Systems and methods for digital affinity-based detection assays - Patents.com

Abstract

Systems and methods for digital affinity-based detection assays are described. In certain embodiments, the method includes associating an analyte, such as a single molecule analyte, with a detectable agent in a sample, flowing the sample containing the analyte associated with the detectable agent through a flow path, outputting excitation light through an interrogation window through a portion of the flow path, and generating an emission signal at a photodetector based on emission light received from the portion of the flow path.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 263,864, filed November 10, 2021, the entire contents of which are incorporated herein by reference.

[0002] (Description of Electronic Sequence Listing) The Sequence Listing XML associated with this application is provided in XML format and is incorporated herein by reference. The name of the XML file containing the Sequence Listing is 3915-P1137WOUWSL. The XML file is 3 pages long, was created on September 29, 2022, and has been submitted with this application via the Patent Center.

[0003] (Government License Rights Statement) This invention was made with Government support under Grant No. UG3TR002874 awarded by the National Institute of Health Sciences. The Government has certain rights in this invention. [Background technology]

[0004] Flow-based analysis of small particles such as extracellular vesicles can present several challenges. A typical flow-based particle analysis involves flowing a large number of particles dispersed in a suspending fluid through a passage. As the size of the particles decreases, they tend to diffuse faster than larger particles. In addition, since the velocity of the fluid flowing through a passage under laminar flow conditions varies radially with the distance from the passage wall, the velocity of the particles flowing through the passage varies accordingly with the distance from the wall. Thus, it is more difficult to distinguish between various smaller particles flowing through a passage, since the particle velocity is dependent on their radial position, which is strongly influenced by their relatively high diffusivity. The problem of detection sensitivity as particles or molecules flow is particularly relevant in digital affinity-based detection assays, where the presence of even a single molecule should be reliably detected and counted as it flows through a detection region or laser probe volume. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is currently a need for devices, systems, and methods for flow-based analysis of small particles that take into account the diffusion-based challenges in identifying and characterizing such small particles. [Means for solving the problem]

[0006] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0007] In an aspect, the disclosure provides a method for a digital affinity-based detection assay. In an embodiment, the method is a method of digital affinity-based assay of an analyte. In an embodiment, the method is a method of digital affinity-based assay of a single molecule analyte. In an embodiment, the method is a method of digital affinity-based assay of a single molecule analyte that does not require and / or does not include performing an amplification step. In an embodiment, the method includes associating an analyte in a sample with a detectable agent, flowing the sample including the analyte associated with the detectable agent through a flow path, outputting excitation light through an inspection window through a portion of the flow path, and generating an emission signal at a photodetector based on emission light received from the portion of the flow path through the inspection window.

[0008] In another aspect, the disclosure provides a system for analyzing a single molecule analyte. In an embodiment, the system for analyzing a single molecule analyte does not include a reagent or structure for performing an amplification step, such as an analyte amplification step. In an embodiment, the system includes a flow path configured to flow a single molecule analyte through a lumen of the flow path and defining an inspection window configured to allow light to pass into and out of the lumen, a light engine configured to output excitation light into the flow path through the inspection window, a detector system positioned to receive emitted light from the flow path and configured to generate a signal based on the received emitted light, a light collection system positioned to collect the emitted light from the flow path and direct the collected emitted light onto the detector system, and a controller operably coupled to the light engine and the detector system, the controller including logic that, when executed by the controller, causes the system to perform operations including outputting the excitation light with the light engine through the inspection window onto a portion of the flow path and generating an emission signal with the detector.

[0009] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0010] [Figure 1A] 1 is a schematic diagram of a flow-based single particle / molecule analysis system using a high numerical aperture (NA) air objective lens according to an embodiment of the present disclosure. [Figure 1B] 1B is a schematic diagram of an emitting fiber bundle head of the system of FIG. 1A according to an embodiment of the present disclosure. [Diagram 2] 1 is a schematic diagram of a detector module of a flow-based single molecule / particle system according to an embodiment of the present disclosure. [Figure 3A] 1 is a schematic diagram of an optical engine and passageway of a flow-based single molecule / particle system according to an embodiment of the present disclosure. [Figure 3B] 3B is a schematic diagram of an inspection window of the passageway of FIG. 3A according to an embodiment of the present disclosure. [Figure 3C] 3B is a schematic diagram of an example of the light engine and passageway of FIG. 3A in accordance with an embodiment of the present disclosure. [Figure 3D] 3B is a schematic diagram of an example of the light engine and passageway of FIG. 3A in accordance with an embodiment of the present disclosure. [Figure 3E] 3B is a schematic diagram of an example of the light engine and passageway of FIG. 3A in accordance with an embodiment of the present disclosure. [Figure 3F] 3B is a schematic diagram of an example of the light engine and passageway of FIG. 3A in accordance with an embodiment of the present disclosure. [Figure 4] 1 is an image of a passageway of a system, according to an embodiment of the present disclosure; [Figure 5A] 13A-13C illustrate diagrammatically emitted light passing through an opening in an optically opaque cover and onto an emission fiber optic bundle head of a detector system, according to an embodiment of the present disclosure; [Figure 5B] 5B illustrates an example of the optically opaque cover of FIG. 5A according to an embodiment of the present disclosure. [Figure 5C]1 is an image of an emission fiber bundle head of a flow-based single molecule / particle system, according to an embodiment of the present disclosure. [Figure 6] 1 is a schematic diagram of a flow-based single molecule / particle system using a high NA air objective lens according to an embodiment of the present disclosure. [Figure 7A] 1 is a schematic diagram of a flow-based single molecule / particle system according to an embodiment of the present disclosure. [Figure 7B] 7B is a schematic diagram of the focusing of a high NA air objective lens on a sample of the system of FIG. 7A on a passageway of the system, according to an embodiment of the present disclosure. [Figure 7C] FIG. 1 is a block diagram illustrating a method for focusing a high NA air objective lens of a flow-based single molecule / particle system according to an embodiment of the present disclosure. [Figure 7D] 1 is a series of images of a passageway taken at several distances between the passageway and a high NA air objective lens and with different amounts of defocus, in accordance with an embodiment of the present disclosure. [Figure 7E] The quantity of focusing quality at various distances between the passageway and the high NA air objective lens according to an embodiment of the present disclosure is illustrated by noting the location of the images in FIG. 7D. [Figure 7F] FIG. 1 is a block diagram illustrating a feedback control loop used to set the focal plane using near-infrared imaging with a high NA air objective lens, according to an embodiment of the present disclosure. [Figure 7G] FIG. 11 is another block diagram illustrating a feedback control loop used to implement near-infrared machine vision and real-time focusing assisted by a high NA air objective lens in accordance with an embodiment of the present disclosure. [Figure 8] 1 is a schematic diagram of a system including a purification subsystem according to an embodiment of the present disclosure. [Figure 9A] 1 is a block diagram of a method according to an embodiment of the present disclosure. [Figure 9B] 1 is a block diagram of a method according to an embodiment of the present disclosure. [Figure 10A] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 10B] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 10C] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 11] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 12A] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 12B] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 13] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 14] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 15] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 16A] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 16B] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 17] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 18A] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 18B] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 18C] 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 19] 1 is a schematic diagram of a system according to an embodiment of the present disclosure. [Figure 20] 1 is a schematic diagram of a system according to an embodiment of the present disclosure. [Figure 21] 1 is a block diagram of a method according to an embodiment of the present disclosure. [Figure 22] 1 is a block diagram of a method according to an embodiment of the present disclosure. [Figure 23A] FIG. 1 is a data trace showing five two-color colocalized events (i.e., analyte, non-fluorescent streptavidin), three unbound detectable agents (i.e., biotin-Alexa647), and two unbound capture agents (i.e., anti-streptavidin-PE antibodies) according to an embodiment of the present disclosure. [Figure 23B] FIG. 23B is a scatter plot showing 372 analyte molecules (i.e., streptavidin) counted using the method from FIG. 23A according to an embodiment of the present disclosure. [Figure 24A] FIG. 1 is a data trace showing seven two-color colocalized events (i.e., non-fluorescent analyte, mouse anti-rabbit IgG), five unbound detectable agents (i.e., goat anti-mouse IgG-PE), and three unbound capture agents (i.e., rabbit anti-human IgG-Alexa647) according to an embodiment of the present disclosure. [Figure 24B] FIG. 24B is a scatter plot showing 765 analyte molecules (i.e., non-fluorescent mouse anti-rabbit IgG) counted using the method from FIG. 24A, according to an embodiment of the present disclosure. [Figure 25A] 1 is a data trace showing eight two-color co-localized non-fluorescent analytes and three unbound capture agents (i.e., rabbit anti-human IgG-Alexa647) according to an embodiment of the present disclosure. [Figure 25B] FIG. 25B is a scatter plot showing 655 analyte molecules counted using the method from FIG. 25A according to an embodiment of the present disclosure. [Figure 26A] Illustrates a data trace of nine two-color colocalization events from a sample prepared without purification indicating the presence of nine analyte molecules counted, and nineteen unbound capture agents (i.e., goat anti-mouse IgG-Alexa647) that did not colocalize with any signal in the fluorescence path from the beads / nanoparticles, according to an embodiment of the present disclosure. [Figure 26B] Illustrates a data trace of eight two-color colocalization events prepared with sample purification indicating the presence of nine analyte molecules counted with only three unbound capture agents (i.e., goat anti-mouse IgG-Alexa647) according to an embodiment of the present disclosure. [Figure 27] Illustrates a data trace showing 12 peaks from Alexa647 fluorescence indicating the presence of 12 analyte molecules (i.e., mouse-IgG) counted in a digital dual affinity protein assay after magnetic bead capture and purification according to an embodiment of the present disclosure. [Figure 28A]A library of nine sample barcodes is illustrated, in which the target barcode exhibited moderate levels of Alexa488 (green fluorescence) and high intensity of Alexa561 (orange fluorescence), according to an embodiment of the present disclosure. [Figure 28B] Three events with colocalization of target barcodes and peaks from the Alexa647 passage (red fluorescence) identified as the presence of mouse IgG, according to an embodiment of the present disclosure. [Figure 29] 1 illustrates a data trace showing seven events involving co-localization of a capture agent and a detectable agent, counted as seven copies of a target or analyte gene molecule, according to an embodiment of the present disclosure. [Diagram 30] 1 illustrates a data trace in which each peak in the Alexa647 pathway data trace represents a single target or analyte gene captured by a capture agent consisting of a nucleic acid molecule associated with a magnetic bead, according to an embodiment of the present disclosure. [Figure 31A] According to an embodiment of the present disclosure, a library of nine model barcodes is illustrated, of which the target barcode exhibited high levels of Alexa488 (green fluorescence) and low intensity Alexa561 (orange fluorescence). [Figure 31B] 1 shows a data trace including three events with co-localization of target barcodes and peaks (red fluorescence) from the Alexa647 passage were detected and counted as target or analyte gene molecules according to an embodiment of the present disclosure. [Diagram 32] 1 illustrates a data trace in which each fluorescent peak from YOYO-3 was counted as a copy of a target gene, according to an embodiment of the present disclosure. [Diagram 33] 1 illustrates a data trace in which each co-localization event from Alexa647 and YOYO-3 indicates the presence of a copy of the target gene, according to an embodiment of the present disclosure. [Figure 34A] 1A-1C are schematic illustrations of a method for labeling and analyzing fluorescently labeled analytes with fluorescent barcoded beads according to an embodiment of the present disclosure. [Figure 34B]FIG. 1 illustrates a data trace showing two triple positive events based on single streptavidin-PE (analyte, orange) molecules captured by fluorescent barcoded beads (blue and purple) and two other events corresponding to unbound beads in the absence of analyte, according to an embodiment of the present disclosure. [Figure 34C] FIG. 1 is a scatter plot showing 566 colocalization events enumerated at 2 minutes, according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure provides systems and methods for flow-based analysis of particles and molecules. In the following description, numerous specific details are set forth to provide a thorough understanding of the embodiments. However, those skilled in the art will recognize that the techniques described herein can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring certain aspects.

[0012] Throughout this specification, a reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Also, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0013] Small particles and molecules tend to diffuse in the fluid flowing through the passage more than larger particles. The radial displacement of particles due to particle diffusion, i.e., displacement of particles in a direction perpendicular to the main flow axis of the passage, can make it difficult to measure the properties of such small particles, such as fluorescence measurements, especially when there are several particles or molecules flowing through the passage. For example, when multiple particles or molecules flow through the passage at the same time, the individual particles or molecules may have different velocities through the passage. If the particles and / or molecules in the passage are measured at various points in the passage, it can be difficult to correlate the signal generated by the photodetector positioned to interrogate the passage at various points along the length of the passage with a single molecule or particle. In fact, as the size of the particles decreases, for example toward the size of extracellular vesicles, or even single molecules, such measurements can become very difficult.

[0014] system Thus, in one aspect, the present disclosure provides a system for analyzing particles, such as single biological nanoparticles, and / or molecules.

[0015] Fiber-bundled emission and excitation In that regard, attention is directed to Figures 1A and 1B, which illustrate a system 100 according to an embodiment of the present disclosure.

[0016] As shown, system 100 includes a passageway 102 configured to flow particles and / or molecules through a lumen 104 of the passageway 102, defining an inspection window 106 configured to allow light to pass in and out of the lumen 104, a light engine 108 configured to output light into the inspection window 106, an emission fiber optic bundle 130 positioned to receive emitted light from the inspection window 106, and a detector system 142 positioned to receive emitted light from the emission fiber optic bundle 130.

[0017] In an embodiment, the system 100 or a portion thereof (e.g., including the passageway 102 and the inspection window 106) comprises a microfluidic chip. A microfluidic chip may be formed from a substrate (e.g., silicon, glass, ceramic, plastic, organosilicon, quartz, polymeric material, or a combination thereof) and may include a network of microfluidic passageways through which fluids flow. Microfluidic devices can be used to process minute amounts of fluid samples and offer advantages over conventional macroscale devices (e.g., by requiring substantially smaller volumes of fluid samples, requiring less reagent use, and reduced processing times compared to macroscale devices). Microfluidic chips provide an attractive and versatile platform for the manipulation, isolation, sorting, and / or transport of particles and / or molecules. Arrays of microfluidic passageways can be easily patterned and integrated into microfluidic devices, making these microfluidic devices an attractive platform for applications involving particles and / or molecules. Microfluidic chips are planar devices and therefore can facilitate detection and analysis of particles and / or molecules by allowing the use of objective lenses with high light collection efficiency, which improves light collection and therefore facilitates detection, analysis, determination and / or identification of particles and / or molecules.

[0018] In some embodiments, the disclosed methods, systems, devices, and apparatus include microfluidic chips that can facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules in motion. Microfluidic chips can be used to process small volumes of fluid samples, offering advantages over traditional macroscale devices (e.g., microfluidic chips require only minute volumes of fluid samples, require fewer reagents, are processed in less time, and increase efficiency compared to macroscale devices). Microfluidic chips are planar devices and therefore can facilitate the detection and analysis of bionanoparticles, and / or can facilitate the detection and analysis of bionanoparticles by enabling the use of high NA (numerical aperture) objectives, such as air objectives with NA of about 0.95 or 0.91-0.99 (e.g., high NA air objectives), lenses, or light collection systems with high numerical apertures that improve light collection and therefore facilitate the detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules. In certain embodiments, microfluidic chips are planar devices, making them more compatible with microscope setups (e.g., having a translation stage on which the microfluidic chip is placed). In addition, microfluidic chips can enable the design and creation of interconnected fluidic networks without dead volumes, which can facilitate detection and manipulation of bionanoparticles and / or molecules (e.g., sorting using flow displacement at the junction of three or more fluidic passages). Dead volumes are a portion of the volume within the microfluidic chip that is outside the flow paths (e.g., a volume into which liquids that may carry sample nanoparticles and / or molecules may diffuse, reducing accuracy). Microfluidic chips, through methods of microfabrication, can enable the creation of passages with cross sections that are non-spherical or non-square (e.g., rectangular), which can facilitate detection, analysis, determination, and / or identification of moving bionanoparticles and / or molecules.Microfluidic chips can facilitate the creation of passages with different widths or heights along the length of the passage (e.g., constrictions in the passages or gradual changes in width and / or height) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules passing through. Microfluidic chips can be formed by bonding to a coverslip (e.g., made from glass or plastic) of a desired thickness and with desired material properties (e.g., refractive index) to facilitate compatibility with highly efficient light collection systems (e.g., high numerical aperture objectives such as high NA air objectives, which require appropriate substrate thickness for maximum light collection) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules during movement. Microfluidic chips provide an attractive and versatile platform for the manipulation, isolation, sorting, and / or transport of bionanoparticles and / or single molecules.

[0019] 1A and 1B, in some embodiments, portions of the system 100, such as the portion including the passageway 102, may be made of a polymeric material (polydimethylsiloxane (PDMS), polyurethane-methacrylate (PUMA), polymethylmethacrylate (PMMA), polyethylene, polyester (PET), polytetrafluoroethylene (PTFE), polycarbonate, parylene, polyvinyl chloride, fluoroethyl ether, etc. The system 100 may be fabricated from materials including, but not limited to, porous membranes, woven or non-woven fibers of wool (such as cloth or mesh), metals (e.g., stainless steel or Monel), glass, paper, or synthetic fibers (e.g., nylon, polypropylene, polycarbonate, parylene, and various polyesters), sintered stainless steel and other metals, and porous inorganic materials such as alumina, silica, or carbon.

[0020] The interrogation window 106 of the passageway 102 allows excitation light, such as from the light engine 108, to pass into the lumen 104 of the passageway 102 and allows emitted light to pass out of the passageway 102 to be received by the detector system 142. Such excitation light and emitted light may include light from a number of wavelength ranges, including, but not limited to, visible light, infrared light, near infrared light, and ultraviolet light, and combinations thereof. In this regard, the interrogation window 106 is suitable for exciting particles and / or molecules flowing through the passageway 102 and allowing light emitted from the interrogation window 106 to be received by the detector system 142 for further analysis.

[0021] As discussed further herein, in some embodiments, the lumen 104 of the passageway 102 within the inspection window 106, or in certain embodiments, the lumen 104 of the passageway 102 adjacent to the inspection window 106, defines a constriction or other narrowing in a cross-section or diameter or other size characteristic of the lumen 104. Such a constriction or narrowing of the lumen 104 is configured to cause particles and / or molecules to flow, particle-by-particle and / or molecule-by-molecule, under laminar flow conditions through the portion of the passageway 102 that includes the inspection window 106.

[0022] In some embodiments, the inspection window 106 comprises a portion of the passageway within the field of view of the objective lens 186 and / or detectable by the detector system 142. In some embodiments, the inspection window 106 comprises a portion of the passageway 102 that defines a constriction relative to other portions of the passageway 102. As described further herein, in such embodiments, the constriction of the inspection window 106 can have dimensions, such as a height, width, cross-sectional area, etc., that are smaller than other immediately adjacent portions of the passageway 102.

[0023] In some embodiments, the constriction has a width less than the widest portion of the microfluidic passage 102. In certain embodiments, the constriction has a width relative to the widest portion of the microfluidic passage 102. In some embodiments, the constriction has a width less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the maximum width of the microfluidic passage. As a non-limiting example, a microfluidic passage 102 having a maximum width of 100 μm can have a constriction that is less than 25% of the maximum width value (i.e., less than 25 μm). In a preferred embodiment, the constriction has a width less than 10% of the maximum width of the microfluidic passage 102.

[0024] In some embodiments, the maximum width of the microfluidic passage 102 is less than 900 μm and more than 0.1 μm, less than 800 μm and more than 0.5 μm, less than 700 μm and more than 1 μm, less than 600 μm and more than 5 μm, less than 500 μm and more than 10 μm, less than 1,000 μm and more than 10 μm, less than 900 μm and more than 10 μm, less than 800 μm and more than 10 μm, less than 700 μm and more than 10 μm, less than 600 μm and more than 10 μm, less than 500 μm and more than 10 μm. In a preferred embodiment, the maximum width of the microfluidic passage 102 has a value of less than 500 μm and more than 10 μm, less than 400 μm and more than 10 μm, less than 300 μm and more than 10 μm, less than 500 μm and more than 0.1 μm, less than 500 μm and more than 1 μm, less than 500 μm and more than 2 μm, less than 500 μm and more than 5 μm, less than 800 μm and more than 0.1 μm, less than 700 μm and more than 0.1 μm, less than 600 μm and more than 0.1 μm, less than 500 μm and more than 0.1 μm, less than 400 μm and more than 0.1 μm, or less than 300 μm and more than 0.1 μm. In a preferred embodiment, the maximum width of the microfluidic passage 102 has a value of less than 500 μm and more than 10 μm.

[0025] In some embodiments, the constriction has a width that is less than the average width of the microfluidic passage. In certain embodiments, the constriction has a width relative to the average width of the microfluidic passage. In some embodiments, the constriction has a width that is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the average width of the microfluidic passage 102.

[0026] In some embodiments, the average width of the microfluidic passage 102 is less than 900 μm and more than 0.1 μm, less than 800 μm and more than 0.5 μm, less than 700 μm and more than 1 μm, less than 600 μm and more than 5 μm, less than 500 μm and more than 10 μm, less than 1,000 μm and more than 10 μm, less than 900 μm and more than 10 μm, less than 800 μm and more than 10 μm, less than 700 μm and more than 10 μm, less than 600 μm and more than 10 μm, less than 500 μm and more than 10 μm. less than 500 μm and more than 0.1 μm, less than 500 μm and more than 1 μm, less than 500 μm and more than 2 μm, less than 500 μm and more than 5 μm, less than 800 μm and more than 0.1 μm, less than 700 μm and more than 0.1 μm, less than 600 μm and more than 0.1 μm, less than 500 μm and more than 0.1 μm, less than 400 μm and more than 0.1 μm, or less than 300 μm and more than 0.1 μm. In a preferred embodiment, the average width of the microfluidic passage 102 has a value less than 500 μm and more than 10 μm.

[0027] In some embodiments, the constriction has a height that is less than the maximum height value (i.e., maximum height) of the microfluidic passage 102. In certain embodiments, the constriction has a height relative to the maximum height of the microfluidic passage. In some embodiments, the constriction has a height that is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the maximum height of the microfluidic passage. As a non-limiting example, a microfluidic passage 102 having a maximum height of 20 μm can have a constriction that is less than 10% of the maximum height value (i.e., less than 2 μm). In a preferred embodiment, the constriction has a height that is less than 25% of the maximum height of the microfluidic passage 102.

[0028] In some embodiments, the maximum height of the microfluidic passage 102 is less than 900 μm and more than 0.1 μm, less than 800 μm and more than 0.5 μm, less than 700 μm and more than 1 μm, less than 600 μm and more than 5 μm, less than 500 μm and more than 10 μm, less than 1,000 μm and more than 10 μm, less than 900 μm and more than 10 μm, less than 800 μm and more than 10 μm, less than 700 μm and more than 10 μm, less than 600 μm and more than 10 μm, less than 500 μm and more than 10 μm. , less than 400 μm and more than 10 μm, less than 300 μm and more than 10 μm, less than 500 μm and more than 0.1 μm, less than 500 μm and more than 1 μm, less than 500 μm and more than 2 μm, less than 500 μm and more than 5 μm, less than 800 μm and more than 0.1 μm, less than 700 μm and more than 0.1 μm, less than 600 μm and more than 0.1 μm, less than 500 μm and more than 0.1 μm, less than 400 μm and more than 0.1 μm, or less than 300 μm and more than 0.1 μm. In a preferred embodiment, the maximum height of the microfluidic passage 102 has a value less than 500 μm and more than 10 μm.

[0029] In some embodiments, the constriction has a height that is less than the average height of the microfluidic passage. In certain embodiments, the constriction has a height relative to the average height of the microfluidic passage. In some embodiments, the constriction has a height that is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the average height of the microfluidic passage.

[0030] In some embodiments, the average height of the microfluidic passages is less than 900 μm and more than 0.1 μm, less than 800 μm and more than 0.5 μm, less than 700 μm and more than 1 μm, less than 600 μm and more than 5 μm, less than 500 μm and more than 10 μm, less than 1,000 μm and more than 10 μm, less than 900 μm and more than 10 μm, less than 800 μm and more than 10 μm, less than 700 μm and more than 10 μm, less than 600 μm and more than 10 μm, less than 500 μm and more than 10 μm , less than 400 μm and more than 10 μm, less than 300 μm and more than 10 μm, less than 500 μm and more than 0.1 μm, less than 500 μm and more than 1 μm, less than 500 μm and more than 2 μm, less than 500 μm and more than 5 μm, less than 800 μm and more than 0.1 μm, less than 700 μm and more than 0.1 μm, less than 600 μm and more than 0.1 μm, less than 500 μm and more than 0.1 μm, less than 400 μm and more than 0.1 μm, or less than 300 μm and more than 0.1 μm. In a preferred embodiment, the average height of the microfluidic channels has a value of less than 500 μm and more than 10 μm.

[0031] In some embodiments, the constriction has a cross-sectional area that is less than the largest cross-sectional area (i.e., the maximum cross-sectional area) of the microfluidic passage. In certain embodiments, the constriction has a cross-sectional area that is relative to the maximum cross-sectional area of ​​the microfluidic passage. In some embodiments, the constriction has a cross-sectional area of ​​less than 50%, 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, or 0.001% of the maximum cross-sectional area of ​​the microfluidic passage. 2 A microfluidic passage having a cross-sectional area less than 10% of the maximum cross-sectional area (i.e., 20 μm 2 In a preferred embodiment, the constriction has a cross-sectional area of ​​10% to 0.01% of the maximum cross-sectional area of ​​the microfluidic passage.

[0032] In some embodiments, the maximum cross-sectional area of ​​a microfluidic passage is greater than or equal to 1,000,000 μm 2 Less than 10μm 2 Ultra, 750,000μm 2 Less than 25μm 2 Ultra, 500,000μm 2 Less than 100μm 2 Ultra, 250,000μm 2 Less than 250μm 2 Ultra, 900,000μm 2 Less than 100μm 2 Ultra, 800,000μm 2 Less than 100μm 2 Ultra, 700,000μm 2 Less than 100μm 2 Ultra, 600,000μm 2 Less than 100μm 2 Ultra, 400,000μm 2 Less than 100μm2 Ultra, 300,000μm 2 Less than 100μm 2 Ultra, 200,000μm 2 Less than 100μm 2 Ultra, 100,000μm 2 Less than 100μm 2 Ultra, 50,000μm 2 Less than 100μm 2 Ultra, 25,000μm 2 Less than 100μm 2 Ultra, 10,000μm 2 Less than 100μm 2 Ultra, 1,000μm 2 Less than 100μm 2 Ultra, 2,000,000μm 2 Less than 250μm 2 Ultra, 1,000,000μm 2 Less than 250μm 2 Ultra, 900,000μm 2 Less than 250μm 2 Ultra, 800,000μm 2 Less than 250μm 2 Ultra, 700,000μm 2 Less than 250μm 2 Ultra, 600,000μm 2 Less than 250μm 2 Ultra, 400,000μm 2 Less than 250μm 2 Ultra, 300,000μm 2 Less than 250μm 2 Ultra, 200,000μm 2 Less than 250μm 2 Ultra, 100,000μm 2 Less than 250μm 2 Ultra, 50,000μm 2 Less than 250μm 2 Ultra, 25,000μm 2 Less than 250μm 2 Ultra, 10,000μm 2 Less than 250μm 2 Ultra, 1,000μm 2 Less than 250μm 2In a preferred embodiment, the maximum cross-sectional area of ​​a microfluidic passage is greater than 250,000 μm 2 Less than 250μm 2 It has a value of greater than .

[0033] In some embodiments, the constriction has a cross-sectional area that is less than the average cross-sectional area of ​​the microfluidic passage. In certain embodiments, the constriction has a cross-sectional area that is relative to the average cross-sectional area of ​​the microfluidic passage. In some embodiments, the constriction has a cross-sectional area that is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.002%, or less than 0.001% of the average cross-sectional area of ​​the microfluidic passage.

[0034] In some embodiments, the average cross-sectional area of ​​the microfluidic channels is greater than or equal to 1,000,000 μm 2 Less than 10μm 2 Ultra, 750,000μm 2 Less than 25μm 2 Ultra, 500,000μm 2 Less than 100μm 2 Ultra, 250,000μm 2 Less than 250μm 2 Ultra, 900,000μm 2 Less than 100μm 2 Ultra, 800,000μm 2 Less than 100μm 2 Ultra, 700,000μm 2 Less than 100μm 2 Ultra, 600,000μm 2 Less than 100μm 2 Ultra, 400,000μm 2 Less than 100μm 2 Ultra, 300,000μm 2Less than 100μm 2 Ultra, 200,000μm 2 Less than 100μm 2 Ultra, 100,000μm 2 Less than 100μm 2 Ultra, 50,000μm 2 Less than 100μm 2 Ultra, 25,000μm 2 Less than 100μm 2 Ultra, 10,000μm 2 Less than 100μm 2 Ultra, 1,000μm 2 Less than 100μm 2 Ultra, 2,000,000μm 2 Less than 250μm 2 Ultra, 1,000,000μm 2 Less than 250μm 2 Ultra, 900,000μm 2 Less than 250μm 2 Ultra, 800,000μm 2 Less than 250μm 2 Ultra, 700,000μm 2 Less than 250μm 2 Ultra, 600,000μm 2 Less than 250μm 2 Ultra, 400,000μm 2 Less than 250μm 2 Ultra, 300,000μm 2 Less than 250μm 2 Ultra, 200,000μm 2 Less than 250μm 2 Ultra, 100,000μm 2 Less than 250μm 2 Ultra, 50,000μm 2 Less than 250μm 2 Ultra, 25,000μm 2 Less than 250μm 2 Ultra, 10,000μm 2 Less than 250μm 2 Ultra, 1,000μm 2 Less than 250μm 2 In a preferred embodiment, the average cross-sectional area of ​​the microfluidic channels is greater than 250,000 μm 2Less than 250μm 2 It has a value of greater than .

