Systems and methods for flow cytometry with adapted discrimination

Abstract

The flow cytometer may include one or more light emitters configured to emit light into an optical path, a rectangular flow cell having a flow cell width generally lateral to the optical path and a flow cell depth generally longitudinal to the optical path, the optical path having an interrogation width at the flow cell narrower than the flow cell width, and a spherical reflector positioned adjacent to the rectangular flow cell, the spherical reflector having a concave reflective surface with a reflection direction positioned generally perpendicular to the optical path such that reflected light is reflected along a reflection path generally perpendicular to the optical path. One or more light absorbing members are positioned at least partially around the reflection path to absorb reflected light at an angle to the reflection path.

Description

[Technical Field]

[0001] The present disclosure relates to flow cytometry using systems and methods that provide tailored discrimination between target analytes (e.g., live cells, proteins, beads, etc.) and non-target particles (e.g., non-viable cells, contaminants, other non-target materials). [Background technology]

[0002] Traditionally, flow cytometry is performed with low flow rates of liquid. Typically, conventional flow cytometry systems can process approximately 100 μL / min, which is quite low. Conventional flow cytometry often also uses sheath flow through an analytical flow cell to ensure that the sample under investigation with light is in the center of the flow, allowing the interrogation light to be directed toward the center of the flow cell. The sheath flow can provide a lensing and focusing effect in the flow cell, allowing the system to acquire data without problems with extraneous signals from the surrounding glass and flow cell walls. Flow cytometry works well when cells are at a sufficiently high concentration that they can be distinguished at low volumetric flow rates.

[0003] However, flow cytometry is deficient in its ability to detect small numbers of detectable analyte entities (e.g., cells) at high flow rates or high volume interrogation rates. For example, when determining whether a fluid is contaminated with microorganisms such as bacteria (e.g., one example of a cell), the concentration limit is lower than with conventional flow cytometry. For example, application of flow cytometry with one detectable analyte entity in 100 mL is not feasible using conventional flow cytometry because the flow rate is too low and the analysis takes too long.

[0004] While flow cytometry has been useful in laboratory settings where liquid samples are being prepared for analysis, the technique is less well established for analyzing the bioburden of liquid samples, such as environmental or purified water samples. The combination of a low flow rate of approximately 100 μL / min and low sensitivity makes flow cytometry unsuitable for bioburden analysis when target microorganisms are present in dilute concentrations. The majority of bioburden tests are performed using heterotrophic plate counting, in which the sample is filtered through a membrane, which is then placed on a Petri dish or agar plate filled with nutrients for microbial growth. These plates are then incubated at 37°C for several days to promote the growth and proliferation of microbial colonies, each containing tens of thousands to millions of cells that may have been present in the original sample, and which can be seen and counted by the human eye. This traditional method, while relatively simple, is extremely time-consuming (e.g., several days) and labor-intensive. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for a flow cytometry system and method of operation that can be useful for analyzing low bioburden in liquids, such as purified water or drinking liquids. It would be beneficial to be able to have higher flow rates to detect low concentrations of detectable analyte entities or cells in high volumes of liquid so that quantification of target analytes (e.g., bioburden) can be determined in a short period of time. [Means for solving the problem]

[0006] In some embodiments, the flow cytometer may include one or more light emitters configured to emit light into the optical path. A rectangular flow cell having a flow cell width is positioned such that the flow cell width is approximately lateral to the optical path and the flow cell depth is approximately longitudinal to the optical path. The optical path has an interrogation width at the flow cell that is narrower than the flow cell width. A spherical reflector is positioned adjacent to the rectangular flow cell. The spherical reflector has a concave reflective surface with a reflection direction positioned approximately perpendicular to the optical path such that reflected light is reflected along a reflection path approximately perpendicular to the optical path. The flow cytometer may include a light absorbing member that prevents reflections from passing through the exit aperture. In some aspects, the flow cytometer may include one or more light absorbing members positioned at least partially around the reflection path to absorb reflected light at an angle relative to the reflection path, one or more light absorbing members positioned between the flow cell and the exit aperture to define an optical conduit therebetween, or one or more light absorbing members that at least partially define the exit aperture. In some embodiments, the one or more light-absorbing members comprise one or more of a tapered light-absorbing surface extending away from the flow cell, a parallel light-absorbing surface that is approximately parallel to the reflection path, an orthogonal light-absorbing surface that is approximately perpendicular to the reflection path, or a light-absorbing material on the surface of a wall of the flow cell.

[0007] In some embodiments, a flow cytometer system may include the flow cytometer of one of the embodiments. The system may also include a sample source. The system may also include an analyte stain bath containing an analyte stain composition, the analyte stain composition including a dye configured to detect a target analyte. The system may also include a counterstain bath containing a counterstain composition, the counterstain composition including a dye configured to detect a non-target analyte. The system may include one or two reactors for staining the sample and a fluid path connecting the flow cytometer to a stain sample preparation system, the stain sample preparation system being connected to the sample source.

[0008] In some embodiments, a kit can include one or more of the flow cytometer embodiments. The kit can also include an analyte stain having a first fluorescent wavelength. Additionally, the kit can include a counterstain having a second fluorescent wavelength.

[0009] In some embodiments, a method for detecting a target analyte in a sample can be performed with a flow cytometer according to one embodiment. The method can include providing a stained sample having an analyte stain and a counterstain, wherein the analyte stain emission wavelength is distinguishable from the counterstain emission wavelength. The stained sample can be analyzed using the flow cytometer, such as by illumination scanning. Potential targets having the analyte stain can be detected with a data analysis computer. The data analysis computer can also identify potential targets as having no counterstain or a counterstain below a counterstain threshold. The data analysis computer can also identify detected potential targets having the analyte stain without a counterstain or a counterstain below a counterstain threshold as target analytes.

[0010] In some embodiments, a flow cytometry system may include a flow cytometer according to one of the embodiments. The system may also include a computer system including one or more processors and one or more non-transitory computer-readable media storing instructions that, when executed by the one or more processors, cause the computer system to perform operations. The operations may include causing a sample preparation system to provide a stained sample having an analyte stain and a counterstain to a flow cytometer, where the analyte stain emission wavelength is distinguishable from the counterstain emission wavelength, causing the flow cytometer to interrogate the stained sample to acquire flow cytometry data, detecting potential targets having the analyte stain from the flow cytometry data, identifying potential targets as having no counterstain or having a counterstain below a counterstain threshold, and identifying detected potential targets having the analyte stain without the counterstain or having a counterstain below a counterstain threshold as target analytes.

[0011] The above summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0012] The above and following information, as well as other features of the present disclosure, will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which: The present disclosure will be described with additional specificity and detail through the use of the accompanying drawings, with the understanding that these drawings illustrate only some embodiments in accordance with the present disclosure and therefore should not be considered limiting of the scope of the disclosure. [Brief explanation of the drawings]

[0013] [Figure 1A] FIG. 1 is a top view illustrating one embodiment of an illumination region in a flow cytometry system. [Figure 1B] FIG. 10 is another horizontal cross-sectional view showing additional areas of the flow cytometry system. [Figure 1C] FIG. 1B is a vertical cross-sectional view (eg, orthogonal to FIG. 1B) of a flow cytometry system. [Figure 1D] 1 shows a light absorbing agent that can be coated around the outer wall of the flow cell. [Figure 1E] 1 is a cross section of a flow cell with a light absorber on the flow cell. [Figure 2A] 1 shows an example of a full assay system including a flow cytometer according to embodiments described herein, a sample source, and a stain sample preparation system having two separate stain reactors. [Figure 2B] 1 shows an example of a full assay system including a flow cytometer according to embodiments described herein, a sample source, and a stain sample preparation system configured for an online system with a single stain reactor. [Figure 3] It includes data showing that the green channel can be used for staining the analyte and the red channel can be used for counterstaining. [Figure 4] Included is data showing an example of a signal that is biological as indicated by having a target analyte stain signal and peaks within size and amplitude parameters within tolerance. [Figure 5] Included are data showing examples of signals that are not viable target analytes (eg, non-biological molecules). [Figure 6] 1 shows graphs from an example matched filter. [Figure 7] 1 illustrates an example of a computing device (eg, a computer) that may be configured to perform the methods (or portions thereof) described herein in some embodiments. [Figure 8A] 1 shows a reactor for staining samples for a flow cytometer protocol. [Figure 8B] 1 shows a piston working with a reactor. [Figure 8C] The piston seal is shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] The elements and components in the figures may be arranged according to one or more of the embodiments described herein, and the arrangement may be modified by one of ordinary skill in the art according to the disclosure provided herein. In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like elements, unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter described herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the figures may be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0015] In general, the present invention relates to flow cytometry systems and methods of operation capable of detecting small numbers of analytes (e.g., cells, proteins, particles, etc.) in high volumes of fluid. For example, the flow cytometry system can detect approximately one detectable analyte entity per 100 mL of fluid (1 / 100 mL) at flows exceeding 5 mL / min. High volume interrogation rates may enable the flow cytometry system to perform a complete analysis in a shorter time, e.g., two hours or less, preferably within about one hour (e.g., 40 minutes using a two-reactor system). Rapid interrogation of high volumes of sample fluid enables the economic feasibility of using flow cytometry for low-concentration samples. However, the flow cytometry and systems described herein can be used in any conventional flow cytometry analysis. Samples can be any type of sample, whether raw or purified, from foods and beverages to manufactured compositions, natural liquids, and various types of water. Another example may include monitoring the rapid release of analytes from manufactured compositions (e.g., zero-order release from drug depots).

[0016] A flow cytometry system can be configured to perform analysis at high flow rates without the use of sheath flow during light (e.g., laser, light emitting diode (LED), high intensity light emitting diode (HLED) high intensity discharge - "HID", halogen, incandescent, etc., and combinations and arrays thereof) interrogation. The flow cell is configured with a size and shape such that the interrogation light does not pass through the sidewalls and sheath flow is not used. Instead, the interrogation light (e.g., one or more light emitters) is positioned such that the light passes through the central region of the flow cell without passing through the side regions of the flow cell. For directional purposes, the light direction defines a longitudinal direction such that the lateral direction can be inclined, such as perpendicular, from the longitudinal direction. The light can be a beam or otherwise shaped to have a width narrower than the flow cell width, and whether it is a laser or other type of light emitter, lenses or other optical components can be used to shape the light. While laser interrogation is described herein, it should be recognized that other light sources, such as shaped HLEDs with lenses, can also be used for interrogation.

[0017] Furthermore, traditional flow cytometry often uses labels (e.g., dyes such as fluorescence) that are specific to the target being analyzed, and the labels can be used in the present flow cytometer. For example, in a laboratory setting, flow cytometers often use specific labels to tag specific types of analytes (e.g., cells), such as a particular target species that has a receptor for a ligand bound to the label. This labeling method can work when the target analyte is known and therefore the label can be configured to specifically bind to the target, but is not useful when the analyte type is unknown, such as in bioburden analysis. One example includes different stains that mark certain types of analytes but not others.

