Light detection system for small particle detection and methods of using the same

Abstract

The present invention includes an optical detection system for small particle detection. The optical detection system includes: an optical adjustment component configured to split a light beam received from a flow cell into a first beam and a second beam, the first beam including light scattered from particles excited within the flow cell, and the second beam including fluorescence from particles excited within the flow cell; a clustered wavelength division (CWD) optical detection subsystem including a first optical receiver configured to transmit the first beam to a first optical detection module, and a second optical receiver configured to transmit the second beam to a second optical detection module, the first optical detection module including a small particle detector; a first optical fiber configured to transmit the first beam from the optical adjustment component to the first optical receiver of the CWD optical detection subsystem; and a second optical fiber configured to transmit the second beam from the optical adjustment component to the second optical receiver of the CWD optical detection subsystem. In embodiments, the optical adjustment component includes a beam splitter or a multimode notch filter. Methods and kits are also provided.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Patent Application Serial No. 19 / 089609, filed March 25, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to an optical detection system for small particle detection and its usage. Background Technology

[0003] Characterization of analytes in biofluids has become an important part of biological research, medical diagnostics, and overall patient health and wellness assessment. Detection of analytes in biofluids (such as human blood or blood-derived products) can provide results that can play a role in determining treatment options for patients with a variety of diseases.

[0004] Flow cytometry is a technique used to characterize biological materials (such as cells in a blood sample or particles of interest in another type of biological or chemical sample) and is often used to sort such materials. A flow cytometer typically includes a sample reservoir for receiving a fluid sample (e.g., a blood sample) and a sheath fluid reservoir containing sheath fluid. The flow cytometer delivers particles (including cells) from the fluid sample as a cell stream into a flow cell, while simultaneously guiding the sheath fluid into the flow cell. To characterize the composition of the flow stream, light is used to illuminate it. Changes in the material within the flow stream (e.g., the presence of morphology or fluorescent labels) can cause changes in the observed light, and these changes allow for characterization and separation. To characterize the components in the flow stream, light must act on the flow stream and be collected. The light source in a flow cytometer can be varied and may include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0005] Parameters measured using a flow cytometer (i.e., a particle analyzer) typically include: light at the excitation wavelength scattered primarily along the forward direction at a narrow angle, called forward scattering (FSC); excitation light scattered by the particle along a direction orthogonal to the excitation laser, called side scattering (SSC); and light emitted from fluorescent molecules or fluorescent dyes. Different particle types can be identified by their light scattering characteristics and fluorescence emission, which is produced by labeling various proteins (e.g., cellular proteins) or other components with antibodies or other fluorescent probes labeled with fluorescent dyes. Forward scattering, side scattering, and fluorescence are detected by photodetectors located within the flow cytometer.

[0006] The development of clustered wavelength division (CWD) optical detection systems (such as those described in U.S. Application Nos. 17 / 870187 and 17 / 159453, each of which is incorporated herein by reference in its entirety) has recently led to improvements in the quality of light collection in flow cytometry. A CWD system includes a wavelength splitter that allows light with a predetermined spectral range to pass through, and an optical detection module that communicates optically with each wavelength splitter. Each optical detection module includes multiple photodetectors and one or more optical components configured to deliver light with a predetermined sub-spectral range to the photodetector. Therefore, the CWD system separates the collected light into spectral ranges and requires less light reflection to generate multiple sub-spectral ranges detected by the photodetectors. Since reflections that generate light with different spectral ranges result in light loss—which in some cases leads to poor detector signal quality (e.g., low signal-to-noise ratio)—the reduction in reflections in the CWD system reduces the amount of light loss and improves signal quality.

[0007] The use of flow cytometry for the optical detection of small particles has also attracted considerable attention. However, the detection of small particles is challenging due to signal attenuation associated with small particles and their corresponding sensitivity to noise. Small particle detection requires highly sensitive optical detection within a system context that can provide a high signal-to-noise ratio. Summary of the Invention

[0008] The inventors have recognized that further improvements are still needed to optical detection systems that include or utilize CWD optical detection systems, for example, as described herein, to improve the ability of these systems to detect, analyze, and / or characterize light scattered by small particles. The inventors have found that it is desirable to combine a high-sensitivity photodetector with a CWD optical detection subsystem, enabling the CWD optical detection subsystem to be used in conjunction with small particle detection. The inventors have also found that optically transmitting light from the flow cell of a flow cytometer to one or more CWD optical detection subsystems using optical fibers enables such CWD optical detection subsystems to be used in conjunction with small particle detection, i.e., by reducing light loss or noise introduction, particularly reducing light loss or noise introduction associated with the high-sensitivity signal generated by light scattering from small particles.

[0009] The term "small particle" refers to any particle that cannot be detected and analyzed using traditional light scattering methods. Small particles of interest include particles as small as 90 nm or smaller, such as 80 nm or smaller, 70 nm or smaller, 60 nm or smaller, 50 nm or smaller, or 40 nm or smaller. Examples of small particles of interest include, but are not limited to: apoptotic bodies with sizes ranging from 500 to 2000 nm, platelets, chylomicrons with sizes ranging from 75 to 1200 nm, microvesicles with sizes ranging from 50 to 1000 nm, certain bacteria, exosomes with sizes ranging from 40 to 160 nm, certain viruses, certain exosome-like particles with a size of approximately 35 nm, or certain lipoproteins (HDL, LDL, VLDL) with sizes ranging from 5 to 80 nm.

[0010] Traditionally, light scattering detection of small particles is unique and distinct from fluorescence detection. While light scattering signals are typically several orders of magnitude higher than fluorescence signals for large particles, light scattering from small particles decreases with the sixth power of the particle radius, resulting in fewer scattered photons compared to fluorescence techniques. Therefore, as described in embodiments of this disclosure, high-sensitivity light scattering detection requires optical filtering and dedicated detectors to improve sensitivity and resolution. Embodiments of this disclosure demonstrate systems and methods for collecting scattered light at independent focal planes, with options for sharing or not sharing fluorescence (FL) optical paths, and refocusing within a CWD light detection subsystem (also known as a Pizza detection system) at a light detection module (also known as Block B), which has independent optics for detection via a shared, low-cost array (e.g., an avalanche photodiode photodetector). Furthermore, embodiments of the light detection module design provide mechano-optical flexibility to further combine / separate / attenuate / filter or focus specific scattering channels. This capability enables light scattering calibration for both small and larger particles (e.g., cells), thereby improving normalization. As described in the embodiments of the present invention, this method of sharing the same collection aperture can also integrate signals from two detectors to create a single detection channel with a larger dynamic range.

[0011] The embodiments of this disclosure offer several advantages over the prior art, including, for example: (1) using optical adjustment components (e.g., beam splitters or multimode notch filters) to separate the fluorescence path and the light scattering path from a single continuous light scattering channel aperture (i.e., where the aperture is upstream of the optical adjustment component), enabling the integration of signals from multiple detectors (e.g., small particle detectors and side-scattering detectors) to create a single detection channel with a larger dynamic range; this method of separating the fluorescence path and the light scattering path using optical components also allows for independent alignment and / or calibration to achieve high-sensitivity light scattering detection without reduced fluorescence sensitivity due to chromatic aberration, and also provides mechatronic flexibility to further combine, separate, attenuate, filter, and / or focus specific scattering channels; (2) the optical adjustment components of the embodiments can be field-upgradeable. (2) Upgradable, meaning that these components can be replaced after system deployment (e.g., after delivery to the customer), such as replacing the first multimode notch filter with a second multimode notch filter with different light transmission characteristics, or replacing the first multimode notch filter with a beam splitter; (3) Directly collecting small particles and separated scattered light onto pinholes / fibers (i.e., the first and second fibers as described herein) allows for modular improvement of signal-to-noise ratio and light transmission to the detector, reducing complexity and / or cost while achieving multimode light scattering collection (rather than just a single mode); (4) The 90-degree fiber bend simplifies or eliminates any need for aperture-fiber-mirror assemblies; and (5) As described in embodiments of this disclosure (i.e., where such apertures are located upstream of optical adjustment components), collecting light of the same wavelength through the same aperture and separating it downstream enables light scattering calibration across detectors, thereby enabling the use of a single continuous light scattering channel.

[0012] Embodiments of this disclosure can achieve these advantages by employing optical adjustment components, such as beam splitters or multimode notch filters. In such embodiments, the optical adjustment components are configured to split a beam received from the flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence from particles excited within the flow cell. Other embodiments of this disclosure can achieve these advantages by configuring the system such that the aperture receiving light emitted from the flow cell is upstream of the optical adjustment components. Other embodiments of this disclosure can achieve these advantages by employing a combination of 800 μm fiber (for fluorescence (FL) signals) and a 200 μm aperture, or, in a more refined alternative, by employing a 90-degree 200 μm fiber to simplify components and allow for independent optical paths while improving the signal-to-noise ratio. Embodiments can utilize multiple fibers to achieve multimode light scattering collection. Embodiments can utilize a single 800 μm fiber pointed to the same Z focal plane for lateral scattering and small particle detection, a method that achieves 80 nm performance using only the fluorescence (FL) optical path. Other methods involve using a single-channel detector at the condenser lens (i.e., without using CWD optical detection subsystem technology, similar to some prior art). As discussed herein, some embodiments of this disclosure can achieve 80nm resolution using avalanche photodiodes without the need for more sensitive detectors.

[0013] Aspects of the present invention include an optical detection system for small particle detection. The optical detection system according to the invention includes: an optical adjustment component configured to split a light beam received from a flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence of particles excited within the flow cell; a clustered wavelength division (CWD) optical detection subsystem including a first optical receiver and a second optical receiver, wherein the first optical receiver is configured to transmit the first beam to a first optical detection module, and wherein the second optical receiver is configured to transmit the second beam to a second optical detection module, wherein the first optical detection module includes a small particle detector; a first optical fiber configured to transmit the first beam from the optical adjustment component to the first optical receiver of the CWD optical detection subsystem; and a second optical fiber configured to transmit the second beam from the optical adjustment component to the second optical receiver of the CWD optical detection subsystem. In embodiments, the optical adjustment component includes a beam splitter or a multimode notch filter.

[0014] The invention also includes a small-particle optical detection module for a clustered wavelength division (CWD) optical detection subsystem, the small-particle optical detection module comprising a small-particle detector, wherein the small-particle optical detection module is configured to receive a light beam from a light receiver. In an embodiment, the small-particle optical detection module further includes a side-scattering detector. The invention also includes methods for practicing the invention and kits having optical detection system components. Attached Figure Description

[0015] This disclosure can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures:

[0016] Figure 1A-1G Different views of a light detection system and its aspects according to certain embodiments are depicted.

[0017] Figure 2 A flow cytometer system according to certain embodiments is presented.

[0018] Figure 3 An image-enabled particle sorter according to certain embodiments is described.

[0019] Figure 4 A functional block diagram of a particle analysis system according to certain embodiments is depicted.

[0020] Figure 5 A functional block diagram of an example control system according to certain embodiments is depicted.

[0021] Figures 6A-6B A schematic diagram of a particle sorting system according to certain embodiments is depicted.

[0022] Figure 7 Aspects of a computer control system according to certain embodiments are described.

[0023] Figure 8 Theoretical results related to the detection of small particles using embodiments of this disclosure are described.

[0024] Figure 9 Empirical results comparing the performance of a particular system with embodiments of this disclosure are described. Detailed Implementation

[0025] Before describing this disclosure in more detail, it should be understood that the invention is not limited to the specific embodiments described, as these embodiments can certainly be varied. It should also be understood that, since the scope of this disclosure will be limited only by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0026] Where a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value (to one-tenth of the lower limit unit) between the upper and lower limits of the range, as well as any other specified value or intermediate value within the range, is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within this disclosure, subject to any specific exclusions within the range. Where the range includes one or both of the included limits, the range excluding one or both of those included limits is also included in this disclosure.

[0027] This article provides certain ranges whose numerical values ​​are preceded by the term "approximately". The term "approximately" is used in this article to provide textual support for the exact number that follows it, as well as for numbers that are close to or approximate to the number that follows the term. In determining whether a number is close to or approximate to a specifically listed number, an unlisted number that is close to or approximate can be a number that provides a basic equivalent to the specifically listed number in the context in which it appears.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of this disclosure, representative illustrative methods and materials are described here.

[0029] All publications and patents referenced in this specification are incorporated herein by reference, as if each individual publication or patent were expressly and individually indicated to be incorporated herein by reference to disclose and describe the methods and / or materials relating to the referenced publication. Any reference to a publication refers to a publication prior to the filing date and should not be construed as an admission that this disclosure has no priority over such publication due to prior art. Furthermore, the publication dates provided may differ from the actual publication dates and may require independent verification.

[0030] It should be noted that, unless the context clearly indicates otherwise, the use of an element in this document and the appended claims without a defined quantity implies the presence of at least one such element. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as "unique," "only," etc., when referencing claim elements or using the "negative" limitation.

[0031] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of this disclosure. Any enumerated methods may be performed in the order of the enumerated events or in any other logically possible order.

[0032] Although the system and method have been or will be described for grammatical fluency and functional interpretation, it should be clearly understood that, unless expressly provided in 35 U.SC §112, the claims should not be construed as necessarily being bound by “means” or “steps,” but rather should be given the full range of meaning and equivalents provided by the definition of the claims in accordance with the doctrine of judicial equivalence, and if the claims are expressly provided in 35 U.SC §112, they should be given the full range of legal equivalents in accordance with 35 U.SC §112.

[0033] Equipment / System Section

[0034] The present invention includes: an optical adjustment component configured to split a light beam received from a flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence of particles excited within the flow cell; a clustered wavelength division (CWD) optical detection subsystem including a first optical receiver and a second optical receiver, wherein the first optical receiver is configured to transmit the first beam to a first optical detection module, and wherein the second optical receiver is configured to transmit the second beam to a second optical detection module, wherein the first optical detection module includes a small particle detector; a first optical fiber configured to transmit the first beam from the optical adjustment component to the first optical receiver of the CWD optical detection subsystem; and a second optical fiber configured to transmit the second beam from the optical adjustment component to the second optical receiver of the CWD optical detection subsystem.

[0035] Optical adjustment components:

[0036] Aspects of the invention include an optical conditioning component configured to split a light beam received from a flow cell into a first beam and a second beam. In an embodiment, the first beam comprises light scattered from particles excited within the flow cell, and the second beam comprises fluorescence from particles excited within the flow cell. Embodiments include an optical conditioning component configured to receive a light beam originating from a flow cell and configured to transmit a first beam to a first optical fiber and a second beam to a second optical fiber, the first beam comprising light scattered from particles excited within the flow cell, and the second beam comprising fluorescence from particles excited within the flow cell. The term “optical conditioning” is used herein in its conventional sense to refer to an optical component that alters or modulates the light propagating to a light scattering detector and a bright-field photodetector. For example, optical conditioning may alter the beam profile, the beam focus, the direction of beam propagation, or the collimation of the beam. As described herein, in some embodiments, optical conditioning includes splitting the beam such that a portion of the collected light (e.g., from a sample in the flow stream) propagates to the first optical fiber, and another portion of the collected light propagates to the second optical fiber. In some embodiments, the optical conditioning component includes a beam splitter or a multimode notch filter.

[0037] As described herein, the light propagating into the first optical fiber in the subject optical detection system (and ultimately to the small particle detector) includes light scattered from particles excited within the flow cell. Furthermore, the amount of light propagating into the first optical fiber in the subject optical detection system (and ultimately to the small particle detector) can vary, wherein, in some embodiments, 50% or less of the collected light is transmitted to the first optical fiber via the optical conditioning element, for example, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, and includes 5% or less of the light collected by the optical detection system being transmitted to the first optical fiber via the optical conditioning element. For example, the amount of collected light (e.g., from a sample in the flow stream) propagating to the first optical fiber via the optical conditioning element can range from 1% to 75%, for example, 2% to 70%, 3% to 65%, 4% to 60%, and includes 5% to 50%.

[0038] As described herein, the light propagating into the second optical fiber in the subject light detection system comprises the fluorescence of particles excited within the flow cell. Furthermore, the amount of light propagating into the second optical fiber via the optical adjustment components can also vary. In some embodiments, 50% or more of the collected light is transmitted to the second optical fiber, for example, 55% or more, 60% or more, 65% or more, 75% or more, 80% or more, 90% or more, and including 95% or more of the light collected by the subject light detection system being transmitted to the second optical fiber via the optical adjustment components. For example, the amount of light propagating into the second optical fiber via the optical adjustment components can range from 25% to 99%, for example, 30% to 95%, 35% to 90%, 40% to 85%, 45% to 80%, and including 50% to 75%. In some embodiments, 10% of the collected light is propagated into the first optical fiber, and 90% of the collected light is propagated into the second optical fiber.

[0039] In some embodiments, light from the optical adjustment component propagates to the first optical fiber through a blocking component. In these embodiments, the blocking component is configured to reduce the amount of light transmitted to the first optical fiber, for example, by reducing the amount of light transmitted to the first optical fiber by 1% or more, such as 5% or more, such as 10% or more, such as 25% or more, such as 40% or more, and including reducing the amount of light transmitted to the first optical fiber by 50% or more. Any convenient blocking method can be employed, including but not limited to optical apertures (e.g., pinholes) or slits. The size of the optical aperture can be varied as needed, wherein the aperture of interest ranges from 0.001 mm to 10 mm, such as from 0.005 mm to 9.5 mm, such as from 0.01 mm to 9 mm, such as from 0.05 mm to 8.5 mm, such as from 0.1 mm to 8 mm, such as from 0.5 mm to 7.5 mm, and includes variations from 1 mm to 5 mm. The width of the blocking slit of interest can also vary within the range of 0.001 mm to 10 mm, for example from 0.005 mm to 9.5 mm, for example from 0.01 mm to 9 mm, for example from 0.05 mm to 8.5 mm, for example from 0.1 mm to 8 mm, for example from 0.5 mm to 7.5 mm, and includes variations within the range of 1 mm to 5 mm. The length of the blocking slit can vary according to the width of the light propagating to the first optical fiber, and can vary within the range of 1 mm to 50 mm, for example from 2 mm to 45 mm, for example from 3 mm to 40 mm, for example from 4 mm to 35 mm, and includes variations within the range of 5 mm to 25 mm.

[0040] In some embodiments, light is also propagated to the second optical fiber through one or more blocking components (e.g., scattering strips or blocking disks). The blocking components used to reduce the amount of light transmitted to the second optical fiber can be of any convenient shape, wherein the cross-sectional shapes of interest include, but are not limited to, straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses; and irregular shapes, such as the parabolic bottom portion coupled to the top portion of a plane. In some embodiments, the blocking component is circular. In other embodiments, the blocking component is elliptical. In still other embodiments, the blocking component is polygonal in shape, such as a square or rectangle. The width of the blocking component can vary, in some cases ranging from 1 mm to 25 mm, for example from 2 mm to 22 mm, for example from 3 mm to 20 mm, for example from 4 mm to 17 mm, and including variations from 5 mm to 15 mm. The length of each shielding component ranges from 1mm to 50mm, for example from 2mm to 45mm, for example from 3mm to 40mm, for example from 4mm to 35mm, for example from 5mm to 30mm, and includes 10mm to 20mm.