[0035] In some embodiments, the constriction has a height of less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, or less than 1 μm. 2 In some embodiments, the constriction has a width of less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, or less than 1 μm.

[0036] In some embodiments, at least one microfluidic passage comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions. In some embodiments, a microfluidic chip comprises a plurality of microfluidic passages, at least a portion of each of which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions, respectively. In some embodiments, a microfluidic chip comprises a plurality of microfluidic passages, a majority of each of which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions, respectively. In some embodiments, a microfluidic chip comprises a plurality of microfluidic passages, each of which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions, respectively.

[0037] In some embodiments, at least a portion of at least one microfluidic passage 102 is 10,000 μm 2 Cross-sectional area less than 5000μm 2 Cross-sectional area less than 3000μm 2 Cross-sectional area less than 1,000μm 2 Cross-sectional area less than 800μm 2 Cross-sectional area less than 600μm 2 Cross-sectional area less than 400μm 2 Cross-sectional area less than 200μm 2Cross-sectional area of ​​less than 100 μm 2 In a preferred embodiment, at least a portion of at least one microfluidic passage has a cross-sectional area of ​​less than 100 μm. 2 , cross-sectional area less than 90 μm 2 , cross-sectional area less than 80 μm 2 , cross-sectional area less than 70 μm 2 , cross-sectional area less than 60 μm 2 , cross-sectional area less than 50 μm 2 , cross-sectional area less than 40 μm 2 , cross-sectional area less than 30 μm 2 , cross-sectional area less than 20 μm 2 , cross-sectional area less than 10 μm 2 , cross-sectional area less than 5 μm 2 , cross-sectional area less than 2 μm 2 , or a cross-sectional area of ​​less than 1 μm 2 In some embodiments, at least one microfluidic passage has a diameter of 250,000 μm. 2 Less than 100,000μm 2 Less than 50,000μm 2 Less than 25,000μm 2 Less than 10,000μm 2 Less than 5,000μm 2 Less than 3,000μm 2 Less than 1,000μm 2 Less than 800μm 2 Less than 600μm 2 Less than 400μm 2 Less than 200μm 2 Less than or equal to 100 μm 2 In some embodiments, at least one microfluidic passage has a maximum cross-sectional area of ​​less than 100 μm 2 Less than 90μm 2 Less than 80μm 2 Less than 70μm 2 Less than 60μm 2 Less than 50μm 2 Less than 40μm 2 Less than 30μm 2 Less than 20μm 2 Less than 10μm 2 Less than 5μm 2 Less than 2μm 2Less than or equal to 1μm 2 In a preferred embodiment, at least a portion of at least one microfluidic passage has a maximum cross-sectional area of ​​less than 1 μm 2 ~100μm 2 In some embodiments, at least one microfluidic passage has a cross-sectional area of ​​100 μm 2 ~10,000μm 2 has a maximum cross-sectional area of

[0038] In certain embodiments, at least a portion of at least one microfluidic passage includes a discontinuous change in at least one of its width or height (e.g., achieved using microfabrication techniques). As used herein, a microfluidic chip may include a microfluidic passage having a step gradient or step change in at least one of its height or width, as opposed to a microfluidic passage including a continuous change in height or width. Passages including a continuous change in height or width are common, for example, in devices including glass tubes, which may be achieved by pulling a heated tube. In specific embodiments, at least a portion of at least one microfluidic passage has heights and widths that vary independently of each other. Independent changes in height and width are in contrast to, for example, glass tubes, where processing to reduce the height is accompanied by a corresponding reduction in width (e.g., stretching and thinning of a glass tube heated to near its melting temperature).

[0039] As mentioned above, the system 100 includes a light engine 108. In the illustrated embodiment, the light engine 108 is shown to include several light sources 110, 114, 118, and 120, each positioned to emit or output excitation light onto respective distinct portions 122, 124, 126, and 128 of the passageway 102 within the inspection window 106. In this regard, the light engine 108 is shown to include a first light source 110 positioned to output a first excitation light 112 onto a first portion 122 of the passageway 102 within the inspection window 106, and a second light source 114 positioned to emit or output a second excitation light 116 onto a second portion 124 of the passageway 102 within the inspection window 106 that is distinct from the first portion 122. The light engine 108 is shown to further include a third light source 118 and a fourth light source 120 positioned to output third and fourth excitation lights, respectively, onto a third portion 126 and a fourth portion 128 of the passage 102 within the inspection window 106.

[0040] In certain embodiments, portions 122, 124, 126, and 128 are defined by a width of the excitation light after being focused by objective lens 186 and entering passage 102. For example, in certain embodiments, portion 122 has a width defined by the width of excitation light 112 entering passage 102. In certain embodiments, the widths of portions 122, 124, 126, and 128 are less than 2.0 μm, 1.5 μm, 1.0 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, or 0.2 μm. In certain embodiments, the widths of portions 122, 124, 126, and 128 are in the range of about 2.0 μm to about 0.2 μm, 1.0 μm to about 0.2 μm, 0.9 μm to about 0.2 μm, 0.8 μm to about 0.2 μm, 0.7 μm to about 0.2 μm, or about 0.6 μm to about 0.2 μm. In certain embodiments, the widths of portions 122, 124, 126, and 128 are in the range of 1 / e of the maximum excitation light intensity. 2 In one embodiment, the widths of portions 122, 124, 126, and 128 are defined as 1 / e of the maximum excitation light intensity.

[0041] If the widths of the portions 122, 124, 126, and 128 are too wide, the signal-to-noise ratio will be too low, for example, for single molecule / particle detection and analysis. In this regard, excitation linewidths that are too wide and too closely spaced (e.g., when there is substantial overlap of two excitation lines) will generate crosstalk between portions of the passage 102. In addition, particularly when the amount of light emitted by a single particle / molecule may be relatively low, an excitation linewidth that is too wide will illuminate a larger portion of the passage 102, creating a larger amount of background light that reduces the signal-to-noise ratio.

[0042] As shown, the light sources 110 and 114 are optically coupled to the optical fibers 164 and 168 such that the excitation light 112 and 116 output therefrom is received and transmitted by the optical fibers 164 and 168. In the illustrated embodiment, the first light source 110 is optically coupled to a proximal end 166 of the first excitation optical fiber 164, and the second light source 114 is optically coupled to a proximal end 170 of the second excitation optical fiber 168.

[0043] Although optically coupled light sources are shown, it will be understood that in certain embodiments, light sources 110, 114, 118, and 120 of light engine 108 are free-space light sources that are not optically coupled to an excitation optical fiber. In this regard, and in certain embodiments, the free-space light sources do not have an optical fiber disposed between the free-space light sources and inspection window 106 positioned to receive the excitation light and output the excitation light into free space. In this regard, the spacing between the excitation light of adjacent light sources is at least partially defined by additional optical components of system 100 that direct and / or shape the excitation light emitted from the light sources. Thus, in certain embodiments, excitation optical fibers, such as excitation optical fibers 164 and 168 of excitation optical fiber bundle 172, are optional. Also, in some embodiments, excitation optical fiber bundle 172 is not present.

[0044] The excitation light output by the light sources 110, 114, 118, and 120 of the light engine 108 can be of any wavelength. In an embodiment, the excitation light of one light source is the same as the excitation light output by another light source of the light engine 108. In an embodiment, the excitation light of one light source is different from the excitation light output by another light source of the light engine 108. In an embodiment, the first excitation light 112 has a wavelength in a first wavelength range and the second excitation light 116 has a wavelength in a second wavelength range that is distinct from the first wavelength range. In an embodiment, the first excitation light 112 has a wavelength in a first wavelength range and the second excitation light 116 has a wavelength in a second wavelength range that is common to the first wavelength range.

[0045] The first excitation light 112 and the second excitation light 116 can be any excitation light suitable for optically exciting a dye or other detectable agent in or on a particle, or associated with a molecule. In an embodiment, the first excitation light 112 and / or the second excitation light 116 comprises coherent light, such as from a laser. In an embodiment, the first light source 110 and the second light source 114 are each independently selected from the group consisting of a solid-state laser, a diode-pumped laser, a light-emitting diode (LED), a lamp, an arc discharge, and natural light.

[0046] In some embodiments, the first excitation light 112 is in the wavelength range of about 350 nm to about 360 nm, about 400 nm to about 410 nm, about 480 nm to about 490 nm, about 530 nm to about 540 nm, about 555 nm to about 565 nm, or about 630 nm to about 690 nm. In some embodiments, the second excitation light 116 is in the wavelength range of about 350 nm to about 360 nm, about 400 nm to about 410 nm, about 480 nm to about 490 nm, about 530 nm to about 540 nm, about 555 nm to about 565 nm, or about 630 nm to about 690 nm.

[0047] As shown, the distal end of the first excitation optical fiber 164 and the distal end of the second excitation optical fiber 168 are disposed within the excitation fiber bundle head 172. As shown, the distal ends of the excitation optical fibers 164 and 168 are spaced apart by a spacing 176. In one embodiment, this spacing 176 at least partially defines the spacing of the excitation lights 112 and 116 output by the excitation fiber bundle head 172 and the light engine 108. Accordingly, in one embodiment, the spacing 176 between the distal end of the first excitation optical fiber 164 and the distal end of the second excitation optical fiber 168 corresponds to the spacing between the first portion 122 and the second portion 124. In this regard, excitation light may be output by the differently positioned light sources 110, 112, 118, and 120 of the light engine 108, may be grouped together by the excitation fiber bundle head 172, and may be output onto positionally distinct portions of the inspection window 106 of the passageway 102 according to the spacing 176 of the distal ends of the excitation optical fibers. Such a configuration of the excitation fiber bundle head 172 may be further manipulated by the excitation optics. Although not shown, see, for example, FIG. 3A. In certain embodiments, the excitation optical fibers 164 and 168 are disposed adjacent to one another in the fiber bundle head, for example, within one fiber diameter (e.g., edge-to-edge distance) of one another.

[0048] In certain embodiments, a fiber bundle, such as excitation fiber bundle 172, refers to optical fibers that are coupled or otherwise proximate at their ends. As discussed further herein, by having the ends of the optical fibers proximate to one another, such as within a fiber diameter width, light output from differently positioned light sources coupled to the optical fibers may be transmitted through the optical fibers and may emerge from the optical fibers in an orientation that is shaped or otherwise defined in part by the orientation of the optical fibers within the fiber optic bundle.

[0049] As discussed above, while an optically coupled light source is illustrated, it will be appreciated that light engine 108 may include a free space light source, as discussed further herein with respect to Figure 6. It will further be appreciated that the portions of interrogation window 106 illuminated by light engine 108 and the spacing therebetween may be manipulated by optical elements positioned between the free space light source and interrogation window 106.

[0050] As shown, the system 100 includes a dichroic mirror 160 positioned to reflect at least a portion of the excitation light 112 and 116 toward the inspection window 106 of the passageway 102. In the illustrated embodiment, the system 100 further includes an objective lens 186, such as an air objective lens 186, positioned to collect the excitation light reflected from the dichroic mirror 160 and configured to focus the excitation light 112 and 116 onto the passageway 102. In this regard, the excitation light from the light source is output onto a spatially separated portion of the passageway 102 within the inspection window 106. As noted above, in certain embodiments, the inspection window 106 is defined, at least in part, by the field of view of the objective lens 186.

[0051] Although a dichroic mirror 160 is illustrated, it will be understood that other optical components may be used to selectively or partially transmit and reflect light. In some embodiments, the dichroic mirror 160 is replaced with a transmitting mirror, such as a 20% reflective / 80% transmitting mirror, or other structure configured to selectively or partially transmit and reflect light.

[0052] As noted above, particles or molecules (such as particles containing one or more detectable agents and / or molecules associated with one or more detectable agents) flowing through passageway 102 may be excited by excitation lights 112 and 116. Such excited particles / molecules may emit emitted lights 146 and 152, which are emitted or otherwise emitted out of interrogation window 106 through dichroic mirror 160. As shown, emitted lights 146 and 152 have the same or similar relative spacing as excitation lights 112 and 116 incident on interrogation window 106.

[0053] The emitted light 146 and 152 are shown entering the light emitting fiber bundle 130 of the system 100. In the illustrated embodiment, the light emitting fiber bundle 130 is shown to include four light emitting optical fibers closely coupled in space. Like the excitation fiber bundle 172, the light emitting fiber bundle 130 brings the ends of the optical fibers, here the light emitting optical fibers 134 and 138, into close proximity and following a particular orientation or arrangement. As discussed further herein, the particular arrangement of the light emitting optical fibers within the fiber bundle head 132 is suitable for positioning the light emitting optical fibers 134 and 138 to receive the emitted light 146 and 152.

[0054] 1A, it will be understood that other numbers and configurations of light emitting optical fibers are possible. In some embodiments, the light emitting optical fiber bundle 130 includes at least three light emitting optical fibers, at least four light emitting optical fibers, at least five light emitting optical fibers, at least six light emitting optical fibers, at least seven light emitting optical fibers, or more light emitting optical fibers. In some embodiments, the light emitting optical fiber bundle 130 includes a first light emitting optical fiber 134 and a second light emitting optical fiber 138, the proximal ends 136 of the first light emitting optical fiber 134 and the second light emitting optical fiber 138 being disposed within the light emitting optical fiber bundle head 132, the proximal end 136 of the first light emitting optical fiber 134 being positioned to receive the first emitted light 146 emitted from the first portion 122, and the proximal end 140 of the second light emitting optical fiber 138 being positioned to receive the second emitted light 152 emitted from the second portion 124.

[0055] In one embodiment, the proximal ends 136 and 140 of each light emitting optical fiber 134 and 138 of the light emitting fiber bundle 130 are disposed within the light emitting fiber bundle head 132. In one embodiment, the proximal ends 136 and 140 of each light emitting optical fiber 134 and 138 of the light emitting fiber bundle 130 are positioned to receive emitted light 146 and 152 emitted from the distinct portions 122 and 124 of the inspection window 106. In one embodiment, the proximal ends 136 and 140 of the light emitting optical fibers 134 and 138 are disposed adjacent to one another, such as within one fiber diameter of each other (e.g., the distance between the edge of one fiber and the nearest edge of an adjacent fiber is within one fiber diameter).

[0056] In one embodiment, the light emitting optical fibers of the light emitting fiber bundle head 132 are positioned to receive, such as by individually or separately receiving, emitted light corresponding to distinct excitation regions or portions of the inspection window 106. As noted above, in one embodiment, the portions of the inspection window 106, such as portions 122 and 124, are defined by the width of the excitation light (such as the width of excitation light 112 and 116) as the excitation light is incident on the inspection window.

[0057] By positioning the proximal ends 136 and 140 of the light emitting optical fibers 134 and 138 proximately within the light emitting fiber bundle head 132, the light emitting optical fibers 134 and 138 are positioned to receive emitted light from different portions of the passageway 102, such as portions of the passageway 102 that are excited by different light sources of the light engine 108. In one embodiment, the spacing between the proximal ends 136 and 140 of the light emitting optical fibers 134 and 138 is based on the spacing of the portions 122 and 124 of the passageway 102, such as based on the spacing of the excitation lights 112 and 116 incident on the inspection window 106.

[0058] In this regard, attention is directed to Figure 1B, which is a schematic diagram of the light emitting fiber bundle head 132 of the system 100. Figure 1B illustrates several configurations for the light emitting optical fibers of the light emitting fiber bundle head 132. In a preferred embodiment, the light emitting optical fibers 134 are arranged in a linear configuration within the light emitting fiber bundle head 132.

[0059] In one embodiment, the spacing 174 and / or arrangement of the proximal ends 136 and 140 of the light emitting optical fibers 134 and 138 correspond to the spacing and / or arrangement of the portion of the inspection window 106 excited by the light source of the light engine 108. As shown, the proximal ends 136 and 140 of the light emitting optical fibers 134 and 138 are arranged in a linear configuration. In this regard, the light emitting optical fibers 134 and 138 are arranged within the light emitting fiber optic bundle head 132 such that the light engine 108 receives emitted light 146 and 152 from the passageway 102, e.g., in a linear configuration, which is configured to emit or output excitation light into the inspection window 106.

[0060] In some embodiments, the spacing 174 is in the range of about 1 μm to about 1,000 μm, in the range of about 100 μm to about 900 μm, in the range of about 1 μm to about 100 μm, in the range of about 10 μm to about 500 μm, or in the range of about 50 μm to about 800 μm. In other embodiments, the distance between the edge of the light emitting optical fiber and the nearest edge of another light emitting optical fiber at the proximal end of the fiber bundle head is approximately zero (i.e., touching) or within a radius of the fibers.

[0061] In one embodiment, the spacing 174 is the distance between the center of one light emitting optical fiber and the center of a different light emitting optical fiber, in one embodiment, the spacing 174 is the distance between the edge of the light emitting optical fiber and the nearest edge of another light emitting optical fiber.

[0062] In an embodiment, the spacing 174 between the proximal end 136 of the first light-emitting optical fiber 134 and the proximal end 140 of the second light-emitting optical fiber 138 corresponds to the spacing between the first portion 122 and the second portion 124 of the inspection window 106. In this embodiment, the spacing is a center-to-center distance. In an embodiment, such correspondence is a direct correspondence, where the spacing between the proximal ends 136 and 140 of the light-emitting optical fibers 134 and 138 and the spacing between the first portion 122 and the second portion 124 match directly or closely after taking into account the magnification of the optical system. In an embodiment, the correspondence is adjusted and / or modified according to the optical system of the system 100, such as the objective lens 186 and any other lenses, mirrors, etc., disposed between the inspection window 106 and the light-emitting optical fiber bundle head 132.

[0063] In an embodiment, the spacing 176 between the proximal end 166 of the first excitation optical fiber 164 and the proximal end 170 of the second excitation optical fiber 168 corresponds to the spacing between the first portion 122 and the second portion 124 of the inspection window 106. In an embodiment, such correspondence is a direct correspondence, where the spacing between the proximal ends 166 and 170 of the excitation optical fibers 164 and 168 and the spacing between the first portion 122 and the second portion 124 match directly or closely after taking into account the magnification of the optics. In this regard, the spacing between the first light source 110 outputting from the distal end 166 and the second light source 114 outputting from the distal end 170 corresponds to the spacing between the first portion 122 and the second portion 124. In an embodiment, the correspondence is adjusted and / or modified according to the optics of the system 100, such as the objective lens 186 and any other lenses, mirrors, etc., disposed between the inspection window 106 and the excitation fiber bundle head 172.

[0064] In one embodiment, the spacing 176 between the proximal end 166 of the first excitation optical fiber 164 and the proximal end 170 of the second excitation optical fiber 168 corresponds to the spacing 174 between the proximal end 136 of the first emission optical fiber 134 and the proximal end 140 of the second emission optical fiber 138. As noted above, such correspondence can be a direct correspondence or a correspondence modified by any optical components that manipulate or direct the emitted light.

[0065] In some embodiments, the spacing 176 is in the range of about 1 μm to about 1,000 μm, in the range of about 1 μm to about 100 μm, in the range of about 250 μm to about 750 μm, in the range of about 1 μm to about 50 μm, or in the range of about 10 μm to about 500 μm. In other embodiments, the distance between the edge of an optical fiber and the nearest edge of another optical fiber in the fiber bundle head is near zero (i.e., touching) or is within a radius of the fibers.

[0066] As described above, system 100 includes detector system 142 positioned to receive emitted light from light emitting fiber bundle 130. As shown, light emitting optical fibers 134 and 138 fan out from light emitting fiber bundle head 132 at their proximal ends 136 and 140, respectively, and terminate at their distal ends 148 and 154 adjacent to and optically coupled to detector modules 144 and 150 of detector system 142. As used herein, a "detector module" refers to a collection of detection structures and / or components configured to generate a signal or set of signals based on light received by the detector module, such as received by one or more detection structures and / or components. Detector systems of the present disclosure, such as detector system 142, may include one or more detector modules and / or one or more individual detectors, such as one or more individual light detectors.

[0067] In certain embodiments, one or more of the detector modules 144 and 150 includes a single photodetector optically coupled and positioned to receive the emitted light 146 and 152. In another embodiment, one or more of the detector modules 144 and 150 includes a plurality of individual photodetectors, as discussed further herein with respect to FIGURE 2. In this regard, each of the detector modules 144 and 150 may be configured to receive the emitted light 146 and 152 and generate a plurality of signals based on the emitted light 146 and 152 (e.g., based on a particular wavelength range within the emitted light 146 and 152).

[0068] In an embodiment, the distal end 148 and 154 of each light emitting optical fiber 134 and 138 is positioned to emit light onto at least one respective detector module 144 and 150. In an embodiment, the detector system 142 is positioned to receive scattered radiation, luminescence radiation, fluorescent radiation, or a combination thereof from the inspection window 106. In an embodiment, the scattered radiation is selected from backscattered light, side scattered light, or forward scattered light.

[0069] Although photodetectors such as the photodetectors in detector modules 144 and 150 are discussed, it will be understood that other types of photodetection structures and components are possible and within the scope of this disclosure. In an embodiment, the photodetectors in detector modules 144 and 150 are selected from the group consisting of a camera, an electron multiplier tube, a charge-coupled device (CCD) image sensor, a photomultiplier tube (PMT), a microchannel plate PMT (MCP), a hybrid PMT detector, an avalanche photodiode (APD), a single-photon avalanche diode (SPAD), a single-photon counting module (SPCM), a silicon photomultiplier (SiPM), and a complementary metal oxide semiconductor (CMOS) image sensor.

[0070] In the illustrated embodiment, a terminal end of each light emitting optical fiber is optically coupled to a detector module for receiving the emitted light. In this regard, the detector system 142 is shown to include a first detector module 144 positioned to receive a first emitted light 146 emitted from a distal end 148 of the first light emitting optical fiber 134, and a second detector module 150 positioned to receive a second emitted light 152 emitted from a distal end 154 of the second light emitting optical fiber 138.

[0071] System 100 is shown to further include a controller 156 operably coupled to light engine 108 and detector system 142. Such controller 156 includes logic configured to govern the operation of these components. Although one controller 156 is shown directly coupled to these components, it will be understood that multiple controllers, such as those wirelessly coupled and / or in a distributed system, are possible and within the scope of the present disclosure.

[0072] In an embodiment, the controller 156 includes logic for implementing some or all aspects of the methods described further herein. In an embodiment, the controller 156 includes logic for outputting excitation light with the light engine 108 into the interrogation window 106 and generating a signal with the detector system 142 based on emission light emanating from the interrogation window 106 and received by the detector system 142. In an embodiment, the controller 156 includes logic that, when executed by the controller 156, causes the system 100 to perform operations including outputting a first excitation light 112 with the first light source 110, outputting a second excitation light 116 with the second light source 114, generating a first emission signal with the first detector module 144 based on the first excitation light 112 received from the first light emission optical fiber 134, and generating a second emission signal with the second detector module 150 based on the second excitation light 116 received from the second light emission optical fiber 138.

[0073] In an embodiment, the controller 156 further includes logic that, when executed by the controller 156, causes the system 100 to perform operations including flowing a suspension of particles and / or a solution of molecules through the passageway 102, such as a suspension fluid in fluid communication with the passageway 102. As used herein, a "particle" refers to a localized object or entity, such as within a surrounding medium. In an embodiment, the particle defines a phase discontinuity with respect to its surroundings, such as when a solid particle is surrounded and suspended in a liquid or gas phase. As discussed further herein, in certain embodiments, the particle is a biological particle, such as a biological nanoparticle, from a subject, derived from a subject, from an organism, derived from an organism, from an environmental sample, etc.

[0074] In some embodiments, flowing the suspension through the passageway 102 includes flowing the suspension through the passageway 102 on a particle-by-particle or molecule-by-molecule basis. Such particle-by-particle or molecule-by-molecule flow is suitable for individually analyzing the particles and / or molecules flowing through the passageway 102. In some embodiments, the interrogation window 106 defines a constriction relative to other portions of the passageway 102, which constriction narrows the lumen 104. In some embodiments, the particle-by-particle and / or molecule-by-molecule flow occurs within the constriction of the lumen 104.

[0075] In an embodiment, the controller 156 includes logic that, when executed by the controller 156, causes the system 100 to perform operations including ranking the particles and / or molecules in the passageway 102. Such ranking may be based on the presence or absence of emitted light associated with the particles and / or molecules, such as the emitted light detected by the detector system 142. In an embodiment, the ranking is based on the intensity of the emitted light and / or the wavelength of the emitted light, as detected by the detector system 142. In an embodiment, the ranking corresponds to a measured emission spectrum of the particles and / or molecules based on one or more of the first emitted light 146 and the second emitted light 152. In an embodiment, the ranking corresponds to a measured excitation spectrum of the particles and / or molecules based on one or more of the first excitation light 112 and the second excitation light 116.

[0076] In certain embodiments, the ranking may include grouping, categorizing, and / or decoding of particles, beads, nanoparticles, or fluorescent probes, such as capture agents and / or detectable agents associated therewith. In certain embodiments, such as digital affinity assay embodiments discussed further herein, the ranking is based at least in part on decoding detectable agent and / or capture agent emission signals associated with the analyte, such as determining the identity of the analyte associated with the detectable agent and / or capture agent, whose respective signals are detected by the systems and methods of the present disclosure.

[0077] As used herein, the term "assignment" refers to the assignment of a quantitative characteristic, qualitative characteristic, or significance of the classification of the particle and / or molecule to the particle and / or molecule to which it is assigned. In one embodiment, a size value may be assigned to the particle. In certain embodiments, the assignment is based on light emitted from the particle or molecule, and the assignment is based on the presence, absence, and / or intensity of such emitted light. As used herein, the term "size value" refers to a relative size value or an actual size value. The size value provides a true or relative measurement of linear distance. In certain embodiments, the assignment is performed by a computer and software representing an assignment algorithm.

[0078] As used herein, the term "ranking" refers to assessing the quantitative, qualitative, or importance of particles and / or molecules by classification. In one embodiment, particles and / or molecules may be ranked as either null (e.g., if the nanoparticles and / or molecules have a luminescence intensity below a detectable threshold) or non-zero (e.g., if the particles and / or molecules are detected). In some embodiments, the ranking is binary. For example, each particle and / or molecule with a detected light intensity above a threshold value is assigned a value of 1, while each measured sample that does not have a detected light intensity above a threshold value is assigned a value of 0, thus forming a binary ranking. In other embodiments, particles and / or molecules may be ranked according to additional categories that correlate, for example, with the identity of the particles and / or molecules, the presence of a detectable property, the presence of a distinguishing feature, etc. The ranking may be assigned any number that corresponds to one of several predefined quantitative or qualitative categories. In other embodiments, the ranking is non-binary, for example, a value is assigned based on the amount of emitted light intensity measured from the particles and / or molecules. In certain embodiments, the ranking is performed by a computer and software representing a ranking algorithm.

[0079] As used herein, a "detectable property" refers to an observable property associated with a particle and / or molecule, such as an optical activity, electrical activity, biological activity, or magnetic property that is associated with or intrinsic to the particle and / or molecule. In certain embodiments, a "detectable property" includes the association of a particle and / or molecule with a detectable agent or biomarker.

[0080] As used herein, "amplification" refers to the use of a molecule, structure, or reagent configured to generate an amplicon or copy of a target analyte, or an amplicon or copy of a molecule that is correlated with the presence of the target analyte. In some embodiments, the molecule that is correlated with the presence of the target analyte is selected from a molecule that requires the presence of the target analyte to be formed or expressed. In some embodiments, the molecule that is correlated with the presence of the target analyte is selected from an amplification product of the target analyte or a portion thereof, a fragment of the target analyte, or a molecule or complex stabilized by the target analyte. In this regard, in certain embodiments, the systems and methods of the present disclosure do not include an amplification step.

[0081] Examples of photoactive properties include, for example, changes in light intensity (optical reflection, scattering, deflection, transmission, absorption, or emission) commonly induced by bioparticle morphology (particle size, internal subcellular structure), fluorescence, luminescence, immunofluorescence, etc. Detection of a photoactive property may report, for example, the size, mass, surface area, volume, protein content, membrane area, lipid content, enzyme content, metabolite content, carbohydrate content, peptide content, nucleic acid content, protein identity, or nucleic acid identity on, within, or associated with a nanoparticle.

[0082] In some embodiments, the ranking corresponds to a measured size value of the particle. In some embodiments, the measured size value is a relative size value. In some embodiments, the measured size value is determined by a difference in detected emitted light intensity. In some embodiments, the measured size value is an actual size value.