[0018] To be useful with samples containing unknown types of analytes, the flow cytometry system can also employ dye combinations useful for detecting a wide range of different types of analytes (e.g., cells of different microbial species) in high-volume samples using high-volume analysis, with differentiation from non-target analytes, such as non-viable cells or other undesirable particles (e.g., plastic, rubber, metal debris). Thus, dye combinations are provided that enable detection of target analytes (e.g., live cells) without highly specific targeting to a particular analyte. In some aspects, the dye combinations provided herein enable detection of low bioburden (e.g., low concentrations of microorganisms) at high volumetric flow rates, with or without a targeting moiety that binds to the target analyte. For example, the dye combination can include a dye that labels cellular material (e.g., a DNA dye) and a counterstain that can only stain damaged or dead cells, such as a counterstain that leaks into cracked or otherwise damaged cells. The presence of the cellular stain (e.g., a DNA dye) indicates a cell, and the presence of the counterstain indicates a dead cell. The detection of the presence of only the DNA stain (or detection below a threshold) can indicate that cells are present and viable. The detection of the presence of only the counterstain indicates that the material bearing the counterstain is not a viable cell. Analyte stains can be used to target analytes, and counterstains can be used to target other materials / particles that are not analytes. While cells are used herein as an example of an analyte, it should be recognized that any reasonable material or particle can be a target analyte that is distinguished from non-target analytes or non-target materials / particles in a sample.

[0019] The dye combination and flow cytometer configuration allows for a detection sensitivity of down to 1 detectable analyte entity per 100 mL. However, it should be recognized that the dimensions and flow rates can be modified to achieve higher flow rates, such as by widening the flow cell or providing greater depth to the optical path in the flow cell so that higher flow rates can be achieved. This may allow for precision of 1 detectable analyte entity per 100 mL, 200 mL, 500 mL, or even up to 1 L.

[0020] In some embodiments, the flow cytometry system is configured to broadly detect living organisms (e.g., live cells) from non-living (e.g., dead cells), non-living matter (e.g., not derived from living organisms), or inert matter (e.g., mechanical particles from wear) in a sample. The dye combination is configured to broadly label live cells so that they are distinguishable from non-living cells and particles, e.g., microscopic particles, such as stainless steel, gasket material, rubber, or other materials. The dye combination is configured to mark live cells differently from dead cells or particles through the analyte stain being distinguishable from the counterstain. This allows for a low detection limit of 1 microorganism out of 10,000 detectable particles, e.g., allowing for the distinction of E. coli from Teflon®. However, modifications are possible to detect 1 microorganism out of 20,000 detectable particles, 30,000 detectable particles, or up to 50,000 detectable particles. However, it should be appreciated that any type of analyte that can be selectively stained relative to other substances / particles in a sample can be targeted for detection and distinguished from other substances / particles. The counterstain can also be configured to enable the target analyte to be distinguished from non-target analytes using the analyte stain. For example, the counterstain may not stain the target analyte, or may be weakly / lightly stained to allow the stained target analyte to be identified from others.

[0021] In various flow cytometry systems and methods, it has traditionally been considered important to detect every photon of a signal. However, it is now known that attempting to detect every photon in a sample in bioburden analysis is not appropriate. Flow cytometry systems can now be configured to reduce the percentage of available photons that are acquired and used in bioburden analysis. The system can acquire a lower percentage of photons to reduce the influence of noise signals or worthless light.

[0022] Many flow cytometry systems use Mie scattering for particle detection. However, the present flow cytometry system is configured to analyze samples without Mie scattering analysis and omit any particle sizing analysis. The present system omits Mie scattering analysis, and therefore, particle sizing data is not acquired and used for particle differentiation. Omission of particle sizing data analysis has been found to reduce confounding of acquired label data. Traditionally, Mie scattering signals are first analyzed for particle size analysis, and then, once particle sizes within a specified range are identified, the system searches for label signals for those particles. This strategy has proven difficult for data analysis. Here, the present flow cytometry system does not perform particle sizing analysis, such as Mie scattering. Therefore, embodiments of the present flow cytometry system omit Mie scattering analysis and use only label detection analysis from dye combinations (e.g., analyte stain vs. counterstain). Omission of Mie scattering or other particle sizing analysis has been found to be beneficial because cells are not solid spheres and do not generate predictable Mie scattering signals. This allows the flow cytometry system to rely on fluorescent detection of labels in dye combinations. Other particles may not be suitable for particle analysis, thereby eliminating particle analysis and associated devices and systems.

[0023] In some embodiments, the flow cytometry system does not use any analysis other than the fluorescent signal analysis of the dye combination, so that when particles or other analytes pass through the flow cell, the system can excite any labels and thereby detect fluorescence. Therefore, to determine whether a particle is a target analyte (e.g., a live cell), it is sufficient to use at least two spectral regions of the analyte stain and counterstain. While a two-dye system can be used, an analyte stain (e.g., that stains the target analyte) and a counterstain (e.g., that does not stain the target analyte), additional dyes can be used to expand the analysis to adaptively distinguish the target analyte from other particles / substances. In some embodiments, the dye combination set can be composed of analyte stains and counterstains in two distinct wavelength ranges.

[0024] Flow cytometry systems can be applied to determining the bioburden of liquids such as food, beverages, pharmaceuticals, biological samples, and ultrapure water. For example, it may be important to have a small number of microorganisms in ultrapure water. Therefore, samples can be filtered so that large particles are already filtered out, thereby reducing the importance of Mie scattering and particle size analysis. Analysis can also be performed between live / viable and dead / nonviable microorganisms, and dead microorganisms may be present (e.g., from the purification process). Dead microorganisms can still be distinguished using analyte stain fluorescence. Thus, the counterstain can be a viability stain; if counterstained, the cell is nonviable. For example, the analyte stain (e.g., a DNA stain) can emit one color, and the counterstain can emit a different color; most combinations, such as yellow, red, green, blue, purple, and orange, are possible. In one example, the color combination can determine whether the counterstain has permeated the cell; if so, the cell is likely nonviable. This allows the system to distinguish cells with both stains from cells with only the cell stain. In another example, a color combination may target a specific particle type with an analyte stain and a different particle type with a counterstain. The less counterstaining there is in the target cell analyte, the more likely the cell is alive and viable. The less counterstaining there is in the particle / substance with the analyte stain, the more likely that particle / substance with the analyte stain is the target. More counterstaining typically indicates a dead cell or the presence of non-target analytes. Therefore, the counterstain is configured to penetrate less well into nearly intact, live cells or to stain less well the target analyte.

[0025] In one example, emission colors can be selected so that one color is a nucleic acid stain or a stain for another substance in the cell, and the counterstain can simply penetrate the cell and stain the cell if the cell membrane is compromised or cracked. In other cases, the counterstain can stain only the outer surface of the cell, thereby reducing the signal. Examples of analyte stains include thiazole orange, GelRed, Hoechst, SYBR (various colors, green, gold), ethidium bromide, acridine orange, etc. Examples of counterstains include Live / Dead stains (various colors) from ThermoFisher Scientific, Live-or-Dye stains from Biotium, ethidium monoazide, DAPI, Sytox Blue, Sytox Green, Sytox Red, ethidium homodimer-1, propidium iodide, 7AAD, PO-Pro-1, YO-Pro-1, Annexin V (various colors), erythrosin B (EB), etc. Staining agents can also be configured for non-cellular applications, such as detection of target proteins, drugs, or other substances.

[0026] In one example, an analyte stain can be used to create a green signal with an excitation peak at approximately a first wavelength, excited by a laser with an excitation wavelength that has a fluorescence response with a peak at a second wavelength, and there can be a small spectral difference between the excitation and emission peaks. Therefore, the system can include a notch filter 137 (FIGS. 1B and 1C) that transmits some light and blocks as much light as possible that is not within the wavelength range window (or other band, if a different dye is used). This provides a color signal of significantly lower intensity than the counterstain, which provides a different color signal. While there is more fluorescence being generated by the dye, this is a smaller spectral region that is collected because the signals begin to confound. This allows for direct focus on only the area around its peak emission, providing better quality information. In some embodiments, the amount or concentration of the analyte stain is significantly lower than the counterstain, such that the analyte stain signal is smaller than the counterstain signal, or vice versa. In some examples, the counterstain can be red, providing an essentially broad-spectrum red channel. The system collects all light from a specific wavelength range that is large compared to the spectrum of the analyte stain channel, which allows for better discrimination between target analytes (e.g., viable cells) and non-target analytes (e.g., viable cells or particles).

[0027] The flow cytometry system can be configured to identify the proportion of counterstain that permeates or otherwise stains detectable target analytes (e.g., viable cells). A counterstain proportion threshold can be established to distinguish between target analytes (e.g., viable cells) and other substances (e.g., non-viable cells), such that target analytes above the counterstain proportion threshold are designated as non-targets (e.g., non-viable cells), and labeled target analytes below the counterstain proportion threshold are designated as target analytes. The counterstain proportion threshold can be defined or set to different values ​​for different samples or different test protocols. It should be clear to those skilled in the art that the area of ​​each signal and other data analytics can equally be used instead of intensity.

[0028] In some embodiments, the ratio of the analyte stain signal to the counterstain signal can be used for discrimination analysis, whereby ratios within a certain range can be determined as target analytes (e.g., viable cells) and ratios outside the certain range can be determined as non-target analytes (e.g., dead cells or other substances such as non-living particles). The ratio can be determined from the adjusted signal of the analyte stain relative to the live signal or counterstain. The area under the curve and / or the ratio of peak intensities can then be used to discriminate between target analytes (e.g., viable cells) and non-target analytes (e.g., non-viable cells).

[0029] In some embodiments, the acquired signal can be processed to omit some of the optical signal data. Instead of acquiring all of the light for analysis, select regions of the optical signal data are acquired for analysis. This processing can improve the desired optical signal while reducing noise. This can enhance the discrimination of target analytes (e.g., viable cells) from non-target analytes (e.g., non-viable cells). This can also allow for empirical optimization of two different signals simultaneously.

[0030] In some embodiments, a flow cytometer can be configured to block total internal reflectance (TIR) ​​in the system so that less background light is collected by the detector. For example, emitted light that strikes the walls can be reduced by coating the internal surface of the device with a light-absorbing material, such as a blackbody material. This can reduce the total light and aid in signal conditioning. Thus, light emitted through the flow cell walls at angles above a threshold angle, such as about 43 degrees, is absorbed and not reflected, so that the photons are not counted (provided no record). This can avoid the mirror effect of light and avoid general light collection from glass, water, and anything else that may emit / reflect light. Adjusting the collected light can be used to better collect light for analysis. For example, by analogy, the light collection configuration can selectively capture a "heart cut" of light, that is, light that is important for the data analysis described herein. Unimportant or unuseful light can be omitted from use by collection and / or absorption.

[0031] The configuration of flow cytometry components is described herein and in the incorporated references, with modifications that reduce the collection of any reflected or non-ideal light by the system. Flow cytometry systems generally use rectangular flow cells with a rectangular internal cell chamber (e.g., a rectangular cross-sectional profile area perpendicular to the sample flow through the flow cell) combined with a spherical reflector (e.g., a hemisphere) and surrounded by a light-absorbing material. The shape is rectangular, and the rectangle can be a perfect rectangle or a substantial rectangle with some variation, such as rounded corners with radii. The overall shape of the internal chamber of a flow cell operating with flow is rectangular, and the corners can be rounded and not perfectly orthogonal. This combination is a non-spherical flow cell with a spherical reflector (e.g., a reflective surface that is concave as the interior of a sphere). The flow cell can be probed with a narrower light beam width so that reflected light is minimized and noise signals can be reduced.

