Flow cytometry linear variable optical filter systems and methods of use thereof

Abstract

Aspects of the present disclosure include systems for detecting light from particles of a flowing stream using spectral discrimination. A light detection system according to certain embodiments includes a wavelength separator assembly that can propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, where each set of linear variable optical filters can pass light having a predetermined sub-spectral range; and a plurality of photodetectors positioned to detect light passing through each sub-spectral range of the linear variable optical filter. The present disclosure also describes a system having a light source for irradiating particles in a flowing stream and a photodetector modulator assembly for binning data signals generated in a plurality of photodetector channels of a light detection system. The present disclosure also provides methods of light detection employing the subject systems and kits having one or more light detection components for light detection according to the subject methods.

Description

Cross-references to related patent applications

[0001] Pursuant to Title 35 of the United States Patent Act, Section 119(e), this application claims the benefit of priority to the filing date of U.S. Provisional Patent Application No. 63 / 346,722, filed on May 27, 2022; the disclosure of U.S. Provisional Patent Application No. 63 / 346,722 is incorporated herein by reference in its entirety and becomes a part of the present invention. Background Art

[0002] When using a sample to diagnose a disease or medical condition, light detection is often used to characterize the composition of a sample (e.g., a biological sample). Samples can scatter, transmit, and (e.g., through fluorescence) emit light when illuminated. Changes in the composition of the sample, such as morphology, absorbance, and the presence of fluorescent labels, can result in changes in the light scattered, transmitted, or emitted by the sample. These changes can be used to characterize and identify the components present in the sample. In order to quantify these changes, light is collected and directed to the surface of a detector. The amount of light reaching the detector affects the overall quality of the light 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.

[0003] Flow cytometry is a technique that uses light detection to characterize components in a sample. Using the data generated from the measured light, the distribution of components can be recorded and the desired substances can be sorted. A flow cytometer generally includes a sample reservoir for receiving a fluid sample (e.g., a blood sample) and a sheath reservoir containing a sheath fluid. The flow cytometer delivers particles (including cells) in the fluid sample as a cell stream to a flow chamber, while also directing a sheath fluid into the flow chamber. Within the flow chamber, a liquid sheath is formed around the cell stream, giving the cell stream a substantially uniform flow rate. The flow chamber fluidically focuses the cells within the flow stream through the center of a light source in the flow chamber. Light from the light source can be detected as scattered light, or detected using transmission spectroscopy, or can be absorbed by one or more components in the sample and re-emitted as luminescence. Summary of the invention

[0004] Various aspects of the present disclosure include systems that use spectral discrimination to detect light from particles in a flowing stream. According to certain embodiments, the light detection system includes: a wavelength separator component that can propagate light between a first group of linear variable optical filters and a second group of linear variable optical filters, wherein each group of linear variable optical filters can allow light having a predetermined sub-spectral range to pass; and multiple photodetectors that are positioned to detect light in each sub-spectral range that passes through the linear variable optical filters. The present disclosure also describes a system having a light source and a photodetector modulator component, wherein the light source is used to irradiate particles in a flowing stream, and the photodetector modulator component is used to bin data signals generated in multiple photodetector channels of the light detection system. The present disclosure also provides a method of performing light detection using the subject system and a kit having one or more light detection components for performing light detection according to the subject method.

[0005] In some embodiments, each set of linear variable optical filters can pass light having a wavelength range of 200 nm-1200 nm (such as 500 nm-800 nm). For example, the first set of linear variable optical filters can pass light having a wavelength range of 500 nm-650 nm, and the second set of linear variable optical filters can pass light having a wavelength range of 650 nm-800 nm. In some instances, 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 instances, there is an offset of a predetermined distance between the long-pass variable bandpass filter and the short-pass variable bandpass filter. In some instances, the sub-spectral range of light that can pass through each set of linear variable optical filters is determined by the offset of the long-pass variable bandpass filter and the short-pass linear variable bandpass filter. In some instances, there is an offset between the long-pass linear variable bandpass filter and the short-pass variable bandpass filter in each set of linear variable filters, so that each sub-spectral range of light has a spectral width of 1 nm-50 nm (such as 5 nm-45 nm, including 10 nm-40 nm). In some embodiments, there is an offset between the long-pass linear variable bandpass filter and the short-pass variable bandpass filter in each set of linear variable optical filters, so that each sub-spectral range of light has a spectral width of 20 nm.

[0006] In some embodiments, light in each spectral range passing through the set of linear variable optical filters has a spectral width of 5 nm-50 nm, such as a spectral width of 10 nm-40 nm (e.g., a spectral width of about 20 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 instances, the wavelength separator assembly can transmit light back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters. In some instances, the wavelength separator assembly is configured so that: the first set of linear variable optical filters can pass light from low wavelength to high wavelength, and the second set of linear variable optical filters can pass light from high wavelength to low wavelength. For example, light from a sample can be transmitted to a first segment of the first set of linear variable optical filters, pass light in a wavelength range of 500 nm-520 nm, and reflect the rest of the light to a first segment of the second set of linear variable optical filters, and the first segment of the second set of linear variable optical filters can pass light in a wavelength range of 800 nm-780 nm and reflect the rest of the light to a second segment of the first set of linear variable optical filters. In certain embodiments, the transfer and passage of light back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters continues throughout the entire length of each set of linear variable optical filters.

[0007] In some embodiments, the light detection system includes a plurality of photodetectors positioned across each set of linear optical filters. In some instances, each photodetector can detect light of a sub-spectral range at each segment of the linear filter. In some instances, each photodetector can detect light of a sub-spectral range having a spectral width of 5 nm-50 nm (e.g., 20 nm) at each segment of the linear optical filter. In some instances, the light detection system includes a first group of photodetectors positioned across a first group of linear variable optical filters, the first group of photodetectors being able to detect light in a sub-spectral range of 500 nm-520 nm, 520 nm-540 nm, 540 nm-560 nm, 560 nm-580 nm, 580 nm-600 nm, 600nm-620 nm, 620 nm-640 nm, and 640 nm-660 nm; and also includes a second group of photodetectors positioned across a second group of linear variable optical filters, the second group of photodetectors being able to detect light in a sub-spectral range of 800nm-780 nm, 780 nm-760 nm, 760 nm-740 nm, 740 nm-720 nm, 720 nm-700 nm, 700 nm-680nm, and 680 nm-660 nm.

[0008] In some instances, the photodetector component of the optical detection system includes a modulator component that can bin data signals from two or more different photodetector channels. In some instances, each photodetector channel can generate a data signal for each sub-spectral range of light. In some instances, the modulator component can bin data signals from adjacent photodetector channels, such as horizontally binning adjacent photodetector channels. In some embodiments, the modulator component includes an output modulator that can modulate the signal output from each photodetector channel. In some embodiments, the output modulator includes one or more of an amplifier (e.g., a transimpedance amplifier), a switching circuit, and a differential amplifier. In some instances, the modulator component can multiplex the generated data signals.

[0009] Various aspects of the present disclosure also include systems that use spectral discrimination to measure light from a sample (e.g., in a flowing stream). In some embodiments, the system includes a light source and a light detection system that can detect a predetermined spectral range of light, the light detection system includes a wavelength separator assembly and a plurality of photodetectors, the wavelength separator assembly can propagate light between a first set of linear variable optical filters and a second set of linear variable optical filters, the first set of linear variable optical filters and the second set of linear variable optical filters can pass light having a predetermined sub-spectral range, and the plurality of photodetectors are positioned to detect light in each sub-spectral range passing through the linear variable optical filters. In some embodiments, the system also includes an optical collection system that propagates light to the light detection system. The light collection system can be a free space optical relay system, and can also include optical fibers, such as a fiber optic optical relay bundle. In some embodiments, the system is a flow cytometer.

[0010] Various aspects of the present disclosure also include methods of irradiating a sample in an interrogation field (e.g., in a flow stream) with a light source, collecting and detecting light from the sample with the subject light detection system, and measuring the detected light at one or more wavelengths. In some embodiments, the light is collected by a free space optical relay system and transmitted to the light detection system. In other embodiments, the light is collected by an optical fiber (e.g., a fiber optic optical relay bundle) and transmitted to the light detection system.

[0011] The present invention also provides a kit including one or more components of the subject light detection system. According to certain embodiments, the kit includes a first group of linear variable optical filters, a second group of linear variable optical filters, and a photodetector. In certain embodiments, one or more of the linear variable optical filters are linear variable bandpass filters, such as a long-pass linear variable bandpass filter or a short-pass linear variable bandpass filter. In some instances, the kit includes an optical collection component, such as a fiber optic optical relay bundle or a free-space optical relay system. The kit may also include one or more photodetectors, such as a photomultiplier tube (such as a metal packaged photomultiplier tube) or a photodiode (such as an avalanche photodiode, APD). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The accompanying drawings include the following figures:

[0013] Figure 1 A first set of linear variable optical filters and photodetectors across a plurality of segments for detecting light are shown in accordance with certain embodiments.

[0014] Figure 2 A second set of linear variable optical filters and photodetectors across multiple segments for detecting light are shown in accordance with certain embodiments.

[0015] Figure 3 A light detection system having a first set of linear variable optical filters and a second set of linear variable optical filters that can transfer light back and forth is shown in accordance with certain embodiments.

[0016] Figure 4A A functional block diagram of a particle analysis system according to certain embodiments is shown. Figure 4B A flow cytometer system with a light detection system according to certain embodiments is shown.

[0017] Figure 5 A functional block diagram of an example of a particle analyzer control system according to certain embodiments is shown.

[0018] Fig. 6A A schematic diagram of a particle sorter system according to certain embodiments is shown.

[0019] Figure 6B A schematic diagram of a particle sorter system according to certain embodiments is shown.

[0020] Figure 7 A block diagram of a computing system according to some embodiments is shown. DETAILED DESCRIPTION

[0021] Various aspects of the present disclosure include systems that use spectral discrimination to detect light from particles in a flowing stream. According to certain embodiments, the light detection system includes: a wavelength separator component that can propagate light between a first group of linear variable optical filters and a second group of linear variable optical filters, wherein each group of linear variable optical filters can allow light having a predetermined sub-spectral range to pass; and multiple photodetectors that are positioned to detect light in each sub-spectral range that passes through the linear variable optical filters. The present disclosure also describes a system having a light source and a photodetector modulator component, wherein the light source is used to irradiate particles in a flowing stream, and the photodetector modulator component is used to bin data signals generated in multiple photodetector channels of the light detection system. The present disclosure also provides a method of performing light detection using the subject system and a kit having one or more light detection components for performing light detection according to the subject method.

[0022] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, which may, of course, vary. It should also be understood that since the scope of the present invention will be limited only by the appended claims, the terms in the present invention are only used to describe specific embodiments and are not intended to be limiting.

[0023] If the present invention provides a range of values, it should be understood that the present invention includes each intermediate value between the upper and lower limits of this range (unless the context clearly stipulates otherwise, it is accurate to one tenth of the unit of the lower limit), as well as any other specified values ​​or intermediate values ​​within this specified range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the scope of the present invention, but are subject to any explicitly excluded limits in the specified range. If the specified range includes one or two of the limits, the present invention also includes a range excluding any or both of these included limits.

[0024] Certain ranges in the present invention are expressed as numerical values ​​preceded by the term "about". The term "about" as used in the present invention is used to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows it. In determining whether a number is close to or approximately a number that is explicitly recited, the close or approximate unrecited number can be a number that is substantially equivalent to the explicitly recited number in the context in which it is presented.

[0025] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods or materials described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described below.

[0026] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference as a part of the present invention and is intended to disclose and describe the methods and / or materials in connection with the cited publication. Citation of any publication is for the purpose of disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, the dates of publication provided may be different from the actual publication dates which need to be independently confirmed.

[0027] It is noted that, as used in the present disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that any optional elements may be excluded when drafting the claims. Therefore, this statement is intended to serve as a precondition for using exclusive terminology such as "only," "only" and the like in connection with the recitation of claim elements or using a "negative" limitation.

[0028] After reading this disclosure, it will be apparent to those skilled in the art that each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other multiple embodiments without departing from the scope or spirit of the invention. Any method recited may be implemented in the order of events recited or in any other order that is logically possible.

[0029] Although methods and compositions have been or will be described for the sake of grammatical fluency and functional explanation, it should be expressly understood that, except as expressly provided in 35 U.S. Patent Act § 112, the claims should not be construed as necessarily being limited in any way to “means” or “steps” but rather be given the full meaning and scope of equivalents provided by the definitions in the claims in accordance with the doctrine of judicial equivalents, and to the extent that a claim is expressly provided in 35 U.S. Patent Act § 112, it should be given its full legal equivalent in accordance with 35 U.S. Patent Act § 112.

[0030] As described above, the present disclosure provides systems and methods for detecting light from particles in a flow stream (e.g., a particle analyzer) using spectral discrimination. In further describing embodiments of the present disclosure, a light detection system is first described in more detail, the light detection system having a first set of linear variable optical filters and a second set of linear variable optical filters and a plurality of photodetectors, the photodetectors being capable of detecting light in a sub-spectral range passing through the linear variable optical filters. Next, a method for performing light detection using spectral discrimination using the subject system is described. A kit having one or more components of the subject spectral discrimination detection system is described.

[0031] System for detecting light from particles in a flowing stream using spectral discrimination

[0032] Various aspects of the present disclosure include systems that detect light from particles in a flowing stream using spectral identification. The term "spectral identification" as used herein refers in its conventional sense to differential detection of a range of wavelengths of light from irradiated particles, wherein, in some instances, differential detection of the spectrum of light 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 can detect multiple different wavelengths of light emitted by the irradiated particles, wherein, in some instances, one or more data signals are generated in response to each different detected wavelength of light. In some embodiments, the subject system can detect the entire wavelength spectrum of light from the irradiated particles.

[0033] In certain embodiments, the system for detecting light from particles using spectral discrimination described herein can improve the accuracy of the photodetector array in capturing the target spectral emission band, for example, the narrow spectrum sampling accuracy is improved by 5% or more, for example 10% or more, for example 15% or more, for example 25% or more, for example 50% or more, for example 75% or more, for example 90% or more, including 95% or more, compared to signal processing without binning the data signal from two or more photodetector channels. According to certain embodiments, the light detection system described herein is capable of generating 10 or more different spectral ranges of light while exhibiting a light loss of 20% or less (e.g., 19% or less, for example, 18% or less, for example, 17% or less, for example, 16% or less, for example, 15% or less), including generating 10 or more different spectral ranges of light while exhibiting a light loss of 10% or less.