[0041] In some cases, optical conditioning of the collected light also includes collimation. The term "collimation," used in its conventional sense, refers to optical conditioning of the collinearity of light propagation or reducing the divergence of light relative to a common propagation axis. In some cases, collimation involves narrowing the spatial cross-section of the beam. In other cases, optical conditioning includes changing the direction of the beam, for example, changing the direction of propagation by 1° or more, such as 5° or more, such as 10° or more, such as 15° or more, such as 20° or more, such as 25° or more, such as 30° or more, such as 45° or more, such as 60° or more, such as 75° or more, and includes changing the direction of light propagation by 90° or more. In still other cases, optical conditioning is a reduction scheme to decrease the size of the light (e.g., the spot size), for example, reducing the size by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, and includes reducing the size by 75% or more.

[0042] The term "beam splitter" is used herein in its conventional sense, referring to an optical component configured to propagate a beam of light along two or more distinct optical paths, such that a predetermined portion of the light propagates along each optical path. Any convenient beam splitting scheme can be employed, such as using triangular prisms, split-mirror prisms, dichroic prisms, and other types of beam splitters. Beam splitters can be formed from any suitable material, provided that the beam splitter can propagate the desired amount and wavelength of light into the first and second optical fibers. For example, beam splitters of interest can be formed from glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glass), silica (e.g., fused silica), quartz, crystals (e.g., CaF2 crystals), zinc selenide (ZnSe), F2, germanium titanate (Ge) (e.g., S-TIH11), or borosilicates (e.g., BK7). In some embodiments, the splitter is formed of a polymeric material, such as, but not limited to, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics, such as PETG (diol-modified polyethylene terephthalate), and other polymeric plastic materials.In some embodiments, the splitter is formed of polyester, wherein the polyester of interest may include, but is not limited to, polyalkylene terephthalates, such as polyethylene terephthalate (PET), bottle-grade PET (a copolymer based on monoethylene glycol, terephthalic acid and other comonomers such as isophthalic acid, cyclohexenedimethyl alcohol, etc.), polybutylene terephthalate (PBT) and polyhexylene terephthalate; and polyalkylene adipate, such as polyethylene adipate, poly(1,4-butyl adipate). Diol esters and polyhexanediol adipate; polyalkylene octanoate, such as polyethylene octanoate; polyalkylene sebacate, such as polyethylene sebacate; poly-ε-caprolactone and poly-β-propiolactone; polyalkylene isophthalate, such as polyethylene isophthalate; polyalkylene 2,6-naphthalene dicarboxylate, such as polyethylene 2,6-naphthalene dicarboxylate; polyalkylene sulfonyl-4,4′-dibenzoate, such as polyethylene sulfonyl-4,4′-dibenzoate; poly(p-phenylene dicarboxylate) Alkylene esters, such as polyethylene p-phenylene dicarboxylate; poly(trans-1,4-cyclohexanediyl dicarboxylate), such as polyethylene trans-1,4-cyclohexanediyl dicarboxylate; poly(1,4-cyclohexanedimethyl dicarboxylate), such as polyethylene 1,4-cyclohexanedimethyl dicarboxylate; poly([2.2.2]-bicyclooctane-1,4-dimethyl dicarboxylate), such as poly([2.2.2]-bicyclooctane-1,4-dimethyl dicarboxylate). Diol esters); lactic acid polymers and copolymers, such as (S)-polylactide, (R,S)-polylactide, polytetramethylglycolic acid and poly(lactide-co-glycolic acid); and polycarbonates of bisphenol A, 3,3′-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A; polyamides, such as poly(p-phenylene terephthalamide); polyethylene terephthalate (e.g., Mylar™ polyethylene terephthalate), combinations thereof, etc.

[0043] In some embodiments, the beamsplitter is a wedge beamsplitter. In these embodiments, the beamsplitter is a beamsplitter with a wedge angle that produces non-collinear back reflection, such that the propagation of the collected light through the wedge beamsplitter causes a small change in the angle of light propagating into one or more of the first and second optical fibers. The wedge beamsplitter according to embodiments of this disclosure has a wedge angle such that a change in the incident angle of the collected light causes a deviation in the angle of the propagated light of 0.001% or more, for example 0.005% or more, for example 0.01% or more, for example 0.05% or more, for example 0.1% or more, for example 0.5% or more, for example 1% or more, for example 2% or more, for example 3% or more, for example 5% or more, and including 10% or more. In some embodiments, the wedge angle of the wedge beamsplitter is from 5 arcminutes to 120 arcminutes, for example, 10 arcminutes to 115 arcminutes, for example, 15 arcminutes to 110 arcminutes, for example, 20 arcminutes to 105 arcminutes, for example, 25 arcminutes to 100 arcminutes, for example, 30 arcminutes to 105 arcminutes, for example, 35 arcminutes to 100 arcminutes, for example, 40 arcminutes to 95 arcminutes, and includes 45 arcminutes to 90 arcminutes. In some embodiments, the wedge beamsplitter has a wedge angle sufficient to reduce or eliminate optical interference. In other embodiments, the wedge beamsplitter has a wedge angle sufficient to reduce or eliminate image artifacts from light, as described herein, where the light is ultimately sensed, measured, or detected by a photodetector.

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

[0045] A beam splitter of interest can be configured to split the amount of light propagating to the first and second optical fibers as needed. In embodiments, the beam splitter may have a beam splitting ratio between the first and second optical fibers (or between the second and first optical fibers) that is 1:99 to 99:1, for example 5:95 to 95:5, for example 10:90 to 90:10, for example 20:80 to 80:20, for example 25:75 to 75:25, and includes a beam splitting ratio of 50:50. In some embodiments, the beam splitter is a 10:90 beam splitter, wherein 10% of the light is propagated to the first optical fiber and 90% of the light is propagated to the second optical fiber.

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

[0047] In embodiments, the notch filter of interest includes a band-stop filter or a band-suppress filter, or other filters configured to allow most frequencies to pass unchanged and remove or attenuate these frequencies to very low levels within a specific range. In embodiments, the notch filter is a bandpass filter with a narrow stopband. In embodiments, a multimode notch filter is a filter configured to remove or attenuate signals at multiple specific frequencies. That is, unlike a conventional notch filter that removes or attenuates a single frequency (e.g., a continuous frequency range), a multimode notch filter removes or attenuates multiple frequencies (e.g., a set of discontinuous frequency ranges). The multimode notch filter of interest includes multiple notch filters, i.e., using multiple individual notch filters, each tuned to the specific frequency to be removed; or, in other cases, includes a multi-stage arrangement of notch filters, i.e., a series of filters, where each stage may be for a different frequency range or type of interference. The multimode notch filter can incorporate any convenient optical separation scheme. In some cases, the multimode notch filter includes one or more notch filters. In other cases, a multimode notch filter includes one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the multimode notch filter includes prisms. In other embodiments, the multimode notch filter includes diffraction gratings. In some embodiments, the multimode notch filter includes dichroic mirrors.

[0048] Further details regarding aspects of the subject light detection system (including optical adjustment components, beam splitters, or multimode notch filters) are described in U.S. Patent No. 10,976,236, the disclosure of which is incorporated herein by reference.

[0049] optical fiber:

[0050] In an embodiment, the optical receiver is configured to receive light passing through an optical collection component. The optical collection component includes, for example, optical fibers. In some cases, the optical collection component includes a fiber optic repeater bundle (e.g., multiple fiber optic components bundled together). In such instances, the optical collection component includes two or more optical fibers, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty-five or more, fifty or more, and even one hundred or more optical fibers.

[0051] In an embodiment, each optical receiver is configured to receive light transmitted through a single optical fiber; that is, a first optical receiver receives a first light beam transmitted by a first optical fiber, and a second optical receiver receives a second light beam transmitted by a second optical fiber. Optical fibers of interest include commercially available optical fibers, such as those available from Thorlabs, Newport Corporation, or FS Inc. The optical fiber includes a light-transmitting material. In some embodiments, the light-transmitting material includes a glass material, such as, but not limited to, silica (e.g., fused silica). In other embodiments, the light-transmitting material includes a polymer material. In such embodiments, the light-transmitting material may include one or more materials, such as, but not limited to, polymethyl methacrylate (PMMA), polystyrene, and poly(perfluorobutylene vinyl ether) (CYTOP).

[0052] The first and second optical fibers can be of the same or different types. In an embodiment, the diameter of the first optical fiber is smaller than the diameter of the second optical fiber. The term "optical fiber diameter" refers (e.g., ultimately) to the inner diameter of the fiber, such as the core diameter or the diameter excluding the cladding or buffer layer. In some cases, the diameter of the first optical fiber can be 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less of the diameter of the second optical fiber. Alternatively, for example, the diameter of the first optical fiber may be 50 µm, 100 µm, 150 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900 µm, 950 µm, or 1000 µm or more smaller than the diameter of the second optical fiber.

[0053] In one embodiment, the first and second optical fibers may be single-mode fibers. In other embodiments, the first and second optical fibers may be multimode fibers. In still other embodiments, one optical fiber may be a single-mode fiber and the other a multimode fiber. The optical fiber of interest includes a near end and a far end, wherein the near end receives light from an optical conditioning element, and the far end transmits light to an optical receiver (e.g., a first optical receiver or a second optical receiver).

[0054] In embodiments, the first optical fiber is configured for an aperture suitable for side-scattered light (or, in some cases, for the aperture suitable for side-scattered light from small particles). In some embodiments, the first optical fiber is configured with a size suitable for the aperture of side-scattered light, or, in some cases, the first optical fiber is configured with a size suitable for the aperture of side-scattered light from small particles. In some embodiments, the diameter of the first optical fiber is configured with a size suitable for the aperture of side-scattered light (or, in some cases, for the aperture of side-scattered light from small particles). In embodiments, the first optical fiber is a 200 µm optical fiber. For example, the diameter of the first optical fiber is 200 µm, for example, the core diameter is 200 µm.

[0055] In embodiments, the first optical fiber is configured to include fiber bends, such as 90-degree bends, 85-95-degree bends, 80-100-degree bends, 75-105-degree bends, 70-110-degree bends, 65-115-degree bends, or 60-120-degree bends. In some embodiments, the first optical fiber is operatively connected to a housing (e.g., a light collection tube) including optical adjustment components, and such housing includes guides or other connectors that cause the first optical fiber to bend. Including bends in the optical fiber simplifies the design of embodiments of the optical detection system and can improve the sensitivity of the system by reducing optical loss or noise. For example, as described herein, including fiber bends eliminates the need for separate optical modulation components (e.g., mirrors, multiple mirrors, or aperture-mirror-fiber assemblies) to guide light from the optical adjustment components to the first optical fiber or along the fiber to the CWD optical detection subsystem.

[0056] In some embodiments, the second optical fiber is configured with an aperture size suitable for fluorescence. In some embodiments, the second optical fiber is configured with an aperture size appropriate for fluorescence. In some embodiments, the diameter of the second optical fiber is configured with an aperture size appropriate for fluorescence. In some embodiments, the second optical fiber is an 800 µm optical fiber. For example, the diameter of the second optical fiber is 800 µm, for example, the core diameter is 800 µm.

[0057] Further details regarding aspects of optical detection systems (including optical fibers and optical fiber systems of interest) are discussed in U.S. Application No. 17,577,639, the disclosure of which is incorporated herein by reference.

[0058] CWD optical detection subsystem:

[0059] As described herein, the CWD optical detection subsystem of interest includes a first optical receiver and a second optical receiver, wherein the first optical receiver is configured to transmit a first light beam to a first optical detection module, and the second optical receiver is configured to transmit a second light beam to a second optical detection module. In an embodiment, the first optical detection module includes a small particle detector. Various aspects of the CWD optical detection subsystem will be described in detail below. Furthermore, other details regarding embodiments of the invention (including the CWD optical detection subsystem and aspects thereof) are discussed in U.S. Application No. 17 / 870,187 and U.S. Patent No. 11,821,830, the disclosures of each of which are incorporated herein by reference in their entirety.

[0060] Optical receiver:

[0061] As described herein, the CWD light detection subsystem according to an embodiment is capable of simultaneously analyzing multiple collected light beams in a single subsystem. The CWD light detection subsystem according to an embodiment includes a first light receiver and a second light receiver. The first light receiver and the second light receiver are respectively configured to receive a first light beam and a second light beam. As discussed herein, a "light receiver" refers to a device configured to receive collected light and propagate said light in a desired direction and / or possess certain characteristics.

[0062] In some embodiments, where the first and second optical receivers include or are mated to an optical collection element (e.g., an optical fiber cable), one or both optical receivers may include a coupler for operatively attaching each respective optical collection element. As described herein, a "coupler" refers to an element configured to engage an optical collection element (e.g., an optical fiber cable) to an optical receiver such that light passing through the optical collection element is transmitted to the receiver. Couplers of interest are configured to engage with an optical fiber connector (e.g., in a mating relationship). In some cases, the coupler includes a recess for receiving the optical collection element, and in some versions, the recess includes a collar for securing the optical collection element relative to the coupler. In other embodiments, the coupler is configured to engage the optical collection element by one or more fasteners (e.g., magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, or combinations thereof).

[0063] The first and second optical receivers can be of the same or different types. In some cases, both the first and second optical receivers include couplers for operatively attaching an optical collection component (i.e., an optical fiber). That is, in an embodiment, the first optical receiver includes a coupler for operatively attaching a first optical fiber, and the second optical receiver includes a coupler for operatively attaching a second optical fiber.

[0064] In some embodiments, one or both of the first and second light receivers further include a beam modulator. As described herein, a “beam modulator” is an optical element configured to alter one or more properties of the light received by the light receiver. Properties of interest may include, but are not limited to, illumination direction, wavelength, beam width, beam intensity, and focal spot. Suitable beam modulators include, for example, lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. In some cases, the received light passes through one or more focusing lenses to reduce the light profile. In other cases, the beam modulator includes one or more collimating lenses for reducing the divergence of the beam delivered to the light detection system. In some cases, the beam modulator is an achromatic double lens.

[0065] The characteristics of the first and second light beams received by the first and second light receivers may be the same or different. For example, in some cases, both the first and second light beams comprise light exhibiting the same wavelength or the same wavelength range. In other embodiments, the first and second light beams comprise light exhibiting different wavelengths or different wavelength ranges. In some embodiments, the first light beam is particle-modulated light generated by irradiating particles in a flow cell with a first light source, and the second light beam is particle-modulated light generated by irradiating particles in a flow cell with the first light source (i.e., the same light source). In other embodiments, the first light beam is particle-modulated light generated by irradiating particles in a flow cell with a first light source, and the second light beam is particle-modulated light generated by irradiating particles in a flow cell with a second light source. For example, in some embodiments, the first light beam comprises light with wavelengths greater than 500 nm. In some versions, the first light beam comprises a concentration of light energy in the blue spectrum (e.g., 400-500 nm). In such versions, light in the ultraviolet (UV), violet, and yellow-green spectra may have been filtered out from the first light beam before being received by the first light receiver. In other versions, the first light beam comprises a concentration of light energy in the yellow-green spectrum (e.g., 500-600 nm). In such versions, light in the ultraviolet (UV), violet, and blue spectra may have been filtered out of the first beam before being received by the first light receiver. In another embodiment, the second beam comprises light with wavelengths in the blue spectrum (e.g., 400-500 nm). In other embodiments, the second beam comprises light with wavelengths greater than 600 nm. In some versions, the second beam comprises light with concentrated energy in the red spectrum (e.g., 600-750 nm). In such versions, light in the ultraviolet (UV), violet, blue, and yellow-green spectra may have been filtered out of the second beam before being received by the second light receiver.

[0066] Wavelength splitter:

[0067] In embodiments, light received from a sample can be divided into spectral ranges by passing it through one or more wavelength splitters. The light in each spectral range generated by the wavelength splitters can be further divided into smaller sub-spectral ranges by optical components, which are detected by one or more detectors in each light detection module. In some embodiments, the light detected from the sample is emitted light, such as fluorescence. In other embodiments, the light detected from the sample is scattered light. The term "scattered light" is used herein in its conventional sense, referring to the propagation of light energy from particles in the sample (e.g., particles flowing in a flow), the light energy being deflected from the incident light path, for example, by beam deflection, reflection, refraction, etc. In some cases, the light detected from the sample is small particle scattered light, i.e., light scattered by small particles.

[0068] In embodiments, the light detection subsystem as described herein is configured to exhibit minimal or no light loss in the light collected from the sample (e.g., light received from the first and second optical fibers). In some embodiments, the light loss due to the transmission of light through the subject light detection subsystem is 25% or less, such as 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, and includes 0.001% or less. In some cases, there is no light loss as the light from the sample propagates through the subject light detection subsystem (i.e., exhibiting immeasurable light loss). For example, when transmitted via a subject light detection system, the amount of light from the sample is reduced by 1 mW / cm² or less, such as 0.5 mW / cm² or less, 0.1 mW / cm² or less, 0.05 mW / cm² or less, 0.01 mW / cm² or less, 0.005 mW / cm² or less, 0.001 mW / cm² or less, 0.0005 mW / cm² or less, 0.0001 mW / cm² or less, 0.00005 mW / cm² or less, and includes 0.00001 mW / cm² or less.

[0069] In some embodiments, the wavelength splitter is configured to generate two or more predetermined spectral ranges of light from a light source (e.g., light from a sample illuminated by light, as described in detail herein), such as generating 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 25 or more, 50 or more, 75 or more, and including 100 or more predetermined spectral ranges of light. In some instances, the CWD light detection subsystem includes a wavelength splitter configured to generate light from a first predetermined spectral range and a second predetermined spectral range from the light source. In other instances, the CWD light detection subsystem includes a wavelength splitter configured to generate light from a first predetermined spectral range, a second predetermined spectral range, and a third predetermined spectral range from the light source.

[0070] In some embodiments, the CWD optical detection subsystem includes two or more wavelength splitters, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty-five or more, fifty or more, seventy-five or more, and even one hundred or more wavelength splitters. The term "wavelength splitter" is used herein in its conventional sense to refer to an optical component configured to separate light collected from a sample into a predetermined spectral range. In some embodiments, the wavelength splitter is configured to separate light collected from a sample into a predetermined spectral range by allowing light having the predetermined spectral range to pass through and reflecting light of one or more remaining spectral ranges. In other embodiments, the wavelength splitter is configured to separate light collected from a sample into a predetermined spectral range by allowing light having the predetermined spectral range to pass through and absorbing light of one or more remaining spectral ranges. In still other embodiments, the wavelength splitter is configured to spatially diffract light collected from a sample into a predetermined spectral range. Each wavelength splitter can be any convenient light separation scheme, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the wavelength splitter is a prism. In other embodiments, the wavelength splitter is a diffraction grating. In some embodiments, the wavelength splitter in a subject light detection system is a dichroic mirror.

[0071] In an embodiment, the wavelength splitter is configured to allow the wavelength range to extend from a first wavelength X. i (Unit: nanometers, nm) to the second wavelength X n Light (unit: nanometers, nm) passes through. In some embodiments, the wavelength splitter is configured to allow the wavelength range from X... i To X nLight can pass through the system, with wavelengths ranging from, for example, 100 nm to 1500 nm, 150 nm to 1450 nm, 200 nm to 1400 nm, 250 nm to 1350 nm, 300 nm to 1300 nm, 350 nm to 1250 nm, 400 nm to 1200 nm, 450 nm to 1150 nm, 500 nm to 1100 nm, and 550 nm to 1050 nm, and also including light with wavelengths ranging from 600 nm to 1000 nm. In some embodiments, the wavelength splitter in the photodetector system of interest is configured to allow light with wavelengths ranging from 360 nm to 960 nm to pass through.