[0032] The configuration of the flow cytometer can reduce the amount of light collected to less than 50% of the total light, less than 40% of the total light, less than 30% of the total light, less than 25% of the total light, or less than 20% of the total light that can be collected without absorption.

[0033] Additionally, flow cytometry systems may include means for reducing the size of the collection area. This reduces the amount of light that can reach the detector. Therefore, an aperture can be placed in the light path to reduce the collection area. For example, the aperture can suppress the light signal and reduce the exit portion of the light that can reach the detector. For example, a 50 mm collection system can be reduced to 25 mm at the exit by an aperture. The aperture and / or light-absorbing material can help reduce the total amount of light collected, thereby reducing the relative noise from reflections relative to the target emission signal from live cells.

[0034] 1A is a top view of one embodiment of an illumination region 102 in a flow cytometry system 100. System 100 shows a rectangular flow cell 104 (with flow channels extending in and out of the plane of the page perpendicular to the plane of the page) along with a spherical reflector 106 and an aperture 108 with a light path hole 110. The flow cell 104 is enlarged on the left side of system 100 to illustrate its shape and dimensions; for example, the flow cell 104 may be 2200-2400 μm wide (W1) relative to the interrogation region 112 (the area affected by the laser). As can be seen, interrogation region 112 is shown to be 2000 μm wide (W2), which is significantly shorter than the width of flow cell 104 (e.g., 200-400 μm narrower, with the interrogation region having a height of 50 μm (H2) and a depth of 500 μm (D2)). However, it should be recognized that if the width of the interrogation light beam is smaller than the width of the flow cell, the dimensions can be adjusted (e.g., + / - 1%, 5%, 10%, 25%, 75%, etc.), such as a narrower or wider flow cell in a plane perpendicular to the interrogation light beam. For example, the light beam width can be shaped to have an edge that is within the flow cell without touching the flow cell wall, e.g., a gap of at least 10 μm, 20 μm, 30 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, or more than 200 μm between the edge of the light beam and the flow cell wall.

[0035] This allows the laser (or other interrogation light) to avoid the capillary wall 104a of the flow cell 104 and only interrogate the central region of the flow cell as the interrogation region 112. The spherical reflector 106 is at the rear of the flow cell 104, while the aperture 108 and light path hole 110 are at the front of the flow cell 104. The system 100 can omit the front reflector, which is typically located where the first absorber 114 is located in the present invention. The spherical reflector 106 is a rear reflector and therefore by definition is positioned for rear light, which is why it has a hemispherical shape in the rear region. The first absorber 114 can be positioned around the light path from the spherical reflector 106. While the first absorber 114 is shown as conical so that light passes through the light path (e.g., hole) of the first absorber 114 and other light is absorbed, it can be another shape.

[0036] The first absorber 114 is configured as a light absorber that absorbs light (photons) that it contacts, thereby preventing reflection. The first absorber 114 and other absorbers described herein can be blackbody materials that absorb photons, so that the illumination area 102 does not experience any reflections from its walls or surfaces.

[0037] The illumination area 102 includes a condenser lens 116 that facilitates focusing the light toward the optical aperture 110. The condenser lens 116 thus collects the light and directs it to exit the aperture 110. A first absorber 114 is adjacent to the condenser lens 116, absorbing the light and blocking reflections, thereby preventing reflected light from contacting the condenser lens 116. The first absorber 114 blocks any non-coaxial light from reaching the condenser lens 116, thereby blocking forward scattered light. In one example, the blackbody material first absorber 114 can be Acktar black coating (Acktar)—a light-absorbing material and coating that provides a solution for suppressing stray light at VIS, SWIR, and MWIR wavelengths. The material can be a superblack material (blackest black), a black foil, or a direct coating for blackening opto-mechanical components. The first absorber 114 optimizes the fluorescence signal by eliminating reflected light, such as forward reflected light; by reducing reflected light, light has loss from reflections and can improve the signal-to-noise ratio. This removes noise and background, making the desired signal more prominent, detectable, and distinguishable.

[0038] The result is a high-quality signal that can be utilized in radiometric analysis of signal intensity. Conventional flow cytometry has a two-dimensional space, and in two-dimensional space, data processing results in a cloud that is significantly scattered due to the distribution of the signal, and the system does not acquire a dense cloud, which typically produces a significantly scattered cloud. Contrary to conventional flow cytometry, the present invention acquires a concentrated region that is less scattered and provides an improved cloud area. The improved signal response area is useful for distinguishing target analytes from non-target analytes, such as distinguishing viable cells from non-viable particles.

[0039] As shown, the interrogation area 112 is narrower in the lateral direction than the width of the flow cell 104 between the capillary walls 104a. The interrogation area 112 may be approximately 2000 μm (2 mm) wide (transverse or horizontal), approximately 200-600 μm thick (or deep if deep) (e.g., approximately 400 μm), and approximately 20-90 μm high (e.g., 50 μm), or + / - 1%, 5%, 10%, 25%, 75%, etc., of these values. This provides a significantly larger interrogation area 112 than typical flow cytometry. This larger interrogation area allows for higher levels of throughput, which may be at high flow rates of approximately 5 mL / min, allowing 100 mL to be analyzed in a short period of time. For example, the system can operate for 20 minutes and interrogate a 100 mL sample. This allows samples to be analyzed at flow rates exceeding 1 mL / min without sheath flow. Eliminating sheath flow significantly reduces supply requirements, waste, system complexity, and other issues. The elimination of sheath flow can be achieved by making the interrogation region 112 smaller than the flow cell 104 so that the capillary walls 104a do not receive the laser light, reflect light, or otherwise adversely affect signal quality from any labeled substances in the sample. The edges of the flow are not interrogated, but the large area of ​​the interrogation region 112 and high flow rate allow for accurate detection. This eliminates any reflections from corners or the capillary walls 104a, reducing light stress or background light noise in the system. For example, the interrogation region 112 can be approximately 100 μm in from the capillary wall 104a, or a gap of at least 10 μm, 20 μm, 30 μm, 50 μm, 75 μm, 150 μm, 200 μm, or more than 200 μm can exist between the nearest wall 104a and the interrogation region.

[0040] In some cases, the thickness of the interrogation region 112 can be reduced to reduce the flow area and thereby the flow rate, however the width of the interrogation region 112, while still wide, is narrower than the width of the flow cell 104.

[0041] As an example, the system reduces the amount of light measured compared to the excitation light. While a laser may emit light at 10-10 photons, the system described herein receives approximately 10-10 photons, a significant reduction. Thus, the blackened surface prevents light reflection, thereby preventing signal degradation.

[0042] 1A also shows an excitation blocking filter 124 downstream from the exit aperture 110, which filters light passing through the exit aperture 110 as described herein. The blocking filter 124 prevents any stray excitation light from reaching the detector down the detection path.

[0043] FIG. 1A also shows that the flow cytometry system 100 uses orthogonal photoactivation of the stain, with the laser light direction at approximately 90 degrees from the collection direction. FIG. 1A shows a laser emitter 120 on one side of the flow cell 104 and a pass-through collector 122 (e.g., coated with a blackened light-absorbing material) on the opposite side of the flow cell 104 from the emitter 120. This arrangement has a laser path at approximately 90 degrees relative to the emission light path through the collection lens 116 and exit aperture 110. This configuration is advantageous in that it eliminates the use of Mie scattering in particle sizing, where the coaxial interrogation laser and fluorescent emission are coaxial. It has been found that an excitation path at approximately 90 degrees from the collection path improves efficiency. A review of orthogonal configurations can be found in U.S. Pat. No. 4,745,285, incorporated herein by specific reference.

[0044] The pass-through collector 122 is configured to absorb and attenuate any laser light (photons) that pass through the flow cell 104. As shown, the pass-through collector 122 is cone-shaped. However, the pass-through collector may be flat or circular, and may be coated with a blackbody material (absorbent), have other shapes with an interior coated with a blackbody material (absorbent), or other excitation light mitigation methods utilized by those skilled in the art.

[0045] FIG. 1B is another horizontal cross-sectional view showing additional regions of the flow cytometry system 100. The flow cell 104, spherical reflector 106, aperture 108, exit aperture 110, first absorber 114, lens 116, laser emitter 120, and laser pass-through collector 122 are shown in FIG. 1A. Additionally, while FIG. 1B shows only two collection channels 130, 132 separated by a beam splitter 136, there can be more collection channels, such as using an additional channel region 134 as shown. However, any number of collection channels can be used. The number of collection channels can be less than, more than, or the same as the number of different stains in the stain combination set. The two collection channels 130, 132 pass different wavelengths of light due to the beam splitter 136. For example, the beam splitter 136 separates green light from red light. The beam splitter can be a filter, as described herein, that selectively separates the green spectrum from the red spectrum. Beam splitter 136 may be at approximately 45 degrees to the light path, with green light reflected into first collection channel 130 and red light traveling to second collection channel 132. First channel detector 130a and second channel detector 132a are shown.

[0046] Laser emitter 120 is shown coupled to laser generator 120a through movable mirror 120b. First collection channel 130 includes a notch filter 137 that reduces the bandwidth, such as to 510-535 nm. Filtered light from notch filter 137 passes through first channel lens 138. First channel absorber 139 (blackbody absorber) is downstream from notch filter 137 and in front of and optionally surrounding first channel lens 138. Second channel 132, in this embodiment, omits the notch filter or similar spectrally constraining filter element, but includes second channel lens 135 and second channel absorber 133 (blackbody absorber).

[0047] FIG. 1C is a vertical cross-sectional view (e.g., orthogonal to FIG. 1B) of the flow cytometry system 100. Here, the sample path 104b is shown with the flow cell 104 stretched along the plane of the page. Here, the shape of the first absorber 114 is expanded to encompass the illumination area 102 from the flow cell 104 to the aperture 108. Here, the first absorber 114 covers the interior wall area of ​​the illumination area, so that off-axis or angled light impinges on the first absorber 114 without reflection. Although the first absorber 114 is shown in cross-section, the light path is clear, and the exit aperture 110 can be coated around the area to be clear without a blackbody material. As shown, the first absorber 114 includes a tapered region 114a, a vertical region 114b, and a horizontal region 114c that forms the exit aperture 110 between a portion of the horizontal region 114c. However, other shapes may be used for the first absorber 114 to cover the inner surface of the illumination area 102 and prevent reflections. Here, the exit hole 110 is approximately 25 mm, but may be 20-30 mm wide, 15-40 mm wide, or 10-50 mm wide.

[0048] 1C further shows a second absorber 140 (blackbody absorber) downstream from the first absorber 114 and the exit aperture 110. The region of the system 100 downstream from the exit aperture 110 is a main channel 142 having sidewalls 144 that are at least partially, and preferably completely, coated with the second absorber 140, which is coated with a light-absorbing material.

[0049] 1D and 1E also show a third absorber 104c that can coat the periphery of the outer wall of the flow cell 104, encapsulating areas of the flow cell where light is emitted, absorbed, and not reflected so that photons are not counted through the flow cell wall at angles above a threshold angle, such as about 43 degrees. FIG. 1D also shows that the flow cell 104 can have absorbers 104c (e.g., third absorbers) in some areas to prevent light from escaping the area. The third absorber 104c can be a black body in the same area as the first absorber 114, can be the same material, and can be a continuous or separate film. The third absorber 104c is positioned in the flow cell holder adjacent to or around the flow cell 104 to block any reflection or refraction from the flow cell wall 104. A third light absorbing body 104c may be part of the device or part of the flow cell 104, so that the area around the interrogation region 112 may be painted black and non-reflective.

