True spectral flow cytometers

Abstract

Methods and apparatuses are disclosed for true spectral flow cytometers that use wide band lasers to provide excitation light to cells, proteins, antibodies, and the like. Light from wide band lasers (emitting light through a continuous, wide bandwidth of wavelengths) is projected into a flow cell as a spread out spectrum to generate a spatially arranged spectrum that can excite a fluorescent response in cells passing through the flow cell to generate at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

Description

TRUE SPECTRAL FLOW CYTOMETERSCLAIM OF PRIORITY

[0001] This patent application claims priority to U.S. provisional patent application no. 63 / 671,729, titled “TRUE SPECTRAL FLOW CYTOMETERS,” and filed on July 15, 2024, herein incorporated by reference in its entirety.INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.FIELD

[0003] Described herein are methods and apparatuses related to flow cytometry, and more particularly to true spectral flow cytometers.BACKGROUND

[0004] Flow cytometry systems are widely used in biomedical research to study cells in the immune system, cancer cells, and other types of cells. Flow cytometry systems can also be used as a diagnostic tool to diagnose cancer, blood diseases and other medical conditions.

[0005] In general, flow cytometry systems use a laser to determine physical and fluorescent properties of cells (or particles) as they flow past a detector in a single-file. The cells may be stained to fluoresce when excited by particular wavelengths of laser light.However, the number of different fluorescent stains that may be detected may be limited by the number of distinct or separate wavelengths that can be used to excite the cells. Generally, additional excitation wavelengths may require additional lasers and / or dichroic filters increasing the cost and complexity of the flow cytometry system.SUMMARY OF THE DISCLOSURE

[0006] Described herein are apparatuses, systems, and methods to perform flow cytometry using wide bandwidth laser light. The wide bandwidth laser light is used as excitation light for cells. The cells may be stained to fluoresce (emit light) in response to particular wavelengths of light. A multispectral spectrometer and highly sensitive photodiodeapparatus, such as a single photon avalanche photodiode (SPAD) camera may be used to detect emission light and excitation light.

[0007] Any of the flow cytometry apparatuses described herein can include a broadspectrum laser, such as a supercontinuum white light laser, configured to emit light having a continuous wavelength of between about 400 and 700 nanometers, a focus and dispersion module configured to illuminate cells in a first axis of a flow cell, that is configured to receive the spread apart spectrum and transport cells through the spread apart spectrum, and a multispectral spectrometer module configured receive at least a portion of the spread apart spectrum passing through the flow cell, wherein the multispectral spectrometer module is configured to generate at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

[0008] In general, the broad-spectrum laser can emit light in a relatively continuous spectrum between a first or upper bound and a second or lower bound. The bounds are typically expressed in wavelengths. For example, the broad-spectrum laser can emit wavelengths of light between (and including) 400 and 700 nanometers. In another example, the broad-spectrum laser can emit wavelengths of light between 300 and 800 nanometers. In general, the broad-spectrum laser can be configured to emit wavelengths of light between any two feasible wavelengths.

[0009] In any of the apparatuses described herein, the multispectral spectrometer module can include a single photon avalanche photodiode camera configured to determine a number of photons associated with at least one wavelength included in the spread apart spectrum. In general, the multispectral spectrometer can be any feasible device that is sensitive to wavelengths of light emitted from the broad-spectrum laser or emitted by a cell (or by a stain / dye on / in the cell).

[0010] In any of the apparatuses described herein, the flow cell may be further configured to align cells (any cells under test) in a single-file line along a first axis. Axes in general may be ordinate directions in one of three orthogonal axes. A conventional example of three orthogonal axes is x, y, and z directions.

[0011] In any of the apparatuses described herein, the dispersion module may be configured to distribute the spread apart light along a second axis aligned at approximately 90 degrees with respect to the first axis.

[0012] In any of the apparatuses described herein, the multispectral spectrometer module may be configured to detect at least a wavelength of laser light from the broad-spectrum laser, a wavelength of light emitted by the cells, or a fluorescence intensity. In general,detection of the at least one wavelength can be based at least in part on spread apart wavelengths in the capture image as part of an image-based technique.

[0013] In any of the apparatuses described herein, the flow cell may be removable.

[0014] Any methods described herein may include generating a broad spectrum laser light, generating, with a dispersion module, a spread apart spectrum from the laser light, transporting, with a flow cell, cells through the spread apart spectrum, and generating, with a multispectral spectrometer module, at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

[0015] In general, in any of the methods or apparatuses, the laser light spectrum can have wavelengths between about 400 and 700 nanometers. These exemplary band limitations are exemplary and not limiting.

[0016] In any of the methods described herein, generating the EEM plot may further include capturing excitation wavelength, emission wavelength, and fluorescence intensity data with a single photon avalanche photodiode camera.

[0017] In any of the methods described herein may further include transmitting, with a multi-channel optical fiber, the spread apart spectrum of light from the broad spectrum laser to the flow cell.

[0018] In some example methods, the spread apart spectrum may be disposed on a first axis and the cells can traverse the spatially arranged spectrum substantially on the first axis.

[0019] In any of the methods described herein, transporting the cells may further include aligning the cells substantially transverse with respect to an axis including the spread apart spectrum.

[0020] In general, the methods and apparatuses described herein may advantageously be used with virtually any fluorescent dye; unlike conventional systems, in which the dye must be chosen to match the limited wavelengths available to the system, the methods and apparatuses described herein may instead be configured to adapt to the wavelength to whatever dye (or dyes) is / are used. This allows the use of a large range of dyes that have not previously been available. Further dyes may be simultaneously activated and the emitted fluorescence sensed over the entire spread apart spectrum. The resulting excitation emission matrix (EEM) may be analyzed to determine a characteristic set of properties. This analysis may be interpreted by a pattern recognition agent (e.g. a trained machine learning agent) trained to recognize characteristic EEM patterns. The agent may be trained on control patterns for known fluorophores.

