Linear Variable Optical Filter System for Flow Cytometry, and Method for Using the Same

Abstract

Aspects of the present disclosure include a system for detecting light from particles in a flow stream by spectral discrimination. The light detection system includes a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, wherein each set of linear variable optical filters is configured to pass light having a predetermined sub-spectrum range; and a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filters. Also provided are a system having a light source for irradiating particles in the flow stream and a photodetector modulator component for binning data signals generated in a plurality of photodetector channels of the light detection system, a method for detecting light using the system, and a kit having one or more components for detecting light by the method.

Description

Background Art

[0001] Introduction Optical detection is often used to characterize the components of a sample, for example, when the sample (e.g., a biological sample) is used for the diagnosis of a disease or condition. When the sample is irradiated, light is not only scattered by the sample and transmitted through the sample, but can also be emitted by the sample (e.g., by fluorescence). Differences in sample components such as morphology, absorption rate, and the presence of fluorescent labels can cause differences in the light scattered, transmitted, or emitted by the sample. These differences can be used to characterize and identify the presence of components within the sample. To quantify these differences, light is collected and directed onto the surface of a detector. The amount of light reaching the detector can affect the overall quality of the optical signal output by the detector. The amount of light reaching the detector can be increased by increasing the surface area of the detector or by increasing the collection of light from the sample.

[0002] One technique that utilizes optical detection to characterize components within a sample is flow cytometry. Using data generated from the detected light, the distribution of components can be recorded and desired materials can be sorted. A flow cytometer typically includes a sample reservoir for receiving a fluid sample such as a blood sample and a sheath reservoir containing sheath fluid. The flow cytometer transfers particles (including cells) within the fluid sample as a cell stream into a flow cell while directing the sheath fluid into the flow cell. Within the flow cell, a liquid sheath is formed around the cell stream to impart a substantially uniform velocity to the cell stream. The flow cell hydrodynamically constricts the cells within the stream to pass through the center of a light source within the flow cell. Light from the light source can be detected as scattered or by transmission spectroscopy, or can be absorbed by one or more components within the sample and re-emitted as luminescence.

Summary of the Invention

[0003] Aspects of the present disclosure include a system for detecting light from particles within a flow stream by spectral discrimination. A light detection system according to certain embodiments includes a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, wherein each set of linear variable optical filters is configured to pass light having a predetermined sub-spectrum range; and a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filters. Also described is a system having a light source for irradiating particles within the flow stream and a photodetector modulator component for binning data signals generated in a plurality of photodetector channels of the light detection system. Also provided are a method for detecting light using the system of the subject matter and a kit having one or more components for detecting light by the method of the subject matter.

[0004] In some embodiments, each set of linear variable optical filters is configured to pass light having wavelengths in the range of 200 nm to 1200 nm, such as 500 nm to 800 nm, across the linear variable optical filter. For example, the first set of linear variable optical filters can be configured to pass light having wavelengths in the range of 500 nm to 650 nm across the linear variable optical filter, and the second set of linear variable optical filters can be configured to pass light having wavelengths in the range of 650 nm to 800 nm across the linear variable optical filter. In some cases, each set of linear variable optical filters includes a long-pass linear variable bandpass filter and a short-pass linear variable bandpass filter. In some cases, the long-pass variable bandpass filter is offset from the short-pass variable bandpass filter by a predetermined distance. In a particular case, the light in the sub-spectrum range that can pass across each set of linear variable optical filters is determined by the offset of the long-pass variable bandpass filter from the short-pass linear variable bandpass filter. In a particular case, the long-pass linear variable bandpass filter is offset from the short-pass variable bandpass filter in each set of linear variable optical filters such that the light in each sub-spectrum range has a spectral width of 1 nm to 50 nm, including, for example, 5 nm to 45 nm and 10 nm to 40 nm. In a particular embodiment, the long-pass linear variable bandpass filter is offset from the short-pass variable bandpass filter in each set of linear variable optical filters such that the light in each sub-spectrum range has a spectral width of 20 nm.

[0005] In some embodiments, the light in each spectral range passing through the set of linear variable optical filters has a spectral width of 5 nm to 50 nm, such as a spectral width of about 20 nm, such as a spectral width of 10 nm to 40 nm. In some embodiments, the first set of linear variable optical filters and the second set of linear variable optical filters are positioned along two parallel planes. In some cases, the wavelength separator component is configured to transmit light to and from between the first set of linear variable optical filters and the second set of linear variable optical filters. In a particular case, the wavelength separator component is configured such that the first set of linear variable optical filters is configured to pass light from a low wavelength to a high wavelength across the linear variable optical filter, and the second set of linear variable optical filters is configured to pass light from a high wavelength to a low wavelength across the linear variable optical filter. For example, the light from the sample is transmitted to the first segment of the first set of linear variable optical filters, passes light having a wavelength in the range of 500 nm to 520 nm, and the remaining portion of the light can be reflected to the first segment of the second set of linear variable optical filters, which is configured to pass light having a wavelength in the range of 800 nm to 780 nm and reflect the remaining portion of the light to the second segment of the first set of linear variable optical filters. The to-and-fro transmission and passage of light between the first set of linear variable optical filters and the second set of linear variable optical filters is continued over the entire length of each set of linear variable optical filters in certain embodiments.

[0006] In some embodiments, the light detection system includes a plurality of photodetectors positioned sandwiching each set of linear optical filters. In some cases, each photodetector is configured to detect light in a sub-spectrum range at each segment of the linear optical filter. In some cases, each photodetector is configured to detect light in a sub-spectrum range having a spectral width of 5 nm to 50 nm (e.g., 20 nm) at each segment of the linear optical filter. In a particular case, the light detection system is positioned sandwiching a first set of linear variable optical filters and is configured to detect light having sub-spectrum ranges of 500 nm to 520 nm, 520 nm to 540 nm, 540 nm to 560 nm, 560 nm to 580 nm, 580 nm to 600 nm, 600 nm to 620 nm, and 620 nm to 640 nm, 640 nm to 660 nm, a first set of photodetectors, and is positioned sandwiching a second set of linear variable optical filters and is configured to detect light having sub-spectrum ranges of 800 nm to 780 nm, 780 nm to 760 nm, 760 nm to 740 nm, 740 nm to 720 nm, 720 nm to 700 nm, 700 nm to 680 nm, and 680 nm to 660 nm, a second set of photodetectors.

[0007] In some cases, the photodetector components of the light detection system include modulator components configured to bin data signals from two or more different photodetector channels. In some cases, each photodetector channel is configured to generate a data signal for each sub-spectrum range of light. In some cases, the modulator components are configured to bin data signals from adjacent photodetector channels, such as horizontal binning of adjacent photodetector channels. In some embodiments, the modulator components include an output modulator configured to modulate the signals output from each photodetector channel. In a particular embodiment, the output modulator includes one or more of an amplifier (e.g., a transimpedance amplifier), a switch circuit, and a differential amplifier. In a particular case, the modulator components are configured to multiplex the generated data signals.

[0008] Aspects of the present disclosure also include a system for measuring light from a sample (e.g., within a flow stream) by spectral discrimination. In certain embodiments, the system includes a light source, a first set of linear variable optical filters and a second set of linear variable optical filters configured to pass light having a predetermined sub-spectrum range across the linear variable optical filters, a wavelength separator component configured to propagate light between the first and second sets of linear variable optical filters, a light detection system configured to detect light in a predetermined spectral range, and a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filters. In some embodiments, the system also includes an optical collection system for propagating light to the light detection system. The optical collection system can be a free space optical relay system and can include optical fibers such as an optical fiber optical relay bundle. In some embodiments, the system is a flow cytometer.

[0009] Aspects of the present disclosure also include a method of irradiating a sample in an interrogation field (e.g., within a flow stream) with a light source, a method of collecting and detecting light from the sample with the subject light detection system, and a method of measuring light detected at one or more wavelengths. In some embodiments, the light is collected and transmitted to the light detection system by a free space optical relay system. In other embodiments, the light is collected by an optical fiber such as an optical fiber optical relay bundle and transmitted to the light detection system.

[0010] Also provided are kits having one or more components of the subject light detection system. A kit according to certain embodiments includes a first set of linear variable optical filters, a second set of linear variable optical filters, and a photodetector. In some embodiments, one or more of the linear variable optical filters are linear variable bandpass filters such as long pass linear variable bandpass filters or short pass linear variable bandpass filters. In some instances, the kit includes an optical collection component such as an optical fiber optic relay bundle or a free space optical relay system. The kit may also include one or more photodetectors such as a photomultiplier tube (e.g., a metal package photomultiplier tube) or a photodiode (e.g., an avalanche photodiode, APD).

[0011] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures.

Brief Description of the Drawings

[0012]

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[0013] Aspects of the present disclosure include systems for detecting light from particles in a flow stream by spectral discrimination. A light detection system according to a particular embodiment includes a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, wherein each set of linear variable optical filters is configured to pass light having a predetermined sub-spectrum range, and a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filters. Also described are systems having a light source for irradiating particles in a flow stream and a photodetector modulator component for binning data signals generated in a plurality of photodetector channels of the light detection system. Also provided are methods for detecting light using the subject systems and kits having one or more components for detecting light by the subject methods.

[0014] Before the present invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, and accordingly, can of course vary. Also, the scope of the present invention is to be limited only by the appended claims, so it should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0015] When a range of values is provided, unless the context clearly dictates otherwise, each intervening value, to the tenth of the unit of the lower limit, is understood to be included between the upper and lower limits of the range and any other stated value or intervening value within the stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are included within the invention, subject to any specific excluded limitations within the stated range. Ranges excluding any one or both of those included limitations, when the stated range includes one or both of the limitations, are also included within the invention.

[0016] A particular range is presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the exact number preceding the term and for numbers that are close to or approximately the number preceding the term. When determining whether a number is close to or approximately the specifically recited number, a number that is close to or approximately the unrecited number may be a number that provides substantial equivalence to the specifically recited number in the context presented.

[0017] Unless defined otherwise, 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, representative, exemplary methods and materials are described herein.

[0018] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention has no right to antedate such publication by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates, which may need to be independently confirmed.

[0019] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any optional element. Accordingly, this description is intended to serve as a basis for the use of exclusive terms such as "solely" and "only" in connection with the recitation of claim elements, or for the use of "negative" limitations.

[0020] As will be apparent to those skilled in the art upon reading this disclosure, each of the distinct embodiments described and illustrated herein can be readily separated from, or combined with, the features of any of the other various embodiments without departing from the scope or spirit of the present invention. Any recited method can be performed in the order of the recited events, or in any other logically possible order.

[0021] Although the apparatus and method are described or may be described in functional terms for reasons of grammatical fluidity, the claims should not be construed as necessarily limited by the "means" or "step" limitation syntax unless expressly recited under 35 U.S.C. § 112, but should be given the full scope of the meaning and equivalents provided by the claim under the doctrine of judicial equivalents, and it should be clearly understood that where the claims are expressly recited under 35 U.S.C. § 112, full statutory equivalents under 35 U.S.C. § 112 should be given.

[0022] As summarized above, the present disclosure provides a system and method for detecting light from particles within a flow stream (e.g., within a particle analyzer) by spectral discrimination. In a further description of embodiments of the present disclosure, a first set of linear variable optical filters, a second set of linear variable optical filters, and a plurality of photodetectors configured to detect light from a sub-spectrum range across the linear variable optical filters are first described in more detail. Next, a method for detecting light by spectral discrimination using the subject system is described. A kit having one or more components of the subject spectral discrimination detection system is described.

[0023] System for Detecting Light from Particles within a Flow Stream by Spectral Discrimination Aspects of the present disclosure include a system for detecting light from particles within a flow stream by spectral discrimination. The term "spectral discrimination" is used herein in its conventional meaning to refer to the differential detection of light of a spectrum of wavelengths from the irradiated particles, and in certain instances, the differential detection of light of a spectrum from the irradiated particles can be used to identify or characterize the particles or their components. As described in more detail below, the subject system is configured to detect light of a plurality of different wavelengths emitted from the irradiated particles, and in some instances, one or more data signals are generated in response to the detected light of each different wavelength. In some embodiments, the subject system provides for the detection of light over the entire spectrum of wavelengths from the irradiated particles.

[0024] In some embodiments, as described herein, a system for detecting light from particles by spectral discrimination provides, for example, increased narrow spectral sampling accuracy, such as greater than 5%, for example greater than 10%, for example greater than 15%, for example greater than 25%, for example greater than 50%, for example greater than 75%, for example greater than 90%, and greater than 95% compared to signal processing, without binning data signals from two or more photodetector channels, and provides an increase in the accuracy of capturing a target spectral emission band by a photodetector array. According to certain embodiments, the light detection system described herein can generate light in different spectral ranges of greater than 10% while exhibiting a light loss of 20% or less, such as 19% or less, such as 18% or less, such as 17% or less, such as 16% or less, such as 15% or less, and includes generating light in different spectral ranges of greater than 10% while exhibiting a light loss of 10% or less.

[0025] In some embodiments, the subject light detection system provides increased resolution of light in a narrow spectral range. For example, the light detection system can provide greater granularity in spectrally separating light from irradiated particles. In some embodiments, the light detection system provides an increase in the signal-to-noise ratio of the data signal, including greater than 5%, for example greater than 10%, for example greater than 15%, for example greater than 25%, for example greater than 50%, for example greater than 75%, for example greater than 90%, and greater than 95%. Generating data signals within a plurality of photodetector channels according to certain embodiments of the present light detection system increases the signal-to-noise ratio by enabling the processing of data signals from more photodetector channels simultaneously. Since more data signals can be processed at a given spectral width, the signal-to-noise ratio of the light detected from the sample increases. Additionally, the light detection system can increase the signal-to-noise ratio in some cases by reducing the amount of interference between the emissions of different fluorophores in the sample.

[0026] A light detection system according to certain embodiments includes a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, each set of linear variable optical filters being configured to pass light having a predetermined sub-spectrum range. The term "wavelength separator" is used herein in its conventional sense to refer to an optical component configured to separate light (e.g., collected from particles irradiated within a flow stream) into predetermined spectral ranges. In some embodiments, the wavelength separator is configured to separate the collected light into a predetermined spectral range by passing light having a predetermined spectral range and reflecting light in one or more remaining spectral ranges. In other embodiments, the wavelength separator is configured to separate the collected light into a predetermined spectral range by passing light having a predetermined spectral range and absorbing light in one or more remaining spectral ranges. In yet other embodiments, the wavelength separator is configured to spatially diffract the collected light into a predetermined spectral range.

