Flow cell with optimized flow passage shape, flow site meter comprising flow cell, and use method thereof

Abstract

To provide a flow cell having an optimized flow passage shape, for a cell sorter, that exhibits high light collection efficiency, and that further, conducts collection of optical images in a satisfactory manner.SOLUTION: A flow cell 100 has a cuvette 101 that transports particles in a flow stream. The cuvette includes a flow passage 102 penetrating the cuvette. The flow passage 102 has a cross section of a rectangular shape whose aspect ratio a / b falls within the range of 1.0-1.4.SELECTED DRAWING: Figure 1A

Description

[Background technology]

[0001] Characterization of analytes in biological fluids has become an important part of biological research, medical diagnostics, and the assessment of a patient's overall health and well-being. Detecting analytes in biological fluids, such as human blood or blood-derived products, can yield results that may be relevant to determining treatment protocols for patients with various medical conditions.

[0002] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells in a blood sample or particles of interest in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer carries particles (including cells) in the fluid sample as a cell stream to a flow cell, while the sheath fluid is directed toward the flow cell. To characterize components in the flow stream, light is irradiated onto the flow stream. Changes in the biological materials in the flow stream, such as morphology or the presence of fluorescent labels, can alter the observed light, enabling characterization and separation. To characterize components in a flow stream, light must be directed onto the flow stream and collected. The light source for a flow cytometer can be a variety of light sources, including one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and optical responses from the illuminated particles are collected and quantified.

[0003] Separation of biological particles has been achieved by adding a sorting or collection function to a flow cytometer. Particles present in the separated stream and detected as having one or more desired properties are individually separated from the sample stream by mechanical or electrical removal. A common flow sorting technique utilizes droplet sorting, in which a fluid stream containing linearly separated particles is split into droplets. Droplets containing particles of interest are electrically charged and deflected into a collection tube by passing through an electric field. Typically, linearly separated particles in a stream are characterized as they pass an observation point directly below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, it is possible to predict the time at which the particle will reach the droplet breakoff point and separate from the stream into droplets. Ideally, the fluid stream is briefly charged just before droplets containing selected particles separate from the fluid stream, and then grounded immediately after the droplets separate. The droplets to be sorted maintain their charge as they separate from the fluid stream, while all other droplets remain uncharged. In some of the above embodiments, the flow cell has square channels with an aspect ratio of 1 for the cell sorter and rectangular channels with an aspect ratio of approximately 2.4 for the analyzer.

[0004] Parameters measured using a flow cytometer generally include light at the excitation wavelength that is scattered by particles primarily in the forward direction at narrow angles, referred to as forward scatter (FSC), excitation light that is scattered by particles in a direction perpendicular to the excitation laser, referred to as side scatter (SSC), and light emitted by fluorescent molecules in one or more detectors that measure signals across a range of spectral wavelengths, or light emitted by fluorescent dyes that are detected primarily by that particular detector or array of detectors. Different cell types can be distinguished by their light scattering properties and fluorescence emission by labeling various cellular proteins or other components with fluorochrome-labeled antibodies or other fluorescent probes. Summary of the Invention [Problem to be solved by the invention]

[0005] An aspect ratio of 1 for cell sorters maximizes fluid stability, which is particularly important for cell sorting, while also providing excellent optical quality for imaging. However, scattering and clipping of collected light significantly reduce light collection efficiency. Additionally, an aspect ratio of 2.4 maximizes light collection efficiency without overly elongating the core stream ellipse. However, this high aspect ratio hinders optical image collection. Therefore, a flow cell with an optimized shape that provides the best results across all these various considerations is desirable. The flow cell, flow cytometer, and method of the present disclosure fulfill this need. [Means for solving the problem]

[0006] Aspects of the present disclosure include flow cells with optimized flow path geometries. Flow cells of interest include a cuvette configured to transport particles in a flow stream, the cuvette having a flow path with a rectangular cross section and an aspect ratio within a range of 1.0 to 1.4 (e.g., about 1.2). In some cases, the length of the rectangular cross section is within a range of 275 μm to 325 μm (e.g., 295 μm to 305 μm). In some cases, the rectangular cross section of the flow path has a width within a range of 225 μm to 275 μm (e.g., 245 μm to 255 μm). In some cases, a surface of the cuvette is spaced apart from the flow path by a distance within a range of 1.9 mm to 2.1 mm (e.g., 2.02 mm to 2.04 mm). In further cases, a surface (e.g., another surface) of the cuvette is spaced apart from the flow path by a distance within a range of 5 mm to 5.2 mm (e.g., 5.07 mm to 5.09 mm). In some embodiments, one or more surfaces of the cuvette may be provided with an anti-reflective coating. In embodiments, the cuvette has a height perpendicular to its rectangular cross section within a range of 5 mm to 9 mm (e.g., 7 mm to 7.2 mm). In some cases, the cuvette has a length within a range of 8 mm to 12 mm (e.g., 10 mm to 10.2 mm). In some cases, the width of the cuvette is within a range of 2 mm to 6 mm (e.g., 4 mm to 4.1 mm). In certain cases, the ratio of the width of the rectangular cross section of the flow channel to the width of the cuvette is within a range of 1:8.9 to 1:21.8 (e.g., 1:16.1 to 1:16.3). In some cases, the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within a range of 1:22.2 to 1:32.7 (e.g., 1:28.2 to 1:28.6). In some cases, the ratio of the length of the rectangular cross section of the flow channel to the length of the cuvette is within a range of 1:29.1 to 1:40 (e.g., 1:33.4 to 1:33.9). In embodiments, the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within a range of 1:18.2 to 1:27.7 (e.g., 1:23.6 to 1:23.7). The cuvette of interest may be constructed of, for example, fused silica. In some variations, the flow cell has a collection efficiency within a range of 20% to 40% (e.g., 23% to 25%). In some cases, the rectangular cross section of the flow channel is symmetrical with respect to the width, length, or both the width and length of the cuvette.In an embodiment, the flow path extends the entire height of the cuvette.

[0007] Aspects of the present disclosure further include flow cytometers including a subject flow cell (e.g., as described above and herein). Flow cytometers of interest include a light source configured to illuminate particles in a flow stream at an interrogation point within the flow cell and a detector configured to collect light emitted from the illuminated particles. In some cases, the flow cytometer is configured as an imaging flow cytometer. In some such cases, the light source includes a light beam generator configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light. For example, the flow cytometer may include an acousto-optic deflector (AOD). In some cases, the flow cytometer includes a sample fluid line configured to deliver particles to the flow cell. In some such cases, the flow cytometer is configured to deliver particles in the flow stream at a velocity within a range of 0.1 m / s to 10 m / s. In some variations, the flow cytometer is configured to operate in an imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a first velocity, and in a non-imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a second velocity higher than the first velocity. In some such variations, the first velocity is within a range of 0.5 m / s to 1.5 m / s (e.g., 0.9 m / s to 1.1 m / s). Similarly, the second velocity may optionally be within a range of 3 m / s to 8 m / s (e.g., 5 m / s to 6 m / s). The ratio of the first velocity to the second velocity may, in certain cases, be within a range of 1:12 to about 1:2. In some embodiments, the flow cytometer further includes a collection lens in optical communication with the flow cell and the detector. Optionally, the collection lens has a collection angle (θ) within a range of 90 degrees to about 110 degrees (e.g., 97 degrees to 103 degrees). In an embodiment, the flow cytometer includes a light source and an objective lens in optical communication with the flow cell.

[0008] Aspects of the present disclosure further include methods of analyzing a sample fluid. The featured methods involve introducing a sample fluid into a flow cytometer of the present disclosure (e.g., as described above and herein) and illuminating particles in the flow stream to analyze the sample fluid. In some cases, the methods involve operating in an imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to a flow cell at a first rate, and in a non-imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a second rate higher than the first rate. In addition to the method of analyzing a sample, the disclosed methods also include a method of assembling a flow cytometer. In some cases, the disclosed methods further include disposing a flow cell of the present disclosure in the flow cytometer. In some cases, the methods further include disposing a collection lens in optical communication with the flow cell and a detector (e.g., coupling the collection lens to a cuvette). In some cases, the methods further include disposing an objective lens in optical communication with a light source and the flow cell. [Brief explanation of the drawings]

[0009] The present disclosure can be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, including:

[0010] [Figure 1A] FIG. 1 illustrates a flow cell with optimized channel geometry according to an embodiment. [Figure 1B] FIG. 1 illustrates a flow cell with optimized channel geometry according to an embodiment. [Figure 1C] FIG. 1 illustrates a flow cell with optimized channel geometry according to an embodiment. [Figure 2] FIG. 1 illustrates a flow cytometry system according to an embodiment. [Figure 3-1] FIG. 1 illustrates an image-enabled particle sorter according to an embodiment. [Figure 3-2] FIG. 1 illustrates an image-enabled particle sorter according to an embodiment. [Figure 4] FIG. 1 is a functional block diagram illustrating a particle analysis system according to an embodiment. [Figure 5] FIG. 1 is a functional block diagram illustrating an example of a control system according to an embodiment. [Figure 6A] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 6B] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 7] FIG. 1 is a block diagram illustrating a computing system according to an embodiment. [Figure 8] 1 is a chart showing a matrix illustrating the status of various flow path dimensions for various considerations. [Figure 9] 10 is a diagram modeling the change in collection efficiency due to bead displacement. [Figure 10A] 10 is a diagram modeling the change in collection efficiency due to bead displacement. [Figure 10B] 10 is a diagram modeling the change in collection efficiency due to bead displacement. DETAILED DESCRIPTION OF THE INVENTION

[0011] A flow cell having an optimized flow path geometry is provided. The flow cell of interest includes a cuvette configured to transport particles in a flow stream, the cuvette having a flow path with a rectangular cross section and an aspect ratio in the range of 1.0 to 1.4. Also provided are flow cytometers including the subject flow cells, as well as methods for using and assembling the same.

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

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

[0014] In this specification, a range is presented with the term "about" before the numerical values. The term "about" is used herein to literally support the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately the number may be a number that, in the context in which the specifically stated number is presented, provides a substantial equivalent to the specifically stated number.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, representative illustrative methods and materials are described.

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

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

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

[0019] Although the systems and methods have been or will be described for grammatical fluidity with functional descriptions, it should be clearly understood that the claims, unless expressly recited under 35 U.S.C. 112, should not be construed as necessarily limited in any way by limitations of "means" or "step" construction, but should be accorded the full scope of the meaning and equivalents of the definition given by the claims under the judicial theory of equivalents, and that if a claim is expressly recited under 35 U.S.C. 112, it should be accorded the full legal equivalents under 35 U.S.C. 112.

[0020] Flow cell Aspects of the present disclosure include flow cells having optimized geometries. An "optimized" geometry means that the geometry of the flow cell enables those skilled in the art of performing flow cytometry to obtain superior results more efficiently than a non-optimized flow cell. For example, a flow cell of the present disclosure may enable high performance across various operating modes of a flow cytometer. As described herein, "operating mode" refers to the particular way a given flow cytometer may be configured to illuminate particles and process signals received from the illuminated particles. In some cases, an operating mode refers to the velocity of the flow stream. Additionally or alternatively, an operating mode refers to the type of flow cytometry performed. For example, an operating mode may refer to an imaging mode (e.g., a mode that acquires images of particles in a flow stream) or a non-imaging mode. In some embodiments, a flow cell of the present disclosure enables high performance across two or more operating modes, three or more operating modes, four or more operating modes, five or more operating modes, six or more operating modes, seven or more operating modes, eight or more operating modes, nine or more operating modes, and ten or more operating modes. "High performance" means that a flow cell of the present disclosure provides optimal results for a certain metric. Metrics of interest may include, but are not limited to, one or more of fluid stability (e.g., maintaining a desired core stream shape), imaging optical quality, light collection efficiency, etc. For example, light collection efficiency may in some cases be in the range of 20%-40%, e.g., 21%-30%, e.g., 23%-25%. In certain cases, flow cells of the present disclosure may be characterized by a light collection efficiency of 24% (or close to 24%).

[0021] Flow cells of interest include cuvettes configured to transport particles in a flow stream. As used herein, the term "flow cell" is used in its conventional sense to refer to an element having a flow path for a liquid flow stream to transport particles in a sheath fluid. Cuvettes of interest include a passageway (i.e., a flow path) extending therethrough. The flow stream may include a liquid sample injected from a sample tube. In some cases, flow cells include optically transparent flow paths. The cuvette may be constructed of, for example, quartz, glass, or clear plastic. In some embodiments, the cuvette is formed from silica, such as fused silica. In some cases, flow cells are configured to be illuminated with light from a light source at one or more interrogation points. The term "interrogation point" used herein refers to an area within the flow cell where particles are illuminated by light from the light source, e.g., for analysis. The size of the interrogation point may vary as desired. For example, if 0 μm represents the axis of light emitted by the light source, the interrogation point may be within a range of -50 μm to 50 μm, e.g., -25 μm to 40 μm, or e.g., -15 μm to 30 μm. Depending on certain considerations (eg, number and placement of lasers), there may be multiple illumination points within the flow cell.

