Dynamic light detection suppression system and method of using same

Abstract

Systems are described for separately detecting light from a sample in a flow stream (e.g., in a flow cytometer) across one or more dimensions. According to embodiments, the light detection system includes a flow cell configured to flow the sample in the flow stream, a light source configured to illuminate the sample in the flow cell, and a detector system including an optical conditioning component and a detector configured to separately detect light from the flow cell without a scatterbar. According to certain embodiments, the system is configured to separately detect light by adjusting one or more components of the optical conditioning component or the detector. Also described are methods for separately detecting light from a sample in a flow stream by a detector unit without a scatterbar. Kits having two or more components for use in the system are also provided.

Description

[Background technology]

[0001] Optical detection is often used to characterize components of a sample (e.g., a biological sample) that are used, for example, to diagnose a disease or health condition. An illuminated sample scatters, transmits, and even emits (e.g., by fluorescence) light. Changes in the morphology, absorbance, changes, presence or absence of fluorescent labels, etc. of sample components can result in changes in the light scattered, transmitted, or emitted by the sample. These changes can be used to characterize and identify the presence or absence of components in the sample. To quantify these changes, the light is collected and sent to the surface of a detector. The amount of light that reaches the detector can affect the overall quality of the optical signal output by the detector. The amount of light that reaches the detector can be increased by increasing the surface area of ​​the detector or by collecting more light from the sample.

[0002] Flow cytometry is one technique that utilizes light detection to characterize components in a sample. Using data generated from the detected light, the distribution of components can be recorded and, if necessary, the material can be sorted. Typically, a flow cytometer includes a sample reservoir that receives a fluid sample, such as a blood sample, and a sheath reservoir that contains a sheath fluid. The flow cytometer conveys particles (including cells) in the fluid sample as a cell stream to a flow cell, while also directing sheath fluid to the flow cell. A liquid sheath is formed around the cell stream in the flow cell so that the cells travel at a substantially constant velocity on the cell stream. The flow cell hydrodynamically focuses the cells in the stream and passes the center of a light source in the flow cell. Light from the light source can be detected as scattered light, or by transmission spectroscopy, or can be absorbed by one or more components in the sample and re-emitted as luminescence. Changes in the material, such as morphology or fluorescent labeling, produce changes in the light observed, which can then be collected and characterized by focusing the light on a light detector. Summary of the Invention

[0003] A system is described for separately detecting light from a sample in a flow stream (e.g., in a flow cytometer) across one or more dimensions. According to embodiments, the light detection system comprises a flow cell configured to flow a sample in the flow stream, a light source configured to illuminate the sample in the flow cell, and a detector system including an optical conditioning component and a detector configured to separately detect light from the flow cell without a scatter bar. According to certain embodiments, the system is configured to separately detect light by adjusting one or more components of the optical conditioning component or the detector. A method of separately detecting light from a sample in a flow stream by the detector unit without a scatter bar is also described. Kits having two or more components for use in the system are also provided.

[0004] Aspects of the present disclosure include light detection systems that separately detect light from an illuminated flow cell without a scatter bar. In certain embodiments, the systems are configured to dynamically block light (e.g., scattered light, fluorescent light, transmitted light, etc.) emanating from a sample. The subject systems include a flow cell configured to flow a sample in a flow stream, a light source configured to illuminate the sample in the flow cell, and a detector unit configured to separately detect light from the flow cell. In some embodiments, the detector unit includes a detector that includes a detector array. In these embodiments, the system is configured to separately detect the light by adjusting the detector. In other embodiments, the detector unit includes an optical adjustment component and a detector. In these embodiments, the detector unit may be configured to separately detect the light by adjusting the optical adjustment component or one or more components of the detector.

[0005] In some embodiments, the detector unit includes a fused fiber optic component (e.g., a tapered fused fiber optic bundle) and a detector (e.g., an array of photodiodes). The optical conditioning component is in physical contact with the detector in certain embodiments. In some cases, the detector unit includes a light-conducting material, such as a gel (e.g., an index-matching gel), between the optical conditioning component and the detector. In some embodiments, the detector unit is configured to separately detect light from the flow cell by disabling a subset of the photodiodes in the array. In some cases, the disabling is reversible. In other cases, the disabling is irreversible. The subsets of disabled photodiodes may form a predetermined pattern, such as one or more rows, a polygonal pattern, a symmetrical pattern, or an asymmetrical pattern.

[0006] In other embodiments, the detector unit includes a photonic micro-electromechanical system (MOEMS), such as a digital micro-mirror device, and a detector. In some cases, the digital micro-mirror device is configured to adjust a subset of mirrors. In one example, the detector unit is configured to tilt a subset of mirrors on the digital micro-mirror device to separately detect light from the flow cell. In another example, the detector unit is configured to disable a subset of mirrors on the digital micro-mirror device to separately detect light from the flow cell. For example, a subset of mirrors on the digital micro-mirror device may be electrically disabled. The adjusted subset of mirrors on the digital micro-mirror device may form a predetermined shape or pattern on the digital micro-mirror surface, such as one or more rows, a polygonal pattern, a symmetrical pattern, or an asymmetrical pattern.

[0007] Aspects of the present disclosure further include a method of separately detecting light from a sample. The method according to certain embodiments includes illuminating a flow cell containing a sample in a flow stream with a light source and detecting light from the flow cell with a detector unit configured to separately detect light from the flow stream without a scatter bar. In some embodiments, the detector unit includes a detector. In these embodiments, the method includes separately detecting the light by adjusting the detector. In other embodiments, the detector unit includes an optical adjustment component and a detector, and the method further includes adjusting one or more components of the optical adjustment component or the detector. In some cases, the detector unit includes a fused fiber optic component (e.g., a tapered fused fiber optic bundle) and an array of photodiodes, and the method includes disabling a subset of the photodiodes in the array to separately detect light from the sample. For example, the subset of the photodiodes in the array may be disabled in a predetermined pattern, such as one or more rows, a polygonal pattern, a symmetrical pattern, or an asymmetrical pattern. In another example, the detector unit includes a digital micromirror device and a detector, and the method includes adjusting a subset of mirrors on the digital micromirror surface to separately detect light from the sample. For example, the subset of mirrors may be disabled, such as tilted, rotated, or electrically disabled. On the digital micromirror surface, the subset of mirrors may be in the form of a predetermined shape or pattern, such as in the form of one or more rows, a polygonal pattern, a symmetrical pattern, or an asymmetrical pattern.

[0008] Kits containing one or more components of the optical detection system are also provided. In certain embodiments, the kit comprises a flow cell and one or more components of the detector unit described above, such as optical conditioning components (e.g., mirrors, lenses, collimators, pinholes, slits, optical fibers), detectors (e.g., photodiodes, photomultipliers, or arrays of photodiodes or photomultipliers).

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

[0010] [Figure 1] 1 illustrates a digital micromirror device according to a specific embodiment. [Diagram 2] 1 illustrates a light collection system configured to separately detect light from a sample in a flow cell using a digital micromirror device, according to certain embodiments. [Diagram 3] 1 illustrates a light detection system configured to separately detect light from a sample in a flow cell with a tuned light detector array, according to certain embodiments. [Figure 4] FIG. 2 illustrates a front view of a photodetector array having photodetectors enabled and disabled in a predetermined pattern, in accordance with a specific embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] A system is described for separately detecting light from a sample in a flow stream (e.g., in a flow cytometer) across one or more dimensions. According to embodiments, the light detection system comprises a flow cell configured to flow a sample in the flow stream, a light source configured to illuminate the sample in the flow cell, and a detector system including an optical conditioning component and a detector configured to separately detect light from the flow cell without a scatter bar. According to certain embodiments, the system is configured to separately detect light by adjusting one or more components of the optical conditioning component or the detector. A method of separately detecting light from a sample in a flow stream by the detector unit without a scatter bar is also described. Kits having two or more components for use in the system are also provided.

[0012] Before describing the invention in detail, it is to be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0013] Where a range of values ​​is given, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, unless the context clearly indicates otherwise, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of such smaller ranges may be individually included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0014] In this specification, a certain range is presented with "about" before the numerical value. In this specification, the term "about" is a statement that supports the numerical value that follows it, and the numerical value that is close to or close to the numerical value that follows it. The determination of whether a numerical value is close to or close to a specifically stated numerical value depends on whether the close or close numerical value that is not stated can have a substantially equivalent effect to the specifically stated numerical value in the context in which it is stated.

[0015] 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary exemplary methods and materials are described below.

[0016] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date of the present application and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0017] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude optional elements. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or for the use of a "negative" limitation.

[0018] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any method described can be carried out in the order of events described or in any other order which is logically possible.

[0019] Although the apparatus and methods are described above and below in a grammatically fluid manner with functional descriptions, the claims are not to be construed as being limited in any way by construction of "means" or "step" limitations, except as expressly set forth in 35 U.S.C. 112, but are expressly understood to conform to the full scope of meaning and equivalents of the definitions provided by the claims under the common law doctrine of equivalents, and where a claim is expressly set forth in 35 U.S.C. 112, it is to be expressly understood to conform to the full statutory equivalents under 35 U.S.C. 112.

[0020] As summarized above, the present disclosure provides an optical detection system for separately detecting light from a sample in a flow cell. To further describe the embodiments of the present disclosure, an optical detection system according to an embodiment of the present invention will first be described in more detail. Then, a method for separately detecting light from a sample in a flow cell will be described. A kit is also provided having a flow cell, an optical adjustment component, and a detector unit for separately detecting light from a sample.

[0021] [Detection system for separately detecting light] Aspects of the present disclosure include an optical detection system configured to separately detect light from a sample in a flow cell. In some embodiments, the system is configured to dynamically distinguish light emitted by a sample flowing through the flow cell. For example, the optical detection system may be configured to dynamically distinguish light from a sample in the flow cell based on intensity. In other cases, the optical detection system is configured to distinguish light emitted by different particle populations in the sample, such as to distinguish different cell populations in the sample.

[0022] In yet another embodiment, the light detection system is configured to dynamically block the incident light from the illumination light source. This is accomplished by reducing the amount of incident light from the illumination light source that is detected by 50% or more, such as 60% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, including reducing the amount of incident light from the illumination light source that is detected by 99.9% or more. Reducing the amount of incident light from the illumination light source that is detected increases the intensity of the detector signals from the fluorescence and scattered light from the sample compared to the detector signals from the fluorescence and scattered light collected by a light collection system that is not configured to detect light separately, as described herein. In some embodiments, the detector signal intensity from the fluorescence and scattered light measured by the light detection system described herein increases by 10% or more, such as 25% or more, 50% or more, 75% or more, 90% or more, including even an increase of 95% or more. That is, an increase of 1.5 times or more, such as 2 times or more, 3 times or more, 5 times or more, etc., and even including an increase of 10 times or more.

