Method and apparatus for flow-based analysis of single particles and / or single molecules

Abstract

Systems and methods are described for analyzing particles flowing in a channel. In one embodiment, the channel is configured to flow particles through a lumen of the channel and defines a search window configured to allow light to pass into and out of the lumen, and the light engine comprises a first light source positioned to output a first excitation light to a first portion of the channel within the search window and a second light source positioned to output a second excitation light to a second portion of the channel within the search window, separate from the first portion. In one embodiment, the system includes an emission fiber bundle comprising a first emission optical fiber and a second emission optical fiber, the proximal ends of the first emission optical fiber and the second emission optical fiber being disposed in an emission fiber bundle head.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 198,748, filed on November 10, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Statement regarding the government's licensing rights This invention was created with government support under authorization number UG3 TR002874 granted by the National Institutes of Health. The government reserves certain rights in this invention. [Background technology]

[0003] Flow-based analysis of small particles, such as extracellular vesicles, can present several challenges. Typical flow-based particle analysis involves flowing several particles dispersed in a fluid suspension through a channel. As particle size decreases, particles tend to diffuse faster than larger particles. Additionally, under laminar flow conditions, the velocity of the fluid flowing through the channel changes radially depending on the distance of the fluid from the channel wall, and therefore the velocity of particles flowing through the channel changes accordingly depending on the distance of the particles from the wall. Consequently, particle velocity depends on the radial position of the particle, making it more difficult to distinguish between various smaller particles flowing through the channel, and thus strongly influenced by the relatively high diffusivity of the particles.

[0004] Therefore, there is currently a need for devices, systems, and methods for flow-based analysis of such small particles that address the diffusion-based challenges in identifying and characterizing them. [Overview of the project] [Means for solving the problem]

[0005] In some embodiments, the disclosure provides devices, systems, and methods for particle and / or molecular flow-based analysis to address these and related issues.

[0006] In one embodiment, the present disclosure provides a system for analyzing particles and / or molecules. In one embodiment, the system comprises: a channel, which defines a search window configured to allow particles and / or molecules to flow through the lumen of the channel and to allow light to pass in and out of the lumen; an optical engine comprising: a first light source positioned to output a first excitation light to a first portion of the channel in the search window; and a second light source, separate from the first portion, positioned to output a second excitation light to a second portion of the channel in the search window; and an emitting fiber bundle comprising a first emitting optical fiber and a second emitting optical fiber. The present invention comprises an emitting fiber bundle in which the proximal ends of two emitting optical fibers are positioned in an emitting fiber bundle head, the proximal end of a first emitting optical fiber is positioned to receive first synchrotron radiation emitted from a first portion, and the proximal end of a second emitting optical fiber is positioned to receive second synchrotron radiation emitted from a second portion; and a detector system comprising a first photodetector positioned to receive first synchrotron radiation emitted from the distal end of a first emitting optical fiber, and a second photodetector positioned to receive second synchrotron radiation emitted from the distal end of a second emitting optical fiber.

[0007] In another embodiment, the Disclosure provides a method for searching for particles and / or molecules, the method comprising: flowing particles and / or molecules through a channel; outputting a first excitation light to a first portion of the channel through a search window; outputting a second excitation light to a second portion of the channel separate from the first portion through the search window; generating a first emission signal using a first photodetector based on first synchrotron radiation received through the proximal end of a first emission optical fiber; and generating a second emission signal using a second photodetector based on second synchrotron radiation received through the proximal end of a second emission optical fiber, the proximal ends of the first and second emission optical fibers being located within an emission optical fiber bundle head.

[0008] In another embodiment, the Disclosure provides a system for analyzing particles and / or molecules, the system comprising: a channel, which defines a search window configured to allow particles and / or molecules to flow through the lumen of the channel and to allow light to pass in and out of the lumen; an optical engine configured to output excitation light to the channel through the search window; a detector system positioned to receive synchrotron radiation emitted from the channel and configured to generate a signal based on the received synchrotron radiation; and a focusing system positioned to collect synchrotron radiation from the channel and to direct the collected synchrotron radiation to the detector system, the focusing system comprising an air objective lens having a numerical aperture in the range of greater than 0.91 to less than 0.99, greater than 0.92 to less than 0.98, greater than 0.93 to less than 0.97, and greater than 0.94 to less than 0.96.

[0009] In yet another aspect, the Disclosure provides a method for analyzing particles or molecules in a fluid sample, the method comprising: flowing a fluid sample containing a plurality of particles / molecules through a channel; illuminating particles / molecules among the plurality of particles / molecules having a hydrodynamic diameter of less than 1 μm within the channel; collecting the synchrotron radiation emitted from the channel using a focusing system equipped with an air objective lens having a numerical aperture in the range of about 0.91 to less than 0.99; generating a signal based on the collected synchrotron radiation emitted from the channel based on the particles / molecules; and assigning values ​​to the particles / molecules based on the signal.

[0010] In another embodiment, a system for self-correcting flow analysis of a single particle / molecule is provided, the system comprising: a channel defining a search window configured to allow particles / molecules to flow through the lumen of the channel and to allow light to pass in and out of the lumen; an optical engine comprising: a first light source positioned to output first excitation light to a first portion of the channel within the search window; and a second light source, separate from the first portion, positioned to output second excitation light to a second portion of the channel within the search window; and a detector system comprising: a first photodetector positioned to receive first synchrotron radiation emitted from the first portion of the channel; and a second photodetector positioned to receive second synchrotron radiation emitted from the second portion. A detector system comprising two photodetectors, a first light source, a second light source, a first photodetector, and a controller operably coupled to the second photodetector, the controller including logic that, when executed by the controller, causes the system to perform operations including: outputting a first excitation light using the first light source; outputting a second excitation light using the second light source; generating a first emission signal using the first photodetector based on first synchrotron radiation received from the first part; generating a second emission signal using the second photodetector based on second synchrotron radiation received from the second part; and determining the velocity of particles / molecules in the channel based on the time difference between the generation of the first emission signal and the second emission signal, and the distance between the first part and the second part.

[0011] In yet another aspect, the Disclosure provides a method for single-molecule, self-correcting flow analysis, the method comprising: flowing a particle / molecule through the lumen of a channel, wherein the channel defines a search window configured to allow light to pass in and out of the lumen; using a first light source to output first excitation light to a first portion of the search window; using a second light source to output second excitation light to a second portion of the search window, separate from the first portion; generating a first emission signal using a first photodetector based on first synchrotron radiation received from the first portion; generating a second emission signal using a second photodetector based on second synchrotron radiation received from the second portion; and determining the velocity of the particle / molecule in the channel based on the time difference between the first and second emission signals and the distance between the first and second portions.

[0012] In another embodiment, the Disclosure provides an alternative method for self-correcting single-molecule flow analysis, the method comprising: flowing a particle / molecule through the lumen of a channel, the channel defining a search window configured to allow light to pass in and out of the lumen; using a light source to emit excitation light to a portion of the search window; generating an emission signal using a photodetector based on the synchrotron radiation received from the portion; acquiring the emission signal at a rate faster than the transport time of the particle / molecule across the portion; and determining the transport time of the particle / molecule across the portion. In one embodiment, acquiring the emission signal is at a rate at least 2 times, at least 5 times, or at least 10 times faster than the transport time of the particle / molecule. In one embodiment, determining the transport time of the particle / molecule across the portion is based on fitting the acquired emission signal track to a function. In one embodiment, the function is a Gaussian curve. In another aspect, the Disclosure provides a method for maintaining focus on a fluid channel, the method comprising: illuminating a search window of the fluid channel with light from a light source; focusing the light onto the search window using an optical component disposed between the channel and a photodetector; generating a lock signal using the photodetector based on the focused light reflected back from the search window in a first time period; generating a test signal using the photodetector based on the focused light reflected back from the search window in a second time period after the first time period; determining whether the test signal is within a predetermined proportion of the lock signal; and, if the test signal is outside the predetermined proportion of the lock signal, moving the fluid channel relative to the photodetector.

[0013] In yet another embodiment, the Disclosure provides a method for focusing an optical component onto a fluid channel, the method comprising: illuminating a search window of the fluid channel with light from a light source; focusing the light onto the search window using an optical component disposed between the channel and a photodetector; generating a lock signal using a photodetector based on the focused light reflected back from the search window at a first time interval; generating a test signal using a photodetector based on the focused light reflected back from the search window at a second time interval after the first time interval; determining whether the test signal is within a predetermined proportion of the lock signal; and, if the test signal is outside the predetermined proportion of the lock signal, moving the fluid channel relative to a high numerical aperture air objective lens. In one embodiment, imaging is performed outside the visible range, more preferably in the near-infrared region. In one embodiment, imaging is performed using an air objective lens with an NA of 0.91 to 0.99. In one embodiment, imaging is performed using an air objective lens with an NA of about 0.95.

[0014] In another aspect, the Disclosure provides a method for maintaining focus of an optical component directed to a fluid channel, the method comprising: illuminating an imaging area of ​​a microfluidic system with light from a light source; generating an image of the imaging area using a camera; determining the amount of defocus in the image; determining whether the amount of defocus is within a predetermined range; and moving the fluid channel relative to the camera if a test signal is outside a predetermined range.

[0015] In yet another aspect, the Disclosure provides a method for single-molecule detection, which includes flowing a plurality of molecules through a channel such that one or more of the plurality of molecules associate with a detectable drug; illuminating the plurality of molecules within the channel; collecting the synchrotron radiation emitted from the channel using a focusing system equipped with an air objective lens having a numerical aperture in the range of about 0.91 to less than 0.99; generating an emission signal based on the collected synchrotron radiation emitted from the channel based on the molecules; and assigning a value to the molecules based on the signal.

[0016] In another embodiment, the present disclosure provides a device having single-molecule detection efficiency, the device comprising: a channel, which defines a search window configured to allow particles or molecules to flow through the lumen of the channel and to allow light to pass in and out of the lumen; an optical engine configured to output excitation light to the channel through the search window; a detector system positioned to receive synchrotron radiation emitted from the channel and configured to generate a signal based on the received synchrotron radiation; a focusing system positioned to collect synchrotron radiation from the channel and to direct the collected synchrotron radiation to the detector system, the focusing system comprising an air objective lens having a numerical aperture in the range of greater than 0.91 to less than 0.99; and light The system comprises an engine and a controller operably coupled to a photodetector, the controller including logic that, when executed by the controller, causes the system to perform operations including: flowing a plurality of molecules and / or a plurality of particles through a channel such that one or more of the plurality of molecules and / or one or more of the plurality of particles associate with a detectable agent; illuminating the molecules or particles within the channel; collecting the synchrotron radiation emitted from the channel using a focusing system; generating an emission signal based on the collected synchrotron radiation emitted from the channel based on the molecules and / or particles; and assigning values ​​to the molecules and / or particles based on the signal.

[0017] This summary is provided in a simplified form to introduce the selection of concepts further described below in modes for carrying out the invention. This summary is not intended to identify the main features of the patented subject matter, nor is it intended to be used as an aid in determining the scope of the patented subject matter. [Brief explanation of the drawing]

[0018] Many of the aforementioned aspects and associated advantages of the present invention will be more easily understood by referring to the following detailed description in conjunction with the attached drawings.

[0019] [Figure 1A] This is a schematic diagram illustrating a flow-based single-particle / molecular analysis system using a high numerical aperture (NA) air objective lens according to one embodiment of the present disclosure. [Figure 1B] This is a schematic diagram illustrating the emission fiber bundle head of the system shown in Figure 1A according to one embodiment of the present disclosure. [Figure 2] This is a schematic diagram illustrating a flow-based single-molecule / particle system detector module according to one embodiment of the present disclosure. [Figure 3A] This is a schematic diagram illustrating a flow-based single-molecule / particle system optical engine and channel according to one embodiment of the present disclosure. [Figure 3B] This is a schematic example diagram of the channel search window in Figure 3A according to one embodiment of the present disclosure. [Figure 3C] This is a schematic diagram illustrating an example of an optical engine and channel according to one embodiment of the present disclosure, as shown in Figure 3A. [Figure 3D] This is a schematic diagram illustrating an example of an optical engine and channel according to one embodiment of the present disclosure, as shown in Figure 3A. [Figure 3E] This is a schematic diagram illustrating an example of an optical engine and channel according to one embodiment of the present disclosure, as shown in Figure 3A. [Figure 3F] This is a schematic diagram illustrating an example of an optical engine and channel according to one embodiment of the present disclosure, as shown in Figure 3A. [Figure 4] This is an image of a system channel according to one embodiment of the present disclosure. [Figure 5A] This invention schematically illustrates synchrotron radiation passing through an opening in an optically opaque cover and over the emission fiber bundle head of a detector system, according to one embodiment of the present disclosure. [Figure 5B] An example of an optically opaque cover shown in Figure 5A, according to one embodiment of the present disclosure, will be illustrated. [Figure 5C] This is an image of a flow-based single-molecule / particle system emission fiber bundle head according to one embodiment of the present disclosure. [Figure 6] This is a schematic diagram illustrating a flow-based single-molecule / particle system using a high-NA air objective lens, according to one embodiment of the present disclosure. [Figure 7A] This is a schematic diagram illustrating a flow-based single-molecule / particle system according to one embodiment of the present disclosure. [Figure 7B] This is a schematic diagram illustrating one embodiment of the present disclosure in which a high NA air objective lens, relative to the sample in the system shown in Figure 7A, is focused onto the channel of the system. [Figure 7C] This 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 one embodiment of the present disclosure. [Figure 7D] A series of images of a channel having different amounts of defocus, taken at several distances between the channel and a high NA air objective lens, according to one embodiment of the present disclosure. [Figure 7E] An embodiment of this disclosure illustrates the amount of focusing quality at various distances between the channel and the high NA air objective lens, focusing on the position in the image shown in Figure 7D. [Figure 7F] This 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 one embodiment of the present disclosure. [Figure 7G] Another block diagram illustrating a feedback control loop used to implement real-time focusing supported by near-infrared machine vision through a high NA airborne objective lens, according to one embodiment of the present disclosure. [Figure 8] One embodiment of this disclosure illustrates, graphically, the signals generated over time in three search windows of a flow-based single-molecule / particle system. [Figure 9A]A graph illustrating a particle passing through a search window of a flow-based single-molecule / particle system according to one embodiment of the present disclosure (left), and the absorption and emission spectra of a particle stained with one type of membrane dye and excited twice at the same excitation wavelength according to one embodiment of the present disclosure (right). [Figure 9B] A graph illustrating a particle passing through a search window of a flow-based single-molecule / particle system according to one embodiment of the present disclosure (left), and the absorption and emission spectra of a particle stained with one type of membrane dye excitable at two excitation wavelengths according to one embodiment of the present disclosure (right). [Figure 9C] A graph illustrating a particle passing through a search window of a flow-based single-molecule / particle system according to one embodiment of the present disclosure (left), and the absorption and emission spectra of a particle stained with two membrane dyes and excited at two different excitation wavelengths according to one embodiment of the present disclosure (right). [Figure 9D] A graph illustrating a particle passing through a search window of a flow-based single-molecule / particle system according to one embodiment of the present disclosure (left), and the absorption and emission spectra of a particle stained with a membrane dye and a volume dye and excited at two different excitation wavelengths according to one embodiment of the present disclosure (right). [Figure 10A] The left image shows the peak emission intensity of a dye associated with particles passing through a portion of the search window of a fluid device, illuminated by a first and second light source according to one embodiment of the present disclosure, and the right image shows a comparison with the peak emission area of ​​a dye associated with particles passing through the search of a fluid device, illuminated by a first and second light source according to one embodiment of the present disclosure and corrected by the method of the present disclosure. [Figure 10B] The left image shows the peak emission intensity of a dye associated with particles passing through a portion of the search window of a fluid device, illuminated by a first and second light source according to one embodiment of the present disclosure, and the right image shows a comparison with the peak emission area of ​​a dye associated with particles passing through the search of a fluid device, illuminated by a first and second light source according to one embodiment of the present disclosure and corrected by the method of the present disclosure. [Figure 11A]This embodiment of the present disclosure shows a comparison of normalized linear velocities between particles in a channel of a fluid system. [Figure 11B] The results of measuring the concentration of extracellular vesicles in a fluid sample passing through a channel of a fluid system according to one embodiment of the present disclosure are shown and were calculated using the comparison illustrated in Figure 11A according to one embodiment of the present disclosure. [Figure 12A] The images show the fluorescence spectra (left), sample tracking in flow (center), and signal intensity distribution (right) of human semen exosomes stained with (12A)di-8-ANEPPS, (12B) single R-phycoerythrin (PE), and (12C) single Alexa647 according to one embodiment of the present disclosure. [Figure 12B] The images show the fluorescence spectra (left), sample tracking in flow (center), and signal intensity distribution (right) of human semen exosomes stained with (12A)di-8-ANEPPS, (12B) single R-phycoerythrin (PE), and (12C) single Alexa647 according to one embodiment of the present disclosure. [Figure 12C] The images show the fluorescence spectra (left), sample tracking in flow (center), and signal intensity distribution (right) of human semen exosomes stained with (12A)di-8-ANEPPS, (12B) single R-phycoerythrin (PE), and (12C) single Alexa647 according to one embodiment of the present disclosure. [Figure 13] One embodiment of the present disclosure demonstrates that background levels acquired and measured using a high numerical aperture air objective lens (0.95 NA at 40X magnification) are very stable during a 2000-second experimental measurement period in two fluorescence channels. [Figure 14A]One embodiment of the present disclosure shows the multicolor colocalization of extracellular vesicles in semen labeled with the di-8-ANEPPS membrane dye, anti-CD63-Alexa647 antibody, and anti-CD81-PE / CF594 antibody, where four laser excitation regions or laser lines were used experimentally, with flow directions of 640 nm first, then 561 nm, 488 nm, and finally the 405 nm excitation region or laser line, where the direction of peak search was opposite to the flow because the last two laser lines were used to detect signals from di-8-ANEPPS. [Figure 14B] One embodiment of the present disclosure shows a subpopulation or subtype of extracellular vesicles of semen based on the colocalization of a di-8-ANEPPS membrane dye, an anti-CD63-Alexa647 antibody, and an anti-CD81-PE / CF594 antibody. [Figure 15] This embodiment of the present disclosure shows the measurement of sperm extracellular vesicle (sEV) concentrations after generating a series of dilutions of the original stock sEV sample. [Figure 16] An embodiment of this disclosure illustrates, graphically, an apparatus for achieving automatic focusing by using near-infrared imaging or machine vision together with a high numerical aperture air objective lens (NA=0.95). [Figure 17A] The fluorescence intensities of semen exosome size-versus-antibodies for CD63+(17A), CD81+(17B), and CD9+(17C) exosomes according to one embodiment of this disclosure are illustrated. [Figure 17B] The fluorescence intensities of semen exosome size-versus-antibodies for CD63+(17A), CD81+(17B), and CD9+(17C) exosomes according to one embodiment of this disclosure are illustrated. [Figure 17C] The fluorescence intensities of semen exosome size-versus-antibodies for CD63+(17A), CD81+(17B), and CD9+(17C) exosomes according to one embodiment of this disclosure are illustrated. [Figure 17D] This is a Venn diagram illustrating the proportion of subpopulations of semen exosomes illustrated in Figures 17A to 17C, according to one embodiment of the present disclosure. [Figure 18A]An example of the intensity distribution of single anti-CD63(18A), anti-CD81(18B), and anti-CD9(18C) antibodies and corresponding antibody-labeled semen exosomes according to one embodiment of the present disclosure is illustrated. [Figure 18B] An example of the intensity distribution of single anti-CD63(18A), anti-CD81(18B), and anti-CD9(18C) antibodies and corresponding antibody-labeled semen exosomes according to one embodiment of the present disclosure is illustrated. [Figure 18C] An example of the intensity distribution of single anti-CD63(18A), anti-CD81(18B), and anti-CD9(18C) antibodies and corresponding antibody-labeled semen exosomes according to one embodiment of the present disclosure is illustrated. [Figure 18D] The copy number distributions of tetraspanins CD63(18D), CD9(18E), and CD81(18F) according to one embodiment of this disclosure are illustrated. [Figure 18E] The copy number distributions of tetraspanins CD63(18D), CD9(18E), and CD81(18F) according to one embodiment of this disclosure are illustrated. [Figure 18F] The copy number distributions of tetraspanins CD63(18D), CD9(18E), and CD81(18F) according to one embodiment of this disclosure are illustrated. [Figure 19A] An example of one embodiment of this disclosure is given of the proportion of membrane-permeable RNA dye (SYTO)-reported vesicles or lipid-containing nanoparticles containing and not containing RNA, reported by membrane dyes, the proportion of RNA-containing vesicles (positive for both membrane dyes and RNA dyes), and the proportion of all vesicles with or without anti-CD63 antibodies (positive for membrane dyes). [Figure 19B] An example of one embodiment of this disclosure illustrates the proportion of RNA-containing particles or molecules (positive for RNA staining) reported by a membrane-permeable RNA dye (SYTO), whether or not they are vesicles, or lipid-containing nanoparticles reported by a membrane dye, and furthermore, the proportion of RNA-containing vesicles (positive for membrane dyes and RNA dyes), regardless of the presence or absence of anti-CD63 antibodies against all RNA-containing particles. [Figure 19C]An example of one embodiment of this disclosure illustrates the proportion of RNA-containing, CD63-positive (i.e., membrane dye and RNA dye-positive) whole vesicles or RNA-containing lipid nanoparticles (i.e., membrane dye and RNA dye-positive) that are RNA-containing. [Figure 19D] An example of the relationship between vesicle size and RNA content of vesicles or lipid-containing nanoparticles in Figure 19A, according to one embodiment of this disclosure, is illustrated. [Figure 20A] The concentration determination of a single fluorescent particle or molecule via a direct coefficient is illustrated, where 20A illustrates the coefficient of a single 200 nm fluorescent bead at the same flow rate, covering a concentration range of approximately four orders of magnitude, so that the lower concentration range can be further extended by increasing the flow rate, and the higher concentration range can be further extended by incorporating Poisson correction, and 20B shows the absolute quantification of the concentration of a single fluorescent protein down to less than 1 fM. [Figure 20B] The concentration determination of a single fluorescent particle or molecule via a direct coefficient is illustrated, where 20A illustrates the coefficient of a single 200 nm fluorescent bead at the same flow rate, covering a concentration range of approximately four orders of magnitude, so that the lower concentration range can be further extended by increasing the flow rate, and the higher concentration range can be further extended by incorporating Poisson correction, and 20B shows the absolute quantification of the concentration of a single fluorescent protein down to less than 1 fM. [Modes for carrying out the invention]

[0020] This disclosure provides systems and methods for flow-based analysis of particles and molecules. Numerous specific details are provided in the following description to provide a complete understanding of the embodiments. However, it will be understood by those skilled in the art that the techniques described herein may be carried out without using one or more of the specific details, or using other methods, components, materials, etc. In other examples, well-known structures, materials, or operations are not illustrated or described in detail so as not to obscure certain aspects.

[0021] Throughout this specification, any reference to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, the occurrence of the phrase “one embodiment” or “an embodiment” in various parts of this specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any preferred manner in one or more embodiments.

[0022] Small particles and molecules tend to diffuse more than larger particles in the fluid flowing through a channel. Radial displacement of particles, i.e., displacement due to diffusion perpendicular to the channel's main flow axis, can make the measurement characteristics of such small particles, such as fluorescence measurements, difficult, especially when multiple particles or molecules are flowing through the channel. For example, when multiple particles or molecules are flowing through a channel simultaneously, individual particles or molecules may have different velocities through the channel. When particles and / or molecules within a channel are measured at various points within the channel, it can be difficult to correlate the signals generated by a photodetector positioned to interrogate the channel at various points along its length with those of a single molecule or particle. Indeed, such measurements can become extremely difficult as particle size decreases, for example, toward the size of an extracellular vesicle or even a single molecule.

[0023] system Accordingly, in one embodiment, the present disclosure provides a system for analyzing particles such as single biological nanoparticles and / or molecules.

[0024] Fiber-bundled radiation and excitation In this regard, we focus on Figures 1A and 1B, where a system 100 according to one embodiment of the present disclosure is illustrated.

[0025] As shown, the system 100 includes a channel 102 configured to allow particles and / or molecules to flow through the lumen 104 of the channel 102, defining a search window 106 configured to allow light to pass in and out of the lumen 104; an optical engine 108 configured to output light to the search window 106; a radiant fiber bundle 130 positioned to receive synchrotron radiation emitted from the search window 106; and a detector system 142 positioned to receive synchrotron radiation emitted from the radiant fiber bundle 130.

[0026] In one embodiment, system 100, or a portion thereof including, for example, channel 102 and exploration window 106, includes a microfluidic chip. The microfluidic chip may be formed from a substrate (e.g., silicon, glass, ceramic, plastic, organosilicon, quartz, polymer material, or a combination thereof) and may include a network of microfluidic channels through which fluid flows. Microfluidic devices can be used to process minute amounts of fluid samples and offer advantages over conventional macroscale devices (e.g., significantly less fluid sample volume required, less reagent usage, and shorter processing times compared to macroscale devices). Microfluidic chips provide an attractive and versatile platform for manipulating, isolating, sorting, and / or transporting particles and / or molecules. The ease with which arrays of microfluidic channels can be patterned and integrated into a microfluidic device makes these microfluidic devices an attractive platform for particle and / or molecular applications. Because microfluidic chips are planar devices, the detection and analysis of particles and / or molecules can be facilitated by using highly efficient objective lenses that enhance light focusing, thereby facilitating the detection, analysis, determination, and / or identification of particles and / or molecules.

[0027] In some embodiments, the methods, systems, devices, and apparatus of the present disclosure include microfluidic chips that facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules in transport. Microfluidic chips can be used to process small amounts of fluid samples and can offer advantages over conventional macroscale devices (for example, microfluidic chips require only small amounts of fluid sample, fewer reagents, shorter processing times, and improved efficiency compared to macroscale devices). Because microfluidic chips are planar devices, they can facilitate the detection and analysis of biological nanoparticles and / or enhance focusing by enabling the use of high-NA objective lenses (e.g., high-NA air objective lenses), lenses with high numerical apertures, or focusing systems, such as air objective lenses with an NA (numerical aperture) of about 0.95 or 0.91-0.99, thereby facilitating the detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules. In some embodiments, the microfluidic chip is a planar device, which enhances the compatibility of these microfluidic chips with microscope setups (e.g., those having a translational stage on which the microfluidic chip is placed). Microfluidic chips can also enable the design and creation of interconnected fluid networks without dead volume, which in turn can facilitate the detection and manipulation of biological nanoparticles and / or molecules (e.g., sorting using fluid displacement at the junctions of three or more fluid channels). Dead volume is a portion of the volume within the microfluidic chip outside the flow path (e.g., a volume in which liquid that may be transporting sample nanoparticles and / or molecules may enter and diffuse, reducing accuracy). Microfluidic chips can also enable the creation of channels with non-spherical or non-square (e.g., rectangular) cross-sections by microfabrication methods, which can facilitate the detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules in transport.Microfluidic chips facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules during transport by facilitating the creation of channels of varying widths or heights along the channel length (e.g., channel constrictions, or stepped changes in width and / or height). Microfluidic chips can be formed by coupling them to a coverslip (e.g., glass or plastic) of a desired thickness and desired material properties (e.g., refractive index) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules during transport, thereby enhancing compatibility with highly efficient focusing systems (e.g., high numerical aperture objective lenses such as high NA air objective lenses requiring appropriate substrate thickness for maximum focusing). Microfluidic chips provide an attractive and versatile platform for the manipulation, isolation, sorting, and / or transport of biological nanoparticles and / or single molecules.