[0034] In certain embodiments, the subject light detection system can improve the resolution of a narrow spectral range of light. For example, when spectrally separating light from irradiated particles, the light detection system can improve granularity. In certain embodiments, the light detection system can improve the signal-to-noise ratio of the data signal by 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including 95% or more. According to certain embodiments of the present light detection system, generating data signals in multiple photodetector channels can simultaneously process data signals from more photodetector channels, thereby improving the signal-to-noise ratio. For a predetermined spectral width, more data signals can be processed, thereby improving the signal-to-noise ratio of light detected from the sample. In addition, in certain instances, the light detection system can improve the signal-to-noise ratio by reducing the amount of interference between the emissions of different fluorophores in the sample.

[0035] The optical detection system according to certain embodiments includes a wavelength separator component that can 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 can pass light having a predetermined sub-spectral range. The term "wavelength separator" as used in the present invention refers to an optical element that can separate light (for example, collected from irradiated particles in a flow stream) into predetermined sub-spectral ranges in its conventional sense. In certain embodiments, the wavelength separator can separate the collected light into predetermined spectral ranges by passing light having a predetermined spectral range and reflecting light of one or more remaining spectral ranges. In other embodiments, the wavelength separator can separate the collected light into predetermined spectral ranges by passing light having a predetermined spectral range and absorbing light of one or more remaining spectral ranges. In other embodiments, the wavelength separator can spatially diffract the collected light into predetermined spectral ranges.

[0036] In some instances, the light detected using the subject system is emitted light, such as fluorescence from irradiated particles. In some cases, the light from irradiated particles 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 from irradiated particles is transmitted light.

[0037] In an embodiment, each set of linear variable optical filters of the wavelength separator assembly can allow light with a wavelength range of 200 nm-1200 nm (e.g., 250 nm-1150 nm, e.g., 300 nm-1100 nm, e.g., 350 nm-1050 nm, e.g., 400 nm-1000 nm, e.g., 450 nm-950 nm, e.g., 500 nm-900 nm, e.g., 550 nm-850 nm. Including 600 nm-800 nm) to pass through the linear variable optical filter. In certain embodiments, each set of linear variable optical filters includes a long-pass variable bandpass filter and a short-pass linear variable bandpass filter. In certain embodiments, the long-pass linear variable bandpass filter is a linear variable bandpass filter that can allow light of multiple 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, and greater than 800 nm to pass through the bandpass filter. In some embodiments, the short-pass linear variable bandpass filter is a linear variable bandpass filter that allows light of multiple wavelengths of 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, and 200 nm or less to pass through the bandpass filter.

[0038] Each set of linear variable optical filters includes two linear variable optical filters arranged adjacent to each other. In each set of linear variable optical filters, the distance between the two linear variable optical filters can vary, for example, 0.001 mm-25 mm, for example, 0.005 mm-20 mm, for example, 0.01 mm-15 mm, for example, 0.05 mm-10 mm, for example, 0.1 mm-10 mm, for example, 0.5 mm-10 mm, for example, 1 mm-9 mm, including 2 mm-8 mm. In some embodiments, the two linear variable optical filters in each set of linear variable optical filters are in physical contact with each other (i.e., there is no gap between the two linear variable optical filters).

[0039] In some examples, there is an offset of a predetermined distance between the long-pass variable bandpass filter and the short-pass variable bandpass filter. In some examples, the sub-spectral range of light that can pass through each set of linear variable optical filters is determined by the offset of the long-pass variable bandpass filter and the short-pass linear variable bandpass filter. In some embodiments, the offset between the long-pass variable bandpass filter and the short-pass variable bandpass filter is 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, including 15 mm or more. In some embodiments, in each set of linear variable optical filters in the wavelength separator component, the offset between the long-pass variable bandpass filter and the short-pass variable bandpass filter is 0.001 mm-25 mm, for example, 0.005 mm-20 mm, for example, 0.01 mm-15 mm, for example, 0.05 mm-10 mm, for example, 0.1 mm-10 mm, for example, 0.5 mm-10 mm, for example, 1 mm-9 mm, including 2 mm-8 mm.

[0040] In some embodiments, each set of linear variable optical filters can generate light in a plurality of spectral ranges, and the light in these spectral ranges reaches the photodetector (described in more detail below) through the linear variable optical filters, such as 3 or more spectral ranges, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 15 or more, such as 25 or more, such as 50 or more, such as 75 or more, including 100 or more spectral ranges of light, and the photodetector can detect the light in each spectral range. In some embodiments, the light in each spectral range that passes through the set of linear variable optical filters and is detected by the photodetector has a spectral width of 5 nm-50 nm, such as a spectral width of 10 nm-40 nm, such as a spectral width of about 20 nm. In some instances, in each set of linear variable optical filters, there is an offset between the long-pass linear variable bandpass filter and the short-pass variable bandpass filter so that light in each sub-spectral range has a spectral width of 1 nm or more (e.g., 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, including 50 nm or more). In some embodiments, in each set of linear variable optical filters, there is an offset between the long-pass linear variable bandpass filter and the short-pass variable bandpass filter, so that the light in each sub-spectral range has a spectral width of 1 nm-50 nm (e.g., 2 nm-49 nm, e.g., 3 nm-48 nm, e.g., 4 nm-47 nm, e.g., 5 nm-46 nm, e.g., 6 nm-45 nm, e.g., 7 nm-44 nm, e.g., 8 nm-43 nm, e.g., 9 nm-42 nm, including 10 nm-40 nm). In some embodiments, there is an offset between the long-pass linear variable bandpass filter and the short-pass variable bandpass filter in each set of linear variable optical filters, so that the light in each sub-spectral range has a spectral width of 20 nm.

[0041] In an embodiment, the first group of linear variable optical filters and the second group of linear variable optical filters are in optical communication with each other so that light is transmitted between each other. In some instances, the wavelength separator assembly can transmit light back and forth between the first group of linear variable optical filters and the second group of linear variable optical filters. In some embodiments, the first group of linear variable optical filters and the second group of linear variable optical filters are positioned along two parallel planes. In other embodiments, in the light detection system, the angles at which the groups of linear variable optical filters are oriented relative to each other are 0.001°-15°, such as 0.005°-14.5°, such as 0.01°-14°, such as 0.05°-13.5°, such as 0.1°-13°, such as 0.5°-12.5°, including 1°-10°.

[0042] In some embodiments, the wavelength separator assembly is configured such that: the first set of linear variable optical filters can pass light from low wavelength to high wavelength, and the second set of linear variable optical filters can pass light from high wavelength to low wavelength. In some instances, light from the sample is transmitted to the first segment of the first set of linear variable optical filters, passes light in the wavelength range of 500 nm-520 nm, and reflects the remaining portion of the light to the first segment of the second set of linear variable optical filters, and the first segment of the second set of linear variable optical filters can pass light in the wavelength range of 800 nm-780 nm, and reflects the remaining portion of the light to the second segment of the first set of linear variable optical filters. In some embodiments, light is continuously transmitted back and forth and passed between the first set of linear variable optical filters and the second set of linear variable optical filters through the length of each set of linear variable optical filters (e.g., 10% or more of the length of each set of linear variable optical filters, such as 15% or more of the length of each set of linear variable optical filters, such as 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, including 99.5% or more). In some instances, light is transmitted back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters through the entire length of the linear variable optical filters.

[0043] A plurality of photodetectors detect light passing through the set of linear variable optical filters. Each photodetector can detect a predetermined spectral width of light passing through the set of linear variable optical filters. For example, each photodetector can be positioned along the length of the set of linear variable optical filters and detect a spectral width of 5 nm-50 nm, such as a spectral width of 10 nm-40 nm, such as a spectral width of about 20 nm. In some instances, each photodetector is positioned to detect light that has passed through a segment of the set of linear variable optical filters and has a spectral width of 1 nm or more (e.g., 2 nm or more, such as 3 nm or more, such as 4 nm or more, such as 5 nm or more, such as 6 nm or more, such as 7 nm or more, such as 8 nm or more, such as 9 nm or more, such as 10 nm or more, such as 11 nm or more, such as 12 nm or more, such as 13 nm or more, such as 14 nm or more, such as 15 nm or more, such as 16 nm or more, such as 17 nm or more, such as 18 nm or more, such as 19 nm or more, such as 20 nm or more, such as 25 nm or more, such as 30 nm or more, such as 35 nm or more, such as 40 nm or more, such as 45 nm or more, including 50 nm or more). In some embodiments, the light detection system includes a photodetector that is positioned to detect light that has passed through the set of linear variable optical filters and has a spectral width of 20 nm.

[0044] In some embodiments, the first set of linear variable optical filters can pass light in the range of 500 nm-650 nm, and the photodetector is positioned to detect light passing through the first set of linear variable optical filters at intervals of 20 nm spectral width. Figure 1 A shows a first set of linear variable optical filters and a photodetector for detecting light passing through a plurality of segments according to some embodiments. Figure 1As shown in Figure A, the linear variable optical filter group 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 through the linear variable optical filter group 100 and reaches photodetectors 103a, 103b, 103c, 103d, 103e, 103f, and 103g, wherein each photodetector can detect a 20 nm spectrum width of light. Photodetector 103a can detect light of 500 nm-520 nm, photodetector 103b can detect light of 520 nm-540 nm, photodetector 103c can detect light of 540 nm-560 nm, photodetector 103d can detect light of 560 nm-580 nm, photodetector 103e can detect light of 580 nm-600 nm, photodetector 103f can detect light of 600 nm-620 nm, and photodetector 103g can detect light of 620 nm-640 nm.

[0045] In some embodiments, the second set of linear variable optical filters can pass light in the range of 650 nm-800 nm, and the photodetector is positioned to detect the light passing through the second set of linear variable optical filters at intervals of 20 nm spectral width. Figure 2 A shows a first set of linear variable optical filters and a photodetector for detecting light passing through a plurality of segments according to some embodiments. Figure 2 As shown in Figure A, the linear variable optical filter group 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 through the linear variable optical filter group 200 and reaches photodetectors 203a, 203b, 203c, 203d, 203e, 203f, and 203g, wherein each photodetector can detect a 20 nm spectrum width of light. Photodetector 203a can detect light of 800 nm-780 nm, photodetector 203b can detect light of 780 nm-760 nm, photodetector 203c can detect light of 760 nm-740 nm, photodetector 203d can detect light of 740 nm-720 nm, photodetector 203e can detect light of 720 nm-700 nm, photodetector 203f can detect light of 700 nm-680 nm, and photodetector 203g can detect light of 680 nm-660 nm.

[0046] In an embodiment, the first group of linear variable optical filters and the second group of linear variable optical filters can transmit light to each other. In some instances, each group of linear variable optical filters can pass light of a certain spectral range and transmit (e.g., by reflection) light of one or more remaining spectral ranges to another group of linear variable optical filters. Light is transmitted back and forth between the first group of linear variable optical filters and the second group of linear variable optical filters through the length of the linear variable optical filters. In one example, the group of linear variable optical filters can receive light from a sample and can pass light of a first spectral range and transmit light of a second spectral range to the second group of linear variable optical filters. The second group of linear variable optical filters can pass light of a third spectral range and transmit light of a fourth spectral range back to the first group of linear variable optical filters. Then, the first group of linear variable optical filters can pass light of a fifth spectral range and return to the second group of linear variable optical filters. This back and forth transmission and passage of light continues between each group of linear variable optical filters, and light of each spectral width is detected by multiple photodetectors.

[0047] Figure 3A light detection system having a first set of linear variable optical filters and a second set of linear variable optical filters that can transmit light back and forth according to certain embodiments is shown. Light is transmitted to 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 the first set of linear variable optical filters 301, which is composed of a short-pass linear variable bandpass filter and a long-pass linear variable bandpass filter. Light with a wavelength of 500 nm-520 nm passes through the first segment of the first set of linear variable optical filters 301 and is detected by a photodetector 303a. Light of the remaining wavelength (i.e., light with a wavelength greater than 520 nm) is reflected and transmitted to the 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 bandpass filter and a long-pass linear variable bandpass filter. Light with a wavelength of 800 nm-780 nm passes through the first segment of the second set of linear variable optical filters 302 and is detected by a photodetector 303h. The remaining wavelengths of light are reflected back to the first set of linear variable optical filters 301, wherein the light having a wavelength of 520 nm-540 nm passes through the second segment of the first set of linear variable optical filters 301 and is detected by the photodetector 303b. The light is reflected back and forth between the first set of linear variable optical filters 301 and the second set of linear variable optical filters 302, and the light is detected by the photodetectors 303b, 303c, 303d, 303e, 303f and 303g after passing through the segments across the first set of linear variable optical filters 301, and is detected by the photodetectors 303i, 303j, 303k, 303l, 303m and 303n after passing through the segments across the second set of linear variable optical filters 302.

[0048] In an embodiment, the light detection system includes a plurality of photodetectors positioned to detect light in each sub-spectral range passing through the linear variable optical filter. In some embodiments, light is transmitted to each photodetector by an optical conditioning component. The term "optical conditioning" as used in the present invention refers to any device that is capable of changing the spatial width of light or some other characteristic (e.g., direction, wavelength, beam width, beam intensity, and focus) of light passing through each segment of the linear variable optical filter in its conventional sense. The optical conditioning scheme can be any convenient device for adjusting one or more characteristics of light, including but not limited to lenses, reflectors, filters, optical fibers, wavelength separators, pinholes, slits, collimation schemes, and combinations thereof. In some embodiments, the target system includes one or more focusing lenses. In one example, the focusing lens can be a magnifying lens. In another example, the focusing lens is a magnifying lens. In other embodiments, the target system includes one or more mirrors. In other embodiments, the target system includes an optical fiber.

[0049] In some embodiments, the optical conditioning component is in physical contact with the photodetector. In other embodiments, the conditioning component is in optical communication with the active surface of the photodetector and can be 0.001 mm or more, 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 10 mm or more, such as 25 mm or more, such as 50 mm or more, including 100 mm or more from the photodetector.

[0050] The photodetector can be releasably connected to each optical adjustment component in the subject light detection module. The photodetector and the optical adjustment component can be connected in any suitable scheme. In some embodiments, the photodetector and the optical component are connected together by mounting the photodetector together with the optical component, or using fasteners (such as hook and loop fasteners, magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, Velcro, non-permanent adhesives or combinations thereof). In some instances, the photodetector is connected to the optical component by fitting the wavelength separator slot into the groove of the light detection module. In other instances, the photodetector is connected to the optical component by one or more screws.

