0084] Processor 300 can also be configured to display the bio-event data inside the door in a different manner than other events in the bio-event data outside the door on display device 306. For example, processor 300 can be configured to present the color of the bio-event data contained inside the door as a different color than the bio-event data outside the door. Display device 306 can be implemented as a monitor, tablet computer, smart phone, or other electronic device configured to present a graphical interface.

[0085] The processor 300 can be configured to receive a door selection signal identifying a door from a first input device. For example, the first input device can be implemented as a mouse 310. The mouse 310 can initiate a door selection signal to the processor 300 to identify a door to be displayed on the display device 306 or manipulated via the display device 306 (e.g., by clicking on or in the desired door when the cursor is at the desired door). In some implementations, the first device can be implemented as a keyboard 308 or other device for providing input signals to the processor 300, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices can include multiple input functions. In such an implementation, the input functions can all be considered input devices. For example, Figure 3 As shown, the mouse 310 can include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0086] A triggering event can cause processor 300 to change the way data is displayed, which portions of the data are actually displayed on display device 306, and / or can cause processor 300 to provide input for further processing, such as selecting a population of interest for particle sorting.

[0087] In some embodiments, processor 300 can be configured to detect when mouse 310 initiates gate selection. Processor 300 can also be configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a particular distribution of bio-event data received by processor 300. In some embodiments, processor 300 expands a first gate so that a second gate is generated (e.g., as described above).

[0088] Processor 300 can be connected to storage device 304. Storage device 304 can be configured to receive and store biological event data from processor 300. Storage device 304 can also be configured to receive and store flow cytometry event data from processor 300. Storage device 304 can also be configured to allow processor 300 to retrieve biological event data, such as flow cytometry event data.

[0089] The display device 306 can be configured to receive display data from the processor 300. The display data can include plots of the biological event data and gates outlining portions of the plots. The display device 306 can also be configured to change the information presented based on input received from the processor 300 in combination with input from the flow cytometer 302, the storage device 304, the keyboard 308, and / or the mouse 310.

[0090] In some implementations, the processor 300 can generate a user interface to receive sample events for sorting. For example, the user interface can include a mechanism for receiving sample events or sample images. Sample events or images or sample gates can be provided before collecting event data for a sample or based on an initial event set for a portion of the sample in the sample.

[0091] Figure 4A is a schematic diagram of a particle sorting system 400 (e.g., flow cytometer 302) according to one embodiment of the present invention. In some embodiments, the particle sorting system 400 is a cell sorting system. Figure 4A As shown, a droplet formation transducer 402 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 401, which can be coupled to a nozzle 403, can include a nozzle 403, or can be a nozzle 403. Within the fluid conduit 401, a sheath fluid 404 hydrodynamically focuses a sample fluid 406 including 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 arranged in a single file to pass through a monitoring area 411 (e.g., where the laser intersects the stream) illuminated by an illumination source 412 (e.g., a laser). The vibration of the droplet formation transducer 402 causes the moving fluid column 408 to break into a plurality of droplets 410, some of which contain particles 409.

[0092] In operation, a detection station 414 (e.g., an event detector) identifies when a particle of interest (or a cell of interest) passes through the monitoring area 411. The detection station 414 feeds a timing circuit 428, which in turn feeds a flash charge circuit 430. At a droplet breakoff point informed by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 408 so that the droplet of interest carries a charge. The droplets of interest can include 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 droplets into a vessel such as a collection tube or a multi-well or micro-well sample plate, where the wells or micro-wells can be associated with droplets of particular interest. As Figure 4A As shown, the droplets can be collected in a drain container 438 .

[0093] A detection system 416 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal when a particle of interest passes through the monitoring area 411. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 416 allows the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 416 can feed into an amplitude signal 420 and / or a phase signal 418, which in turn feeds (via amplifier 422) into an amplitude control circuit 426 and / or a frequency control circuit 424. The amplitude control circuit 426 and / or the frequency control circuit 424 in turn controls the droplet formation transducer 402. The amplitude control circuit 426 and / or the frequency control circuit 424 can be included in a control system.

[0094] In some implementations, the sorting electronics (e.g., detection system 416, detection station 414, and processor 440) can be coupled to a memory configured to store detected events and sorting decisions based on the detected events. The sorting decisions can be included in the event data for the particle. In some embodiments, detection system 416 and detection station 414 can be implemented as a single detection unit or communicatively coupled so that event measurements can be collected by one of detection system 416 or detection station 414 and provided to a non-collecting element.

[0095] Figure 4B is a schematic diagram of a particle sorting system according to one embodiment presented herein. Figure 4B The particle sorting system 400 shown includes deflection plates 452 and 454. Charge can be applied via a stream-charging wire in the barb. This produces a droplet stream 410 containing particles 409 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to produce light scattering and fluorescence information. For example, by sorting electronics or other detection systems ( Figure 4B Deflector plates 452 and 454 can be independently controlled to attract or repel charged droplets to direct the droplets toward a destination collection container (e.g., one of 472, 474, 476, or 478). Figure 4B As shown, deflector plates 452 and 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 a particle of interest (e.g., does not exhibit scattering or illumination information within a particular sorting range), the deflector plates can allow the particle to follow flow path 464. Such uncharged droplets can enter a waste container, for example, via an aspirator 470.

[0096] Sorting electronics can be included to enable collection of measurements, receive fluorescence signals from particles, and determine how to adjust the deflection plates to sort the particles. Figure 4B An example implementation of the illustrated embodiment includes a flow cytometer commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ) BD FACSAria TM series.