[0021] For example, described herein are flow cytometry apparatuses comprising: a broad-spectrum laser configured to emit light having a continuous wavelength of between about 400 and 700 nanometers; a dispersion module configured to spread out the light into a spatially arranged spectrum arranged in a first axis; a seat (e.g., a flow cell seat) configured to hold a flow cell in the first axis, wherein the dispersion module is configured to deliver the spatially arranged spectrum into the flow cell so that cells transported through the flow cell pass through the spatially arranged spectrum; and a multispectral spectrometer module configured receive at least a portion of the spatially arranged spectrum passing through the flow cell, wherein the multispectral spectrometer module is configured to generate at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

[0022] The multispectral spectrometer module may include a single photon avalanche photodiode camera configured to determine a number of photons associated with at least one wavelength included in the spatially arranged spectrum.

[0023] Any of these apparatuses may include a multi-channel optical fiber configured to couple the spatially arranged spectrum passing through the flow cell to the multispectral spectrometer.

[0024] In any of these examples, the seat may be configured to hold the flow cell so that cells passing through the flow cell aligned in a single-file line along the first axis. Any of these apparatuses may include the flow cell as part of the apparatus (e.g., system). The flow cell may be reusable or disposable (e.g., single use). The flow cell may be configured to pass a particle (e.g., cell) for detection in a spatially constrained region in-line with the first axis. For example, any of these apparatuses may include a flow cell that is configured to align cells in a single-file along the first axis.

[0025] Any of these apparatuses may include a controller configured to control a rate of fluid flow through the flow cell. The controller may receive input from the flow cell and / or may provide output to the flow cell or to one or more flow elements (e.g., valves, pumps, fluid lines, etc.) in or in communication with the flow cell.

[0026] The seat may removably hold the flow cell. For example, the seat may be a chamber or surface that holds the flow cell in-line with the first axis, e.g., so that particles (e.g., cells, tissue, viruses, nanoparticles, etc.) passing through the flow cell are in the first axis and can be interrogated by the apparatus (e.g., can receive the spatially distributed light and can have any fluorescence emitted in response detected by the multispectral spectrometer module). The seat may include a lock or securement to hold the flow cell until released (e.g., by actuating a latch, fastener, bolt, etc.). The seat may include one or more keying features toorient the flow cell within the apparatus, confirming and / or maintaining alignment. In some cases, the seat may include one or more sensors to detect seating of a flow cell within the apparatus. The one or more sensors may be connected to the controller. The controller may prevent or limit operation of the apparatus until the flow cell is seated in the seat. Seat may also include an interface for interfacing with one or more controls (valves, connectors, etc.) on the flow cell to regulate operation of the flow cell.

[0027] The controller may receive input from the multispectral spectrometer module (and / or the camera). For example, the controller may include hardware, firmware and / or software. In some cases, the controller may include a trained pattern recognition agent configured to recognize a pattern of excitation wavelength, emission wavelength and fluorescence intensity in the EEM plot. The controller may include or may be coupled to an output (e.g., screen / di splay, memory, etc.). The controller may store, transmit and / or display information about the operation of the apparatus (errors, status, output, etc.).

[0028] Any of these apparatuses may include a multispectral spectrometer module that is configured to detect at least a wavelength of light from the broad-spectrum laser, a wavelength of light emitted by the cells, and / or a fluorescence intensity.

[0029] The dispersion module may comprise any appropriate optical components. For example, the dispersion module may include one or more prisms.

[0030] As mentioned, in any of these apparatuses the flow cell may be included as part of the apparatus. For example, a flow cytometry apparatus may include: a broad-spectrum laser configured to emit light having a continuous wavelength of between about 400 and 700 nanometers; a dispersion module configured to spread out the light into a spatially arranged spectrum arranged in a first axis; a flow cell, configured to receive the spatially arranged spectrum and transport cells through the spatially arranged spectrum; and a multispectral spectrometer module configured receive at least a portion of the spatially arranged spectrum passing through the flow cell, wherein the multispectral spectrometer module is configured to generate at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

[0031] Also described herein are methods of operating any of these apparatuses. These methods may include flow cytometry methods capable of detecting a plurality of emission wavelengths using concurrent excitation wavelengths. For example, a method may include: generating a wide wavelength laser light; generating, with a dispersion module, a spatially arranged spectrum from the wide wavelength laser light within a flow cell; transporting cells within the flow cell through the spatially arranged spectrum; and generating, with a multispectral spectrometer module, at least one excitation emission matrix (EEM) plot,wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

[0032] The wide wavelength laser light may include light having wavelengths between about 400 and 700 nanometers. Generating the EEM plot may further comprise capturing excitation wavelength, emission wavelength, and fluorescence intensity data with a single photon avalanche photodiode camera. In any of these examples, generating the EEM plot may further comprise generating the EEM plot in the controller.

[0033] These methods may include transmitting, with a multi-channel optical fiber, the spatially arranged spectrum to the flow cell. The spatially arranged spectrum may be disposed on a first axis and the cells traverse the spatially arranged spectrum substantially on the first axis.

[0034] Any of these methods may include controlling a flow of cells through the flow cell and the application of the wide wavelength laser light based using a controller. Any of these methods may include placing the flow cell within apparatus, and / or securing the flow cell in the apparatus so that a flow channel within the flow cell, though which the particles (e.g., cells, etc.) may pass in-line with the first axis. Any of these methods may include controlling a rate of fluid flow through the flow cell so that a single cell passes through the spatially arranged spectrum at a time.

[0035] Generating the spatially arranged spectrum with the dispersion module may comprise passing the wide wavelength laser light through a plurality of prisms and one or more lenses configured to separate the wide wavelength light by refraction and project the light into the flow cell.