[0050] FIG. 1E shows a cross-section of a flow cell 104 having a third absorber 104c on the flow cell wall 104a. The open area of ​​the flow cell wall 104a between the two third absorbers 104c allows light to pass for flow cytometry operations. As shown, light greater than a defined angle is absorbed by the third absorber 104c. The interrogation volume defined by the excitation beam is shown for scale, but the dimension between both regions of the third absorber 104c can be modified to fall approximately flush with the interrogation volume defined by the excitation beam. Thus, the angle of the light beam that is absorbed and prevented from exiting the flow cell can be modified to accommodate more or less light as needed or desired for operation and data analysis. The light angle can be approximately a 43° TIR angle. This provides a preferred emission zone 174 between the two regions of the third absorber 104c. Note that the inner wall of the flow cell may also be coated with a light-absorbing material, or both the inner and outer walls of the flow cell may be coated. TIR may be the angle at which total internal reflection occurs. As shown, the clear area of ​​the flow cell extends above and below the excitation beam to the TIR angle of the flow cell. There may be a masked section of a third light absorber 104c.

[0051] In some embodiments, the length of the flow cell optical window can be shortened to be only the length of the preferred emission zone 174, in which case the flow cell can include some regions of light-absorbing material, or the light-absorbing material can be omitted if the flow cell is not so long that it has undesirable angles of light emission. That is, the flow cell length (e.g., the height of the interrogation volume with shaped light) can be the same as the height of the preferred emission zone 174. The height of the preferred emission zone 174 can be the total height of the interrogation zone plus the distance less than the height 178 of each TIR degree (approximately 43°) above and below the light beam axis 176. In one example, if the width of the flow cell is 3,500 μm, the preferred emission zone 174 can have a height of approximately 6,578 μm. However, these dimensions can be varied as needed (e.g., + / - 1%, 5%, 10%, 25%, 75%, etc.). The preferred emission zone 174 can have any desired height. It should be noted that the light emitting zone 174 can be sized as described herein through the use of the third light absorber 104c or other light blocking member.

[0052] It should further be appreciated that some of the dimensions, such as the diameter of the exit hole 110 relative to the dimensions of the main channel 142, can be varied or changed. The main channel 142 can be a larger dimension than the exit hole 110, such as 50 nm to 25 nm, 25 nm to 20 nm, or even 25 nm to 23 nm. The ratio of the diameter of the main channel 142 to the diameter of the exit hole 110 is variable. The exit hole 110 can act as a pinhole for the main channel 142.

[0053] In some embodiments, the spherical reflector 106 is a hemisphere. In some aspects, the spherical reflector 106 is not a parabolic reflector. Without being bound thereto, it is believed that the spherical reflector 106 as a hemisphere, along with the rectangular flow cell 104, provides the advantage of enhancing the columnar "heart-cut" light through the exit aperture 110 compared to other reflectors, such as elliptical or parabolic reflectors. Other reflector concepts tuned to preferentially reflect desired light over background light are contemplated herein.

[0054] In some embodiments, a flow cytometry system is optimized using a stain combination set. The system is optimized and tuned with analyte stains and counterstains, as well as a configuration of collection optics and signal amplification when the signal reaches the photomultiplier tube (PMT). The optimization provides simple discrimination logic using an algorithm.

[0055] The system can be used for flow cytometry as described herein. The sample can be heated to 37°C, and an analyte stain can be added, along with a buffer at a pH value of about pH 7.5 or higher, as may be necessary for optimal fluorescence from the stain employed. The analyte stain provides bioburden staining but is added at the lowest possible concentration to minimize background fluorescence from unbound stain. Reducing the concentration increases the clarity of the analyzed data signal and aids in signal-to-noise ratio. Lower concentrations result in fewer background photons, helping to reduce signal confounds. A counterstain is also added, and the labeled sample is then analyzed, which can be performed in approximately 20 minutes. These stains can be added manually or automatically from reservoirs shown in Figures 2A and 2B. The reservoirs can be connected to the sample reservoirs through conduits not shown. A controller, such as the computer or computing system 600 described herein, can be used to control flow cytometer system functions, from mixing the sample and stain to pumping through the flow cell using a pump (not shown), controlling the laser, and collecting data from detectors for different signals. The controller can then be configured to analyze the data and distinguish target analytes (e.g., viable cells) from non-target analytes (e.g., non-viable cells) and other particles by counting fluorescent hits that have no counterstain or that are below a certain threshold of counterstain and omitting counts of fluorescent hits that have counterstain above a certain threshold. The controller can control all aspects of operation, from temperature control, temperature measurement, flow control, etc., to data analysis.

[0056] In use, a human or automated operator can place a sample bottle with a sample at the input of the flow cytometer. The controller can control the heat management, timing management, stain introduction, pumping speed, and all fluidics, which can be fully automated. Additionally, the controller can sanitize the flow cell and sample vessels between samples, so that reagents, buffers, or anything else does not carry over between samples.

[0057] 2A and 2B show an example of a full system including a flow cytometer, identified in the figure as an analytical module, showing the sample vial autosampler, thermal sample control, reactors, reservoirs, pumps, valves, sample controller, and other components.

[0058] The slow cytometry system 200 may include two reactors 202, as shown in FIG. 2A. In a first cycle, one reactor 202 is processed with a 20-minute incubation of the sample 224 with stain (e.g., stain 204, counterstain 204a, 50 mL), and then the stained sample 212 is provided to the analysis module 206 (e.g., with a flow cell) for examination, which takes approximately 20 minutes. This provides approximately 40 minutes for the initial stained sample 212 to be processed by the flow cytometer 208. If there is only one reactor 202, there is a 40-minute turnaround for the nuclear stained sample 212. However, now, using a second reactor 202, while one reactor 202 pumps the stained sample 212 to the analysis module 206 (not shown), the second reactor 202 is cleaned, primed, and filled with another sample 224 along with stain 204, counterstain 204a, buffer 204b (1 L), or detergent 204c (1 L). Then, once the first stained sample 212 has been processed through the analysis module 206 (e.g., 20 minutes), the second stained sample 212 can be immediately processed through the analysis module 206. This allows two stained samples 212 to be fully processed in 60 minutes for the first two samples. Note that the second stained sample 212 is processed 20 minutes after the first stained sample 212 has been processed. However, it takes only 40 minutes total for both the second stained sample 212 (20 minutes after the first sample) and the third stained sample (20 minutes), and then 20 minutes after each of the next stained samples 112 in the sequence. However, processing of the stained samples 212 is completed every 20 minutes. This is a significant time savings, especially when dealing with more than two samples 224 or a larger number of samples (e.g., 10 or more).

[0059] Thus, while one stained sample 212 is being processed through the flow cytometer 208, the next sample 224 is in another reactor 202 and is being stained with a stain 204 or counterstain 204a. This allows one analysis module 206 to operate and be fed from two reactors 202, maximizing throughput from the automated system 200.

[0060] The reactor 202 includes a pressure transducer (PT) 216 for monitoring system health and reliable operation, including the absence of sample fluid, leaks, or other functions. Reagents can enter through an upper access line 218, and the sample inlet and outlet is a lower access line 220. A distribution valve 222 controls fluid access to provide sample 224, washing solution (cleaner 204c), sanitizing solution, air 204e, or other fluids to the reactor 202. An outlet 226 from the reactor 202 returns to the distribution valve 222 and is transported to an appropriate location, such as a flow cytometer 208, for staining sample or waste 228, washing solution 204c, sanitizing solution, sample collection 240, etc.

[0061] The reactor 202 includes a movable piston 230 (see, e.g., FIGS. 8A-8C ) as a pump. The reactor 202 is configured to perform incubation and assay processes simultaneously with syringe pump function. Thus, the reactor 202 functions as a pump. The movement of the reactor piston 230 draws in the sample 224 and expels the stained sample 212. A pressure transducer 216 monitors pressure to ensure operation is within parameters and can identify the presence of clogs, missing fluid, or other problems. This allows the piston 230 in the reactor 202 to push the stained sample 212 into the flow cytometer 208. Thus, the reactor 202 has the dual function of a reaction vessel and a pump that draws the sample 224 into the reactor 202 for staining (e.g., stain 204, counterstain 204a) and pushes the stained sample 212 out through the flow cell 104 of the flow cytometer 208.

[0062] In some embodiments, an automated system can be operated to perform the method without using the piston reactor shown herein, in which case any type of reactor can be used that uses any type of pumping and conduit system to move fluids between the reservoirs, reactors, and flow cytometer. Thus, one or both reactors shown in FIG. 2A can be replaced with a different type of reactor (e.g., a different reactor system), and any number or type of pumps, valves, or distributors can be used to move fluids, reagents, cleaners, samples, and stains through the system. In some aspects, one or more of the reactors are fixed-volume reactors. In some aspects, one or more reactors are variable-volume reactors. In some aspects, one or more pumps, such as vacuum pumps or fluid pumps, can be used to move fluids to and from the reagent reservoirs, reactors, and flow cytometer, and any number of valves can be used to regulate the flow. In some aspects, a vacuum pump can be used to fill the reactor with sample and / or stain, and then a fluid pump is used to flow the stained sample to the flow cytometer. The vacuum pump and fluid pump can be located anywhere to facilitate the movement of the liquid.

[0063] In one example, the reactor may include a solid cap to provide a constant volume reaction chamber, and a vacuum pump may be connected to the piston / cap to facilitate filling the reaction chamber with reagents, samples, or cleaners. A fluidic gear pump downstream of the flow cytometer 208 may also be used to draw the stained sample through the fluidic pathway into the flow cytometer 208. However, it should be recognized that other modifications to the system, such as the type of equipment or its placement, may be employed under the teachings of the reactor system to arrive at the present invention.

[0064] The reactor 202 also includes a drive (not shown) for the piston and a drive for asymmetric mixing (not shown) that vibrates the reactor 202 to effect mixing of the reagents within the chamber of the reaction 202. A pulley drive for the piston 230 can be used, and other drives can be asymmetric (cam-type) relative to gravity to vibrate the reactor 202 for mixing.

[0065] The staining dye 204 (counterstain 204) can be provided under the control of a metering system, such as a syringe pump (SYR) 232 and a dispensing valve 222. Thus, the analyte stain 204, counterstain 204a, cleaner 204c, washing fluid (WFCC) 204d, and buffer 204b can be provided to the reactor 202 from the upper line 218 through the piston 230 in a top-down injection manner, as shown, thereby minimizing sample contact with the dispensed fluids.

[0066] FIG. 1A also shows an assay system 250 having an automated sample bottle distributor 252 configured to take a sample 224, which is passed to an inlet 254 of the system 200, can pass through a thermal sample control mechanism 256, and then passes through a distribution valve 220 and is transported to one of the reactors 202 for processing with a stain 204 and / or a counterstain 204a as described herein.

[0067] 2B shows an embodiment of the system 200a that omits the automatic sample bottle distributor 252 and includes only one reactor 202. This embodiment is an online version that sequentially samples from the fluid supply. Each run of the stained sample 212 has a run time of approximately 40 minutes.

[0068] In one embodiment, the system may include a flow cytometer, shown as an analytical module, and a pump, whether manual or driven, that can then receive the stained sample and pump it to a flow cell for analytical interrogation.