[0097] Figure 5 Depicted is the overall architecture of an example computing device 500 in accordance with some embodiments. Figure 5 The overall architecture of the computing device 500 depicted in the figure includes an arrangement of computer hardware and software components. However, in order to provide an advantageous disclosure, it is not necessary to show all of these generally conventional elements. As shown, 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 can communicate with each other via a communication bus. The network interface 520 can provide a connection to one or more networks or computing systems. Therefore, the processing unit 510 can receive information and instructions from other computing systems or services via the network. The processing unit 510 can also communicate with the memory 570, and can also provide output information to the optional display 550 via the input / output device interface 540. For example, analysis software (e.g., data analysis software or program, such as a computer program) stored as executable instructions in the non-transitory memory of the analysis system ) can display flow cytometry event data to a user. The input / output device interface 540 can also accept input from an optional 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.

[0098] Memory 570 may contain computer program instructions (grouped into modules or components in some embodiments) that processing unit 510 executes to implement one or more embodiments. Memory 570 typically includes RAM, ROM, and / or other permanent, auxiliary, or non-transitory computer-readable media. Memory 570 may store an operating system 572 that provides computer program instructions for use by processing unit 510 in general management and operation of computing device 500. Data may be stored in data storage device 590. Memory 570 may also include computer program instructions and other information for implementing various aspects of the present disclosure.

[0099] Methods for analyzing samples

[0100] As described above, aspects of the invention include methods of analyzing a sample. Methods of interest include (a) introducing a particulate sample into a flow cytometer comprising a plurality of light sources and an integrated optofluidic interrogation module of the invention, and (b) analyzing the particulate sample using flow cytometry.

[0101] The sample analyzed can be any sample of interest to the user. In certain embodiments, the sample includes a biological component, or the sample is a biological sample. The term "biological sample" is used in its conventional sense to refer to a sample derived from or comprising a whole organism, such as a prokaryotic cell, a eukaryotic cell, a plant, a fungus, or an animal tissue, a subset of a cell or a component, which can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen in some instances. Therefore, "biological sample" refers to a subset of a natural organism or its tissue, and a homogenate prepared from an organism or its tissue, a biological particle (e.g., DNA, RNA, protein, subcellular organelle, etc.) separated, purified, or enriched, and a lysate or extract, an organism or its tissue subset includes, but is not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin slices, respiratory tract, gastrointestinal tract, cardiovascular and urogenital tract, tears, saliva, milk, blood cells, tumors, organs. The biological sample can be any type of biological tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some instances, the sample is a blood sample, including whole blood, such as blood obtained by venipuncture or finger stick blood collection (the blood may or may not be combined with any reagents such as preservatives, anticoagulants, etc. before testing).

[0102] In certain embodiments, the source of the sample is "mammal" or "mammals", wherein these terms are widely used to describe organisms within the class of mammals, including carnivora (e.g., dogs and cats), rodents (e.g., mice, guinea pigs and rats) and primates (e.g., humans, chimpanzees and monkeys). In some instances, the subject is a human. The method can be applied to samples obtained from human subjects of both sexes and at any stage of development (i.e., newborns, infants, teenagers, adolescents, adults), wherein in certain embodiments, human subjects are teenagers, adolescents or adults. Although embodiments of the present disclosure can be applied to samples from human subjects, it should be understood that the method can also be performed on samples from other animal subjects (i.e., "non-human subjects") (e.g., but not limited to birds, mice, rats, dogs, cats, livestock and horses).

[0103] The cells of interest can be targeted and characterized according to a variety of parameters (e.g., phenotypic features identified by attaching specific fluorescent tags to the cells of interest). In certain embodiments, the system is configured to deflect the analysis droplets determined to contain target cells. The subject method can be used to characterize a variety of cells. The 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 erythroid cells. The target cells of interest include cells with convenient cell surface markers or antigens, which can be captured or labeled by convenient affinity agents or their conjugates. For example, the target cell may include 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 α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cell is 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.

[0104] The method of interest may also include using particles in research, laboratory testing or treatment. In certain embodiments, the subject method includes obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject method includes obtaining cells from a fluid or tissue sample to be used as a research or diagnostic sample for a disease such as cancer. Similarly, the subject method includes obtaining cells from a fluid or tissue sample for treatment. A cell therapy protocol is a protocol that can prepare a living cell material including, for example, cells and tissues and introduce the living cell material into a subject as a treatment. Conditions that can be treated by administering a sample sorted by flow cytometry include, but are not limited to, blood disorders, immune system diseases, organ damage, etc.

[0105] Typical cell therapy protocols may include the following steps: sample collection, cell separation, genetic modification, in vitro culture and in vitro amplification, cell harvesting, sample volume reduction and washing, biological preservation, storage and introduction of cells into subjects. The protocol may begin with the collection of living cells or tissues from the source tissue of the subject to produce cell and / or tissue samples. Samples may be collected by any appropriate procedure, and any appropriate procedure includes, for example, administering a cell mobilization agent to the subject, drawing blood from the subject, removing bone marrow from the subject, etc. After collecting the sample, cell enrichment may be performed by several methods, including, for example, centrifugation-based methods, filtration-based methods, elutriation, magnetic separation methods, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells may be genetically modified by any convenient method, such as by gene editing mediated by nucleases. Genetically modified cells may be cultured, activated and amplified in vitro. In some cases, cells are preserved (e.g., cryopreserved) and stored for future use, and in the case of future use, the cells are thawed and then applied to the patient, such as cells may be injected into the patient.