[0051] In an embodiment, each photodetection system includes two or more photodetectors, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 15 or more, such as 25 or more, such as 50 or more, including 100 or more photodetectors. In certain embodiments, the light detection module includes one or more photodetector arrays. The term "photodetector array" refers to an arrangement or series of two or more photodetectors in its conventional sense. In an embodiment, the photodetector array includes 2 or more photodetectors, such as 3 or more photodetectors, such as 4 or more photodetectors, such as 5 or more photodetectors, such as 6 or more photodetectors, such as 7 or more photodetectors, such as 8 or more photodetectors, such as 9 or more photodetectors, such as 10 or more photodetectors, such as 12 or more photodetectors, including 15 or more photodetectors.

[0052] In some embodiments, the light of each sub-spectral range transmitted through the linear variable optical filter is transmitted to one or more photodetectors, for example, the light of each sub-spectral range is transmitted to a single photodetector, 2 different photodetectors, 3 different photodetectors, 4 different photodetectors, including the light of each sub-spectral range is transmitted to 5 or more different photodetectors. In some instances, the light of each sub-spectral range is transmitted to a respective different detector (i.e., a different photodetector can detect the light of each different sub-spectral range). In these embodiments, the light of each sub-spectral range can be collected and transmitted to the photodetector by a different set of optical conditioning components (e.g., for each different sub-spectral range and photodetector, by a different set of collimators and focusing lenses).

[0053] In some instances, light of each sub-spectral range transmitted through the linear variable optical filter is detected in one or more different detector channels (e.g., in a multi-channel photodetector array). In some instances, light of each sub-spectral range is detected in a different detector channel. In other instances, light of each sub-spectral range is detected across multiple detector channels, such as 2 or more detector channels, such as 3 or more, such as 4 or more, such as 8 or more, such as 16 or more, including detecting light of each sub-spectral range transmitted through the linear variable optical filter in 32 or more detector channels.

[0054] The photodetector of the subject light detection system can be any suitable light detection scheme, including but not limited to a photoelectric sensor or photodetector, such as an avalanche photodiode (APD), an active pixel sensor (APS), a quadrant photodiode, an image sensor, a charge coupled device (CCD), an intensified charge coupled device (ICCD), a light emitting diode, a photon counter, a bolometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor or photodiode, and combinations thereof, and other photodetectors. In some embodiments, the photodetector is a photomultiplier tube, such as a photomultiplier tube, having an active detection surface area of ​​0.01 cm per region. 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 , including 1 cm 2 -5 cm 2 ) of the photomultiplier tube.

[0055] In an embodiment of the present disclosure, each photodetector may detect light at 1 or more wavelengths (e.g., 2 or more wavelengths, e.g., 5 or more different wavelengths, e.g., 10 or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths), including measuring light at 400 or more different wavelengths.

[0056] Photodetectors can measure light continuously or at discrete intervals. In some cases, an object detector can measure light continuously. In other cases, an object detector can measure light at discrete intervals, such as every 0.001 ms, 0.01 ms, 0.1 ms, 1 ms, 10 ms, 100 ms (including 1000 ms), or at other intervals.

[0057] During each discrete time interval, the photodetector may measure the light one or more times (e.g., 2 or more times, e.g., 3 or more times, e.g., 5 or more times, including 10 or more times). In some embodiments, the photodetector measures the light 2 or more times, and in some cases the data is averaged. The light from the particles in the flow stream may be detected in one or more photodetector channels (e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 6 or more, e.g., 7 or more, e.g., 8 or more, e.g., 9 or more, including 10 or more photodetector channels).

[0058] In some instances, the light detected from the particles of the sample 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 instances, the light transmitted from the irradiated particles is transmitted light. In some embodiments, the light detected from each particle is emitted light, 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 from two or more light sources. For example, each particle may include 2 or more fluorophores, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, including 10 or more fluorophores. In some instances, each particle includes a fluorophore that emits fluorescence in response to irradiation from a light source. In certain embodiments, the target fluorophore may include, but is not limited to, dyes suitable for analytical applications (e.g., flow cytometry, imaging, etc.), such as acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazo dyes, nitro dyes, etc., triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinoneimine dyes, azine dyes, diaminoaczine dyes, safranin dyes, indamines, indophenol dyes, fluorine dyes, oxazine dyes, oxone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronine dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, as well as dyes combining 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. Many dyes are available commercially from a variety of sources, including Molecular Probes (Eugene, OR), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, MO), Sirigen (Santa Barbara, CA), BD Biosciences (Franklin Lakes, NJ), and Exciton (Dayton, OH).For example, fluorophores may include 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and its derivatives, such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin (APC), phycoerythrin (PE), peridinin-chlorophyll-protein, 5-(2'-aminoethyl)naphthylamine-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl]phenyl]naphthylimide-3,5-disulfonate (fluorescein yellow VS); N-(4-anilino-1-naphthyl)maleimide ; aminobenzamide; brilliant yellow; coumarin and its derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanogen and its derivatives, such as tetrachlorotetrabromofluorescein, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-diamidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallolsulfonphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methyl coumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodiphenylethane-2,2'-disulfonic acid; 4,4'-diisothiocyanato-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazo-4'-isothiocyanate (DABITC); eosin and its derivatives, such as eosin and eosin isothiocyanate; erythrosine and its derivatives, such as erythrosine B and erythrosine 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), chlorotriazinylfluorescein, naphthylfluorescein and QFITC ​​(XRITC); Fluorescein amine; IR144; IR1446; Green fluorescent protein (GFP); Coral reef fluorescent protein (RCFP); Lissamine. TM ; Lissamine rhodamine, fluorescent yellow; malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; p-aminobenzenesulfonic acid; Nile red; Oregon green; phenol red; B-cantharidin; o-phthalaldehyde; pyrene and its derivatives, such as pyrene, pyrene butyrate and 1-pyrene butyric acid succinimidyl ester; Reactive Red 4 (Cibacron TMBrilliant Red 3B-A); Rhodamine and its derivatives, such as 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, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine fluorescein isothiocyanate (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rhodamine and terbium chelate derivatives; xanthene; dye conjugated polymers (i.e., polymer adsorbed dyes), such as fluorescein isothiocyanate-dextran and dyes combining two or more dyes (e.g., tandem dyes or protein complex tandem dyes), such as phycoerythrin (PE) tandem dyes or allophycocyanin (APC) tandem dyes, such as phycoerythrin-CF594 (PE-CF594) tandem, phycoerythrin-cyanine 5 tandem (PE-Cy5), phycoerythrin-cyanine 5.5 tandem (PE-Cy5.5), phycoerythrin-cyanine 7 tandem (PE-Cy7), allophycocyanin-R700 tandem (APC-R700), allophycocyanin-cyanine 7 (APC-Cy7), polymeric dyes having one or more monomeric dye units, and mixtures of two or more of the above dyes or combinations thereof.

[0059] In some instances, the fluorophore is a polydye. In some instances of the method, the polydye comprises a conjugated polymer. A conjugated polymer (CP) is characterized by a delocalized electronic structure comprising a backbone of alternating unsaturated bonds (e.g., double bonds and / or triple bonds) and saturated bonds (e.g., single bonds), wherein π electrons can move from one bond to another. Thus, the conjugated backbone can allow the polydye to have an extended linear structure with limited bond angles between polymer repeating units. For example, while proteins and nucleic acids are also polymeric, in some cases they do not form extended rod-like structures, but rather fold into higher-order three-dimensional shapes. In addition, CPs may form a "rigid rod" polymer backbone and experience limited twist (e.g., torsion) angles between monomer repeating units along the polymer backbone. In some instances, the polydye comprises a CP having a rigid rod structure. The structural features of the polydye can affect the fluorescent properties of the molecule.

[0060] Targeted polydyes include, but are not limited to, those disclosed in Gaylord et al., No. 20040142344, No. 20080293164, No. 20080064042, No. 20100136702, No. 20110256549, No. 20110257374, No. 20120028828, No. 20120252986, No. 20130190193, No. 20160264737, No. 20160266131, No. 20180231530, No. 201 The polymeric dyes described in U.S. Publications No. 80009990, No. 20180009989 and No. 20180163054, the disclosures of which are incorporated herein by reference in their entirety; Gaylord et al., Journal of the American Chemical Society, 2001, 123(26), pp. 6417-6418; Feng et al., Reviews of the Chemical Society, 2010, 39, 2411-2419; Traina et al., Journal of the American Chemical Society, 2011, 133(32), pp. 12600-12607, the disclosures of which are incorporated herein by reference in their entirety.

[0061] The polydye may have one or more desirable spectral properties, such as a specific maximum absorption wavelength, a specific maximum emission wavelength, an extinction coefficient, a quantum yield, etc. (see Chattopadhyay et al., "Brilliant violet fluorophores: novel ultrabright fluorescent compounds for immunofluorescence experiments." Flow Cytometry Part A, 81A(6), 456-466, 2012). In some embodiments, the polydye has an absorption curve between 280 nm and 475 nm. In some embodiments, the polydye has a maximum absorption value (maximum excitation value) in the range of 280 nm and 475 nm. In some embodiments, the polydye absorbs incident light in the wavelength range of 280 nm and 475 nm. In some embodiments, the polydye has a maximum emission wavelength in the range of 400 nm and 850 nm (e.g., 415 nm and 800 nm), wherein specific examples of target maximum emission values ​​include, but are not limited to, 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm. In some instances, the polydye has a maximum emission wavelength in a range selected from the group consisting of 410 nm-430 nm, 500 nm-520 nm, 560 nm-580 nm, 590 nm-610 nm, 640 nm-660 nm, 700 nm-720 nm, and 775 nm-795 nm. In some embodiments, the polydye has a maximum emission wavelength of 421 nm. In some instances, the polydye has a maximum emission wavelength of 510 nm. In some cases, the polydye has a maximum emission wavelength of 570 nm. In some embodiments, the polydye has a maximum emission wavelength of 602 nm. In some instances, the polydye has a maximum emission wavelength of 650 nm. In some cases, the polydye has a maximum emission wavelength of 711 nm. In some embodiments, the polydye has a maximum emission wavelength of 786 nm. In some instances, the polydye has a maximum emission wavelength of 421 nm ± 5 nm. In some embodiments, the polydye has a maximum emission wavelength of 510 nm ± 5 nm. In some instances, the polydye has a maximum emission wavelength of 570 nm ± 5 nm. In some instances, the polydye has a maximum emission wavelength of 602 nm ± 5 nm. In some embodiments, the polydye has a maximum emission wavelength of 650 nm ± 5 nm. In some instances, the polydye has a maximum emission wavelength of 711 nm ± 5 nm. In some cases, the polydye has a maximum emission wavelength of 786 nm ± 5 nm.In certain embodiments, the polydye has an emission maximum selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0062] Specific polydyes that may be used include, but are not limited to, BD Horizon Brilliant TM Dyes, such as BD Horizon Brilliant TM Violet dyes (e.g., BV421, BV480, BV510, BV570, BV605, BV650, BV711, BV786, BV829); BD Horizon Brilliant TM Ultraviolet dyes (e.g., BUV395, BUV496, BUV563, BUV615, BUV661, BUV737, BUV805); BD Horizon Brilliant TM Blue dyes (e.g., BB515, BB630, BB660, BB700, BB755, BB790) (BD Biosciences, San Jose, CA).

[0063] In some embodiments, the optical detection system includes a modulator component that can bin data signals from two different photodetector channels. In some embodiments, the modulator component can bin data signals from two or more different photodetector channels to produce a combined spectral data signal. In some instances, the modulator component bins data signals from non-adjacent photodetectors. In some instances, the modulator component bins data signals from adjacent photodetectors. In some instances, the modulator component can horizontally bin data signals from adjacent photodetectors.

[0064] In some embodiments, the modulator component may bin data signals from three or more different photodetector channels (e.g., 4 or more, 5 or more, 6 or more, 16 or more, 32 or more, including from 64 or more different photodetector channels). In some instances, each photodetector channel may generate a data signal for each sub-spectral range of light. 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.

[0065] In some embodiments, the modulator component can dynamically bin data signals from two or more different photodetector channels in real time. In some instances, 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), or 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 the FPGA includes programming to dynamically bin data signals from different photodetector channels. In some instances, for each photodetector channel, the modulator component is programmed with a mapped bin configuration, such as the modulator component includes an FPGA, and the FPGA has programming to dynamically map the bin configuration in real time.

[0066] In some embodiments, the modulator component includes one or more amplifiers. In some embodiments, the amplifier component includes multiple amplifiers, such as a transimpedance amplifier, a summing amplifier, a differential amplifier, or a combination thereof. In some instances, the modulator component includes an amplifier for each photodetector channel, for example, the modulator component includes 2 or more amplifiers, such as 4 or more, such as 8 or more, such as 12 or more, such as 16 or more, such as 20 or more, such as 24 or more, such as 28 or more, such as 32 or more, such as 36 or more, such as 40 or more, such as 44 or more, such as 48 or more, such as 52 or more, such as 56 or more, such as 60 or more, including 64 or more amplifiers.

[0067] In some embodiments, the modulator component includes a first amplifier component and a second amplifier component, wherein the first amplifier component can amplify the data signal from each photodetector channel, and the second amplifier component can amplify the data signal from the electronic switch component. In some examples, the first amplifier component includes a plurality of transimpedance amplifiers, and the second amplifier component includes a plurality of differential amplifiers.

[0068] In some instances, the modulator assembly includes an electronic switch (e.g., a digital switching circuit) that can bin data signals from two or more different photodetector channels. In some instances, the switch can multiplex or demultiplex the output data signals from each photodetector channel. Depending on the number of photodetector channels and amplifiers used in the optical detection system (as described above), the electronic switch assembly can include 2 or more electronic switches, such as 3 or more electronic switches, such as 4 or more electronic switches, such as 5 or more electronic switches, such as 6 or more electronic switches, such as 7 or more electronic switches, such as 8 or more electronic switches, such as 9 or more electronic switches, such as 10 or more electronic switches, such as 15 or more electronic switches, such as 25 or more electronic switches, such as 50 or more electronic switches, such as 100 or more electronic switches, such as 250 or more electronic switches, such as 500 or more electronic switches, such as 750 or more electronic switches, including 1000 or more electronic switches.

[0069] In some embodiments, the target light detection system includes a photodetector array having N photodetector channels and an amplifier component having N transimpedance amplifiers, where N is an integer between 4 and 1000. In some instances, the light detection system includes a photodetector array having N photodetector channels, a modulator component having N transimpedance amplifiers, and an electronic switch component having an N×N switch array. In some instances, the photodetector array can be a photodiode array having N photodiodes. The modulator component in these embodiments can include an array of N transimpedance amplifiers and 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.