[0072] In this embodiment, each wavelength separator of interest is configured to generate a predetermined spectral range X. s (Unit: nanometers, nm) light. The predetermined spectral range can vary, wherein in some embodiments, the wavelength separator of interest is configured to generate a spectral range (X) spanning from 50 nm to 300 nm. s The light, spanning, for example, from 75 nm to 275 nm, from 100 nm to 250 nm, from 125 nm to 225 nm, and including light with a spectral range from 150 nm to 200 nm. In some embodiments, each wavelength splitter is configured to generate a spectral range spanning 100 nm (i.e., X). s Light with a wavelength of 100 nm.

[0073] In one example, the optical detection system includes a wavelength splitter configured to generate: light in a first predetermined spectral range of 360 nm to 480 nm; light in a second predetermined spectral range of 480 nm to 600 nm; light in a third predetermined spectral range of 600 nm to 720 nm; light in a fourth predetermined spectral range of 720 nm to 840 nm; and light in a fifth predetermined spectral range of 840 nm to 960 nm.

[0074] In another example, the optical detection system includes: a first wavelength splitter configured to provide a wavelength range of 360 nm to 480 nm (i.e., X). s The system includes a second wavelength splitter, configured to allow light with a wavelength range of 480 nm to 600 nm to pass through; a third wavelength splitter, configured to allow light with a wavelength range of 600 nm to 720 nm to pass through; a fourth wavelength splitter, configured to allow light with a wavelength range of 720 nm to 840 nm to pass through; and a fifth wavelength splitter, configured to allow light with a wavelength range of 840 nm to 960 nm to pass through.

[0075] In some embodiments, the CWD optical detection subsystem of interest includes two or more wavelength splitters that communicate optically with each other, for example, these wavelength splitters are positioned to transmit light between each other. In other embodiments, the CWD optical detection subsystem of interest includes three or more wavelength splitters that communicate optically with each other, for example, these wavelength splitters are positioned to transmit light between each other. Wavelength splitters in the CWD optical detection subsystem may be oriented relative to each other (as referenced in the XZ plane) at angles ranging from 10° to 180°, such angles being, for example, 15° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and including 45° to 90°. In some instances, wavelength splitters are positioned along a single plane. In other instances, wavelength splitters are positioned along more than one plane. For example, wavelength splitters may be positioned along two or more parallel planes, such as three or more, four or more, and including positioning along five or more parallel planes. In some cases, wavelength splitters are arranged in geometric configurations, including, but not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregularly shaped configurations. In some embodiments, wavelength splitters are arranged in a pentagonal configuration. In other embodiments, wavelength splitters are arranged in a heptagonal configuration. Wavelength splitters can be spaced apart from each other at any convenient distance. In some instances, adjacent wavelength splitters are spaced 0.001 mm or more, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. In some cases, the distance between adjacent wavelength splitters is constant. In other words, the spacing is the same for any pair of adjacent wavelength splitters. In some cases where the distance between adjacent wavelength splitters is constant across multiple wavelength splitters, this consistency makes the analysis results easier to compare after the signals from optical sensors (such as photodetectors) are digitized.

[0076] In some embodiments, wavelength splitters are configured to transmit light between each other. In some instances, each wavelength splitter is configured to allow light of a spectral range to pass through and transmit (e.g., by reflection) light of one or more remaining spectral ranges to another wavelength splitter. In one example, the light detection system includes two wavelength splitters. In another example, the light detection system includes three wavelength splitters. The first wavelength splitter is configured to receive light from a sample, allow light of a first spectral range to pass through, and transmit light of a second spectral range to the second wavelength splitter. The second wavelength splitter is configured to allow light of a third spectral range to pass through and transmit light of a fourth spectral range to the third wavelength splitter. In some instances, the light of the third spectral range is a portion of the light of the second spectral range, for example, its spectral range span is 90% or less of the light of the second spectral range, such as 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50%. The third wavelength splitter is configured to allow light of a fifth spectral range to pass through. In some instances, the light in the fifth spectral range is a portion of the light in the fourth spectral range, for example, its spectral range span is 90% or less of the light in the fourth spectral range, such as 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50%.

[0077] In another example, the light detection system includes five wavelength splitters. A first wavelength splitter is configured to receive light from a sample, allow light in a first spectral range to pass through, and transmit light in a second spectral range to a second wavelength splitter. A second wavelength splitter is configured to allow light in a third spectral range to pass through and transmit light in a fourth spectral range to a third wavelength splitter. In some instances, the third spectral range is a portion of the second spectral range, for example, its spectral range spanning 90% or less of the second spectral range, such as 85% or less, 70% or less, 65% or less, 55% or less, or 50%. A third wavelength splitter is configured to allow light in a fifth spectral range to pass through and transmit light in a sixth spectral range to a fourth wavelength splitter. In some instances, the light in the fifth spectral range is a portion of the light in the fourth spectral range, for example, its spectral range span is 90% or less of the light in the fourth spectral range, such as 85% or less, 70% or less, 65% or less, 55% or less, or 50%. The fourth wavelength splitter is configured to allow light in the seventh spectral range to pass through and to transmit light in the eighth spectral range to the fifth wavelength splitter. In some instances, the light in the seventh spectral range is a portion of the light in the sixth spectral range, for example, its spectral range span is 90% or less of the light in the sixth spectral range, such as 85% or less, 55% or less, 60% or less, or 55% or less. The fifth wavelength splitter is configured to allow light in the ninth spectral range to pass through. In some instances, the light in the ninth spectral range is a portion of the light in the eighth spectral range, for example, its spectral range span is 90% or less of the light in the eighth spectral range, such as 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50%.

[0078] In some embodiments, the optical detection system includes five wavelength splitters configured to separate light with wavelengths ranging from 360 nm to 960 nm. A first wavelength splitter is configured to allow light with wavelengths ranging from 360 nm to 480 nm to pass through and transmit light with wavelengths ranging from 480 nm to 960 nm to a second wavelength splitter; a second wavelength splitter is configured to allow light with wavelengths ranging from 480 nm to 600 nm to pass through and transmit light with wavelengths ranging from 600 nm to 960 nm to a third wavelength splitter; a third wavelength splitter is configured to allow light with wavelengths ranging from 600 nm to 720 nm to pass through and transmit light with wavelengths ranging from 720 nm to 960 nm to a fourth wavelength splitter; and a fourth wavelength splitter is configured to allow light with wavelengths ranging from 720 nm to 840 nm to pass through and transmit light with wavelengths ranging from 840 nm to 960 nm to a fifth wavelength splitter. In this embodiment, the fifth wavelength splitter is configured to allow light with wavelengths ranging from 840 nm to 960 nm to pass through.

[0079] As discussed herein, a first optical receiver and a second optical receiver are configured to receive a first light beam and a second light beam. In some embodiments, the first light beam is transmitted by a first subset of wavelength splitters, and the second light beam is transmitted by a second subset of wavelength splitters. For example, in one embodiment, the first subset of wavelength splitters includes only one wavelength splitter configured to allow light in the wavelength range of 450–500 nm (i.e., blue) to pass through. In another example, in some cases, the first subset of wavelength splitters includes: a first wavelength splitter configured to allow light in the wavelength range of 450–500 nm (i.e., blue) to pass through; a second wavelength splitter configured to allow light in the wavelength range of 600–675 nm (i.e., red) to pass through; and a third wavelength splitter configured to allow light in the wavelength range of 675–750 nm (i.e., red) to pass through. In other embodiments, a first subset of the wavelength splitters includes: a first wavelength splitter configured to allow light in the wavelength range of 450-500 nm (i.e., blue) to pass through; a second wavelength splitter configured to allow light in the wavelength range of 500-600 nm (i.e., yellow-green) to pass through; a third wavelength splitter configured to allow light in the wavelength range of 600-675 nm (i.e., red) to pass through; and a fourth wavelength splitter configured to allow light in the wavelength range of 675-750 nm (i.e., red) to pass through. In some cases, a second subset of the wavelength splitters includes: a first wavelength splitter configured to allow light in the wavelength range of 600-675 nm (i.e., red) to pass through; and a second wavelength splitter configured to allow light in the wavelength range of 675-750 nm (i.e., red) to pass through. In still other cases, embodiments of the CWD light collection subsystem include a subset of wavelength splitters, wherein wavelength splitters are not applied to either or both of the first and second light detection modules.

[0080] Some embodiments of the CWD optical detection subsystem also include a third optical receiver configured to receive a third beam. In such embodiments, the same CWD optical detection subsystem is configured to analyze the three collected beams and may also include a third subset of wavelength splitters configured to transmit the third beam.

[0081] Light detection module:

[0082] As described above, the CWD optical detection subsystem includes an optical detection module that optically communicates with an optical receiver. For example, a first optical receiver and a second optical receiver optically communicate with each of the first and second optical detection modules, respectively. In some embodiments, each optical detection module also optically communicates with a wavelength splitter. For example, each wavelength splitter in a first subset and a second subset of the wavelength splitters optically communicates with the optical detection module, such that each wavelength splitter is associated with a dedicated optical detection module. In some embodiments, the optical detection module is positioned in physical contact with the wavelength splitter, for example, wherein an opening leading to the optical detection module is physically coupled to the wavelength splitter. In other embodiments, each optical detection module is located 0.001 mm or more from the wavelength splitter, for example, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. For example, each optical detection module can be positioned at a distance of 0.0001 mm to 100 mm from the wavelength splitter, such as 0.0005 mm to 95 mm, 0.001 mm to 90 mm, 0.005 mm to 85 mm, 0.01 mm to 80 mm, 0.05 mm to 75 mm, 0.1 mm to 70 mm, 0.5 mm to 65 mm, 1 mm to 60 mm, 1.5 mm to 55 mm, and including distances of 2 mm to 50 mm. In some embodiments, the wavelength splitter is integrated into the optical detection module, for example, it exists within a single unit.

[0083] The optical detection module can be releasably connected to the wavelength splitter. The term "releasably" is used herein in its conventional sense, meaning that each optical detection module or wavelength splitter can be freely removed and reattached. The optical detection module or wavelength splitter can be connected using any convenient method. In some embodiments, the optical detection module and the wavelength splitter are connected together by fasteners such as magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, non-permanent adhesives, or combinations thereof. In some cases, the optical detection module is connected to the wavelength splitter by fitting a slit into a groove in the optical detection module. In still other cases, the wavelength splitter is connected to the optical detection module by one or more screws.

[0084] In some embodiments, light from each wavelength splitter is transmitted to each optical detection module via an optical collection system. Each optical collection system can be any suitable light collection scheme that collects light across the spectral range of the wavelength splitter and directs that light to the optical detection module. In some embodiments, the optical collection system includes optical fibers, such as fiber optic repeater bundles. In other embodiments, the optical collection system is a free-space optical repeater system.

[0085] In some embodiments, each optical collection system may be physically coupled to the optical detection module, for example, by adhesive, co-molding together, or integrated into each optical detection module. In some embodiments, each optical detection module and the optical collection system are integrated into a single unit. In some cases, each optical detection module is coupled to the optical collection system via a connector that secures the optical collection system to each optical detection module, for example, via hook-and-loop fasteners, magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, non-permanent adhesives, or combinations thereof.

[0086] In other embodiments, each optical detection module and the optical collection system communicate optically but do not physically contact each other. In embodiments, the optical collection system may be located at a distance of 0.001 mm or more from the optical detection module, for example, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 10 mm or more, 25 mm or more, 50 mm or more, and includes a distance of 100 mm or more from the optical detection module.

[0087] In some embodiments, the optical collection system includes optical fibers. For example, the optical collection system may be an optical fiber repeater bundle, and light in the spectral range of the wavelength splitter is transmitted to the optical detection module via the optical fiber repeater bundle. Any optical fiber repeater system can be used to transmit light, and in some embodiments, suitable optical fiber repeater systems include, but are not limited to, those described in, for example, U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0088] In other embodiments, each optical collection system is a free-space optical relay system. In some embodiments, the free-space optical relay system includes a housing having a near end and a far end, the near end being coupled to a light detection module. The free-space optical relay system may include any combination of different optical components, such as one or more lenses, mirrors, slits, pinholes, wavelength splitters, or combinations thereof. For example, in some embodiments, the free-space optical relay system of interest includes one or more focusing lenses. In other embodiments, the free-space optical relay system of this subject includes one or more mirrors. In still other embodiments, the free-space optical relay system includes a collimating lens. In some embodiments, suitable free-space optical relay systems for propagating light from the spectral range of a wavelength splitter include, but are not limited to, those described in, for example, U.S. Patent Nos. 7,643,142, 7,728,974, and 8,223,445, the disclosures of each of which are incorporated herein by reference.

[0089] The light detection modules can be arranged in any geometric configuration (e.g., mounted together) as needed within the subject-matter CWD light detection subsystem. The light detection modules can be arranged along one or more planes. In some embodiments, the light detection modules can be oriented at angles from 0° to 180° relative to each other (as referenced in the XZ plane), such angles being, for example, 10° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and including 45° to 90°. In embodiments, the light detection modules can be arranged at the same or different angles relative to each other depending on the number of light detection modules in the light detection system. For example, in some cases, the angle between the first and second light detection modules is the same as the angle between the second and third light detection modules. In some embodiments, the angle between the first and second light detection modules is different from the angle between the second and third light detection modules. In some embodiments, the light detection modules are positioned in a geometric arrangement, such as a star configuration, a triangle configuration, a square configuration, a rectangle configuration, a trapezoidal configuration, a hexagonal configuration, a heptagonal configuration, an octagonal configuration, a nonagonal configuration, a decagonal configuration, a dodecagonal configuration, a circular configuration, an elliptical configuration, and an irregular shape configuration.

[0090] In some embodiments, the light detection modules have complementary shapes, allowing them to coexist when arranged around a central point. In some embodiments, the light detection modules are substantially wedge-shaped. In other words, if the light detection system is conceptualized as a pie, each constituent light detection module can be considered a slice of the pie. "Substantially wedge-shaped" means that the light detection module may or may not be a perfect wedge, and in some embodiments, the light detection module includes one or more regions where the sides of the module deviate from a straight line. The light detection modules can be positioned very close to each other, for example, with adjacent light detection modules spaced 5 mm or less, such as 4 mm or less, 3 mm or less, 2 mm or less, 1 mm or less, 0.5 mm or less, and including cases where the light detection modules are physically in contact with each other.

[0091] Embodiments of the CWD optical detection subsystem include a first optical detection module configured for small particle detection, the module including a small particle detector and, in some cases, a side-scattering detector. Embodiments of the CWD optical detection subsystem also include a second optical detection module for detecting fluorescence. Embodiments of the CWD optical detection subsystem further include two or more optical detection modules for detecting aspects of light received from the sample (e.g., the spectral range of fluorescence).

[0092] Lateral scattering light detector:

[0093] In an embodiment, the first optical detection module of the CWD optical detection subsystem includes a side-scattering light detector (also referred to as a side-scattering detector). As discussed herein, side-scattering light is light scattered by particles in a direction orthogonal (or substantially orthogonal) to the excitation laser. In some cases, the CWD optical detection subsystem may also include a side-scattering light splitter configured to separate the side-scattering wavelength light from the light received by the first optical receiver. In some cases, the side-scattering light splitter may be positioned between the first optical receiver and the first optical detection module. In some cases, the side-scattering light splitter may be configured to redirect (e.g., backscatter) the side-scattering light at an angle of 1° to 90° (e.g., 30° to 60°, and including 40° to 50°) relative to the optical axis of the light received by the optical receiver. The redirected light is then detected by the side-scattering light detector. Any convenient detector may be used as the side-scattering light detector described herein. In some embodiments, the side-scattering light detector is a photomultiplier tube (PMT). In other embodiments, the side-scattering light detector is a photodiode (e.g., an avalanche photodiode). In some embodiments, the side-scattering light detector is equipped with a focusing aspherical lens that focuses light onto the photosensitive region of the detector. In other embodiments, one or more optical filters may be used instead of the focusing aspherical lens, or as a supplement to it.

[0094] Small particle detector:

[0095] In an embodiment, the first optical detection system includes a small particle detector (also known as a small particle side-scattering light detector or small particle side-scattering detector). As discussed herein, side-scattered light is light scattered by particles in a direction orthogonal (or substantially orthogonal) to the excitation laser, while small particle side-scattering light is light scattered by small particles. In an embodiment, the CWD optical detection subsystem may further include a small particle side-scattering light separator configured to separate the small particle side-scattering wavelength light from the light received by the first optical receiver. In some cases, the small particle side-scattering light separator may be positioned between the first optical receiver and the first optical detection module. In some cases, the small particle side-scattering light separator may be configured to redirect (e.g., backscatter) the small particle side-scattering light at an angle of 1° to 90° (e.g., 30° to 60°, including 40° to 50°) relative to the optical axis of the light received by the optical receiver. The redirected light is then detected by the small particle side-scattering light detector. In an embodiment, the small particle detector is a photodiode (e.g., an avalanche photodiode). In some embodiments, the side-scattering light detector is equipped with a focusing aspherical lens that focuses light onto the photosensitive region of the detector. In other embodiments, one or more optical filters may be used instead of the focusing aspherical lens, or as a supplement to it.

[0096] In one embodiment, the first optical detection module includes a second beam splitter configured to split a first beam into a third beam and a fourth beam. That is, the second beam splitter splits the first beam received from the first optical receiver and the first optical fiber into a third beam and a fourth beam. In another embodiment, the first optical detection module is configured to transmit the third beam to a small particle detector and the fourth beam to a side-scattering detector. In yet another embodiment, the first optical detection module also includes one or more mirrors (in cooperation with the beam splitter) to transmit the third beam to the small particle detector. Furthermore, in another embodiment, the second optical detection module is configured to detect fluorescence. In such embodiments, one or more of the second optical receiver, the wavelength splitter, and the second optical detection module are configured to transmit fluorescence to a fluorescence detector of the second optical detection module.

[0097] Other CWD optical detection subsystems:

[0098] As described above, embodiments of the present invention include a CWD optical detection subsystem. Such a CWD optical detection subsystem includes a first optical detection module that includes a small particle detector. Such a CWD optical detection subsystem also includes a second optical detection module that includes a photodetector configured to detect fluorescence. Such a CWD optical detection subsystem may also include other optical detection modules configured to detect other aspects of light from a sample. In an embodiment, such a CWD optical detection subsystem is configured to detect blue light. That is, the first optical detection module may be configured to detect blue light scattered by small particles in the flow stream, and the second optical detection module may be configured to detect blue fluorescence from particles in the flow stream. Embodiments of the present invention may also include a second CWD optical detection subsystem that includes a third optical receiver and a third optical fiber, wherein the third optical receiver is configured to transmit a third beam to the third optical detection module, and the third optical fiber is configured to transmit the third beam to the third optical receiver of the second CWD optical detection subsystem. In an embodiment, the diameter of the third optical fiber is configured to be an aperture size suitable for fluorescence. In some cases, the diameter of the third optical fiber is 800 µm. In other cases, the second CWD optical detection module is configured to detect purple fluorescence.

[0099] Optical components of the optical detection module:

[0100] In some embodiments, each light detection module includes an optical conditioning element (referred to in some cases as an "optical element") configured to transmit light having a predetermined sub-spectral range to one or more light detectors (e.g., photodetectors). "Optical conditioning" refers to the alteration or conditioning of light as it is transmitted to each photodetector in the light detection module. In some embodiments, optical conditioning includes propagating light having a predetermined sub-spectral range to the photodetector. In some embodiments, each light detection module includes one or more optical conditioning elements configured to separate light from a wavelength splitter into a predetermined sub-spectral range by allowing light having the predetermined sub-spectral range to pass through and reflecting light having one or more remaining spectral ranges. In other embodiments, the optical conditioning element is configured to separate light from a wavelength splitter into a predetermined sub-spectral range by allowing light having the predetermined sub-spectral range to pass through and absorbing light having one or more remaining spectral ranges. In still other embodiments, the optical conditioning element is configured to spatially diffract light from the wavelength splitter into the predetermined sub-spectral range. The optical adjustment component can be any convenient light separation scheme, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the optical adjustment component in the light detection module is a dichroic mirror, which is configured to separate the light transmitted from the wavelength splitter into predetermined spectral ranges.