[0022] The cuvettes of the present disclosure have flow channels with rectangular cross sections and aspect ratios in the range of 1.0 to 1.4. The term "aspect ratio" is used herein in its conventional sense to describe the ratio of length to width. Aspect ratios of interest may be in the range of 1.0 to 1.4, e.g., 1.1 to 1.3, e.g., 1.15 to 1.25, e.g., 1.16 to 1.24, e.g., 1.17 to 1.23, e.g., 1.18 to 1.22, e.g., 1.19 to 1.21. In some cases, the aspect ratio is at least in the range of 1.0 to 1.05, 1.05 to 1.1, 1.1 to 1.15, 1.15 to 1.2, 1.2 to 1.25, 1.25 to 1.3, 1.3 to 1.35, or 1.35 to 1.4. In some cases, the aspect ratio is within the range of 1.0 up to 1.05, 1.05 up to 1.1, 1.1 up to 1.15, 1.15 up to 1.2, 1.2 up to 1.25, 1.25 up to 1.3, 1.3 up to 1.35, or 1.35 up to 1.4. In some cases, the aspect ratio is 1.2 or close to 1.2. In some cases, the aspect ratio may be 1.0, 1.1, 1.2, 1.3, or 1.4, and all intervening values ​​therebetween. The length and width of the rectangular cross-section may, in some cases, vary as long as they correspond to the aspect ratios described above. In some cases, the rectangular cross-section of the channel has a length in the range of 275 μm to 325 μm, such as 290 μm to 310 μm, for example, 295 μm to 305 μm, for example, 296 μm to 304 μm, for example, 297 μm to 303 μm, for example, 298 μm to 302 μm, for example, 299 μm to 301 μm. In some cases, the length of the rectangular cross-section can be 295 μm, 296 μm, 297 μm, 298 μm, 299 μm, 300 μm, 301 μm, 302 μm, 303 μm, 304 μm, or 305 μm, and any intervening value therebetween. In some cases, the length of the rectangular cross-section is 300 μm or close to 300 μm.Furthermore, in some embodiments, the rectangular cross-section of the channel has a width in the range of 225 μm to 275 μm, such as 240 μm to 260 μm, such as 245 μm to 255 μm, such as 246 μm to 254 μm, such as 247 μm to 253 μm, such as 248 μm to 252 μm, or such as 249 μm to 251 μm. In some cases, the width of the rectangular cross-section can be 245 μm, 246 μm, 247 μm, 248 μm, 249 μm, 250 μm, 251 μm, 252 μm, 253 μm, 254 μm, or 255 μm, and any intervening value therebetween. In some cases, the width is 250 μm or close to 250 μm.

[0023] As explained above and demonstrated in the Experimental Section below, using a flow cell matching the above aspect ratios, lengths, and widths can potentially provide high performance across various flow cytometer operating modes, for example, when operating in non-imaging (e.g., analytical) or imaging modes. As summarized above, the present disclosure recognizes that low aspect ratios significantly reduce light collection efficiency due to scattering and clipping of collected light. For example, an aspect ratio of 2.4 maximizes light collection efficiency without excessively elongating the core stream ellipse. The present disclosure further recognizes that fluidic performance in imaging (and sorting) modes deteriorates with increasing aspect ratios for several reasons and is optimal when aspect ratios approach 1. Rectangular channels cause the core stream to be roughly elliptical, and higher aspect ratios "stretch" the ellipse further. The more the core stream ellipse is elongated, the greater the spatial variation in actual particle positions within the core stream. This can result in different particle light being interrogated and / or collected differently, potentially resulting in different illumination due to, for example, a non-uniform (Gaussian) beam or being further out of focus in the focusing lens. A more elongated core stream also results in greater variations in particle velocity due to the parabolic flow velocity profile within the cuvette. The greater the velocity variation, the greater the variation in particle arrival times, potentially creating laser delay stability issues, especially at slower imaging speeds. Traveling the same distance at slower imaging speeds amplifies the difference in arrival times between particles traveling at different velocities. Rectangular channels also experience greater shear forces in the narrow direction than square channels. This can magnify commonly occurring fluid instabilities and further stress particles in the core stream. Rectangular channels also result in a greater pressure drop per unit area due to frictional losses, thus requiring higher pressure to drive the same flow. It has been demonstrated that the flow cell parameters described herein, with aspect ratios in the range of 1.1–1.4, provide an optimal balance between these and other considerations.

[0024] The location of the flow path within the cuvette may vary. In some cases, the surface of the cuvette is spaced from the flow path by a distance within a range of 1.9 mm to 2.1 mm, e.g., 2.00 mm to 2.05 mm, e.g., 2.02 mm to 2.04 mm. In some cases, the surface of the cuvette may be spaced from the flow path by 2.00 mm, 2.01 mm, 2.02 mm, 2.03 mm, 2.04 mm, or 2.05 mm, and any intervening distance therebetween. In some cases, the surface of the cuvette is spaced from the flow path by 2.03 mm. In certain variations, the surface of the cuvette is the surface along the length of the cuvette. In some embodiments, another (e.g., a second) surface of the cuvette is spaced from the flow path by a distance within a range of 5 mm to 5.2 mm, e.g., 5.05 mm to 5.1 mm, e.g., 5.07 mm to 5.09 mm. In some cases, the surface of the cuvette may be spaced apart from the flow channel by 5.05 mm, 5.06 mm, 5.07 mm, 5.08 mm, 5.09 mm, or 5.10 mm, and all intervening values ​​therebetween. In some cases, the surface of the cuvette is spaced apart from the flow channel by 5.08 mm or a value close to 5.08 mm. In some variations, this surface (e.g., the second surface) of the cuvette is the surface of the width of the cuvette. The distance between the flow channel and the surface of the cuvette may be measured from the center of the flow channel or from an edge of the flow channel. In certain cases, the rectangular cross-section of the flow channel is symmetrical across the width, length, or both the width and length of the cuvette. For example, in some variations, the rectangular cross-section of the flow channel is symmetrical across the width of the cuvette. In additional variations, the rectangular cross-section of the flow channel is symmetrical across the length of the cuvette. In yet additional variations, the rectangular cross-section of the flow channel is symmetrical across both the length and width of the cuvette. In some variations, a cuvette having the aforementioned dimensions provides optimal image quality (eg, in combination with optical components such as the objective and condenser lenses described in more detail below).

[0025] In some cases, an anti-reflective coating is applied to one or more surfaces of the cuvette. In some cases, the use of such a coating is sufficient to reduce reflective power losses. In some cases, the anti-reflective coating is a broadband anti-reflective coating. Anti-reflective coatings that may be used include, but are not limited to, tantalum pentoxide (Ta2O5), silicon dioxide (SiO2), magnesium fluoride (MgF2), zirconium dioxide (ZrO2), aluminum oxide (Al2O3), and the like, and combinations thereof. If an anti-reflective coating is applied, the thickness of the anti-reflective coating may vary, and the appropriate coating thickness may be readily determined according to manufacturing methods known to those skilled in the art.

[0026] The dimensions of the cuvette itself may also vary. In some cases, the cuvette has a height perpendicular to its rectangular cross section within the range of 5 mm to 9 mm, e.g., 6 mm to 8 mm, e.g., 7.0 mm to 7.2 mm, e.g., 7.05 mm to 7.15 mm, e.g., 7.08 mm to 7.13 mm, e.g., 7.10 mm to 7.12 mm. In some cases, the height of the cuvette may be 7.05 mm, 7.06 mm, 7.07 mm, 7.08 mm, 7.09 mm, 7.10 mm, 7.11 mm, 7.12 mm, 7.13 mm, 7.14 mm, or 7.15 mm, and any intervening value therebetween. In some cases, the height is at or close to 7.11 mm. In some cases, the cuvette has a length within the range of 8 mm to 12 mm, e.g., 9 mm to 11 mm, e.g., 10.0 mm to 10.2 mm. In some cases, the length of the cuvette may be 5.10 mm, 5.11 mm, 5.12 mm, 5.13 mm, 5.14 mm, 5.15 mm, 5.16 mm, 5.17 mm, 5.18 mm, 5.19 mm, or 5.20 mm, and any intervening values ​​therebetween. In some cases, the length of the cuvette is 5.16 mm or close to 5.16 mm. In certain variations, the width of the cuvette is within a range of 2 mm to 6 mm, e.g., 3 mm to 5 mm, e.g., 4.0 mm to 4.1 mm, e.g., 4.02 mm to 4.08 mm, e.g., 4.05 mm to 4.07 mm. In some embodiments, the width of the cuvette may be 4.0 mm, 4.01 mm, 4.02 mm, 4.03 mm, 4.04 mm, 4.05 mm, 4.06 mm, 4.07 mm, 4.08 mm, 4.09 mm, or 4.10 mm, and all intervening values ​​therebetween.

[0027] The flow cell may be further described by a ratio relating the dimensions of the rectangular cross-section to the dimensions of the cuvette. In some cases, the ratio of the width of the rectangular cross-section of the flow channel to the width of the cuvette is within a range of 1:8.9 to 1:21.8, such as 1:12.5 to 1:19.2, e.g., 1:16.1 to 1:16.3. In embodiments, the ratio of the width of the rectangular cross-section of the flow channel to the height perpendicular to the rectangular cross-section is within a range of 1:22.2 to 1:32.7, e.g., 1:25 to 1:30.1, e.g., 1:28.2 to 1:28.6. In some cases, the ratio of the length of the rectangular cross-section of the flow channel to the length of the cuvette is within a range of 1:29.1 to 1:40, e.g., 1:31 to 1:35.6, e.g., 1:33.4 to 1:33.9. In some cases, the ratio of the length of the rectangular cross section of the channel to the height perpendicular to the rectangular cross section is in the range of 1:18.2 to 1:27.7, such as 1:20.7 to 1:25.8, for example 1:23.6 to 1:23.7.

[0028] In some embodiments, the sample flow stream is emitted from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the orifice of the flow cell may have any suitable shape, with cross-sectional shapes of interest including, but not limited to, rectilinear cross-sectional shapes, e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved cross-sectional shapes, e.g., circular, oval, etc.; and irregular shapes, e.g., a parabolic base joined to a planar top. In certain embodiments, flow cells of interest have circular orifices. The size of the nozzle orifice may vary, and in some embodiments may be in the range of 1 μm to 20,000 μm, such as 2 μm to 17,500 μm, for example, 5 μm to 15,000 μm, 10 μm to 12,500 μm, for example, 15 μm to 10,000 μm, for example, 25 μm to 7,500 μm, for example, 50 μm to 5,000 μm, for example, 75 μm to 1,000 μm, for example, 100 μm to 750 μm, for example, 150 μm to 500 μm. In one embodiment, the nozzle orifice is 100 μm.

[0029] In some embodiments, the flow cell of the present disclosure further includes optical components integrated and / or coupled to the flow cell. In some such embodiments, the flow cell includes a collection lens. The collection lens may be any optical component configured to collect light from one or more interrogation zones of the flow cell and direct the light to one or more detectors or additional optical components (e.g., mirrors, lenses, etc., as needed). In some cases, the collection lens is a collimating lens. For example, in some cases, the collimating lens is attached to the outer surface of the cuvette. The collimating lens may be attached to the outer surface of the cuvette by any suitable means, such as optical adhesive or a press / interference fit. In certain cases, the collimating lens is an injection-compression molded lens. In some cases, the collimating lens is a Fresnel lens. In alternative embodiments, the collection lens is not directly coupled to the flow cell but is considered part of the flow cytometer. The numerical aperture (NA) of the collection lens may vary, and in some cases is within the range of 0.75 to 1.5, e.g., 0.9 to 1.4, e.g., 1.1 to 1.3. In some cases, the NA is 1.2 or close to 1.2. The collection lens may have a variety of collection angles (θ) when used with a cuvette. In some cases, the collection lens has a collection angle that satisfies the following equation: tan(θ / 2)=a / b where a is the length of the rectangular cross section of the flow channel and b is the width of the rectangular cross section of the flow channel. In some cases, the collection angle (θ) is in the range of 90 degrees to 110 degrees, e.g., 95 degrees to 105 degrees, e.g., 97 degrees to 103 degrees. Additional optical components may include, for example, one or more objective lenses configured to focus light from one or more light sources (e.g., lasers) onto one or more interrogation points within the flow cell. The objective lenses may or may not be integral and / or coupled to the cuvette.

[0030] 1A-1C are various views of a flow cell having an optimized geometry according to an embodiment of the present disclosure. FIG. 1A shows a flow cell 100 in the XZ direction. As shown in FIG. 1A, the flow cell 100 includes a cuvette 101 having a flow channel 102 extending therethrough. The flow channel 102 has a rectangular cross-section, a length a, and a width b. The aspect ratio of the rectangular cross-section is designated a / b, which is consistent with the above-mentioned aspect ratio parameters (e.g., in the range of 1.0 to 1.4). Also shown is a collection lens 103, characterized by a collection angle θ. In the example of FIG. 1A, θ is consistent with the following equation: tan(θ / 2)=a / b

[0031] Figure 1B shows flow cell 100 in the YZ direction, with the same elements arranged as described above with respect to Figure 1A. Core stream 107, having an elliptical shape, is also shown in Figure 1B. In addition, Figure 1B shows light 105 that cuvette 101 is configured to receive from one or more light sources (e.g., lasers; not shown).