[0070] A system that detects light from particles in a sample using spectral identification

[0071] Various aspects of the present disclosure include systems for detecting light from particles in a flow stream using spectral discrimination. In certain embodiments, the system can differentially detect a spectrum of wavelengths of light from irradiating particles, wherein, in certain instances, differential detection of the spectrum of light from irradiating particles can be used to identify or characterize particles or components thereof. The subject system can detect multiple different wavelengths of light emitted from irradiating particles, wherein, in certain instances, one or more data signals are generated in response to each different detected wavelength of light.

[0072] In an embodiment, a system includes a light detection system as described in the present invention, wherein the light detection system has: a wavelength separator component that can 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 can pass light having a predetermined sub-spectral range; and a plurality of photodetectors positioned to detect light of each sub-spectral range that passes through the linear variable optical filters. In certain embodiments, the system includes an optical collection system for transmitting light (e.g., from an irradiation flow stream) to the light detection system.

[0073] Each optical collection system can be any suitable light collection scheme that collects light from the irradiated sample and directs the light to a 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 a fiber optic optical repeater bundler. In other embodiments, the optical collection system is a free space optical relay system.

[0074] In an embodiment, each optical collection system may be physically coupled to the light detection system by an adhesive, co-molded with the light detection system, or integrated into each light detection module. In some embodiments, the light detection system and the optical collection system are integrated into a single unit. In some instances, the light detection system is coupled to the optical collection system by a connector (e.g., by hook and loop fasteners, magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, Velcro, non-permanent adhesives, or combinations thereof), wherein the connector secures the optical collection system to each light detection module.

[0075] In other embodiments, the light detection system is in optical communication with the optical collection system but is not in physical contact. In embodiments, the optical collection system may be 0.001 mm or more, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 10 mm or more, 25 mm or more, 50 mm or more, including 100 mm or more from the light detection module.

[0076] In some embodiments, the optical collection system includes an optical fiber. For example, the optical collection system can be a fiber optic light relay bundle, and the light from the sample is transmitted to the first set of linear variable optical filters of the optical detection system through the fiber optic light relay bundle. Any fiber optic light relay system can be used for light transmission, wherein, in some embodiments, suitable fiber optic light relay systems include but are not limited to the fiber optic light relay system described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated by reference in the present invention and becomes a part of the present invention.

[0077] In other embodiments, the optical collection system is a free space optical relay system. The phrase "free space optical relay" as used in the present invention refers to the propagation of light in its conventional sense, which uses a configuration of one or more optical components to guide light from a sample through free space to a light detection system. In certain embodiments, the free space optical relay system includes a housing having a proximal end and a distal end, and the proximal end is coupled to a light detection module. The free space relay system may include any combination of different optical components, such as one or more lenses, reflectors, slits, pinholes, wavelength separators, or combinations thereof. For example, in certain embodiments, the target free space optical relay system includes one or more focusing lenses. In other embodiments, the subject free space optical relay system includes one or more reflectors. In other embodiments, the free space optical relay system includes a collimating lens. In some embodiments, suitable free-space optical relay systems for transmitting light from the sample to the first set of linear variable optical filters of the optical detection system include, but are not limited to, optical relay systems such as those described in U.S. Patent Nos. 7,643,142, 7,728,974, and 8,223,445, the disclosures of which are incorporated herein by reference and become a part thereof.

[0078] The target system for measuring light from a sample includes a light source. In an embodiment, the light source can be any suitable broadband or narrowband light source. Depending on the components in the sample (such as cells, microspheres, non-cellular particles, etc.), the light source can emit light with a variable wavelength range of 200 nm-1500 nm (e.g., 250 nm-1250 nm, such as 300 nm-1000 nm, such as 350 nm-900 nm, including 400 nm-800 nm). For example, the light source may include a broadband light source, which can emit light with a wavelength of 200 nm-900 nm. In other examples, the light source includes a narrowband light source, which can emit light with a wavelength range of 200 nm-900 nm. For example, the light source can be a narrowband LED (1 nm-25 nm), which can emit light with a wavelength range of 200 nm-900 nm.

[0079] In some embodiments, the light source is a laser. The target laser 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 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 , neon copper (NeCu) lasers, copper lasers or gold lasers and combinations thereof; solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs) or frequency doubling or tripling of any of the above lasers.

[0080] In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a light emitting diode, such as a continuous spectrum broadband LED, a superluminescent diode, a semiconductor light emitting diode, a wide spectrum white light LED light source, a multi-LED integrated light source. In some instances, the non-laser light source is a stabilized fiber-coupled broadband light source, a white light source, other light sources, or any combination thereof.

[0081] In some embodiments, the light source is a beam generator that can generate two or more frequency-shifted light beams. In some instances, the beam generator includes a laser, a radio frequency generator, and the radio frequency generator can apply a radio frequency drive signal to the 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 target beam generator 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 (H eSe) lasers, helium silver (HeAg) lasers, strontium lasers, neon copper (NeCu) lasers, copper lasers or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers or cerium-doped lasers and combinations thereof.

[0082] The acousto-optic device can be any suitable acousto-optic scheme that can use an applied sound wave to frequency shift the laser. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system can use light from a laser and an applied RF drive signal to produce an angularly deflected laser beam. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), any waveform generator (AWG), or an electrical pulse generator.

[0083] In an embodiment, the controller may apply an RF drive signal to the acousto-optic device to generate a desired number of angularly deflected laser beams in the output laser beam, for example, 3 or more RF drive signals may be applied, for example, 4 or more RF drive signals, for example, 5 or more RF drive signals, for example, 6 or more RF drive signals, for example, 7 or more RF drive signals, for example, 8 or more RF drive signals, for example, 9 or more RF drive signals, for example, 10 or more RF drive signals, for example, 15 or more RF drive signals, for example, 25 or more RF drive signals, for example, 50 or more RF drive signals, including applying 100 or more RF drive signals.

[0084] In some examples, in order to generate an intensity profile of an angularly deflected laser beam in the output laser beam, the controller may apply a radio frequency drive signal with different amplitudes, for example, about 0.001 V to about 500 V, for example, about 0.005 V to about 400 V, for example, about 0.01 V to about 300 V, for example, about 0.05 V to about 200 V, for example, about 0.1 V to about 100 V, for example, about 0.5 V to about 75 V, for example, about 1 V to about 50 V, for example, about 2 V to about 40 V, for example, about 3 V to about 30 V, including about 5 V to about 25 V. In certain embodiments, the frequency of each applied RF drive signal is about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz, including about 5 MHz to about 50 MHz.

[0085] In some embodiments, the controller has a processor, the processor has a memory, the memory is operably coupled to the processor, so that the memory includes instructions stored thereon, and when the processor executes these instructions, these instructions cause the processor to generate an output laser beam, and the angularly deflected laser beam has a desired intensity profile. For example, the memory may include instructions to generate two or more angularly deflected laser beams with the same intensity, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, including the memory may include instructions to generate 100 or more angularly deflected laser beams with the same intensity. In other embodiments, the memory may include instructions to generate two or more angularly deflected laser beams with different intensities, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 50 or more, including the memory may include instructions to generate 100 or more angularly deflected laser beams with different intensities.

[0086] In certain embodiments, the controller has a processor, the processor has a memory, the memory is operably coupled to the processor, such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases along the horizontal axis from the edge to the center of the output laser beam. In these examples, the intensity of the angularly deflected laser beam at the center of the output beam can be 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, including 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, the processor has a memory, the memory is operably coupled to the processor, such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from an edge to a center of the output laser beam along the horizontal axis. In these examples, the intensity of the angularly deflected laser beam at the edge of the output beam can be 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, including 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 other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having a Gaussian intensity profile along a horizontal axis. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0087] In an embodiment, the target beam generator may generate spatially separated angularly deflected laser beams in the output laser beam. Depending on the applied RF drive signal and the desired output laser beam irradiation profile, the angularly deflected laser beams may be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more, including 5000 μm or more. In some embodiments, the system may generate an angularly deflected laser beam in the output laser beam that overlaps with an adjacent angularly deflected laser beam along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (such as the overlap of beam spots) can be an overlap of 0.001 μm or more, for example an overlap of 0.005 μm or more, for example an overlap of 0.01 μm or more, for example an overlap of 0.05 μm or more, for example an overlap of 0.1 μm or more, for example an overlap of 0.5 μm or more, for example an overlap of 1 μm or more, for example an overlap of 5 μm or more, for example an overlap of 10 μm or more, including an overlap of 100 μm or more.

[0088] In some instances, as described in U.S. Patent Nos. 9,423,353; 9,784,661 and 10,006,852 and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, a beam generator that can generate two or more beams of frequency-shifted light includes a laser excitation module; the disclosures of the U.S. patents and publications are incorporated by reference in the present invention and become a part of the present invention.

[0089] The light source can be positioned at any suitable distance from the sample (e.g., the flow stream in a flow cytometer), such as 0.001 mm or more, 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, including a distance of 100 mm or more from the flow stream. In addition, the light source irradiates the sample at any suitable angle (e.g., relative to the vertical axis of the flow stream), such as an angle ranging from 10°-90°, such as 15°-85°, such as 20°-80°, such as 25°-75°, including 30°-60°, such as an angle of 90°.

[0090] The light source may irradiate the sample continuously or at discrete intervals. In some instances, the system includes a light source that can continuously irradiate the sample, such as using a continuous wave laser to continuously irradiate the flow stream at the interrogation point of the flow cytometer. In other instances, the target system includes a light source that can irradiate the sample at discrete intervals (e.g., every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds or some other interval). When the light source irradiates the sample at discrete intervals, the system may include one or more additional components to intermittently irradiate the sample with the light source. For example, the subject system in these embodiments may include one or more laser beam choppers, manual or computer-controlled beam cutoffs, for blocking the sample and placing it under the light source.

[0091] In some embodiments, the system further includes a flow chamber that can propagate particles in the flow stream. Any suitable flow chamber can be used to propagate the fluid sample to the sample interrogation area, wherein, in some embodiments, the flow chamber includes a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminating in a flat surface (having an orifice transverse to the longitudinal axis). The length of the proximal cylindrical portion (measured along the longitudinal axis) can range from 1 mm to 15 mm, such as 1.5 mm to 12.5 mm, such as 2 mm to 10 mm, such as 3 mm to 9 mm, including 4 mm to 8 mm. The length of the distal truncated conical portion (measured along the longitudinal axis) can also range from 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm, including 4 mm to 7 mm. In some embodiments, the diameter of the flow chamber nozzle cavity can range from 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm, including 4 mm to 7 mm.

[0092] In some cases, the flow chamber does not include a cylindrical portion, and the entire flow chamber inner cavity is truncated conical. In these embodiments, the length of the truncated conical inner cavity (measured along the longitudinal axis transverse to the nozzle orifice) can range from 1 mm to 15 mm, such as 1.5 mm to 12.5 mm, such as 2 mm to 10 mm, such as 3 mm to 9 mm, including 4 mm to 8 mm. The diameter of the proximal portion of the truncated conical inner cavity can range from 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm, including 4 mm to 7 mm.

[0093] In some embodiments, the sample flow stream flows out of an orifice at the distal end of the flow chamber. Depending on the desired flow stream characteristics, the flow chamber orifice may have any suitable shape, wherein the target cross-sectional shape includes, but is not limited to: a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, an ellipse; and an irregular shape, such as a parabolic bottom coupled with a flat top. In some embodiments, the target flow chamber has a circular orifice. In some embodiments, the nozzle orifice size may vary in the range of 1 μm-20000 μm, such as 2 μm-17500 μm, such as 5 μm-15000 μm, such as 10 μm-12500 μm, such as 15 μm-10000 μm, such as 25 μm-7500 μm, such as 50 μm-5000 μm, such as 75 μm-1000 μm, such as 100 μm-750 μm, including 150 μm-500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0094] In certain embodiments, the flow chamber includes an injection port that can provide a sample to the flow chamber. In an embodiment, the injection system can provide a suitable sample flow to the inner cavity of the flow chamber. Depending on the desired flow characteristics, the sample rate transmitted to the inner cavity of the flow chamber through the injection port can be 1 μL / min or more, such as 2 μL / min or more, such as 3 μL / min or more, such as 5 μL / min or more, such as 10 μL / min or more, such as 15 μL / min or more, such as 25 μL / min or more, such as 50 μL / min or more, including 100 μL / min or more, wherein in certain instances, the sample rate transmitted to the inner cavity of the flow chamber through the injection port is 1 μL / sec or more, such as 2 μL / sec or more, such as 3 μL / sec or more, such as 5 μL / sec or more, such as 10 μL / sec or more, such as 15 μL / sec or more, such as 25 μL / sec or more, such as 50 μL / sec or more, including 100 μL / sec or more.

[0095] The injection port can be an orifice located on the inner cavity wall, or it can be a catheter located at the proximal end of the inner cavity. In the case where the injection port is an orifice on the inner cavity wall, the injection port orifice can be of any suitable shape, wherein the target cross-sectional shape includes but is not limited to: a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, ellipse, etc.; and an irregular shape, such as a parabolic bottom coupled with a flat top. In some embodiments, the injection port has a circular orifice. The size of the injection port orifice may vary depending on the shape, and in some instances, its opening range is 0.1 mm-5.0 mm, such as 0.2-3.0 mm, such as 0.5 mm-2.5 mm, such as 0.75 mm-2.25 mm, such as 1 mm-2 mm, including 1.25 mm-1.75 mm, such as 1.5 mm.

[0096] In some instances, the injection port is a conduit located at the proximal end of the lumen of the flow chamber. For example, the injection port can be a conduit having an injection port orifice in line with the flow chamber orifice. In the case where the injection port is a conduit in line with the flow chamber orifice, the cross-sectional shape of the injection tube can be any suitable shape, wherein the target cross-sectional shape includes but is not limited to: a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, ellipse, etc.; and an irregular shape, such as a parabolic bottom coupled with a flat top. The conduit orifice can vary in shape, and in some instances, its opening range is 0.1 mm-5.0 mm, such as 0.2-3.0 mm, such as 0.5 mm-2.5 mm, such as 0.75 mm-2.25 mm, such as 1 mm-2 mm, including 1.25 mm-1.75 mm, such as 1.5 mm. The shape of the injection port tip can be the same as or different from the cross-sectional shape of the injection tube. For example, the injection port orifice may include a beveled tip having a bevel angle in the range of 1°-10°, such as 2°-9°, such as 3°-8°, such as 4°-7°, including a bevel angle of 5°.