[0083] In some embodiments, the system 100 further comprises a flow director, such as one or more valves, configured to direct the flow of the particles and / or molecules in the passage 102. In some embodiments, the flow director is operably coupled to the controller 156, which includes logic that, when executed by the controller 156, causes the system 100 to perform operations including directing the flow of the particles and / or molecules based on the presence or absence of emitted light received through the interrogation window 106 and associated with the particles and / or molecules. In some embodiments, the flow director is operably coupled to the controller 156, which includes logic that, when executed by the controller 156, causes the system 100 to perform operations including directing the flow of the particles and / or molecules based on the ranking. In some embodiments, directing the flow of the particles and / or molecules includes directing the particles and / or molecules into one of two or more sorting passages. In an embodiment, the flow director is operably coupled to a controller 156 that includes logic that, when executed by the controller 156, causes the system 100 to perform operations including quantifying the number of particles and / or molecules associated with the emitted light from the interrogation and determining a concentration of the particles and / or molecules associated with the emitted light from the interrogation window 106. In an embodiment, the concentration is further based on the measured flow rate, as discussed further herein with respect to Figures 11A and 11B.

[0084] In some embodiments, the flow director or flow directing mechanism for directing the flow of particles and / or molecules comprises electrodes, magnetic elements, acoustic elements, electrical actuation elements, and optical actuation elements, electric fields, or magnetic fields. In some embodiments, the mechanism for directing the flow of particles and / or molecules comprises one or more electrically actuated valves or pistons, which control the flow of liquid in at least a first directional flow path that intersects with the first input passage and the two outlet passages at a first junction. In one embodiment, the solenoid piston is a subcomponent of an electrically actuated solenoid valve. In another embodiment, the solenoid piston is embedded in the device by molding. In yet another embodiment, the embedded solenoid piston can be replaced by a solenoid valve in fluid communication via tubing.

[0085] In a particular embodiment, the devices provided herein may include one or more electrodes for tracking and / or manipulating the trajectory or flow of particles and / or molecules, particles, molecules, or fluid samples. In certain embodiments, the electrodes may enhance the separation of nanoparticles and / or molecules based on phenomena such as dielectrophoresis or electroosmotic flow or electrophoresis. In embodiments where the particles and / or molecules have a hydrodynamic diameter of less than 100 nm, sheath flow focusing or acoustic focusing may not be sufficient to adequately manipulate the trajectory of the particles for the methods and devices disclosed herein, such as for directing the trajectory of the particles in the passage 102. See, for example, Optics Express Vol. 15, Issue 10, pp. 6167-6176 (2007), which is incorporated herein by reference. Thus, in some embodiments, the mechanism for focusing particles excludes sheath flow focusing, acoustic flow focusing, or a combination thereof. In some embodiments, the particles are directed, provided that the directing does not involve the use of acoustic focusing, sheath flow focusing, or a combination thereof.

[0086] As discussed further herein, in certain embodiments, the systems and methods of the present disclosure do not include structures or reagents configured to generate, for example, an amplicon or copy of a target analyte, or an amplicon or copy of a molecule that is correlated with the presence of the target analyte. In certain embodiments, the systems and methods of the present disclosure do not include structures or reagents configured to generate an amplicon or copy of a molecule that is correlated with the presence of the target analyte, and the molecule that is correlated with the presence of the target analyte is selected from an amplification product of the target analyte or a portion thereof, a fragment of the target analyte, a molecule or complex stabilized by the target analyte, or a molecule that requires the presence of the target analyte to be formed or expressed. In this regard, in certain embodiments, the systems and methods of the present disclosure are not configured to or do not include structures, reagents, and / or steps for amplification.

[0087] Self-corrected flow-based particle analysis As described above, in one embodiment, the system 100 includes a passageway 102 configured to flow particles through a lumen 104 of the passageway 102, the passageway 102 defining an inspection window 106 configured to allow light to pass in and out of the lumen 104, a first light source 110 positioned to output a first excitation light 112 onto a first portion 122 of the passageway 102 within the inspection window 106, and a light engine 108 positioned to output a first excitation light 112 onto a first portion 122 of the passageway 102 within the inspection window 106 separate from the first portion 122. The optical engine 108 includes a second light source 114 positioned to emit or output a second excitation light 116 onto the first portion 124 of the passageway 102, and a detector system 142 including a first detector module 144 positioned to receive the first emitted light 146 from the first portion 122 of the passageway 102 and a second detector module 150 positioned to receive the second emitted light 152 from the second portion 124. Such an embodiment of the system 100 of the present disclosure is suitable for use in self-corrected, single particle and / or single molecule flow analysis. As discussed further herein, measurements of fluorescence emitted from single particles and / or molecules in a flow stream are highly affected by the flow and laser beam profiles. Thus, accurate quantification of fluorescent particles and / or molecules requires deconvolution of signals from the flow profile and / or laser beam profile.

[0088] To overcome these challenges, the present disclosure provides systems and methods for analyzing single molecules and particles, such as vesicles, viruses, lipoproteins, and macromolecular complexes in a flow stream. Such systems and methods are suitable for accurately 1) colocalizing biomarkers expressed on the same particle and / or molecule flowing through multiple interrogation windows or portions of a single interrogation window 106, 2) identifying and enumerating single particles and / or molecules, 3) obtaining the flow rate sampled by each individual particle and / or molecule, and 4) thus determining the concentration of the analyzed particle and / or molecule.

[0089] Briefly, the spatially separated portions of the interrogation window 106 are arranged in a known spatial pattern, and a property (e.g., fluorescence emission) of a particle and / or molecule is measured twice in two different portions of the interrogation window 106 (see, e.g., FIG. 8). Under laminar flow, the transit time of a particular particle flowing through two adjacent or two different portions of the interrogation window 106 is proportional to the distance between these two portions. Also, due to the nature of laminar flow, the position of a particular particle and / or molecule in the cross-section of the passage 102 remains roughly the same. This is especially so when the distance between two such portions is small, the transit time is short, and, as a result, the diffusion of the particle in the passage is correspondingly small. This property therefore suggests that the particle and / or molecule interacts with the different excitation light of the different light sources at very similar positions in the cross-section of the passage. Given these characteristics, it is possible to identify a single analyte (e.g., a vesicle, or a virion, or a lipid nanoparticle, or a single molecule stained with a fluorescent dye) and use the extracted migration time or extracted location or relative location of the analyte in the cross section of the passage 102 to further co-localize fluorescent signals associated with other biological markers on the analyte, e.g., fluorescent signals from different dye-tagged antibodies bound to each corresponding biological marker, and / or fluorescent signals from different nucleic acid stains and / or other specific fluorescent stains of the analyte or biological nanoparticle (see FIG. 8).

[0090] Thus, in one embodiment, a system 100 for use in self-calibrated, single nanoparticle or single molecule flow analysis includes logic for performing the self-calibrated, single molecule / particle flow analysis method of the present disclosure. In one embodiment, the system 100 includes a controller 156 operably coupled to the light engine 108 and the detector system 142, and includes logic that, when executed by the controller 156, causes the system 100 to perform operations including measuring properties of the particle / molecule at various portions of the interrogation window 106. In one embodiment, the system 100 includes a controller 156 operably coupled to the first light source 110, the second light source 114, the first detector module 144, and the second detector module 150, and includes logic that, when executed by the controller 156, causes the system 100 to perform operations including: outputting a first excitation light 112 at the first light source 110; outputting a second excitation light 116 at the second light source 114; generating a first emission signal at the first detector module 144 based on the first emission light 146 received from the first portion 122; generating a second emission signal at the second detector module 150 based on the second emission light 152 received from the second portion 124; and determining a velocity of the particle in the passage 102 based on a time difference between generation of the first emission signal and generation of the second emission signal and the distance between the first portion 122 and the second portion 124. In one embodiment, the velocity of the particles is used to determine the volumetric flow rate through the lumen 104 .

[0091] In conventional fluid mechanics, the average linear velocity

[0092]

number

[0093] teeth,

[0094]

number

[0095] where V is the volumetric flow rate and A is the cross-sectional area. It is well known that when the flow profile is parabolic, the average linear velocity is half the velocity at the centerline (v max ),

[0096]

number

[0097] Conventionally, the volumetric flow rate is first measured and the average linear velocity is calculated accordingly.

[0098] In our system, the volumetric flow rates are so slow (e.g., about nl / min) that in certain embodiments it is not possible to conveniently measure the volumetric flow rate directly. Instead, in certain embodiments, the flow profile in the passageway is sampled by measuring the linear velocities of many individual particles (e.g., 100, 500, 1,000, 5,000, 10,000 or more particles) and the average observed value is calculated.

[0099]

number

[0100] In one embodiment, when sampling the flow profile, the observed linear velocity is affected by the flow profile, so that

[0101]

number

[0102] is actually different from the mean velocity defined in traditional fluid mechanics (i.e.,

[0103]

number

[0104] In laminar flow, the volume (ΔQ) passing through each lamina cross section in unit time is ΔQ=u(r)·2πr·Δr. Therefore, the number of particles with the same velocity (i.e., u(r)) passing through a cross section in a particular unit time is,

[0105]

number

[0106] where C is the concentration of particles. The average observed linear velocity is then:

[0107]

number

[0108] where r is the radial position in the cross section. For parabolic flow:

[0109]

number

[0110] In this regard, Equation 2 can be rearranged to:

[0111]

number

[0112]

number

[0113] The relationship between V and V is as shown in FIG.

[0114]

number

[0115] It is. Thus, in one embodiment, the volumetric flow rate is determined by the following formula:

[0116]

number

[0117] During the ceremony,

[0118]

number

[0119] is the average observed linear velocity of many individual particles through the passage, R is the radius of the passage. The above analysis assumes a passage having a cylindrical shape, but can be easily adjusted to passages having a rectangular or square shape or any other shape.

[0120] In one embodiment, the controller 156 further includes logic that, when executed by the controller 156, causes the system 100 to perform operations including correlating the first emitted signal and the second emitted signal based on a characteristic or relationship of an excitation or emitted signal shared by the first emitted signal and the second emitted signal.

[0121] Air objective lens In an embodiment, the system 100 further includes a collection system 184 positioned to collect radiation, such as the first radiation light 146 and the second radiation light 152, from the passageway 102 and direct the collected radiation light onto the detector system 142, the collection system 184 comprising an air objective lens 186 having a numerical aperture in the range of greater than 0.91 and less than 0.99. In an embodiment, the objective lens 186, such as the air objective lens 186, has a numerical aperture of about 0.95.

[0122] As used herein, an "air objective" refers to an optical objective or lens in which the space between the objective and its focal plane or focus is occupied, at least in part, by a gas, such as air, and not by an immersion liquid, such as oil or water. In this regard, an air objective is contrasted with an oil or water immersion lens, in which the lens is immersed in oil or water disposed between the lens and the sample, typically between the lens and a cover glass or sample holder.

[0123] As used herein, a "high NA (numerical aperture) air objective" refers to an air objective having an NA of 0.91-0.99, preferably 0.92-0.98, more preferably 0.93-0.97, and even more preferably 0.94-0.96. In certain embodiments, the air objective has an NA of about 0.95. As discussed further herein, such high NA air objectives are suitable for performing single molecule and / or single particle analysis, such as in determining the presence, absence, or concentration of particles / molecules passing through the devices and systems of the present disclosure. As discussed elsewhere herein, high NA air objectives offer numerous advantages over conventional objectives, such as oil or water immersion objectives, such as high light collection efficiency, the ability to scan accurately and efficiently, among many others.

[0124] Air objective lenses are generally easier to scan and more stable than, for example, oil immersion objective lenses. Furthermore, in certain embodiments, the objective lens 186 is used not for its high image quality (e.g., high resolution) but rather for its high light collection efficiency. In this regard, too, air objective lenses are superior to oil immersion objective lenses. Thus, air objective lenses 186 having lower numerical apertures, such as in the range of greater than 0.91 and less than 0.99, are suitable for detection of single particles and / or molecules in the flow channel 102.

[0125] radial multiplexing In one embodiment, a detector module of a system of the present disclosure includes two or more optical detectors each positioned to receive emitted light from a distal end of an emitting optical fiber. In this regard, attention is directed to FIG. 2, which shows a simplified diagram of a detector module 242 of a system according to an embodiment of the present disclosure. In one embodiment, the illustrated detector module 242 is an example of detector module 144 or 150 of detector system 142 illustrated in FIG. 1A.

[0126] In the illustrated embodiment, the detector module 242 is shown to include a number of photodetectors 244, 250A, 250B, and 250C positioned to receive the emission light 246A, shown here as fluorescent emission light 246A, or a portion thereof, from the distal end 248 of the emission optical fiber 234. As shown, the detector module 242 includes a number of dichroic mirrors 260 positioned to receive the emission light 246A emitted from the distal end 248 of the emission optical fiber 234. Such dichroic mirrors 260 are configured to reflect a portion (e.g., one wavelength range) of the emission light 246A and allow a different portion (e.g., different wavelength range) of the emission light to pass through the dichroic mirror 260. In the illustrated embodiment, each dichroic mirror 260 is positioned to reflect a portion of the emitted light 246A toward a photodetector 244, 250A, 250B, 250C configured to generate a signal based on this reflected or transmitted portion of the emitted light 246A.

[0127] In this regard, the detector module 242 is shown to include a dichroic mirror 260 disposed between the distal end 248 of the first light-emitting optical fiber 234 and the first photodetector 244 and positioned to reflect a portion 246B of the first emitted light 246A onto the first photodetector 244. In an embodiment, the detector of a system according to an embodiment of the present disclosure further includes a second detector module optically coupled to the second light-emitting optical fiber, such as a second detector module including a second photodetector, as illustrated in FIG. 1A. In the illustrated embodiment of FIG. 2, the detector module 242 is shown to include a third photodetector 250A, a fourth photodetector 250B, and a fifth photodetector 250C, respectively. In one embodiment, the first photodetector 244 is configured to generate a first emission signal based on a first emission wavelength range of the first emitted light 246B, which includes the emitted light 246B, and the third photodetector 250A, the fourth photodetector 250B, and the fifth photodetector 250C are configured to generate third, fourth, and fifth emission signals based on third, fourth, and fifth emission wavelength ranges of the emitted lights 246C, 246D, and 246E, which are different or substantially different from the first emission wavelength range of the emitted light 246B.

[0128] Although the detector module 242 is shown to include four photodetectors 244, 250A, 250B, and 250C, it will be understood that any number of photodetectors is possible, in some embodiments, the detector module includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more photodetectors.

[0129] In the illustrated embodiment, the detector module 242 further includes a filter 262, such as a bandpass filter 262, configured to filter a portion of the reflected emitted light 246B-246E. In this regard, the photodetectors 244 and 250A-250C are configured and positioned to generate a signal based on the filtered portions 246B-246E of the emitted light 246A. As discussed further herein, in some embodiments, the light engine excites particles and / or molecules in the passage with different wavelengths of light. Also, as discussed further herein, in some embodiments, the particles and / or molecules themselves may be impregnated with or associated with one or more detectable agents configured to emit fluorescence having different wavelength ranges and configured to be excited by the different wavelengths of light. Thus, the configuration of the detector module 242 illustrated in FIG. 2 is suitable for generating a signal based on emitted light having wavelengths within one or more wavelength ranges using an array of photodetectors and corresponding filters. In this regard, the illustrated detector module 242 is suitable for performing emission multiplexing of particles and / or molecules that emit light at a portion of the detector module 242. As used herein, "emission multiplexing" refers to a system or method suitable for analyzing particles, molecules, or other analytes by analyzing light emitted from such particles, molecules, or other analytes at different wavelength ranges.

[0130] In some embodiments, the distal end 248 of the first light emitting optical fiber 234 is configured to emit the first emitted light 246A onto at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more photodetectors. In some embodiments, each of the photodetectors is configured to receive a substantially different spectral portion of the emitted light, such as when received through one or more dichroic mirrors or optical filters.

[0131] The portion of detector module 242 illustrated in FIG. 2 is shown to further include lenses 290 shaped and positioned to focus the reflected and / or transmitted radiation onto respective photodetectors.

[0132] In an embodiment, a system of the present disclosure includes multiple detector modules, such as one or more detector modules 242 as illustrated in Figure 2. In an embodiment, each of the distal ends of the light emitting optical fibers is configured to emit emitted light into a detector module, as illustrated in Figure 2.

[0133] Excitation multiplexing As discussed further herein, the light engine of the systems of the present disclosure can include light sources configured to output light in various wavelength ranges, such as wavelength ranges suitable for exciting one or more detectable agents disposed in or on particles flowing through the passageway. In some embodiments, such wavelength ranges of light overlap. In some embodiments, the wavelength ranges of light are disjoint. In this regard, attention is directed to Figures 3A-3F, which illustrate an embodiment of a light engine 308 and a passageway 302 illuminated by the light engine 308, in accordance with an embodiment of the present disclosure.

[0134] 3A is a schematic diagram of a light engine 308 and a path 302 of a system according to an embodiment of the present disclosure. In one embodiment, the light engine 308 and the path 302 are examples of the light engine 108 and the path 102 of the system 100 of FIG. 1A. In one embodiment, the light engine 308 and the path 302 are suitable for use in conjunction with a portion of the detector module 242 of FIG.

[0135] In the illustrated embodiment, the light engine 308 is shown to include four light sources each coupled to a distal end of an excitation optical fiber. Although four light sources are illustrated, it will be understood that more or fewer light sources are possible and are within the scope of the present disclosure. It will also be understood that free space light sources may be used within the scope of the present disclosure, as discussed further herein. The excitation optical fibers are shown to terminate in an excitation fiber bundle positioned to output excitation light. As shown, the excitation fiber bundle head 372 is positioned to output excitation light onto the dichroic mirror 360 and into the objective lens 386. The excitation light is shown to emanate from the objective lens 386 onto a respective portion of the passageway 302 within the inspection window 306.

[0136] 3B is a schematic diagram of an inspection window 306 of a passageway 302 that defines a lumen 304 configured for particles and / or molecules to flow through. As shown, a first excitation light 312 is directed to a first portion 322 of the passageway 302, a second excitation light 316 is directed to a second portion 324 of the passageway 302 separate from the first portion 322, a third excitation light is directed to a third portion 326 of the passageway 302 separate from the first portion 322 and the second portion 324 of the passageway 302, and a fourth excitation light is directed to a fourth portion 328 of the passageway 302 separate from the first portion 322, the second portion 324, and the third portion 326 of the passageway 302. The first portion 322 and the second portion 324 of the passageway 302 are shown separated by a space 374. In an embodiment, the spacing 374 between the first portion 322 and the second portion 324 corresponds to, and is defined, at least in part, by, the spacing 376 between the distal end 366 of the first excitation optical fiber 364 and the distal end 370 of the second excitation optical fiber 368. In this regard, the spacing 376 between the distal end 366 of the excitation optical fiber 364 and the distal end 370 of the excitation optical fiber 368 determines the spacing 374 between the portions 322 and 324 of the passageway 302 illuminated by the light source of the light engine 308. This is further illustrated by FIG. 4, which is a fluorescent image of the passageway 302 illuminated by the light engine 308, in accordance with an embodiment of the present disclosure.

[0137] As noted above, portions 322, 324, 326, and 328 are separated by spacing 374. In certain embodiments, such spacing is in the range of about 100 nm to about 100 microns, about 10 nm to about 10 microns, about 500 nm to about 10 microns, about 1 micron to about 20 microns, 3 microns to about 30 microns, or 2 microns to about 8 microns.

[0138] In one embodiment, spacing 374 is based on the distance between the center of one excitation light, such as excitation light 312, entering interrogation window 306 and the center of another excitation light, such as excitation light 316, entering interrogation window 306. In another embodiment, spacing 374 is based on the distance between edges of the excitation lights, such as an edge of excitation light 312 and an opposing edge of excitation light 316, entering interrogation window 306.

[0139] 1A, in certain embodiments, portions 322, 324, 326, and 328 have a width defined by the width of excitation light, such as excitation light 312 and 316, that is incident on inspection window 306 after passing through or being focused by high NA air objective lens 386. In certain embodiments, the ratio of spacing 374 to the width of one or more of portions 322, 324, 326, and 328 is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, greater than 20:1, or more. In certain embodiments, the ratio of spacing 374 to the width of one or more of portions 322, 324, 326, and 328 is in the range of about 1:1 to about 20:1, 2:1 to about 20:1, 2:1 to about 10:1, 2:1 to about 5:1. Such a ratio is large enough to generate excitation light from the various portions with minimal crosstalk between emissions from the separate portions 322, 324, 326, and 328, for example, as detected by a detector module of a system of the present disclosure.

[0140] 3C-3F are schematic diagrams of a light engine 308 and a passageway 302 according to the present disclosure. In an embodiment, the light engine 308 and the passageway 302 are examples of the light engine 308 and the passageway 302 of FIG. 3A. As shown, the light engine 308 includes a first light source 310, a second light source 314, a third light source 318, and a fourth light source 320. In the illustrated embodiment, the light sources 310, 314, 318, and 320 are optically coupled to excitation optical fibers 364, 368, etc., whose distal ends are coupled together in an excitation fiber bundle 372. The distal ends 366 and 370 of the first excitation optical fiber 364 and the second excitation optical fiber 368 are arranged to provide a spacing 376.

[0141] In the illustrated embodiment, light sources 310, 314, 318, and 320 include several lasers having the stated wavelengths. As shown, in certain embodiments, two or more of lasers 310, 314, 318, and 320 are configured to output light having a common wavelength. In certain other embodiments, lasers 310, 314, 318, and 320 are configured to output light having different wavelengths.

[0142] In this regard, the light engine 308 can be configured to analyze or manipulate particles and / or molecules passing through the passageway 302 with the same wavelength of light, such as when tracking a particular particle as it moves through the passageway 302. Similarly, in some embodiments, the light engine 308 can be configured to analyze or manipulate particles with different wavelengths of light to aid in determining the presence or absence of particular detectable agents associated with different markers.

[0143] As discussed further herein, the variable configurations and wavelength ranges of the light sources of the light engine 308 are suitable for performing excitation and emission multiplexing. As used herein, "excitation multiplexing" refers to a method of analyzing particles, molecules, or other analytes, including excitable detectable agents associated with such particles, molecules, or other analytes, with excitation light having different wavelength ranges. As discussed further herein, by exciting the detectable agent with excitation light having different wavelength ranges, different qualities or characteristics of the particle, molecule, or other analyte associated with the detectable agent may be determined.

[0144] cover In certain embodiments, the systems of the present disclosure include a cover coupled to the light emitting fiber bundle. In this regard, attention is directed to Figures 5A-5C, which illustrate a cover according to an embodiment of the present disclosure.

[0145] Figure 5A illustrates generally the emitted light passing through openings 594A and 594B of an optically opaque cover 592 onto a system's light emitting fiber bundle 530, according to an embodiment of the present disclosure. Figure 5B illustrates an example of the optically opaque cover 592 of Figure 5A. In one embodiment, light emitting fiber bundle 530 is an example of light emitting fiber bundle 130 of Figure 1A.

[0146] As shown, the lens 590 directs the first emitted light 552 and the second emitted light 546 to the light emitting fiber bundle head 532. In the illustrated embodiment, the cover 592 defines an opening 594A shaped to allow passage of the first emitted light 552 onto the proximal end 536 of the first light emitting optical fiber 534. In this regard, the first emitted light 552 is permitted to pass through the opening 594A and onto the proximal end 536 of the first light emitting optical fiber 534. In an embodiment, the cover 592 is optically opaque. In this regard, light, such as light that is not the first emitted light 552, is unlikely to enter the first light emitting optical fiber 534.

[0147] In the illustrated and partially exploded embodiment, the cover 592 is shown separated from the light emitting fiber bundle head 532. In certain embodiments and uses, the optically opaque cover 592 is coupled to the light emitting fiber bundle head 532 so as to prevent or reduce stray light from entering the light emitting optical fiber. In this regard, the optically opaque cover 592 is suitable for increasing the signal-to-noise ratio of the detector system and / or minimizing or eliminating crosstalk between different emitted lights (e.g., a portion of the first emitted light 552 entering the proximal end 540 of the second light emitting optical fiber 538, and vice versa).

[0148] As shown, the optically opaque cover defines a second opening 594B shaped to allow passage of the second emitted light 546 onto the proximal end 540 of the second light-emitting optical fiber 538. In this regard, the second emitted light 546 is allowed to pass through the cover 592 and enter the second light-emitting optical fiber 538.

[0149] Although four openings, including openings 594A and 594B, are illustrated in a linear fashion, it will be understood that any number of openings in cover 592 may be arranged in various configurations to correspond to the light emitting optical fibers of light emitting fiber bundle 530, such as those further discussed herein with respect to Figures 1A and 1B.

[0150] 5C is an image of the proximal end of the fiber bundle head 532 of the system, according to an embodiment of the present disclosure. As shown, the optical fibers are arranged in a linear configuration such that the openings 594A and 594B align with the optical fibers of the fiber bundle head when the cover 592 of FIG. 5A and FIG. 5B is coupled thereto.

[0151] 6 is a schematic diagram of a system 600 according to an embodiment of the present disclosure described herein. As shown, the system 600 includes a passageway 602 configured to flow particles or molecules through a lumen 604 of the passageway 602, defining an interrogation window 606 configured to allow light to pass in and out of the lumen 604, a light engine 608, an emission fiber optic bundle 630 shaped and positioned to receive emitted light from the interrogation window 606, and a detector system configured to generate a signal based on the collected emitted light.

[0152] In the illustrated embodiment, the light engine 608 includes four light sources positioned to output light onto the passageway 602. In this regard, in an embodiment, the light engine 608 includes a first light source positioned to output a first excitation light 612 onto a first portion of the passageway 602 within the inspection window 606, and a second light source positioned or configured to output a second excitation light 616 onto a second portion of the passageway 602 within the inspection window 606 separate from the first portion. In an embodiment, the light sources are free-space light sources that are not coupled to an excitation optical fiber. In this regard, in an embodiment, the spacing of the excitation lights is defined at least in part by the spacing of the free-space light sources. In another embodiment, the excitation spacing from the free-space light sources is defined at least in part by the manner in which the excitation light output therefrom is combined with dichroics and / or lenses and other optical components, etc. In an embodiment, the light engine 608 includes a fiber-coupled light source, as discussed further herein with respect to FIG. 1A.

[0153] As shown, the excitation light is directed through a lens 690 and enters a dichroic mirror 660, which reflects the excitation light to an objective lens 686. The objective lens 686 collects the excitation light and directs it to an inspection window 606 in the passageway 602.

[0154] Emitted light from passage 602 passes through objective lens 686 and dichroic mirror 660 back into emission fiber bundle 630. As discussed further herein, although a dichroic mirror is illustrated, other partially reflective / transmissive structures are possible within the scope of the present disclosure.

[0155] In the illustrated embodiment, system 600 is shown to further include a mirror positioned to reflect light onto light emitting fiber bundle 630 and a cover 692 configured to block light other than the emitted light from entering light emitting fiber bundle 630.

[0156] The system 600 is shown to include a number of photodetectors optically coupled to the light emitting optical fibers. In this regard, the system 600 is shown to include an emitting optical fiber bundle 630 comprising a first emitting optical fiber 634 and a second emitting optical fiber 638, the proximal ends 636 of which are disposed within an emitting optical fiber bundle head 632, the proximal end 636 of the first emitting optical fiber 634 positioned to receive a first emitted light 646 emitted from a first portion, and the proximal end 640 of the second emitting optical fiber 638 positioned to receive a second emitted light 652 emitted from a second portion. The proximal end of the light emitting optical fiber can refer to a portion of such fiber disposed within an emitting optical fiber bundle, such as the emitting optical fiber bundle 630, as well as a portion adjacent to the emitting optical fiber bundle.

[0157] The detector system 600 is shown to include first photodetectors 644, 658A, and 658B positioned to receive first emitted light 646 emitted from the distal end of the first light-emitting optical fiber 634, and second photodetectors 650A and 650C positioned to receive second emitted light 652 emitted from the distal end of the second light-emitting optical fiber 638.

[0158] 2, and as shown herein, the system 600 further includes a dichroic mirror 660 disposed between the distal end of the first light-emitting optical fiber 634 and the first photodetector 644 and positioned to reflect a portion of the first emitted light 646 onto third photodetectors 658A and 658B. In the illustrated embodiment, each light-emitting optical fiber is optically coupled to the dichroic mirror 660 and a bandpass filter 662, which are in turn optically coupled to second photodetectors 650A, 650B, and 650C and third photodetectors 658B, 658C, 658D, 658E, and 658F. In an embodiment, the first photodetector 644 is configured to generate a first signal based on a first wavelength range of the first emitted light 646, and the third photodetectors 658A and 658B are configured to generate a set of signals based on a set of different wavelength ranges of the first emitted light 646. In an embodiment, the second photodetectors 650A-650C are configured and positioned to generate a set of signals based on emitted light other than the first emitted light 646, such as based on the second emitted light 652. In this regard, the emitted light received by each light emitting optical fiber is configured to be spectrally analyzed by several photodetectors.

[0159] As shown, each light emitting optical fiber is optically coupled to several photodetectors. For example, the first light emitting optical fiber 634 is optically coupled to photodetectors 644, 658A, and 658B. In an embodiment, the photodetectors 644, 658A, and 658B comprise a detector module, such as a detector module as discussed further herein with respect to FIG. 2. Similarly, in an embodiment, the photodetectors 650A and 658C are grouped within a second detector module. In an embodiment, such a detector module comprises a box or other housing that encloses the various photodetectors of the detector module.

[0160] System 600 is shown to further include a controller 656 operably coupled to the light engine 608 and the light detector. As discussed further herein with respect to FIG. 1A, in some embodiments, controller 656 is configured to govern the operation of the light engine 608 and the light detector system, including implementing one or more methods of the present disclosure.