[0027] In some cases, the light detected by the subject system is luminescence such as fluorescence from irradiated particles. In some cases, the light from the irradiated particles is scattered light. In some cases, the scattered light is forward scattered light. In some cases, the scattered light is backscattered light. In some cases, the scattered light is side scattered light. In some cases, the light from the irradiated particles is transmitted light.

[0028] In an embodiment, each set of linear variable optical filters of the wavelength separator component is configured to pass light having wavelengths in the range of 200 nm to 1200 nm, including, for example, 250 nm to 1150 nm, for example, 300 nm to 1100 nm, for example, 350 nm to 1050 nm, for example, 400 nm to 1000 nm, for example, 450 nm to 950 nm, for example, 500 nm to 900 nm, for example, 550 nm to 850 nm, and 600 nm to 800 nm, across the linear variable optical filter. In some embodiments, each set of linear variable optical filters includes a long-pass variable bandpass filter and a short-pass variable bandpass filter. In certain embodiments, the long-pass linear variable bandpass filter is a linear variable bandpass filter that passes light having wavelengths greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, across the bandpass filter. In certain embodiments, the short-pass linear variable bandpass filter is a linear variable bandpass filter that passes light having wavelengths less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm, across the bandpass filter.

[0029] Each set of linear variable optical filters includes two linear variable optical filters that are spaced apart and arranged adjacent to each other. The distance between the two linear variable optical filters in each set of linear variable optical filters can vary, such as 0.001 mm to 25 mm, such as 0.005 mm to 20 mm, such as 0.01 mm to 15 mm, such as 0.05 mm to 10 mm, such as 0.1 mm to 10 mm, such as 0.5 mm to 10 mm, such as 1 mm to 9 mm, such as 2 mm to 8 mm. In certain cases, the two linear variable optical filters within each set of linear variable optical filters are physically in contact with each other (i.e., there is no space between the two linear variable optical filters).

[0030] In some cases, the long-pass variable bandpass filter is positioned offset by a predetermined distance from the short-pass variable bandpass filter. In a particular case, the light in the sub-spectrum range that can pass across each set of the linear variable optical filter is determined by the offset of the long-pass variable bandpass filter from the short-pass linear variable bandpass filter. In some embodiments, the long-pass variable bandpass filter is positioned offset from the short-pass variable bandpass filter by including 0.0001 mm or more, for example 0.0005 mm or more, for example 0.001 mm or more, for example 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 1.5 mm or more, for example 2 mm or more, for example 2.5 mm or more, for example 3 mm or more, for example 3.5 mm or more, for example 4 mm or more, for example 4.5 mm or more, for example 5 mm or more, for example 6 mm or more, for example 7 mm or more, for example 8 mm or more, for example 9 mm or more, for example 10 mm or more, and 15 mm or more. In a particular embodiment, the long-pass variable bandpass filter is positioned offset from the short-pass variable bandpass filter at a distance including 0.001 mm to 25 mm, for example 0.005 mm to 20 mm, for example 0.01 mm to 15 mm, for example 0.05 mm to 10 mm, for example 0.1 mm to 10 mm, for example 0.5 mm to 10 mm, for example 1 mm to 9 mm, and 2 mm to 8 mm in each set of the linear variable optical filters in the wavelength separator component.

[0031] In some embodiments, each set of linear variable optical filters is configured to generate light in a plurality of spectral ranges that pass across the linear variable optical filter and reach a photodetector (as described in more detail below), for example, three or more spectral ranges each detected, for example, four or more, for example five or more, for example six or more, for example seven or more, for example eight or more, for example nine or more, for example ten or more, for example fifteen or more, for example twenty-five or more, for example fifty or more, for example seventy-five or more, and light in spectral ranges of one hundred or more each detected by the photodetector. In some embodiments, the light in each spectral range that passes through the set of linear optical filters and is detected by the photodetector has a spectral width of 5 nm to 50 nm, for example a spectral width of 10 nm to 40 nm, for example a spectral width of about 20 nm. In certain cases, the long-pass linear variable bandpass filter is offset from the short-pass variable bandpass filter in each set of linear variable optical filters such that the light in each sub-spectral range has a spectral width of 1 nm or more, including, for example, 2 nm or more, for example 3 nm or more, for example 4 nm or more, for example 5 nm or more, for example 6 nm or more, for example 7 nm or more, for example 8 nm or more, for example 9 nm or more, for example 10 nm or more, for example 11 nm or more, for example 12 nm or more, for example 13 nm or more, for example 14 nm or more, for example 15 nm or more, for example 16 nm or more, for example 17 nm or more, for example 18 nm or more, for example 19 nm or more, for example 20 nm or more, for example 25 nm or more, for example 30 nm or more, for example 35 nm or more, for example 40 nm or more, for example 45 nm or more, and 50 nm or more. In certain embodiments, the long-pass linear variable bandpass filter is offset from the short-pass variable bandpass filter in each set of linear variable optical filters such that the light in each sub-spectral range has a spectral width of 1 nm to 50 nm, including, for example, 2 nm to 49 nm, for example 3 nm to 48 nm, for example 4 nm to 47 nm, for example 5 nm to 46 nm, for example 6 nm to 45 nm, for example 7 nm to 44 nm, for example 8 nm to 43 nm, for example 9 nm to 42 nm, and 10 nm to 40 nm.In certain embodiments, the long pass linear variable band pass filter is offset from the short pass variable band pass filter in each set of linear variable optical filters such that the light in each sub-spectrum range has a spectral width of 20 nm.

[0032] In embodiments, the first set of linear variable optical filters and the second set of linear variable optical filters are optically in communication with each other such that light is transmitted between them. In some cases, the wavelength separator component is configured to transmit light to and from between the first set of linear variable optical filters and the second set of linear variable optical filters. In some embodiments, the first set of linear variable optical filters and the second set of linear variable optical filters are positioned along two parallel planes. In other embodiments, the sets of linear variable optical filters are oriented at an angle in the range of 0.001° to 15°, including, for example, 0.005° to 14.5°, for example 0.01° to 14°, for example 0.05° to 13.5°, for example 0.1° to 13°, for example 0.5° to 12.5°, and 1° to 10°, relative to each other within the light detection system.

[0033] In some embodiments, the wavelength separator component is configured such that a first set of linear variable optical filters is configured to pass light from a low wavelength to a high wavelength across the linear variable optical filter, and a second set of linear variable optical filters is configured to pass light from a high wavelength to a low wavelength across the linear variable optical filter. In some cases, for example, light from a sample is transmitted to a first segment of the first set of linear variable optical filters, passes light having a wavelength in the range of 500 nm to 520 nm, and reflects the remaining portion of the light to a first segment of the second set of linear variable optical filters, which is configured to pass light having a wavelength in the range of 800 nm to 780 nm and reflect the remaining portion of the light to a second segment of the first set of linear variable optical filters. This back-and-forth transmission and passage of light between the first set of linear variable optical filters and the second set of linear variable optical filters is continued over the length of each set of linear variable optical filters, in certain embodiments, for example, at least 10% of the length of each set of linear variable optical filters, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 50%, such as at least 75%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 99%, and including at least 99.5% of the length of each set of linear variable optical filters. In certain cases, the light is transmitted by causing it to travel back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters across the entire length of the linear variable optical filter.

[0034] Light passing through a set of linear variable optical filters is detected by a plurality of photodetectors. Each photodetector is configured to detect light having a predetermined spectral width that can pass through the set of linear variable optical filters. For example, each photodetector is positioned along the length of the set of linear variable optical filters and can detect a spectral width of, for example, 10 nm to 40 nm, for example, a spectral width of about 20 nm, a spectral width of 5 nm to 50 nm. In a particular case, each photodetector is positioned to detect light passing through a segment of the set of linear variable optical filters having a spectral width of 1 nm or more, including, for example, 2 nm or more, for example, 3 nm or more, for example, 4 nm or more, for example, 5 nm or more, for example, 6 nm or more, for example, 7 nm or more, for example, 8 nm or more, for example, 9 nm or more, for example, 10 nm or more, for example, 11 nm or more, for example, 12 nm or more, for example, 13 nm or more, for example, 14 nm or more, for example, 15 nm or more, for example, 16 nm or more, for example, 17 nm or more, for example, 18 nm or more, for example, 19 nm or more, for example, 20 nm or more, for example, 25 nm or more, for example, 30 nm or more, for example, 35 nm or more, for example, 40 nm or more, for example, 45 nm or more, and 50 nm or more. In a particular embodiment, the light detection system includes a photodetector positioned to detect light passing through a set of linear variable optical filters having a spectral width of 20 nm.

[0035] In some embodiments, the first set of linear variable optical filters is configured to pass light in the range of 500 nm to 650 nm such that, for example, a photodetector is positioned to detect light that passes through the first set of linear variable optical filters at intervals having a spectral width of 20 nm. FIG. 1 shows a photodetector for detecting light across a first set of linear variable optical filters and a plurality of segments, according to a particular embodiment. As shown in FIG. 1, the linear variable optical filter set 100 includes a first linear variable optical filter 101 (e.g., a short-pass linear variable bandpass filter) and a second linear variable optical filter 102 (e.g., a long-pass linear variable bandpass filter). Light passes across the linear variable optical filter set 100 and reaches photodetectors 103a, 103b, 103c, 103d, 103e, 103f, and 103g, and each photodetector is configured to detect light having a spectral width of 20 nm. Photodetector 103a is configured to detect light in the range of 500 nm to 520 nm, photodetector 103b is configured to detect light in the range of 520 nm to 540 nm, photodetector 103c is configured to detect light in the range of 540 nm to 560 nm, photodetector 103d is configured to detect light in the range of 560 nm to 580 nm, photodetector 103e is configured to detect light in the range of 580 nm to 600 nm, photodetector 103f is configured to detect light in the range of 600 nm to 620 nm, and photodetector 103g is configured to detect light in the range of 620 nm to 640 nm.

[0036] In some embodiments, the second set of linear variable optical filters is configured to pass light in the range of 650 nm to 800 nm, such that, for example, a photodetector is positioned to detect light that passes through the second set of linear variable optical filters at intervals having a spectral width of 20 nm. FIG. 2 shows a photodetector for detecting light across a first set of linear variable optical filters and a plurality of segments, according to a particular embodiment. As shown in FIG. 2, the linear variable optical filter set 200 includes a first linear variable optical filter 201 (e.g., a short-pass linear variable bandpass filter) and a second linear variable optical filter 202 (e.g., a long-pass linear variable bandpass filter). Light passes across the linear variable optical filter set 200 and reaches photodetectors 203a, 203b, 203c, 203d, 203e, 203f, and 203g, each of which is configured to detect light having a spectral width of 20 nm. Photodetector 203a is configured to detect light from 800 nm to 780 nm, photodetector 203b is configured to detect light from 780 nm to 760 nm, photodetector 203c is configured to detect light from 760 nm to 740 nm, photodetector 203d is configured to detect light from 740 nm to 720 nm, photodetector 203e is configured to detect light from 720 nm to 700 nm, photodetector 203f is configured to detect light from 700 nm to 680 nm, and photodetector 203g is configured to detect light from 680 nm to 660 nm.

[0037] In an embodiment, a first set of linear variable optical filters and a second set of linear variable optical filters are configured to transmit light between each other. In some cases, each set of linear variable optical filters is configured to pass light in a certain spectral range and transmit (e.g., by reflection) light in one or more remaining spectral ranges to another set of linear variable optical filters. Light is transmitted back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters across the length of the linear variable optical filters. In one example, a set of linear variable optical filters is configured to receive light from a sample, pass light in a first spectral range, and transmit light in a second spectral range to a second set of linear variable optical filters. The second set of linear variable optical filters is configured to pass light in a third spectral range and return light in a fourth spectral range to the first set of linear variable optical filters. The first set of linear variable optical filters is then configured to return light in a fifth spectral range to the second set of linear variable optical filters. This back-and-forth transmission and passage of light continues across the sets of linear variable optical filters, and light of each spectral width is detected by a plurality of photodetectors.

[0038] FIG. 3 shows a light detection system having a first set of linear variable optical filters and a second set of linear variable optical filters configured to transmit light back and forth. Light is propagated into the light detection system using an optical collection component 305 such as an optical fiber or a free space optical relay system. Light from the optical collection component 305 is transmitted to a first set of linear variable optical filters 301 composed of a short-pass linear variable band-pass filter and a long-pass linear variable band-pass filter. Light having a wavelength of 500 nm to 520 nm passes across the first segment of the first set of linear variable optical filters 301 and is detected by a photodetector 303a. Light of the remaining wavelengths (i.e., light having a wavelength exceeding 520 nm) is reflected and transmitted to a second set of linear variable optical filters 302. The second set of linear variable optical filters 302 is composed of a short-pass linear variable band-pass filter and a long-pass linear variable band-pass filter. Light having a wavelength of 800 nm to 780 nm passes across the first segment of the second set of linear variable optical filters 302 and is detected by a photodetector 303h. Light of the remaining wavelengths is reflected to the first set of linear variable optical filters 301, and light having a wavelength of 520 nm to 540 nm passes across the second segment of the first set of linear variable optical filters 301 and is detected by a photodetector 303b. Light travels back and forth and is reflected between the first set of linear variable optical filters 301 and the second set of linear variable optical filters 302, passes through segments across the first set of linear variable optical filters 301, and is then detected by photodetectors 303b, 303c, 303d, 303e, 303f, and 303g, passes through segments across the second set of linear variable optical filters 302, and is then detected by photodetectors 303i, 303j, 303k, 303l, 303m, and 303n.

[0039] In an embodiment, the light detection system includes a plurality of photodetectors positioned to detect light from each sub-spectrum range across a linear variable optical filter. In some embodiments, the light is transmitted to each photodetector via an optical adjustment component. The term "optical adjustment" is used herein in its conventional sense and refers to any device that can change the spatial width of light or other characteristics of the light passing through each segment of the linear variable optical filter, such as, for example, direction, wavelength, beam width, beam intensity, and focus. The optical adjustment protocol can be any convenient device for adjusting one or more characteristics of light, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating protocols, and combinations thereof. In a particular embodiment, the system of interest includes one or more focusing lenses. In one example, the focusing lens can be a non-magnifying lens. In another example, the focusing lens is a magnifying lens. In other embodiments, the system of interest includes one or more mirrors. In still other embodiments, the system of interest includes an optical fiber.