[0097] In some embodiments, the flow chamber further comprises a sheath fluid injection port for providing sheath fluid to the flow chamber. In embodiments, the sheath fluid injection system can provide a sheath fluid flow to the inner cavity of the flow chamber, for example, to generate a layered sheath fluid flow stream around the sample flow stream together with the sample. Depending on the desired flow stream characteristics, the rate at which the sheath fluid is delivered to the flow chamber cavity can be 25 μL / sec or more, for example 50 μL / sec or more, for example 75 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, for example 1000 μL / sec or more, including 2500 μL / sec or more.

[0098] In some embodiments, the sheath injection port is an orifice on the inner cavity wall. The sheath injection port orifice can be in any suitable shape, wherein the target cross-sectional shape includes but is not limited to a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, an ellipse; and an irregular shape, such as a parabolic bottom coupled with a flat top. The size of the injection port orifice can vary depending on the shape, and in some instances, its opening range is 0.1 mm-5.0 mm, such as 0.2-3.0 mm, such as 0.5 mm-2.5 mm, such as 0.75 mm-2.25 mm, such as 1 mm-2 mm, including 1.25 mm-1.75 mm, such as 1.5 mm.

[0099] In certain embodiments, the system further includes a pump in fluid communication with the flow chamber, the pump being used to propagate the flow stream through the flow chamber. Any suitable fluid pump scheme can be used to control the flow stream to flow through the flow chamber. In some cases, the system includes a peristaltic pump, such as a peristaltic pump with a pulse dampener. The pump in the subject system causes the fluid to flow through the flow chamber at a rate suitable for detecting light from a sample in a flow cell flow. In certain instances, the sample flow rate in the flow chamber is 1 μL / min (microliter / minute) or more, such as 2 μL / min or more, such as 3 μL / min or more, such as 5 μL / min or more, such as 10 μL / min or more, such as 25 μL / min or more, such as 50 μL / min or more, such as 75 μL / min or more, such as 100 μL / min or more, such as 250 μL / min or more, such as 500 μL / min or more, such as 750 μL / min or more, including 1000 μL / min or more. For example, the system may include a pump that flows the sample through the flow chamber at a rate ranging from 1 μL / min-500 μL / min (e.g., 1 μL / min-250 μL / min, such as 1 μL / min-100 μL / min, such as 2 μL / min-90 μL / min, such as 3 μL / min-80 μL / min, such as 4 μL / min-70 μL / min, such as 5 μL / min-60 μL / min, including 10 μL / min-50 μL / min). In some embodiments, the flow rate of the flow stream is 5 μL / min-6 μL / min.

[0100] In certain embodiments, the subject system is a flow cytometric system. Suitable flow cytometric systems may include, but are not limited to, those described in the following literature: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology, Vol. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss Press (1995); Virgo et al. (2012), Annals of Clinical Biochemistry, January; 49 (pt. 1):17-28; Linden et al., Seminars in Thrombosis and Hemostasis, October 2004; 30(5):502-11; Alison et al., Journal of Pathology, December 2010; 222(4):335-344; and Herbig et al. (2007), Critical Reviews of Therapeutic Drug Delivery Systems, 24(3):203-255; the disclosures of the above references are incorporated herein by reference. In certain embodiments, the flow cytometry system of interest comprises a BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TMCell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter, BD Biosciences FACSAria TM II cell sorter, BD Biosciences FACSAria TM III cell sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorter, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0101] In certain embodiments, the subject system is a flow cytometric system, such as those described in U.S. Pat. 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; No. 10,302,545; No. 10,145,793; No. 10,113,967; No. 10,006,852; No. 9,952,076; No. 9,933,341; No. 9,726,527; No. 9,453,789; No. 9,200,334; No. 9,097,640; No. 9,0 No. 95,494; No. 9,092,034; No. 8,975,595; No. 8,753,573; No. 8,233,146; No. 8,140,300; No. 7,544,326; No. 7,201,875; No. 7,129,505; No. 6,821,740; No. 6,813,017; No. 6,809 ,804; No. 6,372,506; No. 5,700,692; No. 5,643,796; No. 5,627,040; No. 5,620,842; No. 5,602,039; No. 4,987,086; No. 4,498,766; the disclosures of the aforementioned U.S. patents are incorporated herein by reference in their entirety and become a part of the present invention.

[0102] In certain embodiments, the subject system is a particle sorting system that can sort particles through a closed particle sorting module, such as the particle sorting module described in U.S. Patent Publication No. 2017 / 0299493, the contents of which are incorporated by reference into this invention and become a part of this invention. In certain embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated by reference into this invention and becomes a part of this invention. In certain embodiments, the subject system includes a particle sorting module having a deflection plate, such as described in U.S. Patent Publication No. 2017 / 0299493 filed on March 28, 2017, the disclosure of which is incorporated by reference into this invention and becomes a part of this invention.

[0103] In some cases, the flow cytometry system of the present invention can image particles in the flow cell stream by using fluorescence imaging of radio frequency labeled emission (FIRE), as described in the following references: Diebold et al., Nature Photonics, Vol. 7(10), 806-810 (2013); No. 9,423,353; No. 9,784,661; No. 9,983,132; No. 10,006,852; No. 10,078,045; No. 10,036,699; No. 10,222,316; No. 1 No. 0,288,546; No. 10,324,019; No. 10,408,758; No. 10,451,538; No. 10,620,111; and U.S. Patent Nos. 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894; the disclosures of the said U.S. patents are incorporated herein by reference.

[0104] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4A ) analyze and characterize particles. Figure 4A A functional block diagram of a particle analysis system for performing computation-based sample analysis and particle characterization is shown. In certain embodiments, particle analysis system 401 is a flow system. Figure 4A The particle analysis system 401 shown can fully or partially perform the method described in the present invention. Figure 4AThe particle analysis system 401 shown can perform all or part of the methods described in the present invention. The particle analysis system 401 includes a fluid system 402. The fluid system 402 may include a sample tube 405 and a moving fluid column located in the sample tube or coupled thereto, and particles 403 (e.g., cells) of a sample in the sample tube move along a common sample path 409.

[0105] Particle analysis system 401 includes a detection system 404 that can collect signals from each particle as it passes through one or more detection stations along a common sample path. Detection station 408 is generally referred to as a monitored area 407 of the common sample path. In some embodiments, detection can include detecting light or one or more other characteristics of the particle 403 as it passes through the monitored area 407. Figure 4A , one detection station 408 and one monitoring area 407 are shown. Some embodiments of particle analysis system 401 may include multiple detection stations. In addition, some detection stations may monitor more than one area.

[0106] Each signal has a signal value, 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, including measurements of various properties of the particle. The detection system 404 collects a series of such data points over a first time interval.

[0107] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, amplitude control circuits, and / or frequency control circuits. The control system shown may be operably associated with the fluid system 402. The control system may generate a calculated value of the signal frequency for at least a portion of the first time interval based on the Poisson distribution and number of data points collected by the detection system 404 during the first time interval. The control system 406 may further generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system 406 may also compare the experimental signal frequency with the calculated signal frequency or the experimental signal frequency of the predetermined signal frequency.

[0108] Figure 4B A flow cytometry system according to an illustrative embodiment of the invention is shown. The system includes a flow cytometer 400, a controller / processor 490a, and a memory 495a. 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 collection lens 440.

[0109] Excitation lasers 471, 472, 473, 474, and 475 emit light in the form of laser beams. Figure 4BIn the example system shown, 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. First, the laser beams are guided through one or more beam splitters 471a, 472a, 473a, 474a and 475a. Beam splitter 475a transmits light with wavelengths of 637 nm, 561 nm, 488 nm and 405 nm, and reflects light with a wavelength of 349 nm. Beam splitter 474a transmits light with wavelengths of 637 nm, 561 nm and 488 nm, and reflects light with a wavelength of 405 nm. Beam splitter 473a transmits light with wavelengths of 637 nm and 561 nm, and reflects light with a wavelength of 488 nm. Beam splitter 472a transmits light with a wavelength of 637 nm, and reflects light with a wavelength of 561 nm. Beam splitter 471a reflects light with a wavelength of 637 nm. The laser beam is then directed to a focusing lens 476 which focuses the beam onto the portion of the fluid stream where the sample particles are located in a flow chamber 477. The flow chamber is the portion of the fluid system that directs particles in the stream (usually one at a time) to the focused laser beam for interrogation. The flow chamber may include a flow chamber in a benchtop cytometer or a nozzle tip in an air flow cytometer.

[0110] Light from the laser beam interacts with particles in the sample at various wavelengths by diffraction, refraction, reflection, scattering, and absorption and re-emission, depending on the characteristics of the particles, such as size, internal structure, and the presence or absence of one or more fluorescent molecules attached to or naturally present on or in the particles. Fluorescent emission, as well as diffracted, refracted, reflected, and scattered light, can be directed to one or more of forward scatter detector 480a and side scatter detector 480b, and one or more of light detection systems 478a, 478b, 478c, 478d, and 478e, by fluorescence collection lens 440. In some embodiments, light from flow chamber 477 is collected using an optical collection assembly, such as an optical fiber or free space relay as described above, and propagated to one or more of light detection systems 478a, 478b, 478c, 478d, and 478e.

[0111] The light from particles irradiated by each laser according to certain embodiments is detected using various light detection systems described in the present invention. Figure 3 As shown, each optical detection system includes: a wavelength separator component that can transmit light between a first group of linear variable optical filters and a second group of linear variable optical filters, wherein each group of linear variable optical filters can pass light having a predetermined sub-spectral range; and a plurality of photodetectors that are positioned to detect light of each sub-spectral range that passes through the linear variable optical filters. Figure 4BIn the embodiment, the light detection module 478a can detect light from particles propagating through the flow chamber 477 irradiated by the laser 471 (for example, a 637 nm laser); the light detection module 478b can detect light from particles propagating through the flow chamber 477 irradiated by the laser 472 (for example, a 561 nm laser); the light detection module 478c can detect light from particles propagating through the flow chamber 477 irradiated by the laser 473 (for example, a 488 nm laser); the light detection module 478d can detect light from particles propagating through the flow chamber 477 irradiated by the laser 474 (for example, a 405 nm laser); and the light detection module 478e can detect light from particles propagating through the flow chamber 477 irradiated by the laser 475 (for example, a 349 nm laser). In Figure 4B In the embodiment shown, different light detection modules are used to detect light from the flow stream from each laser line. In embodiments, the light detection modules can be arranged at any position along the flow stream at any angle. In some examples, such as Figure 4B As shown, the light detection modules are arranged in a row along the longitudinal axis of the flow stream. Depending on the irradiation position of each laser, the light detection modules can be spaced 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, including 250 mm or more, from each other along the longitudinal axis of the flow stream.

[0112] The forward scattering detector 480a is slightly offset from the axis of the direct beam through the flow chamber and can detect diffracted light and excitation light that mainly passes through the particles or propagates around the particles in the forward direction. The intensity of the light detected by the forward scattering detector depends on the overall size of the particles. The forward scattering detector may include a photodiode. The side scattering detector 480b can detect refracted light and reflected light from the surface and internal structure of the particles, and tends to increase with the increase of the complexity of the particle structure. The signals detected at the forward scattering detector 480a, the side scattering detector 480b and the fluorescence detector can be converted into electronic signals (voltage) by the detector. This data can provide information about the sample.

[0113] Those skilled in the art will appreciate that the flow cytometer according to the embodiments of the present invention is not limited to Figure 4B The flow cytometer shown, but 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 of various wavelengths and various different configurations.

[0114] During operation, the flow cytometer is controlled by a controller / processor 490, and measurement data from the detector may be stored in a memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive output signals therefrom, and may also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, etc. The system may also have input / output (I / O) capabilities 497. The memory 495, the controller / processor 490, and the I / O 497 may all be integral to the flow cytometer 410. In such embodiments, the display may also constitute part of the I / O capabilities 497 for presenting experimental data to a user of the cytometer 400. Alternatively, the memory 495 and the controller / processor 490 and part or all of the I / O capabilities may be part of one or more external devices (e.g., a general purpose computer). In some embodiments, the memory 495 and part or all of the controller / processor 490 may communicate wirelessly or wired with the flow cytometer 400. Controller / processor 490, together with memory 495 and I / O 497, performs various functions related to the preparation and analysis of flow cytometric experiments.

[0115] I / O 497 can receive data about a flow cytometer experiment, the experiment comprising a set of fluorescent labels and a plurality of cell populations having a plurality of labels, each cell population having a subset of the plurality of labels. I / O 497 can also receive biological data assigning one or more labels to one or more cell populations, label density data, emission spectrum data, data assigning labels to one or more labels, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 495. Controller / processor 490 can evaluate one or more results of the assignment of labels to the labels.

[0116] Figure 5 is a functional block diagram of one example of a particle analyzer control system, such as analysis controller 500, for analyzing and displaying biological events. Analysis controller 500 may implement various processes for controlling the graphical display of biological events.

[0117] The particle analyzer or sorting system 502 may collect biological event data. For example, a flow cytometer may generate flow cytometric event data. The particle analyzer 502 may provide the biological event data to the analysis controller 500. A data communication channel may be provided between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0118] The analysis controller 500 may receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometric event data. The analysis controller 500 may provide a graphical display to a display device 506, including a first chart of the biological event data. For example, the analysis controller 500 may further present a target area as a gate around a set of biological event data overlaid on the first chart as shown by the display device 506. In some embodiments, the gate can be a logical combination of one or more target graphical areas drawn on a single parameter histogram or a bivariate graph. In some embodiments, the display can be used to display particle parameters or saturated detector data.

[0119] Analysis controller 500 may further display the biological event data inside the gate on display device 506, which is different from other events in the biological event data outside the gate. For example, analysis controller 500 may present the biological event data contained inside the gate in a color different from the color of the biological event data outside the gate. Display device 506 may be implemented as a monitor, a tablet computer, a smart phone, or other electronic device that presents a graphical interface.

[0120] The analysis controller 500 can receive a door selection signal from a first input device that identifies a door. For example, the first input device can be implemented as a mouse 510. The mouse 510 can send a door selection signal to the analysis controller 500 to identify the door to be displayed on the display device 506 or manipulated by the display device (for example, when the cursor is on or in the desired door, just click here). In some embodiments, the first device can be implemented as a keyboard 508 or other device for providing input signals to the analysis controller 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function can be considered an input device. For example, if Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, each of which may generate a trigger event.