[0161] The controller 656 is also shown operably coupled to a movable stage 688 that is physically coupled to the passage 602. The passage 602 is shown to be part of a microfluidic chip that defines several passages. In an embodiment, the controller 656 includes logic that, when executed by the controller 656, causes the system 600 to move the microfluidic chip with the movable stage 688. Thus, the focus of the objective lens 686 is changed from a first passage 602 to a second passage of the microfluidic chip. In this regard, the system 600 may be used to analyze particles and / or molecules flowing through several passages, such as several passages flowing suspensions of different particles and / or solutions of different molecules.

[0162] automatic focus adjustment In an embodiment, the system of the present disclosure is suitable for analyzing particles or molecules flowing through the system, such as by using an autofocus process. In this regard, attention is directed to Figures 7A and 7B, which illustrate a system 700 according to an embodiment of the present disclosure. Figure 7A is a schematic diagram of system 700. Figure 7B is a schematic diagram of the focusing of a high NA air objective lens 786 of system 700 onto a passageway 702 of system 700. In an embodiment, system 700 is an example of system 100 of Figure 1A or an example of system 600 of Figure 6.

[0163] As shown, system 700 includes a passageway 702 configured to flow particles and / or molecules through a lumen of the passageway 702, the passageway 702 defining an interrogation window 706 configured to allow light to pass in and out of the passageway 702, a moveable stage 788 coupled to the passageway 702 and configured to move the passageway 702 relative to a light collection system 784, a light engine 708, a detector system, and a controller 756 operably coupled to the light engine 708, the moveable stage 788, and the detector system. As shown, the passageway 702 defines a constriction in the interrogation window 706. As discussed further herein, such a constriction is suitable for providing a particle-by-particle flow of particles and / or a molecule-by-molecule flow of molecules through the passageway 702.

[0164] In the illustrated embodiment, the light engine 708 is shown outputting excitation light 712 onto a dichroic mirror 760, which is reflected into a light collection system 784 and into an inspection window 706 of the passageway 702. Emission light 746 is shown to emit from the inspection window 706, through the dichroic mirror 760, a lens 790A, and an opening 794 in an optically opaque cover 778, and received by an emission fiber bundle 730 including an emission fiber bundle head 732. A distal end of one of the emission optical fibers 734 of the emission fiber bundle 730 is shown terminating adjacent to a photodetector 744 of the detector system. The emission light passes through a lens 790 and a bandpass filter 762 before being incident on the photodetector. The photodetector 744 is configured to generate a signal based on the received emission light 746.

[0165] In an embodiment, the controller 756 includes logic that, when executed by the controller 756, causes the system 700 to perform operations. In an embodiment, such operations include one or more of the methods of focusing an optical component onto the fluid passageway 702 according to an embodiment of the present disclosure. In an embodiment, the operations include illuminating an inspection window 706 of the fluid passageway 702 with light from a light source, focusing the light onto the inspection window 706 with an optical component disposed between the passageway 702 and a photodetector 744, generating a lock signal at the photodetector 744 based on the focused light reflected back from the inspection window 706 at a first time, generating a test signal at the photodetector 744 based on the focused light reflected back from the inspection window 706 at a second time after the first time, determining whether the test signal is within a predetermined percentage of the lock signal, and moving the fluid passageway 702 relative to the high NA air objective lens 786 if the test signal is outside the predetermined percentage of the lock signal. 7B, a high NA air objective lens 786 can be moved relative to the passageway 702 to focus the excitation light 712 into the lumen 704 of the passageway 702. In one embodiment, such movement of the air objective lens 786 is controlled by a movable stage 788, such as in response to instructions received from a controller 756, as discussed further herein with respect to FIG.

[0166] 7C is a block diagram illustrating a method of focusing a high NA air objective lens 786 of system 700 according to an embodiment of the present disclosure. As shown, the block diagram illustrates a feedback loop that controls the movement of the objective lens 786 by comparing the current value of the reflection with the value at a previous time (e.g., 200 ms ago) and with a reference value. If the difference between the measured value and the reference or lock value exceeds a predetermined threshold, a motor is driven to move the fluid passage 702 relative to the high NA air objective lens 786.

[0167] In one embodiment, the objective lens is positioned to collect the focused light reflected back from the inspection window using a collection system 784. In one embodiment, the collection system 784 comprises an air objective lens having a numerical aperture in the range of about 0.91 to less than 0.99, or about 0.95.

[0168] In some embodiments, the light is in the non-visible wavelength range, hi some embodiments, the light is infrared light, such as in the range of about 700 nm to about 2000 nm.

[0169] In an embodiment, the controller 756 includes logic that, when executed by the controller 756, causes the system 700 to perform operations including imaging the passageway 702 with a camera and determining an amount of defocus in the image, such as by determining an amount of defocus in the image based on a structure in the passageway 702, such as a wall of the passageway 702 having a known shape and / or dimension. In an embodiment, the structure may be a separate structure adjacent to the passageway 702, designed to perform this image-based autofocus and / or stage movement to position the passageway within the inspection window. In an embodiment, the operation further includes moving the fluid passageway 702 relative to the collection system 784 if the amount of defocus is outside of a predetermined range.

[0170] In some embodiments, the operations include illuminating an imaging region of the system 700 with light from a light source, generating an image of the imaging region with a camera or other image sensor, determining an amount of defocus of the image, determining whether the amount of defocus is within a predetermined amount of defocus, and moving the fluid passageway 702 relative to the high NA air objective if the test signal is outside the predetermined range. In some embodiments, the passageway is moved relative to the high NA air objective using a movable stage 788. In some embodiments, the operations are iterative in that, for example, the camera periodically generates images to check focus and / or moves the passageway 702 relative to the high NA air objective to adjust focus. In some embodiments, the light source is in a non-visible wavelength range. In some embodiments, the light is near infrared light, such as in the range of about 700 nm to about 2000 nm. In some embodiments, the objective is an air objective. In some embodiments, the objective is an air objective having a NA of 0.91 to 0.99. In some embodiments, the objective lens is an air objective lens having an NA of 0.92 to 0.98. In some embodiments, the objective lens is an air objective lens having an NA of 0.93 to 0.97. In some embodiments, the objective lens is an air objective lens having an NA of 0.94 to 0.96. In some embodiments, the objective lens is an air objective lens having an NA of about 0.95.

[0171] Refining Subsystem 8 illustrates a system 800, according to an embodiment. In one embodiment, system 800 is an example of any of the systems and subcomponents thereof discussed further herein with respect to FIGS. 1A, 1B, 2, 3A-3F, 5A-5C, 6, 7A-7G, 19, and 20. As shown, system 800 includes a flow channel 802 configured to flow an analyte, such as a single molecule analyte, through a lumen of the flow channel 802, the flow channel 802 defining an interrogation window configured to allow light to pass into and out of the lumen; a light engine 808 configured to output excitation light 812 into the flow channel 802 through the interrogation window; a detector system 844 positioned to receive emitted light 846 emitted from the flow channel 802 and configured to generate a signal based on the received emitted light 846; a light collection system 884 positioned to collect the emitted light 846 from the flow channel 802 and direct the collected emitted light 846 onto the detector system 844; and a controller 856 operably coupled to the light engine 808 and the detector system 844.

[0172] As described above, the system 800 includes a flow path 802 configured to flow an analyte, such as a single molecule analyte, through the flow path. In certain embodiments, the flow path is configured to flow a single molecule analyte associated with, for example, a detectable agent and a capture agent or a first detection agent and a second detection agent, through the flow path 802. In this regard, the flow path 802 is configured to flow a single molecule analyte associated with a capture agent that includes, for example, a bead.

[0173] As shown, system 800 optionally further includes a purification subsystem controller 898 configured to govern operation of optional purification subsystem 896. In some embodiments, purification subsystem 896 is a separate system or equipment that is separate and / or physically separate from the other system 800 components described with respect to Figure 8. In some embodiments, operation of system 800 is further described herein with respect to Figures 21 and 22. In some embodiments, operation of system 800 including purification subsystem 896 is further described herein with respect to, for example, Figures 9A and 9B.

[0174] In an embodiment, the controller 856 includes logic that, when executed by the controller 856, causes the system 800 to perform operations including one or more of the methods of the present disclosure. In an embodiment, the controller 856 includes logic that, when executed by the controller 856, causes the system 800 to perform operations including outputting excitation light 812 with the light engine 808 through an interrogation window onto a portion of the flow path 802, generating a detectable agent emission signal with the detector 844 based on the detectable agent emission light 846 from the detectable agent, and optionally flowing a sample through the flow path 802 that includes an analyte associated with the detectable agent.

[0175] In the illustrated embodiment, system 800 is shown to include an optional purification subsystem 896 configured to associate an analyte with a capture agent configured to isolate the analyte from a portion of a sample when the analyte is associated with the capture agent and the capture agent is subjected to an isolation procedure. As used herein, "capture agent" refers to a group of particles, beads, nanoparticles, molecules, association moieties configured to isolate an analyte from a portion of a sample when the analyte is associated with the capture agent and the capture agent is subjected to an isolation procedure.

[0176] In an embodiment, purification subsystem 896 is configured to isolate the analyte associated with the capture agent from a portion of the sample to provide a purified sample, and flowing the analyte associated with the detectable agent through flow path 802 includes flowing the purified sample through flow path 802. As shown, purification subsystem 896 is positioned upstream of flow path 802 and is shown configured to provide the purified sample to flow path 802 for detection in flow path 802.

[0177] As discussed further herein with respect to the methods of the present disclosure, in some embodiments, flowing the analyte associated with the detectable agent through flow path 802 comprises flowing a complex comprising a capture agent and the analyte associated with the detectable agent. In some embodiments, the capture agent comprises a magnetic bead.

[0178] As used herein, the term "bead" refers to a particle, such as a micron or submicron scale particle. In an embodiment, the bead is a nanoparticle. In an embodiment, the bead is sized and shaped to flow through a flow channel of a system according to an embodiment of the present disclosure. In this regard, in an embodiment, the bead has a maximum dimension that is smaller than the cross-section of the lumen of the flow channel, including the cross-section of the lumen within the constriction of the flow channel, such that the bead can pass through the flow channel including the constriction. In an embodiment, the capture agent comprises a primary antibody or a nucleic acid molecule configured to selectively associate with a site of the analyte.

[0179] In some embodiments, the beads are functionalized with moieties configured to specifically associate with an analyte and / or an association agent, hi some embodiments, the beads are functionalized with moieties configured to specifically associate with a detectable agent, such as free detectable agent that is not associated with an analyte.

[0180] In some embodiments, the beads are fluorescent beads. In some embodiments, the beads are fluorescent beads that are encoded as further described herein. In some embodiments, the beads are magnetic beads.

[0181] In some embodiments, the beads have a diameter of less than 1 micrometer. In some embodiments, the beads have a diameter of less than 1 micrometer, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. In some embodiments, the beads have a diameter of less than 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, or 40 nm. In some embodiments, the beads have a diameter of more than 1 micrometer. In some embodiments, the beads have a diameter of 1 to 5 micrometers.

[0182] In certain embodiments, the ratio of capture agent to detectable agent ranges from 10:1 to 1:10, 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2, and 1:1. In certain embodiments, the ratio of capture agent to detectable agent is about 1:1.

[0183] In an embodiment, the purification subsystem 896 comprises a magnet. In the illustrated embodiment, the purification subsystem controller 898 is operably coupled to the purification subsystem 896 and further comprises logic that, when executed by the purification subsystem controller 898, causes the subsystem 896 to perform operations including: associating the analyte associated with the capture agent with a magnet; and removing a portion of the sample not associated with the magnet from the analyte associated with the capture agent to provide a purified sample.

[0184] In an embodiment, the capture agent comprises beads and the purification subsystem 896 comprises a centrifuge. In an embodiment, a purification subsystem controller 898 is operably coupled to the purification subsystem 896 and includes logic that, when executed by the purification subsystem controller 898, causes the subsystem 896 to perform operations including centrifuging the sample including the capture agent associated with the analyte in a centrifuge to provide a supernatant and a precipitate including the analyte associated with the capture agent, and decanting the supernatant to provide a purified sample.

[0185] In one embodiment, the capture agent comprises beads, the purification subsystem 896 comprises a size-exclusion chromatography column, the controller 856 is operably coupled to the purification subsystem 896, and the purification subsystem controller 898 comprises logic that, when executed by the purification subsystem controller 898, causes the subsystem 896 to perform operations including passing a sample including the analyte associated with the capture agent through the size-exclusion chromatography column to provide a purified sample including the analyte associated with the capture agent.

[0186] As discussed further herein with respect to Figure 18C, optional purification subsystem 896 includes a surface, and the capture agent is bound to the surface. In an embodiment, a purification subsystem controller 898 operably coupled to purification subsystem 896 further includes logic that, when executed by the purification subsystem controller 898, causes subsystem 896 to perform an operation including removing a portion of the sample that is not associated with the capture agent bound to the surface. In an embodiment, a purification subsystem controller 898 operably coupled to purification subsystem 896 further includes logic that, when executed by the purification subsystem controller 898, causes subsystem 896 to perform an operation including contacting the surface with an elution buffer to elute the analyte associated with the capture agent from the surface.

[0187] As discussed further herein with respect to the methods of the present disclosure, in certain embodiments, the analyte can be associated with a capture agent, the capture agent configured to emit capture agent emission light upon excitation. In certain embodiments, the controller 856 further includes logic that, when executed by the controller 856, causes the system 800 to perform operations including generating a capture agent emission signal based on the capture agent emission light. Also, as discussed further herein, in certain embodiments, such a capture agent emission signal is useful for detecting an analyte associated with a capture agent, such as an analyte also associated with an emission signal of a detectable agent. In certain embodiments, generating the capture agent emission signal includes irradiating the capture agent with excitation light 812 and detecting the capture agent emission light with a photodetector. In one embodiment, for example, as further illustrated herein with respect to FIG. 10A , the controller 856 further includes logic that, when executed by the controller 856, causes the system 800 to perform operations including outputting a second excitation light 812 through an inspection window onto a second portion of the flow path 802 separate from the first portion, and generating a capture agent emission signal at a second photodetector based on the capture agent emission light received from the second portion of the flow path 802.

[0188] In an embodiment, the system 800 is suitable for use with an encoded capture agent, such as those discussed further herein with respect to FIG. 12A. In an embodiment, the capture agent comprises a plurality of distinct chromophores, each chromophore of the plurality of distinct chromophores comprising a predetermined set of adjustable optical encoding parameters, thereby defining an optically detectable code of the capture agent. In an embodiment, the capture agent is configured to emit capture agent emission light upon excitation, and the controller 856 further comprises logic that, when executed by the controller 856, causes the system 800 to perform operations including: generating, at the photodetector, a capture agent emission signal based on the capture agent emission light; and determining a concentration of analytes associated with the emission light 846 from the interrogation window further based on the number of analytes associated with both the emission light 846 from the detectable agent and the capture agent emission. In an embodiment, the optically detectable code comprises a predetermined emission spectrum of the capture agent, a predetermined absorption spectrum of the capture agent, or a combination thereof.

[0189] As discussed further herein, system 800 is suitable for use with two or more detectable agents, which are configured, for example, to associate with different sites on the analyte and emit distinct detectable agent or capture agent emitted light, such as fluorescence. In an embodiment, the detectable agent is a first detectable agent configured to emit a first emitted light within a first wavelength range upon excitation of the first detectable agent, and controller 856 further includes logic that, when executed by controller 856, causes system 800 to perform operations including exciting a second detectable agent configured to associate with the analyte and emit a second emitted light within a second wavelength range upon excitation of the second detectable agent, the first emitted wavelength range being distinct from the second emitted wavelength range. In an embodiment, the first detectable agent is associated with a first site on the analyte and the second detectable agent is associated with a second site on the analyte that is distinct from the first site. In one embodiment, the controller 856 further includes logic that, when executed by the controller 856, causes the system 800 to perform operations including associating the analyte with an association agent configured to specifically associate with the analyte, where associating the detectable agent with the analyte includes associating the detectable agent with the association agent that is associated with the analyte.

[0190] 14, subsystem 896 includes removal beads and / or is configured to cooperate with removal beads configured to remove detectable agents not associated with the analyte, such as after the detectable agent has had an opportunity to associate with the analyte. Thus, in an embodiment, controller 898 further includes logic that, when executed by controller 898, causes subsystem 896 to perform operations including, after associating an analyte in the sample with a detectable agent, contacting the sample with removal beads configured to selectively associate with any detectable agents not associated with the analyte, and removing removal beads associated with detectable agents not associated with the analyte from the sample.

[0191] In an embodiment, as discussed elsewhere herein, in an embodiment, the flow path 802 defines a constriction. In an embodiment, the flow path 802 within the interrogation window defines a constriction relative to an adjacent portion of the flow path 802. In an embodiment, the controller 856 further includes logic that, when executed by the controller 856, causes the system 800 to perform operations including flowing a plurality of analytes of the sample through the flow path 802. In an embodiment, flowing the analytes through the flow path 802 includes flowing a plurality of analytes of the sample through the flow path 802, the analytes including the analytes associated with a detectable agent, and flowing the analytes associated with the detectable agent through the flow path 802 includes, for each analyte associated with the detectable agent, flowing the analyte associated with the detectable agent through a constriction in the flow path 802. In an embodiment, flowing the analyte through the flow path 802 includes flowing a plurality of analytes of a sample through the flow path 802, the analyte comprising an analyte associated with a detectable agent and a capture agent, and flowing the analyte associated with a detectable agent and a capture agent through the flow path 802 includes, for each analyte associated with a capture and detectable agent, flowing the analyte associated with the detectable agent and the capture agent through a constriction in the flow path 802. In an embodiment, flowing the analyte through the flow path 802 includes flowing a plurality of analytes of a sample through the flow path 802, the analyte comprising an analyte associated with two or more detectable agents, and flowing the analyte associated with the two or more detectable agents through the flow path 802 includes, for each analyte associated with a detectable agent, flowing the analyte associated with the two or more detectable agents through a constriction in the flow path 802.

[0192] In an embodiment, the system 800 is configured to determine a number of analytes flowing through the flow path 802. In an embodiment, the system 800 is further configured to determine a concentration of the analytes in the sample based on the number of analytes flowing through the passageway and a flow rate of the fluid flow through the passageway, etc. In an embodiment, the controller 856 further includes logic that, when executed by the controller 856, causes the system 800 to perform operations including quantifying a number of analytes associated with the emitted light 846 and determining a concentration of analytes associated with the emitted light 846 from the interrogation window based on the number of analytes associated with the emitted light 846 and the volume of liquid flowing through the flow path 802.

[0193] In an embodiment, the capture agent is configured to emit capture agent emission light upon excitation, and the controller 856 includes logic that, when executed by the controller 856, causes the system 800 to perform operations including generating a capture agent emission signal based on the capture agent emission light. In an embodiment, determining the concentration of the analyte is based on a number of detectable agent emission signals associated with the capture agent emission signal or the emission signal of a second detectable agent. In an embodiment, determining the concentration of the analyte is based on a ratio of a number of detectable agent emission signals associated with the capture agent emission signal or the emission signal of a second detectable agent to a number of capture agent emission signals, a number of detectable agent emission signals, a number of second detectable agent emission signals, or a combination thereof. In an embodiment, determining the concentration of the analyte is based on a volume of liquid flowing through the flow path.

[0194] Collinear Excitation In some embodiments, the present disclosure provides systems configured for detection of an analyte through collinear excitation and emission measurement, in which the system illuminates a single portion of a flow path, such as a single cross-section of a lumen of the flow path, rather than multiple portions of the flow path, as illustrated in and discussed with respect to Figures 1A-6 of the present disclosure.

[0195] In certain embodiments, such systems are configured or otherwise suitable for the analysis of single molecule analytes. As used herein, a "single molecule analyte" refers to an analyte, such as a protein or nucleic acid molecule, that comprises a single molecule. Such single molecule analytes are in contrast to conglomerates or aggregates of multiple molecules, such as, for example, granules, vesicles, cells, etc., that contain many molecules associated together. Examples of single molecule analytes include a single protein molecule, or a single nucleic acid molecule, consisting of one or more strands of amino acids, either in a single-stranded unhybridized state or in a double-stranded hybridized state. As further described herein, the systems of the present disclosure are, in certain embodiments, configured to detect, count, analyze, etc., single molecule analytes in a flow based on their specific association with one or more detectable agents and / or capture agents, in order to identify the single molecule analytes in the flow, count them, determine their concentration in the sample, etc. As used herein, "specific association" refers to an association caused by specific molecular recognition and binding, including antibody-antigen binding, nucleic acid hybridization, aptamer-antigen binding, antibody-fragment-antigen binding, peptide-antigen binding, and the like.

[0196] In this regard, attention is directed to FIG. 19 , which illustrates a system 1900 for analyzing an analyte. As shown, the system 1900 includes a flow path 1902, a light engine 1908 configured to illuminate the passageway 1902, a detector 1942, and a controller operably coupled to the light engine 1908 and the detector 1942. In the illustrated embodiment, the passageway 1902 is configured to flow the analyte through a lumen 1904 of the passageway 1902, and the passageway 1902 defines an inspection window 1906 configured to allow light to pass in and out of the lumen 1904. In certain embodiments, the system 1900 can include or be combined with a purification subsystem, such as the purification subsystem 896 discussed further herein with respect to FIG. 8 .

[0197] As described above, the system 1900 includes a passageway 1902 configured to flow an analyte through a lumen 1904. In some embodiments, the passageway 1902 is sized and shaped to flow a single molecule analyte through the passageway 1902. In some embodiments, the passageway 1902 is sized and shaped to flow a single molecule analyte associated with a detectable agent and a capture agent or a first detectable agent and a second detectable agent, as described elsewhere herein. In some embodiments, the passageway 1902 is sized and shaped to flow a single molecule analyte associated with a capture agent that comprises a bead.

[0198] In the illustrated embodiment, the system 1900 is shown to include a pump 1998 fluidly coupled to the flow passage 1902, such as for flowing particles and / or molecules (i.e., analytes) through the flow passage 1902. As shown, the pump 1998 is operably coupled to a controller 1956, which can operate the pump 1998 in pumping fluid through the passage 1902. In some embodiments, the pump 1998 is optional, such as when fluid is flowed through the passage 1902 by gravity or by a driving force due to surface tension.

[0199] As shown, the light engine 1908 is shown to include several light sources 1910, 1914, and 1918 optically coupled to the passageway 1902. In the illustrated embodiment, the light engine 1908 is shown to include a first light source 1910, a second light source 1914, and a third light source 1918. In the particular illustrated embodiment, the first light source 1910 is configured to emit light having a wavelength of about 488 nm, the second light source 1914 is configured to emit light having a wavelength of about 561 nm, and the third light source 1918 is configured to emit light having a wavelength of about 637 nm. Although these particular wavelengths of excitation light are illustrated, it will be understood that other excitation wavelengths may be used, such as for illuminating and exciting particular detectable agents, capture agents, and the like, and are within the scope of the present disclosure.

[0200] A light engine 1908 having several light sources configured to emit excitation light of different light wavelengths that are then combined into a collinear excitation beam 1919 is suitable for exciting, for example, capture agents containing small dye molecules or other detectable agents and / or capture agents with small Stokes shifts. In this regard, by exciting such fluorophores at different wavelength ranges, the fluorophores may be excited separately with the collinear excitation beam 1919, and emitted light 1945 of different wavelength ranges approximately corresponding to the respective excitation wavelengths may be detected by detector 1942, as discussed further herein. In an embodiment, detector 1942 is configured to generate a signal, such as an emission signal, in response to the received emitted light 1945.

[0201] In the illustrated embodiment, the system 1900 includes a dichroic mirror 1960 positioned to combine excitation light, including a first excitation light 1912 and a second excitation light 1916, into a collinear excitation light beam 1919 that is directed to the inspection window 1906. Because the light engine 1908 is configured to illuminate the inspection window 1906 with a collinear beam of excitation light 1919 that is a combination of the excitation light from the light sources 1910, 1914, and 1918 of the light engine 1908, the system 1900 need not, and is not shown as including, an optical fiber bundle as discussed with respect to certain other systems of the present disclosure. In this regard, in one embodiment, the light sources 1910, 1912, and 1914 of the light engine 1908 are free-space laser light sources. In another embodiment, the light sources 1910, 1914, and 1918 of the light engine 1908 are fiber-coupled laser light sources.

[0202] System 1900 is shown to include lenses 1990, which may be a cylindrical lens and a plano-convex lens, positioned between light engine 1908 and collection system 1984 and configured to direct collinear beams 1919 from light engine 1908 to collection system 1984. As shown, collection system 1984 includes an air objective lens 1986, as discussed elsewhere herein.

[0203] The collinear composite excitation beams 1919 are shown illuminating a single portion of the passageway 1902, here shown illuminating a cross section of the lumen 1904 of the passageway 1902. Emission light 1945, such as from detectable agents and / or capture agents associated with analytes in the inspection window 1906 of the flow channel 1902, is received by a collection system 1984 and directed through an optical system 1990, such as a mirror and / or a tube lens, to an aperture 1980 in the optically opaque cover 1978. Such emission light 1945 passing through the aperture 1980 is shown to be received by an optical fiber 1966, although this is optional. Alternatively, without the use of fiber coupling, the fluorescence emerging from the tube lens 1990 can be collimated using a plano-convex lens.

[0204] The emitted light 1945 is shown to be received by a detector 1942, which is configured to generate one or more signals based on the received emitted light 1945. The detector 1942 is shown to include a number of dichroic mirrors 1960 configured to reflect a first portion 1946 of the emitted light 1945 onto a first photodetector 1944 and pass a second portion 1952 of the emitted light 1945. In this regard, the second portion 1952 of the emitted light 1945 is incident on a second dichroic mirror 1960, which reflects the second emitted light 1952 onto a second photodetector 1950. As shown, the detector 1942 includes a third photodetector 1958 positioned to receive a further portion 1952 of the emitted light 1945. Detector 1942 is further shown to include a bandpass filter 1962 configured to filter a portion of the received emitted light 1945, and optics 1990, such as an aspheric lens, each positioned between dichroic mirror 1960 and respective photodetectors 1944, 1950, and 1958. In this regard, detector 1942 is configured to disperse emitted light 1945, for example, according to wavelength ranges, to separately detect wavelength ranges within emitted light 1945. Thus, detector 1942 is configured to detect the presence or absence of a detectable agent and / or capture agent associated with an analyte in interrogation window 1906 of passageway 1902, such as through detection of detectable agent fluorescence and capture agent fluorescence, which may be spectrally combined in the emitted light 1945 received by detector 1942.

[0205] In certain embodiments, the present disclosure provides a system including a single light source for detecting and analyzing analytes in a flow. In this regard, attention is directed to FIG. 20, which illustrates a system 2000 according to an embodiment of the present disclosure.

[0206] As shown, system 2000 includes a flow path 2002, a light engine 2008 configured to illuminate the passageway 2002, a detector 2042, and a controller operably coupled to the light engine 2008 and the detector 2042. In the illustrated embodiment, the passageway 2002 is configured to flow an analyte, such as a single molecule analyte, through a lumen 2004 of the passageway 2002, and the passageway 2002 defines an inspection window 2006 configured to allow light to pass into and out of the lumen 2004. In certain embodiments, the system 2000 can include or be combined with a purification subsystem, such as purification subsystem 896, discussed further herein with respect to FIG.

[0207] In the illustrated embodiment, the system 2000 is shown to include a pump 2098 fluidly coupled to the flow path 2002, such as to flow the analyte through the passageway 2002. As shown, the pump 2098 is operably coupled to a controller that can operate the pump 2098 in pumping the fluid through the passageway 2002. In some embodiments, the pump 2098 is optional, such as in cases where the fluid is driven through the passageway 2002 by gravity or by a driving force due to surface tension.

[0208] As shown, light engine 2008 is shown to include a light source 2010 configured to emit excitation light 2012 through optics 2090, such as including a cylindrical lens and a plano-convex lens, from a dichroic mirror 2060, into a collection system 2084 including an air objective lens 2086, and onto passageway 2002. A single light source system reduces the complexity and cost of system 2000 by combining expensive components such as lasers and source excitation beams and / or fiber coupling to reduce the number of optics 2090 used to illuminate different portions of the inspection window 2006.

[0209] Emitted light 2045, such as light from a detectable agent or capture agent, is collected by collection system 2084 and directed through optics 2090, such as mirrors and a tube lens, to an aperture 2080 in optically opaque cover 2078. Such emitted light 2045 passing through aperture 2080 is shown to be received by optical fiber 2066, although this is optional. Alternatively, without the use of fiber coupling, the fluorescent light emerging from tube lens 2090 may be collimated using a plano-convex lens, as here.

[0210] The emitted light 2045 is shown to be received by a detector 2042. In an embodiment, the detector 2042 is configured to generate an emission signal based on the emitted light 2045 received by the detector 2042. The detector 2042 is shown to include a number of dichroic mirrors 2060 configured to reflect a first portion 2046 of the emitted light 2045 onto a first photodetector 2044 and pass a second portion 2052 of the emitted light 2045. In this regard, the second portion 2052 of the emitted light 2045 is incident on a second dichroic mirror 2060, which reflects the second emitted light 2052 onto a second photodetector 2050. As shown, the detector 2042 includes a third photodetector 2058 positioned to receive a further portion 2052 of the emitted light 2045. Detector 2042 is further shown to include a bandpass filter 2062 configured to filter a portion of the received radiation 2045, and optics 2090, such as an aspheric lens, each positioned between dichroic mirror 2060 and respective photodetectors 2044, 2050, and 2058. In this regard, detector 2042 is configured to disperse radiation 2045 according to wavelength ranges, for example, to separately detect wavelength ranges within radiation 2045, such as through the generation of one or more emission signals based on the received radiation 2045.