[0036] All of the methods and apparatuses described herein, in any combination, are herein contemplated and can be used to achieve the benefits as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0038] FIG. 1 A schematically illustrates an example of a of a conventional single laser flow cytometry technique.

[0039] FIG. IB schematically illustrates an example of a of a conventional multi-laser flow cytometry technique.

[0040] FIG. 2 illustrates an example of an absorption spectrum (shown as a dashed line) and an emission spectra of FITC, typically excited at laser wavelength 488nm.

[0041] FIG. 3 schematically illustrates one example of a diagram of a typical detection module.

[0042] FIG. 4 schematically illustrates one example of a true spectral flow cytometer as described herein.

[0043] FIG. 5 shows one example of a schematic for focus and dispersion optics for a true spectral flow cytometer similar to that shown in FIG. 4.

[0044] FIGS. 6A and 6B show side perspective and top views, respectively, of an EEM plot for cells labeled with FITC.

[0045] FIGS. 7A and 7B show side perspective and top views, respectively of an example full spectrum plot associated (EEM) for cells labeled with seven different dyes.

[0046] FIG. 8 is a simplified diagram of an example true spectral flow cytometer.

[0047] FIG. 9 is a flowchart showing an example method for generating an emission excitation matrix plot.DETAILED DESCRIPTION

[0048] True spectral flow cytometers can achieve increased sensitivity and performance over conventional flow cytometers by using a wide-bandwidth (also referred to herein as broad spectrum, multi -wavelength, and / or continuous wavelength) laser light source. A broad spectrum bandwidth laser light source can provide increased frequencies for the excitation of cells under test, especially when compared to conventional flow cytometers. In addition, true spectral flow cytometers may include a wide band spectral detector that can respond to wide- bandwidth laser light.

[0049] Conventional single laser flow cytometers are limited in their ability to detect more than one marker (e.g., fluorophore). For example, FIG. 1 A shows an example of a conventional single laser flow cytometer, which includes an excitation laser emitting a laser beam 101 with a wavelength that is focused onto a core stream 103, in which sample blood cells 107 are constrained, within a flow cell 105. Cells are introduced to the flow cell 105 and pass across the laser beam one by one. Cells stained with a fluorochrome absorb light at wavelength and emit fluorescence at longer wavelengths 111, if the excitation wavelength is within the absorption band of the fluorochrome. The system include collection optics 109 and a multimode optical fiber 113 to convey the emitted light (as well as at least some of the excitation light) to a detection module 115.

[0050] Although it is possible to modify this arrangement to detect multiple absorption wavelengths, as shown in FIG. IB, in which multiple lasers are used to generate multiplewavelengths 120 in an otherwise conventional flow cytometer that excite different emitted wavelengths, each of which is detected by separate detection modules 121, 123, 125. This arrangement is both expensive and overly complex.

[0051] FIG. 2 shows an example of an absorption (dashed line) and emission spectra of FITC which is frequently used in flow cytometry. Laser light at 488nm is commonly used in conventional flow cytometry. In general, in these apparatuses, as shown in FIGS. 1 A-1B, the collection optics project the interrogation point (where the core stream and laser beam intersect) onto an image plane. One end of a multi-mode optical fiber is placed at the image of the point to collect emission fluorescence originated from the point. The other end of the fiber is connected to a detection module.

[0052] This is schematically illustrated in FIG. 3, which shows a diagram of a typical detection module. Emission fluorescence light 331 at shorter wavelengths (e.g., from a multimode optical fiber) is reflected by a long-pass optical filter 333. The reflected light then passes through a band-pass filter 335 to further reduced fluorescence outside of a predefined spectral range, so that light may be detected by an optical detector 337; this light represents the overall emission fluorescence within this first spectral range. Multiple different optical filters may be connected in parallel. For example, light passing the first long-pass optical filter 333 is split by another long-pass filter 333’, band-pass filter 335’, and optical detector 339 to record overall emission fluorescence within a second spectral range. This arraignment may be repeated multiple ( / ??) times, to achieve so-calledchannel detection module.”

[0053] When m is large enough (e.g., 10 to 20), full emission spectrum of a fluorochrome could be mapped out, which helps in unmixing emission spectrum when a cell is co-stained with more than one fluorochromes. This technology forms the foundation of so called full spectral cytometry. However, convectional multi-laser flow cytometers have more than one laser, with wavelengths i toLaser beams are focused onto the core stream inside flow cell and spatially arranged along the flow direction, as shown in FIG. IB. The spatially arranged interrogation points may be projected onto an image plane. A multimode optical fiber may be arranged to collect emission fluorescence originated from each interrogation point at the corresponding image point. Thus, a cell moving in the flow cell may be interrogated by laser 1 first, and its emission fluoresces induced by the first laser (laser 1) may be recorded by the first detection module 337. The cell may be sequentially interrogated by all the lasers, and the fluorescence is recorded by the corresponding detection modules, respectively.

[0054] A flow cytometer, either conventional or full spectral, can only install limited number of lasers with distinct and fixed wavelengths. This, in turn, limits the selection offluorochromes. The apparatuses and methods described herein may overcome these limitations and may provide many advantages not previously achieved. In general, these apparatuses may use broad spectrum lasers, such as (but not limited to) supercontinuum white light lasers, together with single photon avalanche photodiode (SPAD) camera.

[0055] FIG. 4 is a simplified block diagram of an example true spectral flow cytometer 400. The true spectral flow cytometer 400 may include a broad spectrum (e.g., white light) laser 410, a focus and dispersion optics module 420, a flow cell 430, collection optics 440, a multispectral spectrometer 450 and a single photon avalanche photodiode camera 460. Other examples of true spectral flow cytometers may have other units arranged in other orders, as described herein.