[0032] FIG. 1C is a three-dimensional view of flow cell 100. As shown in FIG. 1C, light 105 from objective lens 104 enters cuvette 101 at surface S1 and illuminates channel 102 at the interrogation point. Light resulting from this interaction exits cuvette 101 at surface S2 and is collected by collection lens 103 (not shown). Cuvette 101 is characterized by a length l, width w, and height h, which correspond to the parameters described above for the length, width, and height of the cuvette. Channel 102 is spaced a distance d1 from surface S1. Additionally, channel 102 is spaced a distance d2 from surface S2. These distances correspond to the parameters described above for the distance separating the surface of the cuvette from the channel.

[0033] In some embodiments, the flow cell has or is configured for use with a sample injection port configured to deliver a sample to the flow cell, hi embodiments, the sample injection system is configured to deliver a suitable flow of sample to the internal chamber (i.e., flow path) of the flow cell. Depending on the desired characteristics of the flow stream, the flow rate of the sample delivered by the sample injection port to the chamber of the flow cell may be 1 μL / min or more, such as 2 μL / min or more, for example 3 μL / min or more, such as 5 μL / min or more, for example 10 μL / min or more, such as 15 μL / min or more, for example 25 μL / min or more, for example 50 μL / min or more, for example 100 μL / min or more, and in some cases the flow rate of the sample delivered by the sample injection port to the chamber of the flow cell is 1 μL / sec or more, such as 2 μL / sec or more, for example 3 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, for example 15 μL / sec or more, for example 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more.

[0034] The sample injection port may be an orifice in the wall of the internal chamber or a tube located at the proximal end of the internal chamber. When the sample injection port is an orifice in the wall of the internal chamber, the orifice may have any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal; curved shapes, such as circular and oval; and irregular shapes, such as a parabolic bottom joined to a flat top. In some embodiments, the sample injection port has a circular orifice. The size of the orifice of the sample injection port may vary depending on the shape, and in some cases may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., a 1.5 mm opening.

[0035] In some cases, the sample injection port is a tube located at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a tube aligned with the orifice of the flow cell. When the sample injection port is a tube aligned with the orifice of the flow cell, the cross-sectional shape of the sample injection tube may have any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes, such as circular and oval; and irregular shapes, such as a parabolic bottom joined to a flat top. The orifice of the tube may vary depending on the shape and may in some cases have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may have a beveled tip with a bevel angle within a range of 1° to 10°, for example, 2° to 9°, for example, 3° to 8°, for example, 4° to 7°, for example, a bevel angle of 5°.

[0036] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid, e.g., along with the sample, into the internal chamber of the flow cell to generate a laminated flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of the sheath fluid delivered to the chamber of the flow cell may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, e.g., 2500 μL / sec or more.

[0037] In some embodiments, the sheath fluid injection port is an orifice in the wall of the internal chamber. The sheath fluid injection port orifice may have any suitable shape, and cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal, curved cross-sectional shapes, such as circular and oval, and irregular shapes, such as a parabolic bottom joined to a flat top. The size of the sheath fluid injection port orifice may vary depending on the shape, and in some cases may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., a 1.5 mm opening.

[0038] flow cytometer Aspects of the present disclosure further include flow cytometers. Flow cytometers of interest include a flow cell of the present disclosure. As described in detail above, flow cells of interest include a cuvette, the cuvette having a flow channel with a rectangular cross-section and an aspect ratio in the range of 1.0 to 1.4. Additionally, flow cytometers of the present disclosure include a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell. The number of light sources in a flow cytometer may vary. In some embodiments, the flow cytometer includes a single light source. Alternatively, the flow cytometer may include multiple light sources in some cases. In some such cases, the number of light sources is in the range of 2 to 10, e.g., 2 to 5, e.g., 2 to 4. Any convenient light source may be used as the light source described herein.

[0039] In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the subject flow cytometer is equipped with a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, lasers of interest include metal vapor lasers, such as helium cadmium (HeCd), helium mercury (HeHg), helium selenium (HeSe), helium silver (HeAg), strontium, neon copper (NeCu), copper, or gold lasers, and combinations thereof. In still other cases, the subject flow cytometers are equipped with solid-state lasers, such as ruby, Nd:YAG, NdCrYAG, Er:YAG, Nd:YLF, Nd:YVO, Nd:YCaO(BO), Nd:YCOB, titanium sapphire, thulium YAG, ytterbium YAG, YbO, or cerium-doped lasers, and combinations thereof.

[0040] In some embodiments, the laser light source may further include one or more optical adjustment components. In some embodiments, the optical adjustment components are disposed between the light source and the flow cell and may include any device capable of changing the spatial width or other characteristics of the illumination from the light source, such as the illumination direction, wavelength, beam width, beam intensity, and focus. The optical adjustment protocol may include any convenient device for adjusting one or more characteristics of the light source, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In some embodiments, the flow cytometer of interest includes one or more focusing lenses. In one example, the focusing lens may be a reduction lens. In yet other embodiments, the flow cytometer of interest includes optical fibers.

[0041] The light source may be positioned at any suitable distance from the flow cell, for example the light source and the flow cell are separated by 0.005 mm or more, such as 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, such as 0.5 mm or more, for example 1 mm or more, for example 5 mm or more, for example 10 mm or more, for example 25 mm or more, such as 100 mm or more. Additionally, the light source may be positioned at any suitable angle relative to the flow cell, for example between 10 degrees and 90 degrees, for example between 15 degrees and 85 degrees, for example between 20 degrees and 80 degrees, for example between 25 degrees and 75 degrees, for example between 30 degrees and 60 degrees, for example at an angle of 90 degrees.

[0042] In some embodiments, the light source of interest includes multiple lasers, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers, e.g., fifteen or more lasers configured to provide laser light for separate illumination of the flow stream. Depending on the desired wavelength of light for illuminating the flow stream, each laser may have a different specific wavelength within a range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. In certain embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0043] In some embodiments, the light source is an optical beam generator configured to generate two or more beams of frequency-shifted light. In some cases, the optical beam generator comprises a laser, a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous wave laser. For example, optical beam generator lasers of interest include gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO lasers, CO lasers, argon fluorine (ArF) excimer lasers, krypton fluorine (KrF) excimer lasers, xenon chlorine (XeCl) excimer lasers, or xenon fluorine (XeF) excimer lasers, or combinations thereof; dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers; metal vapor lasers, such as helium cadmium (HeCd) lasers, helium mercury (HeHg) lasers, and the like. ) laser, helium selenium (HeSe) laser, helium silver (HeAg) laser, strontium laser, neon copper (NeCu) laser, copper laser or gold laser and combinations thereof; solid state lasers, such as ruby ​​laser, Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO4 laser, Nd:YCa4O(BO3)3 laser, Nd:YCOB laser, titanium sapphire laser, thulium YAG laser, ytterbium YAG laser, Yb2O3 laser or cerium doped laser and combinations thereof.

[0044] The acousto-optic device may be any convenient acousto-optic protocol configured to frequency-shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device of the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0045] In an embodiment, the controller is configured to apply high frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams of the output laser beam, for example configured to apply 3 or more high frequency drive signals, for example 4 or more high frequency drive signals, for example 5 or more high frequency drive signals, for example 6 or more high frequency drive signals, for example 7 or more high frequency drive signals, for example 8 or more high frequency drive signals, for example 9 or more high frequency drive signals, for example 10 or more high frequency drive signals, for example 15 or more high frequency drive signals, for example 25 or more high frequency drive signals, for example 50 or more high frequency drive signals, for example configured to apply 100 or more high frequency drive signals.

[0046] In some cases, to generate an intensity profile of the angularly deflected laser beam of the output laser beam, the controller is configured to apply a high frequency drive signal having a varying amplitude within a range, for example, from about 0.001 V to about 500 V, for example, from about 0.005 V to about 400 V, for example, from about 0.01 V to about 300 V, for example, from about 0.05 V to about 200 V, for example, from about 0.1 V to about 100 V, for example, from about 0.5 V to about 75 V, for example, from about 1 V to about 50 V, for example, from about 2 V to about 40 V, for example, from about 3 V to about 30 V, or for example, from about 5 V to about 25 V. The applied high frequency drive signal in some embodiments has a frequency within the range of about 0.001 MHz to about 500 MHz, for example, about 0.005 MHz to about 400 MHz, for example, about 0.01 MHz to about 300 MHz, for example, about 0.05 MHz to about 200 MHz, for example, about 0.1 MHz to about 100 MHz, for example, about 0.5 MHz to about 90 MHz, for example, about 1 MHz to about 75 MHz, for example, about 2 MHz to about 70 MHz, for example, about 3 MHz to about 65 MHz, for example, about 4 MHz to about 60 MHz, for example, about 5 MHz to about 50 MHz.

[0047] In some embodiments, the controller includes a processor to which a memory is operatively coupled, the memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam including an angularly deflected laser beam having a desired intensity profile. For example, the memory may include instructions for generating two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more angularly deflected laser beams of the same intensity, e.g., the memory may include instructions for generating one hundred or more angularly deflected laser beams of the same intensity. In other embodiments, the memory may include instructions for generating two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more angularly deflected laser beams of different intensities, e.g., the memory may include instructions for generating one hundred or more angularly deflected laser beams of different intensities.

[0048] In some embodiments, the controller has a processor to which the memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the center to the edges of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may be in a range of 0.1% to about 99%, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, or may be in a range of about 10% to about 50% of the intensity of the angularly deflected laser beam at the edges of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor to which the memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the edge to the center of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may be in a range of 0.1% to about 99%, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, or may be in a range of about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller has a processor to which a memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam having a Gaussian intensity profile along a horizontal axis.In yet another embodiment, the controller has a processor to which the memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0049] In embodiments, the optical beam generator of interest may be configured to generate spatially separated angularly deflected laser beams of the output laser beam. Depending on the applied high frequency drive signal and the desired irradiance profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more, such as 5000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams of the output laser beam that overlap, for example, adjacent angularly deflected laser beams along a horizontal axis of the output laser beam. The overlap of adjacent angularly deflected laser beams (e.g., overlap of beam spots) may be 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more.

[0050] In some cases, the light beam generator configured to generate two or more beams of frequency-shifted light comprises a laser excitation module as described in U.S. Pat. Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Application Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0051] Additionally, the flow cytometer includes a photodetector configured to collect light emitted from the illuminated particles. The photodetector is configured to detect the particle-modulated light transmitted by the fiber optic collection element and generate a signal based on a characteristic (e.g., intensity) of the light. For example, the one or more particle-modulated light detectors may include one or more side scatter detectors for detecting side-scattered wavelengths of light (i.e., light refracted and reflected by the particle's surface and internal structure). In some embodiments, the flow cytometer includes one side scatter detector. In other embodiments, the flow cytometer includes multiple side scatter detectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more side scatter detectors.

[0052] Any convenient detector for detecting collected light may be used in the side scatter light detectors described herein. Detectors of interest may include, but are not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other detectors. In some embodiments, collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10cm 2 , e.g. 0.05cm 2 ~9cm 2 , e.g., 0.1 cm 2 ~8cm 2 , e.g., 0.5 cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .times. ...

[0053] In embodiments, the subject flow cytometer further comprises a fluorescence detector configured to detect light at one or more fluorescent wavelengths, hi other embodiments, the flow cytometer comprises a plurality of fluorescence detectors, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 10 or more, e.g., 15 or more, e.g., 20 or more fluorescence detectors.

[0054] Any convenient detector for detecting collected light may be used in the fluorescence detectors described herein. Detectors of interest may include, but are not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other detectors. In some embodiments, collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10 cm 2 , e.g., 0.05 cm 2 ~9cm 2 , e.g., 0.1 cm 2 ~8cm 2 , e.g., 0.5 cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .times. ...

[0055] When a subject flow cytometer includes multiple fluorescence detectors, each fluorescence detector may be identical, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, when a subject flow cytometer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-based device and the second fluorescence detector (or image sensor) is a CMOS-based device. In other embodiments, both the first and second fluorescence detectors are CCD-based devices. In still other embodiments, both the first and second fluorescence detectors are CMOS-based devices. In still other embodiments, the first fluorescence detector is a CCD-based device and the second fluorescence detector is a photomultiplier tube (PMT). In still other embodiments, the first fluorescence detector is a CMOS-based device and the second fluorescence detector is a photomultiplier tube. In still other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0056] In embodiments of the present disclosure, a fluorescence detector of interest is configured to measure collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., twenty-five or more different wavelengths, e.g., fifty or more different wavelengths, e.g., one hundred or more different wavelengths, e.g., two or more different wavelengths, e.g., three hundred or more different wavelengths, e.g., four hundred or more different wavelengths, e.g., light emitted from a sample in a flow stream. In some embodiments, two or more detectors of a module as described herein are configured to measure the same or overlapping wavelengths of collected light.