[0211] The system 2000 illustrated in Fig. 20 is suitable for exciting several fluorophores, such as detectable agents and / or capture agents as described herein, that are excited by a common wavelength range and then emit light at different wavelengths. As an example, a polymer dot may have a large Stokes shift with a tunable emission peak, and may be excited by a single light source and then emit at a wide variety of wavelengths that are separately detectable by detector 2042. In this regard, for example, a detectable agent and a capture agent each associated with an analyte may be excited with a single light source of the light engine 2008, and their separate fluorescence may be detected separately by detector 2042, thus confirming, for example, co-localization of the detectable agent and capture agent on the analyte.

[0212] As noted above, systems of the present disclosure, such as systems 1900 and 2000, include controllers 1956 and 2056, respectively. In an embodiment, such controllers 1956 and 2056 are suitable and configured to govern the components of systems 1900 and 2000 operatively coupled thereto. In this regard, in an embodiment, the controllers include logic that, when executed by the controllers, causes the system to perform operations, such as those described with respect to the methods of the present disclosure.

[0213] In this regard, attention is directed to FIG. 21, which illustrates a method 2100 for system control and operation, according to an embodiment of the present disclosure. In an embodiment, the method 2100 is for system control, such as systems 1900 or 2000, discussed further herein with respect to FIG. 19 and FIG. 20, respectively, for example, for a digital affinity assay. Such a system control method may be implemented through execution of logic stored in one or more controllers 1956 and / or 2056 in the systems 1900 and / or 2000. In an embodiment, such logic is stored on a remote server and / or in a distributed system operably coupled or coupleable to the controllers. In an embodiment, the system control method includes use and execution of logic stored on a purification subsystem controller, as further described herein with respect to FIG. 8.

[0214] In one embodiment, method 2100 begins at process block 2101, which includes flowing a sample including an analyte associated with a detectable agent through a flow path. In one embodiment, the analyte is incubated with the detectable agent under conditions and for a time sufficient to cause the analyte to associate with the detectable agent. In one embodiment, the analyte is also associated with a capture agent, such as through incubation of the analyte under conditions and for a time sufficient to cause the analyte to associate with a capture agent. In one embodiment, flowing the analyte through the flow path includes flowing the analyte associated with multiple detectable agents through a constriction in the flow path for each analyte associated with a detectable agent. In one embodiment, flowing the analyte through the flow path includes flowing the analyte associated with multiple capture and detectable agents through a constriction in the flow path for each analyte associated with a capture and detectable agent.

[0215] In certain embodiments, flowing the sample through the passageway is performed through the action of one or more pumps configured to pump a sample, such as a liquid sample, through the flow path.

[0216] In an embodiment, the sample is flowed through the channel through gravity or surface tension driven flow, and in an embodiment, process block 2101 is optional as it relates to a method of system control for a system according to an embodiment of the present disclosure.

[0217] In an embodiment, process block 2101 is followed by, or method 2100 begins with, process block 2103 which includes outputting excitation light through an interrogation window in a portion of the flow path. In an embodiment, outputting excitation light includes operation of a light engine, such as outputting excitation light from a light source of the light engine. Such excitation light can be configured to excite one or more of a detectable agent and / or a capture agent associated with the analyte.

[0218] In an embodiment, following process block 2103 is process block 2105 which includes outputting a second excitation light through an inspection window in the flow path. In an embodiment, outputting the second excitation light occurs through operation of a light engine, such as a light engine including two or more light sources. As discussed with respect to FIG. 19 and FIG. 20, the light engine may include one light source or two or more light sources depending, etc., on the type of detectable agent and / or capture agent used in the digital affinity assay. As discussed with respect to FIG. 20, in an embodiment, the light engine includes only a single light source, and thus, in an embodiment, process block 2105 is optional.

[0219] In one embodiment, following process block 2103 or 2105 is process block 2107 which includes generating a detectable agent emission signal at a photodetector based on detectable agent emission light received from the flow path. As discussed elsewhere herein, the detectable agent is configured to associate with the analyte, such as through specific association with the analyte (e.g., via specific molecular recognition), and generate detectable agent emission light, such as fluorescence, in response to being optically excited, which detectable agent emission light may be detected by a photodetector. In one embodiment, process block 2107 includes generating multiple detectable agent emission signals at a detector based on detectable agent emission light from multiple detectable agents, such as when multiple analytes associated with detectable agents are flowed through the flow path.

[0220] In an embodiment, following process block 2107 is process block 2109 which includes generating a capture agent emission signal based on the capture agent emission light received by the detector. In an embodiment, the analyte is associated with the capture agent, such as through a specific association between the two (e.g., via specific molecular recognition), as discussed elsewhere herein. In an embodiment, the sample containing the analyte includes a capture agent, but the analyte is not associated with the capture agent. In an embodiment, the capture agent is configured to emit capture agent emission light, such as when excited by excitation light from a light source of the light engine. In an embodiment, process block 2109 is optional. In an embodiment, process block 2109 includes generating multiple capture agent emission signals at the detector based on the capture agent emission light from the capture agent, such as when multiple analytes associated with the capture agent are flowed through the flow path.

[0221] In an embodiment, following process block 2107 or process block 2109 is process block 2111, which includes correlating or colocalizing two or more emission signals to determine the presence and / or identity of the analyte. In an embodiment, this includes correlating or colocalizing the emission light of the detectable agent with the capture agent emission light or the emission light of a second detectable agent, such as to determine the presence and / or identity of the analyte. As discussed elsewhere herein, in an embodiment, the detectable agent and capture agent are each configured to specifically associate with the analyte. In this regard, by detecting the presence of the detectable agent and capture agent within the interrogation window, such as by near-simultaneous detection of the detectable agent emission signal and the capture agent emission signal, it can be determined with some degree of confidence that the capture agent and detectable agent are associated with a single analyte molecule or particle. Furthermore, through such association of the detectable agent and capture agent, it can be further inferred that the analyte includes a site with which the detectable agent and capture agent are configured to specifically associate (e.g., via specific molecular recognition). In this regard, the correlation or co-location of the emission signal of the detectable agent and the capture agent emission signal of the capture agent can be used to detect the presence of the analyte and / or identify the identity of the analyte, at least with respect to the site on the analyte with which the detectable agent and capture agent are configured to specifically associate. Although a detectable agent and a capture agent are discussed with respect to process block 2109, it will be understood that similar approaches can be used, for example, where two or more detectable agents and the emission signals of each detectable agent can be used to determine the presence or identity of an analyte.

[0222] In an embodiment, process block 2111 is followed by process block 2113 which includes associating the ratio of the intensities of the emitted signals with the identity of the analyte. In an embodiment, this may include associating the ratio of the intensities of the capture agent emitted light with the identity of the capture agent and the identity of the analyte associated with the capture agent. In an embodiment, this may include associating the ratio of the intensities of the detectable agent emitted light with the identity of the detectable agent and the identity of the analyte associated with the detectable agent. In an embodiment, this may include associating an optical barcode of the capture agent emitted light and / or the detectable agent emitted light with the identity of the analyte associated with the capture agent and the detectable agent. In an embodiment, this may include associating a fluorescent spectral intensity barcode of the capture agent emitted light and / or the detectable agent emitted light with the identity of the analyte associated with the capture agent and the detectable agent. As discussed further herein, in certain embodiments, the capture agent is an encoded capture agent configured to emit encoded capture agent emission light, such as, for example, where the encoded capture agent includes a plurality of distinct chromophores, each chromophore of the plurality of distinct chromophores including a predetermined set of adjustable optical encoding parameters, thereby defining an optically detectable code of the capture agent. In this regard, the capture agent may include a first concentration of a first chromophore and a second concentration of a second chromophore such that the capture agent emits a first fluorescence and a second fluorescence, the ratio of the intensity of the first fluorescence to the intensity of the second fluorescence defining a ratio suitable for identifying the capture agent. Furthermore, since the capture agent is configured to specifically associate with a particular site of the analyte, the analyte may be identified by identifying the capture agent through the ratio of the received emission intensities. In certain embodiments, process block 2113 may include associating an optical barcode of the capture agent emission light and / or the detectable agent emission light with the identity of the capture agent and the analyte associated with the detectable agent. In one embodiment, process block 2113 is optional.

[0223] In one embodiment, following process block 2111 or process block 2113 is process block 2115. In one embodiment, process block 2115 involves quantifying the number of detectable agent emission signals associated with the capture agent emission signal or the second detectable agent emission signal. In one embodiment, process block 2115 involves quantifying the number of detectable agent emission signals associated with two or more detectable agent emission signals. In one embodiment, process block 2115 involves quantifying a ratio of the number of detectable agent emission signals associated with the capture agent emission signal or the second detectable agent emission signal to the number of capture agent emission signals, the number of detectable agent emission signals, the number of second detectable agent emission signals, or a combination thereof. As discussed above with respect to process block 2111, the analyte may be associated with both a detectable agent and a capture agent, or with two detectable agents, in which case two or more emission signals are detected at approximately the same time, such as when using a single excitation of the flow path, indicating that the capture agent and the detectable agent, or two detectable agents, are associated with the analyte. In other scenarios, the detectable agent, the second detectable agent, or the capture agent are not associated with the analyte, in which case only the detectable agent emission signal, the second detectable agent emission signal, or only the capture agent emission signal is detected within a given time period. In this regard, process block 2115 may include quantifying or otherwise determining the number or ratio of analytes associated with the detectable agent and the capture agent / second detectable agent, such as relative to a blank detectable agent, capture agent / second detectable agent (i.e., detectable / capture agent not associated with the analyte).

[0224] In some embodiments, quantifying the number of analytes includes single molecule sensitivity or detection efficiency. In some embodiments, quantifying the number of analytes includes single molecule sensitivity or detection efficiency without amplification of the analyte, for example, without amplifying the analyte to generate an amplicon or copy of the target analyte or an amplicon or copy of a molecule that correlates with the presence of the target analyte. In some embodiments, single molecule sensitivity or detection efficiency includes detecting more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of the molecules flowing through the flow path. In some embodiments, single molecule detection efficiency includes detecting more than 50%, 55%, 60%, 65%, 70%, 75, 80%, 85%, 90%, 95%, or 99% of the single molecules flowing through the flow path.

[0225] In an embodiment, following process block 2115 is process block 2117 which includes determining the concentration of the analyte or the ratio of analyte associated with the emission signal. As discussed elsewhere herein, the concentration of the analyte in the sample can be determined by counting the number of analytes associated with the emission signal and by determining the flow rate through the flow path. In an embodiment, process block 2117 includes determining the concentration of the analyte based on the number of capture agent emission signals associated with the detectable agent emission signals. In an embodiment, determining the concentration of the analyte is based on the volume of liquid flowing through the flow path. In an embodiment, process block 2117 includes determining the concentration of the analyte based on the number of detectable agent emission signals associated with two or more detectable agent emission signals and the volume of liquid flowing through the flow path. In an embodiment, determining the concentration of the analyte is based on the ratio of the number of detectable agent emission signals associated with the capture agent emission signal to the number of capture agent emission signals, the number of detectable agent emission signals, and combinations thereof. In this regard, process block 2117 may include determining a ratio between the number of detectable agent emission signals associated with the capture agent emission signal and the total number of capture agent emission signals (whether associated with a detectable agent emission signal or not), the total number of detectable agent emission signals (whether associated with a capture agent emission signal or not), and combinations thereof. In an embodiment, method 2100 may further include ranking the analytes in the flow path based on the presence or absence of detectable agent and / or capture agent emission light from the interrogation window, such as those discussed further herein with respect to FIG. 1A and FIG. 1B. In an embodiment, the ranking corresponds to the detectable agent emission signal and / or the capture agent emission signal. In an embodiment, the ranking corresponds to the emission spectrum of the analyte-associated capture agent and the detectable agent measured based on one or more of the first emitted light and the second emitted light. In an embodiment, the ranking corresponds to the measured light scattering light of the beads.

[0226] FIG. 22 illustrates a method 2200 for system control and operation, according to an embodiment of the present disclosure. In an embodiment, the method 2200 is for system control, such as systems 1900 or 2000, discussed further herein with respect to FIG. 19 and FIG. 20, respectively, for example, for a digital affinity assay. Such system control may be implemented through execution of logic stored in one or more controllers 1956 and / or 2056 in systems 1900 and / or 2000. In an embodiment, such logic is stored on a remote server and / or in a distributed system operably coupled or coupleable to the controllers. In an embodiment, system control includes use and execution of logic stored on a purification subsystem controller, such as further described herein with respect to FIG. 8.

[0227] In one embodiment, method 2200 begins at process block 2201, which includes flowing a sample containing an analyte associated with a detectable agent through a flow path. In one embodiment, the analyte is incubated with the detectable agent under conditions and for a time sufficient to cause the analyte to associate with the detectable agent. In one embodiment, the analyte is also associated with a capture agent, such as through incubation of the analyte under conditions and for a time sufficient to cause the analyte to associate with the capture agent.

[0228] In certain embodiments, flowing the sample through the passageway is performed through the action of one or more pumps configured to pump a sample, such as a liquid sample, through the flow path.

[0229] In an embodiment, the sample is flowed through the flow path via gravity, and in an embodiment, process block 2201 is optional as it relates to a method of system control for a system according to an embodiment of the present disclosure.

[0230] In an embodiment, process block 2201 is followed by, or method 2200 begins with, process block 2203 which includes outputting excitation light through an interrogation window in a portion of the flow path. In an embodiment, outputting excitation light includes operation of a light engine, such as outputting excitation light from a light source of the light engine. Such excitation light can be configured to excite one or more of a detectable agent and / or a capture agent associated with the analyte.

[0231] In an embodiment, following process block 2203 is process block 2205 which includes outputting a second excitation light through an inspection window in the flow path. In an embodiment, outputting the second excitation light occurs through operation of a light engine, such as a light engine including two or more light sources. As discussed with respect to FIG. 19 and FIG. 20, the light engine may include one light source or two or more light sources depending, etc., on the type of detectable agent and / or capture agent used in the digital affinity assay. As discussed with respect to FIG. 20, in an embodiment, the light engine includes only a single light source, and thus, in an embodiment, process block 2205 is optional.

[0232] In one embodiment, following process block 2203 or 2205 is process block 2207 which includes generating a detectable agent emission signal at a photodetector based on the detectable agent emission received from the flow path. As discussed elsewhere herein, the detectable agent is configured to associate with the analyte, such as through specific association with the analyte, and to generate a detectable agent emission, such as fluorescence, in response to being optically excited, and such fluorescence may be detected by the photodetector.

[0233] In an embodiment, following process block 2207 is process block 2209 which includes generating a capture agent emission signal based on the capture agent emission light. In an embodiment, the analyte is associated with the capture agent, such as through a specific association between the two, as discussed elsewhere herein. In an embodiment, the sample containing the analyte contains a capture agent, but the analyte is not associated with the capture agent. In an embodiment, the capture agent is configured to emit the capture agent emission light, such as when excited by excitation light from a light source of the light engine. In an embodiment, process block 2209 is optional.

[0234] In an embodiment, process block 2209 is followed by process block 2211 which includes correlating or co-localizing two or more emission signals to determine the presence and / or identity of the analyte. As discussed elsewhere herein, in an embodiment, the detectable agent and capture agent are each configured to specifically associate with the analyte. In this regard, by detecting the presence of the detectable agent and capture agent within the interrogation window, such as by near-simultaneous detection of the detectable agent emission signal and the capture agent emission signal, it can be determined with some degree of confidence that the capture agent and the detectable agent are associated with a single analyte molecule or particle. Furthermore, through such association of the detectable agent and the capture agent, it can be further inferred that the analyte includes a site with which the detectable agent and the capture agent are configured to specifically associate. In this regard, the correlation or co-location of the detectable agent emission signal and the capture agent emission signal can be used to identify the identity of the analyte, at least with respect to the site on the analyte with which the detectable agent and the capture agent are configured to specifically associate. With respect to process block 2109, a detectable agent and a capture agent are discussed, however, it will be understood that similar approaches may be used, for example, where two or more detectable agents and the emitted light / signal of each detectable agent may be used to determine the presence or identity of an analyte.

[0235] In certain embodiments, process block 2211 is followed by process block 2213 which includes correlating the ratio of the intensities of the emitted signals to the identity of the analyte. In certain embodiments, this may include correlating the ratio of the intensities of the capture agent emitted light to the identity of the capture agent and the identity of the analyte associated with the capture agent. In certain embodiments, this may include correlating the ratio of the intensities of the detectable agent emitted light to the identity of the detectable agent and the identity of the analyte associated with the detectable agent. As discussed further herein, in certain embodiments, the capture agent is a coded capture agent configured to emit a coded capture agent emitted light, such as where the coded capture agent comprises a plurality of distinct chromophores, each chromophore of the plurality of distinct chromophores comprising a predetermined set of adjustable optical encoding parameters, thereby defining an optically detectable code for the capture agent. In this regard, the capture agent can include a first concentration of a first chromophore and a second concentration of a second chromophore such that the capture agent emits a first fluorescence and a second fluorescence, and a ratio of the intensity of the first fluorescence to the intensity of the second fluorescence defines a capture agent emission intensity ratio suitable for identifying the capture agent, for example. Furthermore, since the capture agent is configured to specifically associate with a particular site of the analyte, the analyte can be identified by identifying the capture agent through the ratio of the received emission intensities. In certain embodiments, process block 2213 is optional.

[0236] In one embodiment, following process block 2211 or process block 2213 is process block 2215 which includes quantifying the number of detectable agent emission signals associated with the capture agent emission signal or the emission signal of a second detectable agent, or a ratio of the number of detectable agent emission signals associated with the capture agent emission signal or the emission signal of a second detectable agent to the number of capture agent emission signals, the number of detectable agent emission signals, the number of second detectable agent emission signals, or a combination thereof. In one embodiment, following process block 2211 or process block 2213 is process block 2215 which includes quantifying the number of detectable agent emission signals associated with two or more detectable agent emission signals. As discussed above with respect to process block 2111, the analyte may be associated with both a detectable agent and a capture agent, or with two detectable agents, in which case two or more emission signals, such as a capture agent emission signal and a detectable agent emission signal, are detected at approximately the same time, indicating that the capture agent and detectable agent, or two detectable agents, are associated with the analyte. In other scenarios, either the detectable agent or the capture agent, or both, are not associated with the analyte, in which case only the detectable agent emission signal or only the capture agent emission signal is detected within a given time period.

[0237] In this regard, process block 2115 may include quantifying or otherwise determining the number or ratio of analytes associated with a detectable agent / capture agent, such as relative to blank detectable agent and / or capture agent (i.e., detectable / capture agent not associated with an analyte).

[0238] In one embodiment, following process block 2215 is process block 2217 which includes determining the concentration of the analyte based on the number of capture agent emission signals associated with the detectable agent emission signals and the volume of liquid flowed through the flow path. In one embodiment, determining the concentration of the analyte is based on a ratio of the number of detectable agent emission signals associated with the capture agent emission signals to the number of capture agent emission signals, the number of detectable agent emission signals, and combinations thereof. In one embodiment, determining the concentration of the analyte is based on the volume of liquid flowing through the flow path.

[0239] As discussed elsewhere herein, the concentration of the analyte in the sample can be determined by counting the number of analytes associated with two or more emission signals, such as a capture agent emission signal and a detectable agent emission signal, and by determining the flow rate through the flow path.

[0240] The order in which some or all of the process blocks appear in each process should not be considered limiting - rather, one of ordinary skill in the art having the benefit of this disclosure will understand that some of the process blocks may be performed in various orders not illustrated, or even in parallel.

[0241] method In another aspect, the present disclosure provides a method for interrogating particles and / or molecules, such as analytes, hi an embodiment, the method comprises the use of a system described herein.

[0242] In some embodiments, the method is a method for single molecule detection. In some embodiments, the method includes the use of light collection with a collection assembly that includes a high NA air objective lens.

[0243] In certain embodiments, the method is a method for the analysis of single molecule analytes. As used herein, a "single molecule analyte" refers to an analyte, such as a protein or nucleic acid molecule, that comprises a single molecule. Such single molecule analytes are in contrast to conglomerates or aggregates of multiple molecules, such as, for example, granules, vesicles, cells, which contain many molecules associated together. Examples of single molecule analytes include a single protein molecule, or a single nucleic acid molecule, consisting of one or more strands of amino acids, either in a single-stranded unhybridized state or in a double-stranded hybridized state. As further described herein, the methods of the present disclosure, in certain embodiments, include the detection, identification, counting, quantification, analysis, concentration determination, etc. of single molecule analytes in a flow based on specific association with one or more detectable agents and / or capture agents, in order to identify single molecule analytes in the flow, count them, determine their concentration in the sample, etc. As used herein, "specific association" refers to an association caused by specific molecular recognition and binding, including antibody-antigen binding, nucleic acid hybridization, aptamer-antigen binding, antibody-fragment-antigen binding, peptide-antigen binding, and the like.

[0244] In an embodiment, the method includes flowing a plurality of molecules associated with a detectable agent through a passageway. In an embodiment, such flowing includes flowing a plurality of molecules associated with a detectable agent through a passageway, and includes flowing a molecule of the plurality of molecules through the passageway molecule by molecule. In this regard, as discussed elsewhere herein, in an embodiment, the molecules of the plurality of molecules pass through a passageway, such as a portion of a passageway that includes a constriction, one at a time. In this regard, the method is suitable for individually irradiating molecules flowing through the passageway.

[0245] As described above, the molecule is associated with a detectable agent. In some embodiments, each molecule is associated with one or more detectable agents. In some embodiments, a molecule of the plurality of molecules is associated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more detectable agents. As discussed elsewhere herein, such detectable agents are configured to generate a signal, such as a fluorescent signal, in response to excitation light. As used herein, a "detectable agent" refers to a molecule, an assembly of molecules, a portion of a molecule, a particle, etc., that is configured to generate a detectable signal, such as a fluorescent emission. In some embodiments, the detectable agent is configured to emit an optically detectable signal, such as fluorescence, that is detectable, such as through the use of a photodetector. In some embodiments, the detectable agent is a fluorescently labeled antibody. In some embodiments, the detectable agent is a fluorescently labeled nucleic acid probe. In some embodiments, the detectable agent comprises a fluorescent moiety, e.g., a fluorescent moiety selected from the group consisting of a fluorescent small molecule, a chromophore semiconducting polymer, a fluorescent polymer dot, a quantum dot, a fluorescent bead, a fluorescent polymer, and combinations thereof. In certain embodiments, the detectable agent is functionalized with a moiety, such as an antibody or nucleic acid probe, configured to specifically associate with the analyte, such as a specific association caused by specific molecular recognition or binding, such as antibody-antigen binding or nucleic acid hybridization.

[0246] In certain embodiments, the molecule is selected from the group consisting of a cell signaling molecule, a cytokine, a chemokine, an antibody, a protein, a nucleic acid, a nucleic acid binding protein, an RNA binding protein, a peptide, a carbohydrate, a drug molecule, and a therapeutic molecule.

[0247] In some embodiments, the method further includes illuminating molecules of the plurality of molecules in the passageway. In some embodiments, illuminating the molecules includes illuminating molecules of the plurality of molecules as the molecules flow through the passageway molecule by molecule. In some embodiments, illuminating the plurality of molecules includes illuminating the molecules with a plurality of light sources, the light being within one or more wavelength ranges. In some embodiments, such one or more light sources are positioned to illuminate spatially distinct portions of the passageway, such as different portions of an interrogation window, as discussed elsewhere herein.

[0248] In some embodiments, the method includes collecting emitted light from more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% of single molecules flowing through the passage. By flowing a plurality of molecules through the passage, molecule by molecule, the molecules can be detected and assessed individually. In this regard, particles such as molecules can be efficiently and accurately detected by the methods and systems of the present disclosure. Such efficient and accurate detection is suitable for accurately determining the concentration of particles and molecules in a large population of particles / molecules. This is particularly important, for example, when assigning a value to a molecule or particle is based on the detection of signals from several detectable agents. If each different detectable agent associated with a molecule or particle is not detected, it is not possible to accurately identify the molecule or particle.

[0249] In certain embodiments, "single molecule sensitivity" refers to the ability to detect greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% of the single molecules flowing through a passageway, preferably greater than 90% of the single molecules flowing through a passageway. In certain embodiments, "single molecule sensitivity" refers to a detection efficiency of greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, preferably greater than 90%.

[0250] In some embodiments, the "detection efficiency" of single molecules and / or particles under flow is the number of molecules / particles detected relative to the number of molecules / particles flowing through the passage (e.g., through the excitation region). In some embodiments, the "single molecule detection efficiency" refers to a detection efficiency of more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, preferably more than 90%. In some embodiments, the "single molecule detection efficiency" refers to the ability to detect more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, preferably more than 90% of the single molecules flowing through the passage. For a given type of fluorescent molecule / particle, having a "single molecule sensitivity" or "single molecule detection efficiency" is a direct indicator of the sensitivity of a flow system or device, and is therefore an important metric for evaluating the sensitivity and performance of a device or instrument.

[0251] In some embodiments, illuminating the molecules of the plurality of molecules in the passage includes outputting excitation light through an interrogation window onto a portion of the passage using line illumination, hi some embodiments, illuminating the molecules of the plurality of molecules in the passage includes outputting excitation light through an interrogation window onto a portion of the passage using a confocal detection geometry or a line confocal detection geometry.

[0252] In an embodiment, illuminating the passageway is accomplished with a tightly focused laser line covering the entire cross section of the passageway to ensure that all molecules passing through the passageway are illuminated and excited with a very high probability, such as greater than 90% probability, preferably close to 100% probability. In an embodiment, a confocal detection geometry is achieved by using an aperture (e.g., a fiber aperture or a slit aperture), which improves detection sensitivity by increasing the signal-to-noise ratio and by minimizing crosstalk between different excitation regions or laser lines. In an embodiment, an apparatus employing a high NA air objective, line illumination using a tightly focused laser line, and confocal detection geometry was used to ensure that all or nearly all molecules or particles flowing through the passageway are detected with high detection efficiency and high single molecule sensitivity as well as high throughput.

[0253] In an embodiment, the method includes collecting the emitted light from the passageway with a collection system including a high NA air objective lens having a numerical aperture in the range of 0.91 to less than 0.99, preferably about 0.95. As discussed elsewhere herein, high NA air objective lenses are particularly suitable for collecting relatively large amounts of light. In addition, such air objective lenses are suitable for accurately scanning a device while maintaining a consistent distance between the air objective lens and the device being imaged. Oil or water immersion objective lenses often cannot maintain a consistent distance between the objective lens and the device being imaged during scanning because they draw oil or water onto the device being imaged.

[0254] In some embodiments, the air objective lens has a numerical aperture of 0.91 to less than 0.99. In some embodiments, the air objective lens has a numerical aperture in the range of about 0.92 to about 0.98, in the range of about 0.93 to about 0.97, or in the range of about 0.94 to about 0.96. In some embodiments, the air objective lens has a numerical aperture of about 0.95.

[0255] In some embodiments, the method includes generating an emission signal based on the collected emission light emitted from the passageway based on the molecule, hi some embodiments, the signal is generated using one or more detector systems, detector modules, and / or photodetectors, as described elsewhere herein.

[0256] In some embodiments, the method includes assigning a value to the analyte based on the signal. In some embodiments, the value is based on one or more fluorescent signals emitted from the particle / molecule. Such a value may be used to sort particles / molecules among a plurality of particles / molecules, such as, for example, when sorting based on the presence and / or absence of one or more detectable moieties disposed on the particle / molecule.

[0257] As described herein above, in certain embodiments, the disclosed methods, systems, devices, and apparatus include a microfluidic chip with microfluidic channels that can facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules. Microfluidic chips with microfluidic channels can be used to process small volumes of fluid samples, providing advantages over traditional macroscale devices (e.g., microfluidic chips require only minute volumes of fluid samples, require fewer reagents, are processed in less time, and increase efficiency compared to macroscale devices). In certain embodiments, the microfluidic chip is a planar device, and thus can facilitate the detection and analysis of bionanoparticles and single molecules, and / or can facilitate the detection and analysis of bionanoparticles by enabling the use of high NA (numerical aperture) objectives (e.g., high NA air objectives), lenses, or light collection systems with high numerical apertures that improve light collection and thus facilitate the detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules in motion. In certain embodiments, microfluidic chips are planar devices, making them more compatible with microscope setups (e.g., having a translation stage on which the microfluidic chip is placed). In addition, microfluidic chips can enable the design and creation of interconnected fluidic networks without dead volumes, which can facilitate detection and manipulation of bionanoparticles and / or molecules (e.g., sorting using flow displacement at the junction of three or more fluidic passages). Dead volumes are a portion of the volume within the microfluidic chip that is outside the flow paths (e.g., a volume into which liquids that may carry sample nanoparticles and / or molecules may diffuse, reducing accuracy). Microfluidic chips, through methods of microfabrication, can enable the creation of passages with cross sections that are non-spherical or non-square (e.g., rectangular), which can facilitate detection, analysis, determination, and / or identification of moving bionanoparticles and / or molecules.Microfluidic chips can facilitate the creation of passages with different widths or heights along the length of the passage (e.g., constrictions in the passage or gradual changes in width and / or height) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules passing through. Microfluidic chips can be formed by bonding to a coverslip (e.g., made from glass or plastic) of a desired thickness and with desired material properties (e.g., refractive index) to facilitate compatibility with highly efficient light collection systems (e.g., high numerical aperture objectives such as high NA air objectives, which require appropriate substrate thickness for maximum light collection) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules in motion. Microfluidic devices allow the generation of many passages (e.g., 96 or 384 passages for 96 or 384 samples) on the same device for high throughput analysis of more samples (e.g., 96 or 384 in a format compatible with a multichannel pipettor). Microfluidic chips provide an attractive and versatile platform for the manipulation, isolation, sorting, and / or transport of bionanoparticles and / or single molecules.