[0106] Methods for assembling a flow cytometer

[0107] Aspects of the present invention also include methods of assembling a flow cytometer. The method of interest includes (a) positioning an integrated optofluidic interrogation module into a flow cytometer including a plurality of light sources, and (b) operably attaching the plurality of fiber optic light transmitter couplers to the plurality of light sources to assemble the flow cytometer. As described in detail above, the integrated optofluidic interrogation module of the present disclosure includes: a flow cell including an optically accessible flow channel for transporting particles in a flow stream; a linear fiber optic array integrated with the flow cell and including a plurality of non-circular core optical fibers, each non-circular core optical fiber being configured to project excitation wavelength light onto a corresponding focus within the flow cell; and a plurality of fiber optic light transmitter couplers for operably attaching the plurality of light sources to the linear fiber optic array.

[0108] As described above, embodiments of the integrated optofluidic interrogation module include a photodetector integrated with a flow cell and an electrical coupler for operably attaching an electrical wire to the photodetector. In some cases, assembling the flow cytometer includes operably attaching the electrical wire to the electrical coupler. In addition, embodiments of the integrated optofluidic interrogation module include multiple fiber optic light collecting element couplers for operably attaching multiple fiber optic light collecting elements to the flow cell. In some instances, assembling the flow cytometer includes operably attaching multiple fiber optic light collecting elements to multiple fiber optic light collecting element couplers. In the case where the module includes a sample fluid input coupler for injecting a flow stream into the flow cell, some embodiments of the disclosed method involve fluidly connecting a sample fluid line of the flow cytometer to the sample fluid input coupler. In the case where the module includes a sheath fluid input coupler for injecting a sheath fluid into the flow cell, some embodiments of the disclosed method involve fluidly connecting a sheath fluid line of the flow cytometer to the sheath fluid input coupler. In the case where the module includes a fluid output coupler for receiving a flow stream from the flow cell, some embodiments of the disclosed method include fluidly connecting a waste line of the flow cytometer to the fluid output coupler.

[0109] However, the methods of assembling a flow cytometer disclosed herein do not include mechanical mounting and alignment. In other words, when the integrated optofluidic interrogation module is located within the flow cytometer and connected to the relevant components (e.g., lasers, fiber optic light collection elements, wires, etc.) by virtue of the integrated nature of the module, the optical components are already precisely optically aligned. In some cases, the methods of the present invention result in assembly and / or alignment times that are 20% or less, such as 30% or less, such as 50% or less, and including 60% or less, than conventional methods that do not involve integrated optics.

[0110] Kits

[0111] As described above, aspects of the invention also include kits. Kits of interest include an integrated optofluidic interrogation module (e.g., as described above). Embodiments of the subject kits include a single integrated optofluidic interrogation module. In some cases, the kits include a plurality of integrated optofluidic interrogation modules. In some such cases, the number of integrated optofluidic interrogation modules in the subject kits can be from 2 to 20, such as from 2 to 10, and including from 2 to 5.

[0112] The kit may also include one or more other components for assembling the flow cytometer. In some cases, the kit includes one or more wires for transmitting signals generated by a light detector integrated with the flow cell. In some cases, the kit includes a plurality of wires, such as 2 or more wires, such as 3 or more wires, such as 4 or more wires, and including 5 or more wires. According to some embodiments, the kit includes a plurality of fiber optic light collecting elements. For example, the number of fiber optic light collecting elements can range from 2 to 20, such as 2 to 10. In some instances, the kit includes fluid components, such as sample fluid lines, sheath fluid lines, and / or waste lines operably attached to the module.

[0113] The kit also includes instructions for practicing the subject method. These instructions can be present in the subject kit in various forms, wherein one or more of these instructions can be present in the kit. A form in which these instructions can exist is as printed information on a suitable medium or substrate, for example, in the packaging of the kit, in a packaging insert etc., one or more sheets of paper with information are printed. Another form of these instructions is a computer-readable medium having recorded information thereon, for example a floppy disk, an optical disc (CD), a portable flash drive etc. Another form in which these instructions can exist is a website address, which can be used for accessing the information at a site removed by the Internet.

[0114] Practicality

[0115] The subject integrated optical fluid interrogation module, flow cytometer and method can be used in various applications, in which it is necessary to improve the resolution and accuracy of the parameter determination of analytes (e.g., cells, particles) in biological samples. For example, the use of the present invention can be beneficial to the assembly of flow cytometers, that is, so that no cumbersome assembly and adjustment are required. Therefore, the subject integrated optical fluid interrogation module, flow cytometer and method can be used for situations where it is necessary to simplify the assembly of complex flow cytometers that usually include cumbersome mechanical installation and alignment. Therefore, the subject integrated optical fluid interrogation module, flow cytometer and method can be used to reduce the cost of flow cytometer assembly and improve the robustness of the results obtained from flow cytometry. In some embodiments, the present invention can be used in situations where it is necessary to have modular flow cytometer components, that is, to enable components to be exchanged for different modules as needed to achieve functions of interest. In other examples, the present invention can be used in situations where it is necessary to improve the collection efficiency of flow cytometers.

