Optical detection module with adjustable sensitivity

Abstract

A light detection system is described for simultaneously measuring scattered light (e.g., in a flowing stream) from particles with diameters differing by 100 nm or more. The light detection system, according to some embodiments, includes a static optical adjustment assembly, a variable optical adjustment assembly, and a photodetector. Systems and methods for measuring scattered light from a sample (e.g., in a flowing stream), and kits having a static optical adjustment assembly, a variable optical adjustment assembly, and a photodetector are also provided.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 938,034, filed November 20, 2019, pursuant to 35 USC §119(e); the disclosure of that application is incorporated herein by reference. Background Technology

[0003] For example, when a sample is used for the diagnosis of a disease or medical condition, optical detection is often used to characterize the components of the sample (e.g., a biological sample). When a sample is illuminated, light can be scattered by the sample, transmitted through the sample, and emitted by the sample (e.g., through fluorescence). Variations in sample components, such as morphology, absorptivity, and the presence of fluorescent labels, can cause changes in the light scattered, transmitted, or emitted by the sample. To quantify these variations, light is collected and directed onto the surface of a detector. The amount of light reaching the detector can affect the overall quality of the optical signal output by the detector. The amount of light reaching the detector can be increased by increasing the surface area of ​​the detector or by increasing the amount of light collected from the sample.

[0004] Flow cytometry is a technique that uses light detection to characterize components in a sample. Using data generated from the detected light, the distribution of components can be recorded, and desired materials can be sorted there. A flow cytometer typically includes a sample container for receiving fluid samples (such as blood samples) and a sheath container containing sheath fluid. The flow cytometer delivers particles (including cells) from the liquid sample as a flow stream to the flow cell, while simultaneously guiding the sheath fluid into the flow cell. Within the flow cell, a liquid sheath forms around the cell stream to impose a substantially uniform velocity on it. The flow cell focuses the cells within the stream in a hydrodynamic manner to pass through the center of the light source within the flow cell. In some cases, the light from the light source can be detected as scattered light from particles in the flow stream. The particle size variation of components in biological samples (e.g., tissue blocks, extracellular components, cells, etc.) can be greater, depending on the source of the sample and the volume of sample processed before flow cytometry analysis. Summary of the Invention

[0005] This disclosure includes a light detection module configured to simultaneously measure scattered light from particles in a flowing stream whose diameters differ by 100 nm or more (e.g., 500 nm or more). According to some embodiments, the light detection module includes a static optical adjustment assembly, a variable optical adjustment assembly, and a light scattering detector. In some embodiments, the light scattering detector is a side-scattering detector. In other embodiments, the light scattering detector is a forward-scattering detector. In other embodiments, the light scattering detector is a back-scattering detector. In some embodiments, the static optical adjustment assembly includes a bandpass filter. In some embodiments, the variable optical adjustment assembly includes a variable neutral density filter. In other embodiments, the variable optical adjustment assembly includes an adjustable beam splitter. In some embodiments, the light detection module includes a plurality of light scattering detectors and a variable optical adjustment assembly including a plurality of adjustable beam splitters. In some cases, the light detection module of interest is configured to simultaneously measure scattered light from particles in a flowing stream illuminated with a low-power light source (such as a laser with a peak power output of 1 mW or less).

[0006] This disclosure also includes a system for measuring scattered light from particles with diameters differing by 100 nm or more. In some embodiments, the system includes a light source and a light detection module having static optical adjustment components, variable optical adjustment components, and a light scattering detector. In some embodiments, the light source is a beam generator that generates multiple frequency-shifted beams (e.g., a first RF-shifted beam and a second RF-shifted beam). In some cases, the beam generator includes an acousto-optic deflector, such as an acousto-optic deflector operably coupled to a direct digital synthesizer RF comb generator. In these cases, the beam generator is configured to generate a local oscillator beam and multiple comb beams (e.g., an RF-shifted local oscillator beam and RF-shifted comb beams). In some embodiments, the light source includes a laser, such as a continuous-wave laser. In some cases, the light detection module includes multiple photodetectors. In some cases, the system also includes an optical collection system for propagating scattered light from a flow stream to the light detection module. The optical collection system may be a free-space light relay system or may include optical fibers, such as fiber optic repeater bundles. In some embodiments, the system is a flow cytometer.

[0007] This disclosure also includes methods for simultaneously measuring scattered light from particles with diameters differing by 100 nm or more. According to some embodiments, the method includes illuminating a sample having particles differing by 100 nm or more (e.g., in a flowing stream) with a light source and measuring the scattered light from the particles in the sample using a subject light detection module. In some embodiments, the method includes characterizing the particles in the sample based on the measured light, such as determining the size of the particles in the sample or determining the abundance of particles with predetermined sizes in the sample. In some embodiments, the method further includes sorting the particles in the sample based on the measured light or the characterization of the particles.

[0008] Kits are also provided that include one or more components of a subject light detection module. According to some embodiments, the kit includes: static optical adjustment components, such as a bandpass filter; variable optical adjustment components, such as a variable neutral density filter or a power beam splitter; and a photodetector. In some embodiments, the kit includes one or more shielding components, such as a diffuser bar, a shielding disk, an optical slit, or a pinhole. The kit may also include one or more lasers (e.g., continuous-wave lasers) and components for a beam generator for generating multiple frequency-shifted beams, such as an acousto-optic deflector and a direct digital synthesizer. Attached Figure Description

[0009] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The following drawings are included in the drawings:

[0010] Figure 1 The method describes the simultaneous measurement of side-scattered light from a sample containing particles with diameters ranging from 100 nm to 1000 nm using a light detection module according to certain embodiments.

[0011] Figure 2A and Figure 2B The configuration of a light detection module according to certain embodiments of the present disclosure is described. Figure 2A The arrangement of a static optical adjustment assembly, a variable optical adjustment assembly, and a photodetector in a light detection module according to certain embodiments is depicted. Figure 2B An arrangement having multiple photodetectors, a static optical adjustment assembly, and a variable optical adjustment assembly according to other embodiments is depicted. Detailed Implementation

[0012] A light detection system is described for simultaneously measuring scattered light (e.g., in a flowing stream) from particles with diameters differing by 100 nm or more. The light detection system, according to some embodiments, includes a static optical adjustment assembly, a variable optical adjustment assembly, and a photodetector. Systems and methods for measuring scattered light from a sample (e.g., in a flowing stream), and kits having a static optical adjustment assembly, a variable optical adjustment assembly, and a photodetector are also provided.

[0013] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as changes are certainly possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0014] When a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value (to one-tenth of the lower limit unit) between the upper and lower limits of the range, and any other value or intermediate value within the range, is included within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and also within the invention, subject to any specific exclusions within the range. Where the range includes one or both of these limit values, the invention also includes ranges that exclude any one or both of those included limit values.

[0015] This article uses certain ranges where numerical values ​​are preceded by the term "approximately". The term "approximately" is used in this article to provide textual support for the exact number preceding it and for numbers that are close to or approximate to the number preceding the term. In determining whether a number is close to or approximate to a specifically listed number, the unlisted number that is close to or approximate can be a number that, in the context in which it appears, provides a substantial equivalence to the specifically listed number.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, representative exemplary methods and materials are described hereafter.

[0017] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated herein by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the methods and / or materials referenced in the said publications. Any reference to any publication is for disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to prior disclosure by virtue of prior invention. Furthermore, the publication date provided may differ from the actual publication date, which may require separate confirmation.

[0018] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. It should also be noted that the drafting of the claims may exclude any optional elements. Therefore, this statement is intended to serve as a prior basis for the use of proprietary terms such as “merely,” “only,” or the use of “negative” limitations in relation to the statement of the claim elements.

[0019] As will be apparent to those skilled in the art upon reading this invention, each of the various embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any method referenced may be performed in the order of the referenced events or in any other logically possible order.

[0020] Although the apparatus and method have been or will be described for grammatical fluency and functional interpretation, it should be clearly understood that, unless expressly stated in accordance with 35 U.SC §112, the claims should not be construed as subject to the necessary limitation of being limited in any way by the interpretation of “means” or “steps,” but should be given the full range of meaning and equivalent meaning as defined by the claim under the doctrine of judicial equivalence, and where the claims are expressly formulated in accordance with 35 U.SC §112, they should be given the full legal equivalent meaning in accordance with 35 U.SC §112.

[0021] As described above, this disclosure provides a light detection module configured to simultaneously measure scattered light from a flow stream containing particles with diameters differing by 100 nm or more. In further describing embodiments of this disclosure, the light detection module according to embodiments of the present invention is first described in more detail. Next, systems and methods for measuring scattered light from samples (e.g., in a flow stream) containing particles with diameters differing by 100 nm or more are described. Kits having static optical adjustment components (e.g., a bandpass filter), variable optical adjustment components (e.g., a variable neutral density filter or a power beam splitter), and a light detector are also described.