[0069] The system may include a barcode system to identify sample bottles, thereby allowing chain of custody of each sample. Used sample bottles are ejected into a waste container (not shown).

[0070] While a system with an automatic sample bottle dispenser 252 can operate automatically and unattended as sequential bottles are sampled, a STAT sampling access door in the autosampler allows a user to process certain samples earlier by lifting the door and manually loading a sample bottle before others in the system.

[0071] The reactor 200 (e.g., RXTR) is configured for improved performance. The thermal sample control mechanism 256 is an implementation of a shell and tube heat exchanger that regulates the temperature of the sample 224, whether heated or cooled, from 5°C to 95°C at 50 mL / min to a correct 37°C at the outlet 226. Lines to the reactor 202 indicate that the sample 224 and stains (204, 204a) are at an operating temperature of 37°C (e.g., physiological temperature). Line 220 indicates the path from the reactor 202 to the analysis module 206.

[0072] System 202a can fill sample 224 at up to 50 mL / min, can quickly fill reactor 200 to 100 mL, and does not require thermal equilibration, allowing reagents to be added and reactions to begin immediately. Thermal sample control system 256, beneficially under the control of a controller (e.g., 600), provides sample to reactor 202 at the appropriate temperature required for the reagents and intended sample particles.

[0073] Lines 270 to and from the reactor indicate the cleaning flow from cleaning fluid 204c to piston 230 within reactor 200. Piston 230 is configured with a dual seal 234 design, allowing fluids such as cleaning fluid 204c to be channeled into ports 236 between seals 234 of piston 230, as shown in FIGS. 8A-8C, to clean and lubricate the seals 234, as shown in FIG. 8B. Port 236 between the two seals 234 provides cleaning fluid 204c, lubricant, or other liquid 240b, which passes between the two seals 234. Cleaning fluid 204c or staining fluid can be passed to prevent contamination and buildup on the seals 234. It also cleans any particulates that may form from the seals 234. Port 236 can be an inlet or an outlet, with another port on the opposite side opposite the first port. One port 236 is an inlet and one is an outlet. The piston head 230a includes fluid conduits 238 for providing reagent fluids (eg, stain 204, counterstain 204a) to the sample 222 within the reactor chamber.

[0074] The ability to load the reactor 202 with a sample 224 that is already at reaction temperature and will immediately react with the dye in the stain 204, 204a, which is also already at reaction temperature, saves time during the protocol. Preconditioning the temperature of the sample can shave up to 15-20 minutes off the entire protocol from start to finish.

[0075] The reactor can include a body with a highly polished surface finish across the entire fluid contact area. Thus, the ports provide lubricant to the seals to enhance their longevity. The lubricant can also clear contaminants from the reactor.

[0076] In some embodiments, the flow cytometer (100) may include one or more light emitters (120) configured to emit light into an optical path. The flow cytometer (100) may include a rectangular flow cell (104) having a flow cell width (W1) generally laterally relative to the optical path and a flow cell depth generally longitudinally relative to the optical path. The optical path has an interrogation width (W2) at the flow cell (104) that is narrower than the flow cell width (W1). The spherical reflector (106) is positioned adjacent to the rectangular flow cell (104) and has a concave reflective surface with a reflection direction positioned generally perpendicular to the optical path such that reflected light is reflected along a reflection path generally perpendicular to the optical path. The illumination area 102 includes one or more light-absorbing members (114) positioned at least partially around the reflection path to absorb reflected light at an angle relative to the reflection path. Each light absorbing member (114) includes one or more of a tapered light absorbing surface (114a) extending away from the flow cell (104), a parallel light absorbing surface (114b) that is approximately parallel to the reflection path, an orthogonal light absorbing surface (114c) that is approximately perpendicular to the reflection path, or a flow cell absorbing surface (104c) on the surface of a wall of the flow cell. Optionally, each light absorbing member (114) forms a light conduit with a light absorbing inner wall.

[0077] The flow cytometer 100 can include an aperture 108 defining an exit aperture 110 positioned such that the reflected path passes through the exit aperture 110. The exit aperture 110 can have a cross-sectional dimension that is smaller than the housing (e.g., the aperture, etc.) in which the exit aperture defines an opening. In some embodiments, the exit aperture 110 has a dimension less than 50 mm, less than 25 mm, or less than 15 mm.

[0078] The flow cytometer (100) may include one or more light-absorbing members (114) positioned between the flow cell (104) and the exit aperture (110) to define an optical conduit therebetween. In some embodiments, the one or more light-absorbing members (114) at least partially define the exit aperture (110).

[0079] In some embodiments, the main conduit (142) is downstream from the injection hole (110), where downstream refers to the reflected light path through the injection hole and into the main conduit. The main conduit (142) may include one or more light absorbing members (114) that cover at least a portion of the inner surface of the main conduit (142).

[0080] In some embodiments, the beam splitter (136) is disposed in the main conduit (142). The light absorbing member (14) is positioned between the exit aperture (110) and the beam splitter (136) to define a light conduit therebetween.

[0081] The flow cytometer (100) may include a first collection channel (130) positioned to receive light reflected from a beam splitter (136) and a second collection channel (132) positioned to receive light passing through the beam splitter (136). At least one of the first collection channel (130) or the second collection channel (132) includes a notch filter (137). In some embodiments, at least one of the first collection channel (130) and the second collection channel (132) includes one or more light-absorbing members (133 / 139).

[0082] The flow cytometer 100 can include a collection lens 116 adjacent to the flow cell 104 such that the flow cell is between the spherical reflector 106 and the collection lens. The spherical reflector 106 can be a rear reflector, and the flow cytometer 100 does not have a front reflector, in which case the absorber 114 replaces any front reflector.

[0083] In some embodiments, the flow cytometer (100) may include or be included in a system (200, 200a) that includes various components. The components may include a sample reservoir or sample collection system (250) that provides a sample (224) for staining (204, 204a). The components may include an analyte stain reservoir (204) containing an analyte stain composition, the analyte stain composition including a dye configured to detect a target analyte. The components may include a counterstain reservoir (204a) containing a counterstain composition, the counterstain composition including a dye configured to detect a non-target analyte.

[0084] The flow cytometer (100 / 208) or system (200, 200a) can include one or more pumps (110) operably coupled to the sample reservoirs, analyte stain reservoirs (204) and counterstain reservoirs (204a), and flow cells (104) in the analysis module 206 of the flow cytometer (100 / 208). (For example, the line 220 supplying the sample 224 can be a sample reservoir, and the sample in the automated sample bottle dispenser 252 can also be a reservoir.) Each pump (110) is configured for a flow rate of at least 1 mL / min, 5 mL / min, 20 mL / min, 50 mL / min, or 100 mL / min.

[0085] In some embodiments, the flow cytometer (100 / 208) lacks one or more of the following: a sheath flow reservoir and / or a sheath flow pump or a particle sizing system so that no sheath flow occurs in the flow cell.

[0086] In some embodiments, a kit may include the flow cytometer (100 / 208) of one of the embodiments. The kit may also include an analyte stain (204) having a first fluorescent wavelength. The kit may also include a counterstain (204a) having a second fluorescent wavelength.

[0087] In some embodiments, a method for detecting a target analyte in a sample is provided, and the method may be performed under the operation of a controller (e.g., computing device 600, computing system, etc.). The target analyte detection method may be performed in an embodiment of a flow cytometer (100 / 208). A sample having an analyte stain and a counterstain may be provided to the flow cytometer (100 / 208). The analyte stain emission wavelength is distinguishable from the counterstain emission wavelength, which may be detected and processed by the controller. The controller may analyze the sample using optical interrogation of the stained sample by the flow cytometer (100 / 208). The controller may analyze sample data of the stained sample from the flow cytometer (100 / 208) to detect potential targets having the analyte stain. The controller may also be a data analysis computer, which may be used to identify potential targets as those without a counterstain or those with a counterstain below a counterstain threshold. The controller can then be used to identify detected potential targets that have an analyte stain without a counterstain or that have a counterstain below the counterstain threshold as target analytes. However, the controller and data analysis computer can be separate computing devices (600). The controller can be in operative communication with any component of the system to control the operation of that component, receive data from, and optionally modify the operation of any component.

[0088] In some embodiments, the flow cytometer has a detection limit of about 1 analyte per 100 mL, and the detection limit may range from 1 analyte per 50 mL to 150 mL, 75 mL to 125 mL, or 90 mL to 120 mL. Absorbance characteristics within the flow cytometer can be used to obtain detection limits outside of this range. The method may include passing the sample through the flow cell at a flow rate of at least 1 mL / min, 5 mL / min, 20 mL / min, 50 mL / min, or 100 mL / min, which may also contribute to a lower detection limit.

[0089] In some embodiments, the analyte stain is specific for staining nucleic acids and the counterstain is capable of penetrating non-viable cells. In some aspects, the sample is selected from a food sample, a beverage sample, an environmental sample, or a purified water sample. In some aspects, the method includes blocking a wavelength range of light emission from the analyte stain or the counterstain using a filter, splitter, or the like.

[0090] In some embodiments, the method includes defining a counterstain percentage threshold. The method may then include determining whether the counterstain emission is below the counterstain percentage threshold. If the counterstain emission is below the counterstain percentage threshold, the analyte is determined to be present or the cell is determined to be viable. If the counterstain emission is above the counterstain percentage threshold, the analyte is determined to be absent or the cell is determined to be non-viable.

[0091] In some embodiments, the method may include defining a threshold analyte stain to counterstain ratio. The method may then include determining whether the analyte stain emission and counterstain emission have an analyte stain to counterstain ratio that exceeds the analyte stain to counterstain ratio threshold. If the ratio exceeds the analyte stain to counterstain ratio threshold, the analyte is determined to be present or the cell is determined to be viable. If the ratio is below the analyte stain to counterstain ratio threshold, the analyte is determined to be absent or the cell is determined to be non-viable.

[0092] In some embodiments, the method may include defining a threshold counterstain to analyte stain ratio. The method may then include determining whether the counterstain emission and the analyte stain emission have a counterstain to analyte stain ratio below the threshold counterstain to analyte stain ratio. If the ratio is below the threshold counterstain to analyte stain ratio, the analyte is determined to be present or the cell is determined to be viable. If the ratio is above the threshold counterstain to analyte stain ratio, the analyte is determined to be absent or the cell is determined to be non-viable.

[0093] In some embodiments, the method can include blocking internal reflections of light with one or more light absorbing members, which can be done using any one or more or all of the light absorbing members in the flow cytometer.

[0094] In some embodiments, the method may include emitting one or more interrogation light beams into a rectangular flow cell using one or more light emitters such that the optical path of the interrogation light beam has an interrogation width at the flow cell that is narrower than the flow cell width. Redirected light from the back wall of the flow cell travels toward a spherical reflector and is reflected back through the flow cell and from the front wall of the flow cell on a reflective path.

[0095] In some embodiments, the method may include absorbing light between the flow cell front wall and the exit aperture at an angle relative to the reflected path with one or more light absorbing members. In some aspects, reflected light at an angle relative to the reflected path is absorbed with one or more light absorbing members. In some aspects, light is not reflected into the exit aperture or along the reflected path upon contact with one or more light absorbing members. In some aspects, light does not pass through a light absorbing member on the flow cell wall. In some aspects, the light conduit uses one or more light absorbing members to absorb light at an angle relative to the reflected path.