[0101] Based on the wavelength of the light transmitted from the wavelength splitter to the optical detection module (as described herein), one or more optical components in the optical detection module may be configured to reduce the wavelength range from a first wavelength Y. i (Unit: nanometers, nm) to the second wavelength Y n Light (in nanometers, nm) is transmitted to the photodetector. In some embodiments, one or more optical components are configured to transmit light with wavelengths ranging from 100 nm to 1500 nm to the photodetector, such as from 150 nm to 1450 nm, from 200 nm to 1400 nm, from 250 nm to 1350 nm, from 300 nm to 1300 nm, from 350 nm to 1250 nm, from 400 nm to 1200 nm, from 450 nm to 1150 nm, from 500 nm to 1100 nm, from 550 nm to 1050 nm, and includes propagating light with wavelengths ranging from 600 nm to 1000 nm to the photodetector.

[0102] In this embodiment, the optical components in each optical detection module are configured to target a predetermined sub-spectral range Y. sLight (in nanometers, nm) is transmitted to each photodetector. The predetermined sub-spectral range transmitted by each optical component can vary, wherein certain optical components of interest are configured to transmit light spanning a sub-spectral range from 5 nm to 50 nm, such as from 6 nm to 49 nm, from 7 nm to 48 nm, from 8 nm to 47 nm, from 9 nm to 46 nm, and including from 10 nm to 45 nm. In some embodiments, the optical components are configured to allow light spanning a spectral range of 20 nm to pass through.

[0103] For example, in one instance, one or more optical components are configured to allow the wavelength range to begin from 360 nm (i.e., Y). i =360 nm) to 480 nm (i.e., Y = 360 nm) to 480 nm (i.e., Y n Light with a wavelength of 480 nm (i.e., Y = 480 nm) at a span of 20 nm (i.e., Y = 480 nm) in the light ... s The light passes through a sub-spectral range of 20 nm. In this embodiment, the light detection module includes: a first optical component configured to transmit light with a wavelength range of 360 nm to 380 nm to a photodetector; a second optical component configured to transmit light with a wavelength range of 380 nm to 400 nm to a photodetector; a third optical component configured to transmit light with a wavelength range of 400 nm to 420 nm to a photodetector; a fourth optical component configured to transmit light with a wavelength range of 420 nm to 440 nm to a photodetector; a fifth optical component configured to transmit light with a wavelength range of 440 nm to 460 nm to a photodetector; and a sixth optical component configured to transmit light with a wavelength range of 460 nm to 480 nm to a photodetector.

[0104] In some embodiments, the optical components in each optical detection module communicate optically with each other, for example, these optical components are positioned to transmit light between each other. The optical components in the optical detection module can be oriented at angles from 10° to 180° relative to each other (as referenced in the XZ plane), such angles being, for example, 15° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and including 45° to 90°. In some instances, the optical components are positioned along a single plane. In other instances, the optical components are positioned along more than one plane. For example, the optical components can be positioned along two or more parallel planes, such as three or more, four or more, and including five or more parallel planes. In some cases, the optical components are arranged in geometric configurations, wherein arrangements of interest include, but are not limited to, square configurations, rectangular configurations, trapezoidal configurations, triangular configurations, hexagonal configurations, heptagonal configurations, octagonal configurations, nonagonal configurations, decagonal configurations, dodecagonal configurations, circular configurations, elliptical configurations, and irregular shape configurations. In some embodiments, the optical components are arranged in a hexagonal configuration. In other embodiments, the optical components are arranged in a heptagonal configuration.

[0105] In some embodiments, the optical components are configured to transmit light between each other. In some instances, each optical component is configured to allow light of a spectral range to pass through and transmit (e.g., by reflection) light of one or more remaining spectral ranges to another optical component. In one example, the light detection module includes three optical components. A first optical component is configured to receive light from a wavelength splitter, allow light of a first sub-spectral range to pass through, and transmit light of a second sub-spectral range to a second optical component. A second optical component is configured to allow light of a third sub-spectral range to pass through and transmit light of a fourth sub-spectral range to a third optical component. In some instances, the light of the third sub-spectral range is a portion of the light of the second sub-spectral range, for example, its sub-spectral range span is 90% or less of the light of the second sub-spectral range, such as 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50%. A third optical component is configured to allow light of a fifth sub-spectral range to pass through. In some instances, the light in the fifth sub-spectral range is a portion of the light in the fourth sub-spectral range, for example, its sub-spectral range spans 90% or less of the light in the fourth sub-spectral range, such as 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50%.

[0106] In another example, the light detection module includes five optical components. A first optical component is configured to receive light from a wavelength splitter, allow light in a first sub-spectral range to pass through, and transmit light in a second sub-spectral range to a second optical component. The second optical component is configured to allow light in a third sub-spectral range to pass through and transmit light in a fourth sub-spectral range to a third optical component. In some instances, the light in the third sub-spectral range is a portion of the light in the second sub-spectral range, for example, its sub-spectral range spanning 90% or less of the light in the second sub-spectral range, such as 85% or less, 75% or less, 65% or less, 55% or less, 50% or less, or 60% or less. The third optical component is configured to allow light in a fifth sub-spectral range to pass through and transmit light in a sixth sub-spectral range to a fourth optical component. In some instances, the light in the fifth sub-spectral range is a portion of the light in the fourth sub-spectral range, for example, its sub-spectral range spanning 90% or less of the light in the fourth sub-spectral range, such as 85% or less, 70% or less, 65% or less, 55% or less, 60% or less, 55% or less, or 50%. The fourth optical component is configured to allow the light in the seventh sub-spectral range to pass through and to transmit the light in the eighth sub-spectral range to the fifth optical component. In some instances, the light in the seventh spectral range is a portion of the light in the sixth spectral range, for example, its spectral range spanning 90% or less of the light in the sixth spectral range, such as 85% or less, 55% or less, 60% or less, or 55% or less, or 50%. The fifth optical component is configured to allow the light in the ninth sub-spectral range to pass through. In some instances, the light in the ninth sub-spectral range is a portion of the light in the eighth sub-spectral range, for example, its sub-spectral range span is 90% or less of the light in the eighth sub-spectral range, such as 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50%.

[0107] As described herein, the CWD light detection subsystem is configured to generate light in multiple sub-spectral ranges from light collected from a sample. In some embodiments, the CWD light detection subsystem is configured to generate two or more different spectral ranges of light for each reflection from an optical component (e.g., a dichroic mirror) in the CWD light detection subsystem, such as three or more, four or more, and includes being configured to generate five or more different spectral ranges of light for each reflection from an optical component in the subject light detection system. In some embodiments, the CWD light detection subsystem is configured to generate 30 different spectral ranges using 10 or fewer reflections from the optical component, such as using 9 or fewer reflections from the optical component. In some cases, the ratio of the generated different spectral ranges to the number of reflections from the optical component in the subject light detection system can be from 2:1 to 10:1, such as from 3:1 to 7:1, and includes 3:1 to 5:1.

[0108] In some cases, optical components are configured to collimate light. The term "collimation," used in its conventional sense, refers to optically adjusting the collinearity of light propagation or reducing the divergence of light relative to a common propagation axis. In some cases, collimation involves narrowing the spatial cross-section of the beam. In other cases, optical components include means for changing the direction of the beam, such as changing the propagation direction of the beam by 1° or more, for example, 5° or more, 10° or more, 15° or more, 20° or more, 25° or more, 30° or more, 45° or more, 60° or more, 75° or more, and including changing the light propagation direction by 90° or more. In still other cases, optical components include reduction means to reduce the size of the light (e.g., the spot size), such as reducing the size by 5% or more, for example, 10% or more, 25% or more, 50% or more, and including reducing the size by 75% or more.

[0109] Light in each sub-spectral range is transmitted to the photodetector by an optical component. In some embodiments, the optical component is in physical contact with the photodetector. In other embodiments, the optical component communicates optically with the photosensitive surface of the photodetector and may be located at a distance of 0.001 mm or more from the photodetector, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 10 mm or more, 25 mm or more, 50 mm or more, and including a distance of 100 mm or more from the photodetector.

[0110] In embodiments, each light detection module includes one or more photodetectors, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty-five or more, fifty or more, and up to one hundred or more photodetectors. In some embodiments, the light detection module includes one or more photodetector arrays. The term "photodetector array" is used in its conventional sense to refer to an arrangement or series connection of two or more photodetectors. In embodiments, a photodetector array may include two or more photodetectors, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, and up to fifteen or more photodetectors. The photodetectors in each array can be arranged in any geometric configuration as needed, including but not limited to square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregularly shaped configurations. The photodetectors in each photodetector array can be oriented at an angle of 10° to 180° relative to another (as referenced in the XZ plane), such angles being, for example, 15° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and including 45° to 90°.

[0111] Photodetector:

[0112] As described herein, the photodetector can be any convenient optical sensor, such as an active pixel sensor (APS), an avalanche photodiode, an image sensor, a charge-coupled device (CCD), an enhancement charge-coupled device (ICCD), a complementary metal-oxide-semiconductor (CMOS) image sensor or an N-type metal-oxide-semiconductor (NMOS) image sensor, a light-emitting diode, a photon counter, a calorimeter, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor, or a combination thereof, as well as other types of photodetectors. In some embodiments, the photodetector includes a photomultiplier tube, such as a metal-encapsulated photomultiplier tube. As described herein, in an embodiment, the small particle detector of the first light detection module is an avalanche photodiode.

[0113] The photodetector of interest is configured to measure light collected at one or more wavelengths, such as at two or more wavelengths, such as at five or more different wavelengths, such as at ten 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 includes measuring light emitted by a sample in the flow stream at 400 or more different wavelengths.

[0114] In embodiments, the photodetector is configured to measure light continuously or at discrete intervals. In some instances, the photodetector of interest is configured to measure the collected light continuously. In other instances, the photodetector 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 some other interval.

[0115] In some embodiments, one or more photodetectors described herein are positioned within a detection block. The detection block of interest is configured to receive one or more photodetectors therein. The detection block described herein can be constructed from any readily available material. In some embodiments, the detection block includes a thermally conductive material. In some embodiments, the thermally conductive material includes metals (e.g., copper or aluminum). In some cases, the detection block is made of copper. In some instances, the detection block is coated with a thermally conductive compound (e.g., thermal paste). Thermally conductive compounds of interest include, but are not limited to, epoxy resins, silicone resins, polyurethanes, and acrylates. One or more photodetectors can be secured to the detector block within the photodetector module, for example, by means of the fixture described in U.S. Provisional Patent Application No. 63,210,390, the disclosure of which is incorporated herein by reference in its entirety.

[0116] Temperature control system:

[0117] In some instances, the CWD photodetector subsystem also includes a temperature control system configured to dissipate heat generated by the photodetector. For example, in one embodiment, the photodetector module includes a thermoelectric cooler in contact with the bottom surface of one or more detection blocks. The term “thermoelectric cooler” is used herein in its conventional sense to refer to a heat pump that transfers heat between two different surfaces (e.g., a “cold” surface and a “hot” surface) in response to the application of an electric current. In some embodiments, the heat flux between the two different surfaces is generated by the Peltier effect, and the thermoelectric cooler of interest is a Peltier heat pump. In some embodiments, the two different surfaces of the thermoelectric cooler (e.g., plates) are formed of different materials (n-type semiconductor, p-type semiconductor), such as narrow bandgap semiconductors and heavy element materials with low thermal conductivity. For example, the surface of the thermoelectric cooler of interest may be formed of a semiconductor (e.g., bismuth telluride, lead telluride, silicon germanium, bismuth-antimony alloys, and combinations thereof). In some instances, the thermoelectric cooler is coated with a thermally conductive compound (e.g., thermal paste). Thermally conductive compounds of interest include, but are not limited to, epoxy resins, silicones, polyurethanes, and acrylates. In some embodiments, thermoelectric coolers of interest include those described in U.S. Patent Publication No. 2004 / 0155251 and U.S. Patent Nos. 6,499,306, 4,581,898, 4,922,822, 5,409,547 and 2,984,077, the disclosures of which are incorporated herein by reference.

[0118] In another embodiment, the light detection module includes a heat sink in thermal contact with the thermoelectric cooler. The heat sink may include, for example, heat sink fins. According to embodiments, the heat sink fins may be any convenient shape configured to dissipate heat from the heat transfer block, and may be cylinders, cubes, cones, hemispheres, stars, triangular prisms, rectangular prisms (cubic prisms), hexagonal prisms, or other suitable polyhedra. For example, the cross-section of the heat sink may be circular, elliptical, semi-circular, crescent-shaped, star-shaped, square, triangular, rhomboid, pentagonal, hexagonal, heptagonal, octagonal, rectangular, or other polygonal. In some embodiments, the heat sink may include multiple heat sink fins, such as two or more, five or more, ten or more, fifteen or more, twenty-five or more, fifty or more, and even one hundred or more heat sink fins.

[0119] In one embodiment, the temperature control system further includes an air inlet at the bottom of each photodetector module. Cool air received from the air inlet passes through a heat sink in each photodetector module. Heat is transferred from the heat sink fins into the air. The resulting hot air is collected from each photodetector module and exhausted.

[0120] Exemplary Example:

[0121] Figure 1A-1C Different views of an optical detection system for small particle detection according to certain embodiments of the present disclosure are shown. Figure 1A A light detection system 100 is depicted. In the light detection system 100, a light collection tube 101 receives a light beam 102 from a flow cell (not shown) of, for example, a flow cytometer (i.e., the light beam 102 propagates from the right side of the figure toward the left side of the figure). As the light beam 102 propagates from right to left, it is incident on an optical adjustment component, which is a beam splitter 103 located in the collection tube 101. The beam splitter 103 splits the light beam 102 received from the flow cell into a first beam 104 and a second beam 105. The first beam 104 includes light scattered from particles excited within the flow cell, and the second beam 105 includes fluorescence from particles excited within the flow cell. The beam splitter 103 directs the first beam 104 to a first optical fiber 106, and the beam splitter 103 directs the second beam 105 to a second optical fiber 108. That is, the proximal end of the first optical fiber 106 is in optical communication with the beam splitter 103, such that the first beam 104 is transmitted from the beam splitter 103 to the first optical fiber 106. Similarly, the near end of the second optical fiber 108 communicates optically with the beam splitter 103, so that the second beam 105 is transmitted from the beam splitter 103 to the second optical fiber 108.

[0122] In an embodiment, the diameter of the first optical fiber 106 is smaller than the diameter of the second optical fiber 108. In the optical detection system 100, the first optical fiber 106 is an aperture for small-particle light, and the second optical fiber 108 is an aperture for fluorescence. Specifically, the first optical fiber 106 is selected to have a diameter that allows it to be an aperture for small-particle light, and the second optical fiber 108 is selected to have a diameter that allows it to be an aperture for fluorescence. In an embodiment, the first optical fiber 106 is a 200µm fiber, and the second optical fiber 108 is an 800µm fiber. The first optical fiber 106 includes an optical fiber bend 107 to redirect the first beam 104. In the illustrated embodiment, the optical fiber bend 107 is approximately a 90-degree bend; however, larger or smaller bends are also considered, and such bends can be varied as needed to facilitate wiring the first optical fiber 106 within the optical detection system 100.

[0123] The first optical fiber 106 is configured to transmit a first beam 104 from the beam splitter 103 to the first optical receiver 111 of the clustered wavelength division (CWD) optical detection subsystem 110. That is, as described above, the near end of the first optical fiber 106 communicates optically with the beam splitter 103, and the far end of the first optical fiber 106 communicates optically with the first optical receiver 111, such that the first optical fiber 106 transmits light from the beam splitter 103 to the first optical receiver 111 of the CWD optical detection subsystem 110. The first optical receiver 111 is positioned to transmit light to the first optical detection module 120 of the CWD optical detection subsystem 110.

[0124] The second optical fiber 108 is configured to transmit the second beam 105 from the beam splitter 103 to the second optical receiver 113 of the CWD optical detection subsystem 110. That is, as described above, the near end of the second optical fiber 108 communicates optically with the beam splitter 103, and the far end of the second optical fiber 108 communicates optically with the second optical receiver 113, such that the second optical fiber 108 transmits light from the beam splitter 103 to the second optical receiver 113 of the CWD optical detection subsystem 110. The second optical receiver 113 is positioned to transmit light to the second optical detection module 130 of the CWD optical detection subsystem 110.

[0125] In this embodiment, the light collecting tube 101 and the optical adjustment components serving as the beam splitter 103 can be located at any convenient distance from the CWD optical detection subsystem 110. In this embodiment, the use of the first optical fiber 106 and the second optical fiber 108 (including by utilizing fiber bending 107) to optically transmit the first beam and the second beams 104, 105 to the CWD optical detection subsystem 110 enables design flexibility in the placement of components around the optical detection system 100 (i.e., spacing, orientation, or relative orientation, etc.), such as the placement of the light collecting tube 101 and the CWD optical detection subsystem 110, and the placement when other CWD optical detector subsystems are present. This also helps to transmit light to the CWD optical detection subsystem while reducing light loss or noise.

[0126] Figure 1B A close-up view of a CWD optical detection subsystem 110 is shown. The CWD optical detection subsystem 110 receives a first light beam 104 transmitted by a first optical fiber 106 at a first optical receiver 111 and a second light beam 105 transmitted by a second optical fiber 108 at a second optical receiver 113. The first and second optical receivers 111 and 113 are attached to a substrate 119 of the CWD optical detection subsystem 110 such that they are fixed relative to each other. In an embodiment, the first optical receiver 111 may include a first coupler 112 configured to operatively attach an optical fiber (e.g., the first optical fiber 106) to the first optical receiver 111; the second optical receiver 113 may include a second coupler 114 configured to operatively attach an optical fiber (e.g., the second optical fiber 108) to the second optical receiver 113. The first and second couplers 112 and 113 may include, for example, threaded connections configured to engage with the housings or connectors of the first and second optical fibers 106 and 108.

[0127] The CWD optical detection subsystem 110 also includes a first optical detection module 120 and a second optical detection module 130. Upon receiving a first beam 104 transmitted from the first optical fiber 106, the first optical receiver 111 directs the first beam 104 to the first optical detection module 120. The first optical detection module 120 is shown in cross-sectional view. The first optical detection module 120 includes a small particle detector 121 (SPD) for detecting light from small particles (e.g., blue scattered light from small particles). The first optical detection module 120 also includes a side-scattering detector (BSSC) for detecting side-scattered light, which is not limited to light scattered by small particles.

[0128] Similarly, upon receiving the second beam 105 transmitted from the second optical fiber 108, the second optical receiver 113 directs the second beam 105 to the second optical detection module 130. The second optical detection module 130 includes one or more detectors for detecting fluorescence (e.g., blue fluorescence). Unlike the first optical detection module 120, the second optical detection module 130 is not shown in cross-sectional view.