[0028] Referring further to Figures 1A and 1B, in one embodiment, the portion of system 100 including channel 102 is made from polymer materials (polydimethylsiloxane (PDMS), polyurethane methacrylate (PUMA), polymethyl methacrylate (PMMA), polyethylene, polyester (PET), polytetrafluoroethylene (PTFE), polycarbonate, parylene, polyvinyl chloride, fluoroethylpropylene, lexane, polystyrene, cyclic olefin polymers, cyclic olefin copolymers, polyurethane, polyester carbonate, polypropylene, polybutylene, polyacrylate, polycaprolactone, polyketone, polyphthalamide, cellulose acetate, polyacrylonitrile, polysulfone, epoxy polymers, thermoplastic resins, fluoropolymers, and polyvinylidene fluoride, polyamide, polyimide), inorganic materials (glass, quartz, silicon, GaAs, silicon nitride), fused silica, ceramics, glass (organic), and / or other materials or combinations thereof. In one embodiment, system 100 includes porous membranes, woven or nonwoven wool fibers (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), metals such as sintered stainless steel, and porous inorganic materials such as alumina, silica, or carbon.

[0029] The search window 106 of channel 102 allows excitation light, such as from the optical engine 108, to pass into the lumen 104 of channel 102, and synchrotron radiation to pass out of channel 102 to be received by the detector system 142. Such excitation light and synchrotron radiation may include, but are not limited to, visible light, infrared light, near-infrared light, and ultraviolet light, as well as combinations thereof, in several wavelength ranges. In this regard, the search window 106 is suitable for exciting particles and / or molecules flowing through channel 102, and for allowing the light emitted from the search window 106 to be received by the detector system 142 for further analysis.

[0030] As will be further discussed herein, in one embodiment, the lumen 104 of channel 102 within the exploration window 106, or in a certain embodiment, the lumen 104 of channel 102 adjacent to the exploration window 106, defines a constriction or other narrowing of the cross-section, diameter, or other size features of the lumen 104. Such a constriction or narrowing of the lumen 104 is configured to allow particles and / or molecules to flow through the portion of channel 102 containing the exploration window 106 under laminar flow conditions, particle by particle and / or molecule by molecule.

[0031] In one embodiment, the search window 106 includes a portion of the channel that is within the field of view of the objective lens 186 and / or detectable by the detector system 142. In one embodiment, the search window 106 includes a portion of the channel 102 that defines a constriction relative to the other portion of the channel 102. As further described herein, in such embodiments, the constriction of the search window 106 may have dimensions such as height, width, cross-sectional area, etc., that are smaller than other directly adjacent portions of the channel 102.

[0032] In some embodiments, the constriction has a width smaller than the widest part of the microfluidic channel 102. In some embodiments, the constriction has a width relative to the widest part of the microfluidic channel 102. In some embodiments, the constriction has a width of 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 width of the microfluidic channel. As a non-limiting example, a microfluidic channel 102 having a maximum width of 100 μm may have a constriction that is less than 25% of the maximum width (i.e., less than 25 μm). In preferred embodiments, the constriction has a width of less than 10% of the maximum width of the microfluidic channel 102.

[0033] In some embodiments, the maximum width of the microfluidic channel 102 is less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm and greater than 10 μm. The values ​​are greater than m, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1 μm, less than 700 μm and greater than 0.1 μm, less than 600 μm and greater than 0.1 μm, less than 500 μm and greater than 0.1 μm, less than 400 μm and greater than 0.1 μm, or less than 300 μm and greater than 0.1 μm. In a preferred embodiment, the maximum width of the microfluidic channel 102 is less than 500 pm and greater than 10 μm.

[0034] In some embodiments, the constriction has a width smaller than the average width of the microfluidic channel. In some embodiments, the constriction has a width relative to the average width of the microfluidic channel. In some embodiments, the constriction has a width of 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 channel 102.

[0035] In some embodiments, the average width of the microfluidic channel 102 is less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm and greater than 10 μm The values ​​are greater than m, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1 μm, less than 700 μm and greater than 0.1 μm, less than 600 μm and greater than 0.1 μm, less than 500 μm and greater than 0.1 μm, less than 400 μm and greater than 0.1 μm, or less than 300 μm and greater than 0.1 μm. In a preferred embodiment, the average width of the microfluidic channel 102 is less than 500 μm and greater than 10 μm.

[0036] In some embodiments, the constriction has a height lower than the greatest height value (i.e., maximum height) of the microfluidic channel 102. In some embodiments, the constriction has a height relative to the maximum height of the microfluidic channel. In some embodiments, the constriction has a height of 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 channel. As a non-limiting example, a microfluidic channel 102 having a maximum height of 20 μm may 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 of less than 25% of the maximum height of the microfluidic channel 102.

[0037] In some embodiments, the maximum height of the microfluidic channel 102 is less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm and greater than 10 μm. The values ​​are greater than μm, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1 μm, less than 700 μm and greater than 0.1 μm, less than 600 μm and greater than 0.1 μm, less than 500 μm and greater than 0.1 μm, less than 400 μm and greater than 0.1 μm, or less than 300 μm and greater than 0.1 μm. In a preferred embodiment, the maximum height of the microfluidic channel 102 is less than 500 μm and greater than 10 μm.

[0038] In some embodiments, the constriction has a height lower than the average height of the microfluidic channel. In some embodiments, the constriction has a height relative to the average height of the microfluidic channel. In some embodiments, the constriction has a height of 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 channel.

[0039] In some embodiments, the average height of the microfluidic channels is less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm and 10 μm The values ​​are greater than, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1 μm, less than 700 μm and greater than 0.1 μm, less than 600 μm and greater than 0.1 μm, less than 500 μm and greater than 0.1 μm, less than 400 μm and greater than 0.1 μm, or less than 300 μm and greater than 0.1 μm. In a preferred embodiment, the average height of the microfluidic channel has a value of less than 500 μm and greater than 10 μm.

[0040] In some embodiments, the constriction has a cross-sectional area smaller than the largest cross-sectional area (i.e., maximum cross-sectional area) of the microfluidic channel. In some embodiments, the constriction has a cross-sectional area relative to the maximum cross-sectional area of ​​the microfluidic channel. In some embodiments, the constriction has a cross-sectional area of ​​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 maximum cross-sectional area of ​​the microfluidic channel. A non-limiting example is 200 μm. 2 A microfluidic channel with a maximum cross-sectional area of ​​less than 10% of the maximum cross-sectional area value (i.e., 20 μm) is defined as having a maximum cross-sectional area of ​​less than 10% (i.e., 20 μm). 2It may have a constriction that is less than). In a preferred embodiment, the constriction has a cross-sectional area that is 10% to 0.01% of the maximum cross-sectional area of the microfluidic channel.

[0041] In some embodiments, the maximum cross-sectional area of the microfluidic channel is 1,000,000 μm 2 less than and 10 μm 2 more than 750,000 μm 2 less than and 25 μm 2 more than 500,000 μm 2 less than and 100 μm 2 more than 250,000 μm 2 less than and 250 μm 2 more than 900,000 μm 2 less than and 100 μm 2 more than 800,000 μm 2 less than and 100 μm 2 more than 700,000 μm 2 less than and 100 μm 2 more than 600,000 μm 2 less than and 100 μm 2 more than 400,000 μm 2 less than and 100 μm 2 more than 300,000 μm 2 less than and 100 μm 2 more than 200,000 μm 2 less than and 100 μm 2 more than 100,000 μm 2 less than and 100 μm 2 more than 50,000 μm 2 less than and 100 μm 2 more than 25,000 μm 2 less than and 100 μm 2 more than 10,000 μm 2 less than and 100 μm 2 more than 1,000 μm 2 less than and 100 μm 2 more than 2,000,000 μm 2 less than and 250 μm 2 more than 1,000,000 μm 2 less than and 250 μm 2 more than 900,000 μm 2 less than and 250 μm2 Ultra, 800,000μm 2 Less than 250 μm 2 Ultra, 700,000μm 2 Less than 250 μm 2 Ultra, 600,000μm 2 Less than 250 μm 2 Ultra, 400,000μm 2 Less than 250 μm 2 Ultra, 300,000μm 2 Less than 250 μm 2 Ultra, 200,000μm 2 Less than 250 μm 2 Ultra, 100,000μm 2 Less than 250 μm 2 Ultra, 50,000μm 2 Less than 250 μm 2 Ultra, 25,000μm 2 Less than 250 μm 2 Ultra, 10,000μm 2 Less than 250 μm 2 Ultra-large, or 1,000 μm 2 Less than 250 μm 2 It has a value greater than . In a preferred embodiment, the maximum cross-sectional area of ​​the microfluidic channel is 250,000 μm². 2 Less than 250 μm 2 It has a value greater than the limit.

[0042] In some embodiments, the constriction has a cross-sectional area smaller than the average cross-sectional area of ​​the microfluidic channel. In some embodiments, the constriction has a cross-sectional area relative to the average cross-sectional area of ​​the microfluidic channel. In some embodiments, the constricted portion has a cross-sectional area of ​​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 channel.

[0043] In some embodiments, the average cross-sectional area of ​​the microfluidic channels is 1,000,000 μm². 2 Less than 10 μm 2 Ultra, 750,000μm 2 Less than 25 μm 2 Ultra, 500,000μm 2 Less than 100 μm 2 Ultra, 250,000μm 2 Less than 250 μm 2 Ultra, 900,000μm 2 Less than 100 μm 2 Ultra, 800,000μm 2 Less than 100 μm 2 Ultra, 700,000μm 2 Less than 100 μm 2 Ultra, 600,000μm 2 Less than 100 μm 2 Ultra, 400,000μm 2 Less than 100 μm 2 Ultra, 300,000μm 2 Less than 100 μm 2 Ultra, 200,000μm 2 Less than 100 μm 2 Ultra, 100,000μm 2 Less than 100 μm 2Greater than 50,000 μm 2 Less than and 100 μm 2 Greater than 25,000 μm 2 Less than and 100 μm 2 Greater than 10,000 μm 2 Less than and 100 μm 2 Greater than 1,000 μm 2 Less than and 100 μm 2 Greater than 2,000,000 μm 2 Less than and 250 μm 2 Greater than 1,000,000 μm 2 Less than and 250 μm 2 Greater than 900,000 μm 2 Less than and 250 μm 2 Greater than 800,000 μm 2 Less than and 250 μm 2 Greater than 700,000 μm 2 Less than and 250 μm 2 Greater than 600,000 μm 2 Less than and 250 μm 2 Greater than 400,000 μm 2 Less than and 250 μm 2 Greater than 300,000 μm 2 Less than and 250 μm 2 Greater than 200,000 μm 2 Less than and 250 μm 2 Greater than 100,000 μm 2 Less than and 250 μm 2 Greater than 50,000 μm 2 Less than and 250 μm 2 Greater than 25,000 μm 2 Less than and 250 μm 2 Greater than 10,000 μm 2 Less than and 250 μm 2 Greater than or 1,000 μm 2 Less than and 250 μm 2 Has a value greater than. In a preferred embodiment, the average cross-sectional area of the microfluidic channel is 250,000 μm 2 Less than and 250 μm 2 Has a value greater than.

[0044] In some embodiments, the constricted portion 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 1 μm 2 In some embodiments, the height is less than 10 μm, the constricted portion 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.

[0045] In some embodiments, at least one microfluidic channel includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions. In some embodiments, a microfluidic chip includes multiple microfluidic channels, at least a portion of which each includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions. In some embodiments, a microfluidic chip includes multiple microfluidic channels, the majority of which each includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions. In some embodiments, a microfluidic chip includes multiple microfluidic channels, each including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 constrictions.

[0046] In some embodiments, at least a portion of at least one microfluidic channel 102 is 10,000 μm 2 Cross-sectional area less than 5,000 μm 2 Cross-sectional area less than 3,000 μm 2 Cross-sectional area less than 1,000 μm 2 Cross-sectional area less than 800 μm² 2 Cross-sectional area less than 600 μm² 2 Cross-sectional area less than 400 μm 2 Cross-sectional area less than 200 μm 2 Cross-sectional area less than 100 μm 2 Having a cross-sectional area of ​​less than 100 μm. In a preferred embodiment, at least a portion of at least one microfluidic channel is 100 μm. 2 Cross-sectional area less than 90 μm 2Cross-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 Cross-sectional area less than 1 μm 2 It has a cross-sectional area of ​​less than 250,000 μm. In some embodiments, at least one microfluidic channel has a cross-sectional area of ​​less than 250,000 μm. 2 Less than 100,000 μm 2 Less than 50,000 μm 2 Less than 25,000 μm 2 Less than 10,000 μm 2 Less than 5,000 μm 2 Less than 3,000 μm 2 Less than 1,000 μm 2 Less than 800 μm 2 Less than 600 μm 2 Less than 400 μm 2 Less than 200 μm 2 Less than 100 μm 2 It has a maximum cross-sectional area of ​​less than 100 μm. In some embodiments, at least one microfluidic channel is 100 μm 2 Less than 90 μm 2 Less than 80 μm 2 Less than 70 μm 2 Less than 60 μm 2 Less than 50 μm 2 Less than 40 μm 2 Less than 30 μm 2 Less than 20 μm 2 Less than 10 μm 2 Less than 5 μm 2 Less than 2 μm 2 Less than 1 μm, or 1 μm 2 It has a maximum cross-sectional area of ​​less than 1 μm. In a preferred embodiment, at least a portion of at least one microfluidic channel is 1 μm 2 ~100μm 2It has a cross-sectional area of ​​. In some embodiments, at least one microfluidic channel is 100 μm 2 ~10,000 μm 2 It has the maximum cross-sectional area.

[0047] In some embodiments, at least a portion of at least one microfluidic channel includes at least one discontinuous change in its width or height (e.g., achieved using microfabrication techniques). Microfluidic chips used herein may include microfluidic channels having at least one step gradient or step change in height or width, in contrast to microfluidic channels including a continuous change in height or width. Channels including a continuous change in height or width are common in devices including, for example, glass tubes, and can be achieved by pulling a heated tube. In certain embodiments, at least a portion of at least one microfluidic channel has a height and width that change independently of each other. Independent changes in height and width are in contrast to, for example, a glass tube, where a process that reduces the height is accompanied by a corresponding reduction in the width (e.g., tensile and thinning of a glass tube heated near its melting point).

[0048] As described above, system 100 includes an optical engine 108. In the illustrated embodiment, the optical engine 108 is shown to include a plurality of light sources 110, 114, 118, and 120, each of which is positioned to emit or output excitation light onto separate portions 122, 124, 126, and 128 of channel 102 in the search window 106. In this regard, the optical engine 108 is shown to include a first light source 110 positioned to output first excitation light 112 onto a first portion 122 of channel 102 in the search window 106, and a second light source 114 positioned to emit or output second excitation light 116 onto a second portion 124 of channel 102 in the search window 106, separate from the first portion 122. The optical engine 108 is further shown to include a third light source 118 and a fourth light source 120, which are positioned to output a third excitation light and a fourth excitation light to a third portion 126 and a fourth portion 128 of channel 102 in the search window 106, respectively.

[0049] In one embodiment, portions 122, 124, 126, and 128 are focused by the objective lens 186, and after impacting the channel 102, are defined by the width of the excitation light. For example, in one embodiment, portion 122 has a width defined by the width of the excitation light 112 impacting the channel 102. In one embodiment, 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 one embodiment, the widths of portions 122, 124, 126, and 128 are in the range of approximately 2.0 μm to approximately 0.2 μm, approximately 1.0 μm to approximately 0.2 μm, approximately 0.9 μm to approximately 0.2 μm, approximately 0.8 μm to approximately 0.2 μm, approximately 0.7 μm to approximately 0.2 μm, or approximately 0.6 μm to approximately 0.2 μm. In one embodiment, the widths of portions 122, 124, 126, and 128 are 1 / e of the maximum excitation light intensity. 2 The widths of portions 122, 124, 126, and 128 are defined as 1 / e of the maximum excitation light intensity.

[0050] If the widths of portions 122, 124, 126, and 128 are too wide, the signal-to-noise ratio will be too low, for example, for the detection and analysis of a single molecule / particle. In this regard, if the excitation linewidths are too wide and the spacing is too close (for example, when two excitation lines substantially overlap), crosstalk will occur between portions of channel 102. Additionally, excessively wide excitation linewidths illuminate larger portions of channel 102, generating more background light, which reduces the signal-to-noise ratio, especially when the amount of light emitted by a single particle / molecule may be relatively low.

[0051] As shown, the light sources 110 and 114 are optically coupled to optical fibers 164 and 168 such that the excitation light 112 and 116 emitted from them is received and transmitted by the optical fibers 164 and 168. In the illustrated embodiment, the first light source 110 is optically coupled to the proximal end 166 of the first excitation optical fiber 164, and the second light source 114 is optically coupled to the proximal end 170 of the second excitation optical fiber 168.

[0052] While optically coupled light sources are shown, it will be understood that in some embodiments, the light sources 110, 114, 118, and 120 of the optical engine 108 are free-space light sources that are not optically coupled to excitation optical fibers. In this regard, and in one embodiment, the free-space light sources do not have optical fibers positioned between the free-space light source and the search window 106 to receive excitation light and output excitation light into free space. In this regard, the spacing of excitation light between adjacent light sources is at least partially defined by additional optical components of the system 100 that direct and / or shape the excitation light emitted from the light sources. Thus, in one embodiment, the excitation optical fibers, such as the excitation optical fibers 164 and 168 of the excitation optical fiber bundle 172, are optional. Also, in some embodiments, the excitation optical fiber bundle 172 is absent.

[0053] The excitation light output by the light sources 110, 114, 118, and 120 of the optical engine 108 may have any wavelength. In one embodiment, the excitation light of one light source is the same as the excitation light output by another light source of the optical engine 108. In one embodiment, the excitation light of one light source is different from the excitation light output by another light source of the optical engine 108. In one embodiment, the first excitation light 112 has wavelengths in a first wavelength range, and the second excitation light 116 has wavelengths in a second wavelength range separate from the first wavelength range. In one embodiment, the first excitation light 112 has wavelengths in a first wavelength range, and the second excitation light 116 has wavelengths in a second wavelength range common to the first wavelength range.

[0054] 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 within or on a particle, or in association with a molecule. In one embodiment, the first excitation light 112 and / or the second excitation light 116 include coherent light from a laser or the like. In one embodiment, the first light source 110 and the second light source 114 are independently selected from the group consisting of solid-state lasers, diode-excited lasers, light-emitting diodes (LEDs), lamps, arc discharges, and natural light.

[0055] In one embodiment, the first excitation light 112 is The wavelength ranges are approximately 350 nm to 360 nm, 400 nm to 410 nm, 480 nm to 490 nm, 530 nm to 540 nm, 555 nm to 565 nm, or 630 nm to 690 nm. In one embodiment, the second excitation light 116 is It is located in the wavelength range of approximately 350nm to 360nm, 400nm to 410nm, 480nm to 490nm, 530nm to 540nm, 555nm to 565nm, or 630nm to 690nm.

[0056] As shown, the distal ends of the first excitation optical fiber 164 and the second excitation optical fiber 168 are located 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 gap 176. In one embodiment, this gap 176 at least partially defines the spacing of the excitation rays 112 and 116 output by the excitation fiber bundle head 172 and the optical engine 108. Correspondingly, in one embodiment, the gap 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, the excitation rays are output by the light sources 110, 112, 118, and 120 of the optical engine 108, which are positioned differently, bundled by the excitation fiber bundle head 172, and can be output to positionally separate portions of the search window 106 of the channel 102 according to the spacing 176 of the distal ends of the excitation optical fibers. Such configuration of the excitation fiber bundle head 172 can be further manipulated by the excitation optical system. See, for example, Figure 3A, although it is not shown. In one embodiment, the excitation optical fibers 164 and 168 are arranged adjacent to each other within the fiber bundle head, such as within one fiber diameter of each other (e.g., edge-to-edge distance).

[0057] In one embodiment, a fiber bundle such as an excitation fiber bundle 172 refers to optical fibers coupled at the ends of optical fibers or otherwise in close proximity. As further discussed herein, by bringing the ends of optical fibers close to each other within the diameter width of the optical fibers, light emitted from light sources coupled to optical fibers at different positions can be transmitted through the optical fibers and emitted from the optical fibers in an orientation partially shaped by the orientation of the optical fibers within the optical fiber bundle or in an orientation otherwise defined.

[0058] As described above, optically coupled light sources are illustrated, but as will be further discussed in this specification with respect to Figure 6, it will be understood that the optical engine 108 may include a free-space light source. Furthermore, it will be understood that the portions of the search window 106 illuminated by the optical engine 108 and the spacing between them are manipulated by an optical element disposed between the free-space light source and the search window 106.

[0059] As shown, system 100 includes a dichroic mirror 160 positioned to reflect at least a portion of the excitation light 112 and 116 toward the search window 106 of channel 102. In the illustrated embodiment, system 100 further includes an objective lens 186, such as an air objective lens 186, which is positioned to collect the excitation light reflected from the dichroic mirror 160 and is configured to focus the excitation light 112 and 116 toward channel 102. In this regard, the excitation light from the light source is output to spatially separate portions of channel 102 within the search window 106. As described above, in one embodiment, the search window 106 is defined at least partially by the field of view of the objective lens 186.

[0060] While 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 one embodiment, the dichroic mirror 160 is replaced by a transmissive mirror, such as a 20% reflect / 80% transmittance mirror, or another structure configured to selectively or partially transmit and reflect light.

[0061] As described above, particles or molecules flowing through channel 102, such as particles containing one or more detectable agents and / or molecules associated with such agents, can be excited by excitation light 112 and 116. Such excited particles / molecules can emit synchrotron radiation 146 and 152, which are either emitted or otherwise emitted from the search window 106 through the dichroic mirror 160. As shown, the synchrotron radiation 146 and 152 have the same or similar relative spacing as the excitation light 112 and 116 that collide with the search window 106.

[0062] Synchrotron radiation 146 and 152 are shown to collide with the emitting fiber bundle 130 of system 100. In the illustrated embodiment, the emitting fiber bundle 130 is shown to include four emitting optical fibers closely coupled in space. Similar to the excitation fiber bundle 172, the emitting optical fiber bundle 130 brings the ends of the optical fibers, in this case the emitting optical fibers 134 and 138, close together and follows a specific orientation or arrangement. As further discussed herein, a specific arrangement of the emitting optical fibers within the fiber bundle head 132 is suitable for positioning the emitting optical fibers 134 and 138 to receive synchrotron radiation 146 and 152.

[0063] Figure 1A shows four emitting optical fibers, but it will be understood that other numbers and configurations of emitting optical fibers are possible. In one embodiment, the emitting fiber bundle 130 includes at least three emitting optical fibers, at least four emitting optical fibers, at least five emitting optical fibers, at least six emitting optical fibers, at least seven emitting optical fibers, or more. In one embodiment, the emitting fiber bundle 130 includes a first emitting optical fiber 134 and a second emitting optical fiber 138, the proximal end 136 of the first emitting optical fiber 134 and the second emitting optical fiber 138 are positioned in the emitting fiber bundle head 132, the proximal end 136 of the first emitting optical fiber 134 is positioned to receive first synchrotron radiation 146 emitted from a first portion 122, and the proximal end 140 of the second emitting optical fiber 138 is positioned to receive second synchrotron radiation 152 emitted from a second portion 124.

[0064] In one embodiment, the proximal ends 136 and 140 of each emitting optical fiber 134 and 138 of the emitting fiber bundle 130 are arranged within the emitting fiber bundle head 132. In one embodiment, the proximal ends 136 and 140 of each emitting optical fiber 134 and 138 of the emitting fiber bundle 130 are positioned to receive synchrotron radiation 146 and 152 emitted from separate portions 122 and 124 of the search window 106. In one embodiment, the proximal ends 136 and 140 of the emitting optical fibers 134 and 138 are arranged adjacent to each other, such that they are within one fiber diameter of each other (for example, the distance from the edge of one fiber to the nearest edge of an adjacent fiber is within one fiber diameter).

[0065] In one embodiment, the emitting optical fibers of the emitting fiber bundle head 132 are configured to individually or separately receive synchrotron radiation corresponding to separate excitation regions or portions of the search window 106. As described above, in one embodiment, portions of the search window 106, such as portions 122 and 124, are defined by the width of the excitation light, such as the width of the excitation light beams 112 and 116, when they collide with the search window.

[0066] By positioning the proximal ends 136 and 140 of the emitting optical fibers 134 and 138 in close proximity to the emitting optical fiber bundle head 132, the emitting optical fibers 134 and 138 are positioned to receive radiant light from different portions of channel 102, such as portions of channel 102, which are excited by different light sources of the optical engine 108. In one embodiment, the spacing between the proximal ends 136 and 140 of the emitting optical fibers 134 and 138 is based on the spacing between portions 122 and 124 of channel 102, such as the spacing between excitation beams 112 and 116 that collide with the search window 106.

[0067] In this regard, we look to Figure 1B, a schematic illustrative diagram of the emission fiber bundle head 132 of system 100. Figure 1B illustrates several configurations of the emission optical fibers of the emission fiber bundle head 132. In a preferred embodiment, the emission optical fiber 134 is arranged in a linear configuration within the emission fiber bundle head 132.

[0068] In one embodiment, the spacing 174 and / or arrangement of the proximal ends 136 and 140 of the emitting optical fibers 134 and 138 corresponds to the spacing and / or arrangement of the portions of the search window 106 excited by the light source of the optical engine 108. As shown, the proximal ends 136 and 140 of the emitting optical fibers 134 and 138 are arranged in a linear configuration. In this regard, the emitting optical fibers 134 and 138 are arranged in an emitting optical fiber bundle head 132 to receive the radiant light 146 and 152 from the channel 102, and the optical engine 108 is configured to emit or output excitation light to the search window 106, for example in a linear configuration.

[0069] In one embodiment, the spacing 174 is in the range of approximately 1 μm to approximately 1,000 μm, approximately 100 μm to approximately 900 μm, approximately 1 μm to approximately 100 μm, approximately 10 μm to approximately 500 μm, and approximately 50 μm to approximately 800 μm. In another embodiment, the distance between the edge of one emitting optical fiber and the nearest edge of another emitting optical fiber at the proximal end of the fiber bundle head is either nearly zero (i.e., in contact) or within the radius of the fiber.

[0070] In one embodiment, the spacing 174 is the distance between the center of one emitting optical fiber and the center of another emitting optical fiber. In another embodiment, the spacing 174 is the distance between the edge of one emitting optical fiber and the nearest edge of another emitting optical fiber.

[0071] In one embodiment, the distance 174 between the proximal end 136 of the first emitting optical fiber 134 and the proximal end 140 of the second emitting optical fiber 138 corresponds to the distance between the first portion 122 and the second portion 124 of the search window 106. In this embodiment, the distance is the distance from center to center. In one embodiment, such a correspondence is a direct correspondence where the distance between the proximal ends 136 and 140 of the emitting optical fibers 134 and 138, and the distance between the first portion 122 and the second portion 124, directly or closely coincide after considering the magnification of the optical system. In one 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 search window 106 and the emitting fiber bundle head 132.

[0072] In one embodiment, the distance 176 between the distal end 166 of the first excitation optical fiber 164 and the distal end 170 of the second excitation optical fiber 168 corresponds to the distance between the first portion 122 and the second portion 124 of the search window 106. In one embodiment, such a correspondence is a direct correspondence where the distance between the distal ends 166 and 170 of the excitation optical fibers 164 and 168, and the distance between the first portion 122 and the second portion 124, directly or closely coincide after considering the expansion of the optical system. In this regard, the distance 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 distance between the first portion 122 and the second portion 124. In one 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 search window 106 and the excitation fiber bundle head 172.