[0022] Light detection module

[0023] Aspects of the present invention include a light detection module configured to simultaneously measure scattered light from a flow stream of particles with diameters differing by 100 nm or more (e.g., 500 nm or more). The term "simultaneously" is used herein in its conventional sense to refer to the simultaneous detection of scattered light from particles of different sizes by a photodetector of the light detection module. In embodiments, the light detection module does not require adjustment of the photodetector's sensitivity to measure light from particles of different sizes, wherein in some cases, the voltage gain of the photodetector is not adjusted to measure light from particles with diameters differing by 100 nm or more. According to certain embodiments, the light detection module includes a static optical adjustment assembly, a variable optical adjustment assembly, and a light scattering detector. In embodiments, the scattered light detector may be a side-scattering detector, a forward-scattering detector, a back-scattering detector, or a combination thereof. The term "light scattering" is used herein in its conventional sense to refer to the propagation of light energy from particles in a sample (e.g., flowing in a flow stream) (e.g., its deflection from the path of an incident beam by reflection, refraction, or deflection of the beam). In some embodiments, the scattered light is not from the emission of a component of the particles (e.g., a fluorophore). In embodiments, the scattered light according to this disclosure is not fluorescence or phosphorescence. In some embodiments, the scattered light used to determine the particle size in the flow stream by a subject method includes Mie scattering through the particles in the flow stream. In other embodiments, the scattered light used to determine the particle size in the flow stream by a subject method includes Rayleigh scattering through the particles in the flow stream. In still other embodiments, the scattered light used to determine the particle size in the flow stream by a subject method includes both Mie scattering and Rayleigh scattering through the particles in the flow stream.

[0024] The photodetector module according to embodiments of this disclosure is configured to simultaneously measure scattered light from particles whose sizes differ by 100 nm or more. For example, the diameters of the particles in the sample may differ by 125 nm or more, such as 150 nm or more, 175 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 275 nm or more, 300 nm or more, 325 nm or more, 350 nm or more, 375 nm or more, 400 nm or more, 425 nm or more, 450 nm or more, 475 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, and including 1000 nm or more. In some embodiments, the light detection module is configured to measure scattered light from a sample having particles with diameters ranging from 1 nm to 5000 nm, for example from 5 nm to 2500 nm, for example from 10 nm to 2000 nm, for example from 15 nm to 1500 nm, for example from 20 nm to 1000 nm, for example from 25 nm to 750 nm, and including 50 nm to 500 nm. In other words, as described in more detail below, the light detection module is capable of simultaneously measuring scattered light from large particles (e.g., particles with a diameter greater than 500 nm) and small particles (e.g., particles with a diameter less than 100 nm). In some embodiments, the light detection module is capable of simultaneously measuring scattered light from large and small particles by amplifying the signal from the scattered light detector, with almost no increase in noise from the scattered light detector.

[0025] Figure 1 The diagram describes the simultaneous measurement of side-scattered light from a sample containing particles having diameters ranging from 100 nm to 1000 nm using a photodetector module according to certain embodiments. Figure 1 As shown, light scattering from silica beads with a diameter of 100 nm and from polystyrene beads with diameters of 300 nm, 500 nm, 800 nm and 1000 nm were measured simultaneously.

[0026] In some embodiments, the light detection module is capable of simultaneously measuring scattered light from particles with a size difference of 100 nm or larger using only one type of detector channel (e.g., a side-scattering detector channel, such as a forward-scattering channel or a backscattering channel). In some cases, the subject light detection channel is capable of measuring scattered light from particles with a size difference of 100 nm or larger using only a single scattered light detector (e.g., a single side-scattering detector, such as a single forward-scattering detector or a single backscattering detector).

[0027] In some embodiments, the subject light detection module is configured to simultaneously measure scattered light from particles with a size difference of 100 nm or larger when illuminated with a laser having a power output of 100 mW or less, such as 50 mW or less, 25 mW or less, 10 mW or less, 5 mW or less, 1 mW or less, 0.5 mW or less, and including lasers with a power output of 0.1 mW or less. In some embodiments, the light detection module is configured to simultaneously measure scattered light from particles with diameters of 500 nm or larger and 100 nm or smaller when illuminated with a laser having a power output of 100 mW or less, such as 50 mW or less, 25 mW or less, 10 mW or less, 5 mW or less, 1 mW or less, 0.5 mW or less, and including lasers with a power output of 0.1 mW or less. For example, the optical detection module can be configured to simultaneously measure scattered light from particles with diameters of 500 nm or larger and 100 nm or smaller when irradiated with a laser having a power output of 1 mW or less.

[0028] In embodiments, the scattered light propagating from the sample to the subject light detection module is light scattered from the sample at an angle relative to the incident light beam, said angle being, for example, 1° or greater, 10° or greater, 15° or greater, 20° or greater, 25° or greater, 30° or greater, 45° or greater, 60° or greater, 75° or greater, 90° or greater, 135° or greater, 150° or greater, and including scattered light propagating from the sample at an angle of 180° or greater relative to the incident light beam. In some cases, the light detection module is configured to detect side-scattered light, for example, a photodetector is positioned to detect scattered light propagating from 30° to 120° relative to the incident light beam, for example, from 45° to 105°, and including from 60° to 90°. In some cases, the light detection module includes a side-scattered light detector positioned at a 90° angle relative to the incident light beam. In other cases, the light detection module is configured to detect forward-scattered light, for example, a photodetector is positioned to detect scattered light propagating at an angle of 120° to 240° relative to the incident light beam, such as 100° to 220°, or 120° to 200°, and including angles of 140° to 180° relative to the incident light beam. In some cases, the light detection module includes a forward-scattered light detector positioned to detect scattered light propagating at an angle of 180° relative to the incident light beam. In still other cases, the light detection module is configured to detect backscattered light, for example, a photodetector is positioned to detect scattered light propagating at an angle of 1° to 30° relative to the incident light beam, such as 5° to 25°, and including angles of 10° to 20° relative to the incident light beam. In some cases, the light detection module includes a backscattered light detector positioned to detect scattered light propagating at an angle of 30° relative to the incident light beam.

[0029] In embodiments of this disclosure, the light detection module includes a static optical adjustment component and a variable optical adjustment component. The term "optical adjustment" is used herein in its conventional sense to refer to optical components that alter or adjust the light propagating to the light scattering detector and the bright-field detector. For example, optical adjustment can change the profile of the light beam, the focal point of the light beam, the direction of light propagation, or collimate the light beam.

[0030] The light detection module includes a static optical adjustment assembly. The term "static" refers to an optics device in which the optical adjustment of scattered light remains constant throughout the use of the light detection module. In other words, the adjustment of light (e.g., changes in beam profile, focus, direction, wavelength, etc.) remains unchanged and cannot be altered without physical or computer manipulation of the static optical adjustment hardware (e.g., when using a digital bandpass filter as described below). In some embodiments, the static optical adjustment assembly is configured to provide static differential masking of light at a specific wavelength. "Differential masking" refers to the masking of one or more wavelengths of light scattered by particles in the sample. In some embodiments, the static optical adjustment component is configured to provide differential masking of one or more different wavelengths of light scattered by particles in the sample, such as two or more different wavelengths, such as three or more different wavelengths, such as four or more different wavelengths, such as five or more different wavelengths, such as ten or more different wavelengths, such as fifteen or more different wavelengths, such as twenty-five or more different wavelengths, such as fifty or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, such as one hundred or more different wavelengths, and including five hundred or more different wavelengths of light scattered by particles in the sample. In other embodiments, the static optical adjustment component is configured to provide differential masking over a wavelength range, such as 5 nm or greater, such as 10 nm or greater, such as 15 nm or greater, such as 25 nm or greater, such as 50 nm or greater, such as 75 nm or greater, such as 100 nm or greater, such as 150 nm or greater, such as 200 nm or greater, such as 250 nm or greater, and including 300 nm or greater. For example, the static optical adjustment component of interest may be configured to provide differential masking over a wavelength range from 2 nm to 500 nm, such as from 3 nm to 450 nm, such as from 4 nm to 400 nm, such as from 5 nm to 350 nm, such as from 10 nm to 300 nm, such as from 15 nm to 250 nm, and including from 20 nm to 200 nm. In still other embodiments, the subject static optical adjustment component is configured to provide differential masking over wavelengths above or below a predetermined wavelength threshold. In one example, light emitted from the sample at wavelengths above 800 nm may be differentially masked. In another example, light emitted from the sample at wavelengths below 400 nm can be differentially blocked. In some cases, static optical adjustment components include one or more bandpass filters, where specific wavelengths are differentially blocked as needed. Static optical adjustment components can include two or more different bandpass filters, such as three or more, four or more, and even five or more different bandpass filters.