[0116] The present disclosure can be used to characterize various types of analytes, particularly analytes relevant to medical diagnosis or patient care regimens, including but not limited to: proteins (including free proteins, proteins, and proteins bound to the surface of structures (e.g., cells), nucleic acids, viral particles, etc. In addition, the sample can be from an in vitro or in vivo source, and the sample can be a diagnostic sample.

[0117] The following is provided by way of example only and not limitation.

[0118] experiment

[0119] Obtained by Figure 1B Irradiance distribution of light emitted by a linear fiber array arranged as shown. The resulting irradiance distribution is Figure 6 As shown in Figure 6 As shown, the irradiance is uniformly and regularly spaced, indicating that the linear fiber array is suitable for irradiating the core flow.

[0120] Notwithstanding the appended claims, the present disclosure is defined by the following terms:

[0121] 1. An integrated optofluidic interrogation module, comprising:

[0122] a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream;

[0123] a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; and

[0124] A plurality of fiber optic transmitter couplers are provided for operably attaching a plurality of light sources to the linear optical fiber array.

[0125] 2. The integrated optofluidic interrogation module of clause 1, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0126] 3. An integrated optofluidic interrogation module according to clause 2, wherein the non-circular core optical fiber is a square core optical fiber.

[0127] 4. An integrated optofluidic interrogation module according to any of the preceding clauses, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile onto the focal point.

[0128] 5. An integrated optofluidic interrogation module according to any of the preceding clauses, wherein the non-circular core optical fibers in the linear optical fiber array are vertically stacked.

[0129] 6. An integrated optofluidic interrogation module according to clause 5, wherein the cores of adjacent non-circular core optical fibers in the linear optical fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm.

[0130] 7. An integrated optofluidic interrogation module according to any of the preceding clauses, wherein the linear optical fiber array is attached to an outer surface of the flow cell.

[0131] 8. An integrated optofluidic interrogation module according to any of the preceding clauses, further comprising a focusing optical system integrated with the flow cell and configured to relay excitation light from the non-circular core optical fiber to the focal point.

[0132] 9. The integrated optofluidic interrogation module of clause 8, wherein the focusing optical system is embedded in the flow cell.

[0133] 10. An integrated optofluidic interrogation module according to clause 8 or 9, wherein the focusing optical system comprises a plurality of microlenses.

[0134] 11. An integrated optofluidic interrogation module according to any of the preceding clauses, further comprising a light detector integrated with the flow cell.

[0135] 12. The integrated optofluidic interrogation module of clause 11, further comprising an electrical coupler for operably attaching electrical wires to the light detector.

[0136] 13. The integrated optofluidic interrogation module of clause 12, further comprising electrical wiring.

[0137] 14. An integrated optofluidic interrogation module according to any of clauses 11 to 13, further comprising an optical filter located between the focal point and the light detector.

[0138] 15. An integrated optofluidic interrogation module according to any of clauses 11 to 14, further comprising an optical mask located between the focal point and the light detector.

[0139] 16. An integrated optofluidic interrogation module according to any of clauses 11 to 15, wherein the light detector is a forward scatter detector.

[0140] 17. An integrated optofluidic interrogation module according to any of clauses 11 to 16, wherein the light detector is attached to an outer surface of the flow cell.

[0141] 18. An integrated optofluidic interrogation module according to any of the preceding clauses, further comprising a plurality of fiber optic light collecting element couplers for operably attaching a plurality of fiber optic light collecting elements to the flow cell.

[0142] 19. The integrated optofluidic interrogation module of clause 18, wherein the fiber optic light collecting element couplers are vertically stacked.

[0143] 20. The integrated optofluidic interrogation module of clause 18 or 19, further comprising said plurality of fiber optic light collection elements.

[0144] 21. An integrated optofluidic interrogation module according to any of the preceding clauses, further comprising a sample fluid input coupler for injecting a sample fluid into the flow cell.

[0145] 22. An integrated optofluidic interrogation module according to any of the preceding clauses, further comprising a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0146] 23. An integrated optofluidic interrogation module according to any preceding clause, further comprising a fluid output coupler for receiving a flow stream from the flow cell.

[0147] 24. An integrated optofluidic interrogation module according to any preceding clause, wherein the flow cell comprises a cuvette.

[0148] 25. The integrated optofluidic interrogation module of clause 24, further comprising a collimating lens attached to an outer surface of the cuvette.

[0149] 26. The integrated optofluidic interrogation module of clause 25, wherein the collimating lens is an injection compression molded microlens.

[0150] 27. An integrated optofluidic interrogation module according to clause 25 or 26, wherein the collimating lens is a Fresnel lens.

[0151] 28. An integrated optofluidic interrogation module according to any preceding clause, wherein the integrated optofluidic interrogation module comprises:

[0152] N non-circular core optical fibers;

[0153] N focal points; and

[0154] N fiber optic transmitter couplers,

[0155] Wherein, N is an integer from 2 to 10.

[0156] 29. An integrated optofluidic interrogation module according to any preceding clause, wherein the flow cell has a width in the range of 75 mm to 125 mm.

[0157] 30. A flow cytometer comprising:

[0158] multiple light sources; and

[0159] An integrated optofluidic interrogation module comprising:

[0160] a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream;

[0161] a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; and

[0162] A plurality of fiber optic transmitter couplers are provided for operably attaching a plurality of light sources to the linear optical fiber array.

[0163] 31. A flow cytometer according to clause 30, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0164] 32. A flow cytometer according to clause 31, wherein the non-circular core optical fiber is a square core optical fiber.