[0073] In one embodiment, the distance 176 between the distal end 166 of the first excitation optical fiber 164 and the distal end 170 of the second excitation optical fiber 168 corresponds to the distance 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 described above, such correspondences may be direct correspondences or correspondences modified by any optical components that manipulate or direct the synchrotron radiation.

[0074] In one embodiment, the spacing 176 is in the range of approximately 1 μm to approximately 1,000 μm, approximately 1 μm to approximately 100 μm, approximately 250 μm to approximately 750 μm, approximately 1 μm to approximately 50 μm, and approximately 10 μm to approximately 500 μm. In another embodiment, the distance between the edge of one optical fiber and the nearest edge of another optical fiber in the fiber bundle head is either nearly zero (i.e., in contact) or within the radius of the fiber.

[0075] As described above, system 100 includes a detector system 142 positioned to receive synchrotron radiation emitted from the emitting fiber bundle 130. As shown, the emitting optical fibers 134 and 138 extend from the 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 with the detector modules 144 and 150 of the detector system 142. As used herein, “detector module” means a collection of detection structures and / or detection components configured to generate a signal or a set of signals based on light received by, for example, one or more detection structures and / or detection components, which are received by the detector module. Detector systems such as the detector system 142 of this disclosure may include one or more detector modules and / or one or more individual detectors, such as one or more individual photodetectors.

[0076] In one embodiment, one or more of the detector modules 144 and 150 include a single photodetector optically coupled and positioned to receive synchrotron radiation 146 and 152. In another embodiment, one or more of the detector modules 144 and 150 include a plurality of individual photodetectors, as further discussed herein with respect to Figure 2. In this regard, each of the detector modules 144 and 150 may be configured to receive synchrotron radiation 146 and 152 and generate a plurality of signals based on that synchrotron radiation 146 and 152, such as based on a specific wavelength range within the synchrotron radiation 146 and 152.

[0077] In one embodiment, the distal ends 148 and 154 of each emitting optical fiber 134 and 138 are positioned to emit light to at least one detector module 144 and 150, respectively. In one embodiment, the detector system 142 is positioned to receive scattered light, luminescent light, fluorescent light, or a combination thereof from the search window 106. In one embodiment, the scattered light is selected from backscattered light, side-scattered light, or forward-scattered light.

[0078] While photodetectors such as those within detector modules 144 and 150 are considered, it will be understood that other types of photodetection structures and photodetection components are possible and within the scope of this disclosure. In one embodiment, the photodetectors within detector modules 144 and 150 are selected from the group consisting of cameras, electron multiplier tubes, charge-coupled device (CCD) image sensors, photomultiplier tubes (PMTs), microchannel plate PMTs (MCPs), hybrid PMT detectors, avalanche photodiodes (APDs), single-photon avalanche diodes (SPADs), single-photon counting modules (SPCMs), silicon photomultiplier tubes (SiPMs), and complementary metal-oxide-semiconductor (CMOS) image sensors.

[0079] In the illustrated embodiment, the terminal end of each emitting optical fiber is optically coupled to a detector module to receive synchrotron radiation. In this regard, the detector system 142 is shown to include a first detector module 144 positioned to receive first synchrotron radiation 146 emitted from the distal end 148 of a first emitting optical fiber 134, and a second detector module 150 positioned to receive second synchrotron radiation 152 emitted from the distal end 154 of a second emitting optical fiber 138.

[0080] System 100 is shown to further include a controller 156 operably coupled to the optical engine 108 and the detector system 142. Such controller 156 includes logic configured to distribute the operation of these components. Although shown as one controller 156 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 this disclosure.

[0081] In one embodiment, the controller 156 includes logic for carrying out some or all aspects of the methods further described herein. In one embodiment, the controller 156 includes logic for outputting excitation light to the search window 106 using the optical engine 108, and for generating a signal using the detector system 142 based on the synchrotron radiation emitted from the search window 106 and received by the detector system 142. In one embodiment, the controller 156 includes logic, which, when executed by the controller 156, causes the system 100 to perform operations including outputting a first excitation light 112 using a first light source 110, outputting a second excitation light 116 using a second light source 114, generating a first emission signal using a first detector module 144 based on the first excitation light 112 received from a first emission optical fiber 134, and generating a second emission signal using a second detector module 150 based on the second excitation light 116 received from a second emission optical fiber 138.

[0082] In one embodiment, the controller 156 further includes logic, which, when executed by the controller 156, causes the system 100 to perform an action including flowing a suspension of particles and / or a solution of molecules, such as a suspension that is in fluid communication with the channel 102, through the channel 102. As used herein, “particle” refers to a localized object or entity, such as in a surrounding medium. In one embodiment, the particle defines a phase discontinuity with respect to its surroundings such that a solid particle is suspended surrounded by a liquid or gas phase. As further considered herein, in some embodiments, the particle is a biological particle, such as a biological nanoparticle, which may be of object origin, biological origin, or environmental sample origin.

[0083] In one embodiment, flowing the suspension through channel 102 includes flowing the suspension through channel 102 particle by particle or molecule by molecule. Such particle by particle or molecule by molecule flow is suitable for individually analyzing the particles and / or molecules flowing through channel 102. In one embodiment, the exploration window 106 defines a constriction relative to the rest of channel 102, and this constriction narrows the lumen 104. In one embodiment, the particle by particle and / or molecule by molecule flow occurs within the constriction of the lumen 104.

[0084] In one embodiment, the controller 156 includes logic that, when executed by the controller 156, causes the system 100 to perform an operation including ranking particles and / or molecules in channel 102. Such ranking may be based on the presence or absence of synchrotron radiation associated with the particles and / or molecules, such as synchrotron radiation detected by the detector system 142. In one embodiment, the ranking is based on the intensity and / or wavelength of the synchrotron radiation, as detected by the detector system 142. In one embodiment, the ranking corresponds to the measured emission spectrum of the particles and / or molecules based on one or more of the first synchrotron radiation 146 and the second synchrotron radiation 152. In one embodiment, the ranking corresponds to the 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.

[0085] As used herein, the term “assignment” refers to specifying a quantitative, qualitative, or significant characteristic of a particle and / or molecule classification to the particles and / or molecules to which the assignment is to be made. In one embodiment, a size value may be assigned to a particle. In one embodiment, 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. A size value provides a true or relative measurement of a linear distance. In one embodiment, the assignment is performed by a computer and software representing an assignment algorithm.

[0086] As used herein, the term “ranking” refers to determining the quantitative, qualitative, or significant characteristics of particles and / or molecules by classification. In one embodiment, particles and / or molecules may be ranked as either null (e.g., when the particles and / or molecules have an emission intensity below a detectable threshold) or non-zero (e.g., when 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 is assigned a value of 1, while each measured sample without a detected light intensity above a threshold is assigned a value of 0, thereby forming a binary ranking. In other embodiments, particles and / or molecules may be ranked according to additional classifications that correlate, for example, with the identity of the particles and / or molecules, the presence of detectable characteristics, the presence of distinctive characteristics, etc. The ranking may be assigned any number corresponding to one of several predetermined quantitative or qualitative categories. In other embodiments, the ranking is non-binary, for example, the values ​​are assigned based on the amount of light intensity emitted from the particles and / or molecules as measured. In one embodiment, ranking is performed by a computer and software representing a ranking algorithm.

[0087] As used herein, “detectable feature” refers to an observable property associated with a particle and / or molecule, such as a photoactive, electroactive, bioactive, or magnetic property that is associated with or inherent to the particle and / or molecule. In some embodiments, “detectable feature” includes the association of a particle and / or molecule with a detectable drug or biomarker.

[0088] Examples of photoactive properties include changes in optical intensity (optical reflection, scattering, deflection, transmission, absorbance, or emission) typically induced by biological particle morphology (particle size, internal intracellular structure), fluorescence, luminescence, immunofluorescence, etc. Detection of photoactive properties can report, for example, 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 nanoparticles.

[0089] In one embodiment, the ranking corresponds to the measured size value of the particles. In one embodiment, the measured size value is a relative size value. In one embodiment, the measured size value is determined by the difference in detected synchrotron radiation intensity. In one embodiment, the measured size value is the actual size value.

[0090] In one embodiment, the system 100 further includes a flow directionator, such as one or more valves configured to direct the flow of particles and / or molecules in the channel 102. In one embodiment, the flow directionator is operably coupled to a controller 156, which includes logic, when executed by the controller 156, to cause the system 100 to perform an operation including directing the flow of particles and / or molecules based on the presence or absence of synchrotron radiation received from the search window 106 and associated with the particles and / or molecules. In one embodiment, the flow directionator is operably coupled to a controller 156, which includes logic, when executed by the controller 156, to cause the system 100 to perform an operation including directing the flow of particles and / or molecules based on ranking. In one embodiment, directing the flow of particles and / or molecules includes directing particles and / or molecules to one of two or more sorting channels. In one embodiment, the flow directionator is operably coupled to a controller 156, which 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 synchrotron radiation from the search and determining the concentration of particles and / or molecules associated with the synchrotron radiation from the search window 106. In one embodiment, the concentration is further based on the measured flow rate, as will be further discussed herein with respect to Figures 11A and 11B.

[0091] In one embodiment, a flow directionor or flow direction mechanism for directing the flow of particles and / or molecules includes electrodes, magnetic elements, acoustic elements, electrooperated elements, optical operating elements, electric fields, or magnetic fields. In some embodiments, the mechanism for directing the flow of particles and / or molecules includes one or more electrooperated valves or pistons, which control the flow of liquid in at least one first directional flow channel that intersects a first input channel and two outlet channels at a first intersection. In one embodiment, the solenoid piston is a subcomponent of an electrooperated solenoid valve. In another embodiment, the solenoid piston is incorporated into the device by molding. In yet another embodiment, the incorporated solenoid piston may be replaced by a solenoid valve fluidly connected via piping.

[0092] In a particular embodiment, the apparatus provided herein may comprise one or more electrodes for tracking and / or manipulating the trajectory or flow of particles and / or molecules, particles, molecules, or fluid samples. In some 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 throttling or acoustic throttling may not be sufficient to adequately manipulate the trajectory of particles for the methods and apparatus disclosed herein, such as directing the trajectory of particles within channel 102. See, for example, Optics Express Vol. 15, Issue 10, pp. 6167-6176 (2007), incorporated herein by reference. Thus, in some embodiments, the mechanism for throttling particles excludes sheath flow throttling, acoustic flow throttling, or a combination thereof. In some embodiments, particles are directed on the condition that the above-mentioned direction does not use acoustic throttling, sheath flow throttling, or a combination thereof.

[0093] Self-correcting, flow-based particle analysis As described above, in one embodiment, the system 100 includes a channel 102 configured to allow particles to flow through a lumen 104 of the channel 102, the channel 102 defining a search window 106 configured to allow light to pass in and out of the lumen 104, the optical engine 108 includes a first light source 110 positioned to output first excitation light 112 onto a first portion 122 of the channel 102 within the search window 106, and a second light source 114 positioned to output second excitation light 116 onto a second portion 124 of the channel 102, separate from the first portion 122, the detector system 142 includes a first detector module 144 positioned to receive first synchrotron radiation 146 emitted from the first portion 122 of the channel 102, and a second detector module 150 positioned to receive second synchrotron radiation 152 emitted from the second portion 124. Such embodiments of System 100 of the present disclosure are suitable for use in self-correcting, single-particle and / or single-molecule flow analysis. As will be further discussed herein, the measurement of fluorescence emitted from single particles and / or molecules in a flow stream is greatly influenced by the flow beam and laser beam profiles. Therefore, accurate quantification of fluorescent particles and / or fluorescent molecules requires deconvolution of the signals from the flow profile and / or laser beam profile.

[0094] 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 precisely 1) colocalizing biomarkers expressed on the same particles and / or molecules flowing through multiple search windows or multiple portions of a single search 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) determining the concentration of the particles and / or molecules thus analyzed.

[0095] In short, the spatially separate portions of the search window 106 are arranged in a known spatial pattern, and the properties of particles and / or molecules (e.g., fluorescence emission) are measured twice in two different portions of the search window 106 (see, for example, Figure 8). Under laminar flow, the transport time of a particular particle flowing through two adjacent or two different portions of the search window 106 is proportional to the distance between these two portions. Also, due to the laminar nature, the position of a particular particle and / or molecule in the cross-section of the channel 102 remains approximately the same. This is especially true when the distance between two such portions is small, the transport time is short, and consequently the diffusion of particles within the channel is correspondingly small. Therefore, this property suggests that particles and / or molecules interact with different excitation light from different light sources at very similar positions in the cross-section of the channel. Considering these characteristics, it is possible to identify a single analyte (e.g., vesicles, or virions, or lipid nanoparticles, or single molecules stained with a fluorescent dye) and to further colocalize fluorescence signals associated with other biological markers of the analyte, such as from different dye-tagged antibodies linked to the corresponding biological marker, and / or from different nucleic acid stains and / or other specific fluorescent stains of the analyte or biological nanoparticles, using the extracted transport time or extraction location or relative location of the analyte in the cross-section of channel 102 (see Figure 8).

[0096] Accordingly, in one embodiment, System 100 for use in self-correcting single nanoparticle or single molecule flow analysis includes the logic of the present disclosure for performing a self-correcting single molecule / particle flow analysis method. In one embodiment, System 100 includes a controller 156 operably coupled to an optical engine 108 and a detector system 142, and includes logic that, when performed by the controller 156, causes System 100 to perform operations including measuring particle / molecule properties in various parts of the search window 106. In one embodiment, the system 100 includes a controller 156 operably coupled to a first light source 110, a second light source 114, a first photodetector module 144, and a second photodetector module 150, the controller 156 including logic that causes the system 100 to perform operations including, when executed by the controller 156, outputting a first excitation light 112 using the first light source 110, outputting a second excitation light 116 using the second light source 114, generating a first emission signal using the first photodetector module 144 based on first synchrotron radiation 146 received from a first section 122, generating a second emission signal using the second photodetector module 150 based on second synchrotron radiation 152 received from a second section 124, and determining the velocity of particles in channel 102 based on the time difference between the generation of the first emission signal and the second emission signal, and the distance between the first section 122 and the second section 124. In one embodiment, the particle velocity is used to determine the volumetric flow rate through the lumen 104.

[0097] In conventional fluid dynamics, the mean linear velocity (

number

number

number

number

number

[0098] In our system, the volumetric flow rate is very slow (e.g., about nl / min), so in some embodiments, it is not possible to conveniently measure the volumetric flow rate directly. Instead, in one embodiment, the flow profile in the channel is sampled by measuring the linear velocity of many individual particles (such as 100, 500, 1,000, 5,000, 10,000 or more particles) and the average observed value

number

number

number

[0099] In laminar flow, the volume (ΔQ) passing through the cross-section of each stack per unit time is:

number

number

number

number

number

number

number

[0100] Therefore, in one embodiment, the volumetric flow rate is given by the following formula:

number

number

[0101] In one embodiment, the controller 156 further includes logic, which, when executed by the controller 156, causes the system 100 to perform an operation that includes correlating the first emission signal and the second emission signal based on emission signal characteristics shared by the first emission signal and the second emission signal.

[0102] Air objective lens In one embodiment, the system 100 further includes a focusing system 184 positioned to collect synchrotron radiation such as a first synchrotron radiation 146 and a second synchrotron radiation 152 from channel 102 and to direct the collected synchrotron radiation to a detector system 142, the focusing system 184 comprising an air objective lens 186 having a numerical aperture in the range of greater than 0.91 to less than 0.99. In one embodiment, the objective lens 186, such as the air objective lens 186, has a numerical aperture of about 0.95.

[0103] As used herein, “air objective lens” refers to an optical objective lens in which the space between the objective lens and its focal plane or focal point is at least partially occupied by a gas such as air, and not occupied by an immersion liquid such as oil or water. In this regard, an air objective lens is in contrast to an oil immersion lens or a water immersion lens in which the lens is immersed in oil or water placed between the lens and the sample, usually between the lens and the coverslip or sample holder.

[0104] As used herein, “high NA (numerical aperture) air objective lens” refers to an air objective lens having an NA of 0.91 to 0.99, preferably 0.92 to 0.98, more preferably 0.93 to 0.97, and even more preferably 0.94 to 0.96. In one embodiment, the air objective lens has an NA of about 0.95. As further discussed herein, such high NA air objective lenses are suitable for performing single-molecule and / or single-particle analysis, such as when determining the presence, absence, or concentration of particles / molecules passing through the devices and systems of the Disclosure. As described elsewhere herein, high NA air objective lenses offer many advantages over conventional objective lenses such as oil immersion or water immersion objective lenses, including, among many other things, high focusing efficiency and the ability to scan accurately and efficiently.

[0105] Air objective lenses are generally easier and more stable to scan than, for example, oil immersion objective lenses. Furthermore, in some embodiments, the objective lens 186 is used not for high image quality (e.g., high resolution), but rather for its high light collection efficiency. In this respect as well, air objective lenses are superior to oil immersion objective lenses. Therefore, air objective lenses 186 with lower numerical apertures, such as in the range of greater than 0.91 to less than 0.99, are suitable for detecting single particles and / or single molecules in the flow channel 102.

[0106] emission multiplexing In one embodiment, the detector module of the system of the present disclosure includes two or more photodetectors, each positioned to receive synchrotron radiation from the distal end of an emitting optical fiber. In this regard, notice Figure 2, which shows a schematic illustrative diagram of a detector module 242 of a system according to one embodiment of the present disclosure. In one embodiment, the shown detector module 242 is an example of a detector module 144 or 150 of a detector system 142 illustrated in Figure 1A.

[0107] In the illustrated embodiment, the detector module 242 is shown to include several photodetectors 244, 250A, 250B, and 250C, which are positioned to receive synchrotron radiation 246A, or a portion thereof, shown here as fluorescent synchrotron radiation 246A, from the distal end 248 of the emitting optical fiber 234. As shown, the detector module 242 includes several dichroic mirrors 260 positioned to receive synchrotron radiation 246A emitted from the distal end 248 of the emitting optical fiber 234. Such dichroic mirrors 260 are configured to reflect a portion of the synchrotron radiation 246A (e.g., a certain wavelength range) and allow a different portion of the synchrotron radiation (e.g., a different wavelength range) to pass through the dichroic mirrors 260. In the illustrated embodiment, each dichroic mirror 260 is positioned to reflect a portion of the synchrotron radiation 246A toward photodetectors 244, 250A, 250B, and 250C, which are configured to generate signals based on the reflected or transmitted portion of the synchrotron radiation 246A.

[0108] In this regard, the detector module 242 is shown to include a dichroic mirror 260 positioned between the distal end 248 of the first emitting optical fiber 234 and the first photodetector 244, and positioned to reflect a portion 246B of the first synchrotron radiation 246A to the first photodetector 244. In one embodiment, the detector of the system according to an embodiment of the present disclosure further includes a second detector module optically coupled to the second emitting optical fiber, such as a second detector module including a second photodetector, as illustrated in Figure 1A. In the illustrated embodiment of Figure 2, the detector module 242 is shown to include third, fourth, and fifth photodetectors 250A, 250B, and 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 synchrotron radiation 246B, including synchrotron radiation 246B, and the third, fourth, and fifth photodetectors 250A, 250B, and 250C are configured to generate third, fourth, and fifth emission signals based on third, fourth, and fifth emission wavelength ranges of synchrotron radiation 246C, 246D, and 246E, which are different from or substantially different from the first emission wavelength range of synchrotron radiation 246B.

[0109] 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 one embodiment, the detector module includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more photodetectors.

[0110] 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 synchrotron radiation 246B–246E. In this regard, photodetectors 244 and 250A–250C are configured and positioned to generate a signal based on the filtered portion of the synchrotron radiation 246A, 246B–246E. As further discussed herein, in one embodiment, the optical engine excites particles and / or molecules in the channel with light of different wavelengths. As also further discussed herein, in one embodiment, the particles and / or molecules themselves can be impregnated with or associated with one or more detectable agents configured to emit fluorescence having different wavelength ranges and to be excited by light of different wavelengths. Thus, the configuration of the detector module 242 illustrated in Figure 2 is suitable for generating a signal based on synchrotron radiation having wavelengths within one or more wavelength ranges, with arranged photodetectors and corresponding filters. In this regard, the illustrated detector module 242 is suitable for emission multiplexing of light-emitting particles and / or molecules in the 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 different wavelength ranges of light emitted from such particles, molecules, or other analytes.

[0111] In one embodiment, the distal end 248 of the first emitting optical fiber 234 is configured to emit the first synchrotron radiation 246A to 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 one embodiment, each photodetector is configured to receive substantially different spectral portions of the synchrotron radiation, for example, when received through one or more dichroic mirrors or optical filters.

[0112] The portion of the detector module 242 illustrated in Figure 2 is shown to further include lenses 290 that are shaped and positioned to focus reflected and / or transmitted synchrotron radiation onto each photodetector.

[0113] In one embodiment, the system of the present disclosure includes a plurality of detector modules, such as one or more detector modules 242 illustrated in Figure 2. In one embodiment, each of the distal ends of the emitting optical fiber is configured to emit synchrotron radiation to the detector modules, as illustrated in Figure 2.

[0114] Excitation multiplexing As will be further discussed herein, the optical engine of the system of the present disclosure may include a light source configured to output light in a variety of wavelength ranges, such as wavelength ranges suitable for exciting one or more detectable agents disposed in or in contact with particles flowing through a channel. In some embodiments, such wavelength ranges of light overlap. In some embodiments, the wavelength ranges of light are separated. In this regard, look to Figures 3A to 3F, which illustrate an embodiment of an optical engine 308 and a channel 302 illuminated by the optical engine 308 according to one embodiment of the present disclosure.

[0115] Figure 3A is a schematic example of the optical engine 308 and channel 302 of a system according to one embodiment of the present disclosure. In one embodiment, the optical engine 308 and channel 302 are examples of the optical engine 108 and channel 102 of system 100 in Figure 1A. In one embodiment, the optical engine 308 and channel 302 are suitable for use in conjunction with a portion of the detector module 242 in Figure 2.

[0116] In the illustrated embodiments, the optical engine 308 is shown to include four light sources, each coupled to the distal end of an excitation optical fiber. While four light sources are illustrated, it will be understood that more or fewer light sources are possible and within the scope of this disclosure. It will also be understood that free-space light sources may be used within the scope of this disclosure, as will be further discussed herein. The excitation optical fiber is shown to be terminated with an excitation fiber bundle positioned to output excitation light. As shown, the excitation fiber bundle head 372 is positioned to output the excitation light into a dichroic mirror 360 and an objective lens 386. The excitation light is shown to be emitted from the objective lens 386 onto each portion of the channel 302 in the search window 306.

[0117] Figure 3B is a schematic illustrative diagram of a search window 306 of channel 302 that defines a lumen 304 through which particles and / or molecules are configured to flow. As shown, a first excitation light 312 is directed to a first portion 322 of channel 302, a second excitation light 316 is directed to a second portion 324 of channel 302, separate from the first portion 322, a third excitation light is directed to a third portion 326 of channel 302, separate from the first portion 322 and the second portion 324, and a fourth excitation light is directed to a fourth portion 328 of channel 302, separate from the first portion 322, the second portion 324, and the third portion 326. The first portion 322 and the second portion 324 of channel 302 are shown separated by a gap of 374. In one embodiment, the spacing 374 between the first portion 322 and the second portion 324 corresponds to, and at least partially defines, 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 channel 302 illuminated by the light source of the optical engine 308. This is further illustrated by Figure 4, which is a fluorescence image of the channel 302 illuminated by the optical engine 308 according to one embodiment of the present disclosure.

[0118] As described above, portions 322, 324, 326, and 328 are separated by an interval of 374. In one embodiment, such intervals are 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.

[0119] In one embodiment, the spacing 374 is based on the distance between the center of one excitation beam, such as excitation beam 312, that strikes the search window 306, and the center of another excitation beam, such as excitation beam 316, that strikes the search window 306. In another embodiment, the spacing 374 is based on the distance between the edges of the excitation beams that strike the search window 306, such as the distance between the edge of excitation beam 312 and the opposing edge of excitation beam 316.

[0120] As will be further discussed herein with respect to Figure 1A, in one embodiment, portions 322, 324, 326, and 328 have widths defined by the widths of excitation light, such as excitation light 312 and 316, which, after passing through the high-NA air objective lens 386 or after being focused by the high-NA air objective lens 386, collide with the search window 306. In one embodiment, the ratio of the 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, 20:1 or more. In one embodiment, the ratio of the 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, and 2:1 to about 5:1. Such ratios are large enough to generate excitation light from various parts having minimal crosstalk between emissions from separate parts 322, 324, 326, and 328, as detected, for example, by the detector module of the system of the present disclosure.

[0121] Figures 3C to 3F are schematic illustrative diagrams of an optical engine 308 and channel 302 according to the present disclosure. In one embodiment, multiple optical engines 308 and multiple channels 302 are examples of the optical engine 308 and channel 302 in Figure 3A. As shown, the optical 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 embodiment illustrated, the light sources 310, 314, 318, and 320 are optically coupled to excitation optical fibers 364, 368, etc., and the distal ends of these fibers 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 gap 376.

[0122] In the illustrated embodiments, the light sources 310, 314, 318, and 320 include several lasers having the wavelengths described. As shown, in one embodiment, two or more of the lasers 310, 314, 318, and 320 are configured to emit light having a common wavelength. In another embodiment, the lasers 310, 314, 318, and 320 are configured to emit light having different wavelengths.

[0123] In this regard, the optical engine 308 can be configured to analyze or manipulate particles and / or molecules passing through channel 302 with light of the same wavelength, such as when tracking specific particles as they move through channel 302. Similarly, in one embodiment, the optical engine 308 can be configured to analyze or manipulate particles with light of different wavelengths to help determine the presence or absence of specific detectable agents associated with different markers.

[0124] As further discussed herein, the variable arrangement and wavelength range of the light source of the optical engine 308 is suitable for performing excitation multiplexing and emission multiplexing. As used herein, “excitation multiplexing” refers to a method of analyzing particles, molecules, or other analytes, including a detectable excitable agent associated with the particles, molecules, or other analytes, using excitation light having different wavelength ranges. As further discussed herein, by exciting the detectable agent with excitation light having different wavelength ranges, different qualities or properties of the particles, molecules, or other analytes associated with the detectable agent can be determined.

[0125] cover In one embodiment, the system of the present disclosure includes a cover coupled to an emission fiber bundle. In this regard, please refer to Figures 5A to 5C, where a cover according to an embodiment of the present disclosure is illustrated.

[0126] Figure 5A schematically illustrates synchrotron radiation passing through openings 594A and 594B of an optically opaque cover 592 and through the system's emitting fiber bundle 530 according to one embodiment of the present disclosure. Figure 5B illustrates an example of the optically opaque cover 592 of Figure 5A. In one embodiment, the emitting fiber bundle 530 is an example of the emitting fiber bundle 130 of Figure 1A.

[0127] As shown, the lens 590 directs the first synchrotron radiation 552 and the second synchrotron radiation 546 toward the fiber bundle head 532. In the illustrated embodiment, the cover 592 defines an aperture 594A that is shaped to allow the first synchrotron radiation 552 to pass through to the proximal end 536 of the first emitting optical fiber 534. In this regard, the first synchrotron radiation 552 is allowed to pass through the aperture 594A and through to the proximal end 536 of the first emitting optical fiber 534. In one embodiment, the cover 592 is optically opaque. In this regard, light other than the first synchrotron radiation 552, such as light, is unlikely to enter the first emitting optical fiber 534.

[0128] In the partially disassembled illustrated embodiment, the cover 592 is shown to be separate from the emitting fiber bundle head 532. In one embodiment, an optically opaque cover 592 is coupled to the emitting fiber bundle head 532 to prevent or reduce stray light from entering the emitting optical fiber during use. 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 synchrotron radiation (e.g., a portion of the first synchrotron radiation 552 entering the proximal end 540 of the second emitting optical fiber 538, and vice versa).