[0040] In some embodiments, the optical adjustment component is in physical contact with the photodetector. In other embodiments, the optical adjustment component is optically in communication with the active surface of the photodetector and can be positioned at least 0.001 mm from the photodetector, including, for example, at least 0.005 mm, for example, at least 0.01 mm, for example, at least 0.05 mm, for example, at least 0.1 mm, for example, at least 0.5 mm, for example, at least 1 mm, for example, at least 10 mm, for example, at least 25 mm, for example, at least 50 mm, and up to 100 mm or more from the photodetector.

[0041] The photodetector can be releasably connected to each optical adjustment component within the subject photodetection module. The photodetector and the optical adjustment component can be connected by any convenient protocol. In certain embodiments, the photodetector and the optical component are connected together by attaching the photodetector with a fastener such as an optical component, or a hook and loop fastener, a magnet, a latch, a notch, a dish hole, a counterbore, a groove, a pin, a tether, a hinge, Velcro®, a non-permanent adhesive, or a combination thereof. In certain cases, the photodetector is connected to the optical component by slotting a wavelength separator into a groove of the photodetection module. In still other cases, the photodetector is connected to the optical component by one or more screws.

[0042] In embodiments, each photodetection system includes two or more photodetectors, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty-five or more, such as fifty or more, and one hundred or more photodetectors. In some embodiments, the photodetection module includes one or more photodetector arrays. The term "photodetector array" is used in its conventional meaning to refer to an arrangement or series of two or more photodetectors. In embodiments, the photodetector array can include two or more photodetectors, such as three or more photodetectors, such as four or more photodetectors, such as five or more photodetectors, such as six or more photodetectors, such as seven or more photodetectors, such as eight or more photodetectors, such as nine or more photodetectors, such as ten or more photodetectors, such as twelve or more photodetectors, including fifteen or more photodetectors.

[0043] In some embodiments, the light of each sub - spectral range transmitted through the linear variable optical filter is transmitted to one or more photodetectors, including being transmitted to a single photodetector, two different photodetectors, three different photodetectors, four different photodetectors, and including the light of each sub - spectral range being transmitted to five or more different photodetectors. In certain instances, the light of each sub - spectral range is transmitted to its own distinct detector (i.e., a distinct photodetector configured to detect light from each different sub - spectral range). In these embodiments, the light from each sub - spectral range can be collected and propagated to the photodetector through different sets of optical adjustment components, such as different sets of collimators and focusing lenses for different sub - spectral ranges and photodetectors.

[0044] In certain instances, the light of each sub - spectral range transmitted through the linear variable optical filter is detected in one or more different detector channels (e.g., a multi - channel photodetector array). In some instances, the light of each sub - spectral range is detected in different detector channels. In other instances, the light of each sub - spectral range is detected across multiple detector channels, including, for example, two or more detector channels, for example three or more, for example four or more, for example eight or more, for example sixteen or more, and including the light of each sub - spectral range transmitted through the linear variable optical filter being detected in thirty - two or more detector channels.

[0045] The light - detection system of the subject system can be any convenient light - detection protocol, including, but not limited to, light sensors or photodetectors such as avalanche photodiodes (APD), active pixel sensors (APS), quadrant photodiodes, image sensors, charge - coupled devices (CCD), intensified charge - coupled devices (ICCD), light - emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum - dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the photodetector is a photomultiplier tube, for example 0.01 cm 2~10 cm 2 、 for example, 0.05 cm 2 ~9 cm 2 、 for example, 0.1 cm 2 ~8 cm 2 、 for example, 0.5 cm 2 ~7 cm 2 in the range of and 1 cm 2 ~5 cm 2 and has a light multiplication tube with an active detection surface area for each region in the range including

[0046] In an embodiment of the present disclosure, each photodetector can be configured to detect light at one or more wavelengths, including, for example, light at two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, and for example, four hundred or more different wavelengths.

[0047] The photodetector can be configured to measure light continuously or at discrete intervals. In some cases, the detector of interest is configured to measure light continuously. In other cases, the detector of interest is configured to measure light at discrete intervals, such as, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at any other interval.

[0048] The photodetector can be configured to measure light one or more times, including, for example, two or more times, for example, three or more times, for example, five or more times, and for example, ten or more times, during each discrete time interval. In a particular embodiment, light is measured two or more times by the photodetector, and in a particular case, the data is averaged. Light transmitted from particles in the flow stream can be detected in one or more photodetector channels, including, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, and for example, ten or more photodetector channels.

[0049] In some cases, the light detected from the particles in the sample is scattered light. In some cases, the scattered light is forward scattered light. In some cases, the scattered light is back scattered light. In some cases, the scattered light is side scattered light. In some cases, the light transmitted from the irradiated particles is transmitted light. In certain embodiments, the light detected from each particle is luminescence such as particle luminescence (i.e., fluorescence or phosphorescence). In these embodiments, each particle may include one or more fluorophores that emit fluorescence in response to irradiation by two or more light sources. For example, each particle may include two or more fluorophores, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, and ten or more fluorophores. In some cases, each particle includes a fluorophore that emits fluorescence in response to irradiation by a light source. In some embodiments, the fluorophores of interest include, but are not limited to, for example, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurosine dyes, safranine dyes, indamine, indophenol dyes, fluorin dyes, oxazine dyes, oxazone dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, and dyes combined from two or more of the above dyes (e.g., tandem), polymeric dyes having one or more monomeric dye units, and mixtures of two or more of the above dyes, and may include dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.). A number of dyes are available from, for example, Molecular Probes (Eugene, OR), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, MO), Sirigen, Inc. (Santa Barbara,(CA) It is commercially available from various suppliers such as Becton Dickinson (BD) and Company (Franklin Lakes, NJ) and Exciton (Dayton, OH). For example, fluorophores include 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and its derivatives such as acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin (APC), phycoerythrin (PE), peridinin-chlorophyll protein, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; brilliant yellow; coumarin, and its derivatives such as 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine, and its derivatives such as cyanosine, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); 5',5”-dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-diethylamino-3(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriaminepentaacetic acid; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin, and its derivatives such as eosin and eosin isothiocyanate; erythrosin, and its derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein, and its derivatives such as 5-carboxyfluorescein (FAM), 5-(4,(6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2’7’-dimethoxy-4’5’-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and derivatives such as QFITC (XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); coral reef fluorescent protein (RCFP); Lissamine (trademark); Lissamine rhodamine; lucifer yellow; malachite green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; para - roaniline; Nile red; Oregon green; phenol red; B - phycoerythrin; o - phthalaldehyde; pyrene, and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 - pyrene butyrate; reactive red 4 (Cibacron (trademark) brilliant red 3B - A); rhodamine, and 6 - carboxy - X - rhodamine (ROX), 6 - carboxyrhodamine (R6G), 4,7 - dichlororhodamine Lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulforhodamine 101 sulfonyl chloride derivative (Texas red), N,N,N’,Derivatives such as N’-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC), riboflavin; rosolic acid, and terbium chelate derivatives; xanthene; dye-conjugated polymers (i.e., polymer-bound dyes) such as fluorescein isothiocyanate dextran, and two or more dyes, for example, phycobiliprotein (PE) tandem dyes or allophycocyanin (APC) tandem dyes, such as phycobiliprotein-CF594 (PE-CF594) tandem, phycobiliprotein-cyanine 5 tandem (PE-Cy5), phycobiliprotein-cyanine 5.5 tandem (PE-Cy5.5), phycobiliprotein-cyanine 7 tandem (PE-Cy7), allophycocyanin-R700 tandem (APC-R700), allophycocyanin-cyanine 7 (APC-Cy7) combined dyes (e.g., tandem dyes or protein complex tandem dyes), polymeric dyes having one or more monomeric dye units, and mixtures of two or more of the aforementioned dyes or combinations thereof may be included.,

[0050] In some cases, the fluorophore is a polymeric dye. In some cases of the method, the polymeric dye includes a conjugated polymer. The conjugated polymer (CP) is characterized by a delocalized electron structure including a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated (e.g., single bond) bonds, and the π electrons can move from one bond to the other. Thus, the conjugated backbone can limit the bond angle between the repeating units of the polymer and impart an extended linear structure to the polymeric dye. For example, proteins and nucleic acids are also polymers, but in some cases, they do not form an extended rod structure but rather fold into a higher-order three-dimensional shape. In addition, the CP can form a "rigid rod" polymer backbone and can experience a limited twist (e.g., torsion) angle between monomeric repeating units along the polymer backbone chain. In some cases, the polymeric dye includes a CP having a rigid rod structure. The structural properties of the polymeric dye can affect the fluorescence properties of the molecule.

[0051] Examples of the polymer dyes to be targeted include, but are not limited to, U.S. Patent Publications Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, 20130190193, 20160264737, 20160266131, 20180231530, 20180009990, 20180009989, and 20180163054, which are hereby incorporated by reference in their entireties; Gaylord et al., J. Am. Chem. Soc., 2001, 123(26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133(32), pp 12600-12607, which are hereby incorporated by reference in their entireties; and the dyes described by Gaylord et al.

[0052] Polymeric dyes can have one or more desirable spectral properties, such as a specific absorption maximum wavelength, a specific emission maximum wavelength, an extinction coefficient, a quantum yield, etc. (see, for example, Chattopadhyay et al., "Brilliant violet fluorophore: A new class of ultrabright fluorescent compounds for immunofluorescence experiments", Cytometry Part A, 81A(6), 456 - 466, 2012). In some embodiments, the polymeric dye has an absorption curve in the range of 280 nm to 475 nm. In certain embodiments, the polymeric dye has an absorption maximum (excitation maximum) in the range of 280 nm to 475 nm. In some embodiments, the polymeric dye absorbs incident light having wavelengths in the range of 280 nm to 475 nm. In some embodiments, the polymeric dye has an emission maximum wavelength in the range of 400 nm to 850 nm, for example, 415 nm to 800 nm. Specific examples of the emission maximum include, but are not limited to, 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm. In some cases, the polymeric dye has an emission maximum wavelength selected from the group consisting of 410 nm to 430 nm, 500 nm to 520 nm, 560 nm to 580 nm, 590 nm to 610 nm, 640 nm to 660 nm, 700 nm to 720 nm, and 775 nm to 795 nm. In a particular embodiment, the polymeric dye has an emission maximum wavelength of 421 nm. In some cases, the polymeric dye has an emission maximum wavelength of 510 nm. In some cases, the polymeric dye has an emission maximum wavelength of 570 nm. In a particular embodiment, the polymeric dye has an emission maximum wavelength of 602 nm. In some cases, the polymeric dye has an emission maximum wavelength of 650 nm. In certain cases, the polymeric dye has an emission maximum wavelength of 711 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 786 nm. In a particular case, the polymeric dye has an emission maximum wavelength of 421 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 510 nm ± 5 nm. In a particular case, the polymeric dye has an emission maximum wavelength of 570 nm ± 5 nm.In some cases, the polymeric dye has an emission peak wavelength of 602 nm ± 5 nm. In some embodiments, the polymeric dye has an emission peak wavelength of 650 nm ± 5 nm. In certain cases, the polymeric dye has an emission peak wavelength of 711 nm ± 5 nm. In some cases, the polymeric dye has an emission peak wavelength of 786 nm ± 5 nm. In certain embodiments, the polymeric dye has an emission peak selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0053] Specific polymeric dyes that can be used include, but are not limited to, BD Horizon Brilliant™ dyes, such as BD Horizon Brilliant™ Violet dyes (e.g., BV421, BV480, BV510, BV570, BV605, BV650, BV711, BV786, BV829), BD Horizon Brilliant™ UV dyes (e.g., BUV395, BUV496, BUV563, BUV615, BUV661, BUV737, BUV805), and BD Horizon Brilliant™ Blue dyes (e.g., BB515, BB630, BB660, BB700, BB755, BB790) (BD Biosciences, San Jose, CA).

[0054] In some embodiments, the light detection system includes a modulator component that bins data signals from different photodetector channels. In some embodiments, the modulator is configured to bin data signals from two or more different photodetector channels to generate a combined spectral data signal. In some cases, the modulator component bins data signals from non-adjacent photodetectors. In some cases, the modulator component bins data signals from adjacent photodetectors. In certain cases, the modulator component is configured for horizontal binning of data signals from adjacent photodetectors.

[0055] In some embodiments, the modulator component is configured to bin data signals from three or more different photodetector channels, such as four or more, such as five or more, such as six or more, such as sixteen or more, such as thirty-two or more, and such as sixty-four or more different photodetector channels. In certain cases, each photodetector channel is configured to generate a data signal for each light in a sub-spectrum range. In some embodiments, the binned data signals are wavelength-separated data signals. In some embodiments, the binned data signals are time-separated data signals. In some embodiments, the binned data signals are time- and wavelength-separated data signals.

[0056] In certain embodiments, the modulator component is configured to dynamically bin data signals from two or more different photodetector channels in real time. In certain cases, the modulator component includes an integrated circuit. In embodiments, the integrated circuit device can be a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), a complex programmable logic device (CPLD), or some other integrated circuit device. For example, the integrated circuit can be a field programmable gate array (FPGA), such as including programming the FPGA to dynamically bin data signals from different photodetector channels. In certain cases, the modulator component is programmed with a binned configuration mapped for each photodetector channel, such as including an FPGA having programming for dynamically mapping the binning configuration in real time.

[0057] In other embodiments, the modulator component includes one or more amplifiers. In some embodiments, the amplifier component includes a plurality of amplifiers such as a transimpedance amplifier, a summing amplifier, a differential amplifier, or a combination thereof. In some cases, the modulator component includes an amplifier for each photodetector channel. For example, the modulator component includes two or more amplifiers, such as four or more amplifiers, such as eight or more amplifiers, such as twelve or more amplifiers, such as sixteen or more amplifiers, such as twenty or more amplifiers, such as twenty-four or more amplifiers, such as twenty-eight or more amplifiers, such as thirty-two or more amplifiers, such as thirty-six or more amplifiers, such as forty or more amplifiers, such as forty-four or more amplifiers, such as forty-eight or more amplifiers, such as fifty-two or more amplifiers, such as fifty-six or more amplifiers, such as sixty or more amplifiers, and sixty-four or more amplifiers.

[0058] In some embodiments, the modulator component includes a first amplifier component configured to amplify a data signal from each photodetector channel and a second amplifier component configured to amplify a data signal from an electronic switch component. In some cases, the first amplifier component includes a plurality of transimpedance amplifiers and the second amplifier component includes a plurality of differential amplifiers.