[0031] In some embodiments, the static optical adjustment component is a computer-operated differential masking system, such as those described in U.S. Patent Application No. 16 / 422,630, filed May 24, 2019, the disclosure of which is incorporated herein by reference.

[0032] The light detection module also includes a variable optical adjustment component. The term "variable" is used to refer to an optical element configured to be altered, for example, where the optical adjustment can be changed during use of the light detection module. In some embodiments, the variable optical adjustment component is a neutral density filter. In one example, the neutral density filter is configured to modify the wavelength of light propagating to the photodetector of the light detection module. For example, the neutral density filter may be an adjustable filter that blocks light of one or more different wavelengths scattered by particles in the sample, such as two or more different wavelengths, three or more different wavelengths, four or more different wavelengths, five or more different wavelengths, ten or more different wavelengths, fifteen or more different wavelengths, twenty-five or more different wavelengths, fifty or more different wavelengths, one hundred or more different wavelengths, one hundred and fifty or more different wavelengths, two hundred and fifty or more different wavelengths, and includes light of five hundred or more different wavelengths scattered by particles in the sample. In other embodiments, the neutral density filter is an adjustable filter that modifies the amount of light propagated to the photodetector of the light detection module. For example, the adjustable neutral density filter is configured to reduce the amount of light by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and includes reducing the amount of light by 95% or more.

[0033] In some embodiments, the variable optical adjustment assembly includes a beam splitter. The term "beam splitter" is used herein in a conventional sense to refer to an optical assembly configured to propagate a beam along two or more distinct optical paths such that a predetermined portion of the light propagates along each optical path. Any convenient beam splitting scheme can be applied to, for example, prisms, beam splitter prisms, dichroic mirror prisms, and other types of beam splitters. The beam splitter can be formed of any suitable material, provided that it can propagate the desired amount and wavelength of light to the light scattering detector and the bright-field detector. For example, the beam splitter or point of interest can be formed of glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glass), silica (e.g., fused silica), quartz, crystals (e.g., CaF2 crystals), zinc selenide (ZnSe), F2, germanium titanate (Ge) (e.g., S-TIH11), or borosilicates (e.g., BK7). In some embodiments, the bundle splitter is formed of a polymeric material, such as, but not limited to, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics (e.g., PETG (ethylene glycol-modified polyethylene terephthalate)), and other polymeric plastic materials.In some embodiments, the splitter is formed of polyester, wherein the polyester of interest may include, but is not limited to: poly(alkyl terephthalate), such as poly(ethylene terephthalate) (PET), bottle-grade PET (a copolymer based on monoethylene glycol, terephthalic acid and other comonomers (e.g., isophthalic acid, cyclohexenedimethyl alcohol, etc.), poly(butylene terephthalate) (PBT) and poly(hexamethylene terephthalate); poly(alkyl adipate), such as poly(vinyl adipate). Poly(1,4-butenyl adipate) and poly(hexamethylene adipate); poly(alkyl octanoate), such as poly(vinyl octanoate); poly(alkyl sebacate), such as poly(vinyl sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkyl isophthalate), such as poly(vinyl isophthalate); poly(alkyl 2,6-naphthalenedicarboxylate), such as poly(vinyl 2,6-naphthalenedicarboxylate); poly(alkyl sulfonyl-4,4′-dibenzoate), such as poly(alkyl sulfonyl-4,4′-dibenzoate). ′-Dibenzoylvinyl ester); poly(p-phenylenealkyl dicarboxylate), such as poly(p-phenylene dicarboxylate); poly(trans-1,4-cyclohexanedialkyl dicarboxylate), such as poly(trans-1,4-cyclohexanedialkyl dicarboxylate); poly(1,4-cyclohexane-dimethyldicarboxylate), such as poly(1,4-cyclohexane-dimethyldicarboxylate); poly([2.2.2]-bicyclooctane-1,4-dimethyldicarboxylate), such as poly([2.2.2]-bicyclooctane-1 Poly(4-dimethyldicarboxylate); lactic acid polymers and copolymers, such as (S)-polylactic acid, (R,S)-polylactic acid, poly(tetramethylglycolic acid) and poly(lactide-co-glycolic acid); and polycarbonates of bisphenol A, 3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A, 3,3′,5,5′-tetramethylbisphenol A; polyamides, such as poly(terephthalamide); polyethylene terephthalate (e.g., Mylar™ polyethylene terephthalate) and combinations thereof.

[0034] In some embodiments, the variable optical adjustment assembly includes a wedge beamsplitter. In these embodiments, the beamsplitter is a beamsplitter with a wedge angle that produces non-collinear back reflection, such that the propagation of the collected light through the wedge beamsplitter causes a small change in the angle of light propagating to one or more photodetectors in the optical detection module. The wedge beamsplitter according to embodiments of this disclosure has a wedge angle, wherein the change in the incident angle of the collected light causes a deviation in the angle of propagation of 0.001% or more, for example 0.005% or more, for example 0.01% or more, for example 0.05% or more, for example 0.1% or more, for example 0.5% or more, for example 1% or more, for example 2% or more, for example 3% or more, for example 5% or more, and including 10% or more. In some embodiments, the wedge beam splitter has a wedge angle ranging from 5 arcminutes to 120 arcminutes, for example, from 10 arcminutes to 115 arcminutes, for example, from 15 arcminutes to 110 arcminutes, for example, from 20 arcminutes to 105 arcminutes, for example, from 25 arcminutes to 100 arcminutes, for example, from 30 arcminutes to 105 arcminutes, for example, from 35 arcminutes to 100 arcminutes, for example, from 40 arcminutes to 95 arcminutes, and including a range from 45 arcminutes to 90 arcminutes. In some embodiments, the wedge beam splitter has a wedge angle sufficient to reduce or eliminate optical interference.

[0035] In some embodiments, the wedge beam splitter has a transparent window ranging from 150 nm to 5 μm; wavelength ranges ranging from 180 nm to 8 μm, from 185 nm to 2.1 μm, from 200 nm to 6 μm, from 200 nm to 11 μm, from 250 nm to 1.6 μm, from 350 nm to 2 μm, from 600 nm to 16 μm, from 1.2 μm to 8 μm, from 2 μm to 16 μm, or some other wavelength ranges.

[0036] In some embodiments, the spatial position of the beam splitter is adjustable, for example, manually (by hand) or by a motor-driven displacement device. For example, the angle of the beam splitter can be adjusted by 5° or more in the subject light detection system, such as 10° or more, 15° or more, 20° or more, 30° or more, 45° or more, 60° or more, and including 75° or more. In some cases, the spatial position of the beam splitter can be adjusted within the light detection module, such as by 1 mm or more, 5 mm or more, 10 mm or more, and including 25 mm or more. Any convenient motor-driven actuator can be used, such as, for example, a motor-driven displacement platform, a motor-driven lead screw assembly, an electric gear actuator employing a stepper motor, a servo motor, a brushless motor, a brushed DC motor, a microstepper drive motor, a high-resolution stepper motor, and other types of motors. In one example, the horizontal or vertical position or orientation angle of the beam splitter can be adjusted using a motor-driven displacement device.

[0037] The photodetector of the subject light detection module can be any suitable light sensor, such as an active pixel sensor (APS), avalanche photodiode, image sensor, charge-coupled device (CCD), enhancement-mode charge-coupled device (ICCD), complementary metal-oxide-semiconductor (CMOS) image sensor or N-type metal-oxide-semiconductor (NMOS) image sensor, light-emitting diode, photon counter, radiometric calorimeter, thermoelectric detector, photoresistor, photovoltaic cell, photodiode, photomultiplier tube, phototransistor, quantum dot photoconductor or photodiode and combinations thereof, and other types of photodetectors. In embodiments, the light scattering photodetector may include one or more light sensors, such as two or more, three or more, five or more, ten or more, and 25 or more light sensors. In some cases, the light scattering photodetector is a photodetector array. The term "photodetector array" is used in its conventional sense to refer to an arrangement or series of two or more photodetectors configured to detect light. In embodiments, the photodetector array may include two or more photodetectors, such as three or more photodetectors, such as four or more photodetectors, such as five or more photodetectors, such as six or more photodetectors, such as seven or more photodetectors, such as eight or more photodetectors, such as nine or more photodetectors, such as ten or more photodetectors, such as twelve or more photodetectors, and including fifteen or more photodetectors. In some embodiments, the photodetector array includes five photodetectors. The photodetectors can be arranged in any geometric configuration as needed, wherein arrangements of interest include, but are not limited to, square configurations, rectangular configurations, trapezoidal configurations, triangular configurations, hexagonal configurations, heptagonal configurations, octagonal configurations, nonagonal configurations, decagonal configurations, dodecagonal configurations, circular configurations, elliptical configurations, and irregular shape configurations. The photodetectors in the light scattering photodetector array can be oriented relative to another (as mentioned in the XZ plane) at an angle ranging from 10° to 180°, for example from 15° to 170°, for example from 20° to 160°, for example from 25° to 150°, for example from 30° to 120°, and including from 45° to 90°.