[0129] As shown, the optically opaque cover defines a second aperture 594B, which is shaped to allow the passage of the second synchrotron radiation 546 to the proximal end 540 of the second emitting optical fiber 538. In this regard, the second synchrotron radiation 546 is allowed to pass through the cover 592 and enter the second emitting optical fiber 538.

[0130] Although four openings, including openings 594A and 594B, are illustrated in a linear arrangement, it will be understood that any number of openings in the cover 592 can be arranged in various configurations to correspond to the emitted optical fibers of the emitted fiber bundle 530, as further discussed herein with respect to Figures 1A and 1B.

[0131] Figure 5C is an image of the proximal end of the fiber bundle head 532 of the system according to one 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 covers 592 in Figures 5A and 5B are coupled to them.

[0132] Figure 6 is a schematic illustrative diagram of a system 600 according to one embodiment of the present disclosure, as described below. As shown, the system 600 includes a channel 602 configured to allow particles or molecules to flow through a lumen 604 of the channel 602, the channel 602 defining a search window 606 configured to allow light to pass in and out of the lumen 604, and includes an optical engine 608, a fiber bundle 630 shaped and positioned to receive synchrotron radiation emitted from the search window 606, and a detector system configured to generate a signal based on the collected synchrotron radiation.

[0133] In the embodiments illustrated, the optical engine 608 includes four light sources positioned to output light to channel 602. In this regard, in one embodiment, the optical engine 608 includes a first light source positioned to output a first excitation light 612 onto a first portion of channel 602 in the search window 606, and a second light source positioned or configured to output a second excitation light 616 onto a second portion of channel 602 in the search window 606, separate from the first portion. In one embodiment, the light sources are free-space light sources not coupled to excitation optical fibers. In this regard, in one embodiment, the excitation spacing is defined at least partially 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 partially by how the excitation light output from the free-space light sources is combined with dichroic and / or lens and other optical components, etc. In one embodiment, the optical engine 608 includes fiber-coupled light sources, as further discussed herein with respect to Figure 1A.

[0134] As shown, the excitation light is directed through lens 690 and strikes a dichroic mirror 660, which reflects the excitation light to the objective lens 686. The objective lens 686 collects the excitation light and directs it to the search window 606 of channel 602.

[0135] Synchrotron radiation emitted from channel 602 returns to the emitting fiber bundle 630 through the objective lens 686 and the dichroic mirror 660. While a dichroic mirror is illustrated as will be further discussed herein, other partial reflection / partial transmission structures are possible within the scope of this disclosure.

[0136] In the illustrated embodiment, the system 600 is shown to further include a mirror positioned to reflect light to the emitting fiber bundle 630, and a cover 692 configured to shield light other than the emitted light from entering the emitting fiber bundle 630.

[0137] System 600 is shown to include several photodetectors optically coupled to an emitting optical fiber. In this regard, System 600 is shown to include an emitting fiber bundle 630 including a first emitting optical fiber 634 and a second emitting optical fiber 638, where the proximal end portion 636 of the first emitting optical fiber 634 and the second emitting optical fiber 638 are positioned in an emitting fiber bundle head 632, with the proximal end 636 of the first emitting optical fiber 634 positioned to receive first synchrotron radiation 646 emitted from the first portion, and the proximal end 640 of the second emitting optical fiber 638 positioned to receive second synchrotron radiation 652 emitted from the second portion. The proximal end of an emitting optical fiber can refer to the portion of such fiber disposed within an emitting fiber bundle such as the emitting fiber bundle 630, and portions adjacent to the emitting fiber bundle.

[0138] The system 600 is shown to include first photodetectors 644, 658A, and 658B positioned to receive first synchrotron radiation 646 emitted from the distal end of a first emitting optical fiber 634, and second photodetectors 650A and 650C positioned to receive second synchrotron radiation 652 emitted from the distal end of a second emitting optical fiber 638.

[0139] With respect to Figure 2, and as shown herein, the system 600 further includes a dichroic mirror 660 positioned between the distal end of a first emitting optical fiber 634 and a first photodetector 644, and configured to reflect a portion of the first synchrotron radiation 646 to third photodetectors 658A and 658B. In the illustrated embodiment, each emitting optical fiber is optically coupled to the dichroic mirror 660, and then to a bandpass filter 662 optically coupled to second photodetectors 650A, 650B, and 650C and third photodetectors 658B, 658C, 658D, 658E, and 658F. In one embodiment, the first photodetector 644 is configured to generate a first signal based on a first wavelength range of the first synchrotron radiation 646, and the third photodetectors 658A and 658B are configured to generate a set of signals based on a different set of wavelength ranges of the first synchrotron radiation 646. In one embodiment, the second photodetectors 650A to 650C are configured and positioned to generate a set of signals based on synchrotron radiation other than the first synchrotron radiation 646, such as based on the second synchrotron radiation 652. In this regard, the synchrotron radiation received by each emitting optical fiber is configured to be spectrally analyzed by several photodetectors.

[0140] As shown, each emitting optical fiber is optically coupled to several photodetectors. For example, the first emitting optical fiber 634 is optically coupled to photodetectors 644, 658A, and 658B. In one embodiment, the photodetectors 644, 658A, and 658B include a detector module, such as the detector module further discussed herein with respect to Figure 2. Similarly, in one embodiment, the photodetectors 650A and 658C are a group within a second detector module. In one embodiment, such a detector module comprises a box or other housing that encloses the various photodetectors of the detector module.

[0141] The system 600 is shown to further include a controller 656 operably coupled to the optical engine 608 and the photodetector. As will be further discussed herein with reference to Figure 1A, in one embodiment the controller 656 is configured to distribute the operation of the optical engine 608 and the photodetector system, such as by carrying out one or more methods of the present disclosure.

[0142] Controller 656 is also shown to be operably coupled to a movable stage 688 that is physically coupled to channel 602. Channel 602 is shown to be part of a microfluidic chip that defines several channels. In one embodiment, controller 656 includes logic that, when executed by controller 656, causes system 600 to move the microfluidic chip on the movable stage 688. Accordingly, the focus of the objective lens 686 is changed from the first channel 602 to the second channel of the microfluidic chip. In this regard, system 600 may be used to analyze particles and / or molecules flowing through several channels, such as several channels through which suspensions of different particles and / or solutions of different molecules flow.

[0143] automatic focusing In one embodiment, the system of the present disclosure is suitable for analyzing particles or molecules flowing through the system using an automated focusing process or the like. In this regard, please note that Figures 7A and 7B illustrate a system 700 according to one embodiment of the present disclosure. Figure 7A is a schematic illustrative diagram of system 700. Figure 7B is a schematic illustrative diagram of focusing the high NA air objective lens 786 of system 700 onto the channel 702 of system 700. In one embodiment, system 700 is an example of system 100 in Figure 1A, or an example of system 600 in Figure 6.

[0144] As shown, the system 700 includes a channel 702 configured to allow particles and / or molecules to flow through the lumen of the channel 702, defining a search window 706 configured to allow light to pass in and out of the channel 702; a movable stage 788 coupled to the channel 702 and configured to move the channel 702 relative to a focusing system 784; an optical engine 708; a detector system; and a controller 756 operably coupled to the optical engine 708, the movable stage 788, and the detector system. As shown, the channel 702 defines a constriction within the search window 706. As will be further discussed herein, such a constriction is suitable for providing a particle-by-particle flow and / or a molecule-by-molecule flow through the channel 702.

[0145] In the illustrated embodiment, the optical engine 708 is shown to output excitation light 712 to a dichroic mirror 760, which is reflected by a focusing system 784 and enters a search window 706 of channel 702. Synchrotron radiation 746 is shown to be emitted from the search window 706 and received by an emitting fiber bundle 730, which includes an emitting fiber bundle head 732, through the dichroic mirror 760, a lens 790A, and an aperture 794 in an optically opaque cover 778. One distal end of one of the emitting optical fibers 734 of the emitting fiber bundle 730 is shown to terminate adjacent to a photodetector 744 of the detector system. The synchrotron radiation passes through the lens 790 and a bandpass filter 762 before colliding with the photodetector. The photodetector 744 is configured to generate a signal based on the received synchrotron radiation 746.

[0146] In one embodiment, the controller 756 includes logic which, when executed by the controller 756, causes the system 700 to perform an action. In one embodiment, such action includes one or more methods of focusing an optical component onto a fluid channel 702 according to one embodiment of the present disclosure. In one embodiment, the action includes illuminating a search window 706 of the fluid channel 702 with light from a light source; focusing the light onto the search window 706 using an optical component disposed between the channel 702 and a photodetector 744; generating a lock signal at the photodetector 744 based on the focused light reflected back from the search window 706 in a first time; generating a test signal at the photodetector 744 based on the focused light reflected back from the search window 706 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 channel 702 relative to a high NA air objective lens 786 if the test signal is outside the predetermined percentage of the lock signal. As shown in Figure 7B, the high NA air objective lens 786 can be moved relative to the channel 702 to focus the excitation light 712 into the lumen 704 of the channel 702. In one embodiment, such movement of the air objective lens 786 is controlled by a movable stage 788, such as by responding to a command received from a controller 756, as will be further discussed herein with respect to Figure 6.

[0147] Figure 7C is a block diagram illustrating a method for focusing a high-NA air objective lens 786 of system 700 according to one 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 a value at a previous point in time (e.g., 200 ms earlier). If the difference between the measured value and a reference or lock value exceeds a predetermined threshold, a motor is driven to move the fluid channel 702 relative to the high-NA air objective lens 786.

[0148] In one embodiment, the objective lens is positioned to collect focused light reflected back from the search window using a focusing system 784. In one embodiment, the focusing 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.

[0149] In one embodiment, the light is in the invisible wavelength range. In another embodiment, the light is infrared light in the range of approximately 700 nm to approximately 2000 nm.

[0150] In one embodiment, the controller 756 includes logic, which, when executed by the controller 756, causes the system 700 to perform an operation that includes imaging the channel 702 with a camera and determining the amount of defocus in the image, such as determining the amount of defocus in the image based on a structure within the channel 702, such as the wall of the channel 702 having a known shape and / or dimensions. In one embodiment, the structure may be a separate structure adjacent to the channel 702 and is designed to perform this image-based autofocus and / or stage movement to position the channel within a search window. In one embodiment, the operation further includes moving the fluid channel 702 relative to the focusing system 784 if the amount of defocus is outside a predetermined range.

[0151] In one embodiment, the operation includes illuminating the imaging area of ​​the system 700 with light from a light source, generating an image of the imaging area with a camera or other imaging sensor, determining the amount of defocus in the image, determining whether the amount of defocus is within a predetermined range, and moving the fluid channel 702 relative to a high-NA air objective lens if the test signal is outside a predetermined range. In one embodiment, the channel is moved relative to the high-NA air objective lens using a movable stage 788. In one embodiment, the operation is iterative in that, for example, the camera periodically generates an image to check the focus and / or moves the channel 702 relative to the high-NA air objective lens to adjust the focus. In one embodiment, the light source is in the invisible wavelength range. In one embodiment, the light is near-infrared, such as in the range of about 700 nm to about 2000 nm. In one embodiment, the objective lens is an air objective lens. In one embodiment, the objective lens is an air objective lens having an NA of 0.91 to 0.99. In one embodiment, the objective lens is an air objective lens having an NA of 0.92 to 0.98. In one embodiment, the objective lens is an air objective lens having an NA of 0.93 to 0.97. In one embodiment, the objective lens is an air objective lens having an NA of 0.94 to 0.96. In one embodiment, the objective lens is an air objective lens having an NA of approximately 0.95.

[0152] method In another embodiment, the Disclosure provides a method for searching for particles and / or molecules. In one embodiment, the method includes using a system described herein.

[0153] Use of high NA (numerical aperture) air objective lenses for single-molecule detection In one embodiment, the method is for single molecule detection. In one embodiment, the method includes the use of focusing, such as using a focusing assembly that includes a high NA air objective lens.

[0154] In one embodiment, the method includes flowing a plurality of molecules associated with a detectable agent through a channel. In one embodiment, such flow includes flowing a plurality of molecules associated with a detectable agent through a channel and flowing the molecules among the plurality of molecules one by one through the channel. In this regard, in one embodiment, as discussed elsewhere herein, the molecules among the plurality of molecules pass through a channel, such as a portion of the channel including a constriction, one at a time. In this regard, the method is suitable for illuminating the molecules flowing through the channel individually.

[0155] As described above, the molecules associate with a detectable agent. In one embodiment, individual molecules associate with one or more detectable agents. In one embodiment, one of the plurality of molecules associates 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 fluorescence signal, in response to excitation light.

[0156] In one embodiment, the molecules are selected from the group consisting of cell signaling molecules, cytokines, chemokines, antibodies, proteins, nucleic acids, nucleic acid binding proteins, RNA binding proteins, peptides, carbohydrates, drug molecules, and therapeutic molecules.

[0157] In one embodiment, the method further includes illuminating the molecules among the plurality of molecules within the channel. In one embodiment, illuminating the molecules includes illuminating the molecules among the plurality of molecules as the molecules flow through the channel one by one. In one embodiment, illuminating the plurality of molecules includes illuminating the molecules with a plurality of light sources having light within one or more wavelength ranges. In one embodiment, one or more such light sources are positioned to irradiate spatially distinct portions of the channel, such as different portions of a search window, as discussed elsewhere herein.

[0158] In embodiments, the method includes collecting more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, and 50% of synchrotron radiation from a single molecule flowing through a channel. By flowing multiple molecules through the channel one by one, molecules can be detected and counted individually. In this regard, as will be further discussed herein with respect to Figures 12A to 12C, 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 concentrations 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 of the different detectable agents associated with the molecule or particle is not detected, it is impossible to accurately identify that molecule or particle.

[0159] In one embodiment, "single-molecule sensitivity" refers to the ability to detect more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, and 50% of single molecules flowing through the channel, preferably more than 90% of single molecules flowing through the channel. In one embodiment, "single-molecule sensitivity" refers to a detection efficiency of more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, and 50%, preferably more than 90%.

[0160] In one embodiment, the “detection efficiency” of single molecules and / or single particles in flow is the number of detected molecules / particles relative to the number of molecules / particles flowing through the channel (e.g., through the excitation region). In one embodiment, “single molecule detection efficiency” refers to a detection efficiency of 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, greater than 50%, preferably greater than 90%. In one embodiment, “single molecule detection efficiency” refers to the ability to detect 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, greater than 50%, preferably greater than 90% of the single molecules flowing through the channel. For a given type of fluorescent molecule / particle, having “single molecule sensitivity” or “single molecule detection efficiency” is a direct indicator of the sensitivity of the fluid system or device, and thus an important metric for evaluating the sensitivity and performance of the device or instrument.

[0161] In one embodiment, illuminating molecules among a plurality of molecules within the channel includes outputting excitation light to a portion of the channel through a search window using line illumination. In one embodiment, illuminating molecules among a plurality of molecules within the channel includes outputting excitation light to a portion of the channel through a search window using confocal detection geometry or line confocal detection geometry.

[0162] In one embodiment, channel illumination is achieved by a tightly focused laser line covering the entire cross-section of the channel, ensuring that all molecules passing through the channel are illuminated and excited with a very high probability, such as a probability of over 90%, preferably close to 100%. In one embodiment, a confocal detection geometry is achieved by using an aperture (e.g., a fiber aperture or slit aperture), which improves detection sensitivity by increasing the signal-to-noise ratio and minimizing crosstalk between different excitation regions or laser lines. In one embodiment, an apparatus employing a high-NA air objective lens, line illumination using a highly focused laser line, and a confocal detection geometry was used to ensure that all or nearly all molecules or particles flowing through the channel are detected with high detection efficiency, high single-molecule sensitivity, and high throughput.

[0163] In one embodiment, the method involves collecting the emitted light from a channel using a focusing system equipped with 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 in this specification, high-NA air objective lenses are particularly suitable for collecting relatively large amounts of light. Additionally, such air objective lenses are suitable for accurately scanning a device while maintaining a consistent distance between the air objective lens and the imaged device. Often, oil immersion or water immersion objective lenses draw oil or water onto the imaged device, making it impossible to maintain a consistent distance between the objective lens and the imaged device during scanning.

[0164] In one embodiment, the air objective lens has a numerical aperture of less than 0.99 between 0.91 and 0.99. In one embodiment, the air objective lens has a numerical aperture in the range of about 0.92 to about 0.98, about 0.93 to about 0.97, and about 0.94 to about 0.96. In one embodiment, the air objective lens has a numerical aperture of about 0.95.

[0165] In one embodiment, the method includes generating an emission signal based on collected synchrotron radiation emitted from a channel based on molecules. In one embodiment, the signal is generated using one or more detector systems, detector modules, and / or photodetectors as described elsewhere in this specification.

[0166] In one embodiment, the method includes assigning values ​​to particles and / or molecules based on signals. In one embodiment, the values ​​are based on one or more fluorescence signals emitted from the particles / molecules. Such values ​​can be used to select particles / molecules from among multiple particles / molecules, for example, when sorting based on the presence and / or absence of one or more detectable portions disposed on the particles / molecules.

[0167] As described above in this specification, in one embodiment, the methods, systems, devices, and apparatus of the Disclosure include a microfluidic chip having a microfluidic channel that can facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules. Microfluidic chips having a microfluidic channel can be used to process small amounts of fluid samples and can offer advantages over conventional macroscale devices (e.g., microfluidic chips require only small amounts of fluid sample, require fewer reagents, have shorter processing times, and are more efficient compared to macroscale devices). In some embodiments, the microfluidic chip is a planar device that can facilitate the detection and analysis of biological nanoparticles and single molecules and / or facilitate the detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules in transport by enabling the use of a high NA (numerical aperture) objective lens (e.g., a high NA air objective lens), lens, or focusing system having a high numerical aperture to enhance focusing. In some embodiments, the microfluidic chip is a planar device, which enhances the compatibility of these microfluidic chips with microscope setups (e.g., having a translational stage on which the microfluidic chip is placed). Microfluidic chips can also enable the design and creation of interconnected fluid networks without dead volume, which in turn can facilitate the detection and manipulation of biological nanoparticles and / or molecules (e.g., sorting using fluid displacement at the junctions of three or more fluid channels). Dead volume is a portion of the volume within the microfluidic chip outside the flow channels (e.g., a volume in which liquid that may be transporting sample nanoparticles and / or molecules may enter and diffuse, reducing accuracy). Microfluidic chips can also enable the creation of channels with non-spherical or non-square (e.g., rectangular) cross-sections by microfabrication methods, which can facilitate the detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules in transport.Microfluidic chips facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules in transport by facilitating the creation of channels of varying widths or heights along the channel length (e.g., channel constrictions, or stepped changes in width and / or height). Microfluidic chips can be formed by coupling them to a coverslip (e.g., glass or plastic) of a desired thickness and desired material properties (e.g., refractive index) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules in transport, thereby improving compatibility with high-efficiency focusing systems (e.g., high numerical aperture objective lenses such as high NA air objective lenses requiring appropriate substrate thickness for maximum focusing). Microfluidic devices make it possible to generate a large number of channels (e.g., 96 or 384 channels for 96 or 384 samples) on the same device to enable high-throughput analysis of more samples (e.g., 96 or 384 samples in a form compatible with multi-channel pipettes). Microfluidic chips offer an attractive and versatile platform for manipulating, isolating, sorting, and / or transporting bio-nanoparticles and / or single molecules.

[0168] Detection of fiber bundle emission In one embodiment, the method includes: passing particles and / or molecules through a channel; outputting a first excitation light to a first portion of the channel through a search window; outputting a second excitation light to a second portion of the channel separate from the first portion through a search window; generating a first emission signal using a first photodetector based on first synchrotron radiation received through the proximal end of a first emission optical fiber; and generating a second emission signal using a second photodetector based on second synchrotron radiation received through the proximal end of a second emission optical fiber, wherein the proximal ends of the first and second emission optical fibers are located within an emission optical fiber bundle head.

[0169] In one embodiment, outputting a first excitation light and a second excitation light includes outputting light using an optical engine, as further discussed herein. In one embodiment, the first and / or second excitation light includes coherent light, such as from a laser. In one embodiment, the first light source and the second light source are independently selected from the group consisting of solid-state lasers, diode-pumped lasers, light-emitting diodes (LEDs), lamps, arc discharges, and natural light.

[0170] In one embodiment, the first and second photodetectors are optically coupled to the emission fiber bundle, as described elsewhere in this specification. In one embodiment, the first photodetector is part of a first detector module, such as a detector module, as further described herein with respect to Figure 2, optically coupled to the first emission optical fiber of the emission fiber bundle. In one embodiment, the second photodetector is part of a second detector module, optically coupled to the second emission optical fiber of the emission fiber bundle, as further described herein with respect to Figure 1A.

[0171] In one embodiment, the method includes receiving first and second synchrotron radiation using an emitting fiber bundle comprising a first emitting optical fiber and a second emitting optical fiber, wherein the proximal ends of the first and second emitting optical fibers are positioned in an emitting fiber bundle head, the proximal end of the first emitting optical fiber is positioned to receive first synchrotron radiation emitted from a first portion, and the proximal end of the second emitting optical fiber is positioned to receive second synchrotron radiation emitted from a second portion.

[0172] In one embodiment, flowing particles and / or molecules through a channel includes flowing a suspension of particles and / or a solution of molecules containing particles and / or molecules through a channel. In one embodiment, the suspension of particles or the solution of molecules is or is derived from a biological sample. In one embodiment, the suspension of particles or the solution of molecules contains or is based on body fluids, or is based on fluids derived from or associated with cells. In one embodiment, the particles are selected from the group consisting of extracellular vesicles, biological nanoparticles, organelles, microvesicles, cell-derived vesicles, lipoproteins, polymeric complexes, exomers, RNA-binding proteins, nucleic acid-binding proteins, biological aggregates containing proteins or nucleic acids, protein aggregates, nucleic acid aggregates, lipid aggregates, single biological molecules, cytokines, chemokines, antibodies, cell signaling molecules, therapeutic molecules, nucleic acids, viruses, bacteria, and exosomes. In one embodiment, the particles are extracellular vesicles. In one embodiment, the body fluid includes serum, plasma, cerebrospinal fluid, saliva, nasopharyngeal fluid, tears, whole blood, urine, sputum, or lymph. In one embodiment, the particles are isolated. In one embodiment, the molecules are isolated. In one embodiment, the particles associate with at least one biomarker.

[0173] In one embodiment, flowing a suspension of particles and / or a solution of molecules through a channel includes flowing the suspension and / or solution through the channel particle by particle and / or molecule by molecule. In some embodiments, at least some of a plurality of particles are detected particle by particle. In some embodiments, at least some of a plurality of molecules are detected molecule by molecule. In some embodiments, at least some of a plurality of particles and / or molecules are illuminated particle by particle and / or molecule by molecule. Particle by particle and / or molecule by molecule means observing a plurality of particles or molecules individually (i.e., one at a time) as they pass through a region (e.g., a light beam of a given width). As a non-limiting example of particle by particle or molecule by molecule, a fluid sample containing a plurality of particles or molecules may flow through a constriction of a microfluidic channel 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., none of the other particles of the plurality are present). As another non-limiting example of particles or molecules, a fluid sample containing multiple particles or molecules may flow through and pass through a microchannel such that one or fewer particles or molecules pass through the light beam at a time without overlapping with any of the other particles or molecules. In some specific embodiments, the majority of particles or molecules pass through the light beam such that one or fewer particles or molecules pass through the light beam at a time without overlapping with any of the other particles or molecules. 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 multiple illuminated particles or molecules are illuminated on a particle-by-particle or molecule-by-molecular basis. In a preferred embodiment, more than 90% of the particles or molecules to be illuminated among a plurality of particles or molecules are illuminated individually, either particle by particle or molecule by molecule.In some embodiments, more than 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the particles or molecules in a plurality of detectable particles or molecules are detected on a particle-by-particle or molecule-by-molecule basis. In preferred embodiments, more than 90% of the particles or molecules in a plurality of detectable particles or molecules are detected on a particle-by-particle or molecule-by-molecule basis.

[0174] Illumination of an individual particle or molecule may refer to a particle or molecule present in a fluid sample containing multiple particles or molecules, which is illuminated in the absence of any of the other particles or molecules. Illumination of an individual particle or molecule is distinct from illumination of two or more particles or molecules randomly colocalized in the illuminated area. Illumination of an individual particle or molecule is distinct from illumination of aggregates of particles or molecules. As a non-limiting example, an individual particle or molecule may pass through a light beam and therefore be illuminated. An individual particle or molecule may pass through a light beam in the absence of any of the other particles or molecules, and therefore an individual particle or molecule is illuminated on its own. In some embodiments, an individual particle or molecule is a single nanoparticle present in a fluid sample that can be searched by a light source in the absence of any of the other particles or molecules (for example, for a given light beam width, the beam contains a single particle or molecule so that the beam can be illuminated in the absence of any of the other particles or molecules).

[0175] While it is described that particles are flowed and detected one by one through a channel, it will be understood that the same concept applies to the method and system of this disclosure, due to its similarity to flowing and detecting molecules one by one. Thus, in one embodiment, the method of this disclosure includes flowing molecules one by one through a channel, for example, through the search window of the channel. In this regard, target molecules, such as those associated with one or more detectable agents, pass through the search one at a time. Thus, in one embodiment, there is only one molecule associated with a detectable agent in the search window at a time. Similarly, in one embodiment, two or more target molecules, each associated with a different detectable agent, do not exist simultaneously in the search window. In one embodiment, other molecules are present in the search window of the channel together with the target molecules associated with the detectable agents. Such molecules may include, for example, solvent molecules that assist the flow of the target molecules.

[0176] In one embodiment, molecules that can pass through the search window one by one are selected from the group consisting of proteins, peptides, antibodies, cytokines, chemokines, signaling molecules, therapeutic molecules, drug molecules, RNA-binding proteins, polymeric complexes, nucleic acids, DNA, RNA, synthetic molecules, aptamers, and the like. In one embodiment, 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 coupled with one or more dyes, a single protein coupled with one or more dyes, or a single nucleic acid molecule coupled with one or more dyes.

[0177] In one embodiment, the first synchrotron radiation and the second synchrotron radiation are independently selected from the group consisting of scattered synchrotron radiation, luminescent synchrotron radiation, fluorescent synchrotron radiation, and combinations thereof.

[0178] In one embodiment, the particles are biological particles. In one embodiment, the biological particles are biological nanoparticles. In one embodiment, the particles are selected from the group consisting of extracellular vesicles, organelles, microvesicles, cell-derived vesicles, lipoproteins, polymeric complexes, exomeres, RNA-binding proteins, nucleic acid-binding proteins, biological aggregates containing proteins or nucleic acids, protein aggregates, nucleic acid aggregates, lipid aggregates, single biological molecules, cytokines, chemokines, antibodies, cell signaling molecules, therapeutic molecules, nucleic acids, nucleic acid-binding proteins, RNA-binding proteins, DNA-binding proteins, therapeutic molecules, viruses, bacteria, and exosomes.

[0179] As described above, in one embodiment, the method of the present disclosure is suitable for analyzing relatively small particles flowing through a channel. In one embodiment, particle size is the hydrodynamic diameter. In a particular embodiment, the hydrodynamic diameter is less than 1000 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 one embodiment, the hydrodynamic diameter is determined by measuring dynamic light scattering (DLS) and refers to the size of a rigid sphere that diffuses light in the same way as the size of the biological nanoparticle being measured.