[0056] In FIG. 4, the excitation light source 410 is a broad band (e.g., 400nm to 700nm) laser. It may be equivalent to a source providing n wavelengths between i =400nm and kn=700nm, where n is infinite. Examples of such lasers (e.g., supercontinuum white light lasers) are commercially available. It this example, the focus and dispersion optics (shown schematically in FIG. 5) may spread apart the wavelengths; at the focal plane (which is configured by the arrangement of the apparatus to be inside flow cell and overlap with core stream), the laser wavelengths spread apart from ki=400 nm to kn=700 nm, gradually and continuously along the flow direction, in contrast with the distinct beam locations for different wavelength in the example shown in FIG. IB. This may be achieved by the focus and dispersion optics 570. The dispersion module optics may include one or more prisms and / or one or more diffusion gratings, one or more lensing and / or one or more mirrors and / or filters.

[0057] The collection optics 440 may include large numerical aperture (NA) optics that may project the section of core stream illuminated by the dispersed white light laser onto an image plane. Emission fluorescence originated from each small section of core stream may fall on a corresponding small section of the image. Thus, an array of collection lines may be arranged in the same plane as the spread apart wavelengths, to emitted light. By analogy, image a single layer fiber ribbon that is placed at the image location of the core stream, so that each fiber would collect fluorescence originated from the corresponding core stream location, and therefore the corresponding excitation wavelength. Similarly, the linearly arranged collection lines may collect corresponding emitted light wavelengths.

[0058] In practice, the dispersion and imaging optics may include a multispectral spectrometer, which may re-image the core stream image onto another plane while spreading the image based on wavelength in a direction orthogonal to the flow direction. This image may be collected by a high-speed camera, such as a single photon avalanche photodiode(SPAD) camera having a high sensitivity. The resulting outcome image is an excitation emission matrix (EEM) plot / image. The EEM is a three-dimensional contour plot of excitation wavelength vs. emission wavelength vs. fluorescence intensity.

[0059] This is illustrated in FIGS. 6A-6B, which shows an example in which cells were stained with FITC (see FIG. 3). The resulting EEM plot / image in FIG. 6 includes a vertical direction (the flow direction) that represents the excitation wavelength (shown for a subregion between 400 and 600 nm). The horizontal direction (which is orthogonal to the flow direction through the flow cell) shows fluorescence wavelength (shown for the region between 400-600 nm). The intensity at a given point represents the emission fluoresce intensity at wavelength indicated by the horizontal reading when the fluorochrome is excited by laser wavelength indicated by the vertical reading. FIG. 6B shows a top view of the resulting EEM plot.

[0060] Since the EEM plot / image contains both emission and excitation information, it helps in differentiating fluorochromes with similar emission spectra (an advantage over full spectral cytometry). The resulting EEM data may be characteristic of marker fingerprint that may be compared and used for classification and identification of multiple markers. Multiple markers may be concurrently used by the system, as illustrated in FIGS. 7A-7B.

[0061] FIG. 7 shows an example EEM plot / image of a combination of seven commonly used fluorochromes: APC, PE, FITC, Pac Blue, mCherry, PerCP, and PE-Cy7. Both conventional and full spectral cytometry rely on detection of emission from each individual fluorochrome to identify sub cell populations. Unmixing of complex emission spectra is sometimes very objective and, therefore, not stable or unique. The use of true spectral flow cytometry as described herein (and shown in FIGS. 7A-7B) may have significant advantages over conventional full spectral cytometry in sub cell population identification, particularly when pattern recognition of EEM data (e.g., images, graphs) is used.

[0062] In general, a white light laser may generate a wide-band laser light that appears white in color. In contrast, conventional flow cytometers may include a narrow band (single wavelength) laser light source. Typically, different biologic stains are often responsive to only a single light wavelength. Therefore, building a flow cytometer that can detect multiple stains has typically required multiple laser light sources or at least multiple laser light filters to provide different laser light wavelengths. Thus, the white light laser can provide multiple laser light wavelengths that, in turn, may be used to detect multiple cells via multiple stains.

[0063] Returning to the schematic shown in FIG. 4, the dispersion module 420 spreads out (e.g., separates) the wide-band laser light from the white light laser 410 into a continuous smear of narrow-band laser light signals. The narrow-band laser light signals are sometimesreferred to as a the spatially arranged spectrum since the wide-band laser light becomes spread out continuous bands of color that are spatially arranged.

[0064] The spread out light signals proceed from the dispersion module 420 to the flow cell 430. The flow cell 430 receives and aligns cells (e.g., cells to be tested) into a single-file orientation. In addition, the flow cell 430 is configured to enable the spread out laser light signal (e.g., a spatially arranged spectrum) to impinge upon and pass through the cells aligned by the flow cell 430. The flow cell can align cells in a single-file. In addition, the flow cell can include a region for the single-file cells to receive the spatially spread out spectrum. The cells that receive light (from the spatially spread out spectrum) can emit light in response based on a cell or stain applied to the cell, antibody, protein, or the like. In some examples, a controller can control the speed with which cells can traverse the spatially spread out spectrum.

[0065] In general, laser light may be used to deliver energy and “excite” one or more stains (dyes, e.g., fluorophores) that may be bound (attached) to cells, antibodies, proteins, or the like (or may trigger excitation of auto-florescence). After exciting a stain, the energy may be released as “emitted” light. This is sometimes referred to as causing a stain to fluoresce. Many stains only fluoresce in response to a particular wavelength of light. In this manner, flow cytometry can detect the presence of cells, antibodies, proteins, etc., by monitoring excitation and emitted light. Laser light from a laser light source is often referred to as excitation light. Light emitted from cells, antibodies, proteins, and the like is referred to as emission light.