[0023] In embodiments, the optical detection system comprises a flow cell configured to flow a sample in a flow stream, a light source configured to illuminate the sample in the flow cell, and a detector unit configured to separately detect light from the flow cell without a scatter bar. In some embodiments, the detector unit comprises an optical conditioning component and a detector. In these embodiments, the optical detection system is configured to adjust one or more components of the optical conditioning component or the detector to separately detect light from the sample. As used herein, the term "optical conditioning" is used in its conventional sense to refer to a component that optically changes light from the sample in the flow cell. For example, the optical conditioning component may change the optical path, direction, collection, or collimation of light from the sample. The optical conditioning component may include, but is not limited to, lenses (e.g., collection, objective, magnification, etc.), collimators, mirrors (e.g., dichroic mirrors), slits, pinholes, filters (e.g., bandpass, interference), diffraction gratings, monochromators, and other types of optical conditioning components. In some embodiments, the optical conditioning component in the detector unit is an optical relay system, such as a fiber optic optical relay system. Any fiber optic optical relay system may be utilized for the propagation of light from the sample to the detector in the detector unit. In certain embodiments, fiber optic optical relay systems suitable for the propagation of light from the sample to the detector include, but are not limited to, fiber optic optical relay systems such as those described in U.S. Pat. No. 6,809,804, the disclosure of which is incorporated herein by reference. In certain embodiments, the optical conditioning component is a fused fiber optic component, such as a tapered fused fiber optic component. In some embodiments, the fiber optic optical component is in physical contact with the detector. In another embodiment, the fused fiber optic component is coupled to the detector by disposing a light conducting material between the fiber optic bundle and the detector surface. Any suitable light conducting material, such as a light conducting gel, may be used. For example, the light conducting material may be an index-matched light conducting gel.

[0024] In a particular embodiment, the optical adjustment component is a micro-optical machine (MOEMS) such as a digital micro-mirror device (DMD). The term digital micro-mirror device as used herein is used in its conventional sense to refer to an optical micro-machine having a substrate (e.g., a planar substrate) on which a number of individually actuatable micro-mirrors are provided on the digital micro-mirror device active surface. By "individually actuatable," it is meant that each micro-mirror can be individually adjusted such that light reflected by the digital micro-mirror device is reflected separately from the active surface. Adjustment of each micro-mirror includes one or more of rotating, tilting, and disabling (e.g., electrically disabling) the mirror such that the reflectivity of each micro-mirror is adjusted as desired. In embodiments, when the micromirror is adjusted by rotating, tilting, or disabling, the desired reflectivity of the micromirror can be reduced by 1% or more, e.g., 2% or more, 3% or more, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, 97% or more, 99% or more, etc., including a 100% reduction in the reflectivity of the micromirror.

[0025] As described in more detail below, in some embodiments, a subset of the mirrors may be adjusted to increase or decrease the amount of light propagating from the sample to the detector. In another embodiment, a subset of the mirrors may be adjusted to block or occlude light (e.g., incident light from an illumination source or light from unwanted sample populations) propagating to the detector. In yet another embodiment, a subset of the mirrors may be adjusted to distinguish light from different particle populations within the sample, such as light emanating from different types of cells within the sample.

[0026] 1 illustrates a digital micromirror device according to one specific embodiment. Digital micromirror device 100 comprises an active surface 101 having a plurality of micromirrors arranged in an array 102. Each micromirror 103 is individually actuatable. This includes tilting, rotating, or disabling the micromirrors to reduce or eliminate light reflection from the adjusted micromirrors.

[0027] The active surface of the subject digital micromirror device may be of any suitable shape, such as a rectilinear shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curvilinear shape (e.g., circular, elliptical), or an irregular shape (e.g., a parabolic bottom joined to a planar top). In certain embodiments, the digital micromirror device is planar and has a rectangular active surface. The subject digital micromirror device may have an active surface with a variable length in the range of 5 mm to 100 mm, e.g., 10 mm to 90 mm, 15 mm to 85 mm, 20 mm to 80 mm, 25 mm to 75 mm, 30 mm to 70 mm, and even including 35 mm to 65 mm. The width of the active surface may also be variable in the range of 5 mm to 100 mm, e.g., 10 mm to 90 mm, 15 mm to 85 mm, 20 mm to 80 mm, 25 mm to 75 mm, 30 mm to 70 mm, and even including 35 mm to 65 mm. In an embodiment, depending on the shape of the digital micromirror device, the active surface may be 5 mm 2 From 1000mm 2 Surface area of, for example, 10 mm 2 From 900mm 2 , 15mm 2 From 800mm 2 , 20mm 2 From 700mm 2 , 25mm 2 From 600mm 2 , 30mm 2 From 500mm 2 , 35mm 2 From 400mm 2 and further having a surface area of ​​40 mm 2 From 300mm2 Also includes.

[0028] Depending on the type of sample flowing through the flow cell and the size of the device, the number of individually actuable mirrors on the active surface of the digital micromirror device can also vary, and can be 50 or more micromirrors, 100 or more micromirrors, 250 or more micromirrors, 500 or more micromirrors, 750 or more micromirrors, 1000 or more micromirrors, 2500 or more micromirrors, 5000 or more micromirrors, 7500 or more micromirrors, 10,000 or more micromirrors, and even including 25,000 or more micromirrors. Each micromirror can have variable dimensions. The lengths of the target micromirrors may be between 1 μm and 25 μm, e.g., between 2 μm and 24 μm, between 3 μm and 23 μm, between 4 μm and 22 μm, between 5 μm and 20 μm, between 6 μm and 19 μm, between 7 μm and 18 μm, between 8 μm and 17 μm, between 9 μm and 16 μm, and even including between 10 μm and 15 μm. The widths of each micromirror may be in the range of between 1 μm and 25 μm, e.g., between 2 μm and 24 μm, between 3 μm and 23 μm, between 4 μm and 22 μm, between 5 μm and 20 μm, between 6 μm and 19 μm, between 7 μm and 18 μm, between 8 μm and 17 μm, between 9 μm and 16 μm, and even including between 10 μm and 15 μm.

[0029] In some embodiments, the light detection system is configured to separately detect light by adjusting a subset of micromirrors on the digital micromirror device. In some cases, the adjustment includes tilting a subset of micromirrors to reduce a desired reflectance by the subset of mirrors. Each micromirror may be tilted 5° to 15°, e.g., 6° to 14°, 7° to 13°, or even including tilting 8° to 12° relative to a surface of the digital micromirror device. In other cases, the adjustment includes rotating a subset of micromirrors to reduce a desired reflectance by the subset of mirrors. Each micromirror may be rotated 5° to 15°, e.g., 6° to 14°, 7° to 13°, or even including rotating 8° to 12°. In yet another embodiment, the light detection system is configured to separately detect light by disabling (e.g., electrically disabling) a subset of micromirrors on the digital micromirror device. Any suitable protocol may be used to disable the subset of micromirrors, such as applying a current to the subset of micromirrors sufficient to reduce or eliminate the reflectivity of the micromirrors, for example, the current applied may be sufficient to reduce the reflectivity of the subset of micromirrors by 50% or more, e.g., 75% or more, 90% or more, 95% or more, 97% or more, 99% or more, including reducing the reflectivity of the subset of micromirrors by 100%.

[0030] The number of micromirrors adjusted to separately detect light from the sample in the flow cell varies depending on the type of light to be separately detected or blocked. In some embodiments, the adjusted subset of micromirrors on the active surface of the digital micromirror device may include 5% or more of the micromirrors on the active surface of the digital micromirror device, such as 10% or more, 25% or more, 50% or more, 75% or more, and may further include 90% or more of the micromirrors on the active surface of the digital micromirror device being adjusted. The adjusted subset of micromirrors may take the form of one or more predefined patterns on the active surface of the digital micromirror device, such as a pattern made up of straight lines (e.g., squares, rectangles, trapezoids, triangles, hexagons, etc.), curved patterns (e.g., circles, ellipses), or asymmetrical, irregular patterns (e.g., a parabolic bottom joined to a planar top). In another embodiment, the adjusted subset of micromirrors to separately detect light from the sample is arranged along one or more rows across the surface of the digital micromirror device.

[0031] In some embodiments, the subject light collection system comprises a subset of micromirrors on the active surface of the digital micromirror device that are adjusted to distinguish light from different particles in the sample, such as light emanating from different cells in the sample. In these embodiments, the subset of micromirrors may be tilted, rotated, or disabled to block light from unwanted components of the sample, such as light scattered from unwanted cell populations, cellular debris, impurities, or non-cellular components in the sample. For example, if it is desired to specifically characterize two different cell populations in a sample, only the micromirrors of the digital micromirror device that correspond to light from these cell populations may be enabled, and the other micromirrors of the digital micromirror device may be disabled to block light from other unwanted cell populations in the sample. In this manner, the detector unit in the subject light collection system may be configured to specifically characterize one or more different cell populations in a sample having multiple different cell populations (e.g., two or more different cell populations, three or more, four or more, five or more, six or more, seven or more, even ten or more different cell populations in a sample having multiple different cell populations).

[0032] In another embodiment, the light collection system of interest comprises a subset of micromirrors on the active surface of the digital micromirror device that are adjusted to block unwanted light from propagating to the detector surface. For example, the subset of micromirrors may be adjusted to block incident light from an illumination source from propagating to the detector surface. In these embodiments, the adjusted subset of micromirrors may be in the form of one or more rows across the digital micromirror surface. This configuration corresponds to scattered or otherwise unblocked incident light from the illumination source.