[0057] In some embodiments, the fluorescence detector of interest is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect a spectrum of light over a range of wavelengths. For example, a flow cytometer may include one or more detectors configured to collect a spectrum of light over one or more wavelength ranges from 200 nm to 1000 nm. In yet other embodiments, the detector of interest is configured to measure light emitted from a sample in the flow stream at one or more specific wavelengths. For example, a module may have one or more detectors configured to measure light at one or more of: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, the one or more detectors may be configured to pair with a particular fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0058] A flow cytometer may have any suitable mechanism or mechanisms for supplying sheath fluid and sample fluid to the sheath fluid input coupler and sample fluid input coupler. For example, the sample fluid input coupler may be fluidly connected to a sample fluid line (e.g., tubing) that is fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidly connected to a sheath fluid line that is fluidly connected to a sheath fluid reservoir. Similarly, a flow cytometer may have any suitable mechanism or mechanisms for managing waste from the flow stream. The fluid output coupler may be fluidly connected to a waste line that is fluidly connected to a waste reservoir. A fluid management system that may be adapted for use in the subject flow cytometer is described in U.S. Patent Application Publication No. 2022 / 0341838, the entire disclosure of which is incorporated herein by reference.

[0059] Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt1):17-28; Linden, et al., Semin Thromb Hemost. 2004 Oct;30(5):502-11; Alison, et al. J Pathol, 2010 Dec;222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst., the disclosures of which are incorporated herein by reference. 24(3):203-255.In some cases, flow cytometry systems of interest include the BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, ...elesta™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and BD Biosciences FACSCalibur™ cell sorter. These include the BD BiosciencesVia™ cell sorter, BD BiosciencesInflux™ cell sorter, BD BiosciencesJazz™ cell sorter, BD BiosciencesAria™ cell sorter, BD BiosciencesFACSAria™ II cell sorter, BD BiosciencesFACSAria™ III cell sorter, BD BiosciencesFACSAria™ Fusion cell sorter, BD BiosciencesFACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, and BD Biosciences FACSDiscover™ cell sorter.

[0060] In some embodiments, the subject system may be configured with a fusion technology similar to that disclosed in U.S. Pat. Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, 10,302,545, 10,145,793, 10,113,967, 10,006,852, 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,099,909, 9,100,100,110,120,130,140,150,160,170,172,180,190,192,194,195,196,197,198,19 ... Nos. 5494, 9092034, 8975595, 8753573, 8233146, 8140300, 7544326, 7201875, 7129505, 6821740, 6813017, 6809804, 6372506, 5700692, 5643796, 5627040, 5620842, 5602039, 4987086, and 4498766.

[0061] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the subject system is a flow cytometry system configured to image particles in a flow stream by fluorescence imaging using radio frequency tag emission (FIRE), as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353, 9,784,661, 10,006,852, U.S. Patent Application Publication No. 2017 / 0133,857, and U.S. Patent Application Publication No. 2017 / 0350,803 (the disclosures of which are incorporated herein by reference). In some embodiments, in which the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter. Image-enabled particle sorters are described in U.S. Provisional Patent Application Nos. 63 / 431,803 and 63 / 465,057, the entire disclosures of which are incorporated herein by reference.

[0062] In certain cases, the flow cytometers of the present disclosure are configured to operate in multiple operating modes. For example, in some cases, the subject flow cytometers are configured to operate in two or more operating modes, e.g., three or more operating modes, e.g., three or more operating modes, e.g., four or more operating modes, e.g., five or more operating modes, e.g., six or more operating modes, e.g., seven or more operating modes, e.g., eight or more operating modes, e.g., nine or more operating modes, e.g., ten or more operating modes. In some cases, the multiple operating modes differ with respect to the velocity of the flow stream. In embodiments, the flow cytometer is configured to operate in at least (a) an imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a first velocity, and (b) a non-imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a second velocity that is higher than the first velocity. In other words, the flow cytometer may be configured to operate in either the imaging mode or the non-imaging mode at a given time and may be configured to switch between these modes as desired. In some cases, the first velocity is in the range of 0.5 m / s to 1.5 m / s, e.g., 0.7 m / s to 1.3 m / s, e.g., 0.9 m / s to 1.1 m / s. In some cases, the first velocity is at or near 1 m / s. Additionally, the second velocity, in some embodiments, is in the range of 3 m / s to 8 m / s, e.g., 4 m / s to 7 m / s, e.g., 5 m / s to 6 m / s. In some cases, the second velocity is at or near 3.3 m / s. In other embodiments, the second velocity is at or near 5.5 m / s. In embodiments, the flow cytometer is configured to operate in multiple imaging and / or non-imaging modes, each of the multiple modes characterized by a different flow rate. In embodiments, flow cells with optimized geometries described herein allow for switching between different operating modes (e.g., imaging and non-imaging modes) while maintaining high performance in a single instrument.

[0063] FIG. 2 illustrates a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 includes a laser 201 configured to illuminate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. While one laser is shown in the example of FIG. 2, it is understood that multiple lasers may also be used. The laser beam from the laser 201 is directed to a focusing lens 202, which focuses the laser beam onto the portion of the fluid stream within the flow cell 210 where the sample particles reside. The flow cell is the part of the fluid system that directs particles in the stream, typically one at a time, into the focused laser beam for interrogation. Alternatively, if the flow cytometer is a stream-in-air cytometer, a nozzle top may be used.

[0064] As shown in FIG. 2 , the flow cell 210 is fluidly connected to a sheath fluid reservoir 203 containing a sheath fluid and a sample fluid reservoir 204 containing a sample fluid. The sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a tube (i.e., a sheath fluid line) 207. Additionally, a sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a tube (i.e., a sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce the particles 211 into the interior of the flow cell 210. The particles 211 are hydrodynamically focused via the sheath fluid flowing from the sheath fluid injection port 208 such that a flow stream 214 is formed downstream of the tapered section 212 of the flow cell 210. Particles emitted at the distal end of flow cell 210 may be discarded and / or collected via any suitable protocol. For example, depending on the type of flow cytometry being performed, particles may be collected at the distal end of flow cell 210, for example, via a waste line. Alternatively, particles may be sorted.

[0065] Light from one or more laser beams interacts with particles 211 in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at a variety of different wavelengths depending on the particle's characteristics, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The fluorescent radiation, as well as the diffracted, refracted, reflected, and scattered light, may be sent to one or more detectors. In particular, forward-scattered light (FSC) is sent to a forward-scattered light detector 223. The forward-scattered light detector 223 is positioned slightly off-axis from the direct beam passing through the flow cell 210 and is configured to detect diffracted light, i.e., excitation light traveling primarily in a forward direction through or around the particle. The intensity of the light detected by the forward-scattered light detector 223 depends on the overall size of the particle. The forward-scattered light detector may include, for example, a photodiode. A scattering bar 222 is positioned between the forward-scattered light detector 223 and the optical filter 221a. The optical filter 221a may be configured to remove at least one wavelength of non-FSC light, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scattered light detector 223.

[0066] Additionally, side-scattered light (SSC) is detected by a side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structures of the particle 211, which tends to increase as the structural complexity of the particle increases. In the example of FIG. 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered light detector 224 while passing non-SSC light (e.g., fluorescent light). An optical filter 221b is configured to prevent at least one wavelength of non-SSC light from being detected by the side-scattered light detector 224. Fluorescence detectors 225a-225c are also shown, each configured to detect fluorescent light of a different wavelength. For example, the dichroic mirror 220b may be configured to reflect fluorescent light (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a while passing light of other wavelengths. Optical filter 221c may be configured to prevent at least one wavelength of light that does not correspond to the first wavelength (or wavelength range) from being detected by fluorescence detector 225a. Similarly, dichroic mirror 220c is configured to reflect FL light corresponding to the second wavelength (or wavelength range) to fluorescence detector 225b, while passing light of a third wavelength (or wavelength range) for detection by fluorescence detector 225c. Optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to the second wavelength (or wavelength range) from being detected by fluorescence detector 225b. Additionally, optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to the third wavelength (or wavelength range) from being detected by fluorescence detector 225c.

[0067] Those skilled in the art will recognize that flow cytometers according to embodiments of the present disclosure are not limited to the flow cytometer shown in FIG. 2 , but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and in a variety of different configurations. For example, while three fluorescence detectors are shown in the embodiment of FIG. 2 for illustrative purposes, it will be understood that any suitable number of fluorescence detectors may be used.

[0068] During operation, the operation of the flow cytometer is controlled by the controller / processor 290, and measurement data from the detectors may be stored in the memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detectors to receive output signals from the detectors, and may further be coupled to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function 297 may also be provided in the system. The memory 295, the controller / processor 290, and the I / O function 297 may be provided entirely as an integral part of the flow cytometer. In such an embodiment, a display may also form part of the I / O function 297 to present experimental data to a user of the flow cytometer 200. Alternatively, some or all of the memory 295 and the controller / processor 290 and the I / O function 297 may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of memory 295 and controller / processor 290 may be in wireless or wired communication with the flow cytometer. Controller / processor 290 in conjunction with memory 295 and I / O functionality 297 may be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0069] The various fluorescent molecules of a panel of fluorescent dyes used in a flow cytometer experiment each emit light in a unique wavelength band. The particular fluorescent labels used in the experiment and their associated fluorescence emission bands may be selected to generally match the filter windows of the detector. I / O function 297 may be configured to receive data regarding a flow cytometer experiment having a panel of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O function 297 may be further configured to receive biological data assigning one or more markers to one or more cell populations, marker concentration data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, may further be stored in memory 295. Controller / processor 290 may be configured to evaluate one or more assignments of labels to markers.

[0070] In some embodiments, the subject system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having a plurality of sorting determination units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module with deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0071] In one embodiment, the system is a fluorescence imaging system using a radio frequency tag emission imaging particle sorter, as shown in FIG. 3. Particle sorter 300 includes an optical illumination unit 300a including a light source 301 (e.g., a 488 nm laser) that generates an output beam of light 301a, which is split into beams 302a and 302b by beam splitter 302. Light beam 302a propagates through an acousto-optic device (e.g., an acousto-optic deflector (AOD)) 303 to generate output beam 303a having one or more angularly deflected beams of light. Optionally, output beam 303a from acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Light beam 302b propagates through an acousto-optic device (e.g., an acousto-optic deflector (AOD)) 304 to generate output beam 304a having one or more angularly deflected beams of light. In some cases, output beam 304a from acousto-optic device 304 includes a local oscillator beam and multiple high-frequency comb beams. Output beams 303a and 304a from acousto-optic device 303 and acousto-optic device 304, respectively, are combined in beam combiner 305 to generate output beam 305a, which is transmitted through optics 306 (e.g., an objective lens) to illuminate particles in flow cell 307. In some embodiments, acousto-optic device 303 (AOD) splits a single laser beam into an array of beamlets, each with a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in beam combiner 305. In some embodiments, the light source and the light irradiation system having the acousto-optical device may further include those described in Schraivogel, et al. (“High-speed fluorescence image-enabled cell sorting” Science (2022), 375(6578): 315-320) and U.S. Patent Application Publication No. 2021 / 0404943, the disclosures of which are incorporated herein by reference.

[0072] Output beam 305a illuminates sample particles 308 propagating through flow cell 307 (e.g., with sheath fluid 309) at illumination region 310. As shown in illumination region 310, multiple beams (e.g., angularly polarized, high-frequency shifted beams of light shown as dots across illumination region 310) overlap with a reference local oscillator beam (shown as a cross-hatched line across illumination region 310). The overlapping beams exhibit beat behavior due to their different optical frequencies, with each beamlet emitting at a different frequency f 1-n transmits sinusoidal modulation.

[0073] Light from the illuminated sample is transmitted to a light detection system 300b having multiple light detectors. The light detection system 300b includes a forward scattered light detector 311 for generating a forward scattered light image 311a and a side scattered light detector 312 for generating a side scattered light image 312a. The light detection system 300b further includes a bright-field light detector 313 for generating a light loss image 313a. In some embodiments, the forward scattered light detector 311 and the side scattered light detector 312 are photodiodes (e.g., avalanche photodiodes (APDs)). In some cases, the bright-field light detector 313 is a photomultiplier tube (PMT). Fluorescence from the illuminated sample is further detected by fluorescence detectors 314-317. In some cases, the light detectors 314-317 are photomultiplier tubes. Light from the illuminated sample is directed via beam splitter 320 to side-scattered light detection channel 312 and fluorescence detection channels 314-317. Light detection system 300b includes bandpass optics 321-324 (e.g., dichroic mirrors) for transmitting light of predetermined wavelengths to photodetectors 314-317, respectively. In some cases, optic 321 is a 534 nm / 40 nm bandpass. In some cases, optic 322 is a 586 nm / 42 nm bandpass. In some cases, optic 323 is a 700 nm / 54 nm bandpass. In some cases, optic 324 is a 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number represents the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, from 500 nm to 520 nm.