[0180] In some embodiments, the hydrodynamic diameter is from 1,000 nanometers to 1 nanometer, from 900 nanometers to 1 nanometer, from 800 nanometers to 1 nanometer, from 700 nanometers to 1 nanometer, from 600 nanometers to 1 nanometer, from 500 nanometers to 1 nanometer, from 400 nanometers to 1 nanometer, from 300 nanometers to 1 nanometer, from 200 nanometers to 1 nanometer, from 100 nanometers to 1 nanometer, from 90 nanometers to 1 nanometer, from 80 nanometers to 1 nanometer, from 70 nanometers to 1 nanometer, from 60 nanometers to 1 nanometer, from 50 nanometers to 10 nanometers, or from 40 nanometers to 1 nanometer. In certain embodiments, the hydrodynamic diameter is from 1,000 nanometers to 800 nanometers, from 800 nanometers to 600 nanometers, from 600 nanometers to 400 nanometers, from 400 nanometers to 200 nanometers, or from 200 nanometers to 10 nanometers. In preferred embodiments, the hydrodynamic diameter is from 200 nanometers to 2 nanometers. In another preferred embodiment, the hydrodynamic diameter is from 200 nanometers to 10 nanometers. In a more preferred embodiment, the hydrodynamic diameter is from 100 nanometers to 20 nanometers.

[0181] In certain embodiments, the size of the particle is the diameter. In particular embodiments, the diameter is less than 1000 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 preferred embodiments, the diameter is less than 100 nanometers. In certain embodiments, the diameter is determined by measurement using electron microscopy (TEM) or super-resolution imaging.

[0182] In some embodiments, the diameter is 1,000 nanometers to 1 nanometer, 900 nanometers to 1 nanometer, 800 nanometers to 1 nanometer, 700 nanometers to 1 nanometer, 600 nanometers to 1 nanometer, 500 nanometers to 1 nanometer, 400 nanometers to 1 nanometer, 300 nanometers to 1 nanometer, 200 nanometers to 1 nanometer, 100 nanometers to 1 nanometer, 90 nanometers to 1 nanometer, 80 nanometers to 1 nanometer, 70 nanometers to 1 nanometer, 60 nanometers to 1 nanometer, 50 nanometers to 10 nanometers, or 40 nanometers to 1 nanometer. In some embodiments, the diameter is 1,000 nanometers to 800 nanometers, 800 nanometers to 600 nanometers, 600 nanometers to 400 nanometers, 400 nanometers to 200 nanometers, or 200 nanometers to 10 nanometers. In a preferred embodiment, the diameter is 200 nanometers to 2 nanometers. In another preferred embodiment, the diameter is 200 nanometers to 10 nanometers. In a more preferred embodiment, the diameter is 100 nanometers to 20 nanometers.

[0183] In one embodiment, the method further includes directing the flow of particles or molecules. In one embodiment, directing the flow of particles or molecules is based on the presence or absence of synchrotron radiation received from the search window and associated with the particles or molecules. In one embodiment, directing the flow of particles or molecules is based on the intensity of synchrotron radiation received by the detector system from the search window. In this regard, the method is suitable, for example, for separating particles or molecules that emit fluorescence and / or scattered excitation light from particles or molecules that do not emit fluorescence and / or scattered excitation light.

[0184] In one embodiment, directing a flow of particles or molecules includes directing particles or molecules into one or more sorting channels, where such two or more channels include channels for particles or molecules that yield a fluorescence or emission signal above a predetermined threshold and channels for particles or molecules that do not. In some embodiments, the method includes sorting the particles or molecules into a concentrated population. In some embodiments, the sorting includes flow-displacement sorting. In some embodiments, the sorting does not involve the use of acoustic focusing or physical barriers. In some embodiments, sorting is determined by size value, presence or absence of a biomarker, detected light intensity, emitted wavelength, multiple emitted wavelengths, identification of particles or molecules, or a combination thereof. In some embodiments, sorting is determined by the presence or absence of a combination of biomarkers. In some embodiments, sorting is determined by the presence or absence of one or more biomarkers and one or more other biomarkers, such as based on an immunophenotype or immunological phenotype (a phenotype based on the presence, absence, or amount of a marker measured by the binding of a combination of antibodies). In some embodiments, the phenotype is determined at least partially by the presence or absence of two or more biomarkers on a particle (e.g., immunophenotype), and may be further indicated or determined by physical characteristics such as particle size or whether the particle contains nucleic acids or lipid molecules. See, for example, Example 9, further discussed herein. In some embodiments, sorting is determined by the number or type of biomarkers present, by setting a sorting threshold.

[0185] In one embodiment, the method includes quantifying or counting the number of particles and / or molecules associated with synchrotron radiation from a search window, and determining the concentration of particles and / or molecules associated with synchrotron radiation from a search window. See, for example, Example 13, further discussed herein.

[0186] In one embodiment, the method includes ranking particles or molecules in a channel based on the presence or absence of synchrotron radiation from a search window. In one embodiment, the ranking corresponds to the measured emission spectrum of the particles or molecules based on one or more of the first and second synchrotron radiation. In one embodiment, the ranking corresponds to the measured size value of the particles. In one embodiment, the measured size value is a relative size value. In one embodiment, the measured size value is determined by the difference in detected light intensity.

[0187] In some embodiments, particles or molecules associate with a detectable agent. In one embodiment, the detectable agent may be, for example, a target molecule (e.g., a protein, nucleic acid, or biomarker on or within an extracellular vesicle) present on or within the particle being analyzed. Alternatively, the detectable agent may be a molecule (e.g., a fluorescent probe or an antibody conjugated to a nucleic acid probe) that associates with the target molecule (e.g., a protein, nucleic acid molecule, or biomarker on or within an extracellular vesicle or biological nanoparticle or polymeric complex) that associates with the particle, thereby enabling the detection of the nanoparticle. In some embodiments, the detectable agent is fluorescent and can therefore be detected by fluorescence-based detection methods known in the art.

[0188] In one embodiment, the particles include at least one biomarker, such as a biomarker associated with one or more detectable drugs. In one embodiment, the method includes determining at least one copy number of the at least one biomarker, as will be further discussed herein with respect to Example 10.

[0189] As used herein, “associate” includes interactions via covalent and / or non-covalent interactions. For example, a detectable agent may be covalently attached to a particle. Alternatively, a detectable agent may be embedded, for example, within the film of the particle and / or within the hydrophobic interior of the particle. In certain embodiments, a detectable agent may be embedded in the film of the particle via non-covalent interactions such as van der Waals forces or electrostatic forces.

[0190] In certain embodiments, the detectable agent associates with the surface of the particle. In some embodiments, the detectable agent may be covalently and / or non-covalently attached to the surface of the particle. In other embodiments, the detectable agent may be embedded within the surface of the particle. In certain embodiments, the detectable agent is surrounded by a membrane dye embedded in the lipid layer of an extracellular vesicle, for example, on the surface of the particle. The relationship between the particle surface and the detectable agent provides information about the particle size. For example, particles with a large surface area will associate with a large number of detectable agents, while particles with a small surface area will associate with fewer detectable agents. The relationship between the number of detectable agents associated with the particle surface provides a correlation between light intensity and nanoparticle surface area. Thus, the amount of emitted light intensity corresponds to the particle size, and specifically, to the particle surface area.

[0191] As will be further discussed herein with respect to Example 11, the particle size determined by means of the fluorescence intensity from the film dye, in conjunction with the number of detectable agents associated with the analyte on the particle surface, can be used to determine whether the particle is intact.

[0192] In other embodiments, the detectable agent associates with the interior of the particle. In some embodiments, the detectable agent is embedded within the particle (e.g., a lipophilic dye embedded in a lipoprotein). In some embodiments, the detectable agent does not associate with the surface of the particle, but is either embedded within the particle or otherwise surrounded by the particle. In certain embodiments, the detectable agent is contained by the particle but does not associate with an internal surface, e.g., a lipid membrane, or with a dye freely suspended in an extracellular vesicle that does not associate internally. Internally detectable agents, such as those embedded in a particle (e.g., a lipophilic dye embedded in a lipoprotein), or those contained within a particle without associating with an internal surface (e.g., a free-floating dye in a vesicle), are also referred to herein as “volume dyes.” The relationship of volume dyes surrounded by a particle provides information about the particle size. 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 a particle provides a correlation between light intensity and nanoparticle volume. Thus, the amount of light intensity emitted corresponds to the size of the particle, and more specifically, to the volume of the particle.

[0193] In some embodiments, the particles include both a volume dye associated with the surface and a detectable agent. Nanoparticles containing both a volume dye and a detectable agent associated with the surface can provide information about both the surface area and volume of the particles. In some embodiments, the volume dye and the detectable agent are the same. In other embodiments, the volume dye and the detectable agent are different. In some embodiments, the volume dye can provide information about the identity or type of the detected or isolated particles. In some embodiments, the use of a volume dye that is a fluorescent substrate can provide information about the identity or type of the detected or isolated particles. In certain embodiments, the use of a volume dye that is a fluorescent substrate for an enzyme specific to particles such as exosomes can further provide information about the identity or type of the detected or isolated particles.

[0194] In some embodiments, particles are labeled with membrane dyes and membrane-permeable nucleic acid dyes, such as membrane-permeable RNA dyes. As further described herein with respect to Example 11, such combinations of detectable agents are suitable for determining whether particles contain nucleic acids such as RNA or DNA, and whether particles contain membranes or lipid molecules. Such particles can be further labeled with detectable agents, such as fluorescently labeled antibodies configured to selectively bind to surface markers, in addition to determining whether particles contain membranes and / or nucleic acids, for determining the immunophenotype of the particles, i.e., the overall phenotype. This overall phenotype includes physical properties (e.g., if lipid membranes and / or nucleic acids are present) and immunophenotype (e.g., if a certain biomarker is present or absent, or if the amount reported by the antibody differs).

[0195] In one embodiment, the detectable agent is selected from fluorescently labeled antibodies, fluorescently labeled proteins, fluorescently labeled nucleic acids, fluorescently labeled lipids, membrane dyes, fluorescent dyes, pigments, polymer dots, and combinations thereof. In another embodiment, the detectable agent is selected from the group consisting of luminescent dyes, fluorescent dyes, fluorescently labeled antibodies, fluorescently labeled proteins, fluorescently labeled nucleic acids, fluorescently labeled lipids, fluorescently labeled carbohydrates, fluorescently labeled small molecules, membrane dyes, fluorescent dyes, pigments, polymer dots, fluorescent substrates for enzymes, membrane-permeable nucleic acid dyes (such as membrane-permeable RNA dyes), or combinations thereof.

[0196] In some embodiments, the detectable agent specifically binds to one or more binding targets associated with the particles. In some embodiments, 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” or “specifically (or selectively) immunoreact with” an antibody, when referring to biological nanoparticles, refer to a binding reaction that determines the presence of the target biological nanoparticles or the presence of a biomarker associated with the target biological nanoparticles, and often occur in a heterogeneous population of nanoparticles and other biomolecules. Therefore, under given immunoassay conditions, the given antibody binds to specific biological nanoparticles at least twice the background, more commonly 10 to 100 times or more the background. Specific binding to an antibody under such conditions requires an antibody selected for its specificity to specific biological nanoparticles or specific biomarkers or specific molecules (e.g., cytokines, chemokines, antibodies, nucleic acids). For example, a polyclonal antibody may be selected to obtain only polyclonal antibodies that are specifically immunoreactive with a selected antigen and not immunoreactive with other proteins. This selection can be achieved by removing antibodies that cross-react with other molecules.

[0197] In one embodiment, the detectable agent is a first detectable agent, and particles associate with a second detectable agent. In one embodiment, the detectable agent is a first detectable agent, and molecules (e.g., cytokines or cell signaling molecules) associate with a second detectable agent. In one embodiment, the first detectable agent has a first emission spectrum in a first emission wavelength range, and the second detectable agent has a second emission spectrum in a second emission wavelength range different from the first emission wavelength range. In one embodiment, the first detectable agent has a first excitation spectrum in a first excitation wavelength range, and the second detectable agent has a second excitation spectrum in a second excitation wavelength range. In one embodiment, the first and second detectable agents have similar, identical, and / or overlapping emission spectra. In one embodiment, the first and second detectable agents have different emission spectra. In one embodiment, the first and second detectable agents have similar, identical, and / or overlapping excitation spectra. In one embodiment, the first and second detectable agents have different excitation spectra.

[0198] In some embodiments, the detectable agent is attached to the surface of the particle, is located within the surface of the particle, is located inside the particle, is located within the particle matrix, or is a combination of these. In some embodiments, the detectable agent is fluorescent, is luminescent, or is any combination of these. In some embodiments, the particle associates with multiple detectable agents. In some embodiments, at least one of the multiple detectable agents is attached to the surface of the particle. In some embodiments, at least one of the multiple detectable agents is attached to the surface of the particle and at least one of the multiple detectable agents is located within the surface of the particle. In some embodiments, at least one of the multiple detectable agents is attached to the surface of the particle and at least one of the multiple detectable agents is located inside the particle. In some embodiments, at least one of the multiple detectable agents is attached to the surface of the particle and at least one of the multiple detectable agents is located within the particle matrix. 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 located within the surface of the particle, at least one of the multiple detectable agents is located inside the particle, or a combination thereof. In some embodiments, the detectable agents among the multiple detectable agents have overlapping emission profiles. In some embodiments, the detectable agents among 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 among the multiple detectable agents have overlapping excitation profiles. In some embodiments, the detectable agents among 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 among the multiple detectable agents include the same detectable agent. In some embodiments, the detectable agents among the multiple detectable agents include two or more types of detectable agents.

[0199] In some embodiments, the detectable agents among a 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, where different emission spectra are used for the detection of different detectable agents. In some embodiments, the detectable agents have different emission lifetimes. In one embodiment, the detectable agents have different emission intensities at a common wavelength.

[0200] In one embodiment, the first detectable agent has a first excitation spectrum in a first excitation wavelength range, and the second detectable agent has a second excitation spectrum in a second excitation wavelength range different from the first excitation wavelength range. In this regard, the detectable agents are suitable for use in excitation multiplexing, thereby allowing different detectable agents to be used by exciting them in different excitation wavelength ranges.

[0201] In one embodiment, the detectable agent is configured to be excited by light in different wavelength ranges. In one embodiment, the detectable agent is configured to be excited in a first amount by a first excitation light in a first wavelength range, and in a second amount by a second excitation light in a second wavelength range different from the first wavelength range. In this regard, the detectable agent is configured to emit synchrotron radiation of a first intensity in response to the first excitation light, and synchrotron radiation of a second intensity in response to the second excitation light. The ratio of the first synchrotron radiation to the second synchrotron radiation can be used to track or otherwise identify particles associated with the detectable agent.

[0202] In some embodiments, peak wavelengths are separated into those greater than 10 nanometers, 20 nanometers, 30 nanometers, 40 nanometers, 50 nanometers, 75 nanometers, 100 nanometers, 120 nanometers, 140 nanometers, 160 nanometers, 180 nanometers, 200 nanometers, 300 nanometers, 400 nanometers, 500 nanometers, 600 nanometers, or 700 nanometers.

[0203] In another embodiment, the Disclosure provides a method for analyzing particles in a fluid sample. In one embodiment, the method includes: flowing a fluid sample containing a plurality of particles and / or molecules through a channel; illuminating particles and / or molecules within the channel; collecting synchrotron radiation emitted from the channel using a focusing system equipped with a high NA air objective lens having a numerical aperture in the range of about 0.91 to less than 0.99; generating a signal based on the collected synchrotron radiation emitted from the channel based on particles or molecules; and assigning values ​​to the particles or molecules based on the signal.

[0204] In some embodiments, detection or imaging employing a fluid device uses a focusing system having an numerical aperture of 0.91 or higher, 0.92 or higher, 0.93 or higher, 0.94 or higher, 0.95 or higher, 0.96 or higher, 0.97 or higher, or 0.98 or higher. In preferred embodiments, the focusing system includes an air objective lens having a numerical aperture of about 0.95. As further discussed 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 focusing 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 involve generating a signal based on emission from a search channel. Such signals are often not conventional image signals, such as signals that produce images of particles or molecules immobilized in the channel, or on the surface or within the matrix. Rather, in many embodiments, the methods of the present disclosure instead rely on the presence, absence, or intensity of light emitted from the channel. In this regard, light-gathering efficiency and synchrotron radiation intensity are more important for the methods of the present disclosure. This is in contrast to conventional imaging applications where image resolution and the absence of optical aberrations (e.g., spherical or chromatic aberration) may be as important as, or even more important than, light-gathering efficiency. Therefore, high NA aerial targets are often suitable, and oil immersion or water immersion objectives are unnecessary and often unsuitable for the methods of the present disclosure.

[0205] In one embodiment, the method is a method for determining the size of a particle, and the value is a size value. In one embodiment, ranking the particles in the channel is based on the presence or absence of synchrotron radiation from the search window. In one embodiment, ranking the particles in the channel is based on the intensity of synchrotron radiation from the search window. In one embodiment, the ranking corresponds to the measured emission spectrum of the particles based on one or more of the first and second synchrotron radiation. In one embodiment, the ranking corresponds to the measured size value of the particles. In one embodiment, the measured size value is a relative size value. In one embodiment, the measured size value is measured by the difference in the intensity of the detected light.

[0206] In some embodiments, particles or molecules associate with a detectable agent. In one embodiment, the detectable agent is a first detectable agent, and the particles or molecules associate with a second detectable agent. In one embodiment, the first detectable agent has a first emission spectrum in a first emission wavelength range, and the second detectable agent has a second emission spectrum in a second emission wavelength range different from the first emission wavelength range. In one embodiment, the detectable agent is a fluorescent detectable agent. In one embodiment, the first detectable has a first emission spectrum in a first emission wavelength range, and the second detectable agent has a second emission spectrum that is identical, similar, and / or overlaps with the first emission wavelength range.

[0207] Self-correcting, flow-based analysis In another embodiment, the disclosure provides a self-correcting single-molecule / particle flow analysis method. Measuring fluorescence emitted from single molecules or particles in a flow stream is highly influenced by the flow beam and / or laser beam profiles. Thus, in one embodiment, 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 signals and their interpretation present many challenges in 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 a flow stream. These challenges include i) co-localization of biomarkers important for phenotyping EVs or other biological nanoparticles, ii) measurement of particle concentrations, iii) examination of biological heterogeneity, often described by the copy number of biomarkers, iv) determination of the copy number of biomarkers associated with EVs or other biological nanoparticles, and v) characterization of physical properties such as the size of vesicles or particles stained with membrane dyes.

[0208] To overcome these challenges, this disclosure provides a suitable method for analyzing single molecules and particles in a flow stream using a self-correcting method. Using such a “self-correcting single molecule / particle” method, it is possible to accurately 1) colocalize biomarkers expressed on the same particle flowing through multiple excitation regions or portions of a channel within a search window, 2) identify and enumerate single particles and / or molecules, 3) obtain the flow rate sampled by each individual particle and / or molecule, and 4) measure the concentration of the analyzed particles and / or molecules. Such a “self-correcting single molecule / particle” method also makes it possible to accurately determine the copy number of biomarkers associated with EVs or other biological nanoparticles. See, for example, Example 10.

[0209] In short, in this method, multiple excitation regions or portions of the channel within the search window are composed of known spatial patterns. A particle or molecule is measured twice in two different portions of the microchannel through which the particle is flowing (see, for example, Figure 8). Because the flow through the microfluidic channel is typically laminar, the transport time of a particular particle flowing through any two adjacent or closely spaced excitation regions or portions of the channel is generally proportional to the distance between these two excitation regions or portions of the channel and the velocity of the molecule. Also, due to the nature of laminar flow and the small separation distance between the excitation regions or portions of the channel, the position of a particular particle in the cross-section of the channel generally remains the same throughout the transport time. Therefore, the particle generally interacts with different laser beams focused on different portions of the channel at very similar positions in the cross-section of the channel. Considering these characteristics, it is possible to identify a single analyte (e.g., vesicles stained with a fluorescent dye or bio-nanoparticles labeled with an antibody) and further co-localize other biological markers using the extracted transport time or particle velocity (see, for example, Figure 8).

[0210] Accordingly, in one embodiment, the method includes: flowing particles through the lumen of a channel, such that the channel defines a search window configured to allow light to pass in and out of the lumen; outputting first excitation light to a first portion of the channel or search window using a first light source; outputting second excitation light to a second portion of the channel or search window, separate from the first portion, using a second light source; generating a first emission signal using a first photodetector based on first synchrotron radiation received from the first portion; generating a second emission signal using a second photodetector based on second synchrotron radiation received from the second portion; and determining the velocity of the particles in the channel based on the time difference between the first and second emission signals and the distance between the first and second portions. In one embodiment, the method includes using any of the systems of the present disclosure. As discussed elsewhere in this specification, in one embodiment, the first photodetector is part of the first detector module, and the second photodetector is part of the second detector module.

[0211] In one embodiment, the method includes detecting light, such as first and second synchrotron radiation, using time bins. The disclosed apparatus and methods for determining the properties of biological nanoparticles can be performed rapidly with short signal integration times or fast bin times. Using bin times, for example, start and stop times for fluorescence searching can be calculated to aid in the selection of information. Time bins (also referred herein to as signal integration times) can disclose a time range of histograms in which events occur or are observed. In some embodiments, detection, measurement, and / or exploration of biological nanoparticles uses time bins. In some embodiments, time bins have ranges 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 ​​of 10ms~0.1ms, 5ms~0.1ms, 1ms~0.1ms, 0.5ms~0.1ms, 0.1ms~1μs, 90μs~1μs, 80ns~1μs, 70ns~1μs, 60ns~1μs, 50ns~1μs, 40ns~1μs, 30ns~1μs, 20ns~1μs, 10μs~0.1μs, 5μs~0.1μs, or 1μs~0.1μs. In preferred embodiments, the time bins have a range of 1μs~2ms.

[0212] In one embodiment, the method includes correlating a first emission signal with a second emission signal based on emission signal characteristics or particle characteristics shared by the first and second emission signals. In one embodiment, the correlation of the first and second emission signals is based on emission signal characteristics or particle characteristics. As shown in Figure 8, a signal detected in a first detection window or first portion of a channel or first excitation line may be detected downstream in a second detection window or second portion of a channel or second excitation line. In this regard, particles may be tracked as they travel through the channel. Additionally, particles may be explored for various different biomarkers. In one embodiment, the detection window is at least partially defined by a portion of the channel or the excitation line of the excitation light, as will be further discussed herein.

[0213] In one embodiment, the method includes correlating a first emission signal with a second emission signal based on emission signal characteristics or particle characteristics. In one embodiment, correlating a first emission signal with a second emission signal includes comparing the intensity of the first emission signal with the intensity of the second emission signal. In one embodiment, the method further includes enumerating the number of particles passing through the channel based on the correlation of the first emission signal with the second emission signal. In one embodiment, the method further includes colocalizing target molecules on particles based on the correlation of the first emission signal with the second emission signal. In one embodiment, the method further includes determining particle concentration based on the correlation of the first emission signal with the second emission signal. Such concentration may be further defined at least in part based on the volumetric flow rate through the channel, as will be further discussed herein with respect to Figures 11A and 11B. In one embodiment, the method further includes determining detection efficiency and recovery rate based on the correlation of the first emission signal with the second emission signal.

[0214] In one embodiment, the particles associate with one or more detectable agents. In one embodiment, the one or more detectable agents are configured to generate one or more signals in response to excitation light. In one embodiment, the particles associate with one or more membrane dyes. In one embodiment, the particles associate with one or more volume dyes. Such detectable agents can be used to detect the presence or absence of a marker in the particles, as will be further discussed herein with respect to Figures 9A to 9D.

[0215] In one embodiment, the method includes detecting each particle and / or molecule, and tagging the particles and / or molecules using their own flow information, which includes the transport time and / or velocity of the particles and / or molecules. In one embodiment, self-correcting flow analysis becomes possible by tagging or assigning the detected particles or molecules with the corresponding flow information (e.g., transport time and / or velocity).

[0216] Figure 9A graphically illustrates (left) particles passing through two excited regions or portions of a channel in a fluid device according to one embodiment of the present disclosure, and shows the absorption and emission spectra of a membrane dye labeling the particles according to one embodiment of the present disclosure (right). In the illustrated embodiment, particles (e.g., extracellular vesicles or liposomes) are labeled with a membrane dye (e.g., di-8-ANEPPS) and excited by two laser lines of the same wavelength (e.g., 488 nm). In this regard, the particles can be tracked by measuring the time between signals emitted from the two illustrated portions of the channel. As will be considered, such tracking is useful in enumerating particles within a population of particles, enumerating particles passing through the channel, and calculating the concentration of a particular type of particle in the suspension passing through the channel by accurately sampling the flow rate of the suspension of particles in the channel. As will be considered, such tracking is also useful for co-localizing different biomarkers on particles labeled with different fluorescent probes.

[0217] Figure 9B graphically illustrates (left) a particle passing through two excitation regions or portions of a channel in a fluid device according to one embodiment of the present disclosure, and (right) shows the absorption and emission spectra of a membrane dye labeling the particle according to one embodiment of the present disclosure. In the illustrated embodiment, the particle (e.g., extracellular vesicles or liposomes) is labeled with a membrane dye (e.g., di-8-ANEPPS) and excited by two laser lines of different wavelengths (e.g., 405 and 488 nm). In one embodiment, the membrane dye is excited by both the first and second wavelengths to varying degrees. In one embodiment, the dye emits a signal of first intensity in response to the first excitation light (e.g., 405 nm laser light) and emits a second signal of second intensity, different from the first intensity, in response to the second excitation light (e.g., 488 nm laser light). Thus, in one embodiment, the method includes measuring the ratio of the generated signals, such as the ratio of signal intensities, at different locations in the channel or excitation region. As long as the pigment is present in or on the particles above a certain level, this ratio can be used to accurately identify the particles.

[0218] Figure 9C graphically illustrates a particle passing through two excitation regions of a fluid device according to one embodiment of the present disclosure (left), and shows the absorption and emission spectra of the particle according to one embodiment of the present disclosure (right). In the illustrated embodiment, a particle (e.g., an extracellular vesicle or liposome) is labeled with a combination of two membrane dyes (e.g., DiO and DiD) and excited by two laser lines having different wavelengths (e.g., 488 and 640 nm). As shown, the dyes provide separate emission signals based on excitation light in distinct wavelength ranges. In this regard, the particle can be tracked by measuring the time between the two signals emitted by the two dyes from two detection windows, i.e., two illustrated portions or excitation regions of a channel.

[0219] Figure 9D graphically illustrates (left) a particle passing through two detection windows of a fluid device according to one embodiment of the present disclosure, and shows the absorption and emission spectra of the particle according to one embodiment of the present disclosure (right). In the illustrated embodiment, the particle (e.g., extracellular vesicles or liposomes) is labeled with a membrane dye (e.g., DiD) in combination with a volume dye (e.g., calcein AM). As shown, the dyes provide separate emission signals based on excitation light in distinct wavelength ranges. In this regard, the particle can be tracked by measuring the time between the two signals emitted by the two dyes from the two illustrated portions or excitation regions (detection windows) of the channel.

[0220] Self-corrected single-molecule / particle counting methods offer numerous advantages over other conventional methods, including significantly improved result quality, as described below.

[0221] Accurate enumeration and colocalization.