[0033] The subset of micromirrors on the active surface of the digital micromirror that is adjusted at any particular time may include any suitable number of micromirrors, such as 10 or more micromirrors, 25 or more micromirrors, 50 or more micromirrors, 100 or more micromirrors, 250 or more micromirrors, 500 or more micromirrors, 1000 or more micromirrors, 2500 or more micromirrors, and even 5000 or more micromirrors. Thus, the subset of micromirrors that is adjusted to separately detect light from a sample in a flow cell may be 5% to 75%, such as 10% to 70%, 15% to 65%, 20% to 60%, or even 25% to 50% of the total number of micromirrors on the active surface of the digital micromirror device. Depending on the overall size of the active surface of the digital micromirror device, the subset of micromirrors that are adjusted to separately detect light from a sample in the flow cell may have a length in the range of 0.01 mm to 25 mm, e.g., 0.05 mm to 20 mm, 0.1 mm to 15 mm, 0.5 mm to 10 mm, 1 mm to 9 mm, and even including 2 mm to 8 mm. The width of the subset of micromirrors that are adjusted to separately detect light from a sample may also be in the range of 0.01 mm to 25 mm, e.g., 0.05 mm to 20 mm, 0.1 mm to 15 mm, 0.5 mm to 10 mm, 1 mm to 9 mm, and even including 2 mm to 8 mm. In an embodiment, depending on the pattern of the subset of micromirrors that are adjusted to separately detect light from a sample, the subset of micromirrors that are adjusted may be less than 0.1 mm of the surface area of ​​the digital micromirror device. 2 From 500mm 2 , e.g., 0.5 mm 2 From 450mm 2 , 1mm 2 From 400mm 2 , 2mm 2 From 350mm 2 , 3mm 2 From 300mm 2 , 4mm2 From 250mm 2 , 5mm 2 From 200mm 2 and may occupy 10 mm 2 From 100mm 2 Also includes.

[0034] In certain embodiments, a subset of micromirrors on the active surface of the digital micromirror device are dynamically adjusted such that one or more of the plurality of micromirrors are tilted, rotated, or disabled for a desired period of time, such as 0.001 μs or more, 0.01 μs or more, 0.1 μs or more, 0.5 μs or more, 1 μs or more, 5 μs or more, 10 μs or more, 25 μs or more, 50 μs or more, 100 μs or more, 500 μs or more, and even including 1000 μs or more. In certain embodiments, a subset of micromirrors on the active surface of the digital micromirror device are dynamically adjusted for an extended period of time, such as 1 second or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 30 seconds or more, 60 seconds or more, 120 seconds or more, 240 seconds or more, 360 seconds or more, 480 seconds or more, and even including 600 seconds or more. In one particular case, a subset of the micromirrors is dynamically adjusted throughout the period during which the sample in the flow cell is illuminated by the light source.

[0035] 2 illustrates a light collection system configured to separately detect light from a sample in a flow cell by adjusting a subset of micromirrors on the active surface of a digital micromirror device, according to a specific embodiment. A light source 201 illuminates a digital micromirror device 200, which reflects the light to a sample in a flow cell 202. Light from the sample in the flow cell 202 (e.g., forward scattered light) propagates back to the digital micromirror device and is reflected to a detector 203. By adjusting the micromirrors illuminated by the light source 201 to only reflect light from the digital micromirror device 200 to the flow cell 202 and not back-reflect it to the detector 203, incident light (e.g., laser scatter) can be blocked from being collected and detected by the detector 203.

[0036] The light collection system configured to separately detect light from the sample in the flow cell according to the embodiment further comprises a detector. The detector of interest may include optical sensors or photodetectors such as, but not limited to, active pixel sensors (APS), avalanche photodiodes, imagers, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultipliers, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other photodetectors. In certain embodiments, the transmitted light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) imager, or an N-type metal-oxide-semiconductor (NMOS) imager.

[0037] In some embodiments, the detector that is to separately detect light from the sample in the flow cell includes multiple detectors. In some cases, the detector unit includes a semiconductor detector, such as a photodiode. In certain cases, the detector unit includes a photodetector array, such as a photodiode array. In these embodiments, the photodetector array may include photodetectors of 4 or more, such as 10 or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, 750 or more, and even 1000 or more. For example, the detector may be a photodiode array having 4 or more photodiodes, such as 10 or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, 750 or more, and even 1000 or more.

[0038] The photodetectors may be arranged in any geometric configuration as desired. Target configurations include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular patterns. The photodetectors in the photodetector array may be oriented at angles between each other (in the XZ plane) ranging from 10° to 180°, such as 15° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and even 45° to 90°. The photodetector array may be of any suitable shape, such as rectilinear (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), curvilinear (e.g., circular, elliptical), or irregular (e.g., a parabolic bottom joined to a planar top). In one particular embodiment, the photodetector array has a rectangular active surface.

[0039] Each photodetector (e.g., photodiode) in the array may have an active surface width in the range of 5 μm to 250 μm, e.g., 10 μm to 225 μm, 15 μm to 200 μm, 20 μm to 175 μm, 25 μm to 150 μm, 30 μm to 125 μm, 50 μm to 100 μm, and a length in the range of 5 μm to 250 μm, e.g., 10 μm to 225 μm, 15 μm to 200 μm, 20 μm to 175 μm, 25 μm to 150 μm, 30 μm to 125 μm, 50 μm to 100 μm, where the surface area of ​​each photodetector (e.g., photodiode) in the array is less than 25 μm. 2 From 10000μm 2 , e.g., 50 μm 2 From 9000μm 2 , 75μm 2 to 8000μm 2 , 100μm 2 From 7000μm 2 , 150μm 2 to 6000μm 2 etc., and further, 200 μm 2 From 5000μm 2 Also includes.

[0040] The size of the photodetector array can vary depending on the amount of light transmitted from the sample in the flow cell, the number of photodetectors, and the desired sensitivity, and can range from 0.01 mm to 100 mm in length, e.g., 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, etc., including 5 mm to 25 mm. The width of the photodetector array can also vary, and can range from 0.01 mm to 100 mm, e.g., 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, etc., including 5 mm to 25 mm. Thus, the active surface of the photodetector array can be as small as 0.1 mm. 2 From 10000mm 2 , e.g., 0.5 mm 2 From 5000mm 2 , 1mm 2 From 1000mm 2 , 5mm 2 From 500mm 2 etc., and further, 2 From 100mm 2 Also includes.

[0041] In some embodiments, each photodetector in the photodetector array may be individually adjusted such that light from the sample is detected separately by the detectors in the array. In some cases, a subset of the photodetectors may be adjusted, such as a group of photodetectors in a particular region of the array or a subset of the photodetectors that form a predetermined pattern or shape, such as a row across the photodetector array.

[0042] For example, each photodetector in the array may be disabled (reversibly or irreversibly) as desired depending on the sample being measured such that different regions of the photodetector array are configured to detect light differently. In some embodiments, one or more photodetectors in the array may be partially or completely disabled such that the photodetectors in a particular region of the array are configured to detect a reduced amount of light, i.e., the photodetector detects 95% or less of the light impinging on the surface of that photodetector in the particular region, e.g., 90% or less, 85% or less, 75% or less, 50% or less, 25% or less, 10% or less, 5% or less, 3% or less, 1% or less, etc., including even 0.1% or less of the light impinging on the surface of the photodetector in the particular region. In certain cases, a subset of the photodetectors in the photodetector array is disabled such that no light is detected by the photodetectors in the subset.

[0043] The number of photodetectors adjusted to separately detect light from the sample in the flow cell varies depending on the type of light to be separately detected or blocked. In some embodiments, the adjusted subset of photodetectors in the photodetector array may be 5% or more of the photodetectors in the photodetector array, such as 10% or more, 25% or more, 50% or more, 75% or more, including 90% or more of the photodetectors in the photodetector array. The subset of photodetectors may take the form of one or more predefined patterns in the photodetector array, such as a pattern made up of lines (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved pattern (e.g., circle, ellipse), or an asymmetrical, irregular pattern (e.g., a parabolic bottom joined to a planar top). In another embodiment, the subset of photodetectors adjusted to separately detect light from the sample is arranged along one or more rows across the photodetector array.

[0044] In some embodiments, the subject light collection system includes a subset of photodetectors in the photodetector array that is adjusted to distinguish light from different particles in the sample, such as light emanating from different cells in the sample. In these embodiments, the subset of photodetectors may be partially or completely disabled to block light from unwanted components of the sample, such as light scattered from unwanted cell populations, cellular debris, impurities, or non-cellular components in the sample. For example, if it is desired to specifically characterize two different cell populations in a sample, only the photodetectors in the photodetector array that correspond to light from these cell populations may be enabled, and the other photodetectors in the photodetector array may be disabled to block light emanating from other unwanted cell populations in the sample. In this manner, the detector units in the subject light collection systems may be configured to specifically characterize one or more different cell populations, e.g., two or more different cell populations, three or more, four or more, five or more, six or more, seven or more, etc., in a sample having multiple different cell populations, including even ten or more different cell populations in a sample having multiple different cell populations.

[0045] In another embodiment, the light collection system of interest includes a subset of photodetectors in the photodetector array that are partially or fully disabled to block unwanted light from being detected by the photodetector array. For example, the subset of photodetectors may be partially or fully disabled to block incident light from an illumination source from being detected by the detectors. In these embodiments, the subset of photodetectors that are disabled may be in the form of one or more rows across the photodetector array that correspond to scattered light or other forms of unblocked incident light from the illumination source.

[0046] The subset of photodetectors in the photodetector array that are partially or completely disabled at any particular time may include any suitable number of photodetectors, such as 10 or more photodetectors, 25 or more photodetectors, 50 or more photodetectors, 100 or more photodetectors, 250 or more photodetectors, 500 or more photodetectors, 1000 or more photodetectors, 2500 or more photodetectors, or even 5000 or more photodetectors. Thus, the subset of photodetectors that are partially or completely disabled to separately detect light from the sample in the flow cell may be 5% to 75%, such as 10% to 70%, 15% to 65%, 20% to 60%, etc., of the total number of photodetectors in the photodetector array, including even 25% to 50% of the total number of photodetectors in the photodetector array. Depending on the overall size of the photodetector array, the subset of photodetectors that are partially or completely disabled to separately detect light from the sample in the flow cell may have a length in the range of 0.01 mm to 25 mm, e.g., 0.05 mm to 20 mm, 0.1 mm to 15 mm, 0.5 mm to 10 mm, 1 mm to 9 mm, etc., including 2 mm to 8 mm. The width of the subset of photodetectors that are partially or completely disabled to separately detect light from the sample may also be in the range of 0.01 mm to 25 mm, e.g., 0.05 mm to 20 mm, 0.1 mm to 15 mm, 0.5 mm to 10 mm, 1 mm to 9 mm, etc., including 2 mm to 8 mm. In an embodiment, depending on the pattern of the subset of photodetectors that are partially or completely disabled to separately detect light from the sample, the subset of photodetectors that are adjusted to have a width of less than 0.1 mm of surface area. 2 From 500mm 2 , e.g., 0.5 mm 2 From 450mm 2 , 1mm 2 From 400mm 2 , 2mm 2 From 350mm 2 , 3mm 2 From 300mm 2 , 4mm 2 From 250mm 2 , 5mm 2 From 200mm 2etc., and further, 10 mm 2 From 100mm 2 Also includes.