[0074] Data signals generated in response to light detected in forward-scattered light detection channel 311, side-scattered light detection channel 312, bright-field light detection channel 313, and fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Based on the data signals generated by processors 350 and 351, images 311a-317a can be generated in each light detection channel. Image-corresponding sorting is performed in response to a sorting signal generated by sorting trigger 352. Sorting unit 300c includes deflection plates 331 for deflecting particles into a sample container 332 or a waste stream 333. In some cases, sorting unit 300c is configured to sort particles using a sealed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In one embodiment, the sorting section 300c includes a sorting determination module having multiple sorting determination units, such as those described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0075] In some embodiments, the system is a particle analyzer, and particle analysis system 401 (FIG. 4) can be used to analyze and characterize particles with or without physical sorting of the particles into a collection vessel. FIG. 4 is a functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, particle analysis system 401 is a flow system. Particle analysis system 401 includes a fluid system 402. Fluid system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, through which particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0076] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. A detection station 408 generally refers to a monitoring region 407 of the common sample path. Detection, in some embodiments, may involve detecting light or one or more other properties of the particle 403 as it passes through the monitoring region 407. In FIG. 4, one detection station 408 is shown with one monitoring region 407. In some embodiments of the particle analysis system 401, multiple detection stations may be provided. Additionally, some detection stations may monitor more than one region.

[0077] Each signal is assigned a signal value, generating a data point for each particle. As described above, this data may be referred to as event data. The data points may be multidimensional data points that include values ​​for each property measured for the particle. The detection system 404 is configured to collect a series of such data points over a first time interval.

[0078] The particle analysis system 401 may further include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system 406 may further compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0079] 5 is a functional block diagram of an example particle analysis control system for analyzing and displaying biological events, such as an analysis controller 500. The analysis controller 500 can be configured to perform various processes for controlling the graphical display of biological events.

[0080] A particle analyzer or particle sorting system 502 may be configured to acquire the biological event data. For example, a flow cytometer may generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or particle sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0081] The analysis controller 500 may be configured to receive biological event data from the particle analyzer or particle sorting system 502. The biological event data received from the particle analyzer or particle sorting system 502 may include flow cytometry event data. The analysis controller 500 may be configured to provide a graphical display including a first plot of the biological event data on the display device 506. The analysis controller 500 may be further configured to render a region of interest as a gate around a population of the biological event data displayed by the display device 506, e.g., overlaid on the first plot. In some embodiments, the gate may be a logical combination of one or more illustrated regions of interest plotted on a histogram or bivariate plot of a parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0082] Analysis controller 500 may further be configured to display the in-gate biological event data on display device 506 differently from other events in the outside-gate biological event data. For example, analysis controller 500 may be configured to render the color of the biological event data included within the gate differently from the color of the outside-gate biological event data. Display device 506 may be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0083] The analysis controller 500 may be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device may be implemented as a mouse 510. The mouse 510 may initiate a gate selection signal to the analysis controller 500 identifying a gate to be displayed or manipulated via the display device 506 (e.g., by clicking on or within the desired gate when the cursor is over the desired gate). In some embodiments, the first device may be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, a pen, a photodetector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, as shown in FIG. 5, the mouse 510 may include a right mouse button and a left mouse button, and the right mouse button and the left mouse button may each generate a trigger event.

[0084] A trigger event can cause the analysis controller 500 to change how the data is displayed, which portions of the data are actually displayed on the display device 506, and / or provide input to further processing, such as selecting a population of interest for particle sorting.

[0085] In some embodiments, the analysis controller 500 may be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 may further be configured to automatically modify the visualization of the plot to facilitate gating. This modification may be made based on a particular distribution of the biological event data received by the analysis controller 500.

[0086] The analysis controller 500 may be connected to a storage device 504. The storage device 504 may be configured to receive and store biological event data from the analysis controller 500. The storage device 504 may be further configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 may be further configured to enable retrieval of biological event data, such as flow cytometry event data, by the analysis controller 500.

[0087] A display device 506 may be configured to receive display data from the analysis controller 500. The display data may include plots of the biological event data and gates outlining sections of the plots. The display device 506 may be further configured to modify the information displayed in response to input received from the analysis controller 500 in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0088] In some embodiments, the analysis controller 500 can generate a user interface for receiving example events for filtering. For example, the user interface can include controls for receiving example events or example images. The example events or images, or example gates, can be provided prior to collection of event data for a sample, or can be provided based on an initial set of events for a portion of the sample.

[0089] FIG. 6A is a schematic diagram illustrating a particle sorting system 600 (e.g., particle analyzer or particle sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in FIG. 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, include, or be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned in a single file and traverse a monitoring region 611 (e.g., where the laser and the stream intersect) that is illuminated by an illumination source 612 (e.g., a laser). Vibration of droplet-forming transducer 602 causes moving fluid column 608 to break up into multiple droplets 610 , some of which contain particles 609 .

[0090] During operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring region 611. The detection station 614 feeds a timing circuit 628, which in turn feeds a flash charge circuit 630. At a droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608 so that the droplet of interest carries a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the charged droplets into a receptacle, such as a collection tube or a multi-well or microwell sample plate, where a well or microwell can be specifically associated with the droplet of interest. As shown in FIG. 6A, the droplets can be collected in a drain receptacle 638.

[0091] A detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal as a particle of interest passes through the monitoring region 611. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 616 enables the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may provide an amplitude signal 620 and / or a phase signal 618, which are then provided (via an amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be provided within the control system.

[0092] In some embodiments, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled to a memory configured to store detected events and sorting decisions based on the detected events. The sorting decisions can be included in the event data for the particles. In some embodiments, detection system 616 and detection station 614 can be implemented as a single detection unit or can be communicatively coupled such that event measurements can be collected by either detection system 616 or detection station 614 and provided to a non-collection element.

[0093] FIG. 6B is a schematic diagram illustrating a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in FIG. 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream of charging wires within the barbs, generating a stream of droplets 610 containing particles 609 for analysis. The particles can be illuminated using one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The information about the particles is analyzed by sorting electronics or other detection systems (not shown in FIG. 6B). Deflection plates 652 and 654 can be independently controlled to attract or repel the charged droplets and direct them toward a desired collection vessel (e.g., one of 672, 674, 676, or 678). 6B, deflector plates 652 and 654 can be controlled to direct particles along a first path 662 toward a container 674 or along a second path 668 toward a container 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector plates may allow the particle to continue along path 664. Such uncharged droplets may be directed into a waste container, such as via an aspirator 670.

[0094] Sorting electronics can be included to initiate the collection of measurements, receive fluorescent signals for the particles, and determine how to adjust the deflection plates to sort the particles. An exemplary implementation of the embodiment shown in Figure 6B includes the BDFACSAria™ line of flow cytometers commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0095] method Aspects of the present disclosure further include methods, including introducing a sample fluid into a flow cytometer having a flow cell of the present disclosure and analyzing the sample fluid by illuminating particles in the flow stream. As described above, the subject flow cytometer includes a flow cell having a cuvette configured to carry particles in a flow stream, the cuvette having a channel with a rectangular cross-section and an aspect ratio in the range of 1.0 to 1.4, a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell, and a detector configured to collect light emitted from the illuminated particles.

[0096] In some embodiments, the disclosed methods operate in (a) an imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a first velocity, and (b) a non-imaging mode, in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a second velocity that is higher than the first velocity. The methods may operate in either the imaging mode or the non-imaging mode, and may switch between these modes.

[0097] In some cases, the sample analyzed in the present method is a biological sample. The term "biological sample" is used in its conventional sense to refer to a whole organism, a plant, a fungus, or a subset of animal tissues, cells, or components, such as may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, or semen, as the case may be. Thus, "biological sample" refers to both a naturally occurring organism or a subset of its tissues, and homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. Biological samples may be any type of organismal tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, etc., and in some cases, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger stick (which may or may not be combined with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0098] In some embodiments, the sample source is a "mammal" or "mammalian," which terms are used broadly to describe organisms belonging to the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., newborn, infant, juvenile, adolescent, adult), and in some embodiments, the human subject is a juvenile, adolescent, or adult. While the present disclosure may be applied to samples from human subjects, it should be understood that the methods may also be performed on samples from other animal subjects (i.e., "non-human subjects"), including, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0099] Cells of interest may be subject to characterization according to various parameters, such as phenotypic characteristics identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analysis droplets determined to contain target cells. A variety of cells may be characterized using the subject methods. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells bearing convenient cell surface markers or antigens that may be internalized or labeled by convenient affinity agents or their conjugates. For example, target cells may comprise cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0100] In practicing the subject methods, a volume of an initial fluid sample is injected into a flow cytometer. The volume of sample injected into the particle sorting module may vary, for example, within the range of 0.001 mL to 1000 mL of sample, e.g., 0.005 mL to 900 mL, e.g., 0.01 mL to 800 mL, e.g., 0.05 mL to 700 mL, e.g., 0.1 mL to 600 mL, e.g., 0.5 mL to 500 mL, e.g., 1 mL to 400 mL, e.g., 2 mL to 300 mL, e.g., 5 mL to 100 mL.

[0101] Methods according to embodiments of the present disclosure enumerate and optionally sort labeled particles (e.g., target cells) in a sample. In practicing the subject methods, a fluid sample containing particles is first introduced into a flow nozzle of the system. Upon exiting the flow nozzle, the particles pass substantially one at a time through a sample interrogation region where each particle is illuminated by a light source, and measurements of light scattering parameters, and optionally fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements), are recorded separately for each particle, as desired. Depending on the characteristics of the flow stream being interrogated, the light may illuminate a flow stream of 0.001 mm or greater, e.g., 0.005 mm or greater, e.g., 0.01 mm or greater, e.g., 0.05 mm or greater, e.g., 0.1 mm or greater, e.g., 0.5 mm or greater, e.g., 1 mm or greater. In certain embodiments, the methods illuminate a planar cross-section of the flow stream within the sample interrogation region, e.g., with a laser (as described above). In another embodiment, the method illuminates a predetermined length of the flow stream within the sample interrogation region to correspond to the illumination profile of a diffuse laser beam or lamp.

[0102] In some embodiments, the method irradiates the flow stream at or near the nozzle orifice of the flow cell. For example, the method may irradiate the flow stream at about 0.001 mm or more from the nozzle orifice, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, for example 1 mm or more from the nozzle orifice. In some embodiments, the method irradiates the flow stream directly adjacent to the nozzle orifice of the flow cell.

[0103] In embodiments of the method, detectors such as photomultiplier tubes (PMTs) are used to record the light passing through each particle (sometimes referred to as forward scattered light), the light reflected perpendicular to the direction of particle flow through the detection region (sometimes referred to as orthogonal or side scattered light), and, if the particles are labeled with one or more fluorescent markers, the fluorescence emitted by the particles as they pass through the detection region and are illuminated by an energy source. Forward scattered light (FSC), side scattered light (SSC), and fluorescent emission each have separate parameters for each particle (or "event"). Thus, for example, two, three, or four parameters may be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle may be analyzed in real time or stored in a data storage and analysis means, such as a computer, as desired.

[0104] In some embodiments, particles are detected and uniquely identified by exposing them to excitation light and measuring the fluorescence of each particle in one or more detection channels, as desired. The fluorescence emitted in the detection channels used to identify particles and their associated binding complexes may be measured after excitation by a single light source, or may be measured separately after excitation by different light sources. When separate excitation light sources are used to excite particle labels, the particle labels may be selected such that all particle labels are excitable by each of the excitation light sources used.

[0105] In some embodiments, the method further includes data collection, analysis, and recording, e.g., using a computer, where multiple data channels record data from each detector regarding light scattering and fluorescence emitted by each particle as it passes through the sample interrogation region of the particle sorting module. In these embodiments, particles are classified and counted during analysis, with each particle present as a set of digitized parameter values. The subject system may be configured to trigger on selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for parameter detection and may be used as a means for detecting when a particle has passed through a light source. Detection of an event exceeding the selected parameter threshold triggers the collection of light scattering and fluorescence data for the particle. Data related to particles or other components in the analysis medium that cause a response below the threshold is not acquired. The trigger parameter may be detection of forward scattered light resulting from a particle passing through a light beam. In this manner, the flow cytometer detects and collects light scattering and fluorescence data for particles.

[0106] Specific subpopulations of interest are then further analyzed by "gating" based on the data collected for the entire population. To select an appropriate gate, the data is plotted to separate the subpopulations as best as possible. This procedure may be performed by plotting forward scatter (FSC) versus side (i.e., orthogonal) scatter (SSC) on a two-dimensional dot plot. A subpopulation of particles (i.e., cells within the gate) is then selected, and particles not within the gate are excluded. If desired, a gate may be selected by drawing a line around the desired subpopulation using a cursor on the computer screen. Only those particles within the gate are then further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to calculate the number of particles of interest in the sample.