[0121] The triggering event may cause the analysis controller 500 to change the manner in which the data is displayed and the portion of the data actually displayed on the display device 506, and / or provide input information for further processing, such as selecting a target population for particle sorting.

[0122] In certain embodiments, analysis controller 500 can detect when a gate selection is initiated by mouse 510. Analysis controller 500 can further automatically modify the chart visualization to facilitate the gating process. The modification can be based on a particular distribution of bio-event data that analysis controller 500 receives.

[0123] Analysis controller 500 may be coupled to storage device 504. Storage device 504 may receive and store biological event data from analysis controller 500. Storage device 504 may also receive and store flow cytometric event data from analysis controller 500. Storage device 504 may further allow retrieval of biological event data, such as flow cytometric event data, by analysis controller 500.

[0124] Display device 506 may receive display data from analysis controller 500. Display data may include a graph of biological event data and gates outlining portions of the graph. Display device 506 may further change the information presented based on input information received from analysis controller 500, in combination with input information from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0125] In some embodiments, the analysis controller 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include a control for receiving sample events or sample images. Sample events or images or sample gates may be provided before collecting event data for a sample, or based on an initial set of events for a portion of the sample.

[0126] Fig. 6A 6A is a schematic diagram of a particle sorter system 600 (e.g., a particle analyzer or sorting system 502) according to an embodiment of the present invention. In some embodiments, the particle sorter system 600 is a cell sorter system. As shown in 6A, a droplet formation sensor 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which can be coupled to a nozzle 603, can include the nozzle 603, or can be the nozzle 603. Within the fluid conduit 601, a sheath fluid 604 fluidically focuses a sample fluid 606 containing particles 609 into a moving liquid column 608 (e.g., a liquid stream). Within the moving liquid column 608, the particles 609 (e.g., cells) are arranged in a single file, traverse a monitoring area 611 (e.g., where a laser and a liquid stream meet), and are irradiated by an irradiation source 612 (e.g., a laser). The droplet formation sensor 602 vibrates, causing the moving liquid column 608 to break into a plurality of droplets 610, some of which contain particles 609.

[0127] During operation, the time when a target particle (or target cell) traverses the monitoring area 611 is determined by a detection station 614 (e.g., an event detector). The detection station 614 feeds into a timing circuit 628, which in turn feeds into a flash charging circuit 630. At the droplet breakoff point, the moving liquid column 608 can be flash charged at the prompt of the timed droplet delay (Δt) to charge the target droplet. The target droplet may include one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the charged droplets into a container such as a collection tube or a multi-well or micro-porous sample plate, in which a well or micro-well can be associated with a specific target droplet. As shown in FIG. Fig. 6A As shown, the droplets may be collected in a discharge container 638 .

[0128] When the target particle passes through the monitoring area 611, the detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated by reference in its entirety herein. The detection system 616 allows the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 can feed an amplitude signal 620 and / or a phase signal 618, which in turn (via an amplifier 622) is fed into an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 in turn controls the droplet formation sensor 602. The amplitude control circuit 626 and / or the frequency control circuit 624 can be included in the control system.

[0129] In some embodiments, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory that can store detected events and sorting decisions based on the events. The sorting decision may be included in the event data of the particle. In some embodiments, the detection system 616 and the detection station 614 may be implemented as a single detection unit or may be coupled in a communication manner so that event measurements can be collected by the detection system 616 or the detection station 614 and provided to a non-collecting component.

[0130] Figure 6B is a schematic diagram of a particle sorter system according to an embodiment of the present invention. Figure 6B The particle sorter system 600 shown includes deflection plates 652 and 654. Charge can be applied through the liquid flow charging wires in the barbs. This forms a droplet stream 610 containing particles 610 to be analyzed. One or more light sources (such as lasers) can be used to irradiate the particles to generate light scattering and fluorescence information. The particles are detected by sorting electronics or other detection systems ( Figure 6BThe deflector plates 652 and 654 can be independently controlled to attract or repel charged droplets and direct the droplets to a destination collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, the deflection plates 652 and 654 can be controlled to direct the particles to flow along the first path 662 to the container 674 or along the second path 668 to the container 678. If the particle is not a target particle (e.g., does not show scattering or illumination information within a specified sorting range), the deflection plates can allow the particle to continue to flow along the flow path 664. Such uncharged droplets can enter a waste container via an aspirator 670 or the like.

[0131] Sorting electronics may be included to initiate measurement collection, receive the fluorescence signal from the particles, and determine how to adjust the deflection plates to achieve particle sorting. Figure 6B Example implementations of the illustrated embodiment include a BD FACSAria TM Serial flow cytometer.

[0132] Computer control system

[0133] Aspects of the present disclosure further include a computer controlled system, wherein the system further includes one or more computers implemented for implementing full automation or partial automation. In certain embodiments, the system includes a computer having a computer readable storage medium having a computer program stored thereon, wherein when loaded on the computer, the computer program includes instructions for irradiating particles propagating along a flow stream through an interrogation zone using a light detection system, the light detection system having: a wavelength separator assembly that can 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 can pass light having a predetermined sub-spectral range; and a plurality of photodetectors positioned to detect light across each sub-spectral range of the linear variable optical filters.

[0134] In some embodiments, the computer control system includes a memory on which instructions for binning data signals from different photodetector channels are stored. 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 instances, the computer program includes instructions for binning data signals from non-adjacent photodetectors. In some instances, the computer program includes instructions for binning data signals from adjacent photodetectors. In some instances, 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 (e.g., 4 or more, e.g., 5 or more, e.g., 6 or more, e.g., 16 or more, e.g., 32 or more, including from 64 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.

[0135] In some embodiments, the computer program includes instructions for amplifying data signals from one or more photodetector channels. In some instances, 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 instances, the computer program includes instructions for binning the multiplexed data signals.

[0136] In an embodiment, the system includes an input module, a processing module, and an output module. The subject system may include hardware and software components, wherein the hardware components may take the form of one or more platforms, such as a server, to implement functional components, i.e., system components that perform specific tasks of the system (e.g., manage the input and output of information, process information, etc.) by executing software applications on one or more typical computer platforms of the system or across the platforms.

[0137] The system may include a display and an operator input device. For example, the operator input device may be a keyboard, a mouse, etc. The processing module includes a processor that can access a memory containing instructions for executing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, an input and output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or may be another processor that is available now or in the future. The processor executes an operating system that interfaces with firmware and hardware in a known manner to help the processor coordinate and execute the functions of various computer programs, which may be in various programming languages, such as Java, Perl, C++, and other high-level or low-level languages ​​and combinations thereof 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 and output control, file and data management, memory management, and communication control and related services, all of which are based on known technologies. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align the light source with the flow stream based on the first and second light signals. In certain embodiments, the processor includes analog electronics that provide feedback control (eg, negative feedback control).

[0138] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly used random access memory (RAM), magnetic media (such as a resident hard disk or tape), optical media (such as a read-write optical disk), flash memory devices, or other memory storage devices. The memory storage device may be any of a variety of known or future devices, including an optical drive, a tape drive, a removable hard drive, or a floppy disk drive. Such memory storage devices typically read and / or write to program storage media (not shown) such as optical disks, tapes, removable hard disks, or floppy disks, respectively. Any of these program storage media, or other program storage media currently in use or that may be developed in the future, may be considered a computer program product. It should be understood that these program storage media typically store computer software programs and / or data. Computer software programs (also known as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0139] In some embodiments, the present invention describes a computer program product including a computer usable medium containing control logic (computer software program, including program code). When executed by a processor and a computer, the control logic causes the processor to perform the functions described in the present invention. In other embodiments, some functions are implemented in hardware, for example, primarily through a hardware state machine. It will be apparent to a person skilled in the relevant art that implementing a hardware state machine can perform the functions described in the present invention.

[0140] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical or solid-state storage device (including a disk, optical disk, tape or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor, which is appropriately programmed by a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communication channel, or previously stored in a computer program product (such as a memory or other portable or fixed computer-readable storage medium using any memory-related device). For example, a disk or optical disk may carry the programming and may be read by a disk writer / reader. The system of the present invention also includes, for example, programming of the algorithm for implementing the above-mentioned method in the form of a computer program product. The programming according to the present invention may be recorded on a computer-readable medium (such as 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 tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and hybrid media of these categories, such as magnetic / optical storage media.

[0141] The processor may also access a communication channel to communicate with a user at a remote location. A remote location is one where the user is not directly interacting with the system and relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., smart phone).

[0142] In some embodiments, the system according to the present disclosure may 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 used 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).

[0143] In one embodiment, the communication interface may include one or more communication ports, such as physical ports or interfaces (e.g., USB ports, RS-232 ports, or any other suitable electrical connection ports) to enable data communications between the subject system and other external devices such as computer terminals for similar supplemental data communications (e.g., in a doctor's office or hospital environment).

[0144] In one embodiment, the communication interface is used for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, so that the subject system can communicate with a computer terminal and / or network, a communication-enabled mobile phone, a personal digital assistant, or other devices, or any other communication device available for use by the user.

[0145] In one embodiment, the communication interface provides a connection for data transfer using the Internet Protocol (IP) through a mobile phone network, a short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet on a WiFi hotspot.

[0146] In one embodiment, the subject system communicates wirelessly with a server device via a communication interface (e.g., using a public standard such as 802.11 or Bluetooth® RF protocol, or an IrDA infrared protocol). The server device may be another portable device, such as a smartphone, a personal digital assistant (PDA), or a laptop; or a larger device, such as a desktop computer, a home appliance, etc. In some embodiments, the server device has a display (e.g., a liquid crystal display (LCD)) and an input device (e.g., a button, a keyboard, a mouse, or a touch screen).

[0147] In certain embodiments, the communication interface automatically or semi-automatically transfers data stored in the subject system (eg, optional data storage unit) via a network or server using one or more of the communication protocols and / or mechanisms described above.

[0148] The output controller may include a controller for any of various known display devices, which are used to present information to a user (whether a person or a machine, whether local or remote). If one of the display devices provides visual information, the information can usually be logically and / or physically organized into a pixel array. A graphical user interface (GUI) controller may include any of various known or future software programs for providing a graphical input and output interface between the system and the user and processing user input information. The functional components of the computer can communicate with each other through a system bus, and in alternative embodiments, some of the communications can be completed using a network or other types of remote communications. The output manager can also provide information generated by the processing module to a user at a remote location according to known techniques, such as through the Internet, telephone or satellite network. The presentation of data by the output manager can be based on various known techniques. For example, the data may include SQL, HTML or XML documents, emails or other files or other forms of data. The data may include an Internet URL address so that the user can retrieve more SQL, HTML, XML or other documents or data from a remote source. Although one or more platforms present in the subject system generally belong to the general category of computers commonly known as servers, they can also be any type of known computer platform or a type developed in the future. However, they may also be mainframes, workstations, or other computer types. These platforms may be networked or otherwise connected by any known or future type of wiring or other communications system, including wireless systems. They may be co-located or physically separate. A variety of operating systems may be applied to any computer platform, which may depend on the type and / or model of computer platform selected. Suitable operating systems include Windows NT ® , Windows XP, Windows 7, Windows 8, iOS, Oracle Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGIIRIX, Siemens Reliant Unix, etc.

[0149] Figure 7 The general architecture of an example computing device 700 is shown in accordance with some embodiments. Figure 7 The general architecture of the computing device 700 shown includes the following computer hardware and software components arranged in a manner similar to those of FIG. Figure 7The components shown are more (or less). However, it is not necessary to show all general conventional components for the purpose of disclosure. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750 and an input device 760, all of which can communicate with each other in the form of a communication bus. The network interface 720 can provide connectivity with one or more networks or computing systems. Therefore, the processing unit 710 can receive information and instructions from other computing systems or services through the network. The processing unit 710 can also communicate with the memory 770, and further provide output information to the optional display 750 through the input / output device interface 740. The input / output device interface 740 can also receive input information from an optional input device 760 (such as a keyboard, a mouse, a digital pen, a microphone, a touch screen, a gesture recognition system, a voice recognition system, a game controller, an accelerometer, a gyroscope or other input device).

[0150] Memory 770 may contain computer program instructions (grouped in modules or components in some embodiments) executed by processing unit 710 to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other permanent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in general management and operation of computing device 700. Memory 770 may further include computer program instructions and other information for implementing various aspects of the present disclosure.

[0151] Method for detecting light from particles in a flowing stream using spectral identification

[0152] Various aspects of the present disclosure also include methods for measuring light from a sample (e.g., in a flow stream of a flow cytometer) using spectral discrimination. In practicing the methods described in accordance with the embodiments, the sample is irradiated with a light source, and the light from the sample is detected with a light detection system having: a wavelength separator assembly that can 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 can pass light having a predetermined sub-spectral range; and a plurality of photodetectors positioned to detect light across each sub-spectral range of the linear variable optical filters, as described above. In certain embodiments, the sample is a biological sample. The term "biological sample" in its conventional sense refers to a subset of a whole organism, plant, fungal, or animal tissue, cell, or component, and in some cases can be present in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Therefore, "biological sample" refers to a primary organism or a subset of its tissues, as well as a homogenate, lysate or extract prepared from an organism or a subset of its tissues, including but not limited to plasma, serum, cerebrospinal fluid, lymph fluid, skin slices, respiratory tract, gastrointestinal tract, cardiovascular, urogenital tract, tears, saliva, milk, blood cells, tumors, organs, etc. A biological sample can be any type of organism tissue, including healthy tissue and diseased tissue (e.g., cancer tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some cases, the sample is a blood sample, including whole blood, such as blood obtained by venipuncture or finger stick (wherein, the blood may be mixed with any reagent such as a preservative, anticoagulant, etc. before analysis, or may not be mixed with any reagent).

[0153] In certain embodiments, the source of the sample is a "mammal" or "mammalian", wherein these terms are broadly used to describe organisms within the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method can be used for samples from human subjects of both sexes and at any stage of development (i.e., newborns, infants, juveniles, adolescents, adults), wherein, in certain embodiments, the human subject is a juvenile, adolescent, or adult. Although the present invention can be used for samples from human subjects, it should be understood that the method can also be used for samples from other animal subjects (i.e., "non-human subjects", such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses).