[0066] Light from the flow cell 430 may be coupled to the multispectral spectrometer 450 through collection optics 440, such as a fiber ribbon. The light from the flow cell 430 can include excitation light (from the dispersion module) and emission light (from one or more cells aligned by the flow cell 430). Emission light may be further characterized by a fluorescence level (an amount of emission light). In some examples, the light from the flow cell 430 may be transported (carried) by collection optics (e.g., a fiber ribbon 440) to the multispectral spectrometer 450 for analysis and output to the camera 460.

[0067] Any appropriate collection optics may be used. For example, in some cases the collection optics 440 may transport light through a multichannel configuration. For example, a fiber ribbon may be a multi-channel ribbon (e.g., a 24-channel ribbon). The number of channels (e.g., the number of fibers in the ribbon) may depend on how many excitation channels, or number of excitation wavelengths, are required. For instance, a typical flow cytometer has four excitation wavelengths, i.e., 405nm, 488nm, 451nm, and 633nm. A 4- fiber ribbon could be used to make a system equivalent to a 4-laser instrument. However, themethods and apparatuses described herein may include significantly more channels (e.g., 5 or more, 8 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 30 or more, 36 or more, 40 or more, 48 or more, 64 or more, etc.).

[0068] The multispectral spectrometer 450 is one example of a spectral detector. Spectral detectors, in general, can detect and / or measure different wavelengths of light. The multispectral spectrometer 450 can detect excitation light and emission light associated with and / or passing through the flow cell 430. The multispectral spectrometer 450 can also quantify an amount of emission light detected and thereby measure fluorescence. Optionally, the camera may be integrated with the multispectral spectrometer. For example, the multispectral spectrometer 450 may include a photon-counting camera (e.g., a SPAD camera 460). The photon-counting camera can be sensitive not only to a wide range of light wavelength, but also sensitive in very low light situations. In some cases, the spectral detector 540 can include relevant circuitry to generate an EEM plot.

[0069] FIG. 8 is a simplified diagram of another example of a true spectral flow cytometer 800. The true spectral flow cytometer 800 may include a wide-bandwidth laser light source 810, a dispersion module 820, a removable flow cell 830, a multispectral spectrometer module, a single-photon avalanche diode camera 840 (which may be part of the multispectral spectrometer module or separate from it), and a controller 850. In this example the flow cell 830 is removably held in the seat 868 (which may secure the flow-cell in-line with the dispersion module 820 and multispectral spectrometer module. The apparatus may optionally include a sample collector / separator 866 that may receive the particles (e.g., cells, tissue, viruses, nanoparticles, etc.) being samples. In some cases, the apparatus may be configured to sort them. The flow cell may pass the particles in a fluid stream (e.g., liquid stream) for detection. Following detection, the fluid stream may optionally be divided into droplets that may be collected and / or sorted by the controller 850, which may coordinate the timing of the emission / excitation (detection) so that the results are linked to the collected samples. Samples may be collected as individual droplets containing individual particles, or as groups of particles.

[0070] The wide-bandwidth laser light source 810 may be an example of the laser light source such as a white light laser 410 of FIG. 4. The wide-bandwidth laser light source 810 may produce laser light having a wide range of wavelengths. For example, the wide- bandwidth laser light source 810 may generate broadband laser light having continuous wavelengths of between 400 and 700 nm. In other examples, the wide-bandwidth laser light source 810 can generate laser light between any feasible wavelengths. The wide-bandwidth laser light source 810 can contemporaneously generate a range of laser light wavelengthsbetween an upper bound (e.g., 400 nm) and a lower band (e.g., 700 nm). The term “white light” is used herein to describe any light (including laser light) that is composed by a relatively wide range of wavelengths. Moreover, any light described as “white light” may be light that includes a plurality of wavelengths and not necessarily a light having a white color.

[0071] The wide-bandwidth laser light source 810 may be controlled by the controller 850. In some examples, the wide-bandwidth laser light source 810 may be pulsed by the controller 650 and thereby generate pulses of laser light. In some aspects, the controller 850 can generate a periodic timing pulse signal that can cause the wide-bandwidth laser light source 610 to emit pulses of laser light.

[0072] The dispersion module 820 can receive light from the wide-bandwidth laser light source 810 and generate a spatially spread out spectrum 821. The dispersion module 820 can spread out light from the wide-band laser light source 810 into a spatially spread out spectrum. The dispersion module 820 can generate the spatially spread out spectrum 821 substantially in a direction of a first axis (the first axis shown as going top to bottom of FIG. 8). For example, the spatially arranged spectrum 821 may be in a vertical direction. The dispersion module 820 may be controlled by the controller 850.

[0073] The flow cell 830 can align cells in a single-file manner and can be configured to transport these cells along the same axis as the spatially arranged spectrum 821. In this manner, the spatially spread out spectrum 821 (arranged on the first axis and traveling on a second axis which is approximately ninety degrees with respect to the first axis) can interact with the cells from the flow cell 830 (cells traveling along the first axis). In some cases, the controller 850 can control the flow rate of cells through the flow cell 830. The controller may limit the number of cells passing through the flow cell over time, so as to provide single cell resolution. In some examples, the flow cell 830 can be removable. That is, the flow cell 830 can be replaced with another flow cell at any time. In some aspects, the flow cell 830 can be disposable.

[0074] The single-photon avalanche diode camera 840 can receive light directly from the spatially spread out spectrum 821, light emitted by cells in response to receiving a particular wavelength of light in and around the flow cell 830, light scattered from cells in and around the flow cell 830, or a combination of these sources. In some examples the single-photon avalanche diode camera 840 may be a photon counting camera. In some other examples, the single-photon avalanche diode camera 840 can be an example of the multispectral spectrometer, a spectral detection module, or a spectral detector. In some cases, the controller 850 can receive photon information from the single-photon avalanche diode camera 840 and generate a EEM data, including but not limited to an EEM plot. For example, the single-photon avalanche diode camera 840 may include a field programmable gate array (FPGA), or other feasible logic circuit, that may be configured or programmed to generate an EEM plot and / or EEM data. In other examples, the controller 850 can generate an EEM plot and / or EEM data. The single-photon avalanche diode camera 840 can advantageously receive a broad spectrum of light and, thereby, detect multiple wavelengths associated with the spatially arranged spectrum and / or multiple stains that have been applied to various cells transported through the flow cell 830. In this manner, a single camera can operate as a multispectral spectrometer and sense multiple laser and light wavelengths.