[0107] Featured methods may also use the particles in research, clinical trials, or treatment. In some embodiments, the subject methods obtain individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods obtain cells from a fluid or tissue sample used as a research or diagnostic specimen for a disease such as cancer. Similarly, the subject methods obtain cells from a fluid or tissue sample used for treatment. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissue, may be prepared and introduced into a subject as a therapeutic treatment. Conditions that may be treated by administration of flow cytometry-sorted samples include, but are not limited to, blood disorders, immune system disorders, organ damage, and the like.

[0108] A typical cell therapy protocol may include the steps of sample collection, cell isolation, genetic modification, culture, in vitro expansion, cell harvesting, sample volume reduction, sample washing, biopreservation, storage, and cell introduction into a subject. A protocol may begin by collecting viable cells and tissue from a tissue source in a subject to generate a cell and / or tissue sample. The sample may be collected by any suitable procedure, including, for example, administering a cell mobilizing agent to the subject, drawing blood from the subject, or removing bone marrow from the subject. After sample collection, cell enrichment may be performed by multiple methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, fluorescence-activated cell sorting (FACS), and the like. In some cases, the enriched cells may be genetically modified by any convenient method, such as nuclease-mediated gene editing. Genetically modified cells may be cultured, activated, and expanded in vitro. In some cases, the cells are preserved, e.g., cryopreserved, and stored for future use. At the time of use, the cells are thawed and then administered to a patient, e.g., the cells may be infused into a patient.

[0109] Aspects of the present disclosure further include methods of assembling a flow cytometer. The methods of the present disclosure include disposing a flow cell of the present disclosure in a flow cytometer. As described above, the flow cell of the present disclosure includes a cuvette configured to carry particles in a flow stream, the cuvette having a flow channel with a rectangular cross-section and an aspect ratio in the range of 1.0 to 1.4. Some embodiments of the methods also include fluidly connecting a sample fluid line to the flow cell, the sample fluid line being configured to supply particles to the flow cell. In some cases, the methods include disposing a collection lens in optical communication with the flow cell and a detector. Some embodiments of the methods include disposing an objective lens in optical communication with a light source and the flow cell.

[0110] Computer Control System Aspects of the present disclosure further include a computer control system, the computer control system comprising one or more computers for full or partial automation. In some embodiments, the system comprises a computer having a non-transitory computer-readable storage medium having a computer program stored thereon, the computer program comprising instructions for performing the methods of the present disclosure when loaded into the computer. For example, the computer may be configured to switch between operating modes of a flow cytometer, such as between (a) an imaging mode, in which the flow cytometer is configured to deliver particles in a flow stream to a flow cell at a first velocity, and (b) a non-imaging mode, in which the flow cytometer is configured to deliver particles in a flow stream to a flow cell at a second velocity higher than the first velocity.

[0111] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access a memory in which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, an input / output controller, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are or become available. The processor executes an operating system that interfaces with firmware and hardware in a well-known manner to facilitate the processor's coordination and execution of functions of various computer programs, which may be written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically cooperates with the processor to coordinate and execute functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0112] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-write compact disk, flash memory device, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, tape drive, or diskette drive. These types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk. Any of these program storage media, or other program storage media now in use or that may be developed in the future, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or on program storage devices used in conjunction with the memory storage devices.

[0113] In some embodiments, a computer program product is described that includes a computer-usable medium having stored thereon control logic (a computer software program including program code). The control logic, when executed by a processor, a computer, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementation of hardware state machines to perform the functions described herein will be apparent to one skilled in the relevant art.

[0114] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, tape, RAM, or any other suitable device, fixed or portable). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium storing the necessary program code. The program may be provided to the processor remotely via a communication channel or pre-recorded on a computer program product, such as a memory, or on other portable or fixed computer-readable storage media using one of these devices connected to the memory. For example, a magnetic or optical disk may store the program and be read by a disk writer / reader. The system of the present disclosure may further include a program, e.g., in the form of a computer program product, an algorithm for use in implementing the method as described above. The program according to the present disclosure may be recorded on a computer-readable medium, e.g., any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media, such as magnetic disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0115] The processor may also access a communication channel to communicate with a user at a remote location, meaning that the user does not have direct contact with the system but instead relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0116] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications such as radio frequency identification (RFID), ZigBee communications protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0117] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communication.

[0118] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, allowing the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that a user may use in conjunction with the device.

[0119] In one embodiment, the communication interface is configured to provide connectivity for data transfer using Internet Protocol (IP) via a cellular network, short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0120] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communications interface using a common standard, such as 802.11, Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0121] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored in the subject system, e.g., any data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0122] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved using a network or other type of remote communication in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, according to known techniques. Presentation of data by the output manager may be performed according to various known techniques. In some examples, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses so that the user can obtain additional SQL, HTML, XML, or other documents or data from remote sources. The platform or platforms present in the subject system are typically a class of computers commonly referred to as servers, but may be any type of computer platform now known or later developed. However, the platforms may also be mainframe computers, workstations, or other computer types. The platforms may be networked or not, and may be connected via any type of cabling, now known or later, or other communication systems, including wireless systems. The platforms may be co-located or physically separated.Various operating systems may be used on any of the computer platforms, depending in some cases on the type and / or configuration of the computer platform selected. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, Windows 10, iOS, macOS, Linux, Ubuntu, Fedora, OS / 400, i5 / OS, IBM i, Android, SGI IRIX, Oracle Solaris, and the like.

[0123] FIG. 7 illustrates a general configuration of an exemplary computing device 700 according to one embodiment. The general configuration of computing device 700 illustrated in FIG. 7 includes an arrangement of computer hardware and software components. However, not all of these typical conventional elements need be shown to provide a useful disclosure. As illustrated, computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and input devices 760, all of which may communicate with each other via a communications bus. Network interface 720 may provide connectivity to one or more networks or computing systems. Thus, processing unit 710 may receive information and instructions from other computing systems or services via a network. Processing unit 710 may further communicate with memory 770 and may further provide output information for an optional display 750 via input / output device interface 740. For example, analysis software (e.g., data analysis software or program, e.g., FlowJo®) stored as executable instructions in non-transitory memory of the analysis system can display flow cytometry event data to a user. The input / output device interface 740 may further accept input from any input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0124] Memory 770 may include computer program instructions (grouped in some embodiments as modules or components) that processing unit 710 executes to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in the general management and operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0125] kit Aspects of the present disclosure further include kits. Kits of interest include one or more flow cells having optimized geometries. In one embodiment, the kit includes one flow cell. In other embodiments, the kit includes multiple flow cells, e.g., one or more flow cells, e.g., two or more flow cells, e.g., three or more flow cells, e.g., four or more flow cells, e.g., five or more flow cells. Kits according to some embodiments further include one or more focusing lenses and / or one or more objective lenses for use with the subject flow cells.

[0126] In addition to the above components, the subject kits may (in some embodiments) further include instructions for use, such as for installing a flow cell of the present disclosure into a flow cytometer. These instructions may be present in the subject kits in a variety of forms, and one or more of these instructions may be present in the kit. One form in which these instructions may be provided is as information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, a package insert, etc. Another form in which these instructions may be provided is as a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Another form in which these instructions may be present is a website address that may be used via the Internet to access the information at a remote location.

[0127] usefulness The subject flow cells, flow cytometers, and methods are used in a variety of applications where it is desirable to analyze components in a sample in a fluid medium. The present disclosure is particularly useful for improving core stream quality in fluid systems. The present disclosure may be used, for example, when it is desirable to operate the same flow cytometer (i.e., a flow cytometer having the same flow cell) across different operating modes (e.g., imaging and non-imaging modes).

[0128] Embodiments of the present disclosure find use in applications where cells prepared from a biological sample may be desirable for use in research, clinical trials, or therapy. In some embodiments, the subject methods and devices may facilitate obtaining and / or analyzing individual cells prepared from a targeted fluid or tissue biological sample. For example, the subject methods and systems facilitate obtaining cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the subject methods and systems may facilitate obtaining cells from fluid or tissue samples used for therapy.

[0129] The following are given by way of example and not limitation.

[0130] experiment A cuvette channel shape that optimally supports the flow cytometer platform is sought. A trade-space analysis was performed on possible analyzer cuvette shapes by imposing design constraints from imaging, light collection, and fluidics considerations. Cuvette channel lengths of 50 to 500 μm in Y and Z were examined, and the aspect ratio was defined as Y / Z. Core velocities of 1 m / s, 3.3 m / s, and 5.5 m / s were investigated. A Zemax model was used to calculate collection efficiency and (flat-top) optical CV. Imaging constraints included a 1 m / s flow velocity, a desire to minimize the aspect ratio, and a 60 μm imaging spot size; therefore, the maximum core constraint was set at 50 μm. Optical constraints included a desire to maximize the aspect ratio for collection efficiency and increased CV for elliptical core streams greater than 30 μm × 40 μm (Z × Y) due to fiber collection clipping. Fluidic constraints include a sheath flow rate greater than 1 ml / min with a sample / sheath ratio greater than 0.1, a sheath flow rate less than 20 ml / min to avoid exceeding historical sheath consumption, and a pressure drop less than 6 psi to accommodate small, low-cost pumps. Ideally, the imaging analyzer can operate at higher speeds and higher throughput modes.

[0131] Fluid and core stream constraints were applied to the Y-channel and Z-channel length matrices, and constraint mismatches were color coded. The results are shown in Figure 8. The uncolored central region represents the only shape that satisfies all constraints. The shape with the largest aspect ratio that satisfied all constraints for imaging was 250 μm × 300 μm. This resulted in a core stream diameter of 35 μm × 50 μm at a velocity of 1 m / s.

[0132] The collection efficiency of the internal pores only was modeled using Zemax, and the results are shown in Table 1 below.

[0133] [Table 1]

[0134] The maximum core stream size was added to model collection for the proposed 250 × 300 geometry flat-top beam. Modeling of the change in collection efficiency due to bead displacement within the 250 μm × 300 μm cuvette hole and the objective lens is shown in Figure 9. The non-uniform response to Z displacement suggests that the Z position should be offset by 15 μm from the nominal value in the model. The calculated CVs from the collection efficiency results are shown in Figures 10A and 10B.

[0135] Regardless of the scope of the appended claims, the present invention may be further defined by the following notes.

[0136] Clause 1. A flow cell comprising a cuvette configured to carry particles in a flow stream, the cuvette having a flow path with a rectangular cross section and an aspect ratio in the range of 1.0 to 1.4; a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell; a detector configured to collect light emitted from the illuminated particles; A flow cytometer comprising:

[0137] Appendix 2. The flow cytometer of Appendix 1, wherein the aspect ratio is within the range of 1.1 to 1.3.

[0138] Appendix 3. The flow cytometer of Appendix 2, wherein the aspect ratio is about 1.2.

[0139] Appendix 4: A flow cytometer according to any one of Appendixes 1 to 3, wherein the rectangular cross section of the flow channel has a length within a range of 275 μm to 325 μm.

[0140] Appendix 5. The flow cytometer according to Appendix 4, wherein the length of the rectangular cross section of the flow channel is within the range of 290 μm to 310 μm.

[0141] Appendix 6. The flow cytometer according to Appendix 5, wherein the length of the rectangular cross section of the flow channel is within the range of 295 μm to 305 μm.

[0142] Appendix 7. The flow cytometer according to any one of Appendixes 1 to 6, wherein the rectangular cross section of the flow channel has a width within the range of 225 μm to 275 μm.

[0143] Appendix 8. The flow cytometer according to Appendix 7, wherein the width of the rectangular cross section of the flow channel is within the range of 240 μm to 260 μm.

[0144] Appendix 9. The flow cytometer according to Appendix 8, wherein the width of the rectangular cross section of the flow channel is within the range of 245 μm to 255 μm.

[0145] Appendix 10. The flow cytometer of any one of appendices 1 to 9, configured as an imaging flow cytometer.

[0146] Appendix 11. A flow cytometer as described in Appendix 10, wherein the light source has a light beam generating unit configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light.

[0147] Clause 12. The flow cytometer of clause 11, further comprising an acousto-optic deflector (AOD).

[0148] Appendix 13. A flow cytometer according to any one of appendices 10 to 12, further comprising a sample fluid line configured to supply particles to the flow cell.

[0149] Clause 14. The flow cytometer of clause 13, configured to deliver particles in the flow stream at a velocity in the range of 0.1 m / s to 10 m / s.

[0150] Clause 15.(a) an imaging mode in which the flow cytometer is configured to deliver particles in a flow stream to a flow cell at a first velocity; and (b) a non-imaging mode in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a second velocity that is greater than the first velocity; 15. The flow cytometer of claim 13 or 14, configured to operate at

[0151] Appendix 16. The flow cytometer of Appendix 15, wherein the first velocity is within the range of 0.5 m / s to 1.5 m / s.

[0152] Appendix 17. The flow cytometer of Appendix 16, wherein the first velocity is within the range of 0.7 m / s to 1.3 m / s.

[0153] Appendix 18. The flow cytometer of Appendix 17, wherein the first velocity is within the range of 0.9 m / s to 1.1 m / s.