[0222] The method of this disclosure is suitable for obtaining or determining a more accurate number of analytes (e.g., extracellular vesicles or biological nanoparticles) because it eliminates or reduces many interfering signals, such as background fluctuations and small aggregates of dyes, in the identification of actual events (e.g., extracellular vesicles or biological nanoparticles). (See, for example, Figures 10A, 10B, 11B, 12, 14, and 15). Colocalization of single-molecule events in a flow stream by statistical methods (e.g., cross-correlation functions) is often adversely affected by interference from spatially close events, contaminants, background fluctuations, and especially linear velocity differences between particles induced by laminar flow profiles. Using this method, it is possible to minimize these interferences and, consequently, improve the quality of enumeration and colocalization. Figure 14A shows multicolor colocalization of extracellular vesicles in semen labeled with di-8-ANEPPS membrane dye, anti-CD63-Alexa647 antibody, and anti-CD81-PE / CF594 antibody according to one embodiment of this disclosure. Figure 14B shows a subpopulation or subtype of extracellular vesicles in semen based on the colocalization of di-8-ANEPPS membrane dye, anti-CD63-Alexa647 antibody, and anti-CD81-PE / CF594 antibody according to one embodiment of the present disclosure. Figure 15 shows the measurement of semen extracellular vesicle (sEV) concentration according to one embodiment of the present disclosure.

[0223] The precise co-localization of biomarkers, such as those expressed on the same biological nanoparticle, underlies many important applications (e.g., immunophenotyping for identifying subtypes of biological nanoparticles or molecules; see Example 9). 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 in different detection windows, or signals from different excitation regions, to a given particle and / or molecule. This is because particles / molecules can flow at a wide range of velocities within the microfluidic channel due to the laminar nature of the microfluidic flow environment and parabolic flow profiles. The methods of this disclosure address these difficulties and enable the precise co-localization of biomarkers on a single biological nanoparticle or molecule (e.g., see Figures 10A, 10B, 11, 12, 14, and 15 and related examples).

[0224] Accurate sampling of flow velocity.

[0225] The self-correcting method of this disclosure provides the transport time for each particle flowing through different locations in the cross-section of the channel. Using known intervals between portions of the channel or detection window illuminated by spatially separated different excitation lights, it is possible to calculate the linear velocity of each particle being inspected and determine the volumetric flow sample accordingly. As shown in Figure 11A, it is possible to convert the average linear velocity through the channel into a volumetric flow rate.

[0226] In microfluidic analysis, knowing the volumetric flow rate is often necessary to determine the volume of the sample analyzed during the experiment. Therefore, the absolute concentration of a single particle / molecule can be measured based on the counting of analytes / molecules / nanoparticles and the volume analyzed. Furthermore, volumetric flow rate is a useful parameter for evaluating sample throughput and consumption. While important, direct measurement of volumetric flow rate is often difficult in microfluidic environments, especially when the volumetric flow rate is very low (e.g., pL~nL / sec), because the excitation region explores the ultra-small volume of the sample. Using the method of this disclosure, volumetric flow rate can be determined using the transport time of each molecule and / or particle flowing through the laser line or excitation region, based on the fact that the flow is laminar in this microfluidic environment. Thus, the linear velocity of each particle and / or molecule can be calculated, and the distance between these laser lines or excitation regions can be determined. From the measured average particle and / or molecular velocity, and by knowing the area of ​​the channel cross-section, the volumetric flow rate can be measured. Therefore, using the method of the present disclosure, the volumetric flow rate can be determined by using the measured transport time and / or velocity of a single particle and / or single molecule (see, for example, Figures 10, 11, 15 and related examples).

[0227] Accurate measurement of concentration.

[0228] By knowing the volumetric flow rate, it is possible to accurately determine the number of single analytes examined within a given time period and obtain the volume of the analyzed sample (often at the nanoliter level); therefore, this method is also suitable for accurately measuring the concentration of an analyte. Figure 11B shows a measurement of the concentration of extracellular vesicles in a fluid sample passing through a channel of the system, calculated using the comparison illustrated in Figure 11A, according to one embodiment of the present disclosure. The method of the present disclosure provides an absolute count of the analyte in a given volume, without relying on parameters obtained from a bulk sample (e.g., extinction coefficient) and any external calibration curve, thus enabling a more accurate determination of the analyte concentration. Figure 15 shows a measurement of the concentration of semen extracellular vesicles (sEVs) according to one embodiment of the present disclosure.

[0229] Accurate determination of detection efficiency and recovery rate.

[0230] Detection efficiency can be defined as the proportion of analytes within the channel or excitation region, or the detection region or detection window, that are counted by the method of this disclosure. If the signal distribution associated with the analytes flowing through the channel follows a known statistical model (e.g., a log-normal distribution commonly seen in flow analysis), detection efficiency can be quantified by knowing the cumulative distribution function (CDF) at the cutoff value.

[0231] Recovery rates, such as the recovery rate defined as the ratio of the counted analyte to the amount of analyte spiked into or present in a given volume, are affected by many other factors in addition to detection efficiency (e.g., accuracy of stock concentration, possible aggregation and decomposition of analyte, surface absorption, etc.). If the stock concentration of the analyte is precisely known, it is also possible to determine the recovery rate accordingly. Figures 12A–12C illustrate that the method of this disclosure is exceptionally sensitive to both single fluorescent dyes and particles (e.g., EVs). In this regard, Figures 12A–12C illustrate that the method of this disclosure is highly sensitive to dyes of both single small molecules (e.g., Alexa647-tagged antibody, see Figure 12B) and proteins (R-phycoerythrin (PE, see Figure 12C)), with a detection efficiency of over 99%. The average SNR of EVs stained with membrane dyes is approximately 54, and the recovery rate exceeds 95%, which is within the range of uncertainty of stock concentration and demonstrates higher sensitivity to particles and molecules than is available by conventional methods.

[0232] Accurate determination of the number of copies As will be 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. Since the method is suitable for detecting an entire population of single molecules / particles present in a sample, or an aliquot of the sample, or a very large proportion (e.g., more than 90%) of such molecules / particles passing through a microchannel, the method can also be used to deconvolve a single-particle intensity distribution using a single-molecule intensity distribution to accurately determine the number of bound antibodies on each particle, and thus the number of corresponding proteins. Such an approach is useful, for example, to determine the number of fluorescently labeled antibodies associated with a particle, such as EVs, to provide quantitative information about the molecular composition of a single particle. As will be further discussed herein with respect to Example 12, this information, in conjunction with other information about particle size, can be used to determine whether the analyzed particle is intact or fragmented, or whether the analyzed particle is hollow (e.g., containing nucleic acids) and non-functional or biologically functional.

[0233] Automatic focusing method In another aspect, the disclosure provides a method for focusing the optical components of a system onto the system's channels. Counting and measuring particles and molecules in flow, such as extracellular vesicles, viruses, lipoproteins, RNA-binding proteins, or cytokines, at the single-molecule / particle level, is often highly sensitive to changes in the environment (e.g., thermal-induced expansion) and changes in instrument configuration (e.g., minute drifts in optical alignment and changes in channel dimensions). To consistently collect data and improve the sensitivity of flow-based devices, the disclosure provides an automated focusing method.

[0234] In one embodiment, excitation light, such as a specific laser beam (e.g., 870 nm), reflected by a microfluidic device is collected via a fiber-coupled confocal scheme, further discussed herein with respect to Figure 7A. The magnitude of this back reflection is externally calibrated and attenuated by a neutral density filter to ensure it is within the dynamic range of the photodetector. In one embodiment, when correct focusing of the detection channel is achieved, a preferred or optimal value of the back-reflected light is determined and set as a reference for "locking" the focus level (see, for example, Figure 7B). In one embodiment, a portion of the microfluidic device, such as a channel including a search window, or an objective lens, is operably coupled to an electrically operated movable stage, such as a stage driven by a piezo or DC motor. In this regard, the focusing system is configured to move relative to the search window of the channel in order to focus the focusing system onto the search window. As detailed in Figure 7C, the movable stage can be controlled based on comparing the current value of the reflection with a value at a previous point in time (e.g., 200 ms earlier) and a reference value.

[0235] Accordingly, in one embodiment, the present disclosure provides a method for focusing excitation light through an optical component on a fluid channel. In one embodiment, the method includes using a system 700, which is further discussed herein with respect to Figures 7A and 7B. In one embodiment, the method includes illuminating a search window or another portion of the fluid channel with light from a light source; focusing the light onto the search window using an optical component disposed between the channel and a photodetector; generating a lock signal using a photodetector based on the focused light reflected back from the search window in a first time; generating a test signal using a photodetector based on the focused light reflected back from the search window in a second time after the first time; determining whether the test signal is within a predetermined proportion of the lock signal; and, if the test signal is outside the predetermined proportion of the lock signal, moving the fluid channel relative to a high-NA air object.

[0236] 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%, about 1% to about 10%, about 2% to about 8%, about 2.5% to about 7.5%, about 3% to about 6%, or about 3% to about 5%.

[0237] In one embodiment, the Disclosure provides a method for maintaining focus on a fluid channel, the method comprising: illuminating an imaging area of ​​a microfluidic system with light from a near-infrared light source; generating an image of the imaging area using a camera; determining the amount of defocus in the image; determining whether the amount of defocus is within a predetermined range; and, if the test signal is outside a predetermined range, moving the fluid channel relative to a high NA air objective lens. In one embodiment, the structure being imaged is adjacent to the channel and, in some embodiments, is a separate structure from the channel. In one embodiment, the structure has a higher level of contrast compared to other parts of the imaging area. In one embodiment, the structure defines an air-filled housing in the microfluidic system. Such an air-filled structure has, for example, a higher contrast than the fluid-filled channel and is suitable for generating an image for determining the amount of defocus in the image.

[0238] Figure 7D is a series of images of a channel with different amounts of defocus, taken at several distances from a high NA air objective lens, according to one embodiment of the present disclosure. Figure 7E illustrates the amount of focusing quality at various distances between the channel and the high NA air objective lens, focusing on the position in the images of Figure 7D, according to one embodiment of the present disclosure. In this embodiment depicted, the focal plane was detected using NIR imaging and an air objective lens with an NA of 0.95. Figure 16 graphically illustrates an apparatus for achieving autofocus by using near-infrared imaging or machine vision with a high numerical aperture air objective lens (NA=0.95), according to one embodiment of the present disclosure.

[0239] As shown, the focal channel includes a constriction. The focusing quality of the constriction was monitored in real time, as shown in Figure 7E. As the objective lens moved upward, the focusing quality improved until it reached a first maximum value indicating that the focal plane was at the bottom of the constriction channel. As the position of the objective lens increased, the focusing quality decreased and then rose to a second maximum value indicating that the focal plane was set at the top of the constriction channel. The four images in Figure 7D show real-time images when the high NA air objective lens was in the four corresponding positions.

[0240] Figure 7F schematically illustrates a feedback control loop used to set the focal plane according to one embodiment of the present disclosure. Figure 7G is another feedback control loop used to perform real-time focusing supported by near-infrared machine vision through a high NA airborne objective lens, according to one embodiment of the present disclosure. Focusing of a channel can be achieved using the focusing methods schematically illustrated in Figures 7F and 7G.

[0241] In one embodiment, the method includes using a focusing system to collect focused light reflected back from the search, the focusing 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.

[0242] In one embodiment, the method includes collecting light using a light-gathering system to generate an image of an imaging area using a camera, wherein the light-gathering system 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.

[0243] In one embodiment, the light is in the range of approximately 700 nm to approximately 1.5 μm. In another embodiment, the light is in the range of approximately 700 nm to approximately 1100 nm. In yet another embodiment, the light is in the invisible wavelength range.

[0244] A process described above is described in terms of computer software and hardware. The described technique, when executed by a machine, may constitute a machine-executable instruction embodied in a tangible or non-temporary machine (e.g., a computer)-readable storage medium that causes the machine to perform the described operation. Additionally, the process may be embodied in hardware such as an application-specific integrated circuit ("ASIC") or otherwise.

[0245] A tangible machine-readable storage medium includes any mechanism that provides (i.e., stores) information in a non-temporary form accessible by a machine (e.g., a computer, network device, personal digital assistant, manufacturing tool, or any device having a set of one or more processors). For example, a machine-readable storage medium includes recordable / non-recordable media (e.g., read-only memory (ROM), random-access memory (RAM), magnetic disk storage media, optical storage media, flash memory devices, etc.).

[0246] The above description of illustrated embodiments of the Invention, including those described in the abstract, is not intended to be exhaustive or to limit the Invention to the exact form disclosed. While specific embodiments and examples of the Invention are described herein for illustrative purposes, various modifications are possible within the scope of the Invention, as will be recognized by those skilled in the art.

[0247] These modifications may be made to the invention in light of the detailed description above. The terms used in the following claims should not be construed as limiting the invention to the specific embodiments disclosed herein. Rather, the scope of the invention should be determined entirely by the following claims, which should be interpreted in accordance with established doctrines of claim interpretation. [Examples]

[0248] Example 1: Multiplexed detection of single particles / molecules using a free-space laser and an emitting fiber bundle. This embodiment describes an apparatus that enables single-molecule and single-particle analysis in a flow using a free-space laser and an emitting fiber bundle. The apparatus (e.g., Figure 6) is configured to detect fluorescence in multiple color channels from spatially separated different excitation regions or detection windows or portions of a channel, and includes two functional modules: an excitation module using a free-space laser and a detector system using an emitting fiber bundle and a detector module.

[0249] The excitation module is formed by combining multiple laser beams to create a desired profile and spatial pattern (e.g., Figure 4). The spacing between excitation regions can be adjusted by a telescope structure, such as two conjugate plano-convex lenses (f=75mm and f=125mm). The laser beam is shaped by a cylindrical lens (f=200mm) to generate four lines (for the four excitation regions / detection windows / parts of the channel), and then transmitted to the rear focal plane of the objective lens by a multiband dichroic mirror or partial reflection mirror. Thus, the four laser lines are focused into each of the four parts of the microfluidic channel that form the four excitation regions or detection windows (Figure 3B), where fluorescently labeled particles or molecules flow and interact with the excitation laser illumination, resulting in fluorescence emitted from each particle or molecule.

[0250] Depending on the application, this excitation module can be configured in various ways. For example, four laser lines with wavelengths of 405, 488, 561, and 640 nm are focused at equal intervals in a microfluidic channel, or alternatively, four laser lines with wavelengths of 488, 488, 561, and 640 nm are focused at equal intervals in a microfluidic channel, or four laser lines with wavelengths of 488, 405, 640, and 561 nm are focused at equal intervals in a microfluidic channel, or four laser lines with wavelengths of 355, 405, 488, and 640 nm are focused at equal intervals in a microfluidic channel. Generally, equal spacing is preferred, but in some applications, unequal spacing, which can be easily achieved by adjusting the excitation module, may be desirable. The number of lasers and excitation regions can be easily adjusted, such as by expanding to include more laser colors.

[0251] The detector system separates and quantitatively measures the fluorescence emitted from four excitation regions. To collect four spatially separated fluorescence signals emitted from particles / molecules as they are transported and interact with four spatially separated laser lines / excitation regions, the inventors use an emission fiber bundle (e.g., Figure 5). Each fiber in the fiber bundle collects fluorescence caused by the excitation of particles / molecules by each of the laser lines / excitation windows, and the fluorescence collected by each fiber can be further spectrally separated and detected by different photodetectors in the detector module (e.g., Figure 2). Here, the fluorescence collected by the objective lens passes through a multiband dichroic mirror and is focused by a tube lens. A microfabricated array of four slits is precisely positioned at the focal plane of the tube lens so that only light from a desired portion of the channel passes through these slits (e.g., Figure 5A). Because this slit array is located at the proximal end of the fiber bundle (e.g., Figure 5A), the four closely spaced fluorescence signals from the four excitation regions / laser lines are therefore separated through the four fibers of the fiber bundle.

[0252] Next, the fluorescence signals from each fiber of the fiber bundle are further spectrally separated into, for example, several color bands by a series of dichroic mirrors and bandpass filters in a detector module (e.g., Figure 2). Using an aspherical lens (f=7.8 mm), the fluorescence corresponding to the spectral region or color band is then focused to a photodetector such as an avalanche photodiode or a single-photon counting module, where the photons are converted into electrical signals. As described in the following embodiments, a high-frequency counting device is used to count the number of photons at a given time interval and store it in a binary file on a computer communicating with the apparatus for further analysis.

[0253] Example 2: Use of a high numerical aperture air objective lens for ultra-sensitive, robust detection of single molecules and particles flowing in a planar microfluidic channel with line illumination and confocal detection. This embodiment describes an apparatus and method for ultra-high sensitivity and efficiency detection of a single molecule flowing through a planar microfluidic channel, equipped with line confocal detection. For a single particle flowing through a planar microfluidic chip or channel, the apparatus and method provide the same ultra-high levels of sensitivity and efficiency as described for a single molecule flowing through a planar microfluidic channel.

[0254] Single-molecule flow analysis is often hampered by a lack of sensitivity, throughput, and robustness. To address these challenges, the apparatus described in Example 1 is configured using a microfluidic device with a high numerical aperture air objective lens and planar channels. To ensure sensitivity for detecting ultrapolymers, it is important to collect as many optical signals as possible, and our apparatus uses a 0.95 NA air objective lens. While the NA of the objective lens may be higher than 1.0 when an immersion medium (e.g., oil or water) is used, such "wet" objective lenses significantly reduce the robustness of the system and are far more susceptible to vibration, drift, and evaporation of the medium. Furthermore, such "wet" objective lenses often make scanning over longer distances difficult due to contamination of the immersion medium, resulting in a significant decrease in throughput and a more complex instrument design. Switching from one planar channel to another by scanning from one planar channel to another in a microfluidic tip equipped with a "wet" objective lens is also difficult.

[0255] In addition to the use of high-NA air objective lenses, planar microfluidic channels are another essential component for enabling ultra-high sensitivity and efficiency in single-molecule flow analysis. Because high-NA objective lenses often require a very limited working distance (e.g., 170 μm for the 40X / 0.95NA objective lens used in this embodiment), typically only planar substrates or tips between the objective lens and the sample are compatible with this working distance. Other widely used flow configurations, such as microcapillaries, often have walls thicker than this very limited working distance and are therefore unsuitable for use with high-NA objective lenses. Additionally, the use of planar microfluidic channels eliminates spherical / cylindrical aberration associated with the use of round cylindrical capillaries, which is another important factor affecting the collection efficiency and focusing quality of high-NA objective lenses. Furthermore, microfluidic chips or microfluidic devices can facilitate the creation of channels having different widths or heights along the channel length (e.g., channel constrictions, or stepped changes in width and / or height), thereby facilitating the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules during transport. Microfluidic chips can be made by microfabrication methods to create channels having non-spherical or non-square (e.g., rectangular) cross-sections, thereby facilitating the detection, analysis, determination, and / or identification of biological nanoparticles and / or molecules during transport. Microfluidic chips can be formed by bonding them to a coverslip (e.g., glass or plastic) of a desired thickness and desired material properties (e.g., refractive index) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles and / or single molecules during transport, thereby improving compatibility with high-NA air objective lenses for maximum light collection. Finally, microfluidic devices make it possible to generate a large number of channels (e.g., 96 or 384 channels for 96 or 384 samples) on the same device, enabling high-throughput analysis of more samples (e.g., 96 or 384 samples in a form compatible with multi-channel pipettes).Microfluidic chips offer an attractive and versatile platform for use with high-NA air objective lenses to achieve ultra-high-sensitivity and efficient analysis of single molecules and / or particles.

[0256] As described in this embodiment, illuminating the channel with a tightly focused laser line covering the entire cross-section of the channel (e.g., Figures 3B and 4) ensures that all molecules passing through the channel are illuminated and excited with nearly 100% probability. Confocal detection geometry using an aperture (e.g., Figure 5A) improves detection sensitivity by increasing the signal-to-noise ratio and minimizing crosstalk between different excitation regions or laser lines. Our apparatus, using a high-NA air objective lens, line illumination with a highly focused laser line, and confocal detection geometry, ensures that all molecules or particles flowing through the channel are detected with high efficiency, high sensitivity, and high throughput. This is in contrast to existing techniques, for example, when examining some or small portions of molecules flowing through a channel using a focused laser spot (rather than a tightly focused laser line). This is because the laser spot has a diameter of only a few micrometers (e.g., 5 microns) and cannot adequately illuminate the entire cross-section of a capillary with a much larger diameter (e.g., 100 microns). A 5-micron diameter laser spot at the center of a 100 μm diameter capillary represents only about 1 / 400th of the cross-section of the channel through which molecules or particles flow. Even assuming that each molecule flowing through the laser spot is detected, this would mean that only about 1 out of 400 molecules in the flow would be detected. To solve this problem, in our apparatus, the laser beam is shaped by a cylindrical lens (f=200 mm) and focused to the center of the constriction of our planar microfluidic channel, forming a series of laser lines that illuminate the entire cross-section of the channel (e.g., Figure 4), so that all molecules in the flow can interact with the laser excitation. Furthermore, the confocal geometry enhances the detection signal versus noise from all molecules in the flow, either through an aperture in the optical fiber or using a slit (Figure 5A), resulting in ultra-high detection efficiency (number of detected single molecules relative to the number of single molecules flowing through the channel).

[0257] The following Example 3 describes in more detail the detection efficiency achieved using the apparatus described in Example 2. In addition to the extremely high single-molecule detection efficiency detailed in the following Example 3, the use of a high-NA air objective lens provides very stable and robust detection of single molecules in a flow, in contrast to oil immersion or water immersion objective lenses. Figure 14 shows a blank sample (PBS buffer) running for 2000 seconds with no change in background noise whatsoever, demonstrating the absence of instability or fluctuation associated with high-NA air objective lenses, which is not typically the case when using oil immersion or water immersion objective lenses.

[0258] Example 3: Achieving high detection efficiency for single molecules in motion. This embodiment demonstrates the ability to detect a single molecule in motion with extremely high detection efficiency using the apparatus described in Examples 1 and 2 (i.e., a 0.95 NA air objective lens and an emission fiber bundle).

[0259] The detection efficiency of a single molecule and / or single particle is the number of molecules / particles detected relative to the number of molecules / particles flowing through the channel (e.g., through the excitation region). For a given type of fluorescent molecule / particle, this metric is a direct indicator of the sensitivity of the flow analyzer and is therefore an important metric for evaluating the sensitivity and performance of the instrument or apparatus.

[0260] In this embodiment, the principle of single-molecule counting is applied to determine detection efficiency. When measuring a population of single molecules, instead of obtaining identical signals from each molecule, a distribution of signals is always obtained. These signal variations are determined by both intrinsic molecular characteristics (e.g., photon emission probability or bleaching probability) and external factors such as those from the measurement process itself. Our research has shown that such distributions can follow a log-normal distribution. Figures 12B and 12C show the signal-to-noise ratio (SNR) distributions for single R-phycoerythrin (PE) and Alexa647, respectively. After fitting to a log-normal distribution, statistical metrics (e.g., coefficient of determination, R) are used to quantify the fitting quality. 2 Apply ).2 If the value is higher than a certain threshold (e.g., 0.98), we can conclude that the distribution of single-molecule SNR follows a log-normal model.

[0261] In signal analysis, the limit of detection (LOD) is often defined as the weakest signal that can be distinguished from a background with an SNR of 3. Using the same definition and the results of log-normal fitting, the cumulative distribution when the SNR exceeds the LOD can be calculated. Using this analysis, Figures 12B and 12C show that the detection efficiency of a single PE and Alexa647 in the flow is over 99% (e.g., 99.5% and 99.8%, respectively).

[0262] Example 4: Self-correcting flow analysis This embodiment describes a method for identifying a population of biological particles that have specific flow information linked to each particle, using self-corrected fluorescence measurements in microfluidic channels.

[0263] Biological nanoparticles are typically described by physical and / or biological parameters. For example, extracellular vesicles (EVs) are lipid bilayer-bound particles released from cells that can be identified by staining with membrane dyes that exhibit strong fluorescence when inserted into a hydrophobic environment (e.g., lipid bilayer). Successful analysis of EVs in a flow (e.g., multicolor colocalization, determination of detection efficiency, measurement of flow profiles, and estimation of concentration) requires the detection of each particle and tagging of the particles by their own flow information (e.g., velocity). Using the apparatus described in these examples above, we describe the tagging of each detected particle by its own flow information (e.g., velocity). Depending on the specific marker used to detect EVs, such tagging can be achieved in different ways. Four examples are given below. 1) Stain the EV with a di-8-ANEPPS film dye and continuously excite it using two 488 nm laser lines or excitation regions. Briefly, 5 × 10 9A 199 μL EV solution with a particle / mL concentration was mixed with 1 μL of 20 μM di-8-ANEPPS dissolved in DMSO and incubated at room temperature for 30 minutes. After incubation, this sample was introduced into a reservoir fluid-connected to a microfluidic channel having a constriction (2 × 2 × 125 μm). The flow was driven by gravity and surface tension. Two laser lines with a wavelength of 488 nm (20 mW) were focused onto this portion of the microfluidic channel to create two excitation regions or detection windows (Figure 9A) separated by 10 μm. As di-8-ANEPPS-stained EVs flowed through these two different excitation regions or detection windows, two fluorescence signals of similar intensity (wavelength range 575–625 nm) were observed sequentially, due to the laminar flow properties within the microfluidic channel. These particles were thus detected and tagged or assigned with corresponding flow information. In this embodiment, this flow information is the transport time between two 488 nm laser-excited regions, and therefore the known velocity between the two excitation regions. This method can be similarly applied to the analysis of single molecules and tagging each detected molecule with its own flow information (e.g., transport time and / or velocity). 2) EV is stained with di-8-ANEPPS and continuously excited with a 405nm laser and a 488nm laser. In short, 5 × 10 9A 199 μL EV solution with a particle / mL concentration was mixed with 1 μL of 20 μM di-8-ANEPPS dissolved in DMSO and incubated at room temperature for 30 minutes. After incubation, this sample was introduced into a reservoir fluid-connected to a microfluidic channel having a constriction (2 × 2 × 125 μm). The flow was driven by gravity and / or surface tension. Based on the absorption spectrum of di-8-ANEPPS (Figure 9B), the inventors focused 405 nm and 488 nm laser outputs at 100 mW and 20 mW, respectively, onto different parts of the microchannel to generate two excitation regions or detection windows (Figure 9B) spaced 10 μm apart. When EVs stained with di-8-ANEPPS flowed through these two different excitation regions, two fluorescence signals with similar intensities were observed sequentially in the 575–625 nm wavelength range, due to the laminar flow properties within the microfluidic channel and the differences in the excitation laser force used. When similar laser excitation forces were used, two fluorescence signals with a known intensity difference were observed sequentially. These particles were thus detected and tagged or assigned with corresponding flow information. In this embodiment, this flow information was the transport time between the two laser excitation regions, and therefore the velocity at which the distance between the two excitation regions was known. 3) EVs are stained with a mixture of two structurally similar membrane dyes, DiO and DiD, and then continuously searched using a 488 nm laser and a 640 nm laser. Briefly, 5 × 10 9A 396 μL particle solution with a concentration of EV / mL was mixed with 2 μL of 20 μM DiO (in DMSO) and 2 μL of 20 μM DiD (in DMSO), and incubated at room temperature for 30 minutes. After incubation, this sample was introduced into a reservoir fluid-connected to a microfluidic channel having a constriction (2 × 2 × 125 μm). The flow was driven by gravity and / or surface tension. Based on the absorption spectra of DiO and DiD (Figure 9C), 488 nm and 640 nm laser outputs were focused to separate portions of the microfluidic channel, creating two excitation regions or detection windows separated by 10 μm. When particles stained with DiO and DiD flowed through these two laser excitation regions, two fluorescence signals were collected at 495 nm–515 nm and 660 nm–700 nm, respectively. These particles were detected and tagged or assigned corresponding flow information. In this embodiment, this flow information was the transport time between the two laser lines, and therefore the speed at which the interval between the two excitation regions was known. 4) Considering that fragments of lipid membranes or solid lipid particles can also be labeled with membrane dyes, intact EVs can be detected by co-staining with a membrane dye (e.g., DiD) and a volume dye (e.g., calcein-AM), and continuously analyzed using 488 nm and 640 nm laser lines (Figure 9D). In short, 5 × 10 9A 396 μL particle solution with a concentration of particles / mL was mixed with 2 μL of 20 μM DiD (in DMSO) and 2 μL of 2 mM calcein-AM (in DMSO), and incubated in the dark at 37°C for 30 minutes. After incubation, this sample was introduced into a microfluidic channel having a constriction (2 × 2 × 125 μm). Based on the absorption spectra of calcein-AM and DiD (Figure 9D), 488 nm and 640 nm laser outputs were focused onto the microfluidic channel at powers of 20 mW and 10 mW, respectively, to create two detection windows or excitation regions separated by 10 μm. When EVs stained with calcein-AM and DiD flowed through these two laser excitation regions, two fluorescence signals were collected at 495 nm–515 nm and 660 nm–700 nm, respectively. These EVs were detected and tagged or assigned with corresponding flow information. In this embodiment, this flow information was the transport time between the two laser lines, and therefore the speed at which the interval between the two excitation regions was known.