[0258] Fiber-bundled emission detection In one embodiment, the method includes flowing particles and / or molecules through a passageway; outputting a first excitation light through an inspection window onto a first portion of the passageway; outputting a second excitation light through the inspection window onto a second portion of the flow path separate from the first portion; generating a first emission signal at a first photodetector based on the first emission light received through a proximal end of the first light-emitting optical fiber; and generating a second emission signal at a second photodetector based on the second emission light received through a proximal end of the second light-emitting optical fiber, wherein the proximal end of the first light-emitting optical fiber and the proximal end of the second light-emitting optical fiber are disposed within a light-emitting fiber bundle head.

[0259] In some embodiments, outputting the first excitation light and the second excitation light includes outputting light with a light engine, as discussed further herein. In some embodiments, the first and / or second excitation light includes coherent light, such as from a laser. In some embodiments, the first light source and the second light source are each independently selected from the group consisting of a solid-state laser, a diode-pumped laser, a light-emitting diode (LED), a lamp, an arc discharge, and natural light.

[0260] In some embodiments, the first and second photodetectors are each optically coupled to an emitting optical fiber bundle, as described elsewhere herein. In some embodiments, the first photodetector is part of a first detector module, such as a detector module described further herein with respect to FIG. 2, optically coupled to a first emitting optical fiber of the emitting optical fiber bundle. In some embodiments, the second photodetector is part of a second detector module, such as a detector module described further herein with respect to FIG. 1A, optically coupled to a second emitting optical fiber of the emitting optical fiber bundle.

[0261] In one embodiment, the method includes receiving the first emitted light and the second emitted light using an emitting fiber bundle including a first emitting optical fiber and a second emitting optical fiber, proximal ends of the first emitting optical fiber and the second emitting optical fiber being disposed within a light emitting fiber bundle head, the proximal end of the first emitting optical fiber being positioned to receive the first emitted light emitted from the first portion, and the proximal end of the second emitting optical fiber being positioned to receive the second emitted light emitted from the second portion.

[0262] In some embodiments, flowing the particles and / or molecules through the passageway comprises flowing a suspension of particles and / or a solution of molecules comprising the particles and / or molecules through the passageway. In some embodiments, the suspension of particles or the solution of molecules is or is derived from a biological sample. In some embodiments, the suspension of particles or the solution of molecules comprises or is based on a body fluid, or is based on a fluid from or associated with a cell. In some embodiments, the particle is selected from the group consisting of an extracellular vesicle, a biological nanoparticle, a cell organelle, a microvesicle, a cell-derived vesicle, a lipoprotein, a macromolecular complex, an exomere, an RNA binding protein, a nucleic acid binding protein, a biological aggregate comprising a protein or a nucleic acid, a protein aggregate, a nucleic acid aggregate, a lipid aggregate, a single biological molecule, a cytokine, a chemokine, an antibody, a cell signaling molecule, a therapeutic molecule, a nucleic acid, a virus, a bacterium, and an exosome. In some embodiments, the particle is an extracellular vesicle. In some embodiments, the bodily fluid comprises serum, plasma, cerebrospinal fluid, saliva, nasopharyngeal fluid, tears, whole blood, urine, sputum, or lymph. In some embodiments, the particle is isolated. In some embodiments, the molecule is isolated. In some embodiments, the particle is associated with at least one biomarker.

[0263] In some embodiments, flowing a suspension of particles and / or a solution of molecules through the passage includes flowing the suspension and / or solution through the passage on a particle-by-particle and / or molecule-by-molecule basis. In some embodiments, at least some of the plurality of particles are detected on a particle-by-particle basis. In some embodiments, at least some of the plurality of molecules are detected on a molecule-by-molecule basis. In some embodiments, at least some of the plurality of particles and / or molecules are illuminated on a particle-by-particle and / or molecule-by-molecule basis. Particle-by-particle or molecule-by-molecule basis refers to the observation of a plurality of particles or molecules passing through a region (e.g., a light beam having a given width) individually (i.e., one at a time). As a non-limiting example of particle-by-particle or molecule-by-molecule basis, a fluid sample containing a plurality of particles or molecules may flow through a constriction of a microfluidic passage and pass through a light beam such that at least some of the plurality of particles or molecules pass through the light beam individually (i.e., in the absence of any of the other particles of the plurality). As another non-limiting example of particle or molecule per particle or molecule, a fluid sample containing a plurality of particles or molecules may flow through a microchannel and pass through a light beam such that only one particle or molecule passes through the light beam at a time without overlapping with any other particle or molecule of the plurality. In some specific embodiments, a majority of the particles or molecules pass through the light beam such that only one particle or molecule passes through the light beam at a time without overlapping with any other particle or molecule of the plurality. In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the particles or molecules in the plurality of illuminated particles or molecules are illuminated per particle or molecule. In a preferred embodiment, more than 90% of the illuminated particles or molecules in the plurality of particles or molecules are illuminated per particle or molecule. In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the particles or molecules in the plurality of detected particles or molecules are detected per particle or molecule.In a preferred embodiment, greater than 90% of the particles or molecules in the plurality of detected particles or molecules are detected particle by particle or molecule by molecule.

[0264] Illumination of an individual particle or molecule may refer to a particle or molecule that is in a fluid sample that contains a plurality of particles or molecules and is illuminated in the absence of any other particles or molecules of the plurality. Illumination of an individual particle or molecule is distinct from illumination of two or more particles or molecules that are randomly co-localized in the illumination area. Illumination of an individual particle or molecule is distinct from illumination of an aggregate of particles or molecules. As a non-limiting example, an individual particle or molecule may pass through a light beam and thus be illuminated. An individual particle or molecule may pass through a light beam in the absence of any other particles or molecules of the plurality and thus be illuminated alone. In some embodiments, an individual particle or molecule is a singular nanoparticle or molecule that can be interrogated by a light source in the absence of any other particles or molecules present in the fluid sample (e.g., for a given light beam width, a single particle or molecule may be present in the beam and thus be illuminated in the absence of any other particles or molecules of the plurality).

[0265] Although flowing particles through a passageway and detecting particles particle by particle is described, it will be understood that the same concepts apply by analogy to flowing and detecting molecules molecule by molecule using the methods and systems of the present disclosure. Thus, in an embodiment, the methods of the present disclosure include flowing molecules molecule by molecule through a passageway, for example, through an inspection window of the passageway. In this regard, molecules of interest, such as those associated with one or more detectable agents, pass through the inspection one at a time. Thus, in an embodiment, there is only one molecule associated with a detectable agent in the inspection window at a time. Similarly, in an embodiment, two or more molecules of interest associated with each detectable agent are not present in the inspection window at the same time. In an embodiment, other molecules are present in the inspection window of the passageway along with the molecules of interest associated with the detectable agents. Such molecules may include, for example, solvent molecules that aid in the flow of the molecules of interest.

[0266] In an embodiment, such molecules that can pass through the interrogation window on a molecule-by-molecule basis are selected from the group consisting of proteins, peptides, antibodies, cytokines, chemokines, signaling molecules, therapeutic molecules, drug molecules, RNA binding proteins, macromolecular complexes, nucleic acids, DNA, RNA, synthetic molecules, aptamers, etc. In an embodiment, the molecules are selected from the group consisting of a single dye molecule, a single protein dye molecule, a single polymer dye molecule, a single Pdot, a single fluorescent probe, a single fluorescent unit, a single antibody conjugated with one or more dyes, a single protein conjugated with one or more dyes, a single nucleic acid molecule conjugated with one or more dyes.

[0267] In certain embodiments, the first emitted light and the second emitted light are independently selected from the group consisting of scattered radiation, luminescent radiation, fluorescent radiation, and combinations thereof.

[0268] In some embodiments, the particle is a biological particle. In some embodiments, the biological particle is a biological nanoparticle. In some embodiments, the particle is selected from the group consisting of an extracellular vesicle, a cellular organelle, a microvesicle, a cell-derived vesicle, a lipoprotein, a macromolecular complex, an exomere, an RNA binding protein, a nucleic acid binding protein, a biological aggregate comprising a protein or a nucleic acid, a protein aggregate, a nucleic acid aggregate, a lipid aggregate, a single biological molecule, a cytokine, a chemokine, an antibody, a cell signaling molecule, a therapeutic molecule, a nucleic acid, a nucleic acid binding protein, an RNA binding protein, a DNA binding protein, a therapeutic molecule, a virus, a bacterium, and an exosome.

[0269] As mentioned above, in some embodiments, the method of the present disclosure is suitable for analyzing relatively small particles flowing through a passageway. In certain embodiments, the size of the particle is the hydrodynamic diameter. In specific embodiments, the hydrodynamic diameter is less than 1,000 nanometers, less than 900 nanometers, less than 800 nanometers, less than 700 nanometers, less than 600 nanometers, less than 500 nanometers, less than 400 nanometers, less than 300 nanometers, less than 200 nanometers, less than 150 nanometers, less than 100 nanometers, less than 90 nanometers, less than 80 nanometers, less than 70 nanometers, less than 60 nanometers, less than 50 nanometers, less than 40 nanometers, or less than 30 nanometers. In a preferred embodiment, the hydrodynamic diameter is less than 100 nanometers. In certain embodiments, the hydrodynamic diameter is determined by measuring dynamic light scattering (DLS) and refers to the size of a hard sphere that scatters light in the same way as the biological nanoparticle being measured.

[0270] In some embodiments, the hydrodynamic diameter is between 1,000 nanometers and 1 nanometer, between 900 nanometers and 1 nanometer, between 800 nanometers and 1 nanometer, between 700 nanometers and 1 nanometer, between 600 nanometers and 1 nanometer, between 500 nanometers and 1 nanometer, between 400 nanometers and 1 nanometer, between 300 nanometers and 1 nanometer, between 200 nanometers and 1 nanometer, between 100 nanometers and 1 nanometer, between 90 nanometers and 1 nanometer, between 80 nanometers and 1 nanometer, between 70 nanometers and 1 nanometer, between 60 nanometers and 1 nanometer, between 50 nanometers and 10 nanometers, or between 40 nanometers and 1 nanometer. In certain embodiments, the hydrodynamic diameter is between 1,000 nanometers and 800 nanometers, between 800 nanometers and 600 nanometers, between 600 nanometers and 400 nanometers, between 400 nanometers and 200 nanometers, or between 200 nanometers and 10 nanometers. In a preferred embodiment, the hydrodynamic diameter is between 200 nanometers and 2 nanometers. In another preferred embodiment, the hydrodynamic diameter is between 200 nanometers and 10 nanometers. In a more preferred embodiment, the hydrodynamic diameter is between 100 nanometers and 20 nanometers.

[0271] In certain embodiments, the size of the particle is the diameter. In specific embodiments, the diameter is less than 1,000 nanometers, less than 900 nanometers, less than 800 nanometers, less than 700 nanometers, less than 600 nanometers, less than 500 nanometers, less than 400 nanometers, less than 300 nanometers, less than 200 nanometers, less than 150 nanometers, less than 100 nanometers, less than 90 nanometers, less than 80 nanometers, less than 70 nanometers, less than 60 nanometers, less than 50 nanometers, less than 40 nanometers, or less than 30 nanometers. In a preferred embodiment, the diameter is less than 100 nanometers. In certain embodiments, the diameter is determined by measuring using electron microscopy (TEM) or super-resolution imaging.

[0272] In some embodiments, the diameter is between 1,000 nanometers and 1 nanometer, between 900 nanometers and 1 nanometer, between 800 nanometers and 1 nanometer, between 700 nanometers and 1 nanometer, between 600 nanometers and 1 nanometer, between 500 nanometers and 1 nanometer, between 400 nanometers and 1 nanometer, between 300 nanometers and 1 nanometer, between 200 nanometers and 1 nanometer, between 100 nanometers and 1 nanometer, between 90 nanometers and 1 nanometer, between 80 nanometers and 1 nanometer, between 70 nanometers and 1 nanometer, between 60 nanometers and 1 nanometer, between 50 nanometers and 10 nanometers, or between 40 nanometers and 1 nanometer. In certain embodiments, the diameter is between 1,000 nanometers and 800 nanometers, between 800 nanometers and 600 nanometers, between 600 nanometers and 400 nanometers, between 400 nanometers and 200 nanometers, or between 200 nanometers and 10 nanometers. In a preferred embodiment, the diameter is between 200 nanometers and 2 nanometers. In another preferred embodiment, the diameter is between 200 nanometers and 10 nanometers. In a more preferred embodiment, the diameter is between 100 nanometers and 20 nanometers.

[0273] In certain embodiments, the method further comprises directing the flow of particles or molecules. In certain embodiments, directing the flow of particles or molecules is based on the presence or absence of emitted light received through the interrogation window and associated with the particles or molecules. In certain embodiments, directing the flow of particles or molecules is based on the intensity of emitted light received by the detector system from the interrogation window. In this regard, the method is suitable, for example, for separating particles or molecules that emit fluorescent and / or scattered excitation light from particles or molecules that do not emit fluorescent and / or scattered excitation light.

[0274] In some embodiments, directing the flow of particles or molecules includes directing the particles or molecules into one or more sorting paths, including paths for particles or molecules that provide a fluorescent or emission signal above a predetermined threshold and paths for particles or molecules that do not. In some embodiments, the method includes sorting the particles or molecules into an enriched population. In some embodiments, the sorting includes flow displacement sorting. In some embodiments, the sorting does not include the use of acoustic focusing or physical barriers. In some embodiments, the sorting is determined by a size value, the presence of a biomarker, the absence of a biomarker, a detected light intensity, an emitted wavelength, a plurality of emitted wavelengths, particle or molecule identity, or a combination thereof. In some embodiments, the sorting is determined by the presence of a combination of biomarkers. In some embodiments, the sorting is determined by the presence of one or more biomarkers and the absence of one or more other biomarkers, such as based on an immunophenotype or immunophenotype (a phenotype based on the presence, absence, or amount of markers measured by binding of a combination of antibodies). In some embodiments, the phenotype is determined, at least in part, by the presence or absence of two or more biomarkers on the particle (e.g., immunophenotype), and may be further informed or determined by physical properties such as particle size, or whether the particle contains nucleic acid, or the amount of lipid molecules that make up the particle. See, for example, Example 9, discussed further herein. In some embodiments, sorting is determined by the number or type of biomarkers present by setting a sorting threshold.

[0275] In one embodiment, the method includes quantifying or counting the number of particles and / or molecules associated with the emitted light from the interrogation window and determining a concentration of the particles and / or molecules associated with the emitted light from the interrogation window.

[0276] In an embodiment, the method includes ranking particles or molecules in the passageway based on the presence or absence of emitted light from the interrogation window. In an embodiment, the ranking corresponds to a measured emission spectrum of the particle or molecule based on one or more of the first emitted light and the second emitted light. In an embodiment, the ranking corresponds to a measured size value of the particle. In an embodiment, the measured size value is a relative size value. In an embodiment, the measured size value is measured by a difference in detected light intensity.

[0277] In some embodiments, the particle or molecule is associated with a detectable agent. In some embodiments, the detectable agent can be, for example, a molecule of interest (e.g., a protein on or in an extracellular vesicle, or a nucleic acid, or a biomarker) present on or in the particle to be analyzed. Alternatively, the detectable agent can be a molecule (e.g., an antibody conjugated to a fluorescent probe or a nucleic acid probe) that associates with a molecule of interest (e.g., a protein on or in an extracellular vesicle, or a biological nanoparticle, or a macromolecular complex, or a nucleic acid molecule, or a biomarker) associated with the particle, thereby allowing the nanoparticle to be detected. In some aspects, the detectable agent is fluorescent and can therefore be detected by fluorescence-based detection methods well known in the art.

[0278] In some embodiments, the particles include at least one biomarker, such as a biomarker associated with one or more detectable agents. In some embodiments, the method includes determining at least one copy number of the at least one biomarker.

[0279] As used herein, "associated" includes interactions through covalent and / or non-covalent interactions. For example, the detectable agent may be covalently attached to a particle or molecule. Alternatively, the detectable agent may be embedded, for example, within the membrane of the particle and / or within the hydrophobic interior of the particle, or may be intercalated into double-stranded DNA or RNA. In certain embodiments, the detectable agent may be embedded in the membrane of the particle via non-covalent interactions, such as van der Waals forces or electrostatic forces. As used herein, the term "specifically associated" or "specifically associated with" refers to the ability of a reagent to associate with a target site, such as based on the affinity and / or avidity of the reagent for the target site. A "specific association" may occur based on specific molecular recognition, such as antibody-antigen association, aptamer-antigen association, peptide-antigen association, and nucleic acid hybridization association.

[0280] In specific embodiments, the detectable agent is associated with the surface of the particle. In some embodiments, the detectable agent can be covalently and / or non-covalently bound to the surface of the particle. In other embodiments, the detectable agent can be embedded within the surface of the particle. In specific embodiments, the detectable agent is surrounded by a membrane dye embedded in the surface of the particle, e.g., lipids of a vesicle. The relationship of the detectable agent associated with the surface of the particle provides information about the size of the particle. For example, a particle with a large surface area will be associated with a large number of detectable agents, while a particle with a small surface area will be associated with fewer detectable agents. The relationship of the number of detectable agents associated with the particle surface provides a correlation between light intensity and nanoparticle surface area. In this way, the amount of light intensity emitted corresponds to the size of the particle, specifically, to the surface area of ​​the particle.

[0281] In certain embodiments, the size of the particle, determined such as through the intensity of fluorescence from a membrane dye, in conjunction with the number of copies of detectable agent associated with the analyte on the surface of the particle, can be used to determine whether the particle is an intact particle.

[0282] In other embodiments, the detectable agent is associated with the interior of the particle. In some embodiments, the detectable agent is embedded within the particle (e.g., a lipophilic dye embedded within a lipoprotein). In some embodiments, the detectable agent is not associated with the surface of the particle, but is embedded within the particle or otherwise surrounded by the particle. In specific embodiments, the detectable agent is contained by the particle, but not associated with the interior surface, e.g., a dye floating freely within an extracellular vesicle that is not internally associated with its lipid membrane. Internal detectable agents, such as those embedded within the particle (e.g., a lipophilic dye embedded within a lipoprotein) or those contained by the particle without association with the interior surface (e.g., a floating dye within a vesicle), are also referred to herein as "volume dyes." The relationship of the volume dyes embedded or surrounded by the particle provides information about the size of the particle. For example, a particle with a large volume will contain a large number of volume dyes, while a particle with a small volume will contain fewer volume dyes. The relationship of the number of volume dyes within the particle provides a correlation between light intensity and nanoparticle volume. Thus, the amount of emitted light intensity corresponds to the size of the particle, and specifically, to the volume of the particle.

[0283] In some embodiments, the particle includes both a volume dye and a detectable agent associated with its surface. Nanoparticles that include both a volume dye and a detectable agent associated with its surface can provide information regarding both the surface area and volume of the particle. In some embodiments, the volume dye and the surface area detectable agent are the same. In other embodiments, the volume dye and the surface area detectable agent are different. In certain embodiments, the volume dye can provide information regarding the identity or type of the particle being detected or isolated. In some embodiments, the use of a volume dye that is a fluorogenic substrate can provide information regarding the identity or type of the particle being detected or isolated. In specific embodiments, the use of a volume dye that is a fluorogenic substrate for an enzyme specific for particles such as exosomes can further provide information regarding the identity or type of the particle being detected or isolated.

[0284] In some embodiments, the particles are labeled with a membrane dye and a membrane-permeable nucleic acid dye, such as a membrane-permeable RNA dye. In some embodiments, such a combination of detectable agents is suitable for determining whether the particles contain nucleic acids, such as RNA or DNA, and whether the particles contain membranes or lipid molecules. Such particles can be further labeled with a detectable agent, such as a fluorescently labeled antibody, that is configured to selectively bind to a surface marker, such as to determine the immunophenotype, i.e., overall phenotype, of the particles, in addition to whether the particles contain membranes and / or nucleic acids, including physical properties (e.g., if lipid membranes and / or nucleic acids are present) and immunophenotype (e.g., if certain biomarkers are present or absent, or in different amounts as reported by antibodies).

[0285] In some embodiments, the detectable agent is selected from a fluorescently labeled antibody, a fluorescently labeled protein, a fluorescently labeled nucleic acid, a fluorescently labeled lipid, a membrane dye, a fluorogenic dye, a dye, a polymer dot, and combinations thereof. In some embodiments, the detectable agent is selected from the group consisting of a luminescent dye, a fluorescent dye, a fluorescently labeled antibody, a fluorescently labeled protein, a fluorescently labeled nucleic acid, a fluorescently labeled lipid, a fluorescently labeled carbohydrate, a fluorescently labeled small molecule, a membrane dye, a fluorogenic dye, a dye, a polymer dot, a fluorogenic substrate for an enzyme, a membrane permeable nucleic acid dye (such as a membrane permeable RNA dye), or combinations thereof.

[0286] In some embodiments, the detectable agent specifically binds to one or more binding targets associated with the particle. In certain aspects, the binding target is a polypeptide, such as a protein, and the detectable agent is a fluorescently labeled antibody that specifically binds to the target polypeptide. The phrases "specifically (or selectively) bind" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a biological nanoparticle, often refer to a binding reaction that determines the presence of a bionanoparticle of interest, or the presence of a biomarker associated with a bionanoparticle of interest, in a heterogeneous population of nanoparticles and other biological materials. Thus, under specified immunoassay conditions, a specified antibody binds to a specific biological nanoparticle that is at least two times greater than background, and more typically 10-100 times greater than background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular biological nanoparticle, or a particular biomarker, or a particular molecule (e.g., cytokine, chemokine, antibody, nucleic acid). For example, a polyclonal antibody can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with a selected antigen and not with other proteins. This selection can be accomplished by subtracting out antibodies that cross-react with other molecules.

[0287] In some embodiments, the detectable agent is a first detectable agent and the particle is associated with a second detectable agent. In some embodiments, the detectable agent is a first detectable agent and the molecule (e.g., a cytokine or cell signaling molecule) is associated with a second detectable agent. In some embodiments, the first detectable agent has a first emission spectrum within a first emission wavelength range and the second detectable agent has a second emission spectrum within a second emission wavelength range that is different from the first emission wavelength range. In some embodiments, the first detectable agent has a first excitation spectrum within a first excitation wavelength range and the second detectable agent has a second excitation spectrum within a second excitation wavelength range. In some embodiments, the first and second detectable agents have similar, the same, and / or overlapping emission spectra. In some embodiments, the first and second detectable agents have different emission spectra. In some embodiments, the first and second detectable agents have similar, the same, and / or overlapping excitation spectra. In some embodiments, the first and second detectable agents have different excitation spectra.

[0288] In some embodiments, the detectable agent is attached to the surface of the particle, the detectable agent is on the surface of the particle, the detectable agent is internal to the particle, the detectable agent is within the matrix of the particle, or a combination thereof. In some embodiments, the detectable agent is fluorescent, the detectable agent is luminescent, or any combination thereof. In some embodiments, the particle is associated with a plurality of detectable agents. In some embodiments, at least one of the plurality of detectable agents is attached to the surface of the particle. In some embodiments, at least one of the plurality of detectable agents is attached to the surface of the particle and at least one of the plurality of detectable agents is on the surface of the particle. In some embodiments, at least one of the plurality of detectable agents is attached to the surface of the particle and at least one of the plurality of detectable agents is internal to the particle. In some embodiments, at least one of the plurality of detectable agents is attached to the surface of the particle and at least one of the plurality of detectable agents is within the matrix of the particle. In some embodiments, at least one of the multiple detectable agents is attached to the surface of the particle, at least one of the multiple detectable agents is on the surface of the particle, at least one of the multiple detectable agents is internal to the particle, or combinations thereof. In some embodiments, the detectable agents of the multiple detectable agents have overlapping emission profiles. In some embodiments, the detectable agents of the multiple detectable agents have the same emission profile. In some embodiments, the emission profiles have the same peak wavelength. In some embodiments, the detectable agents of the multiple detectable agents have overlapping excitation profiles. In some embodiments, the detectable agents of the multiple detectable agents have the same excitation profile. In some embodiments, the excitation profiles have the same peak wavelength. In some embodiments, the detectable agents of the multiple detectable agents include the same detectable agent. In some embodiments, the detectable agents of the multiple detectable agents include two or more types of detectable agents.

[0289] In certain aspects, the detectable agents of the plurality of detectable agents have different emission profiles. In some embodiments, the emission profiles have different peak wavelengths. In this regard, the detectable agents are suitable for use in emission multiplexing, whereby different emission spectra are used in detecting different detectable agents. In certain embodiments, the detectable agents have different emission lifetimes. In certain embodiments, the detectable agents have different emission intensities at a common wavelength.

[0290] In certain embodiments, a first detectable agent has a first excitation spectrum within a first excitation wavelength range and a second detectable agent has a second excitation spectrum within a second excitation wavelength range that is different from the first excitation wavelength range. In this regard, the detectable agents are suitable for use in excitation multiplexing, whereby different detectable agents may be used by exciting them at different excitation wavelength ranges.

[0291] In some embodiments, the detectable agent is configured to be excited by light of different wavelength ranges. In some embodiments, the detectable agent is configured to be excited by a first amount by a first excitation light of a first wavelength range and a second amount by a second excitation light of a second wavelength range different from the first wavelength range. In this regard, the detectable agent is configured to emit emitted light of a first intensity in response to the first excitation light and emit emitted light of a second intensity in response to the second excitation light. The ratio of the first emitted light to the second emitted light can be used to track or otherwise identify a particle associated with the detectable agent.

[0292] In some embodiments, the peak wavelengths are separated by more than 10 nanometers, by more than 20 nanometers, by more than 30 nanometers, by more than 40 nanometers, by more than 50 nanometers, by more than 75 nanometers, by more than 100 nanometers, by more than 120 nanometers, by more than 140 nanometers, by more than 160 nanometers, by more than 180 nanometers, by more than 200 nanometers, by more than 300 nanometers, by more than 400 nanometers, by more than 500 nanometers, by more than 600 nanometers, or by more than 700 nanometers.

[0293] In another aspect, the present disclosure provides a method for analyzing particles in a fluid sample. In an embodiment, the method includes flowing a fluid sample containing a plurality of particles and / or molecules through a passageway, illuminating a particle of the plurality of particles or a molecule of the plurality of molecules in the passageway, collecting radiation emitted from the passageway with a collection system comprising a high NA air objective lens having a numerical aperture in the range of 0.91 to less than 0.99, generating an emission signal based on the collected radiation emitted from the passageway based on the particle or molecule, and assigning a value to the particle or molecule based on the signal.

[0294] In some embodiments, detection or imaging employing a fluidic device uses a light collection system having a numerical aperture of 0.91 or more, 0.92 or more, 0.93 or more, 0.94 or more, 0.95 or more, 0.96 or more, 0.97 or more, or 0.98 or more. In a preferred embodiment, the light collection system includes an air objective lens having a numerical aperture of about 0.95. As discussed further herein, high NA air objective lenses are suitable for single molecule flow detection. Such air objective lenses are generally more stable and easier to scan than oil immersion objective lenses. This is especially true when light collection efficiency is more important than image quality, such as in flow-based analysis and single molecule flow detection. In many embodiments, the methods of the present disclosure include generating a signal based on emission from the interrogation passage. Such a signal is often not a traditional image signal, such as one that generates an image of a particle or molecule in the passage or immobilized on a surface or in a matrix. Rather, in many embodiments, the methods of the present disclosure instead rely on the presence, absence, or intensity of light emitted from the passage. In this regard, light collection efficiency and emitted light intensity are more important for the method of the present disclosure. This is in contrast to conventional imaging applications, where image resolution and lack of optical aberrations (e.g., spherical or chromatic aberration) may be as important or more important than simply light collection efficiency. Therefore, high NA air objectives are often suitable for the method of the present disclosure, and oil or water immersion objectives are unnecessary and often inappropriate.

[0295] In an embodiment, the method is a method for determining a size of a particle, and the value is a size value. In an embodiment, ranking the particles in the passage is based on the presence or absence of emitted light from the inspection window. In an embodiment, ranking the particles in the passage is based on the intensity of emitted light from the inspection window. In an embodiment, the ranking corresponds to a measured emission spectrum of the particle based on one or more of the first emitted light and the second emitted light. In an embodiment, the ranking corresponds to a measured size value of the particle. In an embodiment, the measured size value is a relative size value. In an embodiment, the measured size value is measured by a difference in detected light intensity.