[0096] In some embodiments, the exit hole, through which the light path extends to allow light to exit the illumination region, is an optical aperture defined by a stop. Thus, the exit hole can be from the housing defining an opening. Light on the reflected path enters the main conduit through the exit hole. In some aspects, light between the exit hole and a beam splitter in the main conduit at an angle to the reflected path is absorbed by one or more light absorbing members. Light reflected in the main conduit at an angle to the reflected path is absorbed by one or more light absorbing members in the main conduit. When light comes into contact with one or more light absorbing members in the main conduit, it is not reflected back into the beam splitter or along the reflected path. A light pipe in the main conduit absorbs light at an angle to the reflected path using one or more light absorbing members in the main conduit.

[0097] In some embodiments, one or more beam splitters can be used to split the reflected light into two or more collection channels. Light reflected from the beam splitter into a first collection channel at an angle relative to the beam splitter reflection path is absorbed by one or more light-absorbing members in the first collection channel. Light reflected in the first collection channel at an angle relative to the beam splitter reflection path is absorbed by one or more light-absorbing members in the first collection channel. When light comes into contact with one or more light-absorbing members in the first collection channel, it is not reflected into an optical element in the first collection channel or along the beam splitter reflection path. An optical conduit in the first collection channel uses one or more light-absorbing members in the first collection channel to absorb light at an angle relative to the beam splitter reflection path. Light passed through the beam splitter and directed along the reflection path into a second collection channel at an angle relative to the reflection path is absorbed by one or more light-absorbing members in the second collection channel. Light reflected in the second collection channel at an angle relative to the reflected path is absorbed by one or more light absorbing members in the second collection channel. When light comes into contact with the one or more light absorbing members in the second collection channel, it is not reflected back into the optical elements in the second collection channel or along the reflected path. The optical conduit in the second collection channel uses one or more light absorbing members in the second collection channel to absorb light at an angle relative to the reflected path.

[0098] In some embodiments, the collecting lens directs the light along a reflected path. In some embodiments, the method may include obtaining a sample from a sample vessel, sample bottle, sample input, or other sample source. The analyte stain may be obtained from an analyte stain vessel containing an analyte stain composition, the analyte stain composition including a dye. The counterstain may be obtained from a counterstain vessel containing a counterstain composition, the counterstain composition including a dye. The sample may be stained with the analyte stain and / or counterstain in the reactor. The stained sample may be provided to a flow cell for optical interrogation. The stained sample may flow through the flow cell at a flow rate of at least 1 mL / min, 5 mL / min, 20 mL / min, 50 mL / min, or 100 mL / min.

[0099] In some embodiments, a data analysis computer (e.g., a controller) can be configured to analyze data from a flow cytometer to identify the presence of target analytes. The data analysis computer can be configured to perform matched filter analysis on analyte stain data peaks to identify peaks that are deemed to be target analytes. Matched filter analysis includes applying a matched filter through the raw data to isolate shape-matching information, applying threshold logic to the identified "peaks," identifying peaks if they exceed a detection threshold of a specified minimum / maximum number of records, obtaining peak raw signal intensities for both channels (analyte stain and non-stain), obtaining the number of records above the threshold, obtaining peak intensities for both channels, applying discrimination logic to at least two channels, determining whether the analyte stain channel is within a minimum / maximum intensity range (Ch1 FLF1), determining whether the counterstain channel is within a minimum / maximum intensity range (Ch2 FLF1), and determining the ratio of the analyte stain channel intensity to the counterstain channel intensity (Ch2 FLF2).

[0100] In some embodiments, potential target analytes are then selected based on matched filtering, e.g., the peak must be within a detection threshold range, the analyte stain channel is within a range, the counterstain channel is within a range, and / or the ratio of the analyte stain channel intensity to the counterstain channel is within a range.

[0101] In some embodiments, a flow cytometer or a system for providing stained samples can be configured as described herein. The system can have two reactors for staining two separate samples, and both reactors provide input to a single flow cytometer. The system is configured such that a first sample is heated and stained in the first reactor, the first sample exits the first reactor and passes through the flow cytometer, a second reactor is staining a pre-conditioned second sample while the first sample is passing through the flow cytometer, after the first sample has completely passed through the flow cytometer, the second sample exits the second reactor and passes through the flow cytometer, and the first reactor is staining a pre-conditioned third sample while the second sample is passing through the flow cytometer, and the method continues by alternating between the first and second reactors for the staining and pumping steps to the flow cytometer.

[0102] The methods described herein performed in a flow cytometer or a system providing a stained sample can be under the control of a controller. The controller can include non-transitory memory having computer-executable instructions for performing the method steps described herein. The data analysis methods can be performed on a data analysis computer, which can be the same or a different computing system from the controller. [Example]

[0103] An experiment can involve the analysis of a single 100 mL sample with a target maximum of five bacterial cells. The 100 mL analysis is performed over a 20-minute run, and data collection includes 6.25 million records, each with either a noise band or a signal. In one example, particles were detected by a flow cytometer, with a length of 3 records and noise.

[0104] The data can be picked up as raw data, and analysis is used to find and verify signals that are viable cells and determine which signals to ignore. The protocol runs a matched filter on the data to obtain identification of data peaks that match known viable particle data peaks. The matched filter can be a shape analysis that identifies corresponding data peak shapes with known data peak shapes of identifiable bacteria, other microorganisms, or other target analytes (e.g., labeled viruses, active ingredients, abiotic contaminants, etc.). The analyte stain data signal is used in the matched filter. Once the analyte data signal peak signal is identified as a possible viable cell, further analysis can be performed. Machine learning can be used to learn analyte peaks versus non-target peaks, and then used to select analyte peaks from the data.

[0105] The protocol may include a shape-finding operation using a matched filter, where the match has different criteria for the size of each recorded hit, the minimum number of hits, or the shape of each recorded hit. The data may be analyzed to have a minimum amplitude above the baseline, so if a peak is too small, the system will determine that it is not a viable cell. The analysis may have a maximum amplitude that can be used to disqualify peaks that are too large. If a peak is too large, it may indicate that the peak is something that has come off the gasket, too large to be the target bioburden, and in that case, it will be disqualified as a viable cell. If the emitted fluorescent signal saturates the PMT, it may also indicate that the PMT has been blinded for 1 microsecond, thereby disqualifying the peak from being a viable cell. These parameters are used to determine which peaks qualify for further analysis.

[0106] In one example, the system found 43 viable hits (signals) and 397 other signals that were too short, had insufficient intensity and / or area, or were otherwise unqualified to be viable cells. There were a total of 440 signals determined to be particle events in the cell stain channel, but only 43 were valid viable particles. Comparison of each cell stain peak with the corresponding counterstain peak provides an indication of whether the signal is a viable cell, such as when the percentage of the total peak in the counterstain signal is below a threshold.

[0107] In one example, the signal peak height may be 150 recording units and the discriminator set to 100 units to qualify the peak as a potential viable cell. The signal peak width may have a minimum threshold used to identify peaks that are not viable cells, and the corresponding peaks may be disqualified accordingly.

[0108] The selected 43 valid hits can then be analyzed to determine which hits are viable or non-viable cells (e.g., biotic or non-biotic). The FLF1 channel can be for size discrimination. The FLF2 channel is a ratio of that spectral signal channel to the other channel, e.g., channel 1 is the reference channel and channel 2 is the counterstain channel. The coefficients can be weighted by different factors, such as equally weighted. However, the weighting can be 200% for the counterstain channel (red) versus the analyte stain channel (green). The FLF1 and FLF2 channel parameters can be varied as needed.

[0109] The signal baseline can be dynamic, so that the baseline can track data as it moves in response to dye runs and interactions with the system. Thus, the use of a dynamic baseline can aid in optimal data analysis sensitivity. A dynamic baseline can be obtained by selecting a distribution of the baseline over a period of time and a statistically significant portion of the distribution as the baseline for any particular analysis or comparison.

[0110] As shown in Figure 3, the green channel can be used for the analyte stain and the red channel can be used for the counterstain. In applications using analyte stain dyes, the analysis looks for live cells, but the system can also use two or more analyte stains in two or more channels. The system can also be employed to look for only red cells (e.g., counterstain). Additionally, the system can use a combination of signals and sum them together. The data in Figure 3 shows a bacteria sample run using 100 mL with 15 organisms; the top data points are for the green stain and the bottom data points are for the red stain (e.g., counterstain).

[0111] Figure 4 shows an example of a signal that is biological, as indicated by having a target analyte stain signal and peaks within size and amplitude parameters within tolerances determined to be suitable for the target analyte and counterstain size and spectral profile. The peak intensity is less than the maximum tolerance determined to be suitable in correlation with the target analyte stain size and peak intensity determined to be suitable for the spectral profile for distinguishing viable target analytes (e.g., viable cells). Figure 4 shows a biological with 2% red color.

[0112] Figure 5 shows an example of a signal that is not a live target analyte (e.g., a non-living particle) due to the counterstain peak (e.g., red) exceeding a maximum allowable difference determined to be appropriate relative to the target analyte stain size and peak intensity determined to be a suitable spectral profile for distinguishing live target analytes (e.g., live cells). Figure 5 shows that non-living matter was distinguished due to the intensity of the red channel and the ratio being 117% of live bacteria.

[0113] Figure 6 shows a graph of an example matched filter. The matched filter method can be implemented as follows: A matched filter is applied through the raw data to isolate shape matching information.

[0114] A threshold logic is applied to the identified "peaks." A peak is identified if it exceeds a detection threshold for a specified minimum / maximum number of records.

[0115] The peak raw signal intensities of both channels (analyte stain and non-stain) are acquired. Get the number of records above the threshold. The peak intensities of both channels are obtained.

[0116] Apply discriminant logic (two channel example). It is determined whether the biological channel is within the minimum / maximum intensity range (Ch1 FLF1).

[0117] It is determined whether the abiotic channel is within the minimum / maximum intensity range (Ch2 FLF1). The ratio of biotic to abiotic channel intensity is determined (Ch2 FLF2).

[0118] Potential surviving particles are then selected based on matched filtering, where the peaks in the potential surviving particles are must be within the detection threshold range, The biological channel is within the scope of Abiotic channels are within the scope, and / or The ratio of biotic channel intensity to abiotic channel intensity is within a range.

[0119] In some embodiments, the data and analytical parameters described herein and the resulting determination of whether a target analyte is a viable cell can be used as input data to train an artificial intelligence system. The data can be input and processed through an encoder or other neural network to generate potential data, which can then be decoded into output data. The output data can be a generated predicted determination of whether a particle is a viable cell. The trained artificial intelligence system can then be used to identify which data identify viable cells and which data do not indicate viable cells. The trained artificial intelligence system can then perform data analysis and determine which particles are viable particles based on the results from the training data and the actual data. Thus, methods for distinguishing viable cells from other particles can be implemented as described herein and applied to artificial intelligence systems.

[0120] Those skilled in the art will understand that, with respect to the processes and methods disclosed herein, the functions performed in the processes and methods may be performed in different orders. Furthermore, the outlined steps and operations are provided merely as examples, and some of the steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without departing from the essence of the disclosed embodiments.

[0121] In one embodiment, the method may include aspects implemented in a computing system. As such, the computing system may include a memory device having computer-executable instructions for implementing the method. The computer-executable instructions may be part of a computer program product including one or more algorithms for implementing any of the methods of any of the claims.