[0129] The CWD optical detection subsystem 110 also includes a third optical detection module 131, a fourth optical detection module 132, and a fifth optical detection module 133. Unlike the first optical detection module 120, the third, fourth, and fifth optical detection modules 131, 132, and 133 are not shown in cross-sectional view. Each of the third, fourth, and fifth optical detection modules 131, 132, and 133 receives light originating from the second beam 105 for detecting light within a specific spectral range. After entering the CWD optical detection subsystem 110 via the second optical receiver 113, the second beam 105 is incident on a wavelength splitter 130a associated with the second optical detection module 130. The wavelength splitter 130a allows light with a predetermined spectral range to pass through (i.e., allows light to enter the second optical detection module 130) and directs the remaining light to a wavelength splitter 131a associated with the third optical detection module 131. Wavelength splitter 131a allows light with a predetermined spectral range to pass through (i.e., allows light to enter the third light detection module 131) and directs the remaining light to wavelength splitter 132a associated with the fourth light detection module 132. Wavelength splitter 132a allows light with a predetermined spectral range to pass through (i.e., allows light to enter the fourth light detection module 132) and directs the remaining light to wavelength splitter 133a associated with the fifth light detection module 133.

[0130] Figure 1CA close-up cross-sectional view of the first light detection module 120 is shown. The first light detection module 120 includes a small particle detector 121 for detecting light associated with small particles, and is a dedicated, high-sensitivity detector. In this case, the small particle detector 121 is a dedicated detector for sensing scattered light to analyze small particles. In the illustrated embodiment, the small particle detector 121 is an avalanche photodiode. In another embodiment, the first light detection module 120 also includes another detector, such as a side-scattering detector 122.

[0131] The first optical detection module 120 also includes a second beam splitter 125 configured to split the first beam 104 into a third beam 123 and a fourth beam 124. The first optical detection module 120 is configured (e.g., using a mirror 126) to transmit the third beam 123 to a small particle detector 121 and the fourth beam 124 to a side-scattering detector 122. The first optical detection module 120 also includes a lens 127 and a bandpass filter 128 for adjusting the characteristics of the first beam 104 before it reaches the small particle detector 121 or the side-scattering detector 122.

[0132] Figure 1D It shows in Figure 1A-1C The optical detection system 100 for small particle detection depicted is further extended to detect additional fluorescence and scattered light in a dedicated optical detection module, in this case, the additional fluorescence and scattered light being violet fluorescence and violet scattered light. In this case, the optical detection system 100 also includes a second CWD optical detection subsystem comprising a third optical fiber 141 and a third optical detection module 140. The third optical fiber 141 transmits light from a light collection tube 101 to the third optical detection module 140. The third optical fiber 141 is selected such that it has the aperture for the desired light (in this case, violet fluorescence and violet scattered light), and in this case, the third optical fiber 141 is an 800µm fiber. The third optical detection module 140 is shown in cross-section and includes a detector 142 for sensing and analyzing fluorescence and / or light scattering. Light transmitted through the third optical fiber 141 to the third optical detection module 140 is directed to the detector 142. The third optical fiber 141 transmits light to the second CWD optical detection subsystem by sending light to a third optical receiver (not shown), which in turn transmits the light to a third optical detection module 140, and subsequently to a detector 142 present in the third optical detection module 140. The second CWD optical detection system may include an additional optical detection module for detecting light of a specific spectrum selected using a wavelength splitter, as described herein.

[0133] Figure 1E-1FA close-up view of the light collection tube 101 is shown. The light collection tube 101 is docked to, for example, the flow cell of a flow cytometer, such that it receives light from the flow cell and ultimately transmits it to the small particle detector 121 of the CWD light detection subsystem 110. The light collection tube 101 is mounted using a picomotor to allow fine-tuning of the position of the light beam received from the flow cell and entering the light collection tube 101 along the x, y, and z axes. In embodiments, the light collection tube 101 may be attached to or integrated with the flow cytometer, such that light from the flow cell of the flow cytometer is received by the light collection tube 101 for small particle detection.

[0134] Flow cytometer

[0135] This disclosure also includes aspects of flow cytometers. Flow cytometers of interest include, or are operatively connected to, the optical detection system for small particle detection of this disclosure. As described in detail herein, the optical detection system of interest for small particle detection includes a small particle detector for detecting the presence of small particles in the flow stream or information about the small particles. Furthermore, the flow cytometer includes a light source configured to illuminate particles in the flow stream at a detection point within the flow cell. Light received from the flow stream at the flow cell can be transmitted to optical conditioning components (e.g., a beam splitter or a multimode notch filter) of the optical detection system for small particle detection of this disclosure.

[0136] A flow cell of interest includes a cuvette configured to transport particles in a flow stream. As discussed herein, a “flow cell” generally refers to a component containing a flow channel for transporting particles in a sheath fluid. The cuvette of interest has a passageway (i.e., a flow channel) running through it. The flow stream configured in the flow channel may include a liquid sample injected from a sample tube. In some cases, the flow cell includes a light-accessible flow channel. The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed of silica, for example, fused silica. In some cases, the flow cell is configured to be illuminated with light from a light source at one or more detection points. A “detection point” discussed herein refers to the area within the flow cell where particles are illuminated by light from a light source, for example, for analysis. The size of the detection point can vary as needed. For example, when 0 µm represents the optical axis of the light emitted by the light source, the detection point range can be from -50 µm to 50 µm, for example from -25 µm to 40 µm, and includes -15 µm to 30 µm. Depending on certain considerations (e.g., the number and arrangement of lasers), multiple irradiation points can exist within the flow cell.

[0137] In some embodiments, the flow cell includes or is configured to be used with a sample injection port, which is configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable sample flow into the flow cell cavity (i.e., the flow channel). Depending on the desired flow characteristics, the sample rate delivered from the sample injection port to the flow cell can be 1 µL / min or more, for example 2 µL / min or more, for example 3 µL / min or more, for example 5 µL / min or more, for example 10 µL / min or more, for example 15 µL / min or more, for example 25 µL / min or more, for example 50 µL / min or more, and includes 100 µL / min or more. In some cases, the sample rate delivered from the sample injection port to the flow cell is 1 µL / s or more, for example 2 µL / ss or more, for example 3 µL / ss or more, for example 5 µL / ss or more, for example 10 µL / ss or more, for example 15 µL / ss or more, for example 25 µL / ss or more, for example 50 µL / ss or more, and includes 100 µL / ss or more.

[0138] The sample injection port can be an orifice located in the wall of the lumen or a conduit located proximal to the lumen. When the sample injection port is an orifice located in the wall of the lumen, the orifice can be of any suitable shape. Cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as parabolic bottoms coupled to a flat top. In some embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on its shape; in some cases, the opening is 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0139] In some cases, the sample injection port is a conduit located proximal to the flow cell cavity. For example, the sample injection port may be a conduit positioned so that its orifice is aligned with the flow cell orifice. When the sample injection port is a conduit aligned with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including but not limited to: straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses, etc.; and irregular shapes, such as parabolic bottoms coupled to a flat top. The orifice of the conduit can vary depending on its shape; in some cases, the orifice opening is 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip with a bevel angle of 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, and including a 5° bevel.

[0140] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid into the flow cell cavity, for example, in combination with a sample to generate a laminar flow of sheath fluid around the sample flow. Depending on the desired flow characteristics, the rate of sheath fluid delivered to the flow cell cavity can be 25 µL / s or more, for example 50 µL / s or more, for example 75 µL / s or more, for example 100 µL / s or more, for example 250 µL / s or more, for example 500 µL / s or more, for example 750 µL / s or more, for example 1000 µL / s or more, and includes 2500 µL / s or more.

[0141] In some embodiments, the sheath fluid injection port is an orifice located in the inner wall of the cavity. The sheath fluid injection port orifice can be of any suitable shape, with cross-sectional shapes of interest including but not limited to: straight cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as parabolic bottoms coupled to flat tops. The size of the sheath fluid injection port orifice can vary depending on the shape, and in some cases, the opening is 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0142] The flow cytometer disclosed herein includes a light source configured to irradiate particles in a flow stream at a detection point within a flow cell. The number of light sources in the flow cytometer can vary. In some embodiments, the flow cytometer includes a single light source. Alternatively, in some cases, the flow cytometer may include multiple light sources. In some such cases, the number of light sources ranges from 2 to 10, for example, 2 to 5, including 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 embodiments, 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 cases, 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 cases, the flow cytometers of this subject include dye lasers, such as stilbene, coumarin, or rhodamine lasers. In still other cases, lasers of interest include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, or combinations thereof. In other cases, the flow cytometers of this subject include solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers and combinations thereof.

[0143] The laser source according to some embodiments may also include one or more optical adjustment components. In some embodiments, the optical adjustment components are located between the source and the flow cell and may include any means capable of changing the spatial width of the illumination or some other characteristics of the illumination from the source (e.g., illumination direction, wavelength, beam width, beam intensity, and focal spot). The optical adjustment scheme may include any suitable device for adjusting one or more characteristics of the source, including but not limited to lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation schemes, 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 reducing lens. In other embodiments, the flow cytometer of interest includes optical fibers.

[0144] The light source can be positioned at any suitable distance from the flow cell, for example, 0.005 mm or more, such as 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, and including distances of 100 mm or more. Furthermore, the light source can be positioned at any suitable angle relative to the flow cell, for example, an angle range from 10° to 90°, such as from 15° to 85°, such as from 20° to 80°, such as from 25° to 75°, including from 30° to 60°, and such as at 90°.

[0145] In some embodiments, the light source of interest includes a plurality of lasers configured to provide laser light for discrete illumination of the flowing stream. The plurality of lasers may be, for example, two or more lasers, three or more lasers, four or more lasers, five or more lasers, ten or more lasers, or fifteen or more lasers configured to provide laser light for discrete illumination of the flowing stream. Depending on the wavelength of the light required to illuminate the flowing stream, each laser may have a specific wavelength varying from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including wavelengths from 400 nm to 800 nm. In some embodiments, the lasers of interest may include one or more of 405 nm, 488 nm, 561 nm, and 635 nm lasers.

[0146] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some cases, the beam generator includes a laser or a radio frequency (RF) generator configured to apply an RF drive signal to the acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the lasers in the beam generator of interest include those listed above.

[0147] The acousto-optic device can be any suitable acousto-optic scheme configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in this subject system is configured to generate an angle-deflected laser beam from light from a laser and an applied radio frequency (RF) drive signal. Any suitable RF drive signal source can be used to apply the RF drive signal to the acousto-optic device, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0148] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a laser beam with a desired number of angular deflections in the output laser beam. For example, it is configured to apply three or more radio frequency drive signals, such as four or more radio frequency drive signals, such as five or more radio frequency drive signals, such as six or more radio frequency drive signals, such as seven or more radio frequency drive signals, such as eight or more radio frequency drive signals, such as nine or more radio frequency drive signals, such as ten or more radio frequency drive signals, such as fifteen or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and includes being configured to apply 100 or more radio frequency drive signals.

[0149] In some cases, in order to generate an intensity distribution of an angle-deflected laser beam in the output laser beam, the controller is configured to apply an amplitude-varying radio frequency drive signal, such as from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to 50V, for example from about 2V to 40V, for example from 3V to about 30V, and including from about 5V to about 25V. In some embodiments, each applied radio frequency drive signal has a frequency from about 0.001 MHz to 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and including from about 5 MHz to about 50 MHz.

[0150] In some embodiments, the controller has a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to produce an output laser beam with angular deflections of a desired intensity distribution. For example, the memory may include instructions to produce two or more laser beams with the same intensity, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more; and the memory may include instructions to produce 100 or more laser beams with the same intensity. In other embodiments, the memory may include instructions to produce two or more laser beams with different intensities, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more; and the memory may include instructions to produce 100 or more laser beams with different intensities.

[0151] In some embodiments, the controller has a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with increasing intensity along a horizontal axis from the edge to the center. In these examples, the intensity of the angle-deflected laser beam at the center of the output beam along the horizontal axis can be from 0.1% to about 99%, for example from 0.5% to about 95%, for example from 1% to about 90%, for example from about 2% to about 85%, for example from about 3% to about 80%, for example from about 4% to about 75%, for example from about 5% to about 70%, for example from about 6% to about 65%, for example from about 7% to about 60%, for example from about 8% to about 55%, and includes about 10% to about 50%. In other embodiments, the controller has a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with increasing intensity along a horizontal axis from the edge to the center. In these cases, the intensity of the angle-deflected laser beam at the edge of the output laser beam along the horizontal axis can be from 0.1% to about 99%, for example from 0.5% to about 95%, for example from 1% to about 90%, for example from about 2% to about 85%, for example from about 3% to about 80%, for example from about 4% to about 75%, for example from about 5% to about 70%, for example from about 6% to about 65%, for example from about 7% to about 60%, for example from about 8% to about 55%, and including about 10% to about 50%. In other embodiments, the controller has a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with a Gaussian intensity distribution along the horizontal axis. In still other embodiments, the controller has a processor having a memory operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with a top-hat intensity distribution along the horizontal axis.

[0152] In embodiments, the beam generator of interest can be configured to generate spatially separated angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired illumination distribution of the output laser beam, the angle-deflected laser beams can be spaced 0.001 μm or greater, for example, 0.005 μm or greater, for example, 0.01 μm or greater, for example, 0.05 μm or greater, for example, 0.1 μm or greater, for example, 0.5 μm or greater, for example, 1 μm or greater, for example, 5 μm or greater, for example, 10 μm or greater, for example, 100 μm or greater, for example, 500 μm or greater, for example, 1000 μm or greater, including 5000 μm or greater. In some embodiments, the system is configured to generate overlapping angle-deflected laser beams within the output laser beam, for example, overlapping with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (such as the overlap of beam spots) can be 0.001 μm or greater, for example, 0.005 μm or greater, such as 0.01 μm or greater, 0.05 μm or greater, 0.1 μm or greater, 0.5 μm or greater, 1 μm or greater, 5 μm or greater, 10 μm or greater, including 100 μm or greater.

[0153] In some cases, beam generators configured to produce two or more frequency-shifted beams include, for example, U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, and 10,078,045. The laser excitation modules described in patents 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369, and 11,946,851; the disclosures of these patents are incorporated herein by reference.

[0154] Furthermore, the flow cytometer includes detectors configured to collect light emitted by irradiated particles. The photodetectors are configured to detect particle-modulated light transmitted by an fiber optic light collecting element and generate a signal based on the characteristics of the light, such as intensity. For example, one or more particle-modulated photodetectors may include one or more side-scatter photodetectors for detecting the side-scatter wavelengths of the light (i.e., light refracted and reflected from the surface and internal structure of the particle). In some embodiments, the flow cytometer includes a single side-scatter photodetector. In other embodiments, the flow cytometer includes multiple side-scatter photodetectors, such as two or more, three or more, four or more, and five or more.

[0155] The side-scattering light detector described herein can use any suitable detector for detecting the collected light. Detectors of interest can include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-mode charge-coupled devices (ICCDs), photon counters, calorimeters, photoresistors, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors, or 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, for example, with an effective detection surface area per region ranging from 0.01 cm². 2 Up to 10 cm 2 Photomultiplier tubes, for example, from 0.05 cm 2 Up to 9 cm 2 For example, from 0.1 cm 2 Up to 8 cm 2 For example, from 0.5 cm 2 Up to 7 cm 2 and including from 1 cm 2 up to 5cm 2 .

[0156] In one embodiment, the flow cytometer of this subject also includes a fluorescence detector configured to detect light at one or more fluorescence wavelengths. In other embodiments, the flow cytometer includes a plurality of fluorescence detectors, such as two or more, three or more, four or more, five or more, ten or more, fifteen or more, and including twenty or more.

[0157] Any suitable detector for detecting the collected light can be used in conjunction with the fluorescence detector described herein. Detectors of interest can include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-mode charge-coupled devices (ICCDs), photon counters, calorimeters, photoresistors, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors, or 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, for example, with an effective detection surface area per region ranging from 0.01 cm². 2 Up to 10 cm 2 Photomultiplier tubes, for example, from 0.05 cm 2 Up to 9 cm 2 For example, from 0.1 cm 2 Up to 8 cm 2 For example, from 0.5 cm 2 Up to 7 cm 2 and including from 1 cm 2 Up to 5 cm 2 .

[0158] When the flow cytometer of this subject includes multiple fluorescence detectors, each fluorescence detector can be identical, or the collection of fluorescence detectors can be a combination of detectors of different types. For example, when the flow cytometer of this subject 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, both the first and second fluorescence detectors are CCD-type devices. In still other embodiments, both the first and second fluorescence detectors are CMOS-type devices. In still other embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector is a photomultiplier tube (PMT). In still other embodiments, the first fluorescence detector is a CMOS-type device and the second fluorescence detector is a photomultiplier tube. In still other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0159] In embodiments of this disclosure, the fluorescence detector of interest is configured to measure light collected at one or more wavelengths, such as two or more wavelengths, such as five or more different wavelengths, such as ten or more different wavelengths, such as 25 or more different wavelengths, such as 50 or more different wavelengths, such as 100 or more different wavelengths, such as 200 or more different wavelengths, such as 300 or more different wavelengths, and includes measuring light emitted by a sample in a flowing stream at 400 or more different wavelengths. In some embodiments, two or more detectors in the module as described herein are configured to measure collected light at the same or overlapping wavelengths.

[0160] In some embodiments, the fluorescence detector of interest is configured to measure collected light within a wavelength range (e.g., 200 nm to 1000 nm). In some embodiments, the detector of interest is configured to collect a spectrum within a wavelength range. For example, a flow cytometer can include one or more detectors configured to collect a spectrum within one or more wavelength ranges from 200 nm to 1000 nm. In other embodiments, the detector of interest is configured to measure light emitted by a sample in a flowing stream at one or more specific wavelengths. For example, the module can include one or more detectors configured to measure light at 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In some embodiments, one or more detectors can be configured to pair with a specific fluorophore, such as a fluorophore used with the sample in a fluorescence assay.

[0161] Flow cytometers can include one or more suitable mechanisms for providing sheath fluid and sample flow to the sample fluid input coupler and the sheath fluid input coupler. For example, the sample fluid input coupler can be fluidly connected to a sample fluid line (e.g., tubing) fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler can be fluidly connected to a sheath fluid line fluidly connected to a sheath fluid reservoir. Similarly, flow cytometers can include any suitable mechanism for managing waste from the flow stream. A fluid output coupler can be fluidly connected to a waste line fluidly connected to a waste reservoir. U.S. Patent Application Publication No. 2022 / 0341838 provides a fluid management system applicable to the flow cytometers of the present invention, the disclosure of which is incorporated herein by reference in its entirety.

[0162] Suitable flow cytometry systems may include, but are not limited to, Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), FlowCytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan;49(pt 1):17-28; Linden et al., Semin Throm Hemost. 2004 Oct;30(5):502-11; Alison et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. The public content described in 24(3):203-255 is incorporated herein by reference.In some cases, flow cytometry systems of interest include the BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, BD Biosciences Via™ cell sorter, and BD... Biosciences Influx™ Cell Sorter, BD Biosciences Jazz™ Cell Sorter, BD Biosciences Aria™ Cell Sorter, BD Biosciences FACSAria™ II Cell Sorter, BD Biosciences FACSAria™ III Cell Sorter, BD Biosciences FACSAria™ Fusion Cell Sorter, BD Biosciences FACSMelody™ Cell Sorter, BD Biosciences FACSymphony™ S6 Cell Sorter, BD Biosciences FACSDiscover™ Cell Sorter, or similar products.

[0163] In some embodiments, the subject system is a flow cytometry system, such as the system described in the following U.S. patents: 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, and 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, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,1 The disclosures of the following patents are incorporated herein by reference in their entirety: 46, 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, and 4,498,766.