[0296] In some embodiments, the particle or molecule is associated with a detectable agent. In some embodiments, the detectable agent is a first detectable agent and the particle or molecule is associated with a second detectable agent. In some embodiments, the first detectable agent has a first emission spectrum within a first emission wavelength range and the second detectable agent has a second emission spectrum within a second emission wavelength range that is different from the first emission wavelength range. In some embodiments, the detectable agent is a fluorescent detectable agent. In some embodiments, the first detectable has a first emission spectrum within a first emission wavelength range and the second detectable agent has a second emission spectrum that is common, the same, similar, and / or overlapping with the first emission wavelength range.

[0297] As discussed further herein, in certain embodiments, the methods of the present disclosure do not include an amplification step, for example, to generate an amplicon or copy of an analyte or an amplicon or copy of a molecule that correlates with the presence of the analyte. In this regard, in certain embodiments, the methods of the present disclosure do not include the use of reagents and / or conditions used or required for amplification of a target analyte or a molecule that correlates with the presence of a target analyte, for example, nucleic acid amplification or protein-based amplification of a target analyte or a molecule that correlates with the presence of a target analyte, for example, polymerase chain reaction, enzymatic amplification, isothermal nucleic acid-based amplification, rolling circle amplification, or ELISA.

[0298] Self-correcting flow-based analysis In another aspect, the present disclosure provides a method for self-corrected single molecule / single particle flow analysis. Measurement of fluorescence emitted from single molecules or particles in a flow stream is heavily affected by the flow and / or laser beam profile. Thus, in certain embodiments, accurate quantification of fluorescent molecules requires deconvolution of the signal from the flow profile, which is often difficult, if not impossible. The complex nature of the observed signal and its interpretation poses several challenges for the analysis of particles (e.g., extracellular vesicles (EVs), lipoproteins, RNA-binding proteins, and viruses) or molecules (e.g., cytokines, antibodies, nucleic acid molecules, proteins, peptides, and cell signaling molecules) in the flow stream. The challenges include i) colocalization of biomarkers important for phenotyping EVs or other biological nanoparticles, ii) measuring the concentration of particles, iii) examining biological heterogeneity, often described by the copy number of biomarkers, iv) determining the copy number of biomarkers associated with EVs or other biological nanoparticles, and v) characterizing physical properties such as size of vesicles or particles stained with membrane dyes.

[0299] To overcome these challenges, the present disclosure provides a method suitable for analyzing single molecules and particles in a flow stream in a self-correcting manner. Using such a "self-corrected single molecule / particle" method, it is possible to accurately 1) co-localize biomarkers expressed on the same particle flowing through multiple excitation regions or parts of a passage in an interrogation window, 2) identify and count single particles and / or molecules, 3) obtain the flow rate sampled by each individual particle and / or molecule, and 4) thus measure the concentration of the analyzed particle and / or molecule. Using such a "self-corrected single molecule / particle" method, it is also possible to accurately determine the copy number of biomarkers associated with EVs or other biological nanoparticles.

[0300] Briefly, in this method, multiple excitation regions or portions of the passageway within the interrogation window are arranged in a known spatial pattern. A particle or molecule is measured twice in two different portions of the micropassageway through which the particle is flowing. Since the flow through the microfluidic passageway is typically laminar, the transit time of a particular particle flowing through any two adjacent or closely spaced excitation regions or portions of the passageway is generally proportional to the distance between these two excitation regions or portions of the passageway and the velocity of the particle. Also, due to the nature of laminar flow and the small separation distance between the excitation regions or portions of the passageway, the position of a particular particle within the cross-section of the passageway generally remains the same during the transit time. Thus, that particle generally interacts with different laser beams focused on different portions of the passageway at very similar positions within the cross-section of the passageway. Given these properties, it is possible to identify a single analyte (e.g., a vesicle stained by a fluorescent dye or a bionanoparticle labeled with an antibody) and further co-localize other biological markers using the extracted transit time or particle velocity.

[0301] Thus, in an embodiment, the method includes flowing a particle through a lumen of a passageway, the passageway defining an inspection window configured to allow light to pass in and out of the lumen, outputting a first excitation light with a first light source into a first portion of the passageway or inspection window, outputting a second excitation light with a second light source into a second portion of the passageway or inspection window separate from the first portion, generating a first emission signal with a first detector module based on the first emission light received from the first portion, generating a second emission signal with a second detector module based on the second emission light received from the second portion, and determining a velocity of the particle in the passageway based on a time difference between the first and second emission signals and a distance between the first and second portions. In an embodiment, the method includes use of any of the systems of the present disclosure. As discussed elsewhere herein, in an embodiment, the first photodetector is part of a first detector module and the second photodetector is part of a second detector module.

[0302] In some embodiments, the method includes detecting light, such as the first and second emitted light, using time bins. The disclosed apparatus and methods for determining biological nanoparticle characteristics can be performed quickly, with short signal integration times or fast bin times. Bin times can be used to assess, for example, the start-stop time of the examination of fluorescence to help filter information. Time bins (also referred to herein as signal integration times) can disclose time ranges within a histogram in which events occur or are observed. In some embodiments, the detection, measurement, and / or examination of biological nanoparticles uses time bins. In some embodiments, the time bins have a range of less than 10 ms, less than 5 ms, less than 1 ms, less than 0.5 ms, less than 0.1 ms, less than 90 μs, less than 80 μs, less than 70 μs, less than 60 μs, less than 50 μs, less than 40 μs, less than 30 μs, less than 20 μs, less than 10 μs, less than 5 μs, or less than 1 μs. In some embodiments, the time bins have values ​​between 10 ms and 0.1 ms, between 5 ms and 0.1 ms, between 1 ms and 0.1 ms, between 0.5 ms and 0.1 ms, between 0.1 ms and 1 μs, between 90 μs and 1 μs, between 80 ns and 1 μs, between 70 ns and 1 μs, between 60 ns and 1 μs, between 50 ns and 1 μs, between 40 ns and 1 μs, between 30 ns and 1 μs, between 20 ns and 1 μs, between 10 μs and 0.1 μs, between 5 μs and 0.1 μs, or between 1 μs and 0.1 μs. In a preferred embodiment, the time bins have a range between 1 μs and 2 ms.

[0303] In an embodiment, the method includes correlating the first and second emission signals based on emission signal characteristics or particle characteristics shared by the first and second emission signals. In an embodiment, correlating the first and second emission signals is based on emission signal characteristics or particle characteristics. A signal detected in a first detection window, or a first portion of the passageway, or a first excitation line, can be detected downstream in a second detection window, or a second portion of the passageway, or a second excitation line. In this regard, the particles can be tracked as they travel through the passageway. In addition, the particles can also be interrogated for a variety of different biomarkers. In an embodiment, the detection window includes and / or is at least partially defined by a portion of the passageway, or an excitation line of the excitation light, as discussed further herein.

[0304] In some embodiments, the method includes correlating the first emission signal and the second emission signal based on emission signal characteristics or based on particle characteristics. In some embodiments, correlating the first emission signal and the second emission signal includes comparing an intensity of the first emission signal to an intensity of the second emission signal. In some embodiments, the method further includes enumerating the number of particles passing through the passage based on correlating the first emission signal and the second emission signal. In some embodiments, the method further includes co-localizing the target molecule on the particle based on correlating the first emission signal with the second emission signal. In some embodiments, the method further includes determining a particle concentration based on correlating the first emission signal with the second emission signal.

[0305] Accurate counting and colocalization. The disclosed method is suitable for obtaining or determining a more accurate number of analytes (e.g., extracellular vesicles or biological nanoparticles or molecules) since it removes or reduces many interfering signals, such as background fluctuations and small aggregates of dyes, in identifying real events (e.g., extracellular vesicles or biological nanoparticles). Colocalization of single molecule events in flow streams by statistical methods (e.g., cross-correlation functions) is often adversely affected by interferences due to spatially close events, contamination, background fluctuations, and especially differences in linear velocity between particles induced by laminar flow profiles. Using the present method, it is possible to minimize these interferences and thus improve the quality of counting and colocalization.

[0306] Accurate co-localization of biomarkers, such as those expressed on the same biological nanoparticle, is the basis of many important applications (e.g., immunophenotyping to identify subtypes of biological nanoparticles or molecules). When multiple particles and / or molecules in close proximity in a flow stream pass through an excitation region or detection window, it can be difficult to correctly assign signals observed at different detection windows or from different excitation regions to a given particle and / or molecule, because the particles / molecules may flow at a wide range of velocities in the microfluidic passage due to the laminar and parabolic flow profile of the microfluidic flow environment. Using the methods of the present disclosure, these challenges are addressed to enable accurate co-localization of biomarkers on a single biological nanoparticle or molecule.

[0307] Accurate sampling of flow rates. The self-corrected method of the present disclosure provides the transit time of each particle flowing through different positions within the cross section of the passage. With the known spacing between the portions of the passage or detection window illuminated by the different spatially separated excitation lights, it is possible to calculate the linear velocity of each particle examined and determine the volumetric flow rate sample accordingly. The average linear velocity can be converted to a volumetric flow rate through the passage.

[0308] Knowing the volumetric flow rate in microfluidic-based analysis is often required to determine the volume of the sample analyzed during an experiment. Thus, the absolute concentration of a single particle / molecule can be measured based on the counting and analyzed volume of the analyte / molecule / nanoparticle. The volumetric flow rate is also a useful parameter to evaluate the throughput and consumption of the sample. Although important, direct measurement of the volumetric flow rate is often difficult in a microfluidic environment, especially when the volumetric flow rate is very low (e.g., pL to nL / sec), because extremely small volumes of the sample are interrogated by the excitation region. Using the method of the present disclosure, the volumetric flow rate can be determined using the transit time of each molecule and / or particle flowing through the laser line or excitation region, based on the fact that in this microfluidic environment the flow is laminar. As a result, the linear velocity of each particle and / or molecule can be calculated and the distance between these laser lines or excitation regions can be known. From the measured average particle and / or molecular velocity and by knowing the area of ​​the passage cross section, the volumetric flow rate can be measured. Thus, using the methods of the present disclosure, volumetric flow rates can be determined by using the measured transit times and / or velocities of single particles and / or molecules.

[0309] Accurate measurement of concentration. The method is also suitable for accurately measuring the concentration of analytes, since it is possible to accurately determine the number of single analytes tested in a given time and, by knowing the volumetric flow rate, obtain the volume of the analyzed sample (often at the nanoliter level).The disclosed method provides an absolute count of analytes in a given volume without relying on parameters (e.g., extinction coefficient) obtained from the bulk sample and any external calibration curve, allowing for a more accurate determination of the analyte concentration.

[0310] Accurate determination of detection efficiency and recovery rate. Detection efficiency may be defined as the percentage of analyte counted, such as by the disclosed method, within the analyte flowing through a passage or excitation region, or detection region or detection window. If the distribution of the signal associated with the analyte flowing through the passage follows a known statistical model (e.g., a log-normal distribution commonly found in flow analysis), it is possible to quantify the detection efficiency by knowing the cumulative distribution function (CDF) at the cutoff value.

[0311] Recovery, e.g. defined as the ratio of the counted analyte to the amount of analyte spiked or present in a given volume, is affected by many other factors in addition to detection efficiency, e.g. precision of the stock concentration, possible aggregation and degradation of the analyte, surface absorption, etc. If the stock concentration of the analyte is precisely known, it is also possible to determine the recovery accordingly.

[0312] Accurate copy number determination As further discussed herein with respect to Example 10, the method of the present disclosure is suitable for determining the copy number of a biomarker present on a single particle. Because the method is suitable for detecting the entire population of single molecules / particles present in a sample or an aliquot of a sample, or a very large percentage (e.g., more than 90%) of such molecules / particles passing through a microchannel, the method can also use the single molecule intensity distribution to deconvolute the single particle intensity distribution to accurately determine the number of bound antibodies, and therefore the corresponding proteins, on each particle. Such an approach is useful, for example, to determine the number of fluorescently labeled antibodies associated with a particle such as an EV, providing quantitative information about the molecular composition of a single particle. This information, together with other information about the size of the particle, can be used in determining whether the analyzed particle is intact or fragmented, or whether the analyzed particle is empty (e.g., contains nucleic acid), non-functional, or biologically functional.

[0313] As discussed further herein, in certain embodiments, the methods of the present disclosure do not include an amplification step, for example, to generate an amplicon or copy of an analyte or a molecule that correlates with the presence of the analyte. In this regard, in certain embodiments, the methods of the present disclosure do not include the use of reagents and / or conditions used or required for amplification of a target analyte or a molecule that correlates with the presence of a target analyte, for example, nucleic acid amplification or protein-based amplification of a target analyte or a molecule that correlates with the presence of a target analyte, for example, rolling circle amplification or ELISA.

[0314] Automatic focus adjustment method In another aspect, the present disclosure provides a method for focusing the optical components of a system onto a passageway of the system. Counting and measuring particles and molecules in a flow, such as extracellular vesicles, viruses, lipoproteins, RNA-binding proteins, or cytokines, at the level of a single molecule / particle, is often very sensitive to changes in the environment (e.g., thermally induced expansion) and changes in the instrument configuration (e.g., subtle drifts in optical alignment and variations in passageway dimensions). To consistently collect data and improve the sensitivity of flow-based devices, the present disclosure provides an automatic focusing method.

[0315] In an embodiment, excitation light, such as a particular laser light (e.g., 870 nm), reflected back by the microfluidic device is collected, such as via a fiber-coupled confocal scheme, as further discussed herein with respect to FIG. 7A. The magnitude of such back reflection is externally calibrated and attenuated with neutral density filters to ensure that it is within the dynamic range of the photodetector. In an embodiment, a preferred or optimal value of the back reflected light is determined when correct focusing of the detection passage is achieved and set as a reference for "locking" the focus level (see, e.g., FIG. 7B). In an embodiment, a portion of the microfluidic device, such as the passage containing the inspection window, or the objective lens, is operably coupled to a motorized movable stage, such as a stage driven by a piezoelectric or DC motor. In this regard, the collection system is configured to move relative to the inspection window of the passage to focus the collection system on the inspection window. As detailed in FIG. 7C, the movable stage can be controlled based on comparing the current value of the reflection with a value at a previous time (e.g., 200 ms ago) and with a reference value.

[0316] Thus, in an embodiment, the present disclosure provides a method of focusing excitation light onto a fluid passageway through an optical component. In an embodiment, the method includes using a system 700, discussed further herein with respect to FIGs. 7A and 7B. In an embodiment, the method includes illuminating an inspection window or other portion of the fluid passageway with light from a light source, focusing the light onto the inspection window using an optical component disposed between the passageway and a photodetector, generating a lock signal at the photodetector based on the focused light reflected back from the inspection window at a first time, generating a test signal at the photodetector based on the focused light reflected back from the inspection window at a second time after the first time, determining whether the test signal is within a predetermined percentage of the lock signal, and moving the fluid passageway relative to a high NA air objective lens if the test signal is outside the predetermined percentage of the lock signal.

[0317] As shown in Figures 7C, 7F, and 7G, in one embodiment, the predetermined percentage is about 5%. In one embodiment, the predetermined percentage is in the range of about 0.5% to about 15%, in the range of about 1% to about 10%, in the range of about 2% to about 8%, in the range of about 2.5% to about 7.5%, in the range of about 3% to about 6%, or in the range of about 3% to about 5%.

[0318] In an embodiment, the present disclosure provides a method of maintaining focus on a fluid passage, the method including illuminating an imaging region of a microfluidic system with light from a near infrared light source, generating an image of the imaging region with a camera, determining an amount of defocus of the image, determining whether the amount of defocus is within a predetermined amount of defocus, and moving the fluid passage relative to a high NA air objective lens if the test signal is outside the predetermined range. In an embodiment, the structure imaged is a structure adjacent to the passage, and in certain embodiments, a structure separate from the passage. In an embodiment, the structure has a high level of contrast relative to other portions of the imaging region. In an embodiment, the structure defines an air-filled enclosure in the microfluidic system. Such an air-filled structure has a higher contrast than, for example, a fluid-filled passage, and in this regard is suitable for generating an image and determining the amount of defocus of the image.

[0319] Figure 7D is a series of images of a passageway taken at several distances from a high NA air objective and with different amounts of defocus, according to an embodiment of the present disclosure. Figure 7E illustrates the amount of focusing quality at various distances between the passageway and the high NA air objective, noting the location of the images in Figure 7D, according to an embodiment of the present disclosure. In the depicted embodiment, the focal plane was detected using NIR imaging and a 0.95 NA air objective.

[0320] As shown, the focal passage contains a constriction. The focusing quality of the constriction was monitored in real time, as shown in Figure 7E. As the objective lens was moved up, the focusing quality improved until it reached a first maximum, indicating that the focal plane was at the bottom of the constriction passage. As the objective lens position increased, the focusing quality decreased and then rose to a second maximum, suggesting that the focal plane was set at the top of the constriction passage. The four photographs in Figure 7D show real-time imaging when the high NA air objective lens was in four positions accordingly.

[0321] Figure 7F illustrates a feedback control loop used to set the focal plane according to an embodiment of the present disclosure. Figure 7G illustrates another feedback control loop used to implement near-infrared machine vision assisted real-time focusing through a high NA air objective lens according to an embodiment of the present disclosure. The focusing method illustrated in Figures 7F and 7G can be used to achieve focusing of the passageway.

[0322] In one embodiment, the method includes collecting focused light reflected back from the inspection using a collection system, the collection system comprising an air objective lens having a numerical aperture in the range of about 0.91 to less than 0.99, or about 0.95.

[0323] In some embodiments, the method includes collecting light to generate an image of the imaging area with a camera by collecting light with a collection system, the collection system comprising an air objective lens having a numerical aperture in the range of about 0.91 to less than 0.99, or about 0.95.

[0324] In some embodiments, the light is in the range of about 700 nm to about 1.5 nm. In some embodiments, the light is in the range of about 700 nm to about 1100 nm. In some embodiments, the light is in the non-visible wavelength range.

[0325] Digital affinity-based detection assays In an embodiment, the method is a method for digital affinity-based detection assay. In an embodiment, the digital affinity-based detection assay can be performed using or on one or more of the systems of the present disclosure. As used herein, "digital affinity assay" refers to an assay that includes the detection, identification, and / or enumeration of individual single molecule analytes through specific association with one or more detectable agents and / or capture agents. In an embodiment, such a digital affinity assay includes the detection of individual single protein analyte molecules in the case of a digital protein assay, or the detection of individual single nucleic acid molecules in the case of a digital nucleic acid assay. For example, a digital protein assay can include single molecule detection, identification, and concentration determination of non-fluorescent protein molecules through specific association with one or more fluorescent detectable agents (e.g., fluorescently labeled antibodies) and / or capture agents (e.g., antibody-antigen binding). Similarly, for example, digital nucleic acid assays can include single molecule detection, discrimination, and concentration determination of non-fluorescent nucleic acid molecules through specific association (e.g., nucleic acid hybridization) with one or more fluorescent detectable agents (e.g., fluorescently labeled nucleic acid probes) and / or capture agents. In certain embodiments, such digital affinity assays include detection of individual single protein analyte molecules or individual single nucleic acid molecules without the use of amplification.

[0326] In certain embodiments, the method includes associating an analyte in a sample with a detectable agent, flowing the sample containing the analyte associated with the detectable agent, and in certain embodiments, associated with a capture agent, through a flow path, outputting excitation light through an interrogation window through a portion of the flow path, and generating an emission signal at a photodetector based on emission light received from the portion of the flow path. In this regard, attention is directed to FIG. 9A, which is a block diagram of a method 900A in accordance with an embodiment of the present disclosure.

[0327] In one embodiment, method 900A begins at process block 901A, which includes associating an analyte with an association agent. In one embodiment, the association agent is a primary antibody configured to selectively associate with an analyte, such as a protein or peptide. In one embodiment, the association agent is a nucleic acid probe configured to selectively associate with an analyte that includes a nucleic acid sequence. In one embodiment, the association agent is not configured to generate a detectable signal, such as in response to excitation, such as through illumination with an excitation light. In one embodiment, process block 901A is optional.

[0328] In some embodiments, associating the analyte with the association agent comprises incubating the association agent with the analyte under conditions and for a time sufficient to allow the association agent to associate with the analyte through molecular recognition, antigen-antibody binding, nucleic acid hybridization, covalent binding, non-covalent binding, etc.

[0329] In one embodiment, method 900A begins at process block 903A or process block 901A is followed by process block 903A, which includes associating the analyte with a detectable agent. As discussed further herein, associating the analyte with the detectable agent can make the analyte detectable, such as through detecting a signal produced by the detectable agent and inferring the presence of the analyte based on the time and / or location at which the signal is detected, etc.

[0330] In certain embodiments, allowing the analyte to associate with the detectable agent and / or association agent comprises incubating the detectable agent and / or association agent with the analyte under conditions and for a time sufficient to allow the detectable agent and / or association agent to associate with the analyte through molecular recognition, antigen-antibody binding, nucleic acid hybridization, covalent binding, non-covalent binding, etc.

[0331] In one embodiment, next in process 901A is process block 903A, associating the analyte with the detectable agent includes associating the detectable agent with an association agent that is associated with the analyte. In such an embodiment, the association agent is, for example, a first antibody configured to associate with the analyte and the detectable agent is a second antibody configured to associate with the association agent.

[0332] In certain embodiments, associating the analyte with the detectable agent includes directly associating the analyte with the detectable agent, such as where there is no intermediate component disposed between the analyte and the detectable agent (e.g., a primary antibody where the detectable agent is a secondary antibody). In this embodiment, the primary antibody is, for example, a fluorescently tagged antibody and is thus the detectable agent.

[0333] In certain embodiments, an assay, such as a digital affinity assay, includes several binding events, such as those discussed with respect to the association steps of process blocks 901A-907A. In certain embodiments, a digital affinity assay includes one affinity binding event to a single molecule analyte. In certain embodiments, a digital affinity assay includes two affinity binding events to a single molecule analyte. In certain embodiments, a digital affinity assay includes more than two affinity binding events to a single molecule analyte.

[0334] In an embodiment, following process block 903A is process block 905A, which includes associating the analyte with a second detectable agent. In an embodiment, the detectable agent is a first detectable agent configured to emit a first emitted light in a first wavelength range upon excitation of the first detectable agent, and the method further includes associating with the analyte a second detectable agent configured to emit a second emitted light in a second wavelength range upon excitation of the second detectable agent, the first emitted wavelength range being distinct from the second emitted wavelength range. In such a scenario, the first and second detectable agents associated with the analyte are detectable, e.g., simultaneously or separately. Thus, the analyte can be assayed through the methods of the present disclosure to detect two or more sites or sequences in or on the analyte. For example, in an embodiment, the first detectable agent is associated with a first site on the analyte and the second detectable agent is associated with a second site on the analyte that is distinct from the first site. In some embodiments, the assay is a digital protein assay. In some embodiments, the assay is a digital sandwich assay. In this example, for a digital protein assay (such as a digital sandwich assay), the first site is an epitope on the analyte that binds to a first detectable agent, such as a first antibody, and the second site is a different epitope on the analyte that binds to a second detectable agent, such as a second, different antibody. In this example, for a digital nucleic acid assay, the first site is a first portion of the sequence of the analyte nucleic acid that hybridizes to a first detectable agent, such as a first probe nucleic acid sequence that is complementary to a first portion of the sequence on the analyte nucleic acid, and the second site is a second portion of the sequence on the analyte nucleic acid that is different from the first portion and hybridizes to the first detectable agent, such as a second probe nucleic acid sequence that is complementary to a second portion of the sequence on the analyte nucleic acid. In both of these examples, the first and second antibodies or the first and second nucleic acid probes can be labeled with different fluorescent entities, such as different fluorescent dyes, or different fluorescent barcoded magnetic beads, or a combination thereof.

[0335] In one embodiment, process block 905A is optional.

[0336] In an embodiment, following process blocks 901A, 903A, and / or 905A is process block 907A, which includes associating the analyte with a capture agent. As discussed further herein, in an embodiment, the capture agent is configured to isolate the analyte from a portion of the sample when the analyte is associated with the capture agent and when the capture agent is subjected to an isolation procedure. As discussed further herein, the capture agent can include beads, such as magnetic beads. In an embodiment, the capture agent includes a surface having attached or otherwise associated moieties configured to specifically associate with the analyte.

[0337] In some embodiments, the capture agent comprises a bead. In some embodiments, the bead is magnetic. In some embodiments, the bead is fluorescent. In some embodiments, the bead is fluorescent with a spectral intensity optical barcode. In some embodiments, the bead is both magnetic and fluorescent. In some embodiments, the bead is both magnetic and fluorescent with a spectral intensity optical barcode. In some embodiments, the bead is configured to isolate the capture agent and associated analyte from a portion of a sample through application of a magnetic field via magnetic bead capture, centrifugation, magnetophoresis, etc.

[0338] In some embodiments, the capture agent does not comprise a bead, but is otherwise separable from a portion of the sample. In some embodiments, the capture agent is bound to a surface, as discussed further herein. In such embodiments, the analyte is associated with the surface-bound capture agent, which can then be eluted or otherwise cleaved from the surface.

[0339] In some embodiments, the capture agent is configured to emit capture agent radiation upon excitation. In some embodiments, the bead is a fluorescent bead configured to emit bead fluorescence upon excitation with an excitation light. In some embodiments, the fluorescent bead is a spectral intensity barcoded bead or nanoparticle configured to emit bead fluorescence upon excitation with an excitation light. In some embodiments, the bead comprises a plurality of distinct chromophores, each chromophore of the plurality of distinct chromophores comprising a predetermined set of adjustable optical encoding parameters, thereby defining an optically detectable code for the capture agent. In some embodiments, the optically detectable code comprises a predetermined emission spectrum of the capture agent. In some embodiments, the optically detectable code comprises a predetermined absorption spectrum of the capture agent. In some embodiments, the optically detectable code comprises a predetermined emission and absorption spectrum of the capture agent.

[0340] In some embodiments, the beads have a diameter of less than 1 micrometer. In some embodiments, the beads have a diameter of less than 1 micrometer, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. In some embodiments, the beads have a diameter of less than 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, or 40 nm. In some embodiments, the beads have a diameter of more than 1 micrometer. In some embodiments, the beads have a diameter of 1 to 5 micrometers.

[0341] In certain embodiments, the ratio of capture agent to detectable agent ranges from 10:1 to 1:10, 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2, and 1:1. In certain embodiments, the ratio of capture agent to detectable agent is about 1:1.

[0342] In one embodiment, process block 907A is optional.

[0343] In one embodiment, following process block 907A is process block 909A, which includes isolating the analyte associated with the capture agent from a portion of the sample. In one embodiment, the capture agent includes optically barcoded magnetic beads, and isolating the analyte associated with the capture agent from the sample includes associating the magnetic beads with a magnet and removing a portion of the sample not associated with the magnet from the analyte associated with the capture agent to provide a purified sample. In one embodiment, the capture agent includes beads, and isolating the analyte associated with the capture agent from the sample includes centrifuging the sample including the capture agent associated with the analyte to provide a supernatant and a precipitate including the analyte associated with the capture agent, and decanting or removing the supernatant to provide a purified sample. In one embodiment, the capture agent includes beads, and isolating the analyte bound to the capture agent from the sample includes passing the sample including the analyte associated with the capture agent through a size exclusion chromatography column to provide a purified sample including the analyte associated with the capture agent.

[0344] As described above, in some embodiments, the capture agent is bound to a surface. In some embodiments, the surface is a surface of a well plate, such as a 96- or 384-well plate. In some embodiments, the surface is a surface of a bead. In some embodiments, the surface is a surface of a microfluidic channel. In some embodiments, isolating the analyte associated with the capture agent from the portion of the sample comprises removing the portion of the sample that is not associated with the capture agent bound to the surface.

[0345] In one embodiment, process block 909A is optional.

[0346] In some embodiments, following process block 903A, 905A, 907A, or 909A is process block 911A, which includes contacting the sample with removal beads. In some embodiments, the removal beads are configured to selectively associate with any detectable agent that is not associated with the analyte. In some embodiments, process block 911A further includes removing removal beads associated with detectable agents that are not associated with the analyte from the sample. As discussed further herein, such use of removal beads may be suitable for, or otherwise configured to remove, unbound detectable agents (i.e., detectable agents that are not associated with the analyte). Such unbound or unassociated detectable agents may generate undesirable signals when detected downstream of the interrogation window. Signals from unbound or unassociated detectable agents may complicate the distinction of signals from detectable agents associated with the analyte, and removing unbound or unassociated detectable agents from the sample makes it easier to detect the analyte associated with the detectable agent.

[0347] In one embodiment, process block 911A is optional.

[0348] In an embodiment, following process block 903A, 905A, 907A, 909A, or 911A is process block 913A, which includes flowing a sample including an analyte associated with a detectable agent through the flow path. In an embodiment, flowing a sample including an analyte associated with a detectable agent through the flow path includes flowing a plurality of analytes of the sample including an analyte associated with a detectable agent and a capture agent through the flow path. In an embodiment, flowing a plurality of analytes of the sample including an analyte associated with a detectable agent and a capture agent through the flow path and flowing the analyte associated with a detectable agent and a capture agent through the flow path includes, for each analyte associated with a capture and detectable agent, flowing the analyte associated with the detectable agent and capture agent through a constriction in the flow path. Although a complex including a detectable agent and a capture agent associated with the analyte is described, it is understood and within the scope of the present disclosure that a complex including a first and second detectable agent is prepared and flowed through the flow path for each analyte associated with a first and second detectable agent. Additionally, although a second detectable agent is described, it will be understood that the capture agent can be replaced with a second detectable agent, and it is within the scope of the present disclosure to prepare and flow through a flow path a complex comprising two or more detectable agents for each analyte associated with two or more detectable agents.