[0059] In some cases, the modulator component includes an electronic switch (e.g., a digital switch circuit) configured to bin data signals from two or more different photodetector channels. In certain cases, the switch is configured to multiplex or demultiplex the output data signals from each photodetector channel. Depending on the number of photodetector channels and amplifiers (such as those described above) employed in the photodetection system, the electronic switch component can include two or more electronic switches, e.g., three or more electronic switches, e.g., four or more electronic switches, e.g., five or more electronic switches, e.g., six or more electronic switches, e.g., seven or more electronic switches, e.g., eight or more electronic switches, e.g., nine or more electronic switches, e.g., ten or more electronic switches, e.g., fifteen or more electronic switches, e.g., twenty-five or more electronic switches, e.g., fifty or more electronic switches, e.g., one hundred or more electronic switches, e.g., two hundred and fifty or more electronic switches, e.g., five hundred or more electronic switches, e.g., seven hundred and fifty or more electronic switches, and one thousand or more electronic switches.

[0060] In some embodiments, the target photodetection system includes a photodetector array having N photodetector channels and an amplifier component having N transimpedance amplifiers, where N is an integer from 4 to 1000. In certain cases, the photodetection system includes a photodetector array having N photodetector channels, a modulator component having N transimpedance amplifiers, and an electronic switch component having an array of N×N switches. In some cases, the photodetector array can be a photodiode array having N photodiodes. In these embodiments, the modulator component can include N transimpedance amplifiers and an array of N×N switches. In some embodiments, N is 8. In other embodiments, N is 16. In other embodiments, N is 32. In other embodiments, N is 64. In other embodiments, N is 128.

[0061] System for detecting light from particles in a sample by spectral identification Aspects of the present disclosure include systems for detecting light from particles within a flow stream by spectral discrimination. In certain embodiments, the system provides differential detection of light of a certain spectrum of wavelengths from the irradiated particles, and in certain cases, the differential detection of the spectrum of light from the irradiated particles can be used to identify or characterize the particles or their components. The subject system is configured to detect light of a plurality of different wavelengths emitted from the irradiated particles, and in some cases, one or more data signals are generated in response to the light of each different detected wavelength.

[0062] In an embodiment, the system includes a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, each set of linear variable optical filters being configured to pass light having a predetermined sub-spectrum range, and a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filters, the light detection system described herein. In some embodiments, the system includes an optical collection system for transmitting light, such as from an irradiated flow stream, to the light detection system.

[0063] Each optical collection system can be any suitable optical collection protocol that collects light from the irradiated sample and directs the light towards the first set of linear variable optical filters of the light detection system. In some embodiments, the optical collection system includes an optical fiber, such as an optical fiber light relay bundle. In other embodiments, the optical collection system is a free space optical relay system.

[0064] In an embodiment, each optical collection system can be physically coupled to the light detection system by an adhesive or the like, can be co-molded with each light detection module, or can be integrated into each light detection module. In certain embodiments, the light detection system and the optical collection system are integrated into a single unit. In some cases, the light detection system is coupled to the optical collection system by a connector that clamps the optical collection system to each light detection module, such as a hook and loop fastener, a magnet, a latch, a notch, a dish hole, a counterbore, a groove, a pin, a tether, a hinge, Velcro, a non-permanent adhesive, or a combination thereof.

[0065] In other embodiments, the light detection system and the optical collection system are in optical communication but not in physical contact. In an embodiment, the optical collection system can be positioned at least 0.001 mm, such as at least 0.005 mm, such as at least 0.01 mm, such as at least 0.05 mm, such as at least 0.1 mm, such as at least 0.5 mm, such as at least 1 mm, such as at least 10 mm, such as at least 25 mm, such as at least 50 mm, and at least 100 mm from the light detection system, from the light detection module.

[0066] In certain embodiments, the optical collection system includes an optical fiber. For example, the optical collection system can be an optical fiber optical relay bundle, and light from the sample is transmitted through the optical fiber optical relay bundle to a first set of linear variable optical filters of the light detection system. Any optical fiber optical relay system can be employed to transmit light, and in certain embodiments, suitable optical fiber optical relay systems include, but are not limited to, those described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0067] In other embodiments, the optical collection system is a free-space optical relay system. As used herein, the phrase "free-space optical relay" refers to the propagation of light from a sample through free space and directed to a light detection system using the configuration of one or more optical components in its conventional sense. In certain embodiments, the free-space optical relay system includes a housing having a proximal end and a distal end, the proximal end being coupled to a light detection module. The free-space relay system can include any combination of different optical components such as one or more of lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof. For example, in some embodiments, the free-space optical relay system of interest includes one or more focusing lenses. In other embodiments, the free-space optical relay system includes one or more mirrors. In still other embodiments, the free-space optical relay system includes a collimating lens. In certain embodiments, suitable free-space optical relay systems for propagating light from a sample to a first set of linear variable optical filters of a light detection system include, but are not limited to, light relay systems such as those described in U.S. Pat. Nos. 7,643,142, 7,728,974, and 8,223,445, the disclosures of which are incorporated herein by reference.

[0068] The system of interest for measuring light from a sample includes a light source. In embodiments, the light source can be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, beads, non-cellular particles, etc.), the light source can be configured to emit light having wavelengths that vary in the range of, for example, 250 nm to 1250 nm including 400 nm to 800 nm, 300 nm to 1000 nm, 350 nm to 900 nm, 200 nm to 1500 nm. For example, the light source can include a broadband light source that emits light having wavelengths in the range of 200 nm to 900 nm. In other cases, the light source includes a narrowband light source that emits wavelengths in the range of 200 nm to 900 nm. For example, the light source can be a narrowband LED (1 nm to 25 nm) that emits light having wavelengths in the range of 200 nm to 900 nm.

[0069] In some embodiments, the light source is a laser. The laser of interest may include a pulsed laser or a continuous wave laser. For example, the laser may be a gas laser such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluoride (ArF) excimer laser, a krypton-fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser or a xenon-fluoride (XeF) excimer laser, or a combination thereof, a dye laser such as a stilbene, coumarin, or rhodamine laser, 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, or a gold laser, and a combination thereof, a metal-vapor laser, a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium2O3 laser, or a cerium-doped laser, and a combination thereof, a solid-state laser, a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or an embodiment of frequency doubling or frequency tripling of any of the above lasers.

[0070] In other embodiments, the light source is a non-laser light source including, but not limited to, a halogen lamp, a deuterium arc lamp, a lamp including a xenon arc lamp, a broadband LED having a continuous spectrum, a superluminescent light emitting diode, a semiconductor light emitting diode, a wide-area spectrum LED white light source, a multi-LED integration, and other light emitting diodes. In some cases, the non-laser light source is, among other light sources, a stabilized fiber-coupled broadband light source, a white light source, or any combination thereof.

[0071] In certain embodiments, the light source is an optical beam generator configured to generate two or more beams of frequency-shifted light. In some cases, this optical beam generator includes a laser and a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser in the optical beam generator of interest can be a gas laser such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluoride (ArF) excimer laser, a krypton-fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser or a xenon-fluoride (XeF) excimer laser, or a combination thereof, a dye laser such as a stilbene, coumarin, or rhodamine laser, 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, or a gold laser, and a combination thereof, a solid-state laser such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium 2O3 laser, or a cerium-doped laser, and a combination thereof.

[0072] An acousto-optic device can be any convenient acousto-optic protocol configured to frequency shift a laser beam using an applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device within the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. This high-frequency drive signal can be applied to the acousto-optic device from any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0073] In embodiments, the controller is configured to apply more than 100 high-frequency drive signals, such as more than 4 high-frequency drive signals, such as more than 5 high-frequency drive signals, such as more than 6 high-frequency drive signals, such as more than 7 high-frequency drive signals, such as more than 8 high-frequency drive signals, such as more than 9 high-frequency drive signals, such as more than 10 high-frequency drive signals, such as more than 15 high-frequency drive signals, such as more than 25 high-frequency drive signals, such as more than 50 high-frequency drive signals, such as more than 3 high-frequency drive signals, to apply high-frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams within the output laser beam.

[0074] In some cases, to generate an intensity profile of an angularly deflected laser beam within an output laser beam, the controller is configured to apply a high-frequency drive signal having an amplitude that varies from about 0.001 V to about 500 V, such as from about 5 V to about 25 V, from about 0.005 V to about 400 V, from about 0.01 V to about 300 V, from about 0.05 V to about 200 V, from about 0.1 V to about 100 V, from about 0.5 V to about 75 V, from about 1 V to 50 V, from about 2 V to about 40 V, from 3 V to about 30 V, etc. In some embodiments, each applied high-frequency drive signal has a frequency from about 0.001 MHz to about 500 MHz, such as from about 5 MHz to about 50 MHz, from about 0.005 MHz to about 400 MHz, from about 0.01 MHz to about 300 MHz, from about 0.05 MHz to about 200 MHz, from about 0.1 MHz to about 100 MHz, from about 0.5 MHz to about 90 MHz, from about 1 MHz to about 75 MHz, from about 2 MHz to about 70 MHz, from about 3 MHz to about 65 MHz, from about 4 MHz to about 60 MHz, etc.

[0075] In certain embodiments, the controller has a processor operably coupled to a memory, such that the memory contains instructions stored therein, and the instructions, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam with a desired intensity profile. For example, the memory may contain instructions to generate two or more angularly deflected laser beams having the same intensity, such as three or more, four or more, five or more, ten or more, twenty-five or more, fifty or more, one hundred or more. In other embodiments, the memory may contain instructions to generate two or more angularly deflected laser beams having different intensities, such as three or more, four or more, five or more, ten or more, twenty-five or more, fifty or more, one hundred or more.

[0076] In certain embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory contains instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center along a horizontal axis. In these instances, the intensity of the angularly deflected laser beam at the center of the output beam can be in the range of from about 0.5% to about 95%, such as from about 1% to about 90%, from about 2% to about 85%, from about 3% to about 80%, from about 4% to about 75%, from about 5% to about 70%, from about 6% to about 65%, from about 7% to about 60%, from about 8% to about 55%, from about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory contains instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center along a horizontal axis. In these instances, the intensity of the angularly deflected laser beam at the edge of the output beam can be in the range of from about 0.5% to about 95%, such as from about 1% to about 90%, from about 2% to about 85%, from about 3% to about 80%, from about 4% to about 75%, from about 5% to about 70%, from about 6% to about 65%, from about 7% to about 60%, from about 8% to about 55%, from about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In still other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory contains instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile with a Gaussian distribution along a horizontal axis.In yet other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory contains instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0077] In an embodiment, the light beam generator of interest can be configured to generate angularly deflected laser beams within the output laser beam that are spatially separated. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angularly deflected laser beams can be separated by 0.001 μm or more, such as 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, 100 μm or more, 500 μm or more, 1000 μm or more, including 5000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams within the output laser beam that overlap, such as adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap (such as overlap of beam spots) between adjacent angularly deflected laser beams can be 0.001 μm or more, such as 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, including 100 μm or more.

[0078] In certain instances, a light beam generator configured to generate two or more beams of frequency-shifted light includes a laser excitation module as described in U.S. Pat. Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publications 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0079] The light source can be positioned at any suitable distance from the sample (e.g., the flow stream within a flow cytometer), such as a distance of 0.001 mm or more from the flow stream, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and 100 mm or more. Further, the light source irradiates the sample at any suitable angle (e.g., with respect to the vertical axis of the flow stream), such as an angle ranging from 10° to 90°, including, for example, 15° - 85°, for example, 20° - 80°, for example, 25° - 75°, and 30° - 60°, such as an angle of 90°.

[0080] The light source can be configured to irradiate the sample continuously or at discrete intervals. In some cases, the system includes a light source configured to continuously irradiate the sample, such as a continuous wave laser that continuously irradiates the flow stream at the interrogation point of the flow cytometer. In other cases, the system in question includes a light source configured to irradiate the sample at discrete intervals including, for example, every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other discrete interval of intervals. When the light source is configured to irradiate the sample at discrete intervals, the system may include one or more additional components for providing intermittent irradiation of the sample using the light source. For example, the system of the subject matter in these embodiments may include one or more laser beam choppers that are manual or computer-controlled beam stops for blocking and exposing the sample to the light source.

[0081] In certain embodiments, the system further includes a flow cell configured to propagate a sample within the frost stream. Any convenient flow cell that propagates a fluid sample to the sample inspection region may be employed. In some embodiments, the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal conical portion terminating in a flat surface having an orifice that is transverse to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) can vary in the range of 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, for example, 2 mm to 10 mm, for example, 3 mm to 9 mm, and 4 mm to 8 mm. Similarly, the length of the distal frustoconical portion (measured along the longitudinal axis) can vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, and 4 mm to 7 mm. The diameter of the flow cell nozzle chamber can vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, and 4 mm to 7 mm in some embodiments.

[0082] In certain cases, the flow cell does not include a cylindrical portion and the entire interior chamber of the flow cell is formed in a frustoconical shape. In these embodiments, the length of the frustoconical interior chamber (measured along the longitudinal axis that is transverse to the nozzle orifice) can be in the range of 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, for example, 2 mm to 10 mm, for example, 3 mm to 9 mm, and 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical nozzle chamber can be in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, and 4 mm to 7 mm.

[0083] In some embodiments, the sample flow stream is emitted from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the flow cell orifice can be of any suitable shape, and non-limiting examples of the cross-sectional shape of interest include linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In certain embodiments, the flow cell of interest has a circular orifice. The size of the nozzle orifice can vary in the range of 1 μm to 20,000 μm, including, for example, 2 μm to 17,500 μm, for example 5 μm to 15,000 μm, for example 10 μm to 12,500 μm, for example 15 μm to 10,000 μm, for example 25 μm to 7,500 μm, for example 50 μm to 5,000 μm, for example 75 μm to 1,000 μm, for example 100 μm to 750 μm, and 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0084] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the flow cell interior chamber. Depending on the desired characteristics of the flow stream, the rate of the sample transmitted to the flow cell chamber by the sample injection port can be 1 μL / min or more, including, for example, 2 μL / min or more, for example 3 μL / min or more, for example 5 μL / min or more, for example 10 μL / min or more, for example 15 μL / min or more, for example 25 μL / min or more, for example 50 μL / min or more, and 100 μL / min or more. In some cases, the rate of the sample transmitted to the flow cell chamber by the sample injection port can be 1 μL / sec or more, including, for example, 2 μL / sec or more, for example 3 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, for example 15 μL / sec or more, for example 25 μL / sec or more, for example 50 μL / sec or more, and 100 μL / min or more.