[0165] 33. A flow cytometer according to any of clauses 30 to 32, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile onto the focal point.

[0166] 34. A flow cytometer according to any of clauses 30 to 33, wherein the non-circular core optical fibers in the linear optical fiber array are vertically stacked.

[0167] 35. A flow cytometer according to clause 34, wherein the cores of adjacent non-circular core optical fibers in the linear optical fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm.

[0168] 36. A flow cytometer according to any one of clauses 30 to 35, wherein the linear optical fibre array is attached to an outer surface of the flow cell.

[0169] 37. A flow cytometer according to any of clauses 30 to 36, wherein the integrated optofluidic interrogation module comprises a focusing optical system integrated with the flow cell and configured to relay excitation light from the non-circular core optical fiber to the focus.

[0170] 38. The flow cytometer of claim 37, wherein the focusing optical system is embedded in the flow cell.

[0171] 39. A flow cytometer according to claim 37 or 38, wherein the focusing optical system comprises a plurality of micro lenses.

[0172] 40. A flow cytometer according to any of clauses 30 to 39, wherein the integrated optofluidic interrogation module comprises a light detector integrated with the flow cell.

[0173] 41. A flow cytometer according to clause 40, wherein the integrated optofluidic interrogation module includes an electrical coupler for operably attaching an electrical wire to the optical detector.

[0174] 42. The flow cytometer of clause 41, further comprising electrical wiring.

[0175] 43. A flow cytometer according to any of clauses 40 to 42, wherein the integrated optofluidic interrogation module comprises an optical filter located between the focal point and the light detector.

[0176] 44. A flow cytometer according to any of clauses 40 to 43, wherein the integrated optofluidic interrogation module comprises an optical mask located between the focal point and the light detector.

[0177] 45. A flow cytometer according to any one of clauses 40 to 44, wherein the light detector is a forward scatter detector.

[0178] 46. ​​A flow cytometer according to any one of clauses 40 to 45, wherein the light detector is attached to an outer surface of the flow cell.

[0179] 47. A flow cytometer according to any of clauses 30 to 46, wherein the integrated optofluidic interrogation module comprises a plurality of fiber optic light collecting element couplers for operably attaching a plurality of fiber optic light collecting elements to the flow cell.

[0180] 48. A flow cytometer according to clause 47, wherein the fiber optic light collection element couplers are vertically stacked.

[0181] 49. The flow cytometer of clause 47 or 48, further comprising said plurality of fiber optic light collection elements.

[0182] 50. The flow cytometer of clause 49, further comprising a plurality of light detectors, each light detector operably attached to a fiber optic light collecting element of the plurality of fiber optic light collecting elements.

[0183] 51. A flow cytometer according to any of clauses 30 to 50, wherein the integrated optofluidic interrogation module comprises a sample fluid input coupler for injecting sample fluid into the flow cell.

[0184] 52. A flow cytometer according to any of clauses 30 to 51, wherein the integrated optofluidic interrogation module comprises a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0185] 53. A flow cytometer according to any of clauses 30 to 52, wherein the integrated optofluidic interrogation module comprises a fluidic output coupler for receiving a flow stream from the flow cell.

[0186] 54. A flow cytometer according to any one of clauses 30 to 53, wherein the flow cell comprises a cuvette.

[0187] 55. The flow cytometer of clause 54, wherein the integrated optofluidic interrogation module comprises a collimating lens attached to an outer surface of the cuvette.

[0188] 56. A flow cytometer according to clause 55, wherein the collimating lens is an injection compression molded microlens.

[0189] 57. A flow cytometer according to clause 55 or 56, wherein the collimating lens is a Fresnel lens.

[0190] 58. A flow cytometer according to any one of clauses 30 to 57, wherein the integrated optofluidic interrogation module comprises:

[0191] N non-circular core optical fibers;

[0192] N focal points; and

[0193] N fiber optic transmitter couplers,

[0194] Wherein, N is an integer from 2 to 10.

[0195] 59. A flow cytometer according to any one of clauses 30 to 58, wherein the flow cell has a width in the range of 75 mm to 125 mm.

[0196] 60. A method of analyzing a sample, the method comprising:

[0197] (a) introducing a microparticle sample into a flow cytometer, the flow cytometer comprising:

[0198] multiple light sources; and

[0199] An integrated optofluidic interrogation module comprising:

[0200] a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream;

[0201] a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; and

[0202] a plurality of fiber optic transmitter couplers for operably attaching the plurality of light sources to the linear optical fiber array; and

[0203] (b) analyzing the microparticle sample by flow cytometry.

[0204] 61. The method of clause 60, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0205] 62. The method of clause 61, wherein the non-circular core optical fiber is a square core optical fiber.

[0206] 63. A method according to any one of clauses 60 to 62, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile onto the focal point.

[0207] 64. A method according to any of clauses 60 to 63, wherein the non-circular core optical fibers in the linear optical fiber array are vertically stacked.

[0208] 65. A method according to clause 64, wherein the cores of adjacent non-circular core optical fibers in the linear optical fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm.

[0209] 66. A method according to any one of clauses 60 to 65, wherein the linear optical fiber array is attached to an outer surface of the flow cell.

[0210] 67. A method according to any one of clauses 60 to 66, wherein the integrated optofluidic interrogation module includes a focusing optical system that is integrated with the flow cell and is configured to relay excitation light from the non-circular core optical fiber to the focus.