[0164] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the subject system is a flow cytometry system configured to image particles in a flowing stream using fluorescence imaging with radio frequency labeled emission (FIRE), such as those described by Diebold et al. in Nature Photonics, Vol. 7 (10); those described in 806-810 (2013); and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316. The disclosures of those listed in 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369, and 11,946,851 are incorporated herein by reference. In some embodiments, the flow cytometer is a particle sorter, and the particle sorter is an image-enabled particle sorter. Image-enabled particle sorters are described in the following U.S. Patent Nos. 10,324,019, 10,620,111, 11,105,728, and 11,774,343, and in 18 / 537,103, 18 / 657,618, 18,657,623, and 18 / 657,633, the disclosures of which are incorporated herein by reference in their entirety.

[0165] Figure 1G A system 150 for flow cytometry according to an exemplary embodiment of the present invention is shown. System 150 includes a flow cytometer 192, a controller / processor 190, and a memory 195. The flow cytometer 192 includes one or more excitation lasers 165a-165c, a focusing lens 180, a flow cell 185, a forward-scattering photodetector 193, a light-collecting tube 166 including optical adjustment components, and a light detection system 191.

[0166] The 165a-165c laser emits light in the form of a laser beam. Figure 7In the example system, the laser beams emitted by the excitation lasers 165a-165c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first guided through one or more beam splitters 175a and 175b. Beam splitter 175a transmits 488 nm light and reflects 633 nm light. Beam splitter 175b transmits ultraviolet light (light with wavelengths in the range of 10 to 400 nm) and reflects both 488 nm and 633 nm light.

[0167] The laser beam is then directed to a focusing lens 180, which focuses the beam onto the portion of the sample containing particles within the fluid flow in flow cell 185. Flow cell 185 is part of a fluid system that directs particles (typically one at a time) in the flow to the focused laser beam for detection. The flow cell may comprise a flow cell in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0168] Light from one or more laser beams interacts with particles in a sample through diffraction, refraction, reflection, scattering, and absorption, and is then re-emitted at various wavelengths, depending on the particle characteristics, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on the particles. The optical detection system 191 includes optical detection modules 171a-171e. Fluorescent emission light, as well as diffracted, refracted, reflected, and scattered light, can be routed to one or more of the forward-scattering photodetector 193 and optical detection modules 171a-171e. Optical adjustment components (e.g., multimode notch filters or beam splitters) present in the light collection tube 166 split the beam received from the flow cell 185 into a first beam and a second beam. The optical collection components, namely the first optical fiber cable 173a and the second optical fiber cable 173b, respectively transmit the particle-modulated light received from the flow cell 185 to the first optical receiver 172a and the second optical receiver 172b. That is, the first optical fiber cable 173a transmits the first light beam to the first optical receiver 172a, and the second optical fiber cable 173b transmits the second light beam to the second optical receiver 172b.

[0169] A forward-scattering photodetector 193 is positioned slightly off-axis from the direct beam passing through the flow cell and configured to detect diffracted light, i.e., excitation light that primarily travels forward or surrounds the particle. The intensity of the light detected by the forward-scattering photodetector depends on the overall size of the particle and may include a photodiode. A first light detection module 171d ultimately receives the first beam from a first light receiver 172a and is configured to detect small particle light scattering using small particle detection. The first light detection module 171d is also configured as a side-scattering photodetector (i.e., includes a side-scattering detector) to detect refracted and reflected light from the particle surface and internal structure, which tends to increase with increasing particle structural complexity. The small particle detector may include an avalanche photodiode (APD), and the side-scattering detection may include a photomultiplier tube. The signals detected by the forward scattering photodetector 193, the small particle detector and the side scattering detector in the first optical detection module 171d, and the photodetectors in other optical detection modules 171a, 171b, 171c, and 171e can all be converted into electrical signals (voltages) by the photodetectors. These data can provide information about the sample.

[0170] Those skilled in the art will understand that the flow cytometer according to embodiments of the present invention is not limited to... Figure 1G The flow cytometer shown may include any flow cytometer known in the art. For example, a flow cytometer can have any number of lasers, beam splitters, filters, and photodetectors in various wavelengths and configurations.

[0171] In use, the operation of the cytometer is controlled by a controller / processor 190, and measurement data from the photodetector can be stored in memory 195 and processed by the controller / processor 190. Although not explicitly shown, the controller / processor 190 is coupled to the photodetector to receive its output signal and can also be coupled to the electrical and electromechanical components of the flow cytometer 192 to control the laser, fluid flow parameters, etc. Input / output (I / O) capabilities 197 may also be provided in the system. Memory 195, controller / processor 190, and I / O 197 can be fully integrated into the flow cytometer 192. In this embodiment, a display can also be formed as part of the I / O capability 197 for presenting experimental data to the user of the cytometer 192. Alternatively, some or all of the memory 195, controller / processor 190, and 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 195 and controller / processor 190 can communicate wirelessly or wired with the cytometer 192. The controller / processor 190, combined with memory 195 and I / O 197, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0172] Different fluorescent molecules used in flow cytometry experiments will emit light within their respective characteristic wavelength bands. Specific fluorescent labels used in the experiment and their associated fluorescence emission bands can be selected to approximately coincide with the filter window range of the photodetector. I / O 197 can be configured to receive data related to a flow cytometry experiment having a set of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O 197 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectrum data, data assigning labels to one or more markers, and flow cytometry configuration data. Flow cytometry experiment data, such as label spectral characteristics and flow cytometry configuration data, can also be stored in memory 195. Controller / processor 190 can be configured to evaluate one or more assignment schemes of markers to markers.

[0173] In some embodiments, the subject matter system is a particle sorting system configured to sort particles using closed particle sorting modules, such as those described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (such as cells) in a sample, such as those described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module with deflection plates, such as those described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0174] Figure 2 A system 200 for flow cytometry according to an exemplary embodiment of the present disclosure is shown. The system 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at a detection point 215 within a flow cell 210. Although Figure 2 The example shows a single laser, but it should be understood that multiple lasers can also be used. The laser beam from laser 201 is directed to focusing lens 202, which focuses the laser beam onto the region of the fluid flow containing particles 211 of the sample within flow cell 210. Flow cell 210 is part of a fluid system that directs particles (typically one at a time) in the flow to the focused laser beam for detection. Alternatively, in the case of a flow cytometer that is an airflow cytometer, a nozzle tip can be used.

[0175] like Figure 2 As shown, flow cell 210 is fluidly connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. Sheath fluid from sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a conduit (i.e., a sheath fluid line) 207. Furthermore, a sample flow containing particles 211 from sample fluid reservoir 204 is supplied to sample injection port 206 via a conduit (i.e., a sample fluid line) 205. Sample injection port 206 is fluidly connected to a sample injector 213 (e.g., a sample injection needle), which is configured to introduce particles 211 into the interior of flow cell 210. The sheath fluid entering via sheath fluid injection port 208 is hydrodynamically focused, such that a flow stream 214 is formed downstream of the conical portion 212 of flow cell 210. Particles emitted at the distal end of flow cell 210 can be processed or collected by any suitable method, such as collection at the distal end of flow cell 210, depending on the type of flow cytometer being used, for example, via a waste line. Alternatively, the particles can be sorted.

[0176] Light from one or more laser beams interacts with particles 211 in the sample through diffraction, refraction, reflection, scattering, and absorption, and is then re-emitted at various wavelengths, depending on the particle's characteristics, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The emitted fluorescent light, as well as the diffracted, refracted, reflected, and scattered light, can be routed to one or more detectors. Specifically, forward scattered light (FSC) is routed to forward scattered light detector 223. Forward scattered light detector 223 is located slightly off-center from the axis of the direct beam passing through flow cell 210 and is configured to detect diffracted light, i.e., excitation light that passes primarily forward or surrounds the particle. The intensity of the light detected by forward scattered light detector 223 depends on the overall size of the particle and may include, for example, a photodiode. Between forward scattered light detectors 223 are filters 221a and a scattering baffle 222. The filter 221a can be configured to filter out non-FSC light of at least one wavelength, and the scattering baffle 222 can be configured to prevent incident light (i.e. non-scattered light) from the laser 201 from being detected by the forward scattering detector 223.

[0177] Furthermore, side-scattered light (SSC) is detected by side-scattered light detector 224. In other words, side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of particle 211, which tends to increase with increasing structural complexity of the particle. Figure 2In the example, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to a side-scatter light detector 224 while allowing non-SSC (e.g., fluorescence) light to pass through. A filter 221b is configured to block at least one wavelength of non-SSC light from being detected by the side-scatter light detector 224. A plurality of fluorescence detectors 225a-225c are also shown in the figure, each configured to detect fluorescence at a different wavelength. For example, the dichroic mirror 220b can be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a while allowing light of other wavelengths to pass through. The filter 221c can be configured to block the fluorescence detector 225a from detecting light of at least one wavelength that does not correspond to the first wavelength (or wavelength range). Similarly, the dichroic mirror 220c is configured to reflect FL light corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b while allowing light of a third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. Filter 221d is configured to prevent fluorescence detector 225 from detecting light of at least one wavelength that does not correspond to the second wavelength (or wavelength range). Furthermore, filter 221e is configured to prevent fluorescence detector 225c from detecting light of at least one wavelength that does not correspond to the third wavelength (or wavelength range).

[0178] Those skilled in the art will recognize that the flow cytometers according to embodiments of this disclosure are not limited to... Figure 2 The flow cytometer shown is not limited to any flow cytometer known in the art. For example, a flow cytometer can have any number of lasers, beam splitters, filters, and detectors in various wavelengths and configurations. For example, although... Figure 2 The example shown has three fluorescence detectors for illustrative purposes, but it should be understood that any suitable number of fluorescence detectors can be used.

[0179] During operation, the cytometer is controlled by a controller / processor 290. Measurement data from the detectors can be stored in memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detectors to receive output signals from them and can also be coupled to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. The system is also capable of providing input / output (I / O) capabilities 297. Memory 295, controller / processor 290, and I / O 297 can be provided entirely as integral components of the flow cytometer. In such embodiments, a display can also be provided as part of the I / O function 297 for displaying experimental data to the user of the cytometer 200. Alternatively, some or all of the memory 295, controller / processor 290, and 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 controller / processor 290 are capable of wireless or wired communication with the flow cytometer. The controller / processor 290, combined with memory 295 and I / O 297, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0180] In a flow cytometry experiment, different fluorescent molecules in the fluorophore panel will emit light within their respective characteristic wavelengths. Specific fluorescent labels used in the experiment and their associated fluorescence emission bands can be selected to approximately coincide with the filter window range of the detector. I / O 297 can be configured to receive data related to a flow cytometry experiment having a set of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O 297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning labels to one or more markers, and flow cytometry configuration data. Flow cytometry experiment data, such as label spectral characteristics and flow cytometry configuration data, can also be stored in memory 295. Controller / processor 290 can be configured to evaluate one or more assignment schemes for label assignment to markers.

[0181] In some embodiments, the subject matter system is a particle sorting system configured to sort particles using closed particle sorting modules, such as those described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (such as cells) in a sample, such as those described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module with deflection plates, such as those described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0182] In some embodiments, the system uses radio frequency marker emission images to enable fluorescence imaging of the particle sorter, for example... Figure 3As shown, the particle sorter 300 includes an illumination component 300a, which includes a light source 301 (e.g., a 488nm laser) that generates an output beam 301a. This output beam 301a is split into beam 302a and beam 302b by a beam splitter 302. Beam 302a propagates through an acousto-optic device 303 (e.g., an acousto-optic deflector (AOD)) to generate an output beam 303a with one or more angled deflections. In some examples, the output beam 303a generated from the acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Beam 302b propagates through an acousto-optic device 304 (e.g., an acousto-optic deflector (AOD)) to generate an output beam 304a with one or more angled deflections. In some examples, the output beam 304a generated from the acousto-optic device 304 includes a local oscillator beam and multiple radio frequency comb beams. Output beams 303a and 304a, generated by acousto-optic devices 303 and 304 respectively, are combined by beam splitter 305 to produce output beam 305a, which is transmitted through optical component 306 (e.g., objective lens) to illuminate particles in flow cell 307. In some embodiments, acousto-optic device 303 (AOD) splits a single laser beam into an array of small beams, each with a different optical frequency and angle. A second AOD 304 tunes the optical frequency of a reference beam, which is then overlapped with the array of small beams at beam combiner 305. In some embodiments, the light illumination system having a light source and acousto-optic devices may also include those described in Schraivogel et al., “High-speed fluorescence image-enabled cell sorting”, Science (2022), 375 (6578): 315-320, and U.S. Patent Publication No. 2021 / 0404943, the disclosure of which is incorporated herein by reference in its entirety.

[0183] Output beam 305a irradiates sample particles 308 (e.g., together with sheath fluid 309) propagating through flow cell 307 at irradiation region 310. As shown in irradiation region 310, multiple beams (e.g., RF offset beams with angular deflection appearing as dots on irradiation region 310) overlap with a reference local oscillator beam (appearing as a shaded line on irradiation region 310). Due to their different optical frequencies, the overlapping beams exhibit beat frequency behavior, resulting in each small beam carrying a different frequency f. 1-n Sine modulation.

[0184] Light from the illuminated sample is transmitted to a light detection system 300b comprising multiple photodetectors. The light detection system 300b includes a forward-scattering photodetector 311 for generating a forward-scattering image 311a and a side-scattering photodetector 312 for generating a side-scattering image 312a. The light detection system 300b also includes a bright-field photodetector 313 for generating a light loss image 313a. In some embodiments, the forward-scattering detector 311 and the side-scattering detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the illuminated sample is also detected by fluorescence photodetectors 314-317. In some cases, photodetectors 314-317 are photomultiplier tubes. Light from the illuminated sample is directed by a beamsplitter 320 to the side-scattering detection channel 312 and the fluorescence detection channels 314-317. The optical detection system 300b includes bandpass optics 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optics 321 has a 534 nm / 40 nm bandpass. In some cases, optics 322 has a 586 nm / 42 nm bandpass. In some cases, optics 323 has a 700 nm / 54 nm bandpass. In some cases, optics 324 has a 783 nm / 56 nm bandpass. The first number indicates the center of the spectral band. The second number provides the range of the spectral band. Therefore, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm.

[0185] Data signals generated in response to light detected by scattered light detection channels 311, 312, 313, and 314-317 are digitally processed in real time by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated by processors 350 and 351. Image-enabled sorting is performed in response to a sorting signal generated in sorting trigger 352. Sorting component 300c includes deflection plate 331 for deflecting particles into sample container 332 or waste stream 333. In some cases, sorting component 300c is configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference in its entirety.

[0186] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4 It can be used to analyze and characterize particles, whether or not the particles are physically sorted into a collection container. Figure 4 A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, particle analysis system 401 is a fluid system. Particle analysis system 401 includes fluid system 402. Fluid system 402 may include or be coupled to a sample tube 405 and a fluid column moving within the sample tube, wherein particles 403 (e.g., cells) of the sample move along a common sample path 409 within the sample tube.

[0187] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as it passes through one or more detection stations along a common sample path. Detection station 408 typically refers to a monitoring area 407 along the common sample path. In some embodiments, detection may include detecting light or one or more other characteristics of particle 403 as it crosses the monitoring area 407. Figure 4 The image shows a detection station 408 with a monitoring area 407. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations can monitor more than one area.

[0188] Each signal is assigned a signal value to form a data point for each particle. As described above, this data can be referred to as event data. The data point can be a multi-dimensional data point, including values ​​of various characteristics measured for the particle. The detection system 404 is configured to collect a series of such data points within a first time interval.

[0189] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operationally associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the number of data points collected by the detection system 404 during the first time interval. The control system 406 may also be configured to generate an experimental signal frequency based on the number of data points in that portion of the first time interval. The control system 406 may also additionally compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0190] Figure 5 A functional block diagram of an example particle analyzer control system (e.g., analysis controller 500) for analyzing and displaying biological events is shown. Analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0191] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0192] Analysis controller 500 may be configured to receive biological event data from particle analyzer or sorting system 502. The biological event data received from particle analyzer or sorting system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display including a first graph of the biological event data to display device 506. For example, analysis controller 500 may also be configured to render regions of interest as gates around the cluster of biological event data shown on display device 506, for example, overlaid on the first graph. In some embodiments, a gate may be a logical combination of one or more graphical regions of interest plotted on a single-parameter histogram or bivariate graph. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0193] The analysis controller 500 can be further configured to display the biological event data inside the door on the display device 506 in a manner different from other biological event data outside the door. For example, the analysis controller 500 can be configured to display the biological event data contained inside the door in a different color than the biological event data outside the door. The display device 506 can be a monitor, tablet computer, smartphone, or other electronic device configured to display a graphical interface.

[0194] The analysis controller 500 can be configured to receive a door selection signal from a first input device to identify a door. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a door selection signal to the analysis controller 500 to identify a door to be displayed or manipulated via the display device 506 (e.g., by clicking the door or its interior when the cursor is positioned on the desired door). In some embodiments, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, optical detector, or voice recognition system. Some input devices may include multiple input functions. In these embodiments, each input function can be considered an input device. For example, such as... Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, and each button can generate a trigger event.

[0195] Triggering events may cause the analysis controller 500 to change the way data is displayed, the portion of data actually displayed on the display device 506, and / or provide input for further processing, such as selecting groups of interest for particle sorting.

[0196] In some embodiments, the analysis controller 500 may be configured to detect when the mouse 510 initiates gate selection. The analysis controller 500 may also be configured to automatically modify the drawing visualization to facilitate the gating process. This modification may be based on a specific distribution of the biological event data received by the analysis controller 500.

[0197] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can also be configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0198] Display device 506 can be configured to receive display data from analysis controller 500. This display data may include graphs of biological event data and gates outlining portions of the graphs. Display device 506 can also be configured to change the presented information based on input received from analysis controller 500 and from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0199] In some embodiments, the analysis controller 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include controls for receiving sample events or sample images. The sample events, images, or sample gates may be provided before collecting event data for the samples, or based on an initial set of events from a portion of the samples.

[0200] Figure 6A This is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Figure 6AAs shown, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which can be coupled to a nozzle 603, and may include or be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically aggregates a sample flow 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, particles 609 (e.g., cells) are arranged in a single file to pass through a monitoring area 611 (e.g., a laser-stream intersection) irradiated by an irradiation source 612 (e.g., a laser). Vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to break into multiple droplets 610, some of which contain particles 609.

[0201] In operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses a monitoring area 611. The detection station 614 is fed into a timing circuit 628, which in turn feeds into a flash charge circuit 630. At the droplet break point, notified by a timed droplet delay (Δt), a flash charge can be applied to a moving fluid column 608, causing the droplet of interest to carry a charge. The droplet of interest may include one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect the charged droplet into a container, such as a collection tube or a porous or microporous sample plate, where the droplet of interest can be associated with a pore or micropore. Figure 6A As shown, the droplets can be collected in the discharge container 638.

[0202] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a particle of interest crosses the monitoring region 611. 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 616 enables the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may be fed an amplitude signal 620 and / or a phase signal 618, which are then fed (via amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 further control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0203] In some embodiments, sorting electronics (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions may be included in the event data of the particles. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or communication coupling, such that event measurements can be collected by one of detection system 616 or detection station 614 and provided to non-collection elements.

[0204] Figure 6B This is a schematic diagram of a particle sorting system according to an embodiment of the present document. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via a stream-charging wire in the barbs. This generates a droplet stream 610 containing particles 610 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to produce light scattering and fluorescence information. The particle information is processed by, for example, sorting electronics or other detection systems. Figure 6B (Not shown in the image) is analyzed. Deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, thereby guiding the droplets to a destination collector (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflector plates 652 and 654 can be controlled to allow particles to enter container 674 along a first path 662 or along a second path 668. If the particles are not of interest (e.g., do not exhibit scattering or illumination information within a specified sorting range), the deflector plates can allow the particles to continue flowing along flow path 664. Such uncharged droplets can be introduced into a waste container via, for example, a suction device 670.