[0154] Appendix 19. The flow cytometer of any one of Appendixes 15 to 18, wherein the second velocity is within the range of 3 m / s to 8 m / s.

[0155] 20. The flow cytometer of claim 19, wherein the second velocity is within the range of 4 m / s to 7 m / s.

[0156] Item 21. The flow cytometer of item 20, wherein the second velocity is within the range of 5 m / s to 6 m / s.

[0157] Appendix 22. The flow cytometer of any one of Appendixes 15 to 21, wherein the ratio of the first speed to the second speed is within the range of 1:12 to about 1:2.

[0158] Appendix 23. The flow cytometer of any one of appendices 1 to 22, further comprising a focusing lens in optical communication with the flow cell and the detector.

[0159] Item 24. The flow cytometer of item 23, wherein the focusing lens is coupled to the flow cell.

[0160] Appendix 25. The flow cytometer according to Appendix 23 or 24, wherein the condenser lens has a numerical aperture (NA) in the range of 0.75 to 1.5.

[0161] Appendix 26. The flow cytometer of Appendix 25, wherein the focusing lens has an NA in the range of 0.9 to 1.4.

[0162] Appendix 27. The flow cytometer of Appendix 26, wherein the focusing lens has an NA in the range of 1.1 to 1.3.

[0163] Appendix 28. A flow cytometer according to any one of appendices 23 to 27, wherein the focusing lens has a focusing angle (θ) within the range of 90 degrees to approximately 110 degrees.

[0164] Appendix 29. The flow cytometer of Appendix 28, wherein the focusing lens has a focusing angle in the range of 95 degrees to 105 degrees.

[0165] Appendix 30. The flow cytometer of Appendix 29, wherein the focusing lens has a focusing angle in the range of 97 degrees to 103 degrees.

[0166] Appendix 31. A flow cytometer according to any one of appendices 23 to 30, wherein the surface of the cuvette is separated from the flow path by a distance in the range of 1.9 mm to 2.1 mm.

[0167] Appendix 32. The flow cytometer of Appendix 31, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 2 mm to 2.05 mm.

[0168] Appendix 33. The flow cytometer of Appendix 32, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 2.02 mm to 2.04 mm.

[0169] Appendix 34. The flow cytometer of any one of appendices 23 to 30, further comprising an objective lens in optical communication with the light source and the flow cell.

[0170] Item 35. The flow cytometer of item 34, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5 mm to 5.2 mm.

[0171] Appendix 36. The flow cytometer of Appendix 35, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5.05 mm to 5.1 mm.

[0172] Appendix 37. The flow cytometer of Appendix 36, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 5.07 mm to 5.09 mm.

[0173] Appendix 38. A flow cytometer according to any one of appendices 31 to 37, wherein the surface of the cuvette is provided with an anti-reflective coating.

[0174] Appendix 39. A flow cytometer according to any one of appendices 1 to 38, wherein the cuvette has a height perpendicular to the rectangular cross section in the range of 5 mm to 9 mm.

[0175] Appendix 40. The flow cytometer of Appendix 39, wherein the height of the cuvette is within the range of 6 mm to 8 mm.

[0176] Appendix 41. The flow cytometer of Appendix 40, wherein the height of the cuvette is within the range of 7 mm to 7.2 mm.

[0177] Appendix 42. A flow cytometer according to any one of appendices 1 to 41, wherein the cuvette has a length in the range of 8 mm to 12 mm.

[0178] Appendix 43. The flow cytometer of Appendix 42, wherein the length of the cuvette is within the range of 9 mm to 11 mm.

[0179] Appendix 44. The flow cytometer of Appendix 43, wherein the length of the cuvette is within the range of 10 mm to 10.2 mm.

[0180] Appendix 45. A flow cytometer according to any one of appendices 1 to 44, wherein the width of the cuvette is within the range of 2 mm to 6 mm.

[0181] Appendix 46. The flow cytometer of Appendix 45, wherein the width of the cuvette is within the range of 3 mm to 5 mm.

[0182] Item 47. The flow cytometer of item 46, wherein the width of the cuvette is within the range of 4 mm to 4.1 mm.

[0183] Appendix 48. A flow cytometer according to Appendix 1, wherein the ratio of the width of the rectangular cross section of the flow channel to the width of the cuvette is within the range of 1:8.9 to 1:21.8.

[0184] Appendix 49. A flow cytometer according to Appendix 48, wherein the ratio of the width of the rectangular cross section of the flow channel to the width of the cuvette is within the range of 1:12.5 to 1:19.2.

[0185] Appendix 50. A flow cytometer according to Appendix 49, wherein the ratio of the width of the rectangular cross section of the flow channel to the width of the cuvette is within the range of 1:16.1 to 1:16.3.

[0186] Appendix 51. A flow cytometer according to Appendix 1, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:22.2 to 1:32.7.

[0187] Appendix 52. A flow cytometer according to Appendix 51, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:25 to 1:30.1.

[0188] Appendix 53. A flow cytometer according to Appendix 52, wherein the ratio of the width of the rectangular cross section of the channel to the height perpendicular to the rectangular cross section is within the range of 1:28.2 to 1:28.6.

[0189] Appendix 54. A flow cytometer according to Appendix 1, wherein the ratio of the length of the rectangular cross section of the flow channel to the length of the cuvette is within the range of 1:29.1 to 1:40.

[0190] Appendix 55. A flow cytometer according to Appendix 54, wherein the ratio of the length of the rectangular cross section of the flow channel to the length of the cuvette is within the range of 1:31 to 1:35.6.

[0191] Appendix 56. A flow cytometer according to Appendix 55, wherein the ratio of the length of the rectangular cross section of the flow channel to the length of the cuvette is within the range of 1:33.4 to 1:33.9.

[0192] Appendix 57. A flow cytometer according to Appendix 1, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:18.2 to 1:27.7.

[0193] Appendix 58. A flow cytometer according to Appendix 57, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:20.7 to 1:25.8.

[0194] Appendix 59. A flow cytometer according to Appendix 58, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:23.6 to 1:23.7.

[0195] Item 60. A flow cytometer according to any one of items 1 to 59, wherein the cuvette is constructed of fused silica.

[0196] Appendix 61. A flow cytometer according to any one of appendices 1 to 60, wherein the flow cell has a collection efficiency in the range of 20% to 40%.

[0197] Item 62. The flow cytometer of item 61, wherein the flow cell has a collection efficiency in the range of 21% to 30%.

[0198] Item 63. The flow cytometer of item 62, wherein the flow cell has a collection efficiency in the range of 23% to 25%.

[0199] Appendix 64. A flow cytometer according to any one of appendices 1 to 63, wherein the rectangular cross section of the channel is symmetrical across the width, length, or both the width and length of the cuvette.

[0200] Addendum 65. A flow cytometer according to any one of Addendums 1 to 64, wherein the flow path extends the entire height of the cuvette.

[0201] Appendix 66. A method for analyzing a sample fluid, comprising: (a) a flow cell comprising a cuvette configured to carry particles in a flow stream, the cuvette having a flow path with a rectangular cross section and an aspect ratio in the range of 1.0 to 1.4; a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell; a detector configured to collect light emitted from the illuminated particles; introducing a sample fluid into a flow cytometer comprising: (b) A method for analyzing a sample fluid by irradiating particles in the flow stream.

[0202] Item 67. The method of item 66, wherein the aspect ratio is in the range of 1.1 to 1.3.

[0203] Item 68. The method of item 67, wherein the aspect ratio is approximately 1.2.

[0204] Addendum 69. The method of any one of Addendums 66 to 68, wherein the rectangular cross-section of the channel has a length in the range of 275 μm to 325 μm.

[0205] Appendix 70. The method of Appendix 69, wherein the length of the rectangular cross section of the channel is in the range of 290 μm to 310 μm.

[0206] Appendix 71. The method of Appendix 70, wherein the length of the rectangular cross section of the channel is in the range of 295 μm to 305 μm.

[0207] Addendum 72. The method of any one of Addendums 66-71, wherein the rectangular cross-section of the channel has a width in the range of 225 μm to 275 μm.

[0208] Addendum 73. The method of Addendum 72, wherein the width of the rectangular cross section of the channel is in the range of 240 μm to 260 μm.

[0209] Appendix 74. The method of Appendix 73, wherein the width of the rectangular cross section of the channel is in the range of 245 μm to 255 μm.

[0210] Addendum 75. The method of any one of Addendums 66 to 74, wherein the flow cytometer is configured as an imaging flow cytometer.

[0211] Clause 76. The method of clause 75, wherein the light source comprises a light beam generator configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light.

[0212] Clause 77. The method of clause 76, wherein the flow cytometer further comprises an acousto-optic deflector (AOD).

[0213] Addendum 78. The method of any one of Addendums 75 to 77, wherein the flow cytometer further comprises a sample fluid line configured to supply particles to the flow cell.

[0214] Item 79. The method of item 78, wherein the flow cytometer is configured to deliver particles in the flow stream at a velocity in the range of 0.1 m / s to 10 m / s.

[0215] Clause 80.(a) an imaging mode in which the flow cytometer is configured to deliver particles in a flow stream to a flow cell at a first velocity; and (b) a non-imaging mode in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a second velocity that is greater than the first velocity; 80. The method of claim 78 or 79, wherein the method operates on

[0216] Item 81. The method of item 80, wherein the first velocity is in the range of 0.5 m / s to 1.5 m / s.

[0217] 82. The method of claim 81, wherein the first velocity is in the range of 0.7 m / s to 1.3 m / s.

[0218] 83. The method of claim 82, wherein the first velocity is in the range of 0.9 m / s to 1.1 m / s.

[0219] Addendum 84. The method of any one of Addendums 80 to 83, wherein the second velocity is in the range of 3 m / s to 8 m / s.

[0220] 85. The method of claim 84, wherein the second velocity is in the range of 4 m / s to 7 m / s.

[0221] 86. The method of claim 85, wherein the second velocity is in the range of 5 m / s to 6 m / s.

[0222] Clause 87. The method of any one of clauses 80-86, wherein the ratio of the first speed to the second speed is in the range of 1:12 to about 1:2.

[0223] Addendum 88. The method of any one of Addendums 66 to 87, wherein the flow cytometer further comprises a focusing lens in optical communication with the flow cell and the detector.

[0224] Item 89. The method of item 88, wherein the focusing lens is coupled to the flow cell.

[0225] Addendum 90. The method of Addendum 88 or 89, wherein the focusing lens has a numerical aperture (NA) in the range of 0.75 to 1.5.

[0226] Item 91. The method of item 90, wherein the focusing lens has an NA in the range of 0.9 to 1.4.

[0227] Item 92. The method of item 91, wherein the focusing lens has an NA in the range of 1.1 to 1.3.

[0228] Addendum 93. The method of any one of Addendums 88-92, wherein the focusing lens has a focusing angle (θ) in the range of 90 degrees to about 110 degrees.

[0229] Item 94. The method of item 93, wherein the focusing lens has a focusing angle in the range of 95 degrees to 105 degrees.

[0230] Item 95. The method of item 94, wherein the focusing lens has a focusing angle in the range of 97 degrees to 103 degrees.

[0231] Addendum 96. The method of any one of Addendums 88 to 95, wherein the surface of the cuvette is separated from the flow path by a distance in the range of 1.9 mm to 2.1 mm.

[0232] Item 97. The method of item 96, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 2 mm to 2.05 mm.

[0233] Item 98. The method of item 97, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 2.02 mm to 2.04 mm.

[0234] Addendum 99. The method of any one of Addendums 66 to 98, wherein the flow cytometer further comprises an objective lens in optical communication with the light source and the flow cell.

[0235] Item 100. The method of item 99, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5 mm to 5.2 mm.

[0236] Item 101. The method of item 100, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5.05 mm to 5.1 mm.

[0237] Item 102. The method of item 101, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 5.07 mm to 5.09 mm.

[0238] Addendum 103. The method of any one of Addendums 96 to 102, wherein the cuvette further has an anti-reflective coating on its surface.

[0239] Addendum 104. The method of any one of Addendums 66 to 103, wherein the cuvette has a height perpendicular to the rectangular cross section in the range of 5 mm to 9 mm.

[0240] Addendum 105. The method of Addendum 104, wherein the height of the cuvette is in the range of 6 mm to 8 mm.

[0241] Addendum 106. The method of Addendum 105, wherein the height of the cuvette is within the range of 7 mm to 7.2 mm.

[0242] Addendum 107. The method of any one of Addendums 66 to 106, wherein the cuvette has a length in the range of 8 mm to 12 mm.

[0243] Addendum 108. The method of Addendum 107, wherein the length of the cuvette is within the range of 9 mm to 11 mm.

[0244] Addendum 109. The method of Addendum 108, wherein the length of the cuvette is within the range of 10 mm to 10.2 mm.

[0245] Addendum 110. The method of any one of Addendums 66 to 109, wherein the width of the cuvette is within the range of 2 mm to 6 mm.