[0085] The sample injection port can be an orifice positioned on the wall of the internal chamber or a conduit positioned at the proximal end of the internal chamber. When the sample injection port is an orifice positioned on the wall of the internal chamber, the sample injection port orifice can be of any suitable shape, and the cross-sectional shapes of interest include, but are not limited to, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on the shape, and in certain cases, for example, it has an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0086] In certain instances, the sample injection port is a conduit positioned at the proximal end of the flow cell internal chamber. For example, the sample injection port can be a conduit positioned to have an orifice of the sample injection port along the flow cell orifice. When the sample injection port is a conduit positioned along the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, and examples of the cross-sectional shapes of interest include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. The orifice of the conduit can vary depending on the shape and, in certain instances, has an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, for example, 1.5 mm. The shape of the tip of the sample injection port can be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port can include a slanted tip having a bevel angle in the range of 1° to 10°, including, for example, 2° to 9°, for example, 3° to 8°, for example, 4° to 7°, and 5°.

[0087] In some embodiments, the flow cell also includes a sheath fluid injection port configured to provide a sheath fluid to the flow cell. In an embodiment, the sheath fluid injection system is configured to provide a flow of sheath fluid to the flow cell internal chamber, for example, in combination with the sample, to generate a laminar flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid transmitted to the flow cell chamber can be 25 μL / second or more, including, for example, 50 μL / second or more, for example, 75 μL / second or more, for example, 100 μL / second or more, for example, 250 μL / second or more, for example, 500 μL / second or more, for example, 750 μL / second or more, for example, 1000 μL / second or more, and 2500 μL / second or more.

[0088] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the inner chamber. The sheath fluid injection port orifice can be of any suitable shape, and examples of the cross-sectional shape of interest include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. The size of the sample injection port orifice can vary depending on the shape, and in certain cases, it includes 1.25 mm to 1.75 mm, for example, in the range of 0.1 mm to 5.0 mm such as 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, etc., and has an opening of, for example, 1.5 mm such as 1 mm to 2 mm.

[0089] In some embodiments, the system further includes a pump that is in fluid communication with the flow cell to propagate a flow stream through the flow cell. Any convenient fluid pump protocol may be employed to control the flow of the flow stream through the flow cell. In certain cases, the system includes a peristaltic pump, such as a peristaltic pump having a pulse damper. The pump within the subject system is configured to transfer fluid through the flow cell at a rate suitable for detecting light from a sample within the flow stream. In some cases, the velocity of the sample flow within the flow cell is 1 μL / min (microliters per minute) or more, including, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, for example, 75 μL / min or more, for example, 100 μL / min or more, for example, 250 μL / min or more, for example, 500 μL / min or more, for example, 750 μL / min or more, and 1000 μL / min or more. For example, the system may include a pump configured to flow a sample through the flow cell at a rate in the range of 1 μL / min to 500 μL / min, such as 1 μL / min to 250 μL / min including 10 μL / min to 50 μL / min, such as 1 μL / min to 100 μL / min, such as 2 μL / min to 90 μL / min, such as 3 μL / min to 80 μL / min, such as 4 μL / min to 70 μL / min, such as 5 μL / min to 60 μL / min. In certain embodiments, the flow rate of the flow stream is 5 μL / min to 6 μL / min.

[0090] In certain embodiments, the subject system is a flow cytometry system. Suitable flow cytometry systems may 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. Jan; 49(pt 1):17-28; Linden, et.al., Semin Throm Hemost. 2004 Oct; 30(5):502-11; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are hereby incorporated by reference.In certain cases, the flow cytometry systems in question include the BD Biosciences FACSCanto™ flow cytometer, the BD Biosciences FACSCanto™ II flow cytometer, the BD Accuri™ flow cytometer, the BD Accuri™ C6 Plus flow cytometer, the BD Biosciences FACSCelesta™ flow cytometer, the BD Biosciences FACSLyric™ flow cytometer, the BD Biosciences FACSVerse™ flow cytometer, the BD Biosciences FACSymphony™ flow cytometer, the BD Biosciences LSRFortessa™ flow cytometer, the BD Biosciences LSRFortessa™ X-20 flow cytometer, the BD Biosciences FACSPresto™ flow cytometer, the BD Biosciences FACSVia™ flow cytometer, and the BD Biosciences FACSCalibur™ cell sorter, the BD Biosciences FACSCount™ cell sorter, the BD Biosciences FACSLyric™ cell sorter, and the BD Biosciences Via™ cell sorter, the BD Biosciences Influx™ cell sorter, the BD Biosciences Jazz™ cell sorter, the BD Biosciences Aria™ cell sorter, the BD Biosciences FACSAria™ II cell sorter, the BD Biosciences FACSAria™ III cell sorter, the BD Biosciences FACSAria™ Fusion cell sorter, and the BD Biosciences FACSMelody™ cell sorter, the BD Biosciences FACSymphony™ S6 cell sorter, and the like.

[0091] In some embodiments, the system is a flow cytometric system such as those described in U.S. Patent Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766, the disclosures of which are incorporated herein by reference in their entirety.

[0092] In some embodiments, the subject system is a particle sorting system configured to sort particles using a closed particle sorting module such as those described in U.S. Patent Publication No. 2017 / 0299493, the disclosure of which is incorporated herein by reference. In certain embodiments, sample particles (e.g., cells) are sorted using a sorting determination module having a plurality of sorting determination units such as those described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference. In particular embodiments, the subject system includes a particle sorting module having a deflection plate such as those described in U.S. Patent Publication No. 2017 / 0299493, filed Mar. 28, 2017, the disclosure of which is incorporated herein by reference.

[0093] In certain instances, the flow cytometry system of the present invention is configured to image particles in a flow stream by fluorescence imaging using high-frequency-tagged emission (FIRE), such as those described in Diebold, et al. Nature Photonics Vol.7(10);806-810(2013), and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, and U.S. Patent Publications 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894. These disclosures are incorporated herein by reference.

[0094] In some embodiments, the system is a particle analyzer that can analyze and characterize particles, with or without physically sorting the particles into a collection container, using a particle analysis system 401 (FIG. 4A). FIG. 4A shows a functional block diagram of a particle analysis system for computational-based sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 shown in FIG. 4A can be configured to perform, in whole or in part, the methods described herein. The particle analysis system 401 includes a fluidics system 402. The fluidics system 402 can include or be connected to a sample tube 405 and a moving fluid column within the sample tube through which sample particles 403 (e.g., cells) move along a common sample path 409.

[0095] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as the particles pass through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. In some implementations, detection may include detecting the light of those particles, or one or more other characteristics, as the particles 403 pass through the monitoring area 407. In FIG. 4A, one detection station 408 having one monitoring area 407 is shown. Some implementations of the particle analysis system 401 can include multiple detection stations. Further, some detection stations can monitor two or more regions.

[0096] Each signal is assigned a signal value for forming a data point for each particle. As described above, this data can be referred to as event data. The data point can be a multi-dimensional data point that includes the value of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0097] The particle analysis system 401 can also include a control system 306. The control system 406 can include one or more processors, an amplitude control circuit, and / or a frequency control circuit. The control system shown can be operably associated with the fluidics system 402. The control system can be configured to generate a signal frequency calculated for at least a portion of the first period based on a Poisson distribution and the number of data points collected by the detection system 404 during the first period. The control system 406 can be further configured to generate an experimental signal frequency based on the number of data points in a portion of the first period. The control system 406 can additionally compare the experimental signal frequency to that of the calculated signal frequency or a predetermined signal frequency.

[0098] Figure 4B shows a system for flow cytometry according to an exemplary embodiment of the present invention. This system includes a flow cytometer 400, a controller / processor 490, and a memory 495. The flow cytometer 470 includes one or more excitation lasers 471, 472, 473, 474, and 475, a focusing lens 476, a flow chamber 477, a forward scatter detector 480a, a side scatter detector 480b, and a fluorescence focusing lens 440.

[0099] The excitation lasers 471, 472, 473, 474, and 475 emit light in the form of laser beams. The wavelengths of the laser beams emitted from the excitation lasers 471, 472, 473, 474, and 475 are 637 nm, 561 nm, 488 nm, 405 nm, and 349 nm, respectively, in the exemplary system of Figure 4B. The laser beams are first directed through one or more of the beam splitters 471a, 472a, 473a, 474a, and 475a. The beam splitter 475a transmits light at 637 nm, 561 nm, 488 nm, and 405 nm and reflects light at 349 nm. The beam splitter 474a transmits light at 637 nm, 561 nm, and 488 nm and reflects light at 405 nm. The beam splitter 473a transmits light at 637 nm and 561 nm and reflects light at 488 nm. The beam splitter 472a transmits light at 637 nm and reflects light at 561 nm. The beam splitter 471a reflects light at 637 nm. The laser beams are then directed to the focusing lens 476, which focuses the beams onto the portion of the fluid stream in the flow chamber 477 where the sample particles are located. The flow chamber is part of a fluidics system that directs the particles in the stream, typically one at a time, onto the focused laser beam for investigation. The flow chamber can include a flow cell within a benchtop flow cytometer or a nozzle tip within a stream-in-air cytometer.

[0100] Light from the laser beam interacts with the particles in the sample by diffraction, refraction, reflection, scattering, and absorption with re-emission at various different wavelengths, depending on the properties of the particles, such as the size of the particles, the internal structure, and the presence of one or more fluorescent molecules attached to or naturally occurring on or in the particles. Fluorescent emission, as well as diffracted light, refracted light, reflected light, and scattered light, can be routed through the fluorescence focusing lens 440 to one or more of the forward scatter detector 480a, the side scatter detector 480b, and one or more of the light detection systems 478a, 478b, 478c, 478d, and 478e. In certain embodiments, light from the flow cell 477 is collected by optical collection components such as optical fibers or free space relay devices as described above and propagated to one or more of the light detection systems 478a, 478b, 478c, 478d, and 478e.

[0101] The light from the particles irradiated by each laser according to a particular embodiment is detected by a separate light detection system as described herein. As shown in FIG. 3, each light detection system is a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, wherein each set of linear variable optical filters is configured to pass light having a predetermined sub-spectrum range, a wavelength separator component, and a plurality of light detectors positioned to detect light from each sub-spectrum range across the linear variable optical filter. In FIG. 4B, the light detection module 478a is configured to detect light from particles propagating through the flow cell 477 irradiated by the laser 471 (e.g., a 637 nm laser), the light detection module 478b is configured to detect light from particles propagating through the flow cell 477 irradiated by the laser 472 (e.g., a 561 nm laser), the light detection module 478c is configured to detect light from particles propagating through the flow cell 477 irradiated by the laser 473 (e.g., a 488 nm laser), the light detection module 478d is configured to detect light from particles propagating through the flow cell 477 irradiated by the laser 474 (e.g., a 405 nm laser), and the light detection module 478e is configured to detect light from particles propagating through the flow cell 477 irradiated by the laser 475 (e.g., a 349 nm laser). In the embodiment shown in FIG. 4B, different light detection modules are used to detect light from the flow stream from each laser line. In an embodiment, the light detection modules can be arranged at any position and at any angle along the flow stream. In a particular case, as shown in FIG. 4B, the light detection modules are positioned in a row along the longitudinal axis of the flow stream. Depending on the irradiation position by each laser, the light detection modules can be separated from each other by at least 1 mm, including, for example, at least 5 mm, for example, at least 10 mm, for example, at least 25 mm, for example, at least 50 mm, for example, at least 100 mm, and at least 250 mm, along the longitudinal axis of the flow stream.

[0102] The forward scatter detector 480a is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, excitation light that travels mostly in the forward direction through or around the particles. The intensity of the light detected by the forward scatter detector depends on the overall size of the particles. The forward scatter detector can include a photodiode. The side scatter detector 480b is configured to detect diffracted and reflected light from the surface and internal structure of the particles and tends to increase as the particle structure becomes more complex. The signals detected by the forward scatter detector 480a, the side scatter detector 480b, and the fluorescence detector can be converted by the detectors into electrical signals (voltages). This data can provide information about the sample.

[0103] One of ordinary skill in the art will recognize that a flow cytometer according to an embodiment of the present invention is not limited to the flow cytometer shown in FIG. 4B and can include any flow cytometer known in the art. For example, the flow cytometer can have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in various different configurations.

[0104] During operation, the operation of the flow cytometer is controlled by the controller / processor 490, and the measurement data from the detector is stored in the memory 495 and can be processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive an output signal therefrom, and is also coupled to the electrical and electromechanical components of the flow cytometer 400 to control the laser, fluid flow parameters, etc. An input / output (I / O) function unit 497 may also be provided within the system. The memory 495, the controller / processor 490, and the I / O 497 may be provided as an integral part of the flow cytometer 410. In such an embodiment, the display may also form part of the I / O function unit 497 for presenting experimental data to the user of the cytometer 400. Alternatively, some or all of the memory 495, the controller / processor 490, and the I / O function unit may be part of one or more external devices such as a general-purpose computer. In some embodiments, some or all of the memory 495 and the controller / processor 490 can communicate with the flow cytometer 400 wirelessly or by wire. Together with the memory 495 and the I / O 497, the controller / processor 490 may be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0105] The I / O 497 may be configured to receive data related to flow cytometer experiments having a panel of fluorescent labels and multiple cell populations having multiple markers, each cell population having a subset of the multiple markers. The I / O 497 may also be configured to receive biological data assigning one or more markers to one or more cell populations, marker concentration data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data such as label spectral characteristics and flow cytometer configuration data may also be stored in the memory 495. The controller / processor 490 may be configured to evaluate one or more assignments of labels to markers.

[0106] Figure 5 shows a functional block diagram of an example of a particle analyzer control system, such as analysis controller 500, for analyzing and displaying biological events. Analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0107] Particle analyzer or particle sorting system 502 can be configured to obtain biological event data. For example, a flow cytometer can generate flow cytometric event data. Particle analyzer 502 can be configured to provide biological event data to analysis controller 500. A data communication channel can be included between particle analyzer or particle sorting system 502 and analysis controller 500. Biological event data can be provided to analysis controller 500 via the data communication channel.

[0108] Analysis controller 500 can be configured to receive biological event data from particle analyzer or sorting system 502. The biological event data received from particle analyzer or sorting system 502 can include flow cytometric event data. Analysis controller 500 can be configured to provide a graphical display including a first plot of the biological event data to display device 506. Analysis controller 500 can be further configured to render a target region as a gate around a population of biological event data shown by display device 506, for example, overlaid on the first plot. In some embodiments, the gate can be a logical combination of one or more graphical regions of interest drawn on a histogram of a single parameter or a bivariate plot. In some embodiments, the display can be used to display particle parameters or saturated detector data.