[0047] In certain embodiments, a subset of photodetectors in a photodetector array may be dynamically disabled such that one or more of the photodetectors in the array are partially or fully disabled for a desired period of time (e.g., between laser pulses), such as, for example, 0.001 μs or more, e.g., 0.01 μs or more, 0.1 μs or more, 0.5 μs or more, 1 μs or more, 5 μs or more, 10 μs or more, 25 μs or more, 50 μs or more, 100 μs or more, 500 μs or more, or even 1000 μs or more, etc. In certain embodiments, a subset of photodetectors in a photodetector array may be dynamically disabled for an extended period of time, such as, for example, 1 second or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 30 seconds or more, 60 seconds or more, 120 seconds or more, 240 seconds or more, 360 seconds or more, 480 seconds or more, or even 600 seconds or more. In certain cases, a subset of the photodetectors in the photodetector array are dynamically disabled throughout the period during which the sample in the flow cell is illuminated by the light source.

[0048] 3 illustrates a light detection system in which light from a sample in a flow cell is separately detected by a modulated light detector array, according to a specific embodiment. A sample flowing through a flow cell 302 is illuminated with light from a laser 301. Light emanating from the sample is collected by a tapered fused optical fiber component 303 and propagates to a light detector array 304 having a number of light detectors 304a. The light detector array 304 (front view) includes a subset 304a1 of light detectors that are disabled to reduce the amount of light from the laser 301 that is detected by the light detector array 304. Light detector 304a2 is fully enabled and configured to detect light from sample components from the flow cell 302.

[0049] 4 is a front view of a photodetector array 400 having enabled and disabled photodetectors in a predetermined pattern, according to a specific embodiment. By disabling the photodetectors in a specific pattern, unwanted light (e.g., from an incident illumination source or from unwanted cell populations in a sample) can be blocked. The photodetector array 400 comprises enabled photodetectors 400a and disabled photodetectors 400b arranged in a predetermined distinct pattern corresponding to the light that it is desired to block.

[0050] The subject photodetectors are configured to measure collected light at one or more wavelengths, e.g., 2 or more different wavelengths, 5 or more different wavelengths, 10 or more different wavelengths, 25 or more different wavelengths, 50 or more different wavelengths, 100 or more different wavelengths, 200 or more different wavelengths, 300 or more different wavelengths, etc., including measuring light emitted from samples in the flow stream at 400 or more different wavelengths.

[0051] In some embodiments, the photodetector is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the photodetector of interest is configured to collect a spectrum of light over a range of wavelengths. For example, the system may include one or more detectors configured to collect a spectrum of light over one or more of the wavelength ranges of 200 nm to 1000 nm. In still other embodiments, the detector of interest is configured to measure light from the sample in the flow stream at one or more specific wavelengths. For example, the system may include one or more detectors configured to measure light at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, the photodetector may be configured to be paired with a particular fluorophore, such as one used with a sample for fluorescence analysis.

[0052] In embodiments, the light detection system is configured to measure light continuously or at discrete intervals. In some cases, the target light detector is configured to make continuously collected light measurements. In other examples, the light detection system is configured to measure light 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, etc., including every 1000 milliseconds or some other interval.

[0053] The system of interest for measuring light from a sample includes an illumination light source. In embodiments, the light source may be any suitable broadband or narrowband light source. Depending on the components (e.g., cells, beads, non-cellular particles, etc.) in the sample, the light source may be configured to emit variable wavelength light ranging from 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, etc., including 400 nm to 800 nm. For example, the light source may include a broadband light source that emits light with wavelengths ranging from 200 nm to 900 nm. In another case, 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 may be a narrowband LED (1 nm to 25 nm) that emits light with wavelengths ranging from 200 nm to 900 nm. In some embodiments, the light source is a laser, such as a continuous wave laser. For example, the laser may be a Helium Neon (HeNe) laser. In certain embodiments, the light source is a laser in a flow cytometer.

[0054] In another embodiment, the light source is a non-laser light source, such as, but not limited to, a lamp, such as a halogen lamp, a deuterium arc lamp, a xenon arc lamp, an LED, such as a broadband continuous spectrum LED, an ultra-bright light emitting diode, a semiconductor light emitting diode, a broad spectrum LED white light source, a multi-LED integrated light source, etc. In some cases, the non-laser light source may be a stabilized fiber coupled broadband light source, a white light source, other light sources, or any combination thereof.

[0055] The light source may be positioned at any suitable distance from the sample (e.g., a flow stream in a flow cytometer), such as 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, etc., including distances of 100 mm or more. Furthermore, the light source may illuminate the sample at any suitable angle (e.g., relative to the normal axis of the flow stream), such as, for example, between 10° and 90°, between 15° and 85°, between 20° and 80°, between 25° and 75°, etc., including, for example, between 30° and 60°, such as 90°.

[0056] The light source may be configured to illuminate the sample in the flow cell continuously or at discrete intervals. In some cases, the system includes a light source configured to illuminate the sample continuously, such as by a continuous wave laser that continuously illuminates the flow stream at a measurement point in the flow cytometer. In other examples, the subject systems include a light source configured to illuminate the sample at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 milliseconds, every 10 milliseconds, every 100 milliseconds, etc., including every 1000 milliseconds, or other intervals. Here, when the light source is configured to illuminate the sample at discrete intervals, the system may include one or more additional components to provide intermittent illumination of the sample by the light source. For example, the system in these embodiments may include one or more laser beam choppers, manual or computer controlled beam stops, for blocking and exposing the sample to the light source.

[0057] In some embodiments, the system includes a flow cell configured to flow a sample in a flow stream. Any suitable flow cell may be utilized that flows a fluid sample to a sample measurement region. In some embodiments, the flow cell has a cylindrical proximal portion defining a major axis and a frusto-conical tip portion transverse to the major axis and terminating in a flat surface having an opening. The length of the cylindrical proximal portion (measured along the major axis) may vary from 1 mm to 15 mm, e.g., 1.5 mm to 12.5 mm, 2 mm to 10 mm, 3 mm to 9 mm, etc., including 4 mm to 8 mm. The length of the frusto-conical tip (measured along the major axis) may also vary from 1 mm to 10 mm, e.g., 2 mm to 9 mm, 3 mm to 8 mm, etc., including 4 mm to 7 mm. The diameter of the flow cell nozzle chamber may vary from 1 mm to 10 mm, e.g., 2 mm to 9 mm, 3 mm to 8 mm, etc., including 4 mm to 7 mm, in some embodiments.

[0058] In certain cases, the flow cell does not have a cylindrical portion, and the entire flow cell internal chamber may be frusto-conical. In these embodiments, the length of the frusto-conical internal chamber (measured along the major axis transverse to the nozzle opening) may vary from 1 mm to 15 mm, e.g., 1.5 mm to 12.5 mm, 2 mm to 10 mm, 3 mm to 9 mm, etc., including 4 mm to 8 mm. The diameter of the proximal portion of the frusto-conical internal chamber may vary from 1 mm to 10 mm, e.g., 2 mm to 9 mm, 3 mm to 8 mm, etc., including 4 mm to 7 mm.

[0059] In some embodiments, the sample flow stream originates from an opening at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the flow cell opening may be any shape, including but not limited to a rectilinear cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), or an irregular shape (e.g., a parabolic bottom joined to a planar top). In certain embodiments, the subject flow cell has a circular opening. The size of the nozzle opening may vary in some embodiments from 1 μm to 20,000 μm, such as 2 μm to 17,500 μm, 5 μm to 15,000 μm, 10 μm to 12,500 μm, 15 μm to 10,000 μm, 25 μm to 7,500 μm, 50 μm to 5,000 μm, 75 μm to 1,000 μm, 100 μm to 750 μm, etc., including 150 μm to 500 μm. In a particular embodiment, the nozzle opening is 100 μm.

[0060] In some embodiments, the flow cell includes a sample inlet configured to provide a sample to the flow cell. In embodiments, the sample inlet system is configured to flow the sample into the flow cell chamber accordingly. Depending on the desired characteristics of the flow stream, the rate of the sample delivered by the sample inlet to the flow cell chamber may be 1 μL / min or more, e.g., 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 15 μL / min or more, 25 μL / min or more, 50 μL / min or more, etc., including 100 μL / min or more, and in some cases, the rate of the sample delivered by the sample inlet to the flow cell chamber may be 1 μL / sec or more, e.g., 2 μL / sec or more, 3 μL / sec or more, 5 μL / sec or more, 10 μL / sec or more, 15 μL / sec or more, 25 μL / sec or more, 50 μL / sec or more, etc., including 100 μL / sec or more.

[0061] The sample inlet may be an opening disposed in the wall of the inner chamber, or a conduit disposed at the proximal end of the inner chamber. Where the sample inlet is an opening disposed in the wall of the inner chamber, the sample inlet opening may be any shape, including, but not limited to, a rectilinear cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical, etc.), or an irregular shape (e.g., a parabolic bottom joined to a planar top). In certain embodiments, the sample inlet has a circular opening. The size of the sample inlet opening may vary depending on the shape, and in certain cases, the opening may be in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, etc., including 1.25 mm to 1.75 mm, e.g., 1.5 mm.

[0062] In certain cases, the sample inlet is a conduit located at the proximal end of the flow cell chamber. For example, the sample inlet may be a conduit with the opening of the sample inlet aligned with the flow cell opening. Here, when the sample inlet is a conduit with the opening aligned with the flow cell opening, the cross-sectional shape of the sample inlet tube may be any shape, including, but not limited to, a cross-sectional shape composed of straight lines (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), or an irregular shape (e.g., a parabolic bottom joined to a planar top). The opening of the conduit may vary in shape, and in certain cases, has an opening ranging from 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, etc., including 1.25 mm to 1.75 mm, e.g., 1.5 mm. The shape of the tip of the sample inlet may be the same as or different from the cross-sectional shape of the sample inlet tube. For example, the opening of the sample inlet may have a beveled tip with an inclination angle ranging from 1° to 10°, e.g., 2° to 9°, 3° to 8°, 4° to 7°, etc., including even an inclination angle of 5°.