[0038] The light scattering detector disclosed herein is configured to measure light collected at one or more wavelengths, such as two or more wavelengths, such as five or more different wavelengths, such as ten or more different wavelengths, such as 25 or more different wavelengths, such as 50 or more different wavelengths, such as 100 or more different wavelengths, such as 200 or more different wavelengths, such as 300 or more different wavelengths, and includes measuring light emitted by a sample in a flowing stream at 400 or more different wavelengths.

[0039] In some embodiments, the subject photodetector is configured to measure light collected within a wavelength range (e.g., 200 nm–1000 nm). In some embodiments, the detector of interest is configured to collect the spectrum of light within a wavelength range. For example, the system may include one or more detectors configured to collect the spectrum of light within one or more wavelength ranges of 200 nm–1000 nm. In still other embodiments, the detector of interest is configured to measure light emitted by a sample in a flowing stream at one or more specific wavelengths. In embodiments, the light detection system is configured to measure light continuously or at discrete intervals. In some cases, the detector of interest is configured to continuously measure the collected light. In other cases, the light detection system is configured to perform measurements at discrete intervals, such as every 0.001 ms, every 0.01 ms, every 0.1 ms, every 1 ms, every 10 ms, every 100 ms, and including every 1000 ms or some other interval.

[0040] In some embodiments, the light received by the subject light detection module can be transmitted via an optical collection system. The optical collection system can be any suitable light collection scheme that collects and guides scattered light from the irradiated sample. In some embodiments, the optical collection system includes optical fibers, such as fiber optic repeaters. In other embodiments, the optical collection system is a free-space optical repeater system.

[0041] In embodiments, the optical collection system may be physically coupled to the light detection system, for example, using an adhesive, co-molded together, or integrated into the light detection system. In some embodiments, the optical collection system and the light detection system are integrated into a single unit. In some cases, the optical collection system is coupled to the light detection system using a connector that secures the optical collection system to the light detection system, for example, via hook and loop fasteners, magnets, latches, notches, countersunk holes, counterbores, grooves, pins, straps, hinges, Velcro, non-permanent adhesives, or combinations thereof.

[0042] In other embodiments, the light detection module and the optical collection system are optically communicating but not physically contacting each other. In embodiments, the optical collection system may be positioned at a distance of 0.001 mm or greater from the light detection system, for example, 0.005 mm or greater, 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, 1 mm or greater, 10 mm or greater, 25 mm or greater, 50 mm or greater, and including a distance of 100 mm or greater from the light detection system.

[0043] In some embodiments, the optical collection system includes optical fibers. For example, the optical collection system may be a fiber optic repeater bundle, and light is transmitted to the optical detection system via the fiber optic repeater bundle. Any fiber optic repeater system can be used to propagate light to the optical detection system. In some embodiments, suitable fiber optic repeater systems for propagating light to the optical detection system include, but are not limited to, fiber optic repeater systems (such as those described in U.S. Patent No. 6,809,804), the disclosure of which is incorporated herein by reference.

[0044] In other embodiments, the optical collection system is a free-space optical relay system. The phrase "free-space optical relay" is used herein in its conventional sense to refer to light propagation that employs a configuration of one or more optical components to guide light through free space to a light detection system. In some embodiments, the free-space optical relay system includes a housing having a near end and a far end, the near end being coupled to the light detection system. The free-space relay system may include any combination of different optical adjustment components, such as lenses, mirrors, slits, pinholes, wavelength splitters, or combinations thereof. For example, in some embodiments, the free-space optical relay system of interest includes one or more focusing lenses. In other embodiments, the subject free-space optical relay system includes one or more mirrors. In still other embodiments, the free-space optical relay system includes collimating lenses. In some embodiments, suitable free-space optical relay systems for propagating light to a light detection system include, but are not limited to, those described in, for example, U.S. Patent Nos. 7,643,142, 7,728,974, and 8,223,445, the disclosure of which is incorporated herein by reference.

[0045] Figure 2A and Figure 2B An example configuration of a light detection module according to certain embodiments is depicted. Figure 2A A light detection module 100 is depicted, wherein scattered light (e.g., side-scattered light, forward-scattered light, back-scattered light) from an irradiated sample is propagated by an optical collection system 105 to a static optical adjustment assembly 101 (e.g., a bandpass filter) and transmitted to a photodetector 103 (e.g., a photomultiplier tube) via a variable optical adjustment assembly 102 (e.g., a neutral density filter or an adjustable beam splitter). Figure 2B A light detection module 200 is depicted, which has eight sets of static optical adjustment components, variable optical adjustment components and photodetectors (201c1–201c8) arranged concentrically, and light propagates along the beam path between each set of static optical adjustment components, variable optical adjustment components and photodetectors.

[0046] A system for measuring scattered light from irradiated samples containing particles of different sizes.

[0047] This disclosure also includes a system for measuring light scattered by particles whose size differs from that of a sample (e.g., in a flow stream in a flow cytometer) by 100 nm or more. In some embodiments, the system includes a light source and a light detection module having static optical adjustment components, variable optical adjustment components, and a photodetector. In some embodiments, the light detection module communicates optically with the sample source (e.g., in a flow stream in a flow cytometer) via an optical collection system (e.g., an optical fiber or a free-space optical relay system).

[0048] A system of interest for measuring light scattered by particles whose size differs from the sample by 100 nm or more includes a light source. In embodiments, the light source can be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, small droplets, non-cellular particles, etc.), the light source can be configured to emit light of different wavelengths, ranging from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including 400 nm to 800 nm. For example, the light source can include a broadband light source that emits light with wavelengths ranging from 200 nm to 900 nm. In other cases, the light source includes a narrowband light source that emits light with wavelengths ranging from 200 nm to 900 nm. For example, the light source can be a narrowband LED (1 nm–25 nm) that emits light with wavelengths ranging between 200 nm and 900 nm.

[0049] In some embodiments, the light source is a laser. Lasers of interest may include pulsed lasers or continuous-wave lasers. For example, the laser may be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, or a neon-copper (N) laser. eCu) lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or frequency-doubled or third-doubled embodiments of any of the above lasers.

[0050] In some embodiments, the system includes a laser having a power output of 100 mW or less, such as 50 mW or less, 25 mW or less, 10 mW or less, 5 mW or less, 1 mW or less, 0.5 mW or less, and a laser having a power output of 0.1 mW or less. In some embodiments, the system includes a laser having a power output of 1 mW or less.

[0051] In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to halogen lamps, deuterium arc lamps, xenon arc lamps, and light-emitting diodes (LEDs), such as broadband LEDs with a continuous spectrum, superluminescent LEDs, semiconductor LEDs, broadband LED white light sources, and multi-LED integration. In some cases, the non-laser light source is a stable fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0052] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some instances, the beam generator includes a laser, a radio frequency (RF) generator configured to apply an RF drive signal to the acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser in the beam generator of interest can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, or a helium-selenium (He) laser. Se lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titania-sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium-doped lasers and combinations thereof.

[0053] The acousto-optic device can be any convenient acousto-optic scheme configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angle-deflected laser beam from light from a laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), arbitrary waveform generator (AWG), or electrical pulse generator.

[0054] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angle-deflected laser beams in the output laser beam. For example, it is configured to apply 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and includes being configured to apply 100 or more radio frequency drive signals.

[0055] In some cases, in order to generate an intensity distribution of an angle-deflected laser beam in the output laser beam, the controller is configured to apply an radio frequency drive signal having an amplitude that varies from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to 50V, for example from about 2V to 40V, for example from 3V to about 30V, and including from about 5V to about 25V. In some embodiments, the radio frequency drive signal for each application has a frequency from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and includes frequencies from about 5 MHz to about 50 MHz.

[0056] In some embodiments, the controller has a processor with a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam with a desired intensity distribution. For example, the memory may include instructions for generating two or more laser beams with the same intensity at different angles, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and may also include instructions for generating 100 or more laser beams with the same intensity at different angles. In other embodiments, the memory may include instructions for generating two or more laser beams with different intensities at different angles, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and may also include instructions for generating 100 or more laser beams with different intensities at different angles.