[0109] The analysis controller 500 can be further configured to display biometric event data on the display device 506 within the gate, differently from other events in the biometric event data outside the gate. For example, the analysis controller 500 can be configured to render the color of the biometric event data included within the gate to be distinguishable from the color of the biometric event data outside the gate. The display device 506 can be implemented as a monitor, a tablet computer, a smartphone, or other electronic devices configured to present a graphical interface.

[0110] The analysis controller 500 can be configured to receive a gate selection signal for identifying a gate from a first input device. For example, the first input device can be implemented as a mouse 510. This mouse 510 can initiate a gate selection signal to the analysis controller 500 for identifying a gate that is displayed or operated via the display device 506 (e.g., by clicking on a desired gate when positioning a cursor there). In some implementations, the first device can be implemented as a keyboard 508, or other means for providing an input signal to the analysis controller 500, such as a touch screen, an input pen, a light detector, or a voice recognition system. Some input devices can include multiple input functions. In such implementations, each input function can be considered an input device. For example, as shown in FIG. 5, the mouse 510 can include a right mouse button and a left mouse button, each of which can generate a triggering event.

[0111] The triggering event can cause the analysis controller 500 to change the way data is displayed, which parts of the data are actually displayed on the display device 506, and / or provide an input to further processing, such as the selection of the population of particles to be sorted.

[0112] In some embodiments, the analysis controller 500 can be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the visualization of the plot to facilitate the gating process. This modification can be based on a particular distribution of the biological event data received by the analysis controller 500.

[0113] The analysis controller 500 can be connected to a memory device 504. This memory device 504 can be configured to receive and store biological event data from the analysis controller 500. The memory device 504 can also be configured to receive and store flow cytometric event data from the analysis controller 500. The memory device 504 can be further configured to enable the analysis controller 500 to search for biological event data such as flow cytometric event data.

[0114] The display device 506 can be configured to receive display data from the analysis controller 500. The display data can include a plot of the biological event data and gates that delineate the boundaries of sections of that plot. The display device 506 can be further configured to modify the information presented in accordance with the input received from the analysis controller 500 in conjunction with input from the particle analyzer 502, the memory device 504, the keyboard 508, and / or the mouse 510.

[0115] In some implementations, the analysis controller 500 can generate a user interface and receive exemplary events for sorting. For example, this user interface can include controls for receiving exemplary events or exemplary images. The exemplary events or images, or exemplary gates, can be provided prior to the collection of event data for a sample or based on an initial set of events for a portion of the sample.

[0116] FIG. 6A is a schematic diagram of a particle sorter system 600 (e.g., a particle analyzer or particle sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. As shown in FIG. 6A, a droplet formation transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, include, or be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are lined up in a single file and cross a monitoring area 611 (e.g., where a laser stream intersects) and are irradiated by an irradiation source 612 (e.g., a laser). Due to the vibration of the droplet formation transducer 602, the moving fluid column 608 splits into a plurality of droplets 610, some of which contain particles 609.

[0117] During operation, a detection station 614 (e.g., an event detector) identifies when a target particle (or target cell) crosses the monitoring area 611. The detection station 614 supplies an input to a timing circuit 628, which then supplies an input to a flash charge circuit 630. At a droplet splitting point notified by a time-limited droplet delay (Δt), a flash charge can be applied to the moving fluid column 608, and thus the target droplet carries a charge. The target droplet can contain one or more particles or cells to be sorted. The charged droplet is then sorted by activating a deflection plate (not shown) and deflected into a container such as a collection tube, or a multi-well or microwell sample plate where wells or microwells can be associated with specific target droplets. As shown in FIG. 6A, the droplets can be collected in a drain container 638.

[0118] The detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal when the target particle passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is hereby incorporated by reference in its entirety. The detection system 616 enables the device to accurately calculate the position of each detected particle in the droplet. The detection system 616 can be input-fed to the amplitude signal 620 and / or the phase 618 signal, which are then input-fed (via the amplifier 622) to the amplitude control circuit 626 and / or the frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then controls the droplet formation transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included within the control system.

[0119] In some implementations, the sorting electronics (e.g., the detection system 616, the detection station 614, and the processor 640) can be coupled to a memory configured to store the detected events and the sorting decisions based thereon. The sorting decisions can be included in the particle event data. In some embodiments, the detection system 616 and the detection station 614 can be implemented as a single detection unit or communicatively coupled such that event measurements can be collected by one of the detection system 616 or the detection station 614 and provided to the non-collecting element.

[0120] FIG. 6B is a schematic diagram of a particle sorter system according to an embodiment presented herein. The particle sorter system 600 shown in FIG. 6B includes deflection plates 652 and 654. Charge can be applied via a stream charging wire within the barb. This creates a stream of droplets 610 that includes particles 610 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed, such as by sorting electronics or other detection systems (not shown in FIG. 6B). The deflection plates 652 and 654 are independently controlled to attract or repel charged droplets and direct the droplets towards a desired collection container (e.g., one of 672, 674, 676, or 678). As shown in FIG. 6B, the deflection plates 652 and 654 are controlled to direct the particles along a first path 662 towards container 674 or along a second path 668 towards container 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflection plate may allow the particle to continue to travel along flow path 664. Such uncharged droplets can be transferred into a waste container, such as via aspirator 670.

[0121] The sorting electronics can include starting the collection of measurements, receiving the fluorescence signal regarding the particle, and determining how to adjust the deflection plates to cause sorting of the particle. An exemplary implementation of the embodiment shown in FIG. 6B includes the BD FACSAria™ flow cytometer commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0122] Computer control system Aspects of the present disclosure further include a computer control system, the system further including one or more computers for full automation or partial automation. In some embodiments, the system includes a computer having a computer-readable storage medium storing a computer program, the computer program being configured, when loaded into the computer, to be a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, each set of linear variable optical filters being configured to pass light having a predetermined sub-spectrum range, a wavelength separator component, and a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filter, a light detection system having, and instructions for irradiating particles propagating along a flow stream through an investigation region.

[0123] In some embodiments, the computer control system includes a memory storing instructions for binning data signals from different photodetector channels. In some embodiments, the computer program includes instructions for dynamically binning data signals from two or more different photodetector channels to generate a combined spectral data signal. In some cases, the computer program includes instructions for binning data signals from non-adjacent photodetectors. In some cases, the computer program includes instructions for binning data signals from adjacent photodetectors. In a particular case, the computer program includes instructions for horizontal binning of data signals from adjacent photodetectors. In some embodiments, the computer program includes instructions for binning data signals from three or more different photodetector channels, such as four or more, such as five or more, such as six or more, such as sixteen or more, such as thirty-two or more, and sixty-four or more different photodetector channels. In some embodiments, the binned data signal is a wavelength-separated data signal. In some embodiments, the binned data signal is a time-separated data signal. In some embodiments, the binned data signal is a time- and wavelength-separated data signal.

[0124] In certain embodiments, the computer program includes instructions for amplifying data signals from one or more photodetector channels. In some cases, the computer program includes instructions for amplifying data signals from each photodetector channel and binning the amplified data signals. In some embodiments, the computer program includes instructions for multiplexing the generated data signals. In some cases, the computer program includes instructions for binning the multiplexed data signals.

[0125] In an embodiment, the system includes an input module, a processing module, and an output module. The system of the subject matter may include both hardware components and software components. The hardware components may take the form of one or more platforms, for example, in the form of a server. As a result, the functional elements of the system, i.e., those elements of the system that perform specific tasks (such as managing input and output of information, processing information, etc.), may be executed by the execution of software applications on and across one or more computer platforms represented by the system.

[0126] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses a memory having instructions stored therein for performing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, a memory storage device, and an input / output controller, cache memory, a data backup unit, as well as many other devices. The processor may be a commercially available processor or one of other processors that are available or will be available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to cooperate and execute the functions of various computer programs that may be described in various programming languages such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of 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. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that enable a user to manually align a light source with a flow stream based on a first optical signal and a second optical signal. In some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0127] The system memory can 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 resident hard disks or tapes, optical media such as read / write compact disks, flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices including, for example, a compact disk drive, tape drive, removable hard disk drive, or disk 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 disk. Any of these program storage media, or others currently in use or that may be developed later, can be considered a computer program product. As is understood, these program storage media typically store computer software programs and / or data. The computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or in a program storage device used in conjunction with the memory storage device.

[0128] In some embodiments, a computer program product is described that includes a computer-usable medium having control logic (a computer software program including program code) stored therein. The control logic, when executed by a processor, causes the computer, the processor, to perform the functions described herein. In other embodiments, some of the functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of a hardware state machine for performing the functions described herein will be apparent to those skilled in the relevant art.

[0129] The memory can be any suitable device such as a magnetic, optical, or solid-state storage device (including magnetic or optical disks, or tapes, or RAM, or any other suitable device, either fixed or portable). The processor can include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming can be provided remotely to the processor via a communication channel, or can be pre-stored using any of those devices together with the memory in a computer program product such as a memory or any other portable or fixed computer-readable storage medium. For example, a magnetic or optical disk can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product, and algorithms for use in practicing the above methods. The programming according to the present invention can be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tapes, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0130] The processor can also have access to a communication channel for communicating with a user at a remote location. A remote location means that the user does not directly contact the system, and it means relaying input information from an external device such as a computer connected to any other suitable communication channel including a wide area network ("WAN"), a telephone network, a satellite network, or a mobile phone (i.e., a smartphone) to the input manager.

[0131] In some embodiments, the system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface can be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID)), ZigBee communication protocol, 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).

[0132] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as USB ports, RS-232 ports, or any other suitable electrical connection ports, to enable data communication between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communication.

[0133] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the subject system to communicate with other devices, such as computer terminals and / or networks, communicable mobile phones, personal digital assistants, or any other communication devices that the user may use together.

[0134] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet protocol (IP) via a cellular phone network, short message service (SMS), wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or WiFi connection to the Internet at a WiFi hotspot.

[0135] In one embodiment, the subject system is configured to wirelessly communicate with a server device via a communication interface using a common standard such as, for example, the 802.11 or Bluetooth® RF protocol, or the IrDA infrared protocol. The server device can be another portable device such as a smartphone, a personal digital assistant (PDA), or a notebook computer, or a larger device such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display such as a liquid crystal display (LCD), and an input device such as buttons, a keyboard, a mouse, or a touch screen.

[0136] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored within the subject system, e.g., within an optional data storage unit, with a network or a server device using one or more of the communication protocols and / or mechanisms described above.

[0137] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information can typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. The functional elements of a computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide the user at a remote location, in accordance with known techniques, e.g., via the Internet, telephone, or satellite network, with information generated by the processing module. The presentation of data by the output manager may be implemented in accordance with a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include an Internet URL address so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present within the subject system will typically be of the class of computers commonly referred to as servers, but can be of any type of known computer platform or type to be developed in the future. On the other hand, they can be mainframe computers, workstations, or other computer types. They can be connected via other communication systems, including any known or future type of cable wiring or a wireless system, whether networked or not. They can be located in the same place or physically separated. Optionally, depending on the type and / or configuration of the selected computer platform, various operating systems may be employed on any of the computer platforms.Examples of suitable operating systems include Windows NT (registered trademark), Windows XP, Windows 7, Windows 8, iOS, Oracle Solaris, Linux (registered trademark), OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and the like.

[0138] FIG. 7 shows a general architecture of an exemplary computing device 700 according to a particular embodiment. The general architecture of the computing device 700 shown in FIG. 7 includes the arrangement of computer hardware and software components. The computing device 700 may include more (or fewer) elements than those shown in FIG. 7. However, not all of these generally traditional elements need to be shown in order to provide an effective disclosure. As illustrated, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which may communicate with each other via a communication bus. The network interface 720 may provide a connection to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via the network. The processing unit 710 may also communicate with the memory 770 and may further provide output information for an optional display 750 via the input / output device interface 740. The input / output device interface 740 may also receive input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device.

[0139] Memory 770 may contain computer program instructions (grouped as modules or components in some embodiments) that the processing unit 710 executes in sequence to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of computing device 700. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0140] Method for Detecting Light from Particles in a Frost Stream by Spectrum Identification Aspects of the present disclosure also include a method for measuring light from a sample (e.g., within a flow stream in a flow cytometer). In practicing the method according to an embodiment, the sample is irradiated with a light source, and the light from the sample is a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, wherein each set of linear variable optical filters is configured to pass light having a predetermined sub-spectrum range, and detected by a light detection system having a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filters. In some embodiments, the sample is a biological sample. The term "biological sample" is used in its conventional sense to refer to whole organisms, plants, fungi, or, in certain cases, a subset of animal tissues, cells, or components found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to both a natural organism or a subset of its tissues, as well as a homogenate, lysate, or extract prepared from a subset of a biological or its tissues, including but not limited to plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory, gastrointestinal, cardiovascular, and urinary organs, tears, saliva, milk, blood cells, tumors, and organs. A biological sample can be any type of biological tissue, including both healthy tissue 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 including whole blood, such as blood obtained by venipuncture or finger stick (the blood may or may not be combined with any reagents such as preservatives, anticoagulants, etc. prior to the assay).

[0141] In certain embodiments, the sample source is a "mammal" or "mammalian animal", terms that are widely used to describe organisms within the class of mammals, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method can be applied to samples obtained from human subjects of either sex at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is a juvenile, adolescent, or adult. The present invention can be applied to samples from human subjects, but is not limited to, samples from other animal subjects such as birds, mice, rats, dogs, cats, livestock, and horses (i.e., "non-human subjects").

[0142] In practicing the subject method, a sample (e.g., within the flow stream of a flow cytometer) is irradiated 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, such as including 500 nm or greater, 100 nm or greater, 150 nm or greater, 200 nm or greater, 250 nm or greater, 300 nm or greater, 350 nm or greater, 400 nm or greater, e.g., up to 50 nm or greater. 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 irradiating with a broadband light source, examples of broadband light source protocols of interest include, but are not limited to, among other broadband light sources, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs having a continuous spectrum, ultra-high brightness light emitting diodes, semiconductor light emitting diodes, wide area spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof.

[0143] In other embodiments, the method includes irradiating with a narrow-band light source that emits light at a specific wavelength or a narrow range of wavelengths, such as a light source that emits light at a wavelength in the range of 50 nm or less, 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, including, for example, a light source that emits light of a specific wavelength (i.e., monochromatic light). When the method includes irradiating with a narrow-band light source, examples of the narrow-band light source of interest include, but are not limited to, a laser, a narrow-wavelength LED, a laser diode, or a broadband light source combined with one or more optical band-pass filters, diffraction gratings, monochromators, or any combination thereof.