[0264] By tagging or assigning corresponding flow information (e.g., transport time and / or velocity) to detected particles or molecules, self-correcting flow analysis as described in this embodiment and later embodiments becomes possible. This method can be similarly applied to the analysis of single molecules or other biological nanoparticles or other nanoscale entities in order to tag each detected molecule / nanoparticle / entity with its own flow information (e.g., transport time and / or velocity).

[0265] Example 5: Multicolor colocalization using self-correcting flow analysis This embodiment describes the co-localization of multiple markers (e.g., different dye-tagged antibodies and / or membrane dyes) on extracellular vesicles (EVs) using self-corrected flow analysis. This method can be similarly applied to the analysis of single molecules or other biological nanoparticles or other nanoscale entities.

[0266] The precise co-localization of biomarkers expressed on the same biological nanoparticles is the basis for many important applications (e.g., immunophenotyping for identifying EV subtypes). When multiple particles in close proximity in a flow stream pass through excitation regions or detection windows, it can be difficult to correctly assign signals observed in different detection windows, or signals from different excitation regions, to a given particle. This is because particles can flow at a wide range of velocities within the microfluidic channel due to the laminar nature of the microfluidic flow environment and parabolic flow profiles. In the self-corrected flow analysis described in the above examples, a biomarker (e.g., a membrane dye or a given dye-tagged antibody) is measured twice in two excitation regions or detection windows, but this self-corrected flow analysis is used to detect particles or molecules and tag them with their flow information (i.e., linear velocity and / or transport time). Due to the laminar flow nature in microfluidic systems, the search for the presence and / or absence of other biomarkers (e.g., other dye-tagged antibodies present on EVs) is directed to a very narrow time window (e.g., ±0.1 ms) at a specific location (before or after a specific transport time; see Figure 8), and therefore, the use of self-corrected flow analysis significantly improves the accuracy of colocalization, as shown in Figure 10.

[0267] In this example, human semen exosomes (sEVs) were used and co-stained with CD63-A647, CD81-PE / CF594, and di-8-ANEPPS. Briefly, 5 × 10 9490 μL of sEV solution at a concentration of particles / mL was mixed with 5 μL of 2 μg / mL CD63-A647 and 2.5 μL of CD81-PE / CF594 (100-fold dilution of stock solution) and incubated in the dark at room temperature for 60 minutes. Then, 2.5 μL of 20 μM di-8-ANEPPS (dissolved in DMSO) was added to the sample and incubated for a further 30 minutes. After incubation, this sample was introduced into a microfluidic channel having a constriction (2 × 2 × 125 μm) where detection occurred. To perform multicolor colocalization using self-corrected flow analysis, four laser lines (640, 561, 488, and 405 nm in this sequence) were focused onto a portion of the microfluidic channel to create four excitation regions or detection windows that were continuous along the flow direction. Fluorescence from di-8-ANEPPS was measured using the first two laser lines (405 nm, 100 mW and 488 nm, 20 mW) with the same emission filter set (600 / 50 nm) (Figure 9B).

[0268] The first step was to detect and tag the sEVs stained with di-8-ANEPPS using their respective flow information (i.e., transport time between the 405 nm laser line and the 488 nm laser line). This step can be verified using two film dye signals, both in the absence and in the presence of self-correction, as shown in Figure 10. The fluorescence of di-8-ANEPPS excited at 405 nm from all particles stained with this film dye should colocalize with the corresponding fluorescence excited by the 488 nm laser with near 100% efficiency. Additionally, the intensities of these two signals should be similar (i.e., a difference of ±10%) due to the power settings used in this example (i.e., 100 mW for the 405 nm laser and 20 mW for the 488 nm laser). The left panel of Figure 10B shows the colocalized events using an autocorrelation function, which calculates the "most likely" time window for finding colocalized events. Many events colocalized using this statistical method were false positives due to mismatches in signal intensity between these two channels. After applying self-corrected flow analysis, the scatter plot (right panel of Figure 10B) narrows significantly, indicating that most false positive events were removed. Figure 10A, with a slightly different instrument configuration, shows a similar improvement when self-corrected flow analysis was performed using two 488 nm laser lines.

[0269] In addition to significantly reducing or eliminating false positive rates, accurate co-localization of multiple biomarkers becomes possible by tagging each biological nanoparticle or molecule with a specific transport time and / or rate, which can be approximately constant for each of two adjacent laser excitation regions of the same or similar spacing. Briefly, as shown in Figure 14A, specific sEVs stained with di-8-ANEPPS were initially detected and tagged at a specific transport time (t) between the 405 nm laser line and the 488 nm laser line or between excitation regions. A self-correcting flow analysis algorithm searches for the presence of signals excited by another laser (e.g., the 561 nm laser line) within a very narrow time window (e.g., ±0.1 ms) centered on the transport time (t) between the 488 nm laser line and the 561 nm laser line to find sEVs co-localized with CD81-PE / CF594. Next, at a transport time (2t) between the 488nm and 640nm laser lines, another search window is applied to find sEVs co-localized with CD63-Alexa647. A similar search process can be repeated when more markers and detection windows or excitation regions are examined (Figure 8). After processing the data with self-corrected flow analysis, different subpopulations of sEVs can be determined based on different biomarker combinations, measured by the binding of corresponding antibody combinations to corresponding biomarkers (e.g., immunophenotype or immunological phenotype). In this embodiment, four subpopulations of sEVs are CD81 + 3.7%, CD63 + 32.9%, CD81 + / CD63 + 1.8%, CD81 - / CD63 - The figure was 65.2% (Figure 14B).

[0270] Example 6: Determining volumetric flow rate using single particle / molecule transport time This embodiment describes how to determine the volumetric flow rate using the transport time of a single particle / molecule.

[0271] In microfluidic analysis, knowing the volumetric flow rate is often necessary to determine the volume of the sample analyzed during the experiment. Therefore, the absolute concentration of a single particle / molecule can be measured based on the counting of analytes / molecules / nanoparticles and the volume analyzed. Furthermore, volumetric flow rate is a useful parameter for evaluating sample throughput and consumption. While important, direct measurement of volumetric flow rate is often difficult in microfluidic environments, especially when the volumetric flow rate is very low (e.g., pL~nL / sec), because the excitation region explores the ultra-small volume of the sample.

[0272] In this embodiment, we describe the measurement of volumetric flow rate using the transport time of each analyte / molecule / particle flowing through a laser line or excitation region, based on the fact that the flow is laminar in this microfluidic environment. Thus, the linear velocity of each particle / molecule can be calculated, and the distance between these laser lines or excitation regions can be determined.

[0273] Because this particular channel shape (i.e., 2 × 2 × 150 μm) is close to that of a pipe with a circular cross-section and has a length substantially longer than its diameter, the flow profile can be approximated by the parabolic flow profile in a cylindrical pipe. Based on this simplified assumption, the linear velocity of a single particle / molecule can be converted to the volumetric flow rate of this sample. Briefly, we first calculate the arithmetic mean of the observed linear velocities (

number

number

[0274] The number of particles (i.e., u(r)) passing through the cross-section with the same velocity in a unit time is:

number

number

number

number

[0275] Due to the parabolic flow, the volumetric flow rate (Q) is:

number

number

number

[0276] In summary,

number

[0277] Example 7: Determination of particle / molecular concentration This embodiment describes a method for accurately measuring the absolute concentration of particles / molecules without requiring any external calibration.

[0278] A direct application of volumetric flow rate measurement, as described in Example 6, is to determine the concentration of an analyte (e.g., particles and / or molecules). Briefly, a self-correcting flow analysis is used to enumerate a group of particles flowing through the excitation region or detection window. Due to the ultra-high sensitivity of our instrument, all particles stained with a specific marker (e.g., di-8-ANEPPS in this example) were counted (N). Knowing the volumetric flow rate (Q) determined from the measured linear velocity / transport time (Figure 11A) and experimental measurement time (t) of individual particles, the analyzed volume (V) is determined using V=Qt, and the concentration of the analyte (C) can be calculated as C=N / V=N / (Qt).

[0279] In this example, a sample of human semen exosomes (sEVs) was first diluted 1000-fold by adding 1 μL of stock solution to 999 μL of PBS buffer (pH=7.2). Next, 199 μL of the diluted sample was stained with 1 μL of 20 μM di-8-ANEPPS dissolved in DMSO and incubated at room temperature for 30 minutes. The fluorescence of this membrane dye was detected using 405 nm laser lines (100 mW) and 488 nm laser lines (20 mW) spaced 10.7 μm apart. After incubation, 10 μL of labeled sEVs were introduced into a microfluidic channel with a constriction (dimensions 2 μm width × 2 μm height × 125 μm length), and the laser beam was focused onto the line to illuminate the entire cross-section of the channel constriction. After loading the sample for 3 minutes, data acquisition was started, with each experimental measurement taking 300 seconds. Figure 11B shows the measured concentrations of sEVs that were replicated 35 times.

[0280] To further demonstrate the robustness of this method, the same sEV sample was diluted to obtain a series of concentrations (1 × 10⁻⁶). 7 ~1 × 10 10 Samples (particles / mL) were generated and each was measured using the same method as described above. For lower concentration samples, data collection was performed over a longer period to collect sufficient data points. At least three replications were performed for each concentration. Figure 15 shows the frequency versus dilution ratio of detected sEVs, as well as measured concentration versus calculated concentration, both of which show a linear relationship between measured concentration and dilution ratio over at least three orders of magnitude. These results demonstrate that the measurement of sEV concentrations was very robust.

[0281] Example 8: Automated focusing using near-infrared imaging and a high numerical aperture air objective lens This embodiment describes the apparatus and methods used to determine and maintain the correct focal position before and during the experiment.

[0282] The quality of single-particle / molecular flow analysis depends on how the microfluidic channel is focused relative to the objective lens for two reasons: 1) Centering the focal plane ensures the best excitation quality, so that all molecules flowing through the channel can be explored by the laser beam. 2) It also affects the efficiency of fluorescence collection. Even a 1 μm shift in the focal plane from the desired position can interrupt the measurement. Due to the improved throughput and robustness provided by automation, it is preferable to automatically determine and center the focal plane before each experiment. Additionally, automated focusing methods eliminate user-to-user variability induced by manual processes, thereby improving measurement quality.

[0283] In this embodiment, the single-molecule / particle flow analyzer apparatus was configured to enable automated focusing assisted by near-infrared (NIR) machine vision. Illumination using NIR light was used to minimize interfacing with line confocal fluorescence measurements, which largely reside within the visible light spectral wavelength range. The apparatus shown in Figure 16 consists of an NIR LED (center wavelength 870 nm), a microfluidic device for flow analysis, a high-NA air objective lens mounted on a Z-dimensional stage, a multiband dichroic mirror, a tube lens, a short-path dichroic mirror, and an NIR camera. Briefly, NIR light illuminating the microfluidic channel is collected by a high-NA air objective lens (40X / 0.95NA), reflected by a first dichroic mirror, and then focused by a tube lens. The NIR beam is steered by a short-path dichroic mirror, and the image is finally projected onto the camera's sensor.

[0284] A series of images were recorded at each position, showing the microfluidic channel in a focused or out-of-focus state, as the objective lens was moved up and down (Figure 7D). An edge detection filter was applied to all of these images to detect the boundary of the central channel, allowing the contrast of this channel to be measured. This contrast value is then used to define the focusing quality, with higher contrast indicating a greater degree of focusing of the detected channel. Figure 7D shows the change in focusing quality as a function of the objective lens position relative to the channel, with the two peaks indicating that the bottom (position 2) and top (position 3) of the channel were focused, respectively. Knowing the positions of these two peaks allows the center of the channel to be determined, and as the objective lens moves, the focus moves accordingly to that position. Figure 7F shows the feedback control loop to realize this automated focusing method, supported by NIR machine vision using a high NA airborne objective lens.

[0285] After data acquisition begins, it is also important to maintain the focal plane in the desired position to minimize SNR fluctuations caused by vibration and / or thermal expansion of the microfluidic chip, and / or other factors that may cause drift or fluctuation of the device. In this embodiment, the same automatic focusing mechanism assisted by NIR machine vision is applied to lock the focal plane during the experiment. Since the NIR light source (870 ± 25 nm) does not interfere with fluorescence detection, this automatic focusing scheme can be used in real time in single-molecule / particle flow experiments. Figure 7F shows the feedback control mechanism for this process. Briefly, the focal plane is initialized and locked before the experiment, and the corresponding focusing quality is recorded as a baseline. After the start of the experiment, an NIR image of the chip is captured every 100 ms, and the acquired focusing quality is compared to the baseline. If the difference is within 5%, the focal plane is locked in the same position. Otherwise, the objective lens is moved upward in 50 nm increments with the new measured focusing quality. A smaller difference indicates that the objective lens is moving in the correct direction, while a smaller difference indicates that the objective lens has moved in the opposite direction. Using these iterations and logical controls, focal plane shifts can be detected and corrected in real time during the experiment.

[0286] Example 9: Immunophenotype of single EVs and biological nanoparticles This embodiment describes immunological phenotypic analysis of a single particle using the device and method according to the embodiments of this disclosure.

[0287] Isolation of extracellular vesicles (EVs) and nanoparticles in semen Semen samples were obtained from the University of Washington's Male Fertility Program. Written informed consent was obtained from each donor. All protocols were approved by the University of Washington's Institutional Review Boards and the Fred Hutchinson Cancer Research Center. Briefly, a series of centrifugation steps were employed to isolate semen plasma from the semen samples, and the plasma was then filtered through a 0.22 μm syringe filter. After ultracentrifugation on a sucrose gradient, 30% and 25% sucrose cushions containing semen EV and nanoparticles were pooled and washed by centrifugation through an Amicon Ultracel 100-kDa cellulose centrifuge filter. The semen EV / nanoparticles were then filtered by a size exclusion chromatography column to remove proteins from the solution. The final EV / nanoparticle concentration was approximately 10 based on nanoparticle tracking analysis. 13 It was / mL.

[0288] Flow analysis of extracellular vesicles and nanoparticles in semen Before labeling, the semen EV / nanoparticle suspension was diluted to 10¹¹ / mL in HEPES buffer (20 mM, pH 7.4). To label the tetraspanin, 100 μL of the diluted EV / nanoparticle suspension was incubated with 10 μL of diluted antibody solution. For each antibody, 10 -6 ~10 -1 Titration curves were generated by testing multiple concentrations in the μg / mL range. After incubation with the antibody for 1 hour, the lipid membrane was labeled by adding 1 μL of 20 μM Di-8-ANEPPS in dimethyl sulfoxide to the solution. After incubation with Di-8-ANEPPS for 10 minutes, the solution was centrifuged and rediluted three times using a spin column (Sartorius Vivaspin 500, 300-kDA) in HEPES buffer containing 0.1% bovine serum albumin (BSA) to remove excess antibody.

[0289] According to embodiments of this disclosure, a flow platform was developed based on a line confocal design, comprising four spatially separated laser lines and five avalanche photodiodes. In each experiment, 5 μL of sample was injected into an inlet reservoir on a microfluidic chip. The difference in reservoir fluid levels allowed for initiation of flow without an external pump, resulting in simple and robust operation. The injected sample was flowed through a 2 × 2 μm channel and excited by the four laser lines. This channel geometry provided high sensitivity and high throughput without clogging. The laser lines were more than 10 times the channel width to achieve uniform excitation across the channel. At maximum laser power, the power density inside the channel was approximately 20 kWcm². -2 The photons emitted from each laser line were filtered through aperture and bandpass filters before being focused to the detector. To minimize focus drift, a custom-built automated focusing system was employed, using backscatter from the 640 nm laser line as real-time feedback. Due to high excitation power density, high focusing stability, and reduced excitation and detection volumes, the system provides sufficient sensitivity for detecting a single fluorescent dye molecule.

[0290] As described above, 5 μL of sample was injected into the inlet reservoir of the microfluidic chip for flow measurement. A typical volumetric flow rate was approximately 15 pL / second. The fluorescence signal was collected by an APD at 10 kHz. An automated focusing system, as described in Example 8, was engaged to minimize focus drift during the experiment. To distinguish the signal from noise, the threshold was set as the mean background plus five times the median absolute deviation of the background. The intensity of each event was calculated by integration within a fixed time window after background subtraction.

[0291] The inventors collected the flow trajectories of diluted free antibody and Di-8-ANEPPS-labeled EV / nanoparticles with different excitation forces to determine the optimal signal-to-noise ratio for each channel.

[0292] For high-throughput profiling of tetraspanins, the inventors labeled semen EVs / nanoparticles with membrane dye Di-8-ANEPPS and fluorescent dye molecule-conjugated anti-tetraspanin antibodies: brilliant violet 510 (BV510)-anti-CD9, phycoerythrin (PE)-anti-CD63, and Alexa Fluor647-anti-CD81. Di-8-ANEPPS is nonfluorescent in water but becomes highly fluorescent when inserted into a lipid membrane. The fluorescence intensity of membrane-stained EVs is proportional to the surface area of ​​the lipid membrane. The inventors observed that scaling allowed the distribution of the square root intensity of Di-8-ANEPPS-stained EVs to be superimposed on the EV size distribution determined from dynamic light scattering (DLS). The scaling coefficient allowed for the estimation of EV size from the membrane dye signal detected in flow. Flow trajectories of three diluted free antibodies with different excitation forces were collected to determine the optimal signal-to-noise ratio for each detection channel. With optimal excitation force, we were able to fit a single antibody intensity histogram to a log-normal distribution. Based on the truncation fraction of the intensity histogram fitting, we determined that over 98% of the single antibody was detected in our flow system.

[0293] As EVs labeled with membrane dyes and fluorescently labeled anti-tetraspanin antibodies flowed through the laser lines, the fluorescence signals of each dye were detected by the corresponding detectors. Transport times between the two laser lines were determined by cross-correlation analysis of the trajectories collected by the detection channels. Transport times were used to perform co-localization between different channels. Signals were attributed to EVs only if the antibody peak appeared within a predicted time window near the membrane dye peak, minimizing the influence of free antibodies.

[0294] The square root of the membrane stain intensity was converted to EV size, and EV size versus antibody intensity was plotted (Figures 17A-17C). The resulting scattering plots showed a weak correlation between protein expression levels and EV size for all three tetraspanins. Four-color colocalization analysis identified seven subgroups of semen exosomes (Figure 17D). 15.1% of semen exosomes expressed only CD9, 9.2% expressed only CD63, and 1.5% expressed only CD81. Only 1.1% of semen exosomes contained all three tetraspanins. 53.5% of membrane-stained vesicles, not shown in Figure 17D, did not show significant expression of CD63, CD81, or CD9. These results suggest that while CD63, CD81, and CD9 are all common exosome markers, many semen exosomes express only one or two of these markers, indicating significant heterogeneity in tetraspanin expression levels both among exosomes and among these tetraspanins.

[0295] Example 10: Copy number determination of a single EV and a biological nanoparticle protein Using the semen EV / nanoparticles prepared in Example 9, the copy number distribution of tetraspanins was obtained by deconvolving the intensity distribution of antibody-labeled exosomes using a single antibody intensity distribution (Figures 18A-18C). For each tetraspanin, exosomes were labeled with different concentrations of antibody, and the exosomes in the flow were analyzed to ensure saturation labeling. The mean copy numbers for CD63, CD81, and CD9 were 12.8, 1.6, and 17.0, respectively (Figures 18D-18F).

[0296] Because it can detect less than 1% of the weakest single molecules present, the single-molecule intensity distribution is used to deconvolve the single-EV intensity distribution, allowing for accurate determination of the number of bound antibodies on each EV, and consequently, the number of corresponding proteins. Figures 18A–18C show that both the intensity distribution of a single antibody and single EVs fully labeled with the antibody could be obtained in a single experiment. The mean copy numbers of the three tetraspanins were 12.8 (CD63), 1.6 (CD81), and 17.0 (CD9), respectively, and Figures 18D–18F also show how the copy numbers vary between EVs. This quantitative information on the molecular composition of single EVs is useful for distinguishing between intact and fragmented EVs, subtyping EVs, or studying EV biosynthesis and regulation.

[0297] Example 11: Single nucleic acid-containing nanoparticles and molecular analysis, such as EV or RNA or DNA-binding proteins, RNA or DNA granules, and other RNA or DNA-containing nanoparticles. Figures 19A–19D demonstrate that the single EV / nanoparticle / molecular flow analyzer of this disclosure can detect and track the RNA content of not only individual EVs but also other non-EV biological nanoparticles or molecules, such as RNA-binding proteins. Here, membrane-permeable RNA staining was used, and due to the high sensitivity of the flow analyzer, it was possible to detect RNA contained in EVs, other non-EV vesicles, and other non-vesicular biological nanoparticles and molecules. To perform this measurement, semen EV samples were stained with membrane dye (labeling all EV and non-EV membrane vesicles), anti-CD63 antibody (a classical exosome marker), and membrane-permeable RNA dye (SYTO). The results show that only about 15% of all present membrane vesicles contained RNA (Figure 19A), and only about 35% of all present RNA-containing biological particles were located within membrane vesicles (Figure 19B). However, when RNA was contained within membrane vesicles, about 75% of these vesicles were exosomes defined as CD63 (Figure 19C). No significant correlation was found between EV size and RNA content (Figure 19D).

[0298] This capability, provided by the single EV / nanoparticle / molecular flow analyzer, enables both immunological phenotyping of single EVs / nanoparticles and tracking of RNA content of EVs and other biological nanoparticles.

[0299] Example 12: Single virus particle analysis This embodiment describes how the integrity or functional state of a virus particle is determined by determining the protein copy number and nucleic acid content.

[0300] Proteins and nucleic acids within or on viral particles are labeled as described in previous examples, e.g., Examples 9 and 11. As described above, the single-molecule intensity distribution of fluorescently labeled viral proteins is used to deconvolve the single-viral particle intensity distribution to accurately determine the number of bound antibodies. Furthermore, as described above, the fluorescence intensity of nucleic acid staining reports the presence, absence, or amount of nucleic acids contained in the viral particles. For membrane-contained viral particles, as described above, the fluorescence intensity of membrane-stained viral particles is proportional to the surface area of ​​the lipid membrane, and the size of the viral particles can be determined based on the signal intensity from individual particles.

[0301] Next, the determined particle protein and / or nucleic acid content, and optionally the size, are used to determine whether the analyzed viral particle is a typical intact or functional viral particle, or too small to be intact and rather a part or subunit of a viral particle, or a non-functional viral particle (e.g., an otherwise intact viral particle that is empty and does not contain nucleic acid).

[0302] Example 13: Absolute quantification of concentration by direct counting of fluorescent single particles or single molecules This embodiment demonstrates a method for the absolute quantification of the concentration of fluorescent particles or molecules in a sample by direct counting of such particles or molecules passing through a device, according to an embodiment of the present disclosure.

[0303] As further described herein, the devices of this disclosure are configured to detect essentially all or nearly all (or a large proportion, such as at least over 90%) of fluorescent particles or molecules flowing through a channel. Thus, it is also possible to extract the linear velocity of each particle or portion of a particle with high precision, which, along with knowing the cross-section of the channel, allows for the determination of the corresponding volumetric flow rate. As a result, the devices of this disclosure are configured to provide absolute quantification of EV / biological nanoparticle concentrations in a sample without calibration via single-particle counting (number of counted particles ÷ volume flowing through). This novel capability is found to be widely used by researchers in fields studying biological nanoparticles, including EVs, viruses, lipoproteins (e.g., HDL, LDL, VLDL), and polymeric complexes (e.g., circulating RNA-binding proteins, RNA granules, exomers). For example, this capability allows for the determination of the absorption concentration of a subtype of biological nanoparticles or EVs based on the phenotype of the particles or EVs (e.g., based on the presence and / or absence and / or amount of a certain biomarker (e.g., a protein) and / or nucleic acid, and / or the size of the particles), as described in Examples 9 to 12 above.

[0304] Figures 20A and 20B show the direct counting of fluorescent nanoparticles and fluorescent protein molecules. While fluorescent antibodies are shown here, the method in this example is applicable to other fluorescent proteins, fluorescent DNA, fluorescent RNA, and other fluorescent molecules. Figure 20A shows 1.67 × 10⁻⁶ -18 10 per 1 mL, corresponding to M 6 The measurement values ​​are shown up to 10 fluorescent nanoparticles. This limit is set by the experimental execution time. In this example, the sampling rate was 10 4 The detection limit is events / second, and therefore, even this very low concentration detection limit can be extended by increasing the flow rate. A range of approximately four orders of magnitude, shown in Figure 20A, was achieved using the same flow rate. Figure 20B shows similar absolute quantification of a fluorescent protein molecule (Alexa647-conjugated protein).