[0205] It can include sorting electronics to initiate the collection of measurement results, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to sort the particles. Figure 6B An example implementation of the illustrated embodiment includes the BD FACSAria™ series flow cytometer, commercially available from Becton Dickinson, Inc. (Franklin Lake, New Jersey).

[0206] method

[0207] This disclosure also includes methods comprising: (a) introducing a sample into a system according to embodiments of this disclosure and (b) analyzing the sample for small particles by flow cytometry. Some embodiments also include obtaining results from the system, the results providing information about the presence of small particles in the sample. Other embodiments include: circulating a sample in a flow stream in a detection field; and illuminating the sample in the flow stream in the detection field with light.

[0208] In some instances, the samples analyzed by this method are biological samples. The term "biological sample," used in its conventional sense, refers to a subset of tissues, cells, or components of an organism, plant, fungus, or animal, and in some instances 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. Therefore, "biological sample" refers both to a subset of a natural organism or its tissues and to homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. Biological samples can be any type of biological tissue, including both healthy and diseased tissues (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc. In some instances, the sample is a blood sample, including whole blood, such as blood obtained by venipuncture or finger prick (where the blood may or may not be bound to any reagents such as preservatives, anticoagulants, etc. before testing).

[0209] In some embodiments, the source of the sample is "mammal" or "milk animal," terms that are widely used to describe organisms within the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subject is a human. The method can be applied to samples obtained from human subjects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, teenagers, and adults), wherein in some embodiments, the human subject is an adolescent, teenager, or adult. While this 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).

[0210] Cells of interest can be targeted and characterized based on various parameters, such as phenotypic features identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analytical droplets identified as including target cells. A wide variety of cells can be characterized using the methods of this subject matter. 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. Target cells of interest also include cells with readily available cell surface markers or antigens that can be captured or labeled by readily available affinity agents or conjugates thereof. For example, target cells 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, disialotetrahexosylganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) derived from whole blood, bone marrow, or umbilical cord blood.

[0211] When implementing the methods described in this article, an initial fluid sample is injected into the flow cytometer. The sample volume injected into the particle sorting module can vary, for example, ranging from 0.001 mL to 1000 mL, such as 0.005 mL to 900 mL, 0.01 mL to 800 mL, 0.05 mL to 700 mL, 0.1 mL to 600 mL, 0.5 mL to 500 mL, 1 mL to 400 mL, 2 mL to 300 mL, including samples from 5 mL to 100 mL.

[0212] The method according to embodiments of this disclosure includes counting and optionally sorting labeled particles (such as target cells) in a sample. In implementing this subject matter method, a fluid sample containing particles is first introduced into a flow nozzle of the system. After exiting the flow nozzle, the particles pass substantially one-by-one through a sample detection region, where each particle is illuminated by a light source, and measurements of light scattering parameters for each particle are recorded individually. In some instances, fluorescence emission measurements are also recorded as needed (e.g., two or more light scattering parameter measurements and one or more fluorescence emission measurements). Depending on the characteristics of the detected flow, regions of 0.001 mm or longer in the flow can be illuminated, such as 0.005 mm or longer, 0.01 mm or longer, 0.05 mm or longer, 0.1 mm or longer, 0.5 mm or longer, including regions of 1 mm or longer in the flow that can be illuminated. In some embodiments, the method includes illuminating a planar cross-section of the flow in the sample detection region, for example, using a laser (as described above). In other embodiments, the method includes irradiating a predetermined length region of the flow in the sample detection region, for example, corresponding to the irradiation profile of a diffuse laser beam or lamp.

[0213] In some embodiments, the method includes irradiating the flow at or near the flow cell nozzle orifice. For example, the method may include irradiating the flow at a location approximately 0.001 mm or more from the nozzle orifice, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, including a location 1 mm or more from the nozzle orifice. In some embodiments, the method includes irradiating the flow at a location immediately adjacent to the flow cell nozzle orifice.

[0214] In embodiments of this method, detectors, such as photomultiplier tubes (PMTs), are used to record light passing through each particle (in some cases referred to as forward scattering), light reflected orthogonally to the flow direction of the particle through the detection zone (in some cases referred to as orthogonal or lateral scattering), and fluorescence emitted from the particle as it passes through the detection zone and is illuminated by energy, if the particle is labeled with a fluorescent marker. Each of forward scattering (FSC), lateral scattering (SSC), and fluorescence emission includes a separate parameter for each particle (or each "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle is analyzed in real time as needed or stored in a data storage and analysis device, such as a computer.

[0215] In some embodiments, particles are detected and uniquely identified, as needed, by exposing them to excitation light and measuring the fluorescence of each particle in one or more detection channels. The fluorescence emitted in the detection channels, used to identify the particles and the binding complexes associated with them, can be measured after excitation using a single light source, or separately after excitation using different light sources. If separate excitation sources are used to excite the particle markers, the markers can be selected such that all markers can be excited by each excitation source used.

[0216] In some embodiments, the method also includes data acquisition, analysis, and recording (e.g., using a computer), where multiple data channels record light scattering and fluorescence data from each detector for each particle as it passes through a sample detection area of ​​a particle sorting module. In these embodiments, the analysis includes classifying and counting particles such that each particle exists as a set of digitized parameter values. The subject system can be configured to trigger on selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for the detection parameter, which can be used as a means of detecting particles passing through the light source. Detecting an event exceeding the selected parameter threshold triggers the acquisition of light scattering and fluorescence data for the particles. Data for particles or other components in the analyzed medium that elicit a response below the threshold are not acquired. The trigger parameter could be the detection of forward scattered light caused by particles passing through a light beam. Flow cytometry then detects and collects the light scattering and fluorescence data of the particles.

[0217] The specific subpopulations of interest are then further analyzed by "gating" based on data collected for the entire population. To select a suitable gate, the data is plotted to obtain the best possible subpopulation separation. This process can be performed by plotting forward light scattering (FSC) and lateral (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. The subpopulations of particles (i.e., those cells within the gate) are then selected, and particles not within the gate are excluded. If needed, gates can be selected by drawing lines around the desired subpopulation on the computer screen using a cursor. The particles within the gate are then further analyzed only by plotting other parameters of these particles (e.g., fluorescence). If needed, the above analysis can be configured to generate counts of particles of interest in the sample.

[0218] Methods of interest may also include the use of particles in research, laboratory testing, or treatment. In some embodiments, the subject method includes obtaining single cells prepared from a target fluid or tissue biological sample. For example, the subject method includes obtaining cells from a fluid or tissue sample for use as a research or diagnostic specimen for diseases such as cancer. Similarly, the subject method includes obtaining cells from a fluid or tissue sample for therapeutic purposes. Cell therapy protocols are those that can prepare living cellular materials, including, for example, cells and tissues, and introduce them into a subject for therapeutic treatment. Situations where treatment can be performed by applying samples sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, organ damage, etc.

[0219] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, gene modification, in vitro culture and expansion, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of cells into a subject. The protocol can begin with the collection of live cells and tissues from the subject's source tissue to produce cell and / or tissue samples. Samples can be collected using any suitable procedure, including, for example, administration of cell mobilizing agents to the subject, drawing blood from the subject, or extracting bone marrow from the subject. After sample collection, cells can be enriched using several methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation methods, fluorescence-activated cell sorting (FACS), etc. In some instances, the enriched cells can be gene-modified using any convenient method, such as nuclease-mediated gene editing. Genetically modified cells can then be cultured, activated, and expanded in vitro. In some instances, cells are preserved, for example, cryopreserved, and stored for future use, where the cells are thawed and then administered to the patient, for example, cells can be infused into the patient.

[0220] Computer control system

[0221] This disclosure also includes a computer control system, wherein the system comprises one or more fully automated or partially automated computers. In some embodiments, the system includes a computer having a non-transitory computer-readable storage medium storing a computer program, wherein the computer program, when loaded onto the computer, includes instructions for analyzing data from one or more optical detection systems described herein.

[0222] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, mouse, or similar device. The processing module includes a processor that can access memory storing instructions for performing the steps of the method of the present invention. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or other existing or future-available processors. The processor executes an operating system, which interfaces with firmware and hardware in a known manner to facilitate the processor's coordination and execution of various computer programs written in a variety of programming languages ​​known in the art, such as Java, Perl, C++, Python, other high-level or low-level languages, or combinations thereof. The operating system typically works with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, storage management, and communication control and related services according to known techniques. In some embodiments, the processor includes analog electronic components that provide feedback control, such as negative feedback control.

[0223] System memory can be any of a variety of known or future memory storage devices. Examples include any common random access memory (RAM), magnetic media (such as internal hard disks or magnetic tapes), optical media (such as rewritable optical discs), flash memory devices, or other storage devices. Memory storage devices can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, or floppy disk drives. Such storage devices typically read from and / or write to program storage media (not shown, such as optical discs). Any such program storage media, or other existing or future-developed media, can be considered a computer program product. As those skilled in the art will recognize, these program storage media typically store computer software programs and / or data. Computer software programs (also known as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0224] In some embodiments, a computer program product is described, comprising a computer-usable medium storing control logic (computer software program, including program code). When the computer's processor executes the control logic, the control logic causes the processor to perform the functions described in this disclosure. In other embodiments, some functions are implemented primarily in hardware, for example using a hardware state machine. How to implement a hardware state machine to perform the functions described in this disclosure will be apparent to those skilled in the art.

[0225] The memory can be any suitable device that the processor can store and retrieve data from, such as magnetic, optical, or solid-state storage devices (including disks, optical discs, magnetic tapes, RAM, or any other suitable fixed or portable devices). The processor can include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel or pre-stored in a computer program product using any of these devices connected to the memory, such as memory or other portable or fixed computer-readable storage media. For example, a disk or optical disc can carry the programming and can be read by a disk reader / writer. The system of this disclosure also includes writing algorithms (e.g., in the form of a computer program product) to practice the methods described above. The programming according to this disclosure can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media (such as floppy disks, hard disk storage media, and magnetic tape), optical storage media (such as CD-ROMs), electrical storage media (such as RAM and ROMs), portable flash drives, and mixtures of these categories (such as magnetic / optical storage media).

[0226] The processor can also access communication channels to communicate with users in remote locations. Remote locations mean that users do not have direct contact with the system, but instead relay input information from external devices to the input manager. External devices can be computers connected to a wide area network (WAN), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones).

[0227] In some embodiments, the system described herein may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., radio frequency identification (RFID)), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication (such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM)).

[0228] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces like USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject system and other external devices, such as computer terminals (e.g., computer terminals in a doctor's office or hospital environment), which are configured for similar complementary data communication.

[0229] In one embodiment, the communication interface is configured as infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0230] In one embodiment, the communication interface is configured to provide connectivity for data transmission using the Internet Protocol (IP) via a mobile phone network, short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0231] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, such as using common standards like 802.11 or the Bluetooth® RF protocol, or the IrDA infrared protocol. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop computer, or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display (such as a liquid crystal display (LCD)) and input devices (such as buttons, a keyboard, a mouse, or a touchscreen).

[0232] In some embodiments, the communication interface is configured to automatically or semi-automatically enable data stored in the subject system (e.g., data in an optional data storage unit) to communicate with a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0233] The output controller may include a controller for any of a variety of known display devices used to present information to a user (whether human or machine, local or remote). If one of the display devices provides visual information, this information is typically logically and / or physically organized as an array of image pixels. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. Functional elements of the computer may communicate with each other via a system bus. In alternative embodiments, some of these communications may be implemented via a network or other types of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location, such as via the Internet, telephone, or satellite network, according to known technologies. The way the output manager presents data may be implemented according to a variety of known technologies. For example, the data may include SQL, HTML, or XML documents, emails, or other files, or other forms of data. The data may include Internet URLs, allowing the user to retrieve more SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or type to be developed in the future, although they generally fall into the category of computers commonly referred to as servers. However, they may also be host computers, workstations, or other computer types. They can be connected via any known or future cable or other communication system, including wireless systems, whether networked or otherwise. They can be located in the same place or physically separated. Various operating systems can be deployed on any computer platform, depending on the type and / or model of the chosen platform. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBMi®, Android™, SGIIRIX®, Oracle Solaris®, and others.

[0234] Figure 7 The overall architecture of an example computing device 700 according to certain embodiments is described. Figure 7The overall architecture of the computing device 700 shown includes the arrangement of computer hardware and software components. However, for the sake of providing a feasible disclosure, it is not necessary to show all these generally conventional components. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. The network interface 720 provides connectivity to one or more networks or computing systems. The processing unit 710 can thus receive information and instructions from other computing systems or servers via the network. The processing unit 710 can also communicate to / from memory 770 and further provide output information to an optional display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or programs, such as FlowJo®, etc.) stored as executable instructions in the non-temporary memory of the analysis system can display flow cytometry event data to a user. The input / output device interface 740 can also accept input from optional input devices 760, such as keyboards, mice, digital pens, microphones, touch screens, gesture recognition systems, voice recognition systems, game controllers, accelerometers, gyroscopes, or other input devices.

[0235] Memory 770 may include computer program instructions (grouped into modules or components in some embodiments) that processing unit 710 executes to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions to processing unit 710 in the routine management and operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may also include computer program instructions and other information for implementing aspects of this disclosure.

[0236] kit

[0237] The invention also includes a kit comprising one or more small-particle optical detection modules as described herein. In some embodiments, the kit includes a first optical fiber or a second optical fiber (wherein the diameter of the first optical fiber is smaller than the diameter of the second optical fiber) or other components or aspects of the CWD optical detection subsystem, such as a second optical detection module. In some cases, the kit may include one or more assay components (e.g., the labeling reagents, buffer solutions, etc. described above). In some cases, the kit may also include, as needed, a sample collection device, such as a spray gun or needle configured to puncture the skin to obtain a whole blood sample, a pipette, etc.

[0238] The various assay components of the kit may be present in separate containers, or some or all of them may be pre-assembled. For example, in some embodiments, one or more components of the kit, such as each of a pair of deflection plates in a droplet deflector, are present in a sealed bag, such as a sterile foil bag or envelope.

[0239] In addition to the components described above, the theme kit may also include (in some embodiments) instructions. These instructions may exist in the theme kit in various forms, with one or more forms present within the kit. One form of these instructions is as information printed on a suitable medium or substrate, such as a sheet or more of paper with the information printed on it, kit packaging, inserts, etc. Another form of these instructions is as a computer-readable medium, such as a floppy disk, optical disc (CD), portable flash drive, etc., on which the information is recorded. Yet another form of these instructions is as a URL that can be used to access information on a remote site via the Internet.

[0240] practicality

[0241] The optical detection system, optical detection module, method, and computer system can be used in a variety of applications where it is necessary to detect and, in some cases, analyze or sort particulate components of a sample in a fluid medium (e.g., a biological sample), wherein the detected particles are small particles, such as particles with a diameter of approximately 80 nm; that is, embodiments can achieve 80 nm resolution using an avalanche photodiode detector. Embodiments of the invention are useful in cases where there is a need to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, and expanded particle analysis or collection (i.e., small particle detection).

[0242] The embodiments of this disclosure can be used in applications requiring the preparation of cells from biological samples for research, laboratory testing, or treatment. In some embodiments, the methods and apparatus of this subject matter can facilitate the preparation of single cells from a target fluid or tissue biological sample. For example, the methods and systems of this subject matter facilitate the acquisition of cells from fluid or tissue samples for use as research or diagnostic samples for diseases such as cancer. Similarly, the methods and systems of this subject matter can facilitate the acquisition of cells from fluid or tissue samples for therapeutic purposes.

[0243] The following content is for illustrative purposes only and is not a limitation:

[0244] experiment

[0245] Figure 8Theoretical results related to the detection of small particles using embodiments of this disclosure are presented. Light scattering data for polystyrene particles and vesicles were obtained using violet light at a wavelength of 405 nm and blue light at a wavelength of 488 nm. Theoretical results show that the scattering cross-sectional area increases with increasing cuvette diameter. Results related to irradiation of 80 nm polystyrene particles with violet and blue light are shown as horizontal lines. Figure 9 Empirical results comparing an existing system (S8 (ATG Ops13)) with an embodiment according to this disclosure (S8 + SPD (Ops10)) are shown. These empirical results demonstrate that the embodiment according to this disclosure offers an improved ability to detect small particles (specifically 81 nm polystyrene particles).

[0246] Notwithstanding the appended claims, this disclosure is also defined by the following terms:

[0247] 1. A photodetector system for detecting small particles, the photodetector system comprising:

[0248] An optical adjustment component is configured to split a light beam received from a flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence from particles excited within the flow cell.

[0249] A clustered wavelength division (CWD) optical detection subsystem includes a first optical receiver and a second optical receiver, wherein the first optical receiver is configured to transmit a first light beam to a first optical detection module, and wherein the second optical receiver is configured to transmit a second light beam to a second optical detection module, wherein the first optical detection module includes a small particle detector.

[0250] A first optical fiber, configured to transmit the first light beam from the optical adjustment component to the first optical receiver of the CWD optical detection subsystem; and

[0251] A second optical fiber is configured to transmit the second beam from the optical adjustment component to the second optical receiver of the CWD optical detection subsystem.

[0252] 2. The optical detection system according to Clause 1, wherein the optical adjustment component includes a beam splitter or a multimode notch filter.

[0253] 3. The optical detection system according to any one of the foregoing clauses, wherein the optical adjustment component is field-upgradeable.

[0254] 4. The light detection system according to any one of the preceding clauses, wherein the system is configured such that the optical adjustment component is detachable.

[0255] 5. The optical detection system according to any one of the preceding clauses, wherein the system further includes an aperture located between the optical adjustment component and the flow cell.

[0256] 6. The optical detection system according to any one of the preceding clauses, wherein the optical adjustment component is configured to transmit the first light beam directly onto the first optical fiber and the second light beam directly onto the second optical fiber.

[0257] 7. The optical detection system according to any one of the preceding clauses, wherein the diameter of the first optical fiber is smaller than the diameter of the second optical fiber.

[0258] 8. The optical detection system according to any one of the preceding clauses, wherein the diameter of the first optical fiber is configured as an aperture size suitable for the size of the side-scattered light.

[0259] 9. The optical detection system according to any one of the preceding clauses, wherein the diameter of the first optical fiber is configured to be an aperture size suitable for side-scattered light from small particles.

[0260] 10. The optical detection system according to any one of the preceding clauses, wherein the diameter of the first optical fiber is 200µm.

[0261] 11. The optical detection system according to any one of the preceding clauses, wherein the first optical fiber includes an optical fiber bend.

[0262] 12. The optical detection system according to any one of the preceding clauses, wherein the optical fiber bend is approximately 90 degrees of bend in the first optical fiber.

[0263] 13. The optical detection system according to any one of the preceding clauses, wherein the diameter of the second optical fiber is configured to be an aperture size suitable for fluorescence.

[0264] 14. The optical detection system according to any one of the preceding clauses, wherein the diameter of the second optical fiber is 800µm.

[0265] 15. The light detection system according to any one of the preceding clauses, wherein the first light detection module further includes a side-scattering detector.

[0266] 16. The optical detection system according to Clause 15, wherein the first optical detection module includes a second beam splitter configured to split the first beam into a third beam and a fourth beam.

[0267] 17. The optical detection system according to Clause 16, wherein the first optical detection module is configured to transmit the third beam to the small particle detector and the fourth beam to the side scattering detector.