[0063] In some embodiments, the flow cell further comprises a sheath fluid input configured to provide sheath fluid to the flow cell. In embodiments, the sheath fluid input system is configured to provide a flow of sheath fluid to the flow cell interior chamber, e.g., with the sample, to generate a laminated flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the rate of sheath fluid delivered to the flow cell chamber can be 25 μL / sec or more, e.g., 50 μL / sec or more, 75 μL / sec or more, 100 μL / sec or more, 250 μL / sec or more, 500 μL / sec or more, 750 μL / sec or more, 1000 μL / sec or more, etc., including even 2500 μL / sec.

[0064] In some embodiments, the sheath fluid inlet is an opening disposed in the wall of the inner chamber. The sheath fluid inlet opening may be any shape, including, but not limited to, a rectilinear cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), or an irregular shape (e.g., a parabolic bottom joined to a planar top). The size of the sample inlet opening may vary depending on the shape, and in certain cases, the opening may range from 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, etc., including 1.25 mm to 1.75 mm, e.g., 1.5 mm.

[0065] In certain embodiments, the system is a flow cytometric system that uses the optical detection system described above to detect light emitted from a sample in a flow stream. Suitable flow cytometry systems and methods for analyzing samples include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. January; 49(pt 1):17-28; Linden et al., Semin Thromb Hemost. October 2004; 30(5):502-11; Alison et al., J Pathol, December 2010; 222(4):335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference. In certain cases, flow cytometry systems of interest include BD Biosciences FACSCanto™ flow cytometers, BD Biosciences FACSVantage™, BD Biosciences FACSort™, BD Biosciences FACSCount™, BD Biosciences FACScan™, and BD Biosciences FACSCalibur™ systems, BD Biosciences Influx™ cell sorters, BD Biosciences Aria™ cell sorters, and the like.

[0066] In certain embodiments, the system is configured to incorporate the methods and systems described in U.S. Patent Nos. 3,960,449, 4,347,935, 4,667,830, 4,704,891, 4,770,992, 5,030,002, 5,040,890, 5,047,321, 5,245,318, 5,317,162, 5,464,581, 5,483,469, 5,602,039, 5,620,842, 5,627,040, 5,643,796, 5,700,692, 6,372,506, 6 and 9,097,640 (the disclosures of which are incorporated herein by reference).

[0067] In some embodiments, one or more of the optical adjustment components (e.g., digital micromirror device) and detectors (e.g., photodetector array) may be computer controlled, such that a subset of micromirrors or photodetectors are adjusted (e.g., enabled, disabled, tilted, rotated, etc.) in a fully or partially automated manner. In some embodiments, the system includes a computer having a computer-readable storage medium having a computer program stored thereon, the computer program, when loaded into the computer, includes instructions for adjusting (e.g., tilting, rotating, disabling by applying current, etc.) one or more micromirrors on the active surface of the digital micromirror device. In another embodiment, the system includes a computer having a computer-readable storage medium having a computer program stored thereon, the computer program, when deployed to the computer, includes instructions for enabling or disabling (partially or fully) one or more photodetectors in the photodetector array.

[0068] The system may include both hardware and software components, where the hardware components may take the form of one or more platforms (e.g., in the form of servers), such that the functional elements, i.e., those elements of the system that perform particular tasks of the system (managing the input and output of information, processing information, etc.), may be implemented by executing software applications on and across the one or more computer platforms that represent the system.

[0069] The system may include a display and an operator input device, which may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that has access to a memory in which instructions are stored for adjusting (e.g., tilting, rotating, disabling by applying current, etc.) one or more micromirrors on the active surface of the digital micromirror device, or for enabling or disabling (partially or fully) one or more photodetectors in the photodetector array.

[0070] The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, an input / output controller, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors available now or in the future. The processor executes an operating system, which interfaces with firmware and hardware as is well known, and assists the processor in coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages, e.g., Java, Perl, C++, other high-level or low-level languages, as well as combinations thereof, known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0071] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media, such as a resident hard disk or tape, optical media, such as a read and write compact disk, flash memory device, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, tape drive, removable hard disk drive, or diskette drive. Such types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk, magnetic tape, removable hard disk, or floppy diskette, respectively. Any of these program storage media or others currently in use or that may be developed in the future may be considered computer program products. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or program storage devices used in combination with the memory storage devices.

[0072] In some embodiments, a computer program product is described including a computer usable medium having stored thereon control logic (computer software program including program code). The control logic, when executed by a processor or computer, instructs the processor to perform functions described herein. In other embodiments, some functions are performed primarily in hardware, for example using hardware state machines. Implementation of a hardware state machine to perform functions described herein will be apparent to one skilled in the relevant art.

[0073] The memory may be any suitable device capable of storing and retrieving data by the processor, such as a magnetic device, an optical device, or a solid-state storage device (e.g., a magnetic or optical disk or tape or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor that is suitably programmed from a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communication channel, or may be pre-stored in a computer program product, such as a memory or some other portable or fixed computer-readable storage medium, using any of the devices connected to the memory. For example, a magnetic or optical disk may carry the programming and be readable by a disk writer / reader. The system according to the invention also includes programming, such as algorithms used to implement the method described above, in the form of a computer program product. The programming according to the invention may be recorded on a computer-readable medium, such as any medium that can be read and directly accessed by a computer. Such media include, but are not limited to, magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape, optical storage media, such as CD-ROM, electrical storage media, such as RAM and ROM, portable flash drives, and hybrids of these categories, such as magnetic / optical storage media.

[0074] The processor may also utilize the communication channel to communicate with a remote user, meaning that the user does not have direct contact with the system, but relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, e.g., a mobile phone (i.e., smartphone).

[0075] In some embodiments, a system according to the present disclosure may be configured with a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or other devices. The communication interface may be configured for wired or wireless communication, such as, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee® communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as code division multiple access (CDMA) or global system for mobile communications (GSM®).

[0076] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces, e.g., a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment), configured for similar complementary data communication.

[0077] In one embodiment, the communications interface is configured for infrared communications, Bluetooth® communications, or any other suitable wireless communications protocol to enable communication between the system and other devices, such as computer terminals and / or networks, communications-enabled mobile phones, personal digital assistants, or any other communications devices that a user may use in conjunction with managing the treatment of health conditions such as HIV, AIDS, anemia, etc.

[0078] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing Internet Protocol (IP) via a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0079] In one embodiment, the system is configured to wirelessly communicate with a server device via a communication interface, for example using common standards such as 802.11 or Bluetooth RF protocols or IrDA infrared protocols. The server device can be other portable devices, such as a smartphone, a personal digital assistant (PDA), or a notebook computer, or larger devices, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), as well as input devices, such as buttons, a keyboard, a mouse, or a touch screen.

[0080] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate the system, e.g., data stored in the optional data storage unit, with a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0081] The output controller may include a controller for any of a variety of known display devices to present information to a user, whether human or machine, local or remote. Where one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. A graphical user interface (GUI) controller may include any of a variety of known or future software programs to provide a graphical input / output interface between the system and the user, and to process user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be accomplished in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing modules to a remote user, for example, using the Internet, telephone, or satellite networks, in accordance with known techniques. Presentation of data by the output manager may be performed in accordance with a variety of known techniques. As some examples, the data may include SQL, HTML, XML documents, email or other files, or other forms of data. The data may include Internet URL addresses so that the user may retrieve additional SQL, HTML, XML, or other documents or data from a remote source. The platform or platforms present in the system may be any type of known or future developed computer platform, but they are typically computers of a class commonly referred to as servers. However, they may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cable or other communication system, including networks or otherwise connected wireless systems. They may be co-located or physically separate. Various operating systems may be utilized on any of the computer platforms, possibly depending on the type and / or manufacturer of the computer platform selected.Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and the like.

[0082] [Method of detecting light from an irradiated sample separately] Aspects of the present disclosure further include methods for separately detecting light from a sample (e.g., in a flow stream in a flow cytometer). In carrying out the method of the embodiments, the sample is illuminated by a light source and light from the sample is detected by the light detection system described above. In some embodiments, the sample is a biological sample. The term "biological sample" is used in its traditional sense to refer to a whole organism, a plant, a fungus, or a subset of animal tissues, cells, or components found in certain cases, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Thus, "biological sample" refers to both a natural organism or a subset of its tissues, and homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymph, parts of the skin, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be any type of biological tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or a derivative thereof (e.g., plasma, tears, urine, semen, etc.), and in some cases, the sample is a blood sample comprising whole blood, such as blood obtained from a venipuncture or finger prick (which may or may not be combined with reagents such as preservatives or anticoagulants prior to analysis).

[0083] In certain embodiments, the source of the sample is "mammal" or "mammalian," terms used broadly to describe organisms within the class mammalia, including the order carnivore (e.g., dogs and cats), the order rodentia (e.g., mice, guinea pigs, and rats), and the order primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., neonates, infants, children, adolescents, adults), and in certain embodiments, the human subject is a child, adolescent, or adult. It is understood that while the present invention may be applied to samples from human subjects, the methods may also be performed on samples from other animal subjects (i.e., "non-human subjects"), including, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0084] The method is carried out by illuminating a sample (e.g., in a flow stream of a flow cytometer) with light from a light source. In some embodiments, the light source is a broadband light source that emits light having a wide range of wavelengths, e.g., spanning 50 nm or more, e.g., 100 nm or more, 150 nm or more, 200 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, including even spanning 500 nm or more. For example, one suitable broadband light source emits light having wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having wavelengths from 400 nm to 1000 nm. Where the method includes illuminating with a broadband light source, the subject broadband light source protocol may include, but is not limited to, a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a stabilized fiber-coupled broadband light source, a broadband LED with a continuous spectrum, a superluminescent light emitting diode, a semiconductor light emitting diode, a broad spectrum LED white light source, a white light source with multiple LEDs integrated, or any combination thereof, among other broadband light sources.