[0057] In some embodiments, the controller has a processor with a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases along a horizontal axis from the edge of the output laser beam to the center. In these cases, the intensity of the angularly deflected laser beam located at the center of the output beam can be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, for example, from 0.5% to about 95%, for example, from 1% to about 90%, for example, from about 2% to about 85%, for example, from about 3% to about 80%, for example, from about 4% to about 75%, for example, from about 5% to about 70%, for example, from about 6% to about 65%, for example, from about 7% to about 60%, for example, from about 8% to about 55%, and includes the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis in the range of about 10% to about 55%. In other embodiments, the controller has a processor with a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases along a horizontal axis from the edge to the center of the output laser beam. In these cases, the intensity of the angularly deflected laser beam located at the edge of the output beam can be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, for example, from 0.5% to about 95%, for example, from 1% to about 90%, for example, from about 2% to about 85%, for example, from about 3% to about 80%, for example, from about 4% to about 75%, for example, from about 5% to about 70%, for example, from about 6% to about 65%, for example, from about 7% to about 60%, for example, from about 8% to about 55%, and includes the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis in the range of about 10% to about 55%. In some other embodiments, the controller has a processor, and a memory is operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a Gaussian intensity distribution along a horizontal axis. In other embodiments, the controller has a processor with a memory operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity distribution along a horizontal axis.

[0058] In embodiments, the beam generator of interest can be configured to generate spatially separated, angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired illumination distribution of the output laser beam, the angle-deflected laser beams can be separated by 0.001 μm or greater, for example, 0.005 μm or greater, for example, 0.01 μm or greater, for example, 0.05 μm or greater, for example, 0.1 μm or greater, for example, 0.5 μm or greater, for example, 1 μm or greater, for example, 5 μm or greater, for example, 10 μm or greater, for example, 100 μm or greater, for example, 500 μm or greater, for example, 1000 μm or greater, and including 5000 μm or greater. In some embodiments, the system is configured to generate angle-deflected laser beams within the output laser beam that overlap, for example, with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 0.001 μm or greater, such as 0.005 μm or greater, such as 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 5 μm or greater, such as 10 μm or greater, and includes 100 μm or greater overlap.

[0059] In some cases, beam generators configured to generate two or more frequency-shifted beams include laser excitation modules, as described in U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publications Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0060] In some embodiments, the system further includes a flow cell configured to propagate a sample in the flow stream. Any convenient flow cell that propagates the fluid sample to the sample interrogation region can be employed, wherein in some embodiments, the flow cell includes: a proximal cylindrical portion defining a longitudinal axis; and a distal truncated conical portion terminating on a flat surface having an orifice transverse to the longitudinal axis. The length of the proximal cylindrical portion (as measured along the longitudinal axis) can vary, ranging from 1 mm to 15 mm, for example from 1.5 mm to 12.5 mm, for example from 2 mm to 10 mm, for example from 3 mm to 9 mm, and including from 4 mm to 8 mm. The length of the distal truncated conical portion (as measured along the longitudinal axis) can also vary, ranging from 1 mm to 10 mm, for example from 2 mm to 9 mm, for example from 3 mm to 8 mm, and including from 4 mm to 7 mm. In some embodiments, the diameter of the flow pool nozzle chamber can vary, ranging from 1 mm to 10 mm, for example from 2 mm to 9 mm, for example from 3 mm to 8 mm, and including from 4 mm to 7 mm.

[0061] In some cases, the flow cell does not include a cylindrical portion, and the entire internal chamber of the flow cell is truncated conical. In these embodiments, the length of the truncated conical internal chamber (measured, for example, along a longitudinal axis transverse to the nozzle orifice) can range from 1 mm to 15 mm, such as from 1.5 mm to 12.5 mm, such as from 2 mm to 10 mm, such as from 3 mm to 9 mm, and including from 4 mm to 8 mm. The diameter of the proximal portion of the truncated conical internal chamber can range from 1 mm to 10 mm, such as from 2 mm to 9 mm, such as from 3 mm to 8 mm, and including from 4 mm to 7 mm.

[0062] In some embodiments, the sample flow originates from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow, the flow cell orifice can be of any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as parabolic bottoms connected to a planar top. In some embodiments, the flow cell of interest has a circular orifice. In some embodiments, the nozzle orifice size can vary, ranging from 1 μm to 20,000 μm, for example from 2 μm to 17,500 μm, for example from 5 μm to 15,000 μm, for example from 10 μm to 12,500 μm, for example from 15 μm to 10,000 μm, for example from 25 μm to 7,500 μm, for example from 50 μm to 5,000 μm, for example from 75 μm to 1,000 μm, for example from 100 μm to 750 μm, and including 150 μm to 500 μm. In some embodiments, the nozzle orifice is 100 μm.

[0063] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell. In another embodiment, the sample injection system is configured to provide a suitable sample flow into the internal chamber of the flow cell. Depending on the desired characteristics of the flow, the sample rate delivered to the flow cell chamber through the sample injection port can be 1 μL / min or greater, for example, 2 μL / min or greater, for example, 3 μL / min or greater, for example, 5 μL / min or greater, for example, 10 μL / min or greater, for example, 15 μL / min or greater, for example, 25 μL / min or greater, for example, 50 μL / min or greater, and including 100 μL / min or greater. In some cases, the sample rate delivered to the flow cell chamber through the sample injection port is 1 μL / s or greater, for example, 2 μL / s or greater, for example, 3 μL / s or greater, for example, 5 μL / s or greater, for example, 10 μL / s or greater, for example, 15 μL / s or greater, for example, 25 μL / s or greater, for example, 50 μL / s or greater, and including 100 μL / s or greater.

[0064] The sample injection port can be an orifice located in the wall of the internal chamber, or it can be a conduit located proximal to the internal chamber. When the sample injection port is an orifice located in the wall of the internal chamber, the orifice can be of any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as parabolic bottoms connected to a plane top. In some embodiments, the sample injection port has a circular orifice. In some cases, the size of the sample injection port orifice can vary depending on its shape, with an opening range from 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0065] In some cases, the sample injection port is a conduit located proximal to the internal chamber of the flow cell. For example, the sample injection port may be a conduit positioned such that its orifice aligns with the flow cell orifice. When the sample injection port is a conduit positioned to align with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including but not limited to: linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses; and irregular shapes, such as parabolic bottoms attached to a plane top. In some cases, the orifice of the conduit may vary in shape, with an opening range from 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and including 1.25 mm to 1.75 mm, for example 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip with an angle ranging from 1° to 10°, such as from 2° to 9°, such as from 3° to 8°, such as from 4° to 7°, and including an angle of 5°.

[0066] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to, for example, supply fluid of sheath fluid to the internal chamber of the flow cell along with a sample to generate a laminated flow of sheath fluid surrounding the sample flow. Depending on the desired characteristics of the flow flow, the rate at which the sheath fluid is delivered through the tube to the flow cell chamber can be 25 μL / s or greater, for example 50 μL / s or greater, for example 75 μL / s or greater, for example 100 μL / s or greater, for example 250 μL / s or greater, for example 500 μL / s or greater, for example 750 μL / s or greater, for example 1000 μL / s or greater, and includes 2500 μL / s or greater.

[0067] In some embodiments, the sheath fluid injection port is an orifice located in the wall of an internal chamber. The sheath fluid injection port orifice can be of any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as parabolic bottoms connected to a planar top. In some cases, the size of the sample injection port orifice can vary depending on its shape, with an opening range from 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0068] In some embodiments, the system further includes a pump in fluid communication with the flow cell to propagate the flow stream through the flow cell. Any convenient fluid pumping scheme can be used to control the flow stream through the flow cell. In some cases, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in this subject system is configured to deliver fluid through the flow cell at a rate suitable for detecting light from a sample in the flow stream. In some cases, the sample flows in the flow cell at a rate of 1 μL / min or greater, such as 2 μL / min or greater, such as 3 μL / min or greater, such as 5 μL / min or greater, such as 10 μL / min or greater, such as 25 μL / min or greater, such as 50 μL / min or greater, such as 75 μL / min or greater, such as 100 μL / min or greater, such as 250 μL / min or greater, such as 500 μL / min or greater, such as 750 μL / min or greater, and including 1000 μL / min or greater. For example, the system may include a pump configured to flow a sample through a flow cell at a rate ranging from 1 μL / min to 500 μL / min, such as from 1 μL / min to 250 μL / min, from 1 μL / min to 100 μL / min, from 2 μL / min to 90 μL / min, from 3 μL / min to 80 μL / min, from 4 μL / min to 70 μL / min, from 5 μL / min to 60 μL / min, and from 10 μL / min to 50 μL / min. In some embodiments, the flow rate is from 5 μL / min to 6 μL / min.

[0069] In some embodiments, the subject system is a flow cytometer system employing the aforementioned light detection module for detecting light scattered by particles in the sample within the flowing stream. In some embodiments, the subject system is a flow cytometer system. Suitable flow cytometry systems may include, but are not limited to, those described in the following literature: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49(pt 1): 17-28; Linden et al., Semin Throm Hemost. 2004 Oct; 30(5): 502-11; Alison et al. J Pathol, 2010 Dec; 222(4): 335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the contents of which are incorporated herein by reference. In some cases, flow cytometry systems of interest include BDBiosciences FACSCanto. TM II flow cytometer, BD Accuri TM Flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSL Fortessa TM Flow cytometer, BD Biosciences LSRFortess TM X-20 flow cytometer and BDBiosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCountTM Cell sorting instrument, BDBiosciences FACSLyric TM Cell sorter and BD Biosciences via TM Cell sorting instrument, BDBiosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorting instrument and BD Biosciences FACSMelody TM Cell sorting instruments, etc.