[0349] In an embodiment, flowing the sample including the analyte associated with the detectable agent through the flow path includes flowing a purified sample, such as the purified sample prepared at process block 909A or 911A, through the flow path. In an embodiment, the flow path is a flow path of a device or system further described herein that includes an inspection window. In an embodiment, the device includes one or more portions of a system of the present disclosure. In this regard, in an embodiment, the flow path is a flow path of a system of the present disclosure. In an embodiment, the flow path defines a constriction as defined and described elsewhere herein.

[0350] In some embodiments, flowing the analyte associated with the detectable agent through the flow path includes flowing a complex including the analyte associated with the capture agent and the detectable agent. In some embodiments, the complex includes an optically barcoded bead with the bound analyte, which is further bound to a detectable agent. In some embodiments, the complex is an optically barcoded bead with a capture primary antibody bound to an analyte, such as a protein, via an epitope on the analyte, which is further bound to another fluorescently tagged primary antibody via a different site or epitope. In some embodiments, the complex is an optically barcoded bead with a capture nucleic acid sequence hybridized to the analyte nucleic acid via a first sequence on the analyte nucleic acid, which is further hybridized to another fluorescently tagged nucleic acid probe that serves as the detectable agent via a second sequence different from the first sequence.

[0351] In some embodiments, flowing the sample through the flow channel excludes sheath flow focusing, acoustic flow focusing, or a combination thereof.

[0352] In one embodiment, following process block 913A is process block 915A, which includes outputting excitation light through an interrogation window through a portion of the flow path. In one embodiment, outputting excitation light through the interrogation window through a portion of the flow path includes outputting excitation across a cross-section of a lumen of the flow path. Outputting excitation across a cross-section of a lumen of the flow path is distinct from outputting excitation light only to a portion of the cross-section of the lumen, such that particles or molecules and detectable agents associated therewith can pass through the lumen without being contacted by the excitation light.

[0353] In some embodiments, process block 915A is followed by process block 917A or is simultaneous with process block 917A, which includes outputting a second excitation light through the interrogation window into a second portion of the flow path. Such a second excitation light may be suitable and / or configured to excite, for example, a second detectable agent or capture agent. In some embodiments, process block 917A is optional.

[0354] In one embodiment, following process block 915A and / or 917A is process block 919A, which includes generating a detectable agent emission signal, such as with a photodetector, based on the detectable agent emission light received from a portion of the flow path. In one embodiment, the photodetector is an example of a photodetector as described further herein with respect to the systems of the present disclosure. In one embodiment, the detectable agent emission signal is based on the detectable agent emission light emitted from the detectable agent.

[0355] As noted above, in some embodiments, flowing the sample through the flow path, the sample including the analytes associated with the detectable agent, includes flowing through the flow path a plurality of analytes of the sample including the analytes associated with a detectable agent and a capture agent, In such embodiments, process block 919A may include generating a plurality of detectable agent emission signals based on the detectable agent emission light from the detectable agent.

[0356] As discussed further herein, the emission signal of the detectable agent, such as in combination with other signals, is suitable for determining the presence of an analyte in the flow path.

[0357] In one embodiment, process block 919A is followed by process block 921A which includes generating a capture agent emission signal based on the capture agent emission light. As discussed further herein, in one embodiment, the capture agent is configured to emit light upon excitation, etc. In one embodiment, generating the capture agent emission signal includes illuminating the capture agent with excitation light and detecting the capture agent emission light with a photodetector. In one embodiment, generating the capture agent emission signal, such as with a second photodetector, is based on capture agent emission light received from a second portion of the flow path different from the portion from which the detectable agent emission light is emitted.

[0358] In certain embodiments, the capture agent is optically encoded or otherwise configured to emit a signal that defines tunable optical encoding parameters. As discussed further herein, such encoded capture agents are suitable for detecting several analytes, such as when different encoded capture agents configured to selectively associate with different analytes are used in assaying a sample. In certain embodiments, the capture agent comprises a plurality of distinct chromophores, each chromophore of the plurality of distinct chromophores comprising a predetermined set of tunable optical encoding parameters, thereby defining an optically detectable code for the capture agent. In certain embodiments, the optically detectable code comprises a predetermined emission spectrum of the capture agent, a predetermined absorption or excitation spectrum of the capture agent, or a combination thereof.

[0359] As noted above, in some embodiments, flowing the sample through the flow path, the sample including the analytes associated with the detectable agent, includes flowing through the flow path a plurality of analytes of the sample including the analytes associated with the detectable agent and the capture agent, hi such embodiments, process block 921A includes generating a plurality of capture agent emission signals based on the capture agent emission light from the capture agent.

[0360] In an embodiment, the method 900 includes correlating or co-localizing the emitted light of the detectable agent with the emitted light of the capture agent to determine the presence and / or identity of the analyte. In an embodiment, the method 900 includes correlating or co-localizing the emitted light of the detectable agent with the emitted light of a second detectable agent to determine the presence and / or identity of the analyte. In an embodiment, the method 900 includes correlating or co-localizing the emitted light of the detectable agent with the emitted light of the capture agent and the emitted light of a second detectable agent to determine the presence and / or identity of the analyte. In an embodiment, the method 900 includes correlating or co-localizing the emitted light of two or more detectable agents to determine the presence and / or identity of the analyte.

[0361] In one embodiment, process block 921A is optional. In one embodiment, process blocks 919A and 921A are followed by process block 923A, which includes quantifying the number of detectable agent emission signals associated with the capture agent emission signal or the second detectable agent emission signal, or the ratio of the number of detectable agent emission signals associated with the capture agent emission signal or the second detectable agent emission signal to the number of capture agent emission signals, the number of detectable agent emission signals, the number of second detectable agent emission signals, or a combination thereof.

[0362] In one embodiment, following process blocks 919A and 921A is process block 923A, which includes quantifying the number of detectable agent emission signals associated with the emission signals of one or more additional detectable agents, or the ratio of the number of detectable agent emission signals associated with the emission signals of one or more additional detectable agents to the number of individual detectable agent emission signals, or a combination thereof. As discussed further herein, the methods of the present disclosure are suitable for generating signals associated with single molecule analytes passing through the flow path. Such signals can be counted and associated with such single molecule analytes to further count the number of analytes associated with a detectable agent, capture agent, second detectable agent, additional detectable agent, etc. passing through the flow path, such as over a given period of time.

[0363] In one embodiment, process block 923A is followed by process block 925A which includes determining a concentration of analyte associated with the emission signal. In one embodiment, such determination is based on the number of analytes associated with an emission signal, such as the detectable agent emission signal, the second detectable agent emission signal, and / or the capture agent emission signal. In one embodiment, determining the concentration of the analyte is based on the volume of liquid flowing through the flow path.

[0364] In some embodiments, determining the concentration of the analyte is based on the number of analytes associated with both emitted light from a detectable agent and a capture agent (e.g., determined from a capture agent emitted signal associated with a detectable agent emitted signal), or associated with emitted light from two or more detectable agents (e.g., determined from a detectable agent emitted signal associated with a second detectable agent emitted signal). In some embodiments, determining the concentration of the analyte is based on analytes associated with emitted light. In some embodiments, as discussed above with respect to process block 923A, analytes associated with emitted light include analytes associated with capture agent emitted light and / or detectable agent emitted light. Thus, in some embodiments, determining the concentration of the analyte is based on the number of capture agent emitted signals associated with detectable agent emitted signals. In some embodiments, determining the concentration of the analyte is based on the volume of liquid flowing through the flow path.

[0365] In certain embodiments, determining the concentration of the analyte is based on the ratio of the number of emission signals of the detectable agent associated with the capture agent emission signal to the number of capture agent emission signals. In certain embodiments, determining the concentration of the analyte is based on the ratio of the number of emission signals of the detectable agent associated with the capture agent emission signal to the number of emission signals of the detectable agent. In certain embodiments, determining the concentration of the analyte is based on the ratio of the number of emission signals of the detectable agent associated with the capture agent emission signal to the number of capture agent emission signals, the ratio of the number of emission signals of the detectable agent associated with the capture agent emission signal to the number of emission signals of the detectable agent, or a combination thereof. In this regard, determining the concentration of the analyte in the sample takes into account the emission light of the detectable agent and the capture agent emission light, e.g., co-co-located (e.g., when associated with the analyte) as well as detectable agents and capture agents that are not associated with the analyte.

[0366] In some embodiments, determining the concentration of the analyte associated with the emission signal is based on the ratio of the number of analytes associated with both a detectable agent emission signal and a capture agent emission signal to the number of capture agent emission signals but not a detectable agent emission signal, or the number of detectable agent emission signals but not a capture agent emission signal, or a combination thereof. In some embodiments, determining the concentration of the analyte associated with the emission signal is based on the number of analytes associated with two or more detectable agent emission signals. In some embodiments, determining the concentration of the analyte associated with the emission signal is based on the ratio of the number of analytes associated with two or more detectable agent emission signals to the number of non-analyte associated emission signals.

[0367] In certain embodiments, determining the concentration of the analyte is further based on a flow rate of the sample through the passageway.

[0368] 9B is a block diagram of method 900B according to an embodiment of the present disclosure. In an embodiment, method 900B may be implemented on and / or using one or more components of a system according to an embodiment of the present disclosure, as described elsewhere herein. In an embodiment, method 900B is a digital affinity assay, such as a digital protein or nucleic acid assay. In an embodiment, method 900B is an example of method 900A.

[0369] In one embodiment, method 900B begins with process block 901B, which includes associating an analyte in a sample with a detectable agent. In one embodiment, process block 901B is similar to and / or an example of process block 901A. As discussed further herein with respect to process block 901A specifically, and method 900A generally, in one embodiment, the analyte can include a protein, the detectable agent can include a fluorescently labeled antibody, and / or the detectable agent can include a nucleic acid, the detectable agent can include a fluorescently labeled nucleic acid probe.

[0370] In one embodiment, following process block 901B is process block 903B, which includes associating the analyte with a capture agent. In one embodiment, the capture agent is configured to isolate the analyte from a portion of the sample when the analyte is associated with the analyte and when the capture agent is subjected to an isolation procedure. In one embodiment, process block 903B is similar to and / or an example of process block 907A.

[0371] In one embodiment, following process block 903B is process block 905B, which involves isolating the analyte associated with the capture agent from a portion of the sample. As discussed further herein with respect to the detection schemes of the present disclosure, such isolation may include, for example, the use of a magnet to isolate the capture agent or magnetic beads of the removal beads, centrifugation, size exclusion chromatography, etc. In one embodiment, process block 905B is an example of process block 909A.

[0372] In one embodiment, following process block 901B or process blocks 903B and 905B is process block 907B, which includes flowing a sample including an analyte associated with a detectable agent through the flow path. In one embodiment, flowing a sample including an analyte associated with a detectable agent through the flow path includes flowing a plurality of analytes of the sample including an analyte associated with a detectable agent and a capture agent through the flow path. In one embodiment, the method includes flowing a plurality of analytes of the sample including an analyte associated with a detectable agent and a capture agent through the flow path, and flowing the analytes associated with the detectable agent and the capture agent through the flow path includes, for each analyte associated with a capture and detectable agent, flowing the analyte associated with the detectable agent and the capture agent through a constriction in the flow path. In one embodiment, flowing a sample including an analyte associated with a detectable agent through the flow path includes flowing a plurality of analytes of the sample including an analyte associated with a detectable agent through the flow path. In an embodiment, the method wherein flowing the analyte through the flow path comprises flowing a plurality of analytes of the sample, the analyte being associated with a detectable agent, through the flow path, and wherein flowing the analyte associated with the detectable agent through the flow path comprises, for each analyte associated with a detectable agent, flowing the analyte associated with the detectable agent through a constriction in the flow path. In an embodiment, flowing the analyte through the flow path comprises flowing a plurality of analytes of the sample, the analyte being associated with two or more detectable agents, through the flow path. In an embodiment, the method wherein flowing the analyte through the flow path comprises flowing a plurality of analytes of the sample, the analyte being associated with two or more detectable agents, through the flow path, and wherein flowing the analyte associated with two or more detectable agents through the flow path comprises, for each analyte associated with a detectable agent, flowing the analyte associated with the detectable agent through a constriction in the flow path.

[0373] In one embodiment, process block 907B is an instance of process block 913A.

[0374] In an embodiment, flowing the sample through the flow path comprises flowing the sample through a flow path of a system according to an embodiment of the present disclosure, in an embodiment, the flow path defines a constriction as described elsewhere herein.

[0375] In one embodiment, following process block 907B is process block 909B, which includes outputting excitation light through an interrogation window in a portion of the flow path. In one embodiment, process block 909B is an example of process block 915A. In one embodiment, the excitation light is configured to optically excite the detectable agent and / or capture.

[0376] In one embodiment, process block 909B is followed by process block 911B which includes outputting a second excitation light through an interrogation window in the flow path. In one embodiment, the second excitation light is configured to excite a capture agent and / or a second detectable agent, as described elsewhere herein. In one embodiment, process block 911B is an example of process block 917A. In one embodiment, process block 911A is optional.

[0377] In one embodiment, following process block 909B or 911B is process block 913B, which includes generating a detectable agent emission signal, such as with a photodetector, based on the detectable agent emission light received from the flow path. In one embodiment, the detectable agent emission light is generated in response to the detectable agent receiving excitation light from process block 909B.

[0378] As noted above, in some embodiments, flowing the sample through the flow path includes flowing a plurality of samples through the flow path, the samples including analytes associated with a detectable agent and a capture agent, and in some embodiments, generating a detectable agent emission signal further includes generating a plurality of detectable agent emission signals based on detectable agent emission light from the detectable agent.

[0379] In one embodiment, process block 911B is an instance of process block 919A.

[0380] In one embodiment, following process block 913B is process block 915B, which includes generating a capture agent emission signal based on the capture agent emission light received from the flow path. In one embodiment, generating a capture agent emission signal includes or is part of generating a plurality of capture agent emission signals based on the capture agent emission light from the capture agent. In one embodiment, process block 915B is an example of process block 921A.

[0381] In one embodiment, process block 915B is followed by process block 917B, which includes quantifying the number of detectable agent emission signals associated with the capture agent emission signals or the emission signals of a second detectable agent. In one embodiment, process block 917B includes quantifying a ratio of the number of detectable agent emission signals associated with the capture agent emission signals or the emission signals of a second detectable agent to the number of capture agent emission signals, the number of detectable agent emission signals, the number of second detectable agent emission signals, or a combination thereof. In one embodiment, process block 917B includes quantifying the number of detectable agent emission signals associated with two or more detectable agent emission signals.

[0382] In one embodiment, process block 917B is an instance of process block 923A.

[0383] In one embodiment, following process block 917B is process block 919B, which includes determining a concentration of an analyte associated with emitted light, such as emitted light including a detectable agent emitted light and / or a capture agent emitted light. In one embodiment, process block 919B includes determining a concentration of the analyte based on a number of capture agent emission signals associated with the detectable agent emission signals. In one embodiment, determining the concentration of the analyte is based on a volume of liquid flowing through the flow path. In one embodiment, determining the concentration of the analyte is based on a ratio of a number of detectable agent emission signals associated with the capture agent emission signals to the number of capture agent emission signals. In one embodiment, determining the concentration of the analyte is based on a ratio of a number of detectable agent emission signals associated with the capture agent emission signals to the number of detectable agent emission signals. In an embodiment, determining the concentration of the analyte and / or particles and / or molecules associated with the emission signal is further based on a ratio of the number of particles of the analyte associated with both the detectable agent emission signal and the capture agent emission signal to the number of particles associated with only the capture agent emission signal but not the detectable agent emission signal, or only the detectable agent emission signal but not the capture agent emission signal, or a combination thereof. In an embodiment, determining the concentration of the analyte is based on quantifying the number of detectable agent emission signals associated with the capture agent emission signal. ...

Claims

1. 1. A method for a digital affinity-based single molecule detection assay, said method comprising: associating a single molecule analyte in a sample with a detectable agent; flowing the sample containing the analyte associated with the detectable agent through a flow path; outputting the excitation light through the inspection window through a portion of the flow path; generating an emission signal at a photodetector based on the emission light received from the portion of the flow path through the interrogation window; associating the analyte with a capture agent configured to isolate the analyte from a portion of the sample when the analyte is associated with the capture agent and when the capture agent is subjected to an isolation procedure; isolating the analyte associated with the capture agent from a portion of the sample to provide a purified sample; flowing the analyte associated with the detectable agent through the flow path comprises flowing a complex comprising the capture agent and the analyte associated with the detectable agent; outputting the excitation light through the inspection window and through the portion of the flow path includes outputting the excitation light across a cross-section of a lumen of the flow path; the association between the analyte and the detectable agent and the association between the analyte and the capture agent are based on specific molecular recognition; flowing a plurality of analytes of the sample through the flow path, the analytes being associated with the detectable agent and the capture agent, wherein flowing the analyte associated with the detectable agent and the capture agent through the flow path comprises, for each analyte associated with a capture and detectable agent, flowing the analyte associated with the detectable agent and the capture agent through a constriction in the flow path; generating a plurality of detectable agent emission signals based on the detectable agent emission light from the detectable agent; generating a plurality of capture agent emission signals based on the capture agent emission light from the capture agent; the channel is disposed in a portion of a microfluidic device; the microfluidic device defines a planar portion; The method, wherein the flow path within the inspection window defines a constriction relative to an adjacent portion of the flow path.

2. The method of claim 1, wherein the capture agent comprises beads.

3. The method of claim 2, wherein the beads are magnetic.

4. The method described in claim 2, wherein the beads are fluorescent beads configured to emit bead fluorescence when excited by the excitation light.

5. The method of claim 4, wherein the beads comprise a plurality of distinct chromophores, each chromophore of the plurality of distinct chromophores comprising a predetermined set of adjustable optical encoding parameters, thereby defining an optically detectable code for the capture agent.

6. The method described in claim 4, wherein the fluorescent beads are fluorescent spectral intensity barcoded beads.

7. The method described in claim 2, wherein the beads have a diameter of less than 1 micrometer.

8. The method of claim 1, wherein the capture agent comprises magnetic beads, and isolating the analyte associated with the capture agent from the sample comprises associating the magnetic beads with a magnet and removing a portion of the sample that is not associated with the magnet from the analyte associated with the capture agent to provide the purified sample.

9. The method of claim 1, wherein the capture agent is bound to a surface.

10. The method of claim 9, further comprising removing a portion of the sample that is not associated with the capture agent bound to the surface.

11. The method of claim 1, wherein the association is selected from the group consisting of an antibody-antigen association, an aptamer-antigen association, a peptide-antigen association, and a nucleic acid hybridization association.

12. The method of claim 1, wherein the detectable agent is a first detectable agent configured to emit a first emitted light within a first wavelength range upon excitation of the first detectable agent, and the method further comprises associating with the analyte a second detectable agent configured to emit a second emitted light within a second wavelength range upon excitation of the second detectable agent, wherein the first emitted wavelength range is separate from the second wavelength range.

13. Quantifying the number of capture agent emission signals associated with the detectable agent emission signals; 10. The method of claim 1, further comprising determining the concentration of the analyte based on the number of capture agent emission signals associated with the detectable agent emission signals and the volume of liquid flowed through the flow path.

14. Quantifying the number of capture agent emission signals associated with the detectable agent emission signals. 、 2. The method of claim 1, further comprising determining the concentration of the analyte based on the number of capture agent emission signals associated with the detectable agent emission signals, and based on a ratio of the number of capture agent emission signals associated with the detectable agent emission signals to the number of capture agent emission signals, a ratio of the number of capture agent emission signals associated with the detectable agent emission signals to the number of detectable agent emission signals, or a combination thereof.

15. The method of claim 14, wherein determining the concentration of the analyte associated with the emission signal is further based on the ratio of the number of analytes associated with both a detectable drug emission signal and a capture agent emission signal to the number of capture agent emission signals but not a detectable drug emission signal, or the number of detectable drug emission signals but not a capture agent emission signal, or a combination thereof.

16. The method of claim 1, wherein quantifying the number of analytes includes single molecule sensitivity or detection efficiency.

17. The method described in claim 16, wherein the single molecule detection efficiency includes detecting more than 90% of single molecules flowing through the flow path.

18. The method of claim 1, wherein the detectable agent is selected from the group consisting of a fluorescent antibody, a fluorescent antibody fragment, a fluorescent aptamer, a fluorescent nucleic acid molecule, a fluorescent DNA molecule, a fluorescent peptide, and a fluorescent binder.

19. The method described in claim 1, wherein the digital affinity assay is a digital sandwich immunoassay.

20. The method of claim 19, wherein the sample is a protein.

21. The method described in claim 1, wherein the digital affinity assay is a digital nucleic acid assay.

22. The method of claim 1, wherein the sample comprises a nucleic acid sequence.

23. The method of claim 1, further comprising focusing the emitted light from the inspection window with a focusing system and directing the focused emitted light onto the detector, wherein the focusing system comprises an air objective lens having a numerical aperture in the range of greater than 0.91 and less than 0.

99.

24. The method described in claim 1, wherein the method does not include an amplification step for amplifying the target analyte or for amplifying a molecule that correlates with the presence of the target analyte.

25. The method of claim 24, wherein the method does not include an amplification step selected from polymerase chain reaction, enzymatic amplification, isothermal nucleic acid-based amplification, rolling circle amplification, and combinations thereof.

26. A system for analyzing single molecule analytes, the system comprising: a flow path configured to flow a single molecule analyte through a lumen of the flow path, the flow path defining an inspection window configured to allow light to pass into and out of the lumen; a light engine configured to output excitation light through the interrogation window into the flow path; a detector system positioned to receive radiation emitted from the flow path and configured to generate a signal based on the received radiation; a collection system positioned to collect the emitted light from the flow path and direct the collected emitted light onto the detector system; a controller operably coupled to the light engine and the detector system, the controller, when executed by the controller, providing the system with: outputting excitation light with the light engine through the interrogation window onto a portion of the flow path; generating an emission signal at the detector; and The controller, when executed by the controller, provides the system with: further comprising logic for performing operations including flowing a sample through the flow path, the sample including an analyte associated with a detectable agent; generating an emission signal at the detector based on detectable agent emission from the detectable agent; flowing the analyte associated with the detectable agent through the flow path comprises flowing a complex comprising the capture agent and the analyte associated with the detectable agent; the capture agent is configured to emit capture agent radiation upon excitation; the capture agent comprises a plurality of distinct chromophores, each chromophore of the plurality of distinct chromophores comprising a predetermined set of adjustable optical encoding parameters, thereby defining an optically detectable code for the capture agent; the controller further comprising logic that, when executed by the controller, causes the system to perform operations including generating a capture agent emission signal based on the capture agent emission light; generating the capture agent emission signal includes illuminating the capture agent with the excitation light and detecting the capture agent emission light with the photodetector; outputting the excitation light through the inspection window and through the portion of the flow path includes outputting the excitation light across a cross-section of the lumen of the flow path; the controller further includes logic that, when executed by the controller, causes the system to perform operations including flowing a plurality of analytes of the sample through the flow path, the analytes including the analytes associated with a detectable agent and a capture agent; flowing the plurality of analytes through the flow path includes, for each analyte associated with a capture and detectable agent, flowing the analytes associated with the plurality of capture and detectable agents through a constriction in the flow path; the capture agent is configured to emit capture agent emission light upon excitation, and the controller, when executed by the controller, causes the system to generate, at the detector, a plurality of detectable agent emission signals based on the detectable agent emission light; generating, at the detector, a plurality of capture agent emission signals based on the capture agent emission light; the channel is disposed in a portion of a microfluidic device; the microfluidic device defines a planar portion; The system, wherein the flow path within the inspection window defines a constriction relative to an adjacent portion of the flow path.

27. ​​The system of claim 26, wherein the optically detectable code comprises a predetermined emission spectrum of the capture agent, a predetermined absorption spectrum of the capture agent, or a combination thereof.

28. The system described in claim 26, wherein the capture agent comprises spectral intensity barcoded fluorescent beads.

29. The system described in claim 26, wherein the capture agent comprises spectral intensity barcoded fluorescent magnetic beads.

30. The system described in claim 26, wherein the capture agent comprises beads having a diameter of less than 1 micrometer.

31. The controller, when executed by the controller, causes the system to: outputting a second excitation light through the interrogation window onto a second portion of the flow path separate from the first portion; 27. The system of claim 26, further comprising logic that causes the system to perform an operation including generating the capture agent emission signal at a second photodetector based on capture agent emission light received from the second portion of the flow path.

32. The system of claim 26, wherein the detectable agent is a first detectable agent configured to emit a first radiation within a first wavelength range upon excitation of the first detectable agent, and the controller further includes logic that, when executed by the controller, causes the system to perform an operation including associating with the analyte a second detectable agent configured to emit a second radiation within a second wavelength range upon excitation of the second detectable agent, wherein the first radiation wavelength range is distinct from the second wavelength range.

33. The system described in claim 32, wherein the first detectable agent associates with a first site of the specimen and the second detectable agent associates with a second site of the specimen that is separate from the first site.

34. The controller, when executed by the controller, causes the system to: quantitating the number of capture agent emission signals associated with the detectable agent emission signals; 27. The system of claim 26, further comprising logic that performs an operation including determining the concentration of the analyte based on the number of capture agent emission signals associated with the detectable agent emission signal and the volume of liquid flowed through the flow path.

35. The controller, when executed by the controller, causes the system to: quantitating the number of capture agent emission signals associated with the detectable agent emission signals; 27. The system of claim 26, further comprising logic that performs operations including determining the concentration of the analyte based on the number of capture agent emission signals associated with the detectable agent emission signals and based on the ratio of the number of capture agent emission signals associated with the detectable agent emission signals to the number of capture agent emission signals, the number of detectable agent emission signals, and combinations thereof.

36. The controller, when executed by the controller, causes the system to:

27. The system of claim 26, further comprising logic to perform operations including correlating or co-localizing the detectable agent emission signal with the capture agent emission signal to determine the presence and / or identity of the analyte.

37. The controller, when executed by the controller, causes the system to:

27. The system of claim 26, further comprising logic to perform an operation including correlating a ratio of the intensities of the capture agent emission signals with the identity of the capture agent and the identity of the analyte associated with the capture agent.

38. The controller, when executed by the controller, causes the system to:

27. The system of claim 26, further comprising logic for performing operations including correlating a ratio of the intensities of the emission signals of the detectable agents with the identity of the detectable agents and the identity of the analyte associated with the detectable agents.

39. The controller, when executed by the controller, causes the system to:

27. The system of claim 26, further comprising logic to perform operations including associating an optical barcode of the capture agent emission signal and / or the detectable agent emission signal with the identity of the analyte associated with the capture agent and the detectable agent.

40. The system described in claim 26, wherein quantifying the number of analytes includes single molecule sensitivity or detection efficiency.

41. The system described in claim 40, wherein quantifying the number of analytes includes single molecule sensitivity or detection efficiency without amplification of the analyte.

42. The system described in claim 40 or 41, wherein single molecule sensitivity or detection efficiency includes detecting greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of the molecules flowing through the flow path.

43. The system described in claim 26, further comprising a light emitting fiber bundle including a first light emitting optical fiber and a second light emitting optical fiber, wherein the proximal ends of the first light emitting optical fiber and the second light emitting optical fiber are disposed within a light emitting fiber bundle head, the proximal end of the first light emitting optical fiber is positioned to receive first radiation light emitted from the first portion, and the proximal end of the second light emitting optical fiber is positioned to receive second radiation light emitted from the second portion.

44. The optical engine, a first light source positioned to output a first excitation light onto a first portion of the flow path within the interrogation window; a second light source positioned to output a second excitation light onto a second portion of the flow path within the interrogation window separate from the first portion, wherein the detector system comprises: a first photodetector positioned to receive the first radiation emitted from the distal end of the first light-emitting optical fiber; a second photodetector positioned to receive the second emitted light emitted from the distal end of the second light-emitting optical fiber.

45. The controller, when executed by the controller, causes the system to: outputting the first excitation light from the first light source; outputting the second excitation light from the second light source; generating a first emission signal at the first photodetector based on the first excitation light received from the first light-emitting optical fiber; 45. The system of claim 44, further comprising logic that causes the system to perform operations including: generating a second emission signal at the second photodetector based on the second excitation light received from the second light-emitting optical fiber.

46. The system described in claim 44, further comprising a dichroic mirror disposed between the distal end of the first light-emitting optical fiber and the first photodetector and positioned to reflect a portion of the first emitted light to a third photodetector.

47. The system of claim 43, wherein the distal end of the first light-emitting optical fiber is configured to emit the first emitted light onto at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more photodetectors.

48. The system described in claim 46, further comprising a bandpass filter disposed between the dichroic mirror and the third photodetector and configured to filter a portion of the first emitted light.

49. The system described in claim 44, wherein the first photodetector is configured to generate a first radiation signal based on a first radiation wavelength range of the first radiation light, and the third photodetector is configured to generate a third radiation signal based on a third radiation wavelength range of the first radiation light that is different from the first radiation wavelength range.

50. The system described in claim 26, wherein flowing the sample through the flow path does not include sheath flow focusing, acoustic flow focusing, or a combination thereof.

51. The system described in claim 26, further comprising a focusing system positioned to focus the radiation received from the inner cavity of the flow path through the inspection window and direct the focused radiation onto the detector system, wherein the focusing system comprises an air objective lens having a numerical aperture in the range of greater than 0.91 and less than 0.99.

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