[0075] In some examples, the single-photon avalanche diode camera 840 can capture excitation and / or emission light. The emission light may be associated with a stain that has been applied to a biologic. Thus, the emission light may be a fluorescence of the stain in response to laser light from the dispersion module 820. In some other examples, the emission light may be an autofluorescence. An autofluorescence may be the nature response of a cell or the like to laser light. Any of these apparatuses may include a dispersion module, such as a spectrometer, in front of camera 840. A 1 -bit digital camera can capture whole EEM, e.g., for a single cycle (e.g., pulse) of the white light laser.

[0076] In some examples, “exposure” of the single-photon avalanche diode camera 840 may be controlled, at least in part, by the timing pulse signal from the controller 850. For example, a window of excitation and / or emission light may be applied. Thus, the controller 850 may capture light based on the timing pulse signal. The controller 850 can have the single-photon avalanche diode camera 840 capture excitation light.

[0077] In some examples, the flow rate in the flow cell 830 may be selected in conjunction with a light beam spread of the spatially spread out spectrum 821 provided by the dispersion module 820. If a cell is about 10 microns in diameter, and it is desired to have thirty excitation points (thirty cells) then the beam spread should be about 300 microns. The flow rate in the flow cell 830 may be adjusted so that 30 cells traverse 300 microns during each pulse of laser light. In some applications, the flow rate can be between 5 meters / second and 1 centimeter / second. In other examples, other flow rates are possible.

[0078] Horizontal resolution may be between 10 and 20 nanometers (to accommodate a single-file arrangement of cells). Vertical resolution can be between 20 - 30 wavelengths. Other horizontal and vertical resolutions are possible.

[0079] An EEM plot may be provided by the controller or the single-photon avalanche diode camera (e.g., shown in FIG. 8). In other examples, the EEM plot may be provided by any processor, processing unit (or similar device) having access to light excitation and emission data. As applied to spectral flow cytometry, the EEM plot may conveniently displayinformation associated with cells (or other objects) that have been stained with one or more particular stains. The stains may attach to and indicate particular proteins, antibodies, cell structures, or the like. The stains may respond to specific wavelength of light. In some cases, experimental or research data may be obtained by detecting the presence of the stains by detecting fluorescence or excitation light.

[0080] As described herein, light that “activates” a stain can be referred to as excitation light. As a stain is activated, the stain absorbs energy from the excitation light. The absorbed energy is released and can be observed as emitted light. Thus, the presence of emitted light can indicate the presence of the stain. Furthermore, the intensity of the emitted light (sometimes referred to as fluorescence intensity) can be proportional to the number of stained objects that respond to the excitation light.

[0081] In the EEM plot, excitation light is plotted on the x-axis and emission light is plotted on the y-axis. In addition, fluorescence intensity is plotted on the z-axis. Fluorescence may refer to the amount of emitted light that is detected. An EEM plot may show multiple peaks, each of which may correspond to a different stains and / or different stained objects. By way of example and not limitation, a first peak may correspond to 8-hydroxypyrene-l',3,6- trisulfonic acid, a second peak may correspond to Oregon green 514, a third peak may correspond to Rhodamine B, a fourth peak may correspond to Tris(4,7- diphenyl- 1,10- phenanthroline) ruthenium dichloride, and a fifth peak may correspond to Thionin acetate.

[0082] Generally, in spectral flow cytometry, emitted light is of lower energy (longer wavelength) than the excitation light. Therefore, reliably distinguishing between excitation light and emission light can affect the results of the spectral flow cytometry. In any of the true spectral flow cytometers described herein, a controller may generate a periodic timing signal that controls the output of the laser light source. Thus, the timing of the laser light (the excitation light) is known. The controller can receive data from any feasible spectral detector, and since the controller has timing information associated with the generation of the laser light (e.g., has access to the timing signal), the controller can distinguish between detected excitation and emission light.

[0083] Returning now to FIG. 2, FIG. 2 shows an example full spectrum plot associated with a full spectrum flow cytometer. The full spectrum plot is a two-dimension (2D) graph that displays a detected emission between 400 and 600 nanometers with a peak around 520 nanometers. The full spectrum plot in FIG. 2 only shows the emission associated with one stain or dye. Thus, the full spectrum plot shows much less information compared to the EEM plot of FIGS. 7A-7B. An equivalent EEM plot of the data shown in the full spectrum plot of FIG. 2 would show excitation frequencies that are quite narrow centered about 500nanometers. Since a detection of EEM is equivalent to detection of emission spectrum at multiple excitation wavelengths, it provides both the absorption and emission information. This may help in differentiating fluorochromes with similar emission spectra.

[0084] FIGS. 6A-6B show an EEM plot of a true spectral flow cytometer output associated with the same stain or dye used for the data in FIG. 2. Significantly, since the light source of the true spectral flow cytometer has more available wavelengths, and since the spectral detector of the true spectral flow cytometer can be sensitive across more wavelengths, the EEM plot shown in FIGS. 6A-6B can provide a more detailed response regarding any particular stain. The excitation / emission data of the EEM plot shows data that is not limited as shown in FIG. 2.