[0211] 68. A method according to clause 67, wherein the focusing optical system is embedded in the flow cell.

[0212] 69. A method according to clause 67 or 68, wherein the focusing optical system comprises a plurality of micro lenses.

[0213] 70. The method of any of clauses 60 to 69, wherein the integrated optofluidic interrogation module comprises a light detector integrated with the flow cell.

[0214] 71. The method of clause 70, wherein the integrated optofluidic interrogation module comprises an electrical coupler for operably attaching electrical wires to the optical detector.

[0215] 72. The method of clause 71, wherein the flow cytometer further comprises electrical wires.

[0216] 73. A method according to any of clauses 60 to 72, wherein the integrated optofluidic interrogation module comprises an optical filter located between the focal point and the light detector.

[0217] 74. A method according to any of clauses 70 to 73, wherein the integrated optofluidic interrogation module comprises an optical mask located between the focal point and the light detector.

[0218] 75. A method according to any one of clauses 70 to 74, wherein the light detector is a forward scatter detector.

[0219] 76. A method according to any one of clauses 70 to 75, wherein the light detector is attached to an outer surface of the flow cell.

[0220] 77. The method of any of clauses 60 to 76, wherein the integrated optofluidic interrogation module comprises a plurality of fiber optic light collecting element couplers for operably attaching a plurality of fiber optic light collecting elements to the flow cell.

[0221] 78. The method of clause 77, wherein the fiber optic light collecting element couplers are vertically stacked.

[0222] 79. A method according to clause 77 or 78, wherein the flow cytometer further comprises a plurality of fiber optic light collection elements.

[0223] 80. The method of clause 79, wherein the flow cytometer further comprises a plurality of light detectors, each light detector being operably attached to a fiber optic light collecting element of the plurality of fiber optic light collecting elements.

[0224] 81. The method of any of clauses 60 to 80, wherein the integrated optofluidic interrogation module comprises a sample fluid input coupler for injecting sample fluid into the flow cell.

[0225] 82. The method of any of clauses 60 to 81, wherein the integrated optofluidic interrogation module comprises a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0226] 83. The method of any of clauses 60 to 82, wherein the integrated optofluidic interrogation module comprises a fluidic output coupler for receiving a flow stream from the flow cell.

[0227] 84. A method according to any one of clauses 60 to 83, wherein the flow cell comprises a cuvette.

[0228] 85. The method of clause 84, wherein the integrated optofluidic interrogation module comprises a collimating lens attached to an outer surface of the cuvette.

[0229] 86. The method of clause 85, wherein the collimating lens is an injection compression molded microlens.

[0230] 87. A method according to clause 85 or 86, wherein the collimating lens is a Fresnel lens.

[0231] 88. A method according to any of clauses 60 to 87, wherein the integrated optofluidic interrogation module comprises:

[0232] N non-circular core optical fibers;

[0233] N focal points; and

[0234] N fiber optic transmitter couplers,

[0235] Wherein, N is an integer from 2 to 10.

[0236] 89. A method according to any one of clauses 60 to 88, wherein the flow cell has a width in the range of 75 mm to 125 mm.

[0237] 90. A method of assembling a flow cytometer, the method comprising:

[0238] (a) positioning an integrated optofluidic interrogation module into a flow cytometer comprising a plurality of light sources, wherein the integrated optofluidic interrogation module comprises:

[0239] a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream;

[0240] a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; and

[0241] a plurality of fiber optic transmitter couplers for operably attaching a plurality of light sources to the linear optical fiber array; and

[0242] (b) operably attaching the plurality of fiber optic light transmitter couplers to the plurality of light sources to assemble the flow cytometer.

[0243] 91. The method of clause 90, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0244] 92. The method of clause 91, wherein the non-circular core optical fiber is a square core optical fiber.

[0245] 93. A method according to any one of clauses 90 to 92, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile onto the focal point.

[0246] 94. A method according to any one of clauses 90 to 93, wherein the non-circular core optical fibers in the linear optical fiber array are vertically stacked.

[0247] 95. A method according to clause 94, wherein the cores of adjacent non-circular core optical fibers in the linear optical fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm.

[0248] 96. A method according to any one of clauses 90 to 95, wherein the linear optical fiber array is attached to an outer surface of the flow cell.

[0249] 97. A method according to any one of clauses 90 to 96, wherein the integrated optofluidic interrogation module includes a focusing optical system that is integrated with the flow cell and is configured to relay excitation light from the non-circular core optical fiber to the focus.

[0250] 98. A method according to clause 97, wherein the focusing optical system is embedded in the flow cell.

[0251] 99. A method according to clause 97 or 98, wherein the focusing optical system includes a plurality of microlenses.

[0252] 100. The method of any of clauses 90 to 99, wherein the integrated optofluidic interrogation module further comprises a light detector integrated with the flow cell.

[0253] 101. The method of clause 100, wherein the integrated optofluidic interrogation module comprises an electrical coupler for operably attaching electrical wires to the optical detector.

[0254] 102. The method of clause 101, further comprising operably attaching the electrical wire to the electrical coupler.

[0255] 103. The method of any of clauses 100 to 102, wherein the integrated optofluidic interrogation module comprises an optical filter located between the focal point and the light detector.

[0256] 104. The method of any of clauses 100 to 103, wherein the integrated optofluidic interrogation module comprises an optical mask located between the focal point and the light detector.