[0268] 18. The optical detection system according to any one of the preceding clauses, wherein the small particle detector comprises an avalanche photodiode.

[0269] 19. The optical detection system according to any one of the preceding clauses, wherein the first optical detection module further includes an optical component configured to transmit light having a predetermined sub-spectral range for detection.

[0270] 20. The optical detection system according to Clause 19, wherein the optical component includes a bandpass filter.

[0271] 21. The light detection system according to Clause 19, wherein the optical component includes a dichroic mirror.

[0272] 22. The optical detection system according to any one of the preceding clauses, wherein the second optical detection module is configured to detect fluorescence.

[0273] 23. The optical detection system according to any one of the preceding clauses, wherein the first optical receiver and the second optical receiver are in fixed positions relative to each other.

[0274] 24. The optical detection system according to any one of the preceding clauses, wherein the CWD optical detection subsystem includes a plurality of wavelength splitters configured to allow light having a predetermined spectral range from the first optical receiver and the second optical receiver to pass through.

[0275] 25. The optical detection system according to Clause 24, wherein each of the first optical detection module and the second optical detection module optically communicates with a wavelength splitter among a plurality of wavelength splitters.

[0276] 26. The optical detection system according to any one of clauses 24 to 25, wherein the wavelength splitters are configured to transmit light between each other.

[0277] 27. The light detection system according to any one of clauses 24 to 26, wherein the wavelength splitter comprises a dichroic mirror.

[0278] 28. The optical detection system according to any one of clauses 24 to 27, wherein the second beam is transmitted from a subset of the wavelength splitter.

[0279] 29. The optical detection system according to any one of the preceding clauses, wherein the first optical receiver includes a first coupler for operably attaching to the first optical fiber, and the second optical receiver includes a second coupler for operably attaching to the second optical fiber.

[0280] 30. The light detection system according to any one of the preceding clauses, wherein the first light receiver and the second light receiver each include a beam modulator.

[0281] 31. The light detection system according to Clause 30, wherein the beam modulator is a lens.

[0282] 32. The optical detection system according to any one of the preceding clauses, wherein the CWD optical detection subsystem comprises three or more optical detection modules.

[0283] 33. The light detection system according to Clause 32, wherein the light detection modules are arranged in a polygonal configuration.

[0284] 34. The light detection system according to Clause 33, wherein the polygonal configuration is a heptagonal configuration.

[0285] 35. The light detection system according to any one of the preceding clauses further includes:

[0286] The third optical fiber is configured to transmit the third beam to the third optical receiver of the second CWD optical detection subsystem; and

[0287] The second CWD light detection subsystem includes a third light receiver configured to transmit the third light beam to the third light detection module.

[0288] 36. The optical detection system according to Clause 35, wherein the diameter of the third optical fiber is configured to be an aperture size suitable for fluorescence.

[0289] 37. The optical detection system according to any one of clauses 35 to 36, wherein the diameter of the third optical fiber is 800µm.

[0290] 38. The light detection system according to any one of the preceding clauses, wherein the light beam is blue light.

[0291] 39. The light detection system according to any one of the preceding clauses, wherein the flow cell is the flow cell of a flow cytometer.

[0292] 40. The light detection system according to any one of the preceding clauses, wherein the system is configured such that the optical adjustment component receives side-scattered light from the flow cell.

[0293] 41. The light detection system according to any one of the preceding clauses, wherein the system is configured such that the optical adjustment component receives fluorescence from the flow cell.

[0294] 42. The light detection system according to any one of the preceding clauses, wherein the optical adjustment component is configured to receive a light beam from an optical collection component that is in optical communication with the flow cell.

[0295] 43. The light detection system according to Clause 42 further includes a plurality of miniature motors operatively connected to the optical collection component.

[0296] 44. The optical detection system according to Clause 43, wherein the plurality of micro motors correspond to an adjustment shaft.

[0297] 45. The light detection system according to any one of clauses 42 to 44, wherein the optical adjustment component is integrated with the optical collection component.

[0298] 46. ​​The light detection system according to any one of the preceding clauses, wherein the optical adjustment component is positioned at a specified distance from the flow cell.

[0299] 47. A small particle optical detection module for a clustered wavelength division (CWD) optical detection subsystem, the small particle optical detection module comprising a small particle detector, wherein the small particle optical detection module is configured to receive a light beam from an optical receiver.

[0300] 48. The small particle light detection module according to Clause 41, wherein the light beam comprises light transmitted from a flow cell via an optical adjustment component, the optical adjustment component being configured to split the light received from the flow cell into a first light beam and a second light beam.

[0301] 49. The small particle light detection module as described in Clause 41 further includes a side scattering detector.

[0302] 50. The small particle light detection module according to Clause 42 further includes a beam splitter configured to split a light beam received from the light receiver into a first beam and a second beam.

[0303] 51. The small particle light detection module according to Clause 43, wherein the small particle light detection module is configured to transmit the first light beam to the small particle detector and the second light beam to the side scattering detector.

[0304] 52. The small particle light detection module according to any one of clauses 41 to 44, wherein the small particle detector comprises an avalanche photodiode.

[0305] 53. The small particle light detection module according to any one of clauses 41 to 45 further includes an optical component configured to transmit light having a predetermined sub-spectral range for detection.

[0306] 54. The small particle light detection module according to Clause 46, wherein the optical component includes a bandpass filter.

[0307] 55. The small particle light detection module according to Clause 46, wherein the optical component includes a dichroic mirror.

[0308] 56. The small particle light detection module according to any one of clauses 41 to 48, wherein the small particle light detection module optically communicates with a wavelength splitter, wherein the wavelength splitter is configured to allow light of a predetermined spectral range from the light receiver to pass through.

[0309] 57. The small particle light detection module according to any one of clauses 41 to 49, wherein the light receiver communicates optically with an optical fiber.

[0310] 58. The small particle light detection module according to Clause 50, wherein the diameter of the optical fiber is configured to be an aperture size suitable for side-scattered light.

[0311] 59. The small particle light detection module according to any one of clauses 50 to 51, wherein the diameter of the optical fiber is configured to be an aperture size suitable for the side-scattered light of the small particle.

[0312] 60. The small particle light detection module according to any one of clauses 50 to 52, wherein the diameter of the optical fiber is 200µm.

[0313] 61. A method comprising:

[0314] (a) Introducing a sample into a system, the system comprising:

[0315] An optical adjustment component is configured to split a light beam received from a flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence from particles excited within the flow cell.

[0316] A clustered wavelength division (CWD) optical detection subsystem includes a first optical receiver and a second optical receiver, wherein the first optical receiver is configured to transmit a first light beam to a first optical detection module, and wherein the second optical receiver is configured to transmit a second light beam to a second optical detection module, wherein the first optical detection module includes a small particle detector.

[0317] A first optical fiber, configured to transmit the first light beam from the optical adjustment component to the first optical receiver of the CWD optical detection subsystem; and

[0318] A second optical fiber, configured to transmit the second beam from the optical adjustment component to the second optical receiver of the CWD optical detection subsystem; and

[0319] (b) Analyze the small particles in the sample by flow cytometry.

[0320] 62. The method according to Clause 61, wherein the optical adjustment component includes a beam splitter or a multimode notch filter.

[0321] 63. The method according to clause 61 or 62, wherein the optical adjustment component is field-upgradeable.

[0322] 64. The method according to any one of clauses 61 to 63, wherein the system is configured such that the optical adjustment component is detachable.

[0323] 65. The method according to any one of clauses 61 to 64, wherein the system further includes an aperture located between the optical adjustment component and the flow cell.

[0324] 66. The method according to any one of clauses 61 to 65, wherein the optical adjustment component is configured to transmit the first beam directly onto the first optical fiber and the second beam directly onto the second optical fiber.

[0325] 67. The method according to any one of clauses 61 to 66, wherein the diameter of the first optical fiber is smaller than the diameter of the second optical fiber.

[0326] 68. The method according to any one of clauses 61 to 67 further includes obtaining a result from the system, the result providing information about the presence of small particles in the sample.

[0327] 69. The method according to any one of clauses 61 to 68, wherein the system further includes a light source.

[0328] 70. The method according to any one of clauses 61 to 69 further comprises:

[0329] To make the sample flow in the flow stream in the detection field; and

[0330] The sample in the flow of light in the detection field is illuminated.

[0331] 71. The method according to Clause 70, wherein the flow cell is configured to receive light emitted from a sample irradiating the flow stream.

[0332] 72. The method according to Clause 70, wherein the light beam received from the flow cell comprises light emitted from irradiating a sample in the flow stream.

[0333] 73. The method according to any one of clauses 61 to 59, wherein the diameter of the first optical fiber is configured to be an aperture size suitable for side-scattered light.

[0334] 74. The method according to any one of clauses 61 to 60, wherein the diameter of the first optical fiber is configured to be an aperture size suitable for side-scattered light from small particles.

[0335] 75. The method according to any one of clauses 61 to 61, wherein the diameter of the first optical fiber is 200µm.

[0336] 76. The method according to any one of clauses 61 to 62, wherein the first optical fiber includes optical fiber bending.

[0337] 77. The method according to any one of clauses 61 to 63, wherein the fiber bend is an approximately 90-degree bend of the first fiber.

[0338] 78. The method according to any one of clauses 61 to 64, wherein the diameter of the second optical fiber is configured to be an aperture size suitable for fluorescence.

[0339] 79. The method according to any one of clauses 61 to 65, wherein the diameter of the second optical fiber is 800µm.

[0340] 80. The method according to any one of clauses 61 to 66, wherein the first optical detection module further includes a side-scattering detector.

[0341] 81. The method according to Clause 80, wherein the first optical detection module includes a second beam splitter configured to split the first beam into a third beam and a fourth beam.

[0342] 82. The method according to Clause 81, wherein the first optical detection module is configured to transmit the third beam to the small particle detector and the fourth beam to the side scattering detector.

[0343] 83. The method according to any one of clauses 61 to 82, wherein the small particle detector comprises an avalanche photodiode.

[0344] 84. The method according to any one of clauses 61 to 83, wherein the first optical detection module further includes an optical component configured to transmit light having a predetermined sub-spectral range for detection.

[0345] 85. The method according to clause 84, wherein the optical component includes a bandpass filter.

[0346] 86. The method according to Clause 84, wherein the optical component comprises a dichroic mirror.

[0347] 87. The method according to any one of clauses 61 to 86, wherein the first optical receiver and the second optical receiver are in fixed positions relative to each other.

[0348] 88. The method according to any one of clauses 61 to 87, wherein the CWD optical detection subsystem includes a plurality of wavelength splitters configured to allow light having a predetermined spectral range from the first optical receiver and the second optical receiver to pass through.

[0349] 89. The method according to Clause 88, wherein each of the first optical detection module and the second optical detection module optically communicates with a wavelength splitter among the plurality of wavelength splitters.

[0350] 90. The method according to any one of clauses 88 to 89, wherein the wavelength splitters are configured to transmit light to each other.

[0351] 91. The method according to any one of clauses 88 to 90, wherein the wavelength splitter comprises a dichroic mirror.

[0352] 92. The method according to any one of clauses 88 to 91, wherein the second beam is transmitted from a subset of the wavelength splitter.

[0353] 93. The method according to any one of clauses 61 to 92, wherein the first optical receiver includes a first coupler for operably attaching to the first optical fiber, and the second optical receiver includes a second coupler for operably attaching to the second optical fiber.

[0354] 94. The method according to any one of clauses 61 to 93, wherein the first optical receiver and the second optical receiver each include a beam modulator.

[0355] 95. The method according to clause 94, wherein the beam adjuster is a lens.

[0356] 96. The method according to any one of clauses 61 to 95, wherein the CWD optical detection subsystem comprises three or more optical detection modules.

[0357] 97. The method according to Clause 96, wherein the light detection module is arranged in a polygonal configuration.

[0358] 98. The method according to Clause 97, wherein the polygonal configuration is a heptagonal configuration.

[0359] 99. The method according to any one of clauses 61 to 98, wherein the system further comprises:

[0360] The third optical fiber is configured to transmit the third beam to the third optical receiver of the second CWD optical detection subsystem; and

[0361] The second CWD light detection subsystem includes a third light receiver configured to transmit the third light beam to the third light detection module.

[0362] 100. The method according to Clause 99, wherein the diameter of the third optical fiber is configured to be an aperture size suitable for fluorescence.

[0363] 101. The method according to any one of clauses 99 to 100, wherein the diameter of the third optical fiber is 800µm.

[0364] 102. The method according to any one of clauses 61 to 101, wherein the light beam is blue light.

[0365] 103. The method according to any one of clauses 61 to 102, wherein the flow cell is the flow cell of a flow cytometer.

[0366] 104. The method according to any one of clauses 61 to 103, wherein the system is configured such that the beam splitter receives side-scattered light from the flow cell.

[0367] 105. The method according to any one of clauses 61 to 104, wherein the system is configured such that the beam splitter receives fluorescence from the flow cell.

[0368] 106. The method according to any one of clauses 61 to 105, wherein the beam splitter is configured to receive a light beam from an optical collection component in optical communication with the flow cell.

[0369] 107. The method according to Clause 106, wherein the system further comprises a plurality of micromotors operatively connected to the optical collecting component.

[0370] 108. The method according to Clause 107, wherein the plurality of micro motors correspond to the adjustment shaft.

[0371] 109. The method according to any one of clauses 106 to 108, wherein the beam splitter is integrated with the optical collecting component.

[0372] 110. The method according to any one of clauses 61 to 109, wherein the beam splitter is positioned at a specified distance from the flow cell.

[0373] 111. A kit comprising:

[0374] A small particle optical detection module for a clustered wavelength division (CWD) optical detection subsystem, the small particle optical detection module including a small particle detector, wherein the small particle optical detection module is configured to receive a light beam.

[0375] 112. The kit as described in Clause 111 further includes:

[0376] An optical adjustment component is configured to split a light beam received from a flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence from particles excited within the flow cell.

[0377] 113. The kit according to Clause 111, wherein the optical adjustment component includes a beam splitter or a multimode notch filter.

[0378] 114. The kit according to any one of clauses 111 to 113 further includes a first optical fiber configured to transmit the light beam to the small particle light detection module.

[0379] 115. The kit according to any one of clauses 111 to 114, wherein the diameter of the first optical fiber is configured to be an aperture size suitable for side-scattered light.

[0380] 116. The kit according to any one of clauses 111 to 115, wherein the diameter of the first optical fiber is configured to be an aperture size suitable for the side-scattered light of small particles.

[0381] 117. The kit according to any one of clauses 111 to 116, wherein the diameter of the first optical fiber is 200µm.

[0382] 118. The kit according to any one of clauses 111 to 117, wherein the small particle light detection module further includes a side scattering detector.

[0383] 119. The kit according to any one of clauses 111 to 118 further includes a second light detection module.

[0384] 120. The kit according to Clause 119 further includes a second optical fiber configured to transmit a second beam to the second optical detection module, wherein the diameter of the first optical fiber is smaller than the diameter of the second optical fiber.

[0385] 121. The kit according to Clause 120, wherein the diameter of the second optical fiber is configured to be an aperture size suitable for fluorescence.

[0386] 122. The kit according to any one of clauses 120 to 121, wherein the diameter of the second optical fiber is 800µm.

[0387] 123. The kit according to any one of clauses 111 to 122, wherein the small particle detector comprises an avalanche photodiode.

[0388] Although the invention has been described in detail by way of illustrations and examples for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0389] Therefore, the foregoing only illustrates the principles of the invention. It should be understood that those skilled in the art will be able to design various devices that, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to help the reader understand the principles of the invention and the concepts contributed by the inventors to further developments in the field, and should be understood as not being limited to these specifically listed examples and conditions. In addition, all statements herein referencing the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover their structural and functional equivalents. Furthermore, such equivalents are contemplated to include both currently known equivalents and future-developed equivalents (i.e., any element developed that performs the same function, regardless of its structure). Moreover, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly stated in the claims.

[0390] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, with respect to the limitation in the claims, 35 USC §112(f) or 35 USC §112(6) is explicitly defined as being invoked only when such limitation in the claims begins to refer to the exact phrase “means for…” or the exact phrase “step for…”; if such an exact phrase is not used in the limitation in the claims, then 35 USC §112(f) or 35 USC §112(6) is not invoked.

Claims

1. A photodetector system for detecting small particles, the photodetector system comprising: An optical adjustment component is configured to split a light beam received from a flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence from particles excited within the flow cell. A clustered wavelength division (CWD) optical detection subsystem includes a first optical receiver and a second optical receiver, wherein the first optical receiver is configured to transmit a first light beam to a first optical detection module, and wherein the second optical receiver is configured to transmit a second light beam to a second optical detection module, wherein the first optical detection module includes a small particle detector. A first optical fiber, configured to transmit the first light beam from the optical adjustment component to the first optical receiver of the CWD optical detection subsystem; and A second optical fiber is configured to transmit the second beam from the optical adjustment component to the second optical receiver of the CWD optical detection subsystem.

2. The optical detection system according to claim 1, wherein, The optical adjustment components include a beam splitter or a multimode notch filter.

3. The light detection system according to any one of the preceding claims, wherein, The optical adjustment components are field-upgradeable.

4. The light detection system according to any one of the preceding claims, wherein, The system is configured such that the optical adjustment component is detachable.

5. The light detection system according to any one of the preceding claims, wherein, The system also includes an aperture located between the optical adjustment component and the flow cell.

6. The light detection system according to any one of the preceding claims, wherein, The optical adjustment component is configured to transmit the first beam directly onto the first optical fiber and the second beam directly onto the second optical fiber.

7. The light detection system according to any one of the preceding claims, wherein, The diameter of the first optical fiber is smaller than the diameter of the second optical fiber.

8. The light detection system according to any one of the preceding claims, wherein, The diameter of the first optical fiber is configured to be an aperture size suitable for side-scattered light.

9. The light detection system according to any one of the preceding claims, wherein, The diameter of the first optical fiber is configured to be an aperture size suitable for the side-scattered light of small particles.

10. The light detection system according to any one of the preceding claims, wherein, The first optical detection module also includes a side scattering detector.

11. The optical detection system according to claim 10, wherein, The first optical detection module includes a second beam splitter configured to split the first beam into a third beam and a fourth beam.

12. The light detection system according to any one of the preceding claims further comprises: The third optical fiber is configured to transmit the third beam to the third optical receiver of the second CWD optical detection subsystem; as well as The second CWD light detection subsystem includes the third light receiver, wherein the third light receiver is configured to transmit the third light beam to the third light detection module.

13. A small particle optical detection module for a clustered wavelength division (CWD) optical detection subsystem, the small particle optical detection module comprising a small particle detector, wherein, The small particle light detection module is configured to receive a light beam from a light receiver.

14. A method comprising: (a) Introducing a sample into a system, the system comprising: An optical adjustment component is configured to split a light beam received from a flow cell into a first beam and a second beam, wherein the first beam includes light scattered from particles excited within the flow cell, and wherein the second beam includes fluorescence from particles excited within the flow cell. A clustered wavelength division (CWD) optical detection subsystem includes a first optical receiver and a second optical receiver, wherein the first optical receiver is configured to transmit a first light beam to a first optical detection module, and wherein the second optical receiver is configured to transmit a second light beam to a second optical detection module, wherein the first optical detection module includes a small particle detector. A first optical fiber, configured to transmit the first light beam from the optical adjustment component to the first optical receiver of the CWD optical detection subsystem; and A second optical fiber, configured to transmit the second beam from the optical adjustment component to the second optical receiver of the CWD optical detection subsystem; and (b) Analyze the small particles in the sample by flow cytometry.

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