[0070] In some embodiments, the subject system is a particle sorting system, such as those described in the following U.S. patents: U.S. Patent Nos. 10,006,852, 9,952,076, 9,933,341, 9,784,661, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, and 8,753. 573, 8,233,146, 8,140,300, 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039; all of their publicly available content is incorporated herein by reference.

[0071] In some cases, the subject system is a flow cytometer system that is also configured to image particles in a flowing stream using fluorescence imaging with radio frequency labeled emission (FIRE), such as those described in: Nature Photonics, Diebold et al., Vol. 7 (10); 806-810 (2013), and U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publications 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0072] A method for measuring light from an irradiated sample containing particles of different sizes.

[0073] This disclosure also includes methods for simultaneously measuring scattered light from particles with diameters differing by 100 nm or more. A method according to some embodiments includes illuminating a sample having particles with diameters differing by 100 nm or more (e.g., in a flowing stream) with a light source and measuring the scattered light from the particles in the sample using a photodetector module as described above. In some embodiments, the sample is a biological sample. The term "biological sample" in its conventional sense refers to a subset of tissues, cells, or components of a whole organism, plant, fungus, or animal, and in some cases can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic sac blood, urine, vaginal fluid, and semen. Therefore, "biological sample" refers to a subset of a protist or its tissues, and homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. Biological samples can be any type of organic tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In some embodiments, biological samples are liquid samples, such as blood or derivatives thereof, such as plasma, tears, urine, semen, etc., and in some cases, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger prick (where the blood may or may not be bound to any reagents, such as preservatives, anticoagulants, etc., before testing).

[0074] In some embodiments, the sample source is "mammal" or "milk animal," terms that are broadly used to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is human. The method can be applied to samples obtained from both sexes and human subjects at any developmental stage (i.e., newborns, infants, adolescents, teenagers, and adults), wherein, in some embodiments, the human subject is an adolescent, teenager, or adult. While the invention can be applied to samples from human subjects, it should be understood that the method can also be performed on samples from other animal subjects (i.e., in "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0075] In carrying out the described method, a sample is illuminated with light from a light source (e.g., in the flow stream of a flow cytometer). In some embodiments, the light source is a broadband light source that emits light with a wide wavelength range, for example, light spanning 50 nm or greater, such as 100 nm or greater, such as 150 nm or greater, such as 200 nm or greater, such as 250 nm or greater, such as 300 nm or greater, such as 350 nm or greater, such as 400 nm or greater, and including light spanning 500 nm or greater. For example, a suitable broadband light source emits light with wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with wavelengths from 400 nm to 1000 nm. When the method includes illumination with a broadband light source, the broadband light source schemes of interest may include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with continuous spectra, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof.

[0076] In other embodiments, the method includes illumination with a narrowband light source emitting a specific wavelength or a narrow range of wavelengths, such as illumination with a light source emitting light within a narrow range of wavelengths, for example, 50 nm or less, 40 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, and light sources emitting light of a specific wavelength (i.e., monochromatic light). When the method includes illumination with a narrowband light source, the narrowband light source schemes of interest include, but are not limited to, narrow-wavelength LEDs, laser diodes, or broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0077] In some embodiments, the method includes irradiating the flowing stream with one or more lasers. As discussed above, the type and number of lasers will vary depending on the sample and the desired light collected, and can be pulsed lasers or continuous-wave lasers. For example, the lasers can be gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof; dye lasers, such as stilbene, coumarin, or rhodamine lasers; metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, and neon-copper (N) lasers. eCu) lasers, copper lasers, or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or frequency-doubled or third-doubled embodiments of any of the above lasers.

[0078] In some embodiments, the method includes irradiating the sample with a laser having a power output of 100 mW or less, such as 50 mW or less, 25 mW or less, 10 mW or less, 5 mW or less, 1 mW or less, or 0.5 mW or less, and also includes irradiating the sample with a laser having a power output of 0.1 mW or less. In some embodiments, the method includes irradiating the sample with a laser having a power output of 1 mW or less.

[0079] The sample can be irradiated with one or more of the above-described light sources, such as two or more light sources, three or more light sources, four or more light sources, five or more light sources, and including ten or more light sources. The light source can include any combination of light source types. For example, in some embodiments, the method includes irradiating the sample in the flowing stream with a laser array (e.g., an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers).

[0080] The sample can be illuminated with wavelengths ranging from 200 nm to 1500 nm (e.g., from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, and including from 400 nm to 800 nm). For example, in the case where the light source is a broadband light source, the sample can be illuminated with wavelengths from 200 nm to 900 nm. In other cases where the light source includes multiple narrowband light sources, the sample can be illuminated with specific wavelengths ranging from 200 nm to 900 nm. For example, the light source can be multiple narrowband LEDs (1 nm–25 nm), each emitting light independently in the wavelength range of 200 nm to 900 nm. In other embodiments, the narrowband light source includes one or more lasers (e.g., a laser array) and illuminates the sample with specific wavelengths ranging from 200 nm to 700 nm, for example, with a laser array having gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers as described above.

[0081] When using more than one light source, the sample can be illuminated simultaneously, sequentially, or in combination by the light sources. For example, the sample can be illuminated simultaneously by each light source. In other embodiments, the flow is illuminated sequentially by each light source. When illuminating the sample sequentially by more than one light source, the illumination time of each light source can be independently 0.001 microseconds or longer, for example, 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 30 microseconds or longer, and including 60 microseconds or longer. For example, the method can include illuminating the sample with a light source (e.g., a laser) for a duration from 0.001 microseconds to 100 microseconds, for example, from 0.01 microseconds to 75 microseconds, for example, from 0.1 microseconds to 50 microseconds, for example, from 1 microsecond to 25 microseconds, and including durations from 5 microseconds to 10 microseconds. In embodiments where the sample is illuminated sequentially by two or more light sources, the illumination time of each light source can be the same or different.

[0082] The time period between each light source illumination can also be varied as needed, and can be independently separated by a delay of 0.001 microseconds or longer, such as a delay of 0.01 microseconds or longer, a delay of 0.1 microseconds or longer, a delay of 1 microsecond or longer, a delay of 5 microseconds or longer, a delay of 10 microseconds or longer, a delay of 15 microseconds or longer, a delay of 30 microseconds or longer, and a delay including 60 microseconds or longer. For example, the time period between each light source illumination can range from 0.001 microseconds to 60 microseconds, such as from 0.01 microseconds to 50 microseconds, such as from 0.1 microseconds to 35 microseconds, such as from 1 microsecond to 25 microseconds, and including a range from 5 microseconds to 10 microseconds. In some embodiments, the time period between each light source illumination is 10 microseconds. In embodiments where the sample is sequentially illuminated by two or more (i.e., three or more) light sources, the delay between each light source illumination can be the same or different.

[0083] The sample may be irradiated continuously or at discrete intervals. In some cases, the method involves irradiating the sample with a light source continuously. In other cases, the sample is irradiated with a light source at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0084] Depending on the light source, the sample can be illuminated at varying distances, such as 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, 1 mm or greater, 2.5 mm or greater, 5 mm or greater, 10 mm or greater, 15 mm or greater, 25 mm or greater, and including 50 mm or greater. Furthermore, the angle or illumination can also vary, ranging from 10° to 90°, such as from 15° to 85°, from 20° to 80°, from 25° to 75°, and including from 30° to 60°, such as at an angle of 90°.

[0085] As discussed above, in embodiments, light from the irradiated sample is transmitted to a light detection module as described herein and measured by one or more light detectors. In practicing this subject matter method, light is propagated to the light detection module. The light is further propagated to a light scattering detector and a bright-field light detector via optical conditioning components. In some embodiments, the method includes measuring light collected within a wavelength range (e.g., 200 nm–1000 nm). For example, the method may include: collecting the spectrum of light within one or more wavelength ranges of 200 nm–1000 nm. In other embodiments, the method includes measuring light collected at one or more specific wavelengths.

[0086] The collected light can be measured continuously or at discrete intervals. In some cases, the method involves measuring the light continuously. In others, the light is measured at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0087] The collected light can be measured once or multiple times during the subject method, for example, two or more times, three or more times, five or more times, and including ten or more times. In some embodiments, light propagation is measured two or more times, and in some cases the data is averaged.

[0088] In some embodiments, the method includes further conditioning the light before measuring it with the subject light detection module. For example, light from a sample source may pass through one or more lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. In some cases, the collected light passes through one or more focusing lenses, for example, to reduce the distribution of light directed to the light detection system or optical collection system as described above. In other cases, light emitted from the sample passes through one or more collimators to reduce beam divergence transmitted to the light detection system.