[0144] In certain embodiments, the method includes irradiating a sample with one or more lasers. As described above, the type and number of lasers can vary depending on the sample and the desired light to be collected, and can be gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluoride (ArF) excimer lasers, krypton-fluoride (KrF) excimer lasers, xenon chloride (XeCl) excimer lasers, xenon-fluoride (XeF) excimer lasers, or combinations thereof. In other cases, the method includes irradiating a flow stream with a dye laser such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, the method includes irradiating a flow stream 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, or a gold laser, and combinations thereof. In still other cases, the method includes irradiating a flow stream with a solid laser such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a slim YAG laser, a ytterbium YAG laser, a ytterbium 2O3 laser, or a cerium-doped laser, and combinations thereof.

[0145] The sample can be irradiated with two or more light sources, such as three or more light sources, four or more light sources, five or more light sources, including ten or more light sources, with one or more of the light sources described above. The light sources can include any combination of light source types. For example, in some embodiments, the method includes irradiating a sample in a 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 lasers.

[0146] The sample can be irradiated with wavelengths in the range of 250 nm to 1250 nm including 400 nm to 800 nm, such as 300 nm to 1000 nm, 350 nm to 900 nm, 200 nm to 1500 nm. For example, when the light source is a broadband light source, the sample can be irradiated with wavelengths of 200 nm to 900 nm. In other cases, when the light source includes a plurality of narrowband light sources, the sample can be irradiated with specific wavelengths in the range of 200 nm to 900 nm. For example, the light source can be a plurality of narrowband LEDs (1 nm to 25 nm) that independently emit light having wavelengths in the range of 200 nm to 900 nm. In other embodiments, the narrowband light source includes one or more lasers (such as a laser array), and the sample is irradiated with specific wavelengths in the range of 200 nm to 700 nm by a laser array having a gas laser, an excimer laser, a dye laser, a metal vapor laser, and a solid laser as described above.

[0147] When two or more light sources are employed, the sample can be irradiated simultaneously, sequentially, or in a combination thereof by the light sources. For example, the sample can be irradiated simultaneously by each of the light sources. In other embodiments, the flow stream is irradiated sequentially by each of the light sources. When two or more light sources are used to irradiate the sample sequentially, the irradiation time of each light source can be independently, for example, 0.001 microseconds or more, including, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, and 60 microseconds or more. For example, the method can include irradiating the sample with a light source (such as a laser) for a duration in the range of 0.001 microseconds to 100 microseconds, including, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In embodiments where the sample is irradiated sequentially by two or more light sources, the duration for which the sample is irradiated by each light source can be the same or different.

[0148] Also, the periods between irradiations by each light source can be independently separated by a delay of 0.001 microseconds or more, such as 60 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 15 microseconds or more, 30 microseconds or more, and can be different as needed. For example, the periods between irradiations by each light source can be in the range of 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microseconds to 25 microseconds, including 5 microseconds to 10 microseconds. In a certain specific embodiment, the period between irradiations by each light source is 10 microseconds. In embodiments where the sample is sequentially irradiated by more than two (i.e., three or more) light sources, the delays between irradiations by each light source can be the same or different.

[0149] The sample can be irradiated continuously or at discrete intervals. In some cases, the method includes continuously irradiating the sample within the sample with a light source. In other cases, the sample is irradiated by the light source at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds, or at some other intervals.

[0150] Depending on the light source, the sample can be irradiated from different distances, such as 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, including 50 mm or more. Also, the angle of irradiation can also be different in the range of 10° to 90°, such as 15° to 85°, 20° to 80°, 25° to 75°, including 30° to 60°, for example, at an angle of 90°.

[0151] As discussed above, in embodiments, light from the irradiated sample is transmitted to a light detection system and detected by a plurality of photodetectors as described herein. The light can be measured continuously or at discrete intervals in the light detection system. In some cases, the method includes continuously measuring the light. 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, every 1000 milliseconds, or at some other interval.

[0152] Measurements of the collected light can be made one or more times during the method of the subject, such as two or more times, such as three or more times, such as five or more times, and such as ten or more times. In certain embodiments, the light propagation is measured two or more times, and in certain cases, the data is averaged.

[0153] In some embodiments, the method includes adjusting the light before detecting the light with the light detection system of the subject. For example, light from the sample source can pass through one or more lenses, mirrors, pinholes, slits, gratings, photorefractive devices, and any combination thereof. In some cases, the collected light passes through one or more focusing lenses to reduce the profile of the light directed to the light detection system or the optical collection system as described above. In other cases, light emitted from the sample passes through one or more collimators to reduce the divergence of the light beam being transmitted to the light detection system.

[0154] Kit Also provided are kits having one or more components of the subject light detection system. Kits according to certain embodiments include a first set of linear variable optical filters, a second set of linear variable optical filters, and a light detector. In some embodiments, one or more of the linear variable optical filters are linear variable bandpass filters, such as long pass linear variable bandpass filters or short pass linear variable bandpass filters. In some instances, the kit includes an optical collection component, such as an optical fiber light relay bundle or a free space optical relay system. The kit may also include one or more light detectors, such as a photomultiplier tube (e.g., a metal package photomultiplier tube) or a photodiode (e.g., an avalanche photodiode, APD).

[0155] The kit may also include a flow cell nozzle and a cuvette for irradiating the flow stream. The kit may also include optical conditioning components, such as lenses (e.g., focusing lenses, objective lenses), mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating protocols, and combinations thereof. In certain instances, the kit includes one or more light sources, such as a laser, an LED, a broadband light source, or combinations thereof.

[0156] In addition to the above components, the subject kit may further include (in certain embodiments) instructions for implementing the subject method. These instructions may be present in the subject kit in a variety of forms, one or more of which may be present within the kit. One form in which these instructions may be present is, for example, as printed information on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, within the package of the kit, in an attached document, etc. Another form of these instructions is a computer-readable medium on which the information is recorded, such as a diskette, a compact disk (CD), a portable flash drive, etc. Yet another form of these instructions that may be present is a website address that may be used via the Internet to access the information at a remote site.

[0157] Utility The subject methods, systems, and computer systems find use in a variety of applications where it is desirable to optimize the identification, characterization, and sorting of particles. The subject methods and systems provide for detecting light using spectral identification and for identifying or characterizing particles within a flow stream. The present disclosure also finds use in flow cytometry, where it is desirable to provide a flow cytometer having improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In embodiments, the present disclosure reduces the need for user input or manual adjustment during sample analysis by a flow cytometer. In certain embodiments, the subject methods and systems provide a fully automated protocol such that adjustment of the flow cytometer in use requires little to no human input, even with any human input present.

[0158] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses. 1. A light detection system comprising: a wavelength separator component configured to propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, each set of linear variable optical filters being configured to pass light having a predetermined sub-spectral range across the linear variable optical filter; and a plurality of photodetectors positioned to detect light from each sub-spectral range across the linear variable optical filter. 2. The light detection system of clause 1, wherein each set of linear variable optical filters is configured to pass light having wavelengths from 200 nm to 1200 nm across the linear variable optical filter. 3. The light detection system of clause 1, wherein each set of linear variable optical filters is configured to pass light having wavelengths from 500 nm to 800 nm across the linear variable optical filter. 4. The optical detection system according to clause 3, wherein the first set of linear variable optical filters is configured to pass light having a wavelength of 500 nm to 650 nm across the linear variable optical filter.

[0159] 5. The optical detection system according to clause 3, wherein the second set of linear variable optical filters is configured to pass light having a wavelength of 650 nm to 800 nm across the linear variable optical filter. 6. The optical detection system according to any one of clauses 1 to 5, wherein each set of linear variable optical filters includes a long-pass linear variable band-pass filter and a short-pass linear variable band-pass filter. 7. The optical detection system according to clause 6, wherein the long-pass variable band-pass filter is offset from the short-pass linear variable band-pass filter by a predetermined distance in each set of linear variable optical filters. 8. The optical detection system according to clause 7, wherein the light in the sub-spectrum range across each set of linear variable optical filters is determined by the offset of the long-pass variable band-pass filter from the short-pass linear variable band-pass filter. 9. The optical detection system according to any one of clauses 1 to 6, wherein the light in each sub-spectrum range passing across the set of linear variable optical filters includes a spectral width of 5 nm to 50 nm.

[0160] 10. The optical detection system according to clause 9, wherein the light in each sub-spectrum range passing across the set of linear variable optical filters includes a spectral width of about 20 nm. 11. The optical detection system according to any one of clauses 1 to 10, wherein the first set of linear variable optical filters and the second set of linear variable optical filters are positioned along two parallel planes. 12. The optical detection system according to clause 11, wherein the wavelength separator component is configured to transmit light to and from between the first set of linear variable optical filters and the second set of linear variable optical filters. 13. The wavelength separator component is A first set of linear variable optical filters is configured to allow light to pass from a low wavelength to a high wavelength across the linear variable optical filter, The optical detection system according to any one of clauses 1 to 12, wherein a second set of linear variable optical filters is configured to allow light to pass from a high wavelength to a low wavelength across the linear variable optical filter. 14. The optical detection system according to any one of clauses 1 to 13, wherein a plurality of photodetectors are positioned sandwiching each set of linear variable optical filters.

[0161] 15. The optical detection system according to clause 14, wherein each photodetector is configured to detect light in a sub-spectrum range having a spectral width of 5 nm to 50 nm. 16. The optical detection system according to clause 15, wherein each photodetector is configured to detect light in a sub-spectrum range having a spectral width of about 20 nm. 17. The optical detection system includes A first set of photodetectors positioned sandwiching the first set of linear variable optical filters, a) 500 nm to 520 nm, b) 520 nm to 540 nm, c) 540 nm to 560 nm, d) 560 nm to 580 nm, e) 580 nm to 600 nm, f) 600 nm to 620 nm, g) 620 nm to 640 nm, h) The first set of photodetectors configured to detect light having a sub-spectrum range of 640 nm to 660 nm, and A second set of photodetectors positioned sandwiching the second set of linear variable optical filters, a) 800 nm to 780 nm, b) 780 nm to 760 nm, c) 760 nm to 740 nm, d) 740 nm to 720 nm, e) 720 nm to 700 nm, f) 700 nm to 680 nm, g) a second set of photodetectors configured to detect light having a sub - spectral range of 680 nm to 660 nm, the photodetection system according to any one of clauses 14 to 16, comprising

[0162] 18. The photodetection system according to any one of clauses 1 to 17, wherein the plurality of photodetectors comprises one or more photomultiplier tubes (PMTs). 19. The photodetection system according to any one of clauses 1 to 17, wherein the plurality of photodetectors comprises one or more photodiodes. 20. The photodetection system according to any one of clauses 1 to 17, wherein the plurality of photodetectors comprises one or more avalanche photodiodes (APDs).

[0163] 21. The photodetection system according to any one of clauses 1 to 20, wherein the plurality of photodetectors comprises a modulator component configured to bin data signals from two or more different photodetector channels. 22. The photodetection system according to clause 21, wherein each photodetector channel is configured to generate a data signal for each sub - spectral range of light. 23. The photodetection system according to clause 21 or 22, wherein the modulator component is configured to bin data signals from adjacent photodetector channels. 24. The photodetection system according to clause 23, wherein the modulator component is configured for horizontal binning of adjacent photodetector channels.

[0164] 25. The photodetection system according to any one of clauses 22 to 24, wherein the modulator component is configured to bin data signals from three or more different photodetector channels. 26. The photodetection system according to any one of clauses 21 to 25, wherein the modulator component comprises an output modulator configured to modulate the signals output from each photodetector channel. 27. The photodetection system according to clause 26, wherein the output modulator comprises an amplifier. 28. The optical detection system according to clause 27, wherein the amplifier is a transimpedance amplifier. 29. The optical detection system according to any one of clauses 26 to 28, wherein the output modulator includes a switch circuit. 30. The optical detection system according to any one of clauses 26 to 29, wherein the output modulator includes a differential amplifier. 31. The optical detection system according to any one of clauses 21 to 30, wherein the modulator component is configured to multiplex the generated data signal.

[0165] 32. A system comprising: a light source configured to irradiate particles propagating through a flow stream; an optical detection system comprising: a wavelength separator component configured to propagate light between a first set of linearly variable optical filters and a second set of linearly variable optical filters, each set of linearly variable optical filters being configured to pass light having a predetermined sub-spectrum range across the linearly variable optical filter; a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linearly variable optical filter. 33. The system according to clause 32, wherein the system is a particle analyzer. 34. The system according to clause 33, wherein the system is a flow cytometer. 35. The system according to any one of clauses 32 to 34, wherein each set of linearly variable optical filters is configured to pass light having a wavelength of 200 nm to 1200 nm across the linearly variable optical filter. 36. The system according to any one of clauses 32 to 35, wherein each set of linearly variable optical filters is configured to pass light having a wavelength of 500 nm to 800 nm across the linearly variable optical filter.

[0166] 37. The system according to clause 36, wherein a first set of linear variable optical filters is configured to pass light having a wavelength of 500 nm to 650 nm across the linear variable optical filters. 38. The system according to clause 36, wherein a second set of linear variable optical filters is configured to pass light having a wavelength of 650 nm to 800 nm across the linear variable optical filters. 39. The system according to any one of clauses 32 to 38, wherein each set of linear variable optical filters includes a long-pass linear variable band-pass filter and a short-pass linear variable band-pass filter. 40. The system according to clause 39, wherein the long-pass variable band-pass filter is offset from the short-pass linear variable band-pass filter by a predetermined distance in each set of linear variable optical filters. 41. The system according to clause 40, wherein the light in each sub-spectrum range across the set of linear variable optical filters is determined by the offset of the long-pass variable band-pass filter from the short-pass linear variable band-pass filter.

[0167] 42. The system according to any one of clauses 32 to 41, wherein the light in each sub-spectrum range passing across the set of linear variable optical filters includes a spectral width of 5 nm to 50 nm. 43. The system according to clause 42, wherein the light in each sub-spectrum range passing across the set of linear variable optical filters includes a spectral width of about 20 nm. 44. The system according to any one of clauses 32 to 43, wherein the first set of linear variable optical filters and the second set of linear variable optical filters are positioned along two parallel planes. 45. The system according to clause 44, wherein the wavelength separator component is configured to transmit light to and from between the first set of linear variable optical filters and the second set of linear variable optical filters. 46. The wavelength separator component is A first set of linear variable optical filters is configured to pass light from a low wavelength to a high wavelength across the linear variable optical filter, A second set of linear variable optical filters is configured to pass light from a high wavelength to a low wavelength across the linear variable optical filter, a system according to any one of clauses 32 to 45.