[0085] Returning to FIGS. 7A-7B, these graphs show an example EEM plot from a true spectral flow cytometer. Because true spectral flow cytometers use a wide-bandwidth laser light source and also use a wide-bandwidth spectral detector, the related EEM plot can easily show spectral data associated with multiple stains or dyes. The spectral data may be relatively continuous compared to more conventional flow cytometers. For example, EEM plot shows both excitation and emission wavelength information continuously between 400 and 850 nm. Grouping of fluorescence data can be quickly identified from the EEM plot. Each group may be associated with a different stain, which may be associated with different cells, antibodies, or the like. In some examples, execution of a trained neural network (applied artificial intelligence) can be used to recognize patterns within any EEM plot and determine constituent cells, antibodies, proteins, or the like. FIG. 7A shows a perspective view of the EEM plot shown in FIG. 7B. The amount of detected fluorescence is shown in the height of the curves extending in the z-axis (in this representation up and away from the emission and excitation axes).

[0086] The trained pattern matching agent may be an artificial intelligence agent, including a machine learning agent. The machine learning agent may be a deep learning agent. In some examples, the trained pattern matching agent may be trained neural network. Any appropriate type of neural network may be used, including generative neural networks. The neural network may be one or more of: perceptron, feed forward neural network, multilayer perceptron, convolutional neural network, radial basis functional neural network, recurrent neural network, long short-term memory (LSTM), sequence to sequence model, modular neural network, etc. In some examples a trained pattern matching agent may be trained using a training data set

[0087] As used herein, a processor may include hardware that runs the computer program code. Specifically, the term ‘processor’ may include a controller and may encompass not onlycomputers having different architectures such as single / multi-processor architectures and sequential (Von Neumann) / parallel architectures but also specialized circuits such as field- programmable gate arrays (FPGA), application specific circuits (ASIC), signal processing devices and other devices.

[0088] FIG. 9 is a flowchart showing an example method 900 for generating an EEM plot. Some examples may perform the operations described herein with additional operations, fewer operations, operations in a different order, operations in parallel, and some operations differently. The method 900 is described below with respect to the true spectral flow cytometer of FIG. 4, however, the method 900 may be performed by any other suitable system or device.

[0089] The method may begin as the laser light source generates a wide wavelength laser light illumination. The illumination may be generated based on a timing pulse signal provided by a controller. The wide wavelength laser light may span a range of wavelengths of light. In some examples, the laser light wavelengths may be continuous between 400 and 700 nanometers. In some other examples, the laser light may be between any feasible wavelengths.

[0090] Next 904, a spatially spread apart spectrum from the wide wavelength laser light is projected into the flow cell. The flow cell can transport cells through the spatially spread apparat spectrum 906. In some examples, the controller can control speed of the cells through the flow cell. The flow cell can arrange cells in a single-file.

[0091] Next, the spectral detector can generate at least one excitation emission matrix (EEM) plot 908, wherein the EEM plot includes excitation wavelength, emission wavelength, and fluorescence intensity. As described herein, the EEM plot may graphically display excitation wavelengths, emission wavelengths, and fluorescence intensity. In some examples, light data captured by the spectral detector can be provided to the controller and the controller can generate the EEM plot. In some examples, the spectral detector may include a single photon avalanche photodiode camera configured to capture excitation wavelength, emission wavelength, and fluorescence intensity.

[0092] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0093] The process parameters and sequence of steps described and / or illustrated herein are given by way of example only and can be varied as desired. For example, while the steps illustrated and / or described herein may be shown or discussed in a particular order, thesesteps do not necessarily need to be performed in the order illustrated or discussed. The various example methods described and / or illustrated herein may also omit one or more of the steps described or illustrated herein or include additional steps in addition to those disclosed.

[0094] Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor to control perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like. For example, any of the methods described herein may be performed, at least in part, by an apparatus including one or more processors having a memory storing a non-transitory computer-readable storage medium storing a set of instructions for the processes(s) of the method.

[0095] While various embodiments have been described and / or illustrated herein in the context of fully functional computing systems, one or more of these example embodiments may be distributed as a program product in a variety of forms, regardless of the particular type of computer-readable media used to actually carry out the distribution. The embodiments disclosed herein may also be implemented using software modules that perform certain tasks. These software modules may include script, batch, or other executable files that may be stored on a computer-readable storage medium or in a computing system. In some embodiments, these software modules may configure a computing system to perform one or more of the example embodiments disclosed herein.

[0096] As described herein, the computing devices and systems described and / or illustrated herein broadly represent any type or form of computing device or system capable of executing computer-readable instructions, such as those contained within the modules described herein. In their most basic configuration, these computing device(s) may each comprise at least one memory device and at least one physical processor.

[0097] The term “memory” or “memory device,” as used herein, generally represents any type or form of volatile or non-volatile storage device or medium capable of storing data and / or computer-readable instructions. In one example, a memory device may store, load, and / or maintain one or more of the modules described herein. Examples of memory devices comprise, without limitation, Random Access Memory (RAM), Read Only Memory (ROM), flash memory, Hard Disk Drives (HDDs), Solid-State Drives (SSDs), optical disk drives,caches, variations or combinations of one or more of the same, or any other suitable storage memory.

[0098] In addition, the term “processor” or “physical processor,” as used herein, generally refers to any type or form of hardware-implemented processing unit capable of interpreting and / or executing computer-readable instructions. In one example, a physical processor may access and / or modify one or more modules stored in the above-described memory device. Examples of physical processors comprise, without limitation, microprocessors, microcontrollers, Central Processing Units (CPUs), Field-Programmable Gate Arrays (FPGAs) that implement softcore processors, Application-Specific Integrated Circuits (ASICs), portions of one or more of the same, variations or combinations of one or more of the same, or any other suitable physical processor.

[0099] Although illustrated as separate elements, the method steps described and / or illustrated herein may represent portions of a single application. In addition, in some embodiments one or more of these steps may represent or correspond to one or more software applications or programs that, when executed by a computing device, may cause the computing device to perform one or more tasks, such as the method step.