[0085] In other embodiments, the method includes illuminating with a narrowband light source emitting a specific wavelength or narrow range of wavelengths, for example, with a light source emitting light in a narrow range of wavelengths, such as 50 nm or less (including light sources emitting specific wavelengths of light (i.e., monochromatic light), such as 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, etc.) When the method includes illuminating with a narrowband light source, the subject narrowband light source protocol may include, but is not limited to, a narrow wavelength LED, laser diode, or broadband light source coupled with one or more optical bandpass filters, diffraction gratings, or monochromators, or any combination thereof.

[0086] In certain embodiments, the method includes irradiating the sample with one or more lasers. As discussed above, the type and number of lasers will depend on the sample and the desired light to be collected, and may include Helium Neon lasers, Argon lasers, Krypton lasers, Xenon lasers, Nitrogen lasers, CO lasers, etc. 2 The laser may be a gas laser, such as a CO laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, a xenon fluorine (XeF) excimer laser, or a combination thereof. In another example, the method includes irradiating the flow stream with a dye laser, such as a stilbene, coumarin, or rhodamine laser. In yet another example, the method includes irradiating the sample in the flow cell with a metal vapor laser, such as a helium cadmium (HeCd) laser, a helium mercury (HeHg) laser, a helium selenium (HeSe) laser, a helium silver (HeAg) laser, a strontium laser, a neon copper (NeCu) laser, a copper laser, a gold laser, and combinations thereof. In yet another example, the method includes irradiating the sample in the flow cell with a metal vapor laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO 4 Laser, Nd:YCa 4 O(BO 3 ) 3Laser, Nd:YCOB laser, Titanium sapphire laser, Thulium YAG laser, Ytterbium YAG laser, Ytterbium Yb 2 O 3 This involves irradiating the flow stream with a solid state laser, such as a laser, or a cerium doped laser, and combinations thereof.

[0087] The sample may be illuminated by one or more of the light sources mentioned above, for example, two or more light sources, three or more light sources, four or more light sources, five or more light sources, etc., including ten or more light sources. The light source may include any combination of light source types. For example, in some embodiments, the method includes illuminating the sample in the flow stream with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid state lasers.

[0088] The sample may be illuminated with a wavelength ranging from 200 nm to 1500 nm, such as 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, etc., including 400 nm to 800 nm. For example, if the light source is a broadband light source, the sample may be illuminated with a wavelength ranging from 200 nm to 900 nm. In another example, if the light source includes multiple narrowband light sources, the sample may be illuminated with a specific wavelength ranging from 200 nm to 900 nm. For example, the light source may be multiple narrowband LEDs (1 nm to 25 nm), each independently emitting light having a wavelength range from 200 nm to 900 nm. In another embodiment, the narrowband light source includes one or more lasers (such as a laser array), and the sample is illuminated with a specific wavelength ranging from 200 nm to 700 nm by a laser array having the gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers described above.

[0089] When two or more light sources are used, the sample may be illuminated by the light sources simultaneously, sequentially, or a combination thereof. For example, the sample may be illuminated by both light sources simultaneously. In other embodiments, the flow stream is illuminated sequentially by both light sources. When two or more light sources are used to illuminate the sample sequentially, the time for which each light source illuminates the sample may independently be 0.001 microseconds or more, e.g., 0.01 microseconds or more, 0.1 microseconds or more, 1 microseconds or more, 5 microseconds or more, 10 microseconds or more, 30 microseconds or more, including even 60 microseconds or more. For example, the method may include illuminating the sample with a light source (e.g., a laser) for 0.001 microseconds to 100 microseconds, e.g., 0.01 microseconds to 75 microseconds, 0.1 microseconds to 50 microseconds, 1 microseconds to 25 microseconds, or even 5 microseconds to 10 microseconds. In embodiments in which the sample is illuminated by two or more light sources in sequence, the amount of time the sample is illuminated by each light source may be the same or different.

[0090] Also, the time between illumination by each light source may vary as desired and may be independently separated by a delay of 0.001 microseconds or more, e.g., 0.01 microseconds or more, 0.1 microseconds or more, 1 microseconds or more, 5 microseconds or more, 10 microseconds or more, 15 microseconds or more, 30 microseconds or more, etc., including 60 microseconds or more. For example, the time between illumination by each light source may be 0.001 microseconds to 60 microseconds, e.g., 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microsecond to 25 microseconds, etc., including 5 microseconds to 10 microseconds. In certain embodiments, the time between illumination by each light source is 10 microseconds. In embodiments in which the sample is illuminated by more than two (i.e., three or more) light sources in sequence, the delay between illumination by each light source may be the same or different.

[0091] The sample may be illuminated continuously or at discrete intervals. In some cases, the method includes illuminating the sample in the sample continuously with the light source. In other cases, the sample is illuminated with the light source at discrete intervals, such as, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 milliseconds, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds, or some other interval.

[0092] Depending on the light source, the sample may be illuminated from different distances such as 0.01 mm or more, e.g., 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, including even 50 mm or more. The illumination angle may also vary from 10° to 90°, e.g., 15° to 85°, 20° to 80°, 25° to 75°, etc., including even 30° to 60°, e.g., an angle of 90°.

[0093] As mentioned above, in embodiments, light from the illuminated sample is conveyed to a light detection system as described herein and measured by one or more photodetectors. In certain embodiments, in the practice of the method, light is conveyed from the sample in the flow cell to an optical conditioning component. In certain embodiments, the optical conditioning component is utilized to optically change one or more properties of the light, such as the light path, direction, focusing or collimation of the light. The optical conditioning component may include, but is not limited to, lenses (e.g., focusing, objective, magnifying, etc.), collimators, mirrors (e.g., dichroic mirrors), slits, pinholes, filters (e.g., bandpass, interference), diffraction gratings, monochromators, and other types of optical conditioning components. In some embodiments, the light emanating from the sample is conveyed to a fused optical fiber component, such as a tapered fused optical fiber component. In some embodiments, the fused optical fiber component is coupled to the detector by an optically conductive material, such as a gel, between the fused optical fiber bundle and the detector surface.

[0094] In some embodiments, the method includes transmitting light from the sample to a micro-electromechanical system (MOEMS), such as a digital micromirror device, with multiple micromirrors disposed on a digital micromirror device active surface. In some embodiments, the method further includes adjusting one or more of the multiple micromirrors by tilting, rotating, or disabling the micromirror to separately detect the light from the illuminated sample. The micromirror may be adjusted such that the reflectivity of the adjusted mirror is reduced by 1% or more, e.g., 2% or more, 3% or more, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, 97% or more, 99% or more, etc., including even reducing the reflectivity of the micromirror by 100%.

[0095] In these embodiments, a subset of the micromirrors of the digital micromirror device may be adjusted. In carrying out the method, the number of micromirrors adjusted may vary depending on the size of the digital micromirror device and the amount of light desired to be blocked, and may be 10 or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 2500 or more, including even 5000 or more. As mentioned above, the subset of micromirrors adjusted to separately detect light from the sample in the flow cell may be 5% to 75%, for example, 10% to 70%, 15% to 65%, 20% to 60%, etc., of the total number of micromirrors on the active surface of the digital micromirror device, including even 25% to 50% of the total number of micromirrors on the active surface of the digital micromirror device. The adjusted subset of micromirrors may take the form of one or more predefined patterns on the digital micromirror device active surface, such as a pattern made up of straight lines (e.g., squares, rectangles, trapezoids, triangles, hexagons, etc.), curvilinear patterns (e.g., circles, ellipses), or asymmetric, irregular patterns (e.g., a parabolic shaped base joined to a planar top). In other embodiments, the method includes adjusting one or more rows of micromirrors across the digital micromirror device to separately detect light from a sample.

[0096] In some embodiments, to adjust the subset of micromirrors, the method includes tilting each micromirror to reduce a desired reflectance of the mirror. Each micromirror may be tilted 5° to 15°, e.g., 6° to 14°, 7° to 13°, etc., with respect to a surface of the digital micromirror device, and even including tilted 8° to 12° with respect to a surface of the digital micromirror device. In another embodiment, the method includes rotating the subset of micromirrors to reduce a desired reflectance by the subset of mirrors. Each micromirror may be rotated 5° to 15°, e.g., 6° to 14°, 7° to 13°, etc., and even including rotated 8° to 12°. In yet another embodiment, the method includes disabling (electrically disabling) a subset of micromirrors on the digital micromirror device. Disabling the subset of micromirrors may use any suitable protocol, such as applying a current to the subset of micromirrors sufficient to reduce or eliminate the reflectance of the micromirrors. For example, the applied current may be sufficient to reduce the reflectivity of a subset of the micromirrors by 50% or more, e.g., 75% or more, 90% or more, 95% or more, 97% or more, 99% or more, etc., including reducing the reflectivity of the subset of the micromirrors by 100%.

[0097] In certain embodiments, the method includes adjusting a subset of micromirrors on the active surface of the digital micromirror device to distinguish light from different particles in a sample, such as a sample having a plurality of different cell types. In some cases, the method includes tilting, rotating, or disabling a subset of micromirrors to block light from unwanted components of the sample, such as light scattered from unwanted cell populations, cellular debris, impurities, or non-cellular components in the sample. For example, if it is specifically desired to characterize two different cell populations in a sample, the method includes adjusting (e.g., tilting, rotating, or disabling) micromirrors of the digital micromirror device that correspond to light from the unwanted cell populations in the sample, and maintaining active micromirrors of the digital micromirror device that correspond to light from the desired cell population. The method may include characterizing one or more different cell populations (e.g., two or more different cell populations, three or more, four or more, five or more, six or more, seven or more, etc., including ten or more different cell populations in a sample having a plurality of different cell populations).

[0098] In another embodiment, the method includes adjusting a subset of micromirrors on the digital micromirror device to block unwanted light (e.g., incident illumination light) from propagating to the detector surface. For example, the subset of micromirrors may be tilted, rotated, or disabled to block light in one or more rows or in a predetermined pattern or shape across the digital micromirror surface.

[0099] In practicing the method, a subset of micromirrors on the active surface of the digital micromirror device are dynamically adjusted such that one or more of the micromirrors are tilted, rotated, or disabled for a desired period of time, such as, for example, 0.001 μs or more, e.g., 0.01 μs or more, 0.1 μs or more, 0.5 μs or more, 1 μs or more, 5 μs or more, 10 μs or more, 25 μs or more, 50 μs or more, 100 μs or more, 500 μs or more, or even 1000 μs or more, etc. In certain embodiments, a subset of micromirrors on the active surface of the digital micromirror device are dynamically adjusted for an extended period of time, such as, for example, 1 second or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 30 seconds or more, 60 seconds or more, 120 seconds or more, 240 seconds or more, 360 seconds or more, 480 seconds or more, or even 600 seconds or more, etc. In certain cases, the method includes dynamically adjusting a subset of the micromirrors over a period during which a sample in the flow cell is illuminated by a light source.