[0122] In one embodiment, any of the operations, processes, or methods described herein may be performed or caused to be performed in response to the execution of computer-readable instructions stored on a computer-readable medium and executable by one or more processors. The computer-readable instructions may be executed by processors in a wide range of computing systems, from desktop computing systems to portable computing systems, tablet computing systems, handheld computing systems, and network elements and / or any other computing device. The computer-readable medium is non-transitory. The computer-readable medium is a physical medium having computer-readable instructions stored thereon such that the computer-readable medium is physically readable from the physical medium by a computer / processor.

[0123] There are various means by which the processes and / or systems and / or other techniques described herein may be implemented (e.g., hardware, software, and / or firmware), and the preferred means may vary with the context in which the processes and / or systems and / or other techniques are deployed. For example, if the implementer determines that speed and accuracy are paramount, the implementer may select a primarily hardware and / or firmware implementation; if flexibility is paramount, the implementer may select a primarily software implementation; or again, alternatively, the implementer may select some combination of hardware, software, and / or firmware.

[0124] The various operations described herein may, individually and / or collectively, be implemented virtually by a wide range of hardware, software, firmware, or any combination thereof. In one embodiment, some portions of the subject matter described herein may be implemented via an application specific integrated circuit (ASIC), field programmable gate array (FPGA), digital signal processor (DSP), or other integrated format. However, some aspects of the embodiments disclosed herein may equally be implemented in whole or in part in an integrated circuit as one or more computer programs running on one or more computers (e.g., as one or more programs running on one or more computer systems), as one or more programs running on one or more processors (e.g., as one or more programs running on one or more microprocessors), as firmware, or as virtually any combination thereof; it is possible in light of this disclosure to design circuitry and / or write software and / or firmware code. Additionally, the mechanisms of the subject matter described herein can be distributed as program products in a variety of forms, and exemplary embodiments of the subject matter described herein apply regardless of the particular type of signal-bearing medium used to actually accomplish the distribution. Examples of physical signal-bearing media include, but are not limited to, recordable types of media such as floppy disks, hard disk drives (HDDs), compact discs (CDs), digital versatile discs (DVDs), digital tape, computer memory, or any other physical medium that is not transitory or transmission-oriented.Examples of physical media carrying computer-readable instructions exclude transitory or transmission-type media such as digital and / or analog communications media (e.g., fiber optic cables, wired communications links, wireless communications links, etc.).

[0125] Describing devices and / or processes as described herein is general, and thus integrating such described devices and / or processes into a data processing system employs engineering practice. That is, at least a portion of the devices and / or processes described herein can be integrated into a data processing system through a reasonable amount of experience. A typical data processing system generally includes one or more of: a system unit housing; a video display device; memory, such as non-volatile and volatile memory; a processor, such as a microprocessor and a digital signal processor; an operating system; drives; computing entities, such as a graphical user interface and application programs; one or more interaction devices, such as a touchpad or screen; and / or a control system, including feedback loops and control motors (e.g., feedback for sensing position and / or velocity; control motors for moving and / or adjusting components and / or quantities). A typical data processing system can be implemented utilizing any suitable commercially available components, such as those commonly found in data computing / communication and / or network computing / communication systems.

[0126] The subject matter described herein may depict different components contained within or connected to different other components. Such illustrated architectures are merely exemplary, and in fact, many other architectures that achieve the same functionality may be implemented. In a conceptual sense, components of any configuration that achieve the same functionality are effectively "associated" such that a desired functionality is achieved. Thus, as used herein, any two components that are coupled to achieve a particular functionality, regardless of architecture or intermediate components, may be viewed as being "associated" with one another such that the desired functionality is achieved. Similarly, any two components so associated may be viewed as being "operably connected" or "operably coupled" to one another to achieve the desired functionality, and any two components so associateable may also be viewed as being "operably couplable" with one another to achieve the desired functionality. Examples of operably couplable include, but are not limited to, physically matable and / or physically interacting components, wirelessly interacting and / or wirelessly interacting components, and / or logically interacting and / or logically interacting components.

[0127] 7 illustrates an example computing device 600 (e.g., a computer) that may be configured to perform the methods (or portions thereof) described herein in some embodiments. In a very basic configuration 602, the computing device 600 generally includes one or more processors 604 and a system memory 606. A memory bus 608 may be used for communication between the processors 604 and the system memory 606.

[0128] Depending on the desired configuration, the processor 604 may be of any type, including, but not limited to, a microprocessor (μP), a microcontroller (μC), a digital signal processor (DSP), or any combination thereof. The processor 604 may include one or more levels of caching, such as a level 1 cache 610 and a level 2 cache 612, a processor core 614, and registers 616. An example processor core 614 may include an arithmetic logic unit (ALU), a floating point unit (FPU), a digital signal processing core (DSP core), or any combination thereof. An example memory controller 618 may be coupled with the processor 604, or in some implementations, the memory controller 618 may be an internal part of the processor 604.

[0129] Depending on the desired configuration, the system memory 606 may be of any type, including but not limited to, volatile memory (such as RAM), non-volatile memory (such as ROM, flash memory, etc.), or any combination thereof. The system memory 606 may include an operating system 620, one or more applications 622, and program data 624. The applications 622 may include a decision application 626 configured to perform operations as described herein, including those described with respect to the methods described herein. The decision application 626 may obtain data such as pressure, flow rate, and / or temperature and then determine changes to the system to alter the pressure, flow rate, and / or temperature.

[0130] The computing device 600 may have additional features or functionality and additional interfaces to facilitate communications between the basic configuration 602 and any required devices and interfaces. For example, a bus / interface controller 630 may be used to facilitate communications between the basic configuration 602 and one or more data storage devices 632 via a storage interface bus 634. The data storage device 632 may be a removable storage device 636, a non-removable storage device 638, or a combination thereof. Examples of removable and non-removable storage devices include magnetic disk devices such as floppy disk drives and hard disk drives (HDDs), optical disk drives such as compact disk (CD) drives or digital versatile disk (DVD) drives, solid state drives (SSDs), and tape drives, to name a few. Examples of computer storage media may include volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules, or other data.

[0131] System memory 606, removable storage 636, and non-removable storage 638 are examples of computer storage media including, but not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired information and that can be accessed by computing device 600. Any such computer storage media may be part of computing device 600.

[0132] The computing device 600 may also include an interface bus 640 that facilitates communication from various interface devices (e.g., output devices 642, peripheral interface 644, and communication devices 646) to the basic configuration 602 via a bus / interface controller 630. Examples of output devices 642 include a graphics processing unit 648 and an audio processing unit 650, which may be configured to communicate with various external devices such as a display or speakers via one or more A / V ports 652. Examples of peripheral interfaces 644 include a serial interface controller 654 or a parallel interface controller 656, which may be configured to communicate with external devices such as input devices (e.g., keyboard, mouse, pen, voice input device, touch input device, etc.) or other peripherals (e.g., printer, scanner, etc.) via one or more I / O ports 658. An example of a communication device 646 is a network controller 660, which may be configured to facilitate communication with one or more other computing devices 662 over a network communication link via one or more communication ports 664.

[0133] A network communication link may be an example of a communication medium. Communication media may typically be embodied by computer-readable instructions, data structures, program modules, or other data in a modulated data signal, such as a carrier wave or other transport mechanism, and may include any information transmission media. A "modulated data signal" may be a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media may include wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, radio frequency (RF), microwave, infrared (IR) and other wireless media. As used herein, the term computer-readable media may include both storage media and communication media.

[0134] Computing device 600 may be implemented as part of a small form factor portable (or mobile) electronic device such as a mobile phone, a personal data assistant (PDA), a personal media player device, a wireless web watch device, a personal headset device, an application-specific device, or a hybrid device including any of the above functionality. Computing device 600 may be implemented as a personal computer, including both laptop and non-laptop computer configurations. Computing device 600 may also be any type of network computing device. Computing device 600 may also be an automated system as described herein.

[0135] The embodiments described herein may involve the use of special purpose or general purpose computers containing various computer hardware or software modules. Embodiments within the scope of the present invention also include computer-readable media for carrying or having computer-executable instructions or data structures stored thereon. Such computer-readable media may be any available media that can be accessed by a general-purpose or special-purpose computer. By way of example, and not limitation, such computer-readable media may include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to carry or store desired program code means in the form of computer-executable instructions or data structures and that can be accessed by a general-purpose or special-purpose computer. When information is transferred or provided over a network or another communications connection (either hardwired, wireless, or a combination of hardwired or wireless) to a computer, the computer properly views the connection as a computer-readable medium. Thus, any such connection is properly termed a computer-readable medium. Combinations of the above should also be included within the scope of computer-readable media.

[0136] Computer-executable instructions comprise, for example, instructions and data that cause a general-purpose computer, special-purpose computer, or special-purpose processing device to perform a particular function or group of functions. Although the subject matter has been described in language specific to structural features and / or methodological acts, it should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims.

[0137] In some embodiments, a computer program product may include a non-transitory tangible memory device having computer-executable instructions that, when executed by a processor, cause the computer program product to perform a method, the method may include providing a dataset having object data for an object and state data for a state, processing the object data of the dataset to obtain latent object data and latent object-state data with an object encoder, processing the state data of the dataset to obtain latent state data and latent state-object data with a state encoder, processing the latent object data and latent object-state data with an object decoder to obtain generated object data, processing the latent state data and the latent state-object data with a state decoder to obtain generated state data, comparing the latent object-state data to the latent state data to identify differences, processing the latent object data and the latent object-state data or the latent state-object data with a discriminator to obtain a discriminator value, selecting a selected object from the generated object data based on differences between the generated object data, the generated state data, and the latent object-state data and the latent state-object data, and the latent state-object data, and The non-transitory tangible memory device may also have other executable instructions for any of the methods or method steps described herein. The instructions may also be instructions for performing non-computational tasks, such as synthesis of a molecule and / or an experimental protocol for validating a molecule. Other executable instructions may be provided.

[0138] The present disclosure should not be limited to the specific embodiments described herein, which are intended as illustrations of various aspects. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from the spirit and scope of the present disclosure. Functionally equivalent methods and apparatuses within the scope of the present disclosure, in addition to those recited herein, will become apparent to those skilled in the art from the above description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure should be limited only by the terms of the appended claims, along with the full range of equivalents to which such claims are entitled. It is to be understood that the present disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0139] With respect to the use of nearly any plural and / or singular term herein, those skilled in the art can translate from plural to singular and / or from singular to plural as appropriate to the situation and / or application. For clarity, various singular / plural permutations may also be explicitly set forth herein.

[0140] In general, those skilled in the art will understand that the terms used in this specification, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "comprising" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including, but not limited to," etc.). Furthermore, where a specific number is intended by the introductory claim recitation, such intention will be clearly recited in the claim; in the absence of such recitation, those skilled in the art will understand that no such intention exists. For example, as an aid to understanding, the following appended claims may include the introductory phrases "one or more" and "one or more" to introduce claim recitation. However, the use of such phrases should not be construed as suggesting that when a claim recitation is introduced by the indefinite article "a" or "an," any particular claim including such introduced claim recitation is limited to claims containing only one such recitation, even if the same claim contains both an introductory phrase such as "one or more" or "one or more" and the indefinite article "a" or "an" (e.g., "a" and / or "an" should generally be interpreted to mean "one or more" or "one or more"). The same applies when a definite article is used to introduce claim recitation. In addition, those skilled in the art will recognize that even when a specific number is explicitly stated in an introduced claim recitation, such a statement should generally be interpreted to mean at least the recited number (e.g., a statement simply stating "two recitations" without any other modifiers generally means at least two recitations or more than two recitations).Furthermore, when notation similar to "one or more of A, B, and C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the notation (e.g., "a system having one or more of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). When notation similar to "one or more of A, B, or C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the notation (e.g., "a system having one or more of A, B, or C" includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). Moreover, those skilled in the art will understand that generally, any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either of those terms, or both of those terms. For example, the phrase "A or B" will generally be understood to include the possibilities of "A" or "B" or "A and B."