[0100] In addition, one or more of the devices described herein may transform data, physical devices, and / or representations of physical devices from one form to another. Additionally, or alternatively, one or more of the modules recited herein may transform a processor, volatile memory, non-volatile memory, and / or any other portion of a physical computing device from one form of computing device to another form of computing device by executing on the computing device, storing data on the computing device, and / or otherwise interacting with the computing device.

[0101] The term “computer-readable medium,” as used herein, generally refers to any form of device, carrier, or medium capable of storing or carrying computer-readable instructions. Examples of computer-readable media comprise, without limitation, transmission-type media, such as carrier waves, and non-transitory-type media, such as magnetic-storage media (e.g., hard disk drives, tape drives, and floppy disks), optical-storage media (e.g., Compact Disks (CDs), Digital Video Disks (DVDs), and BLU-RAY disks), electronic-storage media (e.g., solid-state drives and flash media), and other distribution systems.

[0102] A person of ordinary skill in the art will recognize that any process or method disclosed herein can be modified in many ways. The process parameters and sequence of the steps described and / or illustrated herein are given by way of example only and can be varied as desired. For example, while the steps illustrated and / or described herein may be shown ordiscussed in a particular order, these steps do not necessarily need to be performed in the order illustrated or discussed.

[0103] The various exemplary methods described and / or illustrated herein may also omit one or more of the steps described or illustrated herein or comprise additional steps in addition to those disclosed. Further, a step of any method as disclosed herein can be combined with any one or more steps of any other method as disclosed herein.

[0104] The processor as described herein can be configured to perform one or more steps of any method disclosed herein. Alternatively, or in combination, the processor can be configured to combine one or more steps of one or more methods as disclosed herein.

[0105] When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected,” “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected,” “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0106] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0107] Spatially relative terms, such as “under,” “below,” “lower,” “over,” “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will beunderstood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly,” “downwardly,” “vertical,” “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0108] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.

[0109] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0110] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and / or steps may alternatively be exclusive and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps, [oni] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understoodto include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0112] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0113] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of theabove embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMSWhat is claimed is:

1. A flow cytometry apparatus, the apparatus comprising: a broad-spectrum laser configured to emit light having a continuous wavelength of between about 400 and 700 nanometers; a dispersion module configured to spread out the light into a spatially arranged spectrum arranged in a first axis; a seat configured to hold a flow cell in the first axis, wherein the dispersion module is configured to deliver the spatially arranged spectrum into the flow cell so that cells transported through the flow cell pass through the spatially arranged spectrum; and a multispectral spectrometer module configured receive at least a portion of the spatially arranged spectrum passing through the flow cell, wherein the multispectral spectrometer module is configured to generate at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

2. The apparatus of claim 1, wherein the multispectral spectrometer module includes a single photon avalanche photodiode camera configured to determine a number of photons associated with at least one wavelength included in the spatially arranged spectrum.

3. The apparatus of claim 1, further comprising a multi-channel optical fiber configured to couple the spatially arranged spectrum passing through the flow cell to the multispectral spectrometer.

4. The apparatus of claim 1, wherein the flow cell is further configured to align cells in a single-file along the first axis.

5. The apparatus of claim 1, wherein the multispectral spectrometer module is configured to detect at least a wavelength of light from the broad-spectrum laser, a wavelength of light emitted by the cells, or a fluorescence intensity.

6. The apparatus of claim 1, wherein the seat is configured to removably hold the flow cell.

7. The apparatus of claim 1, further comprising a controller configured to control a rate of fluid flow through the flow cell.

8. The apparatus of claim 7, wherein the controller comprises a trained pattern recognition agent configured to recognize a pattern of excitation wavelength, emission wavelength and fluorescence intensity in the EEM plot.

9. The apparatus of claim 1, wherein the dispersion module comprises one or more prisms.

10. The apparatus of claim 1, further comprising the flow cell.

11. A flow cytometry apparatus, the apparatus comprising: a broad-spectrum laser configured to emit light having a continuous wavelength of between about 400 and 700 nanometers; a dispersion module configured to spread out the light into a spatially arranged spectrum arranged in a first axis; a flow cell, configured to receive the spatially arranged spectrum and transport cells through the spatially arranged spectrum; and a multispectral spectrometer module configured receive at least a portion of the spatially arranged spectrum passing through the flow cell, wherein the multispectral spectrometer module is configured to generate at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

12. A method, the method comprising: generating a wide wavelength laser light; generating, with a dispersion module, a spatially arranged spectrum from the wide wavelength laser light within a flow cell; transporting cells within the flow cell through the spatially arranged spectrum; and generating, with a multispectral spectrometer module, at least one excitation emission matrix (EEM) plot, wherein the EEM plot includes excitation wavelength, emission wavelength and fluorescence intensity.

13. The method of claim 12, wherein the wide wavelength laser light include light having wavelengths between 400 and 700 nanometers.

14. The method of claim 12, wherein generating the EEM plot further comprises capturing excitation wavelength, emission wavelength, and fluorescence intensity data with a single photon avalanche photodiode camera.

15. The method of claim 12, further comprising controlling a flow of cells through the flow cell and the application of the wide wavelength laser light based using a controller.

16. The method of claim 15, wherein generating the EEM plot further comprises generating the EEM plot in the controller.

17. The method of claim 12, further comprising transmitting, with a multi-channel optical fiber, the spatially arranged spectrum to the flow cell.

18. The method of claim 12, wherein the spatially arranged spectrum is disposed on a first axis and the cells traverse the spatially arranged spectrum substantially on the first axis.

19. The method of claim 12, wherein generating the spatially arranged spectrum with the dispersion module comprises passing the wide wavelength laser light through a plurality of prisms and one or more lenses configured to separate the wide wavelength light by refraction and project the light into the flow cell.

20. The method of claim 12, further comprising controlling a rate of fluid flow through the flow cell so that a single cell passes through the spatially arranged spectrum at a time.

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