[0257] 105. A method according to any one of clauses 100 to 104, wherein the light detector is a forward scatter detector.

[0258] 106. A method according to any one of clauses 100 to 105, wherein the light detector is attached to an outer surface of the flow cell.

[0259] 107. The method of any of clauses 90 to 106, wherein the integrated optofluidic interrogation module comprises a plurality of fiber optic light collecting element couplers for operably attaching a plurality of fiber optic light collecting elements to the flow cell.

[0260] 108. The method of clause 107, wherein the fiber optic light collecting element couplers are vertically stacked.

[0261] 109. The method of clause 107 or 108, further comprising operably attaching the plurality of fiber optic light collecting elements to the plurality of fiber optic light collecting element couplers.

[0262] 110. The method of any of clauses 90 to 109, wherein the integrated optofluidic interrogation module comprises a sample fluid input coupler for injecting sample fluid into the flow cell.

[0263] 111. The method of clause 110, further comprising fluidly connecting a sample fluid line of the flow cytometer to the sample fluid input coupler.

[0264] 112. The method of any of clauses 90 to 111, wherein the integrated optofluidic interrogation module comprises a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0265] 113. The method of clause 112, further comprising connecting a sheath fluid line fluid of the flow cytometer to the sheath fluid input coupler.

[0266] 114. The method of any of clauses 90 to 113, wherein the integrated optofluidic interrogation module comprises a fluidic output coupler for receiving a flow stream from the flow cell.

[0267] 115. The method of clause 114, further comprising connecting a waste line fluid of the flow cytometer to the fluid output coupler.

[0268] 116. A method according to any one of clauses 90 to 115, wherein the flow cell comprises a cuvette.

[0269] 117. The method of clause 116, wherein the integrated optofluidic interrogation module comprises a collimating lens attached to an outer surface of the cuvette.

[0270] 118. The method of clause 117, wherein the collimating lens is an injection compression molded microlens.

[0271] 119. A method according to clause 117 or 118, wherein the collimating lens is a Fresnel lens.

[0272] 120. The method of any of clauses 90 to 119, wherein the integrated optofluidic interrogation module comprises:

[0273] N non-circular core optical fibers;

[0274] N focal points; and

[0275] N fiber optic transmitter couplers,

[0276] Wherein, N is an integer from 2 to 10.

[0277] 121. A method according to any one of clauses 90 to 120, wherein the width of the flow cell ranges from 75 mm to 125 mm.

[0278] 122. A kit comprising:

[0279] An integrated optofluidic interrogation module comprising:

[0280] a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream;

[0281] a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; and

[0282] A plurality of fiber optic transmitter couplers are provided for operably attaching a plurality of light sources to the linear optical fiber array.

[0283] 123. A kit according to clause 122, wherein the non-circular core optical fiber is a rectangular core optical fiber.

[0284] 124. A kit according to clause 123, wherein the non-circular core optical fiber is a square core optical fiber.

[0285] 125. A kit according to any of clauses 122 to 124, wherein the non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile onto the focal point.

[0286] 126. A kit according to any of clauses 122 to 125, wherein the non-circular core optical fibers in the linear optical fiber array are vertically stacked.

[0287] 127. A kit according to clause 126, wherein the cores of adjacent non-circular core optical fibers in the linear optical fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm.

[0288] 128. A kit according to any of clauses 122 to 127, wherein the linear optical fiber array is attached to an outer surface of the flow cell.

[0289] 129. A kit according to any of clauses 122 to 128, wherein the integrated optofluidic interrogation module includes a focusing optical system that is integrated with the flow cell and is configured to relay excitation light from the non-circular core optical fiber to the focus.

[0290] 130. A kit according to claim 129, wherein the focusing optical system is embedded in the flow cell.

[0291] 131. A kit according to clause 129 or 130, wherein the focusing optical system comprises a plurality of microlenses.

[0292] 132. A kit according to any one of 122 to 131, wherein the integrated optofluidic interrogation module further includes a light detector integrated with the flow cell.

[0293] 133. A kit according to clause 132, wherein the integrated optofluidic interrogation module further comprises an electrical coupler for operably attaching an electrical wire to the optical detector.

[0294] 134. The kit according to clause 133 further comprising electrical wiring.

[0295] 135. A kit according to any of clauses 132 to 134, wherein the integrated optofluidic interrogation module comprises an optical filter located between the focal point and the light detector.

[0296] 136. A kit according to any of clauses 132 to 135, wherein the integrated optofluidic interrogation module comprises an optical mask located between the focal point and the light detector.

[0297] 137. A kit according to any one of clauses 132 to 136, wherein the light detector is a forward scatter detector.

[0298] 138. A kit according to any one of clauses 132 to 137, wherein the light detector is attached to an outer surface of the flow cell.

[0299] 139. A kit according to any of clauses 122 to 138, wherein the integrated optofluidic interrogation module comprises a plurality of fiber optic light collection element couplers for operably attaching a plurality of fiber optic light collection elements to the flow cell.

[0300] 140. A kit according to clause 139, wherein the fiber optic light collection element couplers are vertically stacked.

[0301] 141. The kit of clause 139 or 140, further comprising a plurality of fiber optic light collection elements.

[0302] 142. A kit according to any of clauses 122 to 141, wherein the integrated optofluidic interrogation module comprises a sample fluid input coupler for injecting sample fluid into the flow cell.

[0303] 143. A kit according to any of clauses 122 to 142, wherein the integrated optofluidic interrogation module comprises a sheath fluid input coupler for injecting sheath fluid into the flow cell.