[0100] In some embodiments, the method includes separately detecting light from the sample with a photodetector array. As described above, the photodetector array may include a plurality of photodetectors, such as 4 or more photodetectors, e.g., 10 or more photodetectors, 25 or more photodetectors, 50 or more photodetectors, 100 or more photodetectors, 250 or more photodetectors, 500 or more photodetectors, 750 or more photodetectors, and even 1000 or more photodetectors. To separately detect light with the photodetector array, a subset of the photodetectors may be disabled (e.g., reversibly or irreversibly) such that different regions of the photodetector array are configured to perform different light detection. The method may include completely disabling one or more of the plurality of photodetectors in the subset. In another embodiment, the method may include partially disabling one or more of the plurality of photodetectors in the subset. For example, the method may include partially or completely disabling one or more of the plurality of photodetectors such that photodetectors in a particular region of the array are configured to detect a reduced amount of light. That is, a photodetector detects 95% or less of the light impinging on the surface of the photodetector in a particular area, e.g., 90% or less, 85% or less, 75% or less, 50% or less, 25% or less, 10% or less, 5% or less, 3% or less, 1% or less, or even 0.1% or less. Depending on the number of photodetectors in the array, the method may disable 5% or more of the photodetectors in the photodetector array, e.g., 10% or more, 25% or more, 50% or more, 75% or more, or even 90% or more. The disabled photodetectors may take the form of one or more predefined patterns in the photodetector array, such as a pattern made up of lines (e.g., squares, rectangles, trapezoids, triangles, hexagons, etc.), curved patterns (e.g., circles, ellipses), or asymmetrical or irregular patterns (e.g., a parabolic base joined to a planar top). In another embodiment, the method includes disabling a subset of the photodetectors along one or more columns across the photodetector array.

[0101] In some embodiments, the method includes disabling a subset of the photodetectors in the photodetector array to distinguish between light from different particles in the sample, such as light emanating from different cells in the sample. In these embodiments, the method includes partially or fully disabling a subset of the photodetectors to block light from unwanted components of the sample, such as light scattered from unwanted cell populations, cellular debris, impurities, or non-cellular components in the sample. For example, if it is desired to specifically characterize two different cell populations in a sample, only the photodetectors in the photodetector array that correspond to light from those cell populations may be enabled, and other photodetectors in the photodetector array may be disabled to block light emanating from other unwanted cell populations in the sample.

[0102] In another embodiment, the method includes disabling a subset of the photodetectors in the photodetector array to block unwanted light from being detected by the photodetector array. For example, the method may include partially or completely disabling a subset of the photodetectors to block incident light from an illuminating light source from being detected by the detectors. In these embodiments, the method may partially or fully disable a subset of the photodetectors in the form of one or more rows across the photodetector array that correspond to scattered light from the illuminating light source or other forms of unblocked incident light.

[0103] The light striking the surface of each effective photodetector in the photodetector array may be measured at 1 or more different wavelengths, 5 or more different wavelengths, 10 or more different wavelengths, 25 or more different wavelengths, 50 or more different wavelengths, 100 or more different wavelengths, 200 or more different wavelengths, 300 or more different wavelengths, etc., including measuring light collected at 400 or more different wavelengths.

[0104] In some embodiments, the method includes measuring the detected light over a range of wavelengths (e.g., 200 nm to 1000 nm). For example, the method may include collecting a spectrum of light over one or more of the wavelength ranges of 200 nm to 1000 nm. In yet another embodiment, the method includes measuring the collected light from one or more specific wavelengths. For example, the collected light may be measured at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof.

[0105] The detected light may be measured continuously or at discrete intervals. In some cases, the method includes obtaining measurements of the light continuously. In other cases, 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, etc., including every 1000 milliseconds or some other interval.

[0106] The collected light measurements may be taken one or more times in the present methods, including, for example, two or more, three or more, five or more, and even ten or more times. In certain embodiments, the light propagation may be measured two or more times and the data may optionally be averaged.

[0107] kit Aspects of the invention further include kits. The kits include a flow cell configured to flow a sample in a flow stream, optical conditioning components (e.g., beam splitters, collimating lenses, mirrors, wavelength separators, pinholes, etc.), and a detector unit configured to separately detect light from the flow stream without a scatterbar as described above. In some embodiments, the kits include optical fibers, such as tapered fused optical fiber bundles. In another embodiment, the kits may include a digital micromirror device. In yet another embodiment, the kits may include a photodetector array, such as an avalanche photodiode array.

[0108] In some embodiments, the kit includes fluid components such as digestive enzyme components or buffers. Exemplary buffers include, but are not limited to, PBS (phosphate) buffer, acetate buffer, N,N-bis(2-hydroxyethyl)glycine (bicine) buffer, 3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS) buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, citrate buffer, tris(hydroxymethyl)methylamine (tris) buffer, N-tris(hydroxymethyl)methylglycine (tricine) buffer, 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPS) buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, citrate buffer, tris(hydroxymethyl)methylamine (tris) buffer, N-tris(hydroxymethyl)methylglycine (tricine) buffer, 2-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid (2-hydroxypropanesulfonic acid) ... Examples of suitable buffers include trifluoroacetate (TAPSO) buffer, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) buffer, 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES) buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, dimethylarsinic acid (cacodylate) buffer, saline sodium citrate (SSC) buffer, 2(R)-2-(methylamino)succinic acid (succinic acid) buffer, potassium phosphate buffer, and N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer. In certain cases, the fluid component is a cytometer grade solution.

[0109] In yet other embodiments, the kit comprises a labeling reagent composition. For example, the labeling reagent composition can comprise a fluorophore, a chromophore, an enzyme, a redox label, a radiolabel, an acoustic label, a Raman (SERS) tag, a mass tag, an isotope tag, a magnetic particle, a microparticle, and a nanoparticle, or a combination thereof. In some cases, the labeling reagent comprises a labeled biomolecule, such as a polypeptide, a nucleic acid, a polysaccharide, etc., labeled with a fluorophore, a chromophore, an enzyme, a redox label, a radiolabel, an acoustic label, a Raman (SERS) tag, a mass tag, an isotope tag, a magnetic particle, a microparticle, and a nanoparticle, or a combination thereof.

[0110] The various assay components of the kit may be 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 a flow cell, a digital micromirror device, a photodetector array, are contained in a sealed pouch, such as a sterile foil pouch or packaging material.

[0111] In addition to the above components, the kit may further include instructions (in certain embodiments) for carrying out the method. These instructions may be included in the kit in various forms, one or more of which may be included in the kit. One form in which these instructions may be provided includes information printed on a suitable medium or substrate (e.g., a sheet or sheets of paper on which the information is printed), in the kit's packaging, insert, etc. Another form in which these instructions may be provided includes a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), portable flash drive, etc. Another form in which these instructions may be provided includes a website address that may be used to access the information at a remote site via the Internet.

[0112] Usability The present light collection system for separately detecting light from a sample in a flow cell is used in a variety of applications. In certain embodiments, the light collection system is used to enhance the measurement of light from a sample (e.g., fluorescent or scattered light from a sample in a flow stream of a flow cytometer). Embodiments of the present disclosure are used wherever it is desired to improve the effectiveness of emitted light and scattered light measurements in flow cytometry, such as for research, high throughput laboratory testing, etc. The present disclosure is also used wherever it is desired to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting.

[0113] The present disclosure also finds use in applications where cells prepared from biological samples may be desired for research and laboratory testing or for use in therapy. In some embodiments, the methods and devices can facilitate obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the methods and systems facilitate obtaining cells from a fluid or tissue sample that is to be used as a research or diagnostic sample for a disease such as cancer. Similarly, the methods and systems facilitate obtaining cells from a fluid or tissue sample that is to be used in therapy. The methods and devices of the present disclosure allow for the separation and collection of cells from biological samples (e.g., organs, tissues, tissue slices, fluids) with enhanced efficiency and lower costs compared to conventional flow cytometry systems.

[0114] Notwithstanding the appended claims, the present disclosure is also defined by the following appended claims. 1. A flow cell configured to flow a sample within a flow stream; a light source configured to illuminate the sample in the flow cell; a detector unit configured to separately detect light from the flow cell without a scatterbar; A system comprising: 2. The system of claim 1, wherein the detector unit includes a detector. 3. The system of claim 2, wherein the detector includes a detector array. 4. The system of claim 3, wherein the detector array includes an array of photodiodes. 5. The system of claim 4, wherein the detector unit is configured to separately detect light from the flow stream by disabling a subset of the photodiodes in the array.

[0115] 6. The system of claim 5, wherein the subset of disabled photodiodes form a predetermined pattern. 7. The system of claim 6, wherein the subset of disabled photodiodes form one or more columns across the photodiode array. 8. The system of claim 6, wherein the subsets of disabled photodiodes form a symmetrical or asymmetrical pattern. 9. The system of claim 8, wherein the subsets of disabled photodiodes form a symmetric pattern. 10. The system of claim 8, wherein the subset of disabled photodiodes form an asymmetric pattern.

[0116] 11. The system of any one of claims 5 to 10, wherein disabling of the subset of photodiodes is reversible. 12. The system of any one of claims 5 to 10, wherein disabling of the subset of photodiodes is irreversible. 13. The detector unit includes an optical adjustment component; 2. The system of claim 1, wherein the detector unit separately detects light from the flow stream by adjusting one or more components of the optical adjustment component or detector. 14. The system of claim 13, wherein the optical conditioning component includes a fused optical fiber component and the detector includes a detector array. 15. The system of claim 14, wherein the fused optical fiber component includes a tapered fused optical fiber bundle.

[0117] 16. The system of claim 14 or 15, wherein the detector unit further includes a light-conducting material disposed between the fused optical fiber component and the detector array. 17. The system of claim 16, wherein the photoconductive material comprises a gel. 18. The system of claim 17, wherein the optically conductive material comprises an index matching gel. 19. The system of claim 13, wherein the optical adjustment component includes one or more lenses. 20. The system of any one of claims 13 to 19, wherein the detector array includes an array of photodiodes.