[0141] Additionally, when features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0142] As will be understood by those of skill in the art, for all purposes, including as provided in a written description, all ranges disclosed herein encompass all possible subranges and combinations thereof. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be divided into at least equal 2, 3, 4, 5, 10, etc. divisions. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, upper third, etc. As will also be understood by those of skill in the art, all terms such as "up to," "at least," etc., refer to ranges that are inclusive of the recited number and can be subsequently divided into subranges as previously discussed. Finally, as will be understood by those of skill in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0143] From the foregoing, it will be understood that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0144] All references cited herein are hereby incorporated by specific reference in their entirety: U.S. Pat. No. 4,745,285, U.S. Pat. No. 6,184,990, and U.S. Patent Application No. 2017 / 0038299.

Claims

1. A flow cytometer, one or more light emitters configured to emit light into the optical path; a rectangular flow cell having a flow cell width generally lateral to the optical path and a flow cell depth longitudinal to the optical path, the optical path having an interrogation width at the flow cell that is narrower than the flow cell width; a spherical reflector positioned adjacent to the rectangular flow cell, the spherical reflector having a concave reflective surface with a reflection direction positioned generally perpendicular to the optical path such that reflected light is reflected along a reflection path generally perpendicular to the optical path; a main conduit downstream from the injection hole opposite the flow cell, the main conduit comprising one or more main conduit light absorbing members covering at least a portion of an inner surface of the main conduit.

2. one or more light absorbing members positioned at least partially around the reflective path to absorb reflected light at an angle relative to the reflective path; 10. The flow cytometer of claim 1, further comprising one or more light absorbing members positioned between the flow cell and the exit aperture to define an optical conduit between the flow cell and the exit aperture, and one or more light absorbing members at least partially defining the exit aperture.

3. The one or more light absorbing members include: a tapered absorbing surface extending away from the flow cell; a parallel absorbing surface that is substantially parallel to the reflection path; an orthogonal light-absorbing surface that is substantially orthogonal to the reflection path; 3. The flow cytometer of claim 2, further comprising one or more of: a) on a surface of a wall of the flow cell;

4. 10. The flow cytometer of claim 1, further comprising an aperture defining an exit aperture, the exit aperture being positioned such that the reflected path passes through the exit aperture, the exit aperture having a smaller cross-sectional dimension than a housing through which the exit aperture defines an opening in the housing.

5. 10. The flow cytometer of claim 1, further comprising a beam splitter in the main conduit, wherein one or more light absorbing members are positioned between the exit aperture and the beam splitter to define an optical conduit between the exit aperture and the beam splitter.

6. 6. The flow cytometer of claim 5, further comprising: a first collection channel positioned to receive light reflected from the beam splitter; and a second collection channel positioned to receive light passing through the beam splitter, wherein at least one of the first and second collection channels comprises a notch filter.

7. 7. The flow cytometer of claim 6, further comprising one or more collection channel absorbing members in at least one of the first collection channel or the second collection channel.

8. 1. A flow cytometry system, comprising: The flow cytometer of claim 1; a sample source; an analyte stain bath containing an analyte stain composition, the analyte stain composition including a dye configured to detect a target analyte; 1. A flow cytometry system comprising: a counterstain reservoir containing a counterstain composition, the counterstain composition comprising a dye configured to detect a non-target analyte.

9. A kit comprising: The flow cytometer of claim 1; an analyte stain having a first fluorescent wavelength; a counterstain having a second fluorescent wavelength.

10. 1. A method for detecting a target analyte in a sample, comprising: Providing a flow cytometer according to claim 1; providing a stained sample having an analyte stain and a counterstain, wherein the analyte stain emission wavelength is distinguishable from the counterstain emission wavelength; examining the stained sample with light in the flow cytometer; detecting potential targets with the analyte stain; determining the potential targets as being either free of the counterstain or having the counterstain below a counterstain threshold; identifying the detected potential targets having the analyte stain without the counterstain or having the counterstain below the counterstain threshold as the target analyte; defining a threshold value for the percentage of counterstain; determining whether the counterstain emission is below a threshold counterstain percentage, wherein if the counterstain emission is below the counterstain percentage threshold, the analyte is determined to be present or the cell is viable, and if the counterstain emission is above the counterstain percentage threshold, the analyte is determined to be absent or the cell is non-viable.

11. 11. The method of claim 10, further comprising implementing a detection protocol, wherein the detection protocol comprises a lower detection limit of about 1 analyte / 100 mL.

12. 11. The method of claim 10, further comprising passing the stained sample through the flow cell at a flow rate of at least 1 mL / min, 5 mL / min, 20 mL / min, 50 mL / min, or 100 mL / min.

13. defining a threshold ratio of the analyte stain to the counterstain; 11. The method of claim 10, further comprising determining whether the analyte stain emission and the counterstain emission have an analyte stain to counterstain ratio above a threshold analyte stain to counterstain ratio, wherein if the ratio is above the analyte stain to counterstain ratio threshold, the analyte is determined to be present or the cell is viable, and if the ratio is below the analyte stain to counterstain ratio threshold, the analyte is determined to be absent or the cell is non-viable.

14. defining a threshold ratio of the counterstain to the analyte stain; 11. The method of claim 10, further comprising determining whether the counterstain emission and the analyte emission have a counterstain to analyte stain ratio below a threshold counterstain to analyte stain ratio, wherein if the ratio is below the counterstain to analyte stain ratio threshold, the analyte is determined to be present or the cell is determined to be viable, and if the ratio is above the counterstain to analyte stain ratio threshold, the analyte is determined to be absent or the cell is non-viable.

15. 11. The method of claim 10, further comprising using one or more light absorbing members to block internal reflections of light in the flow cytometer.

16. using the one or more light emitters to emit one or more interrogation light beams into the rectangular flow cell such that the optical path of the interrogation light beams has an interrogation width at the flow cell that is narrower than the flow cell width; 11. The method of claim 10, wherein redirected light from the back wall of the flow cell travels towards the spherical reflector and is reflected back through the flow cell and from the front wall of the flow cell on the reflective path.

17. light between the flow cell front wall and the exit aperture at an angle to the reflected path is absorbed by the one or more light absorbing members; the reflected light at an angle relative to the reflected path is absorbed by the one or more light absorbing members; light is not reflected back to the exit aperture or along the reflection path upon contact with the one or more light absorbing members; the light does not pass through a light absorbing material on the flow cell wall; and a light pipe using the one or more light absorbing members to absorb light at an angle relative to the reflected path.

18. light between the exit aperture and a beam splitter in the main conduit at an angle to the reflected path is absorbed in one or more main conduit light absorbing members; absorbing light reflected from the main conduit at an angle to the reflected path at the one or more main conduit light absorbing members in the main conduit; when light contacts the one or more main conduit light absorbing members in the main conduit, the light is not reflected back to the beam splitter or along the reflection path; a light pipe in the main conduit absorbing light at an angle to the reflected path using the one or more main conduit light absorbing members in the main conduit.

19. light reflected from the beam splitter and directed into a first collection channel at an angle relative to the beam splitter reflection path is absorbed by one or more collection channel light absorbing members in the first collection channel; absorbing reflected light in the first collection channel at an angle relative to the beam splitter reflection path at the one or more collection channel light absorbing members in the first collection channel; when light contacts one or more collection channel light absorbing members in the first collection channel, the light is not reflected back to an optical element in the first collection channel or along the beam splitter reflection path; 20. The method of claim 18, further comprising one or more of the steps of: a light conduit in the first collection channel absorbing light at an angle relative to the beam splitter reflection path using one or more collection channel light absorbing members in the first collection channel.

20. the light that is passed through the beam splitter and directed along the reflected path to a second collection channel is absorbed by one or more collection channel light absorbing members in the second collection channel; the reflected light in the second collection channel at an angle relative to the reflected path is absorbed by the one or more collection channel light absorbing members in the second collection channel; upon contacting the one or more collection channel light absorbing members in the second collection channel, the light is not reflected back to an optical element in the second collection channel or along the reflection path; 20. The method of claim 19, further comprising one or more of: a light conduit in the second collection channel absorbing light at an angle relative to the reflected path using the one or more collection channel light absorbing members in the second collection channel.

21. obtaining a sample from a sample source; obtaining an analyte stain from an analyte stain bath containing an analyte stain composition, the analyte stain composition including a dye; obtaining a counterstain from a counterstain reservoir containing a counterstain composition, the counterstain composition comprising a different dye; staining the sample with the analyte stain and counterstain in a reactor to obtain the stained sample; The method of claim 10, further comprising providing the stained sample to the flow cell for optical interrogation.

22. 22. The method of claim 21, further comprising performing a matched filter analysis on the analyte stain data peaks to identify peaks that are candidates for being target analytes.

23. Potential target analytes are selected based on the matched filter analysis, and the peaks identified as candidates must: a) be within a detection threshold range; b) the analyte stain channel is within range; c) the counterstain channel is within range, or 23. The method of claim 22, wherein d) the ratio of the analyte stain channel intensity to the counterstain channel is within a range.

24. The system having the flow cytometer comprises two reactors for staining two separate samples, and both reactors provide input to a single flow cytometer, and the system is configured such that the method comprises: heating a first sample to a temperature in a first reactor and dyeing the first sample to obtain a first dyed sample; passing the first stained sample from the first reactor through the flow cytometer; a second reactor being heated to the temperature to stain a preconditioned second sample while the first sample is being passed through the flow cytometer to obtain a second stained sample; after the first stained sample has been completely passed through the flow cytometer, passing the second stained sample from the second reactor through the flow cytometer; 11. The method of claim 10, further comprising: while the second stained sample is being passed through the flow cytometer, the first reactor is staining a third sample that has been preconditioned by being heated to the temperature.

25. 1. A flow cytometry system, comprising: The flow cytometer of claim 1; 1. A computer system comprising: one or more processors; one or more non-transitory computer-readable media storing instructions that, in response to being executed by the one or more processors, cause the computer system to perform operations, the operations comprising: causing a sample preparation system to provide to the flow cytometer a stained sample having an analyte stain and a counterstain, wherein the analyte stain emission wavelength is distinguishable from the counterstain emission wavelength; interrogating the stained sample with the flow cytometer to obtain flow cytometry data; detecting potential targets having said analyte stain from said flow cytometry data; determining the potential targets as being either free of the counterstain or having the counterstain below a counterstain threshold; identifying the detected potential targets having the analyte stain without the counterstain or having the counterstain below the counterstain threshold as target analytes; determining whether the emission of the counterstain is below a threshold counterstain percentage, wherein if the emission of the counterstain is below the threshold counterstain percentage, the analyte is determined to be present or the cell is viable, and if the emission of the counterstain is above the threshold counterstain percentage, the analyte is determined to be absent or the cell is non-viable.

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