[0089] In some embodiments, the method includes irradiating a sample with two or more frequency-shifted laser beams. As described above, a beam generator assembly may be employed, having a laser and an acousto-optic device for frequency-shifting the laser. In these embodiments, the method includes irradiating the acousto-optic device with a laser. Depending on the desired wavelength of light generated in the output laser beam (e.g., for irradiating a sample in a flowing stream), the laser may have a specific wavelength ranging from 200 nm to 1500 nm, for example from 250 nm to 1250 nm, for example from 300 nm to 1000 nm, for example from 350 nm to 900 nm, and including variations from 400 nm to 800 nm. The acousto-optic device may be irradiated with one or more lasers, for example two or more lasers, for example three or more lasers, for example four or more lasers, for example five or more lasers, and including ten or more lasers. The lasers may include any combination of various types of lasers. For example, in some embodiments, the method includes irradiating the acousto-optic device with a laser array (e.g., an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers).

[0090] When multiple lasers are used, the acousto-optic device can be irradiated simultaneously, sequentially, or in combination with the lasers. For example, the acousto-optic device can be irradiated simultaneously with each laser. In other embodiments, the acousto-optic device is irradiated sequentially with each laser. When more than one laser is used to irradiate the acousto-optic device sequentially, the irradiation time of each laser can be independently 0.001 microseconds or longer, for example, 0.01 microseconds or longer, for example, 0.1 microseconds or longer, for example, 1 microsecond or longer, for example, 5 microseconds or longer, for example, 10 microseconds or longer, for example, 30 microseconds or longer, and including 60 microseconds or longer. For example, the method can include irradiating the acousto-optic device with lasers for durations from 0.001 microseconds to 100 microseconds, for example, from 0.01 microseconds to 75 microseconds, for example, from 0.1 microseconds to 50 microseconds, for example, from 1 microsecond to 25 microseconds, and including from 5 microseconds to 10 microseconds. In embodiments where two or more lasers sequentially irradiate the acousto-optic device, the duration for which each laser irradiates the acousto-optic device can be the same or different.

[0091] The time period between each laser irradiation can also be varied as needed, and can be independently separated by a delay of 0.001 microseconds or longer, such as a delay of 0.01 microseconds or longer, a delay of 0.1 microseconds or longer, a delay of 1 microsecond or longer, a delay of 5 microseconds or longer, a delay of 10 microseconds or longer, a delay of 15 microseconds or longer, a delay of 30 microseconds or longer, and including a delay of 60 microseconds or longer. For example, the range of the time period between irradiations of each light source can be from 0.001 microseconds to 60 microseconds, such as from 0.01 microseconds to 50 microseconds, such as from 0.1 microseconds to 35 microseconds, such as from 1 microsecond to 25 microseconds, and including from 5 microseconds to 10 microseconds. In some embodiments, the time period between irradiations of each laser is 10 microseconds. In embodiments where the acousto-optic device is sequentially irradiated by two or more (i.e., three or more) lasers, the delay between irradiations of each laser can be the same or different.

[0092] The acousto-optic device can be illuminated continuously or at discrete intervals. In some cases, the method includes illuminating the acousto-optic device continuously with a laser. In other cases, the acousto-optic device is illuminated with a laser at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0093] Depending on the laser, the acousto-optic device can be illuminated at varying distances, such as 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, 1 mm or greater, 2.5 mm or greater, 5 mm or greater, 10 mm or greater, 15 mm or greater, 25 mm or greater, and including 50 mm or greater. Furthermore, the angle or illumination can also be varied, ranging from 10° to 90°, such as from 15° to 85°, from 20° to 80°, from 25° to 75°, and including from 30° to 60°, such as at an angle of 90°.

[0094] In an embodiment, the method includes applying radio frequency (RF) drive signals to an acousto-optic device to generate an angle-deflected laser beam. Two or more RF drive signals (e.g., three or more RF drive signals, four or more RF drive signals, five or more RF drive signals, six or more RF drive signals, seven or more RF drive signals, eight or more RF drive signals, nine or more RF drive signals, ten or more RF drive signals, fifteen or more RF drive signals, twenty-five or more RF drive signals, fifty or more RF drive signals, and including one hundred or more RF drive signals) can be applied to the acousto-optic device to generate an output laser beam with the desired number of angle deflections.

[0095] Each angle-deflected laser beam generated by a radio frequency (RF) drive signal has an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal having an amplitude sufficient to generate a laser beam with an angle deflection of the desired intensity. In some cases, the RF drive signal for each application independently has an amplitude ranging from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to 50V, for example from about 2V to 40V, for example from 3V to about 30V, and including from about 5V to about 25V. In some embodiments, the radio frequency drive signal for each application has a frequency from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and includes frequencies from about 5 MHz to about 50 MHz.

[0096] In these embodiments, the angle-deflected laser beams in the output laser beam are spatially separated. Depending on the applied RF drive signal and the desired illumination distribution of the output laser beam, the angle-deflected laser beams can be separated by 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, and including separations of 5000 μm or more. In some embodiments, the angle-deflected laser beams overlap, for example, with angle-deflected laser beams adjacent to each other along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 0.001 μm or greater, such as 0.005 μm or greater, such as 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 5 μm or greater, such as 10 μm or greater, and includes 100 μm or greater overlap.

[0097] kit

[0098] The invention also includes a kit comprising a light-scattering photodetector, static optical adjustment components (e.g., one or more bandpass filters), and variable optical adjustment components (e.g., a neutral density filter or an adjustable beam splitter). The kit may also include other optical adjustment components as described herein, such as shielding components including optical apertures, slits, and shielding disks, as well as scattering stripes. According to some embodiments, the kit also includes optical components for guiding light to the light-scattering photodetector, such as collimating lenses, mirrors, wavelength splitters, pinholes, etc. The kit may also include optical collection components, such as optical fibers (e.g., fiber optic repeater bundles) or components for free-space repeater systems. In some cases, the kit also includes one or more photodetectors, such as photomultiplier tubes (e.g., metal-encapsulated photomultiplier tubes). In some embodiments, the kit includes one or more components of a beam generator, such as a direct digital synthesizer, an acousto-optic deflector, a beam combining lens, and a Powell lens.

[0099] In some cases, the kit may include one or more analytical components (e.g., labeled reagents, buffers, etc. as described above). In some cases, the kit may also include sample collection equipment, such as a gun or needle configured to prick the skin to obtain a whole blood sample, a pipette, etc., as needed.

[0100] The various analytical components of the kit may be present in separate containers, or some or all of them may be pre-assembled. For example, in some cases, one or more components of the kit (such as two or more light scattering detectors) are contained in a sealed pouch, such as a sterile foil pouch or envelope.

[0101] In addition to the components described above, the theme kit may also include (in some embodiments) instructions for implementing the theme method. These instructions may exist in various forms within the theme kit, including one or more forms. One possible form of these instructions is as printed information on a suitable medium or substrate (e.g., one or more sheets of paper containing printed information), present in the kit's packaging, as a insert, etc. Another form of these instructions is as a computer-readable medium, such as a floppy disk, optical disc (CD), portable flash drive, etc., on which information is recorded. Yet another form of these instructions may be as a website address that can be used via the Internet to access information at the removed site.

[0102] utility

[0103] Subject-based optical detection systems can be used when it is necessary to characterize samples by optical properties, particularly when it is necessary to identify and differentiate cells in the sample. In some embodiments, the systems and methods described herein can be used for flow cytometry characterization of biological samples. In some embodiments, the systems and methods can be used for spectral analysis of scattered light. Embodiments of this disclosure can be used where there is a need to enhance the effectiveness of measurements in flow cytometry, such as for use in research and high-throughput laboratory testing. This disclosure is also intended for situations where it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more precise particle charging, and enhanced particle deflection during cell sorting.

[0104] This disclosure can also be used in applications where cells prepared from biological samples may be intended for research, laboratory testing, or therapeutic use. In some embodiments, the subject methods and apparatus facilitate the preparation of single cells from target fluid or tissue biological samples. For example, the subject methods and systems facilitate the acquisition of cells from fluid or tissue samples for use as research or diagnostic samples for diseases such as cancer. Similarly, the subject methods and systems facilitate the acquisition of cells from fluid or tissue samples for therapeutic purposes. Compared to conventional flow cytometry systems, the methods and apparatus of this disclosure allow for the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with greater efficiency and lower cost.

[0105] Notwithstanding the appended claims, this disclosure is also limited by the following terms:

[0106] 1. A light detection module configured to simultaneously measure scattered light in a flowing stream from particles with diameters differing by 100 nm or more.

[0107] 2. The optical detection module according to Clause 1, wherein the optical detection module is configured to simultaneously measure scattered light in a flowing stream from particles with diameters differing by 500 nm or more.