[0168] 47. A system according to any one of clauses 32 to 46, wherein a plurality of photodetectors are positioned sandwiching each set of linear variable optical filters. 48. A system according to clause 47, wherein each photodetector is configured to detect light in a sub - spectral range having a spectral width of 5 nm to 50 nm. 49. A system according to clause 48, wherein each photodetector is configured to detect light in a sub - spectral range having a spectral width of approximately 20 nm. 50. The photodetection system is A first set of photodetectors positioned sandwiching the first set of linear variable optical filters, i) 500 nm to 520 nm, j) 520 nm to 540 nm, k) 540 nm to 560 nm, l) 560 nm to 580 nm, m) 580 nm to 600 nm, n) 600 nm to 620 nm, and o) 620 nm to 640 nm, p) configured to detect light having a sub - spectral range of 640 nm to 660 nm, the first set of photodetectors, A second set of photodetectors positioned sandwiching the second set of linear variable optical filters, h) 800 nm to 780 nm, i) 780 nm to 760 nm, j) 760 nm to 740 nm, k) 740 nm to 720 nm, l) 720 nm to 700 nm, m) 700 nm to 680 nm, and n) a second set of photodetectors configured to detect light having a sub - spectral range of 680 nm to 660 nm, and the system according to any one of clauses 47 to 49, comprising the same.

[0169] 51. The system according to any one of clauses 32 to 50, wherein the plurality of photodetectors comprise one or more photomultiplier tubes (PMTs). 52. The system according to any one of clauses 32 to 50, wherein the plurality of photodetectors comprise one or more photodiodes. 53. The system according to any one of clauses 32 to 50, wherein the plurality of photodetectors comprise one or more avalanche photodiodes (APDs). 54. The system according to any one of clauses 32 to 53, wherein the plurality of photodetectors comprise a modulator component configured to bin data signals from two or more different photodetector channels. 55. The system according to clause 54, wherein each photodetector channel is configured to generate a data signal for each light in the sub - spectral range.

[0170] 56. The system according to clause 54 or 55, wherein the modulator component is configured to bin data signals from adjacent photodetector channels. 57. The system according to clause 56, wherein the modulator component is configured for horizontal binning of adjacent photodetector channels. 58. The system according to any one of clauses 54 to 57, wherein the modulator component is configured to bin data signals from three or more different photodetector channels. 59. The system according to any one of clauses 54 to 58, wherein the modulator component comprises an output modulator configured to modulate the signal output from each photodetector channel. 60. The system according to clause 59, wherein the output modulator comprises an amplifier.

[0171] 61. The system according to clause 60, wherein the amplifier is a trans - impedance amplifier. 62. The system according to any one of clauses 59 to 61, wherein the output modulator comprises a switch circuit. 63. The system according to any one of clauses 59 to 62, wherein the output modulator comprises a differential amplifier. 64. The system according to any one of clauses 54 to 63, wherein the modulator component is configured to multiplex the generated data signal.

[0172] 65. A method comprising: detecting light from a flow stream with an optical detection system, the optical detection system comprising: a wavelength separator component configured to propagate light between a first set of linearly variable optical filters and a second set of linearly variable optical filters, each set of linearly variable optical filters being configured to pass light having a predetermined sub-spectrum range across the linearly variable optical filter; and a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linearly variable optical filter. 66. The method according to clause 65, further comprising irradiating a sample in a flow stream in an investigation field with a light source. 67. The method according to clause 65 or 66, wherein the flow stream is irradiated with a light source having a wavelength in the range of 200 nm to 800 nm. 68. The method according to any one of clauses 65 to 67, wherein the light source is a laser. 69. The method according to any one of clauses 65 to 68, wherein light from the flow stream is transmitted to the optical detection system by an optical collection component.

[0173] 70. The method according to clause 69, wherein the optical collection component comprises an optical fiber. 71. The method according to clause 70, wherein the optical collection component comprises an optical fiber optical relay bundle. 72. The method according to clause 69, wherein the optical collection component comprises a free space optical relay system. 73. The method according to any one of clauses 65 to 72, wherein each set of linear variable optical filters is configured to allow light having a wavelength of 200 nm to 1200 nm to pass through across the linear variable optical filter. 74. The method according to any one of clauses 65 to 72, wherein each set of linear variable optical filters is configured to allow light having a wavelength of 500 nm to 800 nm to pass through across the linear variable optical filter.

[0174] 75. The method according to clause 74, wherein the first set of linear variable optical filters is configured to allow light having a wavelength of 500 nm to 650 nm to pass through across the linear variable optical filter. 76. The method according to clause 74, wherein the second set of linear variable optical filters is configured to allow light having a wavelength of 650 nm to 800 nm to pass through across the linear variable optical filter. 77. The method according to any one of clauses 65 to 76, wherein each set of linear variable optical filters comprises a long-pass linear variable band-pass filter and a short-pass linear variable band-pass filter. 78. The method according to clause 77, wherein the long-pass variable band-pass filter is offset from the short-pass linear variable band-pass filter by a predetermined distance in each set of linear variable optical filters. 79. The method according to clause 78, wherein the light in the sub-spectrum range across each set of linear variable optical filters is determined by the offset of the long-pass variable band-pass filter from the short-pass linear variable band-pass filter.

[0175] 80. The method according to any one of clauses 65 to 79, wherein the light in each sub-spectrum range passing through across the set of linear variable optical filters includes a spectral width of 5 nm to 50 nm. 81. The method according to clause 80, wherein the light in each sub-spectrum range passing through across the set of linear variable optical filters includes a spectral width of about 20 nm. The method according to any one of clauses 65 to 81, wherein a first set of linear variable optical filters and a second set of linear variable optical filters are positioned along two parallel planes. 83. The method according to clause 82, wherein the wavelength separator component is configured to transmit light to and from between a first set of linear variable optical filters and a second set of linear variable optical filters. 84. The wavelength separator component is configured such that a first set of linear variable optical filters is configured to pass light from a low wavelength to a high wavelength across the linear variable optical filter, and a second set of linear variable optical filters is configured to pass light from a high wavelength to a low wavelength across the linear variable optical filter, the method according to any one of clauses 65 to 83.

[0176] 85. The method according to any one of clauses 65 to 84, wherein a plurality of photodetectors are positioned sandwiching each set of linear variable optical filters. 86. The method according to clause 85, wherein each photodetector is configured to detect light in a sub - spectral range having a spectral width of 5 nm to 50 nm. 87. The method according to clause 86, wherein each photodetector is configured to detect light in a sub - spectral range having a spectral width of about 20 nm. 88. The photodetection system is a first set of photodetectors positioned sandwiching a first set of linear variable optical filters, q) 500 nm to 520 nm, r) 520 nm to 540 nm, s) 540 nm to 560 nm, t) 560 nm to 580 nm, u) 580 nm to 600 nm, v) 600 nm to 620 nm, and w) 620 nm to 640 nm, x) configured to detect light having a sub - spectral range of 640 nm to 660 nm, the first set of photodetectors, A second set of photodetectors positioned sandwiching a second set of linear variable optical filters, o) 800 nm to 780 nm, p) 780 nm to 760 nm, q) 760 nm to 740 nm, r) 740 nm to 720 nm, s) 720 nm to 700 nm, t) 700 nm to 680 nm, and u) A second set of photodetectors configured to detect light having sub - spectral ranges of 680 nm to 660 nm, the method according to any one of clauses 84 - 87, comprising:

[0177] 89. The method according to any one of clauses 65 - 88, wherein the plurality of photodetectors comprises one or more photomultiplier tubes (PMTs). 90. The method according to any one of clauses 65 - 89, wherein the plurality of photodetectors comprises one or more photodiodes. 91. The method according to any one of clauses 65 - 89, wherein the plurality of photodetectors comprises one or more avalanche photodiodes (APDs). 92. The method according to any one of clauses 65 - 91, wherein the plurality of photodetectors comprises a modulator component configured to bin data signals from two or more different photodetector channels. 93. The method according to clause 92, wherein each photodetector channel is configured to generate a data signal for each sub - spectral range of light. 94. The method according to clause 92 or 93, wherein the modulator component is configured to bin data signals from adjacent photodetector channels.

[0178] 95. The method according to clause 94, wherein the modulator component is configured for horizontal binning of adjacent photodetector channels. 96. The method according to any one of clauses 92 - 95, wherein the modulator component is configured to bin data signals from three or more different photodetector channels. 97. The method according to any one of clauses 92 to 96, wherein the modulator component includes an output modulator configured to modulate the signal output from each photodetector channel. 98. The method according to clause 97, wherein the output modulator includes an amplifier. 99. The method according to clause 98, wherein the amplifier is a transimpedance amplifier. 100. The method according to any one of clauses 97 to 99, wherein the output modulator includes a switch circuit. 101. The method according to any one of clauses 97 to 100, wherein the output modulator includes a differential amplifier. 102. The method according to any one of clauses 97 to 101, wherein the modulator component is configured to multiplex the generated data signal.

[0179] 103. A kit comprising: a first set of linear variable optical filters; a second set of linear variable optical filters; and a photodetector. 104. The kit according to clause 103, wherein the first set of linear variable optical filters includes a long-pass linear variable bandpass filter and a short-pass linear variable bandpass filter. 105. The kit according to clause 103 or 104, wherein the first set of linear variable optical filters is configured to pass light having a wavelength of 500 nm to 650 nm across the linear variable optical filter. 106. The kit according to any one of clauses 103 to 105, wherein the second set of linear variable optical filters includes a long-pass linear variable bandpass filter and a short-pass linear variable bandpass filter. 107. The kit according to any one of clauses 103 to 106, wherein the second set of linear variable optical filters is configured to pass light having a wavelength of 650 nm to 800 nm across the linear variable optical filter.

[0180] 108. The kit according to any one of clauses 103 to 107, further comprising a light source. 109. The kit according to clause 108, wherein the light source comprises a laser. 110. The kit according to any one of clauses 103 to 109, further comprising an optical collection component. 111. The kit according to clause 110, wherein the optical collection component comprises an optical fiber. 112. The kit according to clause 111, wherein the optical collection component comprises an optical fiber optical relay bundle. 113. The kit according to clause 110, wherein the optical collection component comprises a free space optical relay system.

[0181] The above invention has been described in some detail by way of illustration and example for the purpose of clear understanding. However, it will be readily apparent to those skilled in the art that, in light of the teachings of the present invention, certain changes and modifications can be made to those inventions without departing from the spirit or scope of the appended claims.

[0182] Therefore, the above merely illustrates the principles of the present invention. It will be understood by those skilled in the art that, although not explicitly described or illustrated herein, various arrangements can be devised that embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language set forth herein are principally intended to assist the reader in understanding the principles of the present invention and the concepts contributed by the inventors to advance the art, and are to be construed as not being limited to such specifically recited examples and conditions. Additionally, all descriptions in this specification of 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. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function regardless of structure. Furthermore, what is disclosed herein is not intended to be dedicated to the public whether or not such disclosure is explicitly recited in the claims.

[0183] Accordingly, 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 are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is explicitly defined as being invoked for a claim limitation only if the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such a limitation in the claim, and such exact phrases are not used in the claim limitation, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is not invoked.

[0184] Cross - reference to related applications This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 346,722, filed May 27, 2022, under 35 U.S.C. § 119(e), and the entire disclosure of that application is incorporated herein by reference.

Claims

1. An optical detection system comprising: A wavelength separator component configured to propagate light between a first set of linearly variable optical filters and a second set of linearly variable optical filters, wherein each set of linearly variable optical filters is configured to pass light having a predetermined sub-spectrum range across the linearly variable optical filter; a wavelength separator component; A plurality of photodetectors positioned to detect light from each sub-spectrum range across the linearly variable optical filter. An optical detection system comprising:

2. The optical detection system according to claim 1, wherein each set of linearly variable optical filters comprises a long-pass linearly variable band-pass filter and a short-pass linearly variable band-pass filter.

3. The optical detection system according to claim 2, wherein the long-pass variable band-pass filter is offset from the short-pass linearly variable band-pass filter by a predetermined distance in each set of linearly variable optical filters.

4. The optical detection system according to claim 3, wherein the light in the sub-spectrum range across each set of linearly variable optical filters is determined by the offset of the long-pass variable band-pass filter from the short-pass linearly variable band-pass filter.

5. The optical detection system according to any one of claims 1 to 4, wherein the light in each sub-spectrum range passing across the set of linearly variable optical filters includes a spectral width of 5 nm to 50 nm.

6. The optical detection system according to any one of claims 1 to 5, wherein the first set of linearly variable optical filters and the second set of linearly variable optical filters are positioned along two parallel planes.

7. The optical detection system according to claim 6, wherein the wavelength separator component is configured to transmit light back and forth between the first set of linearly variable optical filters and the second set of linearly variable optical filters.

8. The wavelength separator component is configured such that: The first set of linearly variable optical filters is configured to pass light from low wavelength to high wavelength across the linearly variable optical filter. The optical detection system according to any one of claims 1 to 7, wherein the second set of the linear variable optical filter is configured such that light passes through the linear variable optical filter from a high wavelength to a low wavelength.

9. The optical detection system according to any one of claims 1 to 8, wherein the plurality of photodetectors are positioned sandwiching each set of the linear variable optical filter.

10. The optical detection system according to any one of claims 1 to 9, wherein the plurality of photodetectors comprise a modulator component configured to bin data signals from two or more different photodetector channels.

11. The optical detection system according to claim 10, wherein each photodetector channel is configured to generate a data signal for each light in a sub-spectrum range.

12. The optical detection system according to claim 10 or 11, wherein the modulator component is configured to bin data signals from adjacent photodetector channels.

13. A system, a light source configured to irradiate particles propagating through a flow stream, an optical detection system, a wavelength separator component configured to propagate light between a first set of a linear variable optical filter and a second set of the linear variable optical filter, wherein each set of the linear variable optical filter is configured to pass light having a predetermined sub-spectrum range across the linear variable optical filter, the wavelength separator component, a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filter, an optical detection system, comprising the system.

14. A method, detecting light from a flow stream with an optical detection system, the optical detection system comprising: a wavelength separator component configured to propagate light between a first set of a linear variable optical filter and a second set of the linear variable optical filter, wherein each set of the linear variable optical filter is configured to pass light having a predetermined sub-spectrum range across the linear variable optical filter, the wavelength separator component, a plurality of photodetectors positioned to detect light from each sub-spectrum range across the linear variable optical filter, comprising detecting.

15. A kit, A first set of linear variable optical filters, a second set of linear variable optical filters, and a photodetector, a kit comprising.

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