[0154] When implementing the subject method, the sample (e.g., in the flow stream of a flow cytometer) is irradiated with light from a light source. In certain embodiments, the light source is a broadband light source, emitting light with a wide wavelength range, such as a wavelength span of 50 nm or more, such as 100 nm or more, such as 150 nm or more, such as 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more, including a span of 500 nm or more. For example, a suitable broadband light source emits light with a wavelength of 200 nm-1500 nm. For another example, a suitable broadband light source includes a light source that emits light with a wavelength of 400 nm-1000 nm. When the method includes irradiation with a broadband light source, the target broadband light source scheme may include, but is not limited to, a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a stabilized fiber-coupled broadband light source, a continuous spectrum broadband LED, a superluminescent diode, a semiconductor light emitting diode, a broadband light source such as a wide spectrum LED white light source, a multi-LED integrated white light source, or any combination thereof.

[0155] In other embodiments, the method includes irradiating with a narrowband light source that emits a specific wavelength or a narrow wavelength range, such as using a light source that emits light in a narrow wavelength range (e.g., 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less), including a light source that emits light of a specific wavelength (i.e., monochromatic light). When the method includes irradiating with a narrowband light source, the target narrowband light source scheme may include, but is not limited to, a narrow wavelength LED, a laser diode, a broadband light source coupled to one or more optical bandpass filters, a diffraction grating, a monochromator, or any combination thereof.

[0156] In some embodiments, the method includes irradiating the sample with one or more lasers. As described above, the type and number of lasers will vary depending on the sample and the desired collection light, and 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. In other instances, the method includes irradiating the flow stream with a dye laser, such as a stilbene, coumarin, or rhodamine laser. In other cases, the method includes irradiating the 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 other cases, the method includes irradiating the flow stream with a solid-state laser (e.g., 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, an ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, and combinations thereof).

[0157] The sample may be irradiated with one or more of the above light sources, such as 2 or more light sources, such as 3 or more light sources, such as 4 or more light sources, such as 5 or more light sources, including 10 or more light sources. The light sources may include any combination of various types of light sources. For example, in some embodiments, the method includes irradiating the sample in the flow stream with a laser array (e.g., an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers).

[0158] The sample may be irradiated at a wavelength in the range of 200 nm-1500 nm (e.g., 250 nm-1250 nm, e.g., 300 nm-1000 nm, e.g., 350 nm-900 nm, including 400 nm-800 nm). For example, when the light source is a broadband light source, the sample may be irradiated at a wavelength in the range of 200 nm-900 nm. In other cases, if the light source includes a plurality of narrow-band light sources, the sample may be irradiated at a specific wavelength in the range of 200 nm-900 nm. For example, the light source may be a plurality of narrow-band LEDs (1 nm-25 nm), each of which may independently emit light having a wavelength in the range of 200 nm-900 nm. In other embodiments, the narrow-band light source includes one or more lasers (e.g., a laser array) to irradiate the sample at a specific wavelength in the range of 200 nm-700 nm, such as the above-mentioned laser array having a gas laser, an excimer laser, a dye laser, a metal vapor laser, and a solid-state laser.

[0159] In the case of using more than one light source, the light sources or combinations thereof may be used to irradiate the sample simultaneously or sequentially. For example, each light source may be used to irradiate the sample simultaneously. In other embodiments, each light source is used sequentially to irradiate the flow stream. In the case of using more than one light source to irradiate the sample sequentially, the time for each light source to irradiate the sample may be independently 0.001 microseconds or more, such as 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 30 microseconds or more, including 60 microseconds or more. For example, the method may include irradiating the sample using a light source (such as a laser) for a duration ranging from 0.001 microseconds to 100 microseconds, such as 0.01 microseconds to 75 microseconds, such as 0.1 microseconds to 50 microseconds, such as 1 microsecond to 25 microseconds, including 5 microseconds to 10 microseconds. In embodiments where two or more light sources are used sequentially to irradiate the sample, the duration for each light source to irradiate the sample may be the same or different.

[0160] The time period between the irradiation of each light source can also be changed as needed, and can be separated independently by a delay of 0.001 microseconds or more (e.g., 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 15 microseconds or more, such as 30 microseconds or more, including 60 microseconds or more). For example, the time period between the irradiation of each light source can range from 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, such as 0.1 microseconds to 35 microseconds, such as 1 microsecond to 25 microseconds, including 5 microseconds to 10 microseconds. In some embodiments, the time period between the irradiation of each light source is 10 microseconds. In embodiments where two or more (i.e., 3 or more) light sources are used sequentially to irradiate the sample, the delay between the irradiation of each light source can be the same or different.

[0161] The sample may be irradiated continuously or at discrete intervals. In some cases, the method includes irradiating the sample in the sample continuously using the light source. In other cases, the sample is measured at discrete intervals (e.g., 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 other intervals) using the light source.

[0162] Depending on the light source, the distance of the irradiated sample can be different, for example, a distance of 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, 2.5 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 15 mm or more, for example, 25 mm or more, including 50 mm or more. In addition, the irradiation angle can also be changed, and the angle range is 10°-90°, for example, 15°-85°, for example, 20°-80°, for example, 25°-75°, including 30°-60°, for example, 90°.

[0163] As described in detail above, in embodiments, light from the irradiated sample is transmitted to the light detection system described in the present invention and detected by the plurality of photodetectors. The light detection system can be used to measure the light continuously or at discrete intervals. In some cases, the method includes measuring the light continuously. In other cases, the light is measured at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds (including every 1000 milliseconds), or at other intervals.

[0164] During the implementation of the subject method, the collected light may be measured one or more times (e.g., 2 or more, 3 or more, 5 or more, including 10 or more). In some embodiments, 2 or more light propagation measurements are made, and in some instances, the data are averaged.

[0165] In some embodiments, the method includes conditioning the light before detecting the light using the subject light detection system. For example, the light from the sample source may pass through one or more lenses, reflectors, pinholes, slits, gratings, light refractors, and any combination thereof. In some cases, for example, to reduce the profile of the light directed to the above-mentioned light detection system or optical collection system, the collected light passes through one or more focusing lenses. In other cases, in order to reduce the divergence of the light beam transmitted to the light detection system, the light emitted from the sample passes through one or more collimators.

[0166] Kits

[0167] The present invention also provides a kit including one or more components of the subject light detection system. According to certain embodiments, the kit includes a first group of linear variable optical filters, a second group of linear variable optical filters, and a photodetector. In certain embodiments, one or more of the linear variable optical filters are linear variable bandpass filters, such as a long-pass linear variable bandpass filter or a short-pass linear variable bandpass filter. In some instances, the kit includes an optical collection component, such as a fiber optic optical relay bundle or a free-space optical relay system. The kit may also include one or more photodetectors, such as a photomultiplier tube (such as a metal packaged photomultiplier tube) or a photodiode (such as an avalanche photodiode, APD).

[0168] The kit may also include a flow chamber nozzle and a small glass tube for irradiating the flow stream. The kit may also include optical adjustment elements, such as lenses (e.g., focusing lenses, objective lenses), mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation schemes, and combinations thereof. In some examples, the kit includes one or more light sources, such as lasers, LEDs, broadband light sources, or combinations thereof.

[0169] In addition to the above components, the subject kit may further include (in some embodiments) instructions for implementing the subject method. These instructions may be present in the subject kit in various forms, wherein one or more forms may be present in the kit. One form of these instructions is information printed on a suitable medium or substrate (e.g., one or more sheets of paper with information printed on them), in the kit packaging, in a package instruction, etc. Another form of these instructions is a computer-readable medium containing information, such as a floppy disk, a compact disk (CD), a portable flash drive, etc. Another form of these instructions is a website address that can be used to access the information on the removed site via the Internet.

[0170] Useful functions

[0171] The subject methods, systems, and computer systems can be used in a variety of applications where it is expected to optimize particle identification, characterization, and sorting. The subject methods and systems can detect light using spectral discrimination and can identify or characterize particles in a flow stream. The present disclosure can also be used in flow cytometry where it is expected to provide a flow cytometer that can improve cell sorting accuracy, enhance particle collection, reduce energy consumption, increase particle charging efficiency, improve particle charging accuracy, and enhance particle deflection during cell sorting. In an embodiment, the present disclosure reduces the need for user input or manual adjustments when analyzing samples using a flow cytometer. In certain embodiments, the subject methods and systems provide a fully automated protocol for adjusting a flow cytometer with little to no human input during use.

[0172] Notwithstanding the appended claims, the present disclosure is defined by the following terms:

[0173] 1. A light detection system, comprising:

[0174] a wavelength separator assembly operable 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 operable to pass light having a predetermined sub-spectral range;

[0175] A plurality of photodetectors are positioned to detect light in each sub-spectral range that passes through the linear variable optical filter.

[0176] 2. The optical detection system according to clause 1, wherein each set of linear variable optical filters can pass light in the wavelength range of 200 nm-1200 nm.

[0177] 3. The optical detection system according to clause 1, wherein each set of linear variable optical filters can pass light in the wavelength range of 500 nm-800 nm.

[0178] 4. The optical detection system according to clause 3, wherein the first set of linear variable optical filters can pass light in the wavelength range of 500 nm-650 nm.

[0179] 5. The optical detection system according to clause 3, wherein the second set of linear variable optical filters can pass light in the wavelength range of 650 nm-800 nm.

[0180] 6. A light detection system according to any of clauses 1 to 5, wherein each set of linear variable optical filters comprises a long-pass linear variable bandpass filter and a short-pass linear variable bandpass filter.

[0181] 7. The optical detection system according to clause 6, wherein in each set of linear variable optical filters, there is an offset of a predetermined distance between the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

[0182] 8. The light detection system according to clause 7, wherein the sub-spectral range of light passing through each set of linear variable optical filters is determined by the offset of the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

[0183] 9. The light detection system according to any of clauses 1 to 6, wherein light of each sub-spectral range passed through the set of linear variable optical filters comprises a spectral width of 5 nm-50 nm.

[0184] 10. The light detection system of clause 9, wherein light of each sub-spectral range passed through the set of linear variable optical filters comprises a spectral width of approximately 20 nm.

[0185] 11. A light detection system according to any 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.

[0186] 12. The light detection system of clause 11, wherein the wavelength separator assembly can transfer light back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters.

[0187] 13. A light detection system according to any one of clauses 1 to 12, wherein the wavelength separator assembly is configured such that:

[0188] The first set of linear variable optical filters can pass light from low wavelength to high wavelength;

[0189] The second set of linear variable optical filters can pass light from high wavelengths to low wavelengths.

[0190] 14. A light detection system according to any of clauses 1 to 13, wherein the plurality of photodetectors are positioned across each set of linear variable optical filters.

[0191] 15. The light detection system according to clause 14, wherein each photodetector can detect light in a sub-spectral range with a spectral width of 5 nm-50 nm.

[0192] 16. A light detection system according to clause 15, wherein each photodetector can detect light in a sub-spectral range having a spectral width of approximately 20 nm.

[0193] 17. A light detection system according to any one of clauses 14 to 16, wherein the light detection system comprises:

[0194] A first set of photodetectors is positioned across the first set of linear variable optical filters, wherein the first set of photodetectors can detect light having the following sub-spectral ranges:

[0195] a) 500 nm-520 nm;

[0196] b) 520 nm-540 nm;

[0197] c) 540 nm-560 nm;

[0198] d) 560 nm-580 nm;

[0199] e) 580 nm-600 nm;

[0200] f) 600 nm-620 nm;

[0201] g) 620 nm-640 nm;

[0202] h) 640 nm-660 nm;

[0203] A second set of photodetectors is positioned across the second set of linear variable optical filters, wherein the second set of photodetectors can detect light having the following sub-spectral ranges:

[0204] a) 800 nm-780 nm;

[0205] b) 780 nm-760 nm;

[0206] c) 760 nm-740 nm;

[0207] d) 740 nm-720 nm;

[0208] e) 720 nm-700 nm;

[0209] f) 700 nm-680 nm;

[0210] g) 680 nm-660 nm.

[0211] 18. The light detection system of any of clauses 1-17, wherein the plurality of photodetectors comprises one or more photomultiplier tubes (PMTs).

[0212] 19. A light detection system according to any of clauses 1-17, wherein the plurality of photodetectors comprises one or more photodiodes.

[0213] 20. The light detection system of any of clauses 1-17, wherein the plurality of photodetectors comprises one or more avalanche photodiodes (APDs).

[0214] 21. A light detection system according to any of clauses 1 to 20, wherein the plurality of photodetectors comprises a modulator component operable to bin data signals from two or more different photodetector channels.

[0215] 22. The light detection system of clause 21, wherein each photodetector channel is operable to generate a data signal for each sub-spectral range of light.

[0216] 23. A light detection system according to any of clauses 21-22, wherein the modulator component is operable to bin data signals from adjacent photodetector channels.

[0217] 24. The light detection system of clause 23, wherein the modulator assembly is operable to horizontally bin adjacent photodetector channels.

[0218] 25. A light detection system according to any of clauses 22-24, wherein the modulator component can bin data signals from three or more different photodetector channels.

[0219] 26. A light detection system according to any of clauses 21 to 25, wherein the modulator assembly comprises an output modulator operable to modulate the signal output from each photodetector channel.

[0220] 27. The light detection system of clause 26, wherein the output modulator comprises an amplifier.

[0221] 28. The light detection system of clause 27, wherein the amplifier is a transimpedance amplifier.

[0222] 29. A light detection system according to any of clauses 26-28, wherein the output modulator comprises a switching circuit.

[0223] 30. A light detection system according to any of clauses 26-29, wherein the output modulator comprises a differential amplifier.

[0224] 31. A light detection system according to any of clauses 21-30, wherein the modulator component is operable to multiplex the generated data signals.

[0225] 32. A system comprising:

[0226] a light source to irradiate particles propagating through the flow stream;

[0227] Light detection system, comprising:

[0228] a wavelength separator assembly operable 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 operable to pass light having a predetermined sub-spectral range;

[0229] A plurality of photodetectors are positioned to detect light in each sub-spectral range that passes through the linear variable optical filter.

[0230] 33. A system according to clause 32, wherein the system is a particle analyzer.

[0231] 34. A system according to clause 33, wherein the system is a flow cytometer.

[0232] 35. A system according to any of clauses 32-34, wherein each set of linear variable optical filters can pass light in the wavelength range of 200 nm-1200 nm.

[0233] 36. A system according to any of clauses 32-35, wherein each set of linear variable optical filters can pass light in the wavelength range of 500 nm-800 nm.

[0234] 37. The system of clause 36, wherein the first set of linear variable optical filters allows light to pass in the wavelength range of 500 nm-650 nm.

[0235] 38. The system of clause 36, wherein the second set of linear variable optical filters allows light to pass in the wavelength range of 650 nm-800 nm.