[0118] 21. The system of claim 20, wherein the detector unit is configured to separately detect light from the flow stream by disabling a subset of the photodiodes in the array. 22. The system of claim 21, wherein the subset of disabled photodiodes form a predetermined pattern. 23. The system of claim 22, wherein the subset of disabled photodiodes form one or more columns across the photodiode array. 24. The system of claim 22, wherein the subsets of disabled photodiodes form a symmetrical or asymmetrical pattern. 25. The system of claim 24, wherein the subsets of disabled photodiodes form a symmetric pattern.

[0119] 26. The system of claim 24, wherein the subset of disabled photodiodes form an asymmetric pattern. 27. The system of any one of claims 21 to 26, wherein disabling of a subset of the photodiodes is reversible. 28. The system of any one of claims 21 to 26, wherein disabling of the subset of photodiodes is irreversible. 29. The system of claim 13, wherein the optical adjustment component includes an optical micromachine. 30. The system of claim 29, wherein the optical adjustment component includes a digital micromirror device.

[0120] 31. The system of claim 30, wherein the detector system is configured to separately detect light from the flow stream by adjusting a subset of mirrors on the digital micromirror device. 32. The system of claim 31, wherein the detector system is configured to separately detect light from the flow stream by tilting a subset of mirrors on the digital micromirror device. 33. The system of claim 31, wherein the detector system is configured to separately detect light from the flow stream by disabling a subset of mirrors on the digital micromirror device. 34. The system of claim 33, wherein a subset of the mirrors are electrically disabled. 35. The system of claim 33 or 34, wherein a subset of the disabled mirrors form a predetermined pattern.

[0121] 36. The system of any one of claims 33 to 35, wherein the subset of disabled mirrors form one or more rows across the digital micromirror device. 37. The system of any one of notes 33-36, wherein the subset of disabled mirrors forms a symmetric or asymmetric pattern. 38. The system of claim 37, wherein a subset of the disabled mirrors form a symmetric pattern. 39. The system of claim 37, wherein a subset of the disabled mirrors form an asymmetric pattern. 40. A system described in any one of appendices 1 to 39, wherein the system is a flow cytometer.

[0122] 41. Illuminating a flow cell containing a sample in a flow stream with a light source; detecting light from the flow cell with a detector unit configured to separately detect light from the flow cell without a scatter bar; A method comprising: 42. The method of claim 41, wherein the detector unit includes a detector. 43. The method of claim 42, wherein the detector comprises a detector array. 44. The method of claim 43, wherein the detector array includes an array of photodiodes. 45. The method of claim 44, further comprising disabling a subset of the photodiodes in the array.

[0123] 46. ​​The method of claim 45, comprising disabling a subset of the photodiodes in a predetermined pattern. 47. The method of claim 46, comprising disabling photodiodes to form one or more columns of disabled photodiodes across the photodiode array. 48. The method of claim 46, comprising disabling the photodiodes to form a symmetric or asymmetric pattern. 49. The method of claim 48, wherein the subsets of disabled photodiodes form a symmetric pattern. 50. The method of claim 48, wherein the subset of disabled photodiodes forms an asymmetric pattern.

[0124] 51. The method of any one of claims 45 to 50, further comprising re-enabling one or more of the disabled photodiodes. 52. The detector unit includes an optical adjustment component and a detector; 52. The method of claim 51, further comprising adjusting one or more components of an optical adjustment component or detector. 53. The method of claim 52, wherein the optical conditioning component includes a fused optical fiber component and the detector includes a detector array. 54. The method of claim 53, wherein the fused optical fiber component comprises a tapered fused optical fiber bundle. 55. The method of any one of claims 52-54, wherein the detector unit further includes a photoconductive material disposed between the fused optical fiber component and the detector array.

[0125] 56. The method of claim 55, wherein the photoconductive material comprises a gel. 57. The method of claim 56, wherein the optically conductive material comprises an index matching gel. 58. The method of claim 52, wherein the optical adjustment component includes one or more lenses. 59. The method of any one of claims 53 to 57, wherein the detector array comprises an array of photodiodes. 60. The method of claim 59, further comprising disabling a subset of the photodiodes in the array.

[0126] 61. The method of claim 60, comprising disabling a subset of the photodiodes in a predetermined pattern. 62. The method of claim 61, comprising disabling photodiodes to form one or more columns of disabled photodiodes across the photodiode array. 63. The method of claim 61, comprising disabling photodiodes to form a symmetric pattern of disabled photodiodes on the photodiode array or an asymmetric pattern of disabled photodiodes on the photodiode array. 64. The method of claim 63, wherein the subsets of disabled photodiodes form a symmetric pattern. 65. The method of claim 63, wherein the subset of disabled photodiodes forms an asymmetric pattern.

[0127] 66. The method of any one of claims 60 to 65, further comprising re-enabling one or more of the disabled photodiodes. 67. The method of claim 52, wherein the optical adjustment component includes an optical micromachine. 68. The method of claim 52, wherein the optical adjustment component includes a digital micromirror device. 69. The method of claim 68, further comprising adjusting a subset of mirrors on the digital micromirror device. 70. The method of claim 69, wherein adjusting includes tilting a subset of mirrors on the digital micromirror device.

[0128] 71. The method of claim 69, wherein adjusting includes disabling a subset of mirrors on the digital micromirror device. 72. The method of claim 71, wherein a subset of mirrors on the digital micromirror device are electrically disabled. 73. The method of claim 71 or 72, comprising disabling a subset of the micromirrors in the form of a predetermined pattern. 74. The method of any one of clauses 71-73, comprising disabling micromirrors to form one or more rows of disabled micromirrors across the digital micromirror device. 75. The method of any one of clauses 71-74, comprising disabling micromirrors to form a symmetric or asymmetric pattern of disabled micromirrors on the digital micromirror device.

[0129] 76. The method of claim 75, comprising disabling micromirrors to form a symmetrical pattern of disabled micromirrors on the digital micromirror device. 77. The method of claim 75, comprising disabling micromirrors to form an asymmetric pattern of disabled micromirrors on the digital micromirror device. 78. The method of any one of claims 71-77, further comprising re-enabling one or more of the disabled micromirrors. 79. The method of any one of claims 42 to 78, wherein one or more components of the optical adjustment component or detector are adjusted prior to irradiating the sample with the light source. 80. The method of any one of claims 42 to 78, wherein one or more components of the optical adjustment component or detector are adjusted while irradiating the sample with the light source.

[0130] 81. The method of any one of claims 41 to 80, wherein the sample comprises cells. 82. The method of claim 81, further comprising identifying one or more distinct types of cell populations in the sample. 83. A flow cell configured to flow a sample in a flow stream; An optical adjustment component; a detector unit configured to separately detect light from the flow cell without a scatterbar; A kit comprising: 84. The detector unit includes an optical adjustment component and a detector; 84. The kit of claim 83, wherein the detector unit separately detects light from the flow cell by adjusting one or more components of the optical adjustment component or detector. 85. The kit of claim 84, wherein the optical adjustment component includes a fused optical fiber component.

[0131] 86. The kit of claim 85, wherein the optical adjustment component includes a tapered fused optical fiber bundle. 87. The kit of claim 84, wherein the optical adjustment component includes a digital micromirror device. 88. The kit of any one of claims 84 to 87, wherein the detector unit includes a detector array. 89. The kit of claim 88, wherein the detector array comprises an array of photodiodes.

[0132] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those skilled in the art, in light of the teachings of this disclosure, that certain changes or modifications can be made to the invention without departing from the spirit or scope of the appended claims.

[0133] Accordingly, the foregoing description merely illustrates the principles of the present invention. It is understood that those skilled in the art may devise various configurations that embody the principles of the present invention, although not expressly described or shown herein, and that such configurations are within the spirit and scope of the present invention. Moreover, all examples, conditional phrases and phrases described herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventor to the advancement of the art, and are understood to be without limitation to such specific described examples and conditions. Moreover, all statements herein that describe the principles, aspects and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future (i.e., any elements developed that perform the same function, regardless of structure). Moreover, nothing disclosed herein is made available to the public, whether or not such disclosure is expressly set forth in the claims.

[0134] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. With respect to the claims, the provisions of 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly defined to apply to a limitation in a claim only if the term "means for" or "step for" is mentioned at the beginning of the limitation in the claim, and if these terms are not used in the limitation in the claim, the provisions of 35 U.S.C. 112(f) or 35 U.S.C. 112(6) do not apply.

[0135] [CROSS REFERENCE TO RELATED APPLICATIONS] Under 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Application No. 62 / 620,916, filed January 23, 2018, the disclosure of which is incorporated herein by reference.

Claims

1. a flow cell configured to flow a sample in a flow stream; a light source configured to illuminate the sample in the flow cell; a detector unit configured to separately detect light from the flow cell without a scatter bar, the detector unit including an optical conditioning component and a detector, the detector unit separately detecting light from the flow stream by adjusting one or more components of the optical conditioning component or the detector to block light received from one portion of the flow cell and allow detection of light received from another portion of the flow cell; A system comprising:

2. The system of claim 1 , wherein the detector unit includes the detector comprising a detector array.

3. The system of claim 2 , wherein the detector array comprises an array of photodiodes.

4. The system of claim 3 , wherein the detector unit is configured to separately detect light from the flow stream by disabling a subset of the photodiodes in the array.

5. The system of claim 1 , wherein the optical conditioning component comprises a fused optical fiber component and the detector comprises a detector array.

6. The system of claim 5 , wherein the fused optical fiber component comprises a tapered fused optical fiber bundle.

7. The system of claim 5 or 6, wherein the detector unit further comprises a light-conducting material disposed between the fused optical fiber component and the detector array.

8. The system of claim 1 , wherein the optical conditioning component comprises one or more lenses.

9. The system of claim 5 , wherein the detector array comprises an array of photodiodes.

10. The system of claim 1 , wherein the optical conditioning component comprises an optical micromachine.

11. The system of claim 10 , wherein the optical conditioning component comprises a digital micromirror device.

12. The system of claim 1 , wherein the system is a flow cytometer.

13. illuminating a flow cell containing a sample within a flow stream with a light source; Detecting light from the flow cell by a detector unit according to any one of claims 1 to 11 configured to detect light from the flow cell separately without a scatter bar. A method comprising:

14. a flow cell configured to flow a sample in a flow stream; An optical adjustment component; A detector unit according to any one of claims 1 to 11 configured to detect light from the flow cell separately, without a scatterbar; A kit comprising:

Images