[0305] While illustrative embodiments are described, it should be understood that various modifications can be made therein without departing from the spirit and scope of the invention. Embodiments of the present invention in which an exclusive right or privilege is claimed are defined as follows: In embodiments of the present invention, for example, the following items are provided. (Item 1) A system for analyzing particles and / or molecules, wherein the system is A channel comprising a channel configured to allow particles and / or molecules to flow through the lumen of the channel, and defining a search window configured to allow light to pass in and out of the lumen, It is an optical engine, A first light source positioned to output first excitation light to a first portion of the channel within the search window, An optical engine comprising: a second light source positioned to output a second excitation light to a second portion of the channel in the search window, separate from the first portion; An emission fiber bundle comprising a first emission optical fiber and a second emission optical fiber, wherein the proximal ends of the first emission optical fiber and the second emission optical fiber are positioned in an emission fiber bundle head, the proximal end of the first emission optical fiber is positioned to receive first synchrotron radiation emitted from the first portion, and the proximal end of the second emission optical fiber is positioned to receive second synchrotron radiation emitted from the second portion, A detector system, A first photodetector positioned to receive the first synchrotron radiation emitted from the distal end of the first emitting optical fiber, A detector system comprising: a second photodetector positioned to receive the second synchrotron radiation emitted from the distal end of the second emitting optical fiber; and a detector system comprising: (Item 2) The system further comprises a controller operably coupled to the optical engine and the detector system, wherein when the controller is executed, it controls the system Outputting the first excitation light using the first light source, Outputting the second excitation light using the second light source, Based on the first synchrotron radiation received from the first emitting optical fiber, a first emission signal is generated using the first photodetector, The system according to item 1, comprising logic causing to perform an operation including generating a second emission signal using the second photodetector based on the second synchrotron radiation received from the second emission optical fiber. (Item 3) The system according to item 1, wherein the first excitation light has a wavelength in a first wavelength range, and the second excitation light has a wavelength in a second wavelength range separate from the first wavelength range. (Item 4) The system according to item 1, wherein the first excitation light has a wavelength in a first wavelength range, and the second excitation light has a wavelength in a second wavelength range common to the first wavelength range. (Item 5) The system as described in item 1, wherein the first excitation light is in the wavelength range of approximately 350 nm to approximately 360 nm, approximately 400 nm to approximately 410 nm, approximately 480 nm to approximately 490 nm, approximately 530 nm to approximately 540 nm, approximately 555 nm to approximately 565 nm, or approximately 630 nm to approximately 690 nm. (Item 6) The system as described in item 1, wherein the second excitation light is in the wavelength range of approximately 350 nm to approximately 360 nm, approximately 400 nm to approximately 410 nm, approximately 480 nm to approximately 490 nm, approximately 530 nm to approximately 540 nm, approximately 555 nm to approximately 565 nm, or approximately 630 nm to approximately 690 nm. (Item 7) The system according to item 1, further comprising a dichroic mirror, the dichroic mirror being disposed between the distal end of the first emitting optical fiber and the first photodetector, and positioned to reflect a portion of the first synchrotron radiation to a third photodetector. (Item 8) The system according to item 7, further comprising a bandpass filter, the bandpass filter being disposed between the dichroic mirror and the third photodetector and configured to filter the portion of the first synchrotron radiation. (Item 9) The system according to item 7, wherein the first photodetector is configured to generate a first emission signal based on a first emission wavelength range of the first synchrotron radiation, and the third photodetector is configured to generate a third emission signal based on a third emission wavelength range of the first synchrotron radiation, which is different from the first emission wavelength range. (Item 10) The system according to item 1, wherein the distal end of the first emitting optical fiber is configured to emit the first synchrotron radiation to 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, each of which is configured to receive substantially different spectral portions of the synchrotron radiation. (Item 11) The system according to item 1, wherein one or both of the first light source and the second light source are free-space light sources. (Item 12) The system according to item 1, wherein 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, and an arc discharge. (Item 13) The first light source is optically coupled to the proximal end of the first excitation optical fiber. The system according to item 1, wherein the second light source is optically coupled to the proximal end of the second excitation optical fiber. (Item 14) The system according to item 13, wherein the distal end of the first excitation optical fiber and the distal end of the second excitation optical fiber are located within an excitation fiber bundle head. (Item 15) The system according to item 13, wherein the distance between the distal end of the first excitation optical fiber and the distal end of the second excitation optical fiber corresponds to the distance between the first portion and the second portion. (Item 16) The system according to item 13, wherein the distance between the distal end of the first excitation optical fiber and the distal end of the second excitation optical fiber corresponds to the distance between the proximal end of the first emission optical fiber and the proximal end of the second emission optical fiber. (Item 17) The system according to item 1, wherein the distance between the first light source and the second light source corresponds to the distance between the first part and the second part. (Item 18) The system according to item 1, further comprising an optically opaque cover coupled to the emission fiber bundle head, wherein the optically opaque cover defines an opening shaped to allow the passage of the first emitted light to the proximal end of the first emission optical fiber. (Item 19) The system according to item 18, wherein the optically opaque cover defines a second opening shaped to allow the passage of the second synchrotron radiation to the proximal end of the second emitting optical fiber. (Item 20) The system according to item 1, wherein the emitting fiber bundle comprises at least three emitting optical fibers, at least four emitting optical fibers, at least five emitting optical fibers, at least six emitting optical fibers, at least seven emitting optical fibers, or more. (Item 21) The system according to item 20, wherein the proximal end of each emitting optical fiber in the emitting fiber bundle is disposed within the emitting fiber bundle head. (Item 22) The system according to item 20, wherein the proximal end of each emitting optical fiber in the emission fiber bundle is positioned to receive synchrotron radiation emitted from a separate portion of the search window. (Item 23) The system described in item 20, wherein the distal end of each emitting optical fiber is positioned to emit light into its respective detector module. (Item 24) The system according to item 1, wherein the lumen of the channel within the exploration window defines a constricted portion with respect to an adjacent portion of the channel. (Item 25) The system according to item 1, wherein the channel is arranged in part of a microfluidic device. (Item 26) The system according to item 25, wherein the microfluidic device defines a planar portion. (Item 27) The system according to item 1, wherein the detector system is positioned to receive scattered synchrotron radiation, luminescent synchrotron radiation, fluorescence synchrotron radiation, or a combination thereof from the search window. (Item 28) The controller further includes logic, and when the logic is executed by the controller, the system The system according to item 2, which causes an operation to be performed, including flowing a suspension of particles and / or a solution of molecules through the channel. (Item 29) The system according to item 28, wherein flowing the suspension through the channel includes flowing the suspension particle by particle through the channel. (Item 30) The system according to item 28, wherein flowing the solution through the channel includes flowing the solution of the target molecule through the channel molecule by molecule. (Item 31) The controller further includes logic, and when the logic is executed by the controller, the system The system described in item 2, which causes the system to perform an operation including ranking particles and / or molecules in the channel based on the presence or absence of synchrotron radiation. (Item 32) The controller further includes logic, and when the logic is executed by the controller, the system Ranking of particles and / or molecules within the channel based on the intensity of synchrotron radiation. A system described in item 2 that performs the operations including those described above. (Item 33) The system according to item 31 or 32, wherein the ranking corresponds to the measured emission spectra of the particles and / or molecules based on one or more of the first synchrotron radiation and the second synchrotron radiation. (Item 34) The system according to item 31 or 32, wherein the ranking corresponds to the measured size value of the particles. (Item 35) The system described in item 34, wherein the measured size value is a relative size value. (Item 36) The system described in item 34, wherein the measured size value is determined by the difference in detected synchrotron radiation intensity. (Item 37) The system described in item 34, wherein the measured size value is the actual size value. (Item 38) The system according to item 2, further comprising a flow directionator configured to direct the flow of particles and / or molecules within the channel. (Item 39) The flow directioner is operably coupled to the controller, and the controller includes logic, and when the logic is executed by the controller, the system The system according to item 38, which causes an operation to be performed that includes directing the flow of particles and / or molecules based on the presence or absence of synchrotron radiation received from the search window and associated with the particles and / or molecules. (Item 40) The flow directioner is operably coupled to the controller, and the controller includes logic, and when the logic is executed by the controller, the system The system according to item 38, which causes an operation to be performed that includes directing the flow of the particles and / or molecules based on the ranking of the particles and / or molecules. (Item 41) The system according to item 39, wherein directing the flow of the particles and / or molecules includes directing the particles and / or molecules into one or more sorting channels. (Item 42) The flow directioner is operably coupled to the controller, and the controller includes logic, and when the logic is executed by the controller, the system A system as described in item 38, which performs an operation including quantifying the number of particles and / or molecules associated with synchrotron radiation from a search. (Item 43) The controller includes logic, and when the logic is executed by the controller, the system The system according to item 42, which causes the system to perform an operation including determining the concentration of particles and / or molecules associated with the synchrotron radiation from the search window. (Item 44) The controller includes logic, and when the logic is executed by the controller, the system Item 42 involves performing an operation that includes identifying a subset of particles and / or molecules based on the presence, absence, or amount of biomarkers associated with the particles and / or molecules. The system described. (Item 45) The system according to item 42, wherein the quantification of the number of particles and / or molecules includes the sensitivity or detection efficiency of a single molecule. (Item 46) The system according to item 45, wherein the sensitivity or detection efficiency of a single molecule includes detecting more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of the single molecule flowing through the channel. (Item 47) The system according to item 45, wherein the single-molecule detection efficiency is such that it detects more than 90% of the single molecules flowing through the channel. (Item 48) The system according to item 1, further comprising a focusing system positioned to collect the first synchrotron radiation and the second synchrotron radiation from the channel and to direct the collected synchrotron radiation to the detector system, wherein the focusing system comprises an air objective lens having a numerical aperture in the range of greater than 0.91 to less than 0.99. (Item 49) The system according to item 1, wherein the air objective lens has an numerical aperture of approximately 0.95. (Item 50) A method for searching for particles and / or molecules, wherein the method is The flow of particles and / or molecules through a channel, The first excitation light is output to the first portion of the channel through the search window, The second excitation light is output through the search window to a second portion of the channel that is separate from the first portion, Based on the first synchrotron radiation received through the proximal end of the first emitting optical fiber, a first emission signal is generated using a first photodetector, This includes generating a second emission signal using a second photodetector based on a second synchrotron radiation received through the proximal end of a second emission optical fiber, A method in which the proximal end of the first emitting optical fiber and the proximal end of the second emitting optical fiber are arranged within an emitting optical fiber bundle head. (Item 51) The method described in item 50, wherein the method includes using a system described in any one of items 1 to 49. (Item 52) The method of item 50, further comprising directing the flow of particles and / or molecules based on the presence or absence of synchrotron radiation received from the search window and associated with the particles and / or molecules. (Item 53) The method according to item 50, wherein directing the flow of the particles and / or molecules includes directing the particles and / or molecules into one or more sorting channels. (Item 54) The method according to item 50, wherein flowing the particles and / or molecules through the channel includes flowing a suspension of particles containing the particles and / or a solution of molecules containing the molecules through the channel, and flowing the suspension of particles and / or a solution of molecules through the channel includes flowing the suspension and / or solution particle by particle and / or molecule by molecule through the channel. (Item 55) Quantifying the number of particles and / or molecules associated with synchrotron radiation from the search, The method of item 50, further comprising determining the concentration of the particles and / or molecules associated with the synchrotron radiation from the search window. (Item 56) The method according to item 50, wherein the first synchrotron radiation and the second synchrotron radiation are independently selected from the group consisting of scattered synchrotron radiation, luminescent synchrotron radiation, fluorescent synchrotron radiation, and combinations thereof. (Item 57) The method according to item 50, further comprising ranking particles and / or molecules in the channel based on the presence or absence of synchrotron radiation from the search window. (Item 58) The method according to item 57, wherein the ranking corresponds to the measured emission spectra of the particles and / or molecules based on one or more of the first synchrotron radiation and the second synchrotron radiation. (Item 59) The method according to item 57, wherein the ranking corresponds to the measured size value of the particles. (Item 60) The method according to item 59, wherein the measured size value is a relative size value. (Item 61) The method according to item 59, wherein the measured size value is determined by the difference in detected light intensity. (Item 62) The method according to item 55, wherein the number of particles and / or molecules is quantified, including the sensitivity or detection efficiency of a single molecule. (Item 63) The method according to item 62, wherein the sensitivity or detection efficiency of a single molecule is such that it detects more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of the single molecule flowing through the channel. (Item 64) The method according to item 62, wherein the single-molecule detection efficiency is such that it detects more than 90% of the single molecules flowing through the channel. (Item 65) The method according to item 50, wherein the particles and / or molecules associate with a detectable agent. (Item 66) The method according to item 65, wherein the detectable agent is a first detectable agent, and the particles and / or molecules associate with a second detectable agent. (Item 67) The method according to item 66, wherein the first detectable agent has a first emission spectrum in a first emission wavelength range, and the second detectable agent has a second emission spectrum in a second emission wavelength range different from the first emission wavelength range. (Item 68) The method according to item 65, wherein the detectable agent is a detectable fluorescent agent. (Item 69) The method according to item 50, wherein the channel is disposed in part of a microfluidic device. (Item 70) The method according to item 69, wherein the microfluidic device defines a planar portion. (Item 71) The method according to item 69, wherein the channel within the search window defines a constricted portion with respect to an adjacent portion of the channel. (Item 72) A system for analyzing particles and / or molecules, wherein the system is A channel comprising a channel configured to allow particles and / or molecules to flow through the lumen of the channel, and defining a search window configured to allow light to pass in and out of the lumen, A photon engine configured to output excitation light to the channel through the search window, A detector system positioned to receive synchrotron radiation emitted from the channel and configured to generate a signal based on the received synchrotron radiation, A system comprising: a focusing system positioned to collect the synchrotron radiation from the channel and to direct the collected synchrotron radiation to the detector system, wherein the focusing system comprises an air objective lens having a numerical aperture in the range of greater than 0.91 to less than 0.99. (Item 73) The system according to item 72, wherein the focusing system has an numerical aperture of approximately 0.95. (Item 74) The system according to item 72, further comprising an emission fiber bundle having a first emission optical fiber and a second emission optical fiber, wherein the proximal ends of the first emission optical fiber and the second emission optical fiber are positioned in an emission fiber bundle head, the proximal end of the first emission optical fiber is positioned to receive first synchrotron radiation emitted from the first portion, and the proximal end of the second emission optical fiber is positioned to receive second synchrotron radiation emitted from the second portion. (Item 75) The aforementioned optical engine, A first light source positioned to output first excitation light to a first portion of the channel within the search window, The system comprises a second light source, which is separate from the first portion and positioned to output a second excitation light to a second portion of the channel within the search window, The detector system, A first photodetector positioned to receive the first synchrotron radiation emitted from the distal end of the first emitting optical fiber, The system according to item 72, comprising: a detector system, a second photodetector positioned to receive the second synchrotron radiation emitted from the distal end of the second emitting optical fiber; (Item 76) The system further comprises a controller operably coupled to the optical engine and the detector system, wherein when the controller is executed, it controls the system Outputting the first excitation light using the first light source, Outputting the second excitation light using the second light source, Based on the first excitation light received from the first emission optical fiber, a first emission signal is generated using the first photodetector, The system according to item 75, comprising logic causing to perform an operation including generating a second emission signal using the second photodetector based on the second excitation light received from the second emission optical fiber. (Item 77) The system according to item 75, wherein the first excitation light has a wavelength in a first wavelength range, and the second excitation light has a wavelength in a second wavelength range separate from the first wavelength range. (Item 78) The system according to item 75, wherein the first excitation light has a wavelength in a first wavelength range, and the second excitation light has a wavelength in a second wavelength range common to the first wavelength range. (Item 79) The system further comprises a dichroic mirror, the dichroic mirror being disposed between the distal end of the first emitting optical fiber and the first photodetector, and the first emission The system described in item 74, which is positioned to reflect a portion of the light to a third photodetector. (Item 80) The system according to item 74, wherein the distal end of the first emitting optical fiber is configured to emit the first synchrotron radiation to 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. (Item 81) The system according to item 79, further comprising a bandpass filter, the bandpass filter being positioned between the dichroic mirror and the third photodetector and configured to filter the portion of the first synchrotron radiation. (Item 82) The system according to item 74, wherein the first photodetector is configured to generate a first emission signal based on a first emission wavelength range of the first synchrotron radiation, and the third photodetector is configured to generate a third emission signal based on a third emission wavelength range of the first synchrotron radiation, which is different from the first emission wavelength range. (Item 83) The system according to item 72, wherein the channel is disposed in part of a microfluidic device. (Item 84) The system according to item 83, wherein the microfluidic device defines a planar portion. (Item 85) The system according to item 83, wherein the channel within the search window defines a constricted portion with respect to an adjacent portion of the channel. (Item 86) A method for analyzing particles and / or molecules in a fluid sample, Flowing a fluid sample containing multiple particles and / or multiple molecules through a channel, Within the channel, the particles among the plurality of particles and / or the molecules among the plurality of molecules are illuminated, The synchrotron radiation emitted from the channel is collected using a focusing system equipped with an air objective lens having a numerical aperture in the range of approximately 0.91 to less than 0.99. To generate a signal based on the collected synchrotron radiation emitted from the channel based on the aforementioned particles and / or molecules, A method comprising assigning values ​​to the particles and / or molecules based on the aforementioned signals. (Item 87) The method according to item 86, wherein the focusing system has a numerical aperture of approximately 0.95. (Item 88) The method according to item 86, wherein the method is a method for determining the size of the particle, and the value is the size value. (Item 89) The method according to item 86, wherein the method is a method for determining the concentration of a target particle or molecule in the fluid sample. (Item 90) The method according to item 86, wherein the method is a method for quantifying the copy number of biomolecules associated with target particles in the fluid sample. (Item 91) The method according to item 86, wherein the method is a method for determining the phenotype of particles in the plurality of particles. (Item 92) The method according to item 86, wherein the synchrotron radiation is selected from the group consisting of scattered synchrotron radiation, luminescent synchrotron radiation, fluorescent synchrotron radiation, and combinations thereof. (Item 93) The method according to item 86, further comprising ranking particles and / or molecules in the channel based on the presence or absence of synchrotron radiation from the search window. (Item 94) The method according to item 86, further comprising ranking particles and / or molecules in the channel based on the intensity of synchrotron radiation from the search window. (Item 95) The means described in item 93 or 94, wherein the ranking corresponds to the measured emission spectra of the particles and / or molecules based on one or more of the first synchrotron radiation and the second synchrotron radiation. (Item 96) The method according to any one of items 93 to 95, wherein the ranking corresponds to the measured size value of the particles. (Item 97) The method according to item 96, wherein the measured size value is a relative size value. (Item 98) The method according to item 96, wherein the measured size value is determined by the difference in detected light intensity. (Item 99) The analysis of the particles includes determining the size of the particles, and the analysis is A signal is generated based on the collected synchrotron radiation emitted from the channel based on the aforementioned particles, The method of item 86, comprising assigning a size value to the particle based on the signal. (Item 100) The method according to item 86, wherein the particles and / or molecules associate with a detectable agent. (Item 101) The method according to item 100, wherein the detectable agent is a first detectable agent, and the particles and / or molecules associate with a second detectable agent. (Item 102) The method according to item 101, wherein the first detectable agent has a first emission spectrum in a first emission wavelength range, and the second detectable agent has a second emission spectrum in a second emission wavelength range different from the first emission wavelength range. (Item 103) The method according to item 101, wherein the first detectable agent has a first excitation spectrum in a first excitation wavelength range, and the second detectable agent has a second excitation spectrum in a second excitation wavelength range different from the first excitation wavelength range. (Item 104) The method according to item 99, wherein the detectable agent is a detectable fluorescent agent. (Item 105) The method described in item 86, wherein the method includes using the system described in any one of items 59 to 69. (Item 106) The method according to item 86, wherein the channel is disposed in part of a microfluidic device. (Item 107) The method according to item 106, wherein the microfluidic device defines a planar portion. (Item 108) The method according to item 106, wherein the channel within the search window defines a constricted portion with respect to an adjacent portion of the channel. (Item 109) The method according to item 86, wherein the channel is disposed in part of a microfluidic device. (Item 110) The method according to item 109, wherein the microfluidic device defines a planar portion. (Item 111) The method according to item 109, wherein the channel within the search window defines a constricted portion with respect to an adjacent portion of the channel. (Item 112) A system for self-correcting flow analysis of single molecules and / or single particles, wherein the system is A channel comprising a channel configured to allow partic...

Claims

1. A system for analyzing particles and / or molecules, wherein the system is A microfluidic device comprising a channel configured to allow particles and / or molecules to flow through the lumen of the channel, wherein the channel defines a search window configured to allow light to pass in and out of the lumen, and the channel is disposed on a planar portion of the microfluidic device, It is an optical engine, A first light source positioned to output first excitation light to the first portion of the channel within the search window, An optical engine comprising: a second light source positioned to output a second excitation light to a second portion of the channel in the search window, separate from the first portion; An emission fiber bundle comprising a first emission optical fiber and a second emission optical fiber, wherein the proximal ends of the first emission optical fiber and the second emission optical fiber are positioned in an emission fiber bundle head, the proximal end of the first emission optical fiber is positioned to receive first synchrotron radiation emitted from the first portion, and the proximal end of the second emission optical fiber is positioned to receive second synchrotron radiation emitted from the second portion, A detector system, A first photodetector positioned to receive the first synchrotron radiation emitted from the distal end of the first emitting optical fiber, A detector system comprising: a second photodetector positioned to receive the second synchrotron radiation emitted from the distal end of the second emitting optical fiber; and a focusing system positioned to collect the first synchrotron radiation and the second synchrotron radiation from the channel and to direct the collected synchrotron radiation to the detector system, wherein the focusing system comprises an air objective lens having a numerical aperture in the range of greater than 0.91 to less than 0.

99. A system equipped with these features.

2. The system further comprises a controller operably coupled to the optical engine and the detector system, wherein when the controller is executed, it controls the system Outputting the first excitation light using the first light source, Outputting the second excitation light using the second light source, Based on the first synchrotron radiation received from the first emitting optical fiber, a first emission signal is generated using the first photodetector, The system according to claim 1, comprising logic for causing the system to perform an operation including generating a second emission signal using the second photodetector based on the second synchrotron radiation received from the second emission optical fiber.

3. The system according to claim 1, wherein the first excitation light has a wavelength in a first wavelength range, and the second excitation light has a wavelength in a second wavelength range separate from the first wavelength range.

4. The system according to claim 1, wherein the first excitation light has a wavelength in a first wavelength range, and the second excitation light has a wavelength in a second wavelength range common to the first wavelength range.

5. The system according to claim 1, wherein the first excitation light is in the wavelength range of approximately 350 nm to approximately 360 nm, approximately 400 nm to approximately 410 nm, approximately 480 nm to approximately 490 nm, approximately 530 nm to approximately 540 nm, approximately 555 nm to approximately 565 nm, or approximately 630 nm to approximately 690 nm.

6. The system according to claim 1, wherein the second excitation light is in the wavelength range of approximately 350 nm to approximately 360 nm, approximately 400 nm to approximately 410 nm, approximately 480 nm to approximately 490 nm, approximately 530 nm to approximately 540 nm, approximately 555 nm to approximately 565 nm, or approximately 630 nm to approximately 690 nm.

7. The system according to claim 1, further comprising a dichroic mirror, wherein the dichroic mirror is disposed between the distal end of the first emitting optical fiber and the first photodetector, and is positioned to reflect a portion of the first synchrotron radiation to a third photodetector.

8. The system according to claim 7, further comprising a bandpass filter, wherein the bandpass filter is disposed between the dichroic mirror and the third photodetector and is configured to filter the portion of the first synchrotron radiation.

9. The system according to claim 7, wherein the first photodetector is configured to generate a first emission signal based on a first emission wavelength range of the first synchrotron radiation, and the third photodetector is configured to generate a third emission signal based on a third emission wavelength range of the first synchrotron radiation, which is different from the first emission wavelength range.

10. The system according to claim 1, wherein the distal end of the first emitting optical fiber is configured to emit the first synchrotron radiation to 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, each of which is configured to receive substantially different spectral portions of the synchrotron radiation.

11. The system according to claim 1, wherein one or both of the first light source and the second light source are free-space light sources.

12. The system according to claim 1, wherein the first light source and the second light source are each independently selected from the group consisting of a solid-state laser, a diode-excited laser, a light-emitting diode (LED), a lamp, and an arc discharge.

13. The first light source is optically coupled to the proximal end of the first excitation optical fiber. The system according to claim 1, wherein the second light source is optically coupled to the proximal end of the second excitation optical fiber.

14. The system according to claim 13, wherein the distal end of the first excitation optical fiber and the distal end of the second excitation optical fiber are located within an excitation fiber bundle head.

15. The system according to claim 13, wherein the distance between the distal end of the first excitation optical fiber and the distal end of the second excitation optical fiber corresponds to the distance between the first portion and the second portion.

16. The system according to claim 13, wherein the distance between the distal end of the first excitation optical fiber and the distal end of the second excitation optical fiber corresponds to the distance between the proximal end of the first emission optical fiber and the proximal end of the second emission optical fiber.

17. The system according to claim 1, wherein the distance between the first light source and the second light source corresponds to the distance between the first portion and the second portion.

18. The system according to claim 1, further comprising an optically opaque cover coupled to the emission fiber bundle head, wherein the optically opaque cover defines an opening formed to the proximal end of the first emission optical fiber, which allows the passage of the first emitted light.

19. The system according to claim 18, wherein the optically opaque cover defines a second opening shaped to allow the passage of the second synchrotron radiation to the proximal end of the second emitting optical fiber.

20. The system according to claim 1, wherein the emitting fiber bundle comprises at least three emitting optical fibers, at least four emitting optical fibers, at least five emitting optical fibers, at least six emitting optical fibers, at least seven emitting optical fibers, or more.

21. The system according to claim 20, wherein the proximal end of each emitting optical fiber in the emitting fiber bundle is disposed within the emitting fiber bundle head.

22. The system according to claim 20, wherein the proximal end of each emitting optical fiber in the emitting fiber bundle is positioned to receive synchrotron radiation emitted from a separate portion of the search window.

23. The system according to claim 20, wherein the distal end of each emitting optical fiber is positioned to emit light to its respective detector module.

24. The system according to claim 1, wherein the lumen of the channel within the exploration window defines a constricted portion with respect to an adjacent portion of the channel.

25. The system according to claim 1, wherein the detector system is positioned to receive scattered synchrotron radiation, luminescent synchrotron radiation, fluorescence synchrotron radiation, or a combination thereof from the search window.

26. The controller further includes logic, and when the logic is executed by the controller, the system The system according to claim 2, wherein an operation is performed which includes flowing a suspension of particles and / or a solution of molecules through the channel.

27. The system according to claim 26, wherein flowing the suspension through the channel includes flowing the suspension through the channel particle by particle.

28. The system according to claim 26, wherein flowing the solution through the channel includes flowing the solution of the target molecule through the channel molecule by molecule.

29. The controller further includes logic, and when the logic is executed by the controller, the system The system according to claim 2, which causes the system to perform an operation that includes ranking particles and / or molecules in the channel based on the presence or absence of synchrotron radiation.

30. The controller further includes logic, and when the logic is executed by the controller, the system Ranking of particles and / or molecules within the channel based on the intensity of synchrotron radiation. The system according to claim 2, which causes the system to perform an operation including the operation described above.

31. The system according to claim 29 or 30, wherein the ranking corresponds to the measured emission spectra of the particles and / or molecules based on one or more of the first synchrotron radiation and the second synchrotron radiation.

32. The system according to claim 29 or 30, wherein the ranking corresponds to the measured size value of the particles.

33. The system according to claim 32, wherein the measured size value is a relative size value.

34. The system according to claim 32, wherein the measured size value is determined by the difference in detected synchrotron radiation intensity.

35. The system according to claim 32, wherein the measured size value is the actual size value.

36. The system according to claim 2, further comprising a flow directionator configured to direct the flow of particles and / or molecules within the channel.

37. The flow directioner is operably coupled to the controller, and the controller includes logic, and when the logic is executed by the controller, the system The system according to claim 36, which causes an operation to be performed that includes directing the flow of particles and / or molecules based on the presence or absence of synchrotron radiation received from the search window and associated with the particles and / or molecules.

38. The flow directioner is operably coupled to the controller, and the controller includes logic, and when the logic is executed by the controller, the system The system according to claim 36, which causes an operation to be performed that includes directing the flow of the particles and / or molecules based on the ranking of the particles and / or molecules.

39. The system according to claim 37, wherein directing the flow of the particles and / or molecules includes directing the particles and / or molecules to one or more sorting channels.

40. The flow directioner is operably coupled to the controller, and the controller includes logic, and when the logic is executed by the controller, the system The system according to claim 36, which performs an operation including quantifying the number of particles and / or molecules associated with synchrotron radiation from the search.

41. The controller includes logic, and when the logic is executed by the controller, the system The system according to claim 40, which includes performing an operation that determines the concentration of the particles and / or molecules associated with the synchrotron radiation from the search window.

42. The controller includes logic, and when the logic is executed by the controller, the system The system according to claim 40, which performs an operation that includes identifying a subset of particles and / or molecules based on the presence, absence, or amount of biomarkers associated with the particles and / or molecules.

43. The system according to claim 40, wherein quantifying the number of particles and / or molecules includes the sensitivity or detection efficiency of a single molecule.

44. The system according to claim 43, wherein the sensitivity or detection efficiency of a single molecule includes detecting more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of the single molecule flowing through the channel.

45. The system according to claim 43, wherein the single-molecule detection efficiency includes detecting more than 90% of the single molecules flowing through the channel.

46. The system according to claim 1, wherein the air objective lens has an numerical aperture of approximately 0.

95.

47. The system according to claim 1, wherein the air objective lens is a microscope objective lens.

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