[0236] 39. A system according to any of clauses 32-38, wherein each set of linear variable optical filters comprises a long-pass linear variable bandpass filter and a short-pass linear variable bandpass filter.

[0237] 40. The system of clause 39, wherein in each set of linear variable optical filters, there is an offset of a predetermined distance between the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

[0238] 41. A system according to clause 40, wherein the sub-spectral range of light passing through each set of linear variable optical filters is determined by the offset of the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

[0239] 42. A system according to any of clauses 32-41, wherein each sub-spectral range of light passed through the set of linear variable optical filters comprises a spectral width of 5 nm-50 nm.

[0240] 43. A system according to clause 42, wherein light in each sub-spectral range passed through the set of linear variable optical filters comprises a spectral width of approximately 20 nm.

[0241] 44. A system according to any of clauses 32-43, wherein the first set of linear variable optical filters and the second set of linear variable optical filters are positioned along two parallel planes.

[0242] 45. The system of clause 44, wherein the wavelength separator assembly can transfer light back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters.

[0243] 46. ​​A system according to any of clauses 32-45, wherein the wavelength separator assembly is configured such that:

[0244] The first set of linear variable optical filters can pass light from low wavelength to high wavelength;

[0245] The second set of linear variable optical filters can pass light from high wavelengths to low wavelengths.

[0246] 47. A system according to any of clauses 32-46, wherein the plurality of photodetectors are positioned across each set of linear variable optical filters.

[0247] 48. A system according to clause 47, wherein each photodetector can detect light in a sub-spectral range with a spectral width of 5 nm-50 nm.

[0248] 49. A system according to clause 48, wherein each photodetector can detect light in a sub-spectral range with a spectral width of approximately 20 nm.

[0249] 50. A system according to any of clauses 47-49, wherein the light detection system comprises:

[0250] A first set of photodetectors is positioned across the first set of linear variable optical filters, wherein the first set of photodetectors can detect light having the following sub-spectral ranges:

[0251] i) 500 nm-520 nm;

[0252] j) 520 nm-540 nm;

[0253] k) 540 nm-560 nm;

[0254] l) 560 nm-580 nm;

[0255] m) 580 nm-600 nm;

[0256] n) 600 nm-620 nm;

[0257] o) 620 nm-640 nm;

[0258] p) 640 nm-660 nm;

[0259] A second set of photodetectors is positioned across the second set of linear variable optical filters, wherein the second set of photodetectors can detect light having the following sub-spectral ranges:

[0260] h) 800 nm-780 nm;

[0261] i) 780 nm-760 nm;

[0262] j) 760 nm-740 nm;

[0263] k) 740 nm-720 nm;

[0264] l) 720 nm-700 nm;

[0265] m) 700 nm-680 nm;

[0266] n) 680 nm-660 nm;

[0267] 51. The system of any of clauses 32-50, wherein the plurality of photodetectors comprises one or more photomultiplier tubes (PMTs).

[0268] 52. The system of any of clauses 32-50, wherein the plurality of photodetectors comprises one or more photodiodes.

[0269] 53. The system of any of clauses 32-50, wherein the plurality of photodetectors comprises one or more avalanche photodiodes (APDs).

[0270] 54. The system of any of clauses 32-53, wherein the plurality of photodetectors comprises a modulator component operable to bin data signals from two or more different photodetector channels.

[0271] 55. The system of clause 54, wherein each photodetector channel is operable to generate a data signal for each sub-spectral range of light.

[0272] 56. A system according to any of clauses 54-55, wherein the modulator component can bin data signals from adjacent photodetector channels.

[0273] 57. The system of clause 56, wherein the modulator assembly is operable to horizontally bin adjacent photodetector channels.

[0274] 58. A system according to any of clauses 54-57, wherein the modulator component can bin data signals from three or more different photodetector channels.

[0275] 59. A system according to any of clauses 54-58, wherein the modulator component includes an output modulator that modulates the signal output from each photodetector channel.

[0276] 60. The system of clause 59, wherein the output modulator comprises an amplifier.

[0277] 61. The system of clause 60, wherein the amplifier is a transimpedance amplifier.

[0278] 62. The system of any of clauses 59-61, wherein the output modulator comprises a switching circuit.

[0279] 63. A system according to any of clauses 59-62, wherein the output modulator comprises a differential amplifier.

[0280] 64. A system according to any of clauses 54-63, wherein the modulator component is operable to multiplex the generated data signals.

[0281] 65. A method comprising:

[0282] Light from the flow stream is detected using a light detection system, the light detection system comprising:

[0283] a wavelength separator assembly operable 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 operable to pass light having a predetermined sub-spectral range;

[0284] A plurality of photodetectors are positioned to detect light in each sub-spectral range that passes through the linear variable optical filter.

[0285] 66. The method of clause 65, further comprising irradiating the sample in the flow stream of the interrogation field with a light source.

[0286] 67. A method according to any of clauses 65-66, wherein the flow stream is irradiated with a light source at a wavelength of 200 nm to 800 nm.

[0287] 68. A method according to any of clauses 65-67, wherein the light source is a laser.

[0288] 69. A method according to any of clauses 65-68, wherein an optical collection assembly is used to transmit light from the flow stream to a light detection system.

[0289] 70. The method of clause 69, wherein the optical collection assembly comprises an optical fiber.

[0290] 71. The method of clause 70, wherein the optical collection assembly comprises a fiber optic light relay bundle.

[0291] 72. The method of clause 69, wherein the optical collection assembly comprises a free space optical relay system.

[0292] 73. A method according to any of clauses 65-72, wherein each set of linear variable optical filters can pass light in the wavelength range of 200 nm-1200 nm.

[0293] 74. A method according to any of clauses 65-72, wherein each set of linear variable optical filters can pass light in the wavelength range of 500 nm-800 nm.

[0294] 75. The method of clause 74, wherein the first set of linear variable optical filters allows light to pass in the wavelength range of 500 nm-650 nm.

[0295] 76. The method of clause 74, wherein the second set of linear variable optical filters allows light to pass in the wavelength range of 650 nm-800 nm.

[0296] 77. A method according to any of clauses 65-76, wherein each set of linear variable optical filters comprises a long-pass linear variable bandpass filter and a short-pass linear variable bandpass filter.

[0297] 78. A method according to clause 77, wherein in each set of linear variable optical filters, there is an offset of a predetermined distance between the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

[0298] 79. A method according to clause 78, wherein the sub-spectral range of light passing through each set of linear variable optical filters is determined by the offset of the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

[0299] 80. A method according to any of clauses 65-79, wherein each sub-spectral range of light passed through the set of linear variable optical filters comprises a spectral width of 5 nm-50 nm.

[0300] 81. A method according to clause 80, wherein light in each sub-spectral range passing through the set of linear variable optical filters comprises a spectral width of approximately 20 nm.

[0301] 82. A method according to any of clauses 65-81, wherein the first set of linear variable optical filters and the second set of linear variable optical filters are positioned along two parallel planes.

[0302] 83. The method of clause 82, wherein the wavelength separator assembly is operable to transfer light back and forth between the first set of linear variable optical filters and the second set of linear variable optical filters.

[0303] 84. A method according to any of clauses 65-83, wherein the wavelength separator assembly is configured such that:

[0304] The first set of linear variable optical filters can pass light from low wavelength to high wavelength;

[0305] The second set of linear variable optical filters can pass light from high wavelengths to low wavelengths.

[0306] 85. A method according to any of clauses 65-84, wherein the plurality of photodetectors are positioned across each set of linear variable optical filters.

[0307] 86. A method according to clause 85, wherein each photodetector can detect light in a sub-spectral range with a spectral width of 5 nm-50 nm.

[0308] 87. A method according to clause 86, wherein each photodetector can detect light in a sub-spectral range with a spectral width of approximately 20 nm.

[0309] 88. A method according to any of clauses 84 to 87, wherein the light detection system comprises:

[0310] A first set of photodetectors is positioned across the first set of linear variable optical filters, wherein the first set of photodetectors can detect light having the following sub-spectral ranges:

[0311] q) 500 nm-520 nm;

[0312] r) 520 nm-540 nm;

[0313] s) 540 nm-560 nm;

[0314] t) 560 nm-580 nm;

[0315] u) 580 nm-600 nm;

[0316] v) 600 nm-620 nm;

[0317] w) 620 nm-640 nm;

[0318] x) 640 nm-660 nm;

[0319] A second set of photodetectors is positioned across the second set of linear variable optical filters, wherein the second set of photodetectors can detect light having the following sub-spectral ranges:

[0320] o) 800 nm-780 nm;

[0321] p) 780 nm-760 nm;

[0322] q) 760 nm-740 nm;

[0323] r) 740 nm-720 nm;

[0324] s) 720 nm-700 nm;

[0325] t) 700 nm-680 nm;

[0326] u) 680 nm-660 nm;

[0327] 89. The method of any of clauses 65-88, wherein the plurality of photodetectors comprises one or more photomultiplier tubes (PMTs).

[0328] 90. A method according to any of clauses 65-89, wherein the plurality of photodetectors comprises one or more photodiodes.

[0329] 91. A method according to any of clauses 65-89, wherein the plurality of photodetectors comprises one or more avalanche photodiodes (APDs).

[0330] 92. A method according to any of clauses 65-91, wherein the plurality of photodetectors comprises a modulator component that can bin data signals from two or more different photodetector channels.

[0331] 93. A method according to clause 92, wherein each photodetector channel can generate a data signal for each sub-spectral range of light.

[0332] 94. A method according to any of clauses 92-93, wherein the modulator component is operable to bin data signals from adjacent photodetector channels.

[0333] 95. The method of clause 94, wherein the modulator assembly is operable to horizontally bin adjacent photodetector channels.

[0334] 96. A method according to any of clauses 92-95, wherein the modulator component can bin data signals from three or more different photodetector channels.

[0335] 97. A method according to any of clauses 92-96, wherein the modulator component includes an output modulator that can modulate the signal output from each photodetector channel.

[0336] 98. The method of clause 97, wherein the output modulator comprises an amplifier.

[0337] 99. The method of clause 98, wherein the amplifier is a transimpedance amplifier.

[0338] 100. A method according to any of clauses 97-99, wherein the output modulator comprises a switching circuit.

[0339] 101. A method according to any of clauses 97-100, wherein the output modulator comprises a differential amplifier.

[0340] 102. A method according to any of clauses 97-101, wherein the modulator component is operable to multiplex the generated data signals.

[0341] 103. A kit comprising:

[0342] A first set of linear variable optical filters;

[0343] a second set of linear variable optical filters;

[0344] Photodetector.

[0345] 104. A 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.

[0346] 105. A kit according to any of clauses 103-104, wherein the first set of linear variable optical filters allows light to pass in the wavelength range of 500 nm-650 nm.

[0347] 106. A kit according to any of clauses 103-105, 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.

[0348] 107. A kit according to any of clauses 103-106, wherein the second set of linear variable optical filters allows light to pass in the wavelength range of 650 nm-800 nm.

[0349] 108. A kit according to any of clauses 103-107, further comprising a light source.

[0350] 109. A kit according to clause 108, wherein the light source comprises a laser.

[0351] 110. A kit according to any of clauses 103-109, further comprising an optical collection assembly.

[0352] 111. The kit of clause 110, wherein the optical collection assembly comprises an optical fiber.

[0353] 112. The kit of clause 111, wherein the optical collection assembly comprises a fiber optic light relay bundle.

[0354] 113. A kit according to clause 110, wherein the optical collection component includes a free space optical relay system.

[0355] Although the above invention has been described in some detail through illustration and example for the purpose of clarity of understanding, it is obvious to those skilled in the art that certain changes and modifications may be made thereto in accordance with the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0356] Therefore, the foregoing only illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in the present invention, embody the principles of the present invention and are included in its spirit and scope. In addition, all embodiments and conditional language described in the present invention are mainly intended to help readers understand the principles of the present invention and the concepts provided by the inventors to deepen the field, and should be interpreted as not being limited to these specifically listed examples and conditions. In addition, all statements recording the principles, aspects and embodiments of the present invention and their specific examples in the present invention are intended to cover their structural and functional equivalents. In addition, it is intended that such equivalents include currently known equivalents and equivalents developed in the future, that is, any part of the developed performance of the same function (regardless of its structure). In addition, any content disclosed by the present invention, whether or not it is explicitly listed in the claims, is not intended to be open exclusively to the public.

[0357] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the appended claims embody the scope and spirit of the present invention. In the claims, 35 U.S. Patent Act Section 112(f) or 35 U.S. Patent Act Section 112(6) is expressly defined as a limitation in a claim only if such limitation begins with the exact phrase "means" or the exact phrase "step" in the claim; if such exact phrase is not used in the claim, 35 U.S. Patent Act Section 112(f) or 35 U.S. Patent Act Section 112(6) is not cited.

Claims

1. A light detection system, comprising: a wavelength separator assembly 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 allow light having a predetermined sub-spectral range to pass through the linear variable optical filters; and A plurality of photodetectors are positioned to detect light in each sub-spectral range that passes through the linear variable optical filter.

2. The optical detection system according to claim 1, wherein: Each set of linear variable optical filters includes a long-pass linear variable bandpass filter and a short-pass linear variable bandpass filter.

3. The optical detection system according to claim 2, wherein: In each set of linear variable optical filters, there is an offset of a predetermined distance between the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

4. The optical detection system according to claim 3, wherein: The sub-spectral range of light passing through each set of linear variable optical filters is determined by the offset of the long-pass variable bandpass filter and the short-pass linear variable bandpass filter.

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

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

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

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

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

10. The optical detection system according to any one of claims 1 to 9, wherein: The plurality of photodetectors includes 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 light of each sub-spectral range.

12. The optical detection system according to any one of claims 10 to 11, wherein: The modulator component is configured to bin data signals from adjacent photodetector channels.

13. A system comprising: a light source configured to irradiate particles propagating through the flow stream; and Light detection system, comprising: a wavelength separator assembly 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 allow light having a predetermined sub-spectral range to pass through the linear variable optical filters; and A plurality of photodetectors are positioned to detect light in each sub-spectral range that passes through the linear variable optical filter.

14. A method comprising: Light from the flow stream is detected using a light detection system, the light detection system comprising: a wavelength separator assembly 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 allow light having a predetermined sub-spectral range to pass through the linear variable optical filters; and A plurality of photodetectors are positioned to detect light in each sub-spectral range that passes through the linear variable optical filter.

15. A kit comprising: A first set of linear variable optical filters; a second set of linear variable optical filters; and Photodetector.

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