[0304] 144. The kit of any of clauses 122 to 143, wherein the integrated optofluidic interrogation module comprises a fluidic output coupler for receiving a flow stream from the flow cell.

[0305] 145. A kit according to any one of clauses 122 to 144, wherein the flow cell comprises a cuvette.

[0306] 146. A kit according to clause 145, wherein the integrated optofluidic interrogation module includes a collimating lens attached to an outer surface of the cuvette.

[0307] 147. A kit according to clause 146, wherein the collimating lens is an injection compression molded microlens.

[0308] 148. A kit according to clause 146 or 147, wherein the collimating lens is a Fresnel lens.

[0309] 149. The kit of any of clauses 122 to 148, wherein the integrated optofluidic interrogation module comprises:

[0310] N non-circular core optical fibers;

[0311] N focal points; and

[0312] N fiber optic transmitter couplers,

[0313] Wherein, N is an integer from 2 to 10.

[0314] 150. A kit according to any one of clauses 122 to 149, wherein the flow cell has a width in the range of 75 mm to 125 mm.

[0315] 151. A kit according to any of clauses 122 to 150, wherein the kit comprises a plurality of integrated optofluidic interrogation modules.

[0316] Although the above invention has been described in some detail by way of illustration and example for purposes of clear understanding, it will be apparent to one skilled in the art based on the teachings of the present invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0317] Therefore, the above only illustrates the principle of the present invention.It should be understood that, although not explicitly described or shown here, those skilled in the art will be able to design various devices, which embody the principle of the present invention and are included in the spirit and scope of the present invention.In addition, all examples and conditional languages ​​described herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to promote the development of the art, and are interpreted as not being limited to these specifically enumerated examples and conditions.In addition, all statements citing the principles, aspects and embodiments of the present invention and its specific examples herein are intended to cover their structural and functional equivalents.In addition, it is expected that such equivalents include currently known equivalents and equivalents developed in the future (that is, any element of the same function developed regardless of the structure).In addition, regardless of whether such disclosure is clearly recorded in the claims, any content disclosed herein is intended to be disclosed to the public.

[0318] Therefore, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. not limited to this (f) or 35 U.S.C. not limited to this (6) is expressly defined as invoking 35 U.S.C. only step (f) or 35 U.S.C. only step (6) only when the exact phrase "device for..." or the exact phrase "step for..." is recited at the beginning of such limitation of the claim; if such exact phrase is not used in the limitation in the claim, then 35 U.S.C. not limited to this (f) or 35 U.S.C. not limited to this (6) is not invoked.

Claims

1. An integrated optofluidic interrogation module, comprising: a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream; a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; as well as A plurality of fiber optic transmitter couplers are provided for operably attaching a plurality of light sources to the linear optical fiber array.

2. The integrated optofluidic interrogation module of claim 1, wherein: The non-circular core optical fiber is a rectangular core optical fiber.

3. The integrated optofluidic interrogation module of claim 2, wherein: The non-circular core optical fiber is a square core optical fiber.

4. An integrated optofluidic interrogation module according to any one of the preceding claims, wherein: The non-circular core optical fiber is configured to project excitation wavelength light having a top-hat profile onto the focal point.

5. An integrated optofluidic interrogation module according to any one of the preceding claims, wherein: The non-circular core optical fibers in the linear optical fiber array are vertically stacked.

6. The integrated optofluidic interrogation module of claim 5, wherein: The cores of adjacent non-circular core optical fibers in the linear optical fiber array are spaced apart from each other by a distance of 0.1 mm to 0.3 mm.

7. An integrated optofluidic interrogation module according to any one of the preceding claims, wherein: The linear optical fiber array is attached to an outer surface of the flow cell.

8. The integrated optofluidic interrogation module of any preceding claim, further comprising a focusing optical system integrated with the flow cell and configured to relay excitation light from the non-circular core optical fiber to the focal point.

9. The integrated optofluidic interrogation module of claim 8, wherein: The focusing optical system is embedded in the flow cell.

10. The integrated optofluidic interrogation module of claim 8 or 9, wherein: The focusing optical system includes a plurality of micro lenses.

11. An integrated optofluidic interrogation module according to any preceding claim, further comprising a light detector integrated with the flow cell.

12. The integrated optofluidic interrogation module of claim 11 further comprising an optical filter positioned between the focal point and the light detector.

13. The integrated optofluidic interrogation module according to any one of claims 11 to 12, wherein: The light detector is attached to an outer surface of the flow cell.

14. A flow cytometer, comprising: Multiple light sources; as well as An integrated optofluidic interrogation module comprising: a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream; a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; and A plurality of fiber optic transmitter couplers are provided for operably attaching the plurality of light sources to the linear optical fiber array.

15. A method for analyzing a sample, the method comprising: (a) introducing a microparticle sample into a flow cytometer, the flow cytometer comprising: multiple light sources; and An integrated optofluidic interrogation module comprising: a flow cell comprising a light-accessible flow channel for transporting particles in a flow stream; a linear fiber array integrated with the flow cell and comprising a plurality of non-circular core optical fibers, each non-circular core optical fiber configured to project excitation wavelength light to a corresponding focal point within the flow cell; and a plurality of fiber optic transmitter couplers for operably attaching the plurality of light sources to the linear optical fiber array; and (b) analyzing the microparticle sample by flow cytometry.

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