[0108] 3. The optical detection module according to any one of Clauses 1 to 2, wherein the optical detection module is configured to measure one or more of the side-scattered light and forward-scattered light from particles in the flowing stream.

[0109] 4. The light detection module according to any one of clauses 1 to 3, wherein the light detection module is configured to simultaneously measure scattered light from particles in a flowing stream illuminated by a low-power light source.

[0110] 5. The optical detection module according to Clause 4, wherein the light source is a low-power laser.

[0111] 6. The optical detection module according to Clause 5, wherein the light source is a laser having a peak power output of 1mW or less.

[0112] 7. The light detection module according to any one of clauses 1 to 6, wherein the light detection module comprises:

[0113] Static optical adjustment components;

[0114] Variable optical adjustment components; and

[0115] Photodetector.

[0116] 8. The light detection module according to Clause 7, wherein the static optical adjustment component includes a bandpass filter.

[0117] 9. The light detection module according to any one of Clauses 7 to 8, wherein the variable optical adjustment component includes a variable neutral density filter.

[0118] 10. The optical detection module according to any one of Clauses 7 to 8, wherein the variable optical adjustment component includes a power beam splitter.

[0119] 11. The optical detection module according to Clause 10, wherein the optical detection module includes a plurality of power beam splitters and a plurality of optical detectors.

[0120] 12. The light detection module according to any one of clauses 7 to 11, wherein the light detector comprises a photomultiplier tube.

[0121] 13. A system comprising:

[0122] Light source; and

[0123] A light detection module configured to simultaneously measure scattered light in a flowing stream from particles with diameters differing by 100 nm or more.

[0124] 14. The system according to Clause 13, wherein the optical detection module is configured to simultaneously measure scattered light in a flowing stream from particles with diameters differing by 500 nm or more.

[0125] 15. The system according to any one of Clauses 13 to 14, wherein the optical detection module is configured to measure one or more of the side-scattered light and forward-scattered light from particles in the flowing stream.

[0126] 16. The system according to any one of clauses 13 to 15, wherein the light source is a low-power light source.

[0127] 17. The system according to Clause 16, wherein the light source is a low-power laser.

[0128] 18. The system according to Clause 17, wherein the light source is a laser having a peak power output of 1 mW or less.

[0129] 19. The system according to any one of clauses 13 to 18, wherein the light detection module comprises:

[0130] Static optical adjustment components;

[0131] Variable optical adjustment components; and

[0132] Photodetector.

[0133] 20. The system according to Clause 19, wherein the static optical adjustment assembly includes a bandpass filter.

[0134] 21. The system according to any one of the clauses 19 to 20, wherein the variable optical adjustment assembly includes a variable neutral density filter.

[0135] 22. The system according to any one of Clauses 19 to 20, wherein the variable optical adjustment assembly includes a power beam splitter.

[0136] 23. The system according to Clause 22, wherein the optical detection module includes a plurality of power beam splitters and a plurality of optical detectors.

[0137] 24. The system according to any one of clauses 19 to 23, wherein the photodetector comprises a photomultiplier tube.

[0138] 25. The system according to any one of clauses 19 to 24 further includes an optical collection module for propagating the scattered light to the light detection module.

[0139] 26. The system according to Clause 25, wherein the optical collection module includes an optical fiber.

[0140] 27. The system according to Clause 26, wherein the optical collection module includes an optical fiber repeater bundle.

[0141] 28. The system according to Clause 25, wherein the optical collection module comprises a free-space optical relay system.

[0142] 29. A method comprising simultaneously measuring scattered light from particles irradiated in a flowing stream whose diameters differ by 100 nm or more.

[0143] 30. The method according to Clause 29, wherein the method includes simultaneously measuring scattered light from particles irradiated in a flowing stream whose diameters differ by 500 nm or more.

[0144] 31. The method according to any one of clauses 29 to 30, wherein the method comprises measuring one or more of the side-scattered light and the forward-scattered light from particles in the flowing stream.

[0145] 32. The method according to any one of clauses 29 to 31, wherein the method comprises irradiating particles in the flowing stream with a low-power light source.

[0146] 33. The method according to Clause 32, wherein the light source is a low-power laser.

[0147] 34. The method according to Clause 33, wherein the light source is a laser having a peak power output of 1 mW or less.

[0148] 35. The method according to any one of clauses 29 to 34, wherein the method includes detecting light from the flowing stream using a light detection module, the light detection module comprising:

[0149] Static optical adjustment components;

[0150] Variable optical adjustment components; and

[0151] Photodetector.

[0152] 36. The method according to clause 35, wherein the static optical adjustment assembly includes a bandpass filter.

[0153] 37. The method according to any one of clauses 35 to 36, wherein the variable optical adjustment assembly includes a variable neutral density filter.

[0154] 38. The method according to any one of clauses 35 to 36, wherein the variable optical adjustment assembly includes a power beam splitter.

[0155] 39. The method according to Clause 38, wherein the optical detection module includes a plurality of power beam splitters and a plurality of optical detectors.

[0156] 40. The method according to any one of clauses 35 to 39, wherein the photodetector comprises a photomultiplier tube.

[0157] 41. The method according to any one of clauses 35 to 40 further comprises: using an optical collection module to propagate light from the irradiated flow to the light detection module.

[0158] 42. The method according to clause 41, wherein the optical collection module comprises an optical fiber.

[0159] 43. The method according to clause 42, wherein the optical collection module includes an optical fiber repeater bundle.

[0160] 44. The method according to clause 42, wherein the optical collection module includes a free-space optical relay system.

[0161] 45. A kit comprising:

[0162] Static optical adjustment components;

[0163] Variable optical adjustment components; and

[0164] Photodetector.

[0165] 46. ​​The kit according to Clause 45, wherein the static optical adjustment assembly includes a bandpass filter.

[0166] 47. The kit according to any one of clauses 45 to 46, wherein the variable optical adjustment assembly includes a variable neutral density filter.

[0167] 48. The kit according to any one of clauses 45 to 46, wherein the variable optical adjustment assembly includes a power beam splitter.

[0168] 49. The kit described in any of Clauses 45 to 48 also includes a light source.

[0169] 50. The kit as described in Clause 49, wherein the light source is a laser.

[0170] 51. The kit according to Clause 50, wherein the laser is a continuous wave laser.

[0171] 52. The kit described in any of Clauses 50 to 51 also includes an acousto-optic deflector.

[0172] 53. The kit described in Clause 52 also includes a direct digital synthesizer (DDS).

[0173] Although the invention has been described in some detail by way of illustrations and examples for clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0174] Therefore, the above description only illustrates the principles of the invention. It should be understood that those skilled in the art will be able to design various arrangements, although not explicitly described or shown herein, which embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language described herein are primarily intended to assist the reader in understanding the principles of the invention and are not intended to limit the scope of these specifically described examples and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to include their structural and functional equivalents. Furthermore, such equivalents are contemplated to include both currently known equivalents and those developed in the future, i.e., any element developed that performs the same function, regardless of its structure. Therefore, the scope of the invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims.

Claims

1. A photodetector module configured to simultaneously measure scattered light from a flow stream of particles with diameters ranging from 1 nm to 5000 nm and diameters differing by 100 nm or more, the module comprising: Photodetector; as well as A variable optical adjustment component that alters or adjusts the amount of light propagating to the photodetector, wherein the variable optical adjustment component includes a variable neutral density filter or a power beam splitter.

2. The light detection module of claim 1, wherein, The optical detection module is configured to simultaneously measure scattered light from a flow stream of particles with diameters differing by 500 nm or more.

3. The light detection module of claim 1, wherein, The optical detection module is configured to measure one or more of the side-scattered and forward-scattered light from particles in the flow.

4. The optical detection module according to claim 1, wherein, The optical detection module is configured to simultaneously measure scattered light from particles in the flow that are illuminated by a low-power light source.

5. The light detection module of claim 4, wherein, The light source is a low-power laser.

6. The light detection module of claim 5, wherein, The light source is a laser with a peak power output of 1 mW or less.

7. The light detection module according to any one of claims 1 to 6, wherein, The optical detection module includes a static optical adjustment component.

8. The light detection module of claim 7, wherein, The static optical adjustment assembly includes a bandpass filter.

9. The optical detection module according to claim 1, wherein, The optical detection module includes multiple power beam splitters and multiple optical detectors.

10. The light detection module of any one of claims 1 to 6, wherein, The photodetector includes a photomultiplier tube.

11. A system comprising: light source; as well as The light detection module according to any one of claims 1 to 10.

12. A method comprising simultaneously measuring, using a light detection module according to any one of claims 1 to 10, the scattered light of particles irradiated in a flowing stream with diameters ranging from 1 nm to 5000 nm and diameters differing by 100 nm or more.

13. A kit comprising a light detection module according to any one of claims 1 to 10.

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