[0246] Addendum 111. The method of Addendum 110, wherein the width of the cuvette is in the range of 3 mm to 5 mm.

[0247] Addendum 112. The method of Addendum 111, wherein the width of the cuvette is in the range of 4 mm to 4.1 mm.

[0248] Addendum 113. The method described in Addendum 66, wherein the ratio of the width of the rectangular cross section of the channel to the width of the cuvette is within the range of 1:8.9 to 1:21.8.

[0249] Addendum 114. The method according to Addendum 113, wherein the ratio of the width of the rectangular cross section of the channel to the width of the cuvette is within the range of 1:12.5 to 1:19.2.

[0250] Addendum 115. The method according to Addendum 114, wherein the ratio of the width of the rectangular cross section of the channel to the width of the cuvette is within the range of 1:16.1 to 1:16.3.

[0251] Addendum 116. The method of Addendum 66, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:22.2 to 1:32.7.

[0252] Addendum 117. The method according to Addendum 116, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is in the range of 1:25 to 1:30.1.

[0253] Addendum 118. The method according to Addendum 117, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:28.2 to 1:28.6.

[0254] Addendum 119. The method described in Addendum 66, wherein the ratio of the length of the rectangular cross section of the channel to the length of the cuvette is within the range of 1:29.1 to 1:40.

[0255] Addendum 120. The method described in Addendum 119, wherein the ratio of the length of the rectangular cross section of the channel to the length of the cuvette is within the range of 1:31 to 1:35.6.

[0256] Addendum 121. The method described in Addendum 120, wherein the ratio of the length of the rectangular cross section of the channel to the length of the cuvette is within the range of 1:33.4 to 1:33.9.

[0257] Addendum 122. The method of Addendum 66, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:18.2 to 1:27.7.

[0258] Addendum 123. The method according to Addendum 122, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:20.7 to 1:25.8.

[0259] Addendum 124. The method according to Addendum 123, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:23.6 to 1:23.7.

[0260] Addendum 125. The method of any one of Addendums 66 to 124, wherein the cuvette is made of fused silica.

[0261] Addendum 126. The method of any one of Addendums 66 to 125, wherein the flow cell has a collection efficiency in the range of 20% to 40%.

[0262] Item 127. The method of item 126, wherein the flow cell has a collection efficiency in the range of 21% to 30%.

[0263] Item 128. The method of item 127, wherein the flow cell has a collection efficiency in the range of 23% to 25%.

[0264] Addendum 129. The method of any one of Addendums 66 to 128, wherein the rectangular cross-section of the channel is symmetrical across the width, length, or both width and length of the cuvette.

[0265] Addendum 130. The method of any one of Addendums 66 to 129, wherein the flow path extends the entire height of the cuvette.

[0266] Addendum 131. A flow cell comprising a cuvette configured to transport particles in a flow stream, the cuvette having a flow path with a rectangular cross-section and an aspect ratio in the range of 1.0 to 1.4.

[0267] Item 132. The flow cell of item 131, wherein the aspect ratio is in the range of 1.1 to 1.3.

[0268] Item 133. The flow cell of item 132, wherein the aspect ratio is about 1.2.

[0269] Appendix 134. A flow cell according to any one of appendices 131 to 133, wherein the rectangular cross section of the flow channel has a length in the range of 275 μm to 325 μm.

[0270] Appendix 135. The flow cell according to Appendix 134, wherein the length of the rectangular cross section of the flow channel is in the range of 290 μm to 310 μm.

[0271] Appendix 136. The flow cell according to Appendix 135, wherein the length of the rectangular cross section of the flow channel is in the range of 295 μm to 305 μm.

[0272] Addendum 137. A flow cell according to any one of Addendums 131 to 136, wherein the rectangular cross section of the flow channel has a width in the range of 225 μm to 275 μm.

[0273] Appendix 138. The flow cell according to Appendix 137, wherein the width of the rectangular cross section of the flow channel is in the range of 240 μm to 260 μm.

[0274] Appendix 139. The flow cell according to Appendix 138, wherein the width of the rectangular cross section of the flow channel is in the range of 245 μm to 255 μm.

[0275] Item 140. A flow cell according to any one of items 131 to 139, wherein the surface of the cuvette is separated from the flow path by a distance in the range of 1.9 mm to 2.1 mm.

[0276] Item 141. The flow cell of item 140, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 2 mm to 2.05 mm.

[0277] Item 142. The flow cell of item 141, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 2.02 mm to 2.04 mm.

[0278] Addendum 143. A flow cell according to any one of Addendums 131 to 139, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5 mm to 5.2 mm.

[0279] Item 144. The flow cell of item 143, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5.05 mm to 5.1 mm.

[0280] Item 145. The flow cell of item 144, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 5.07 mm to 5.09 mm.

[0281] Addendum 146. A flow cell according to any one of Addendums 140 to 145, wherein the surface of the cuvette is provided with an anti-reflective coating.

[0282] Addendum 147. The flow cell of any one of Addendums 131 to 146, wherein the cuvette has a height perpendicular to the rectangular cross section in the range of 5 mm to 9 mm.

[0283] Item 148. The flow cell of item 147, wherein the height of the cuvette is within the range of 6 mm to 8 mm.

[0284] Item 149. The flow cell of item 148, wherein the cuvette height is within the range of 7 mm to 7.2 mm.

[0285] Addendum 150. A flow cell according to any one of Addendums 131 to 149, wherein the cuvette has a length in the range of 8 mm to 12 mm.

[0286] Item 151. The flow cell of item 150, wherein the length of the cuvette is within the range of 9 mm to 11 mm.

[0287] Item 152. The flow cell of item 151, wherein the length of the cuvette is within the range of 10 mm to 10.2 mm.

[0288] Addendum 153. A flow cell according to any one of Addendums 131 to 152, wherein the width of the cuvette is within the range of 2 mm to 6 mm.

[0289] Item 154. The flow cell of item 153, wherein the width of the cuvette is within the range of 3 mm to 5 mm.

[0290] Item 155. The flow cell of item 154, wherein the width of the cuvette is within the range of 4 mm to 4.1 mm.

[0291] Addendum 156. A flow cell according to Addendum 131, wherein the ratio of the width of the rectangular cross section of the flow channel to the width of the cuvette is within the range of 1:8.9 to 1:21.8.

[0292] Appendix 157. A flow cell according to Appendix 156, wherein the ratio of the width of the rectangular cross section of the flow channel to the width of the cuvette is within the range of 1:12.5 to 1:19.2.

[0293] Appendix 158. A flow cell according to Appendix 157, wherein the ratio of the width of the rectangular cross section of the flow channel to the width of the cuvette is within the range of 1:16.1 to 1:16.3.

[0294] Appendix 159. A flow cell according to Appendix 131, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:22.2 to 1:32.7.

[0295] Appendix 160. A flow cell according to Appendix 159, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:25 to 1:30.1.

[0296] Appendix 161. The flow cell according to Appendix 160, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:28.2 to 1:28.6.

[0297] Appendix 162. The flow cell according to Appendix 131, wherein the ratio of the length of the rectangular cross section of the flow channel to the length of the cuvette is within the range of 1:29.1 to 1:40.

[0298] Appendix 163. The flow cell according to Appendix 162, wherein the ratio of the length of the rectangular cross section of the flow channel to the length of the cuvette is within the range of 1:31 to 1:35.6.

[0299] Appendix 164. The flow cell according to Appendix 163, wherein the ratio of the length of the rectangular cross section of the flow channel to the length of the cuvette is within the range of 1:33.4 to 1:33.9.

[0300] Appendix 165. A flow cell according to Appendix 131, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:18.2 to 1:27.7.

[0301] Appendix 166. A flow cell according to Appendix 165, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:20.7 to 1:25.8.

[0302] Addendum 167. A flow cell according to Addendum 166, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:23.6 to 1:23.7.

[0303] Item 168. The flow cell of any one of items 131 to 167, wherein the cuvette is constructed of fused silica.

[0304] Clause 169. The flow cell of any one of clauses 131-168, having a collection efficiency in the range of 20% to 40%.

[0305] 17. The flow cell of claim 169, having a collection efficiency in the range of 0.21% to 30%.

[0306] 171. The flow cell of claim 170, having a collection efficiency in the range of 23% to 25%.

[0307] Addendum 172. The flow cell of any one of Addendums 131 to 171, wherein the rectangular cross-section of the flow channel is symmetrical across the width, the length, or both the width and the length of the cuvette.

[0308] Addendum 173. A flow cell according to any one of Addendums 131 to 172, wherein the flow path extends the entire height of the cuvette.

[0309] Appendix 174. A method for assembling a flow cytometer, comprising: a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell; a detector configured to collect light emitted from the illuminated particles; placing a flow cell having a cuvette configured to carry particles in a flow stream in a flow cytometer comprising: The cuvette has a flow channel with a rectangular cross section and an aspect ratio in the range of 1.0 to 1.4.

[0310] Item 175. The method of item 174, wherein the aspect ratio is in the range of 1.1 to 1.3.

[0311] Item 176. The method of item 175, wherein the aspect ratio is approximately 1.2.

[0312] Addendum 177. The method of any one of Addendums 174 to 176, wherein the rectangular cross-section of the channel has a length in the range of 275 μm to 325 μm.

[0313] Addendum 178. The method of Addendum 177, wherein the length of the rectangular cross section of the channel is in the range of 290 μm to 310 μm.

[0314] Addendum 179. The method of Addendum 178, wherein the length of the rectangular cross section of the channel is in the range of 295 μm to 305 μm.

[0315] Addendum 180. The method of any one of Addendums 174 to 179, wherein the rectangular cross-section of the channel has a width in the range of 225 μm to 275 μm.

[0316] Addendum 181. The method of Addendum 180, wherein the width of the rectangular cross section of the channel is in the range of 240 μm to 260 μm.

[0317] Addendum 182. The method of Addendum 181, wherein the width of the rectangular cross section of the channel is in the range of 245 μm to 255 μm.

[0318] Addendum 183. The method of any one of Addendums 174 to 182, wherein the flow cytometer is configured as an imaging flow cytometer.

[0319] Clause 184. The method of clause 183, wherein the light source comprises a light beam generator configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light.

[0320] Clause 185. The method of clause 184, further comprising an acousto-optic deflector (AOD).

[0321] Clause 186. The method of any one of clauses 183-185, wherein a sample fluid line configured to supply particles to the flow cell is fluidly connected to the flow cell.

[0322] Clause 187. The method of clause 186, wherein the flow cytometer is configured to deliver particles in the flow stream at a velocity in the range of 0.1 m / s to 10 m / s.

[0323] Appendix 188. Flow cytometers are (a) an imaging mode in which the flow cytometer is configured to deliver particles in a flow stream to a flow cell at a first velocity; and (b) a non-imaging mode in which the flow cytometer is configured to deliver particles in the flow stream to the flow cell at a second velocity that is greater than the first velocity; 188. The method of claim 186 or 187, wherein the method is configured to operate on

[0324] Clause 189. The method of clause 188, wherein the first velocity is in the range of 0.5 m / s to 1.5 m / s.

[0325] Addendum 190. The method of Addendum 189, wherein the first velocity is in the range of 0.7 m / s to 1.3 m / s.

[0326] Clause 191. The method of clause 190, wherein the first velocity is in the range of 0.9 m / s to 1.1 m / s.

[0327] Addendum 192. The method of any one of Addendums 188 to 191, wherein the second velocity is in the range of 3 m / s to 8 m / s.

[0328] Clause 193. The method of clause 192, wherein the second velocity is in the range of 4 m / s to 7 m / s.

[0329] Clause 194. The method of clause 193, wherein the second velocity is in the range of 5 m / s to 6 m / s.

[0330] Clause 195. The method of any one of clauses 188-194, wherein the ratio of the first speed to the second speed is within the range of 1:12 to about 1:2.

[0331] Clause 196. The method of any one of clauses 174-195, further comprising positioning a focusing lens in optical communication with the flow cell and the detector.

[0332] Item 197. The method of item 196, wherein a focusing lens is coupled to the cuvette.

[0333] Addendum 198. The method of Addendum 196 or 197, wherein the focusing lens has a numerical aperture (NA) in the range of 0.75 to 1.5.

[0334] Item 199. The method of item 198, wherein the focusing lens has an NA in the range of 0.9 to 1.4.

[0335] Item 200. The method of item 199, wherein the focusing lens has an NA in the range of 1.1 to 1.3.

[0336] Addendum 201. The method of any one of Addendums 196-200, wherein the focusing lens has a focusing angle (θ) in the range of 90 degrees to about 110 degrees.

[0337] Item 202. The method of item 201, wherein the focusing lens has a focusing angle in the range of 95 degrees to 105 degrees.

[0338] Item 203. The method of item 202, wherein the focusing lens has a focusing angle in the range of 97 degrees to 103 degrees.

[0339] Addendum 204. The method of any one of Addendums 196 to 203, wherein the surface of the cuvette is separated from the flow path by a distance in the range of 1.9 mm to 2.1 mm.

[0340] Item 205. The method of item 204, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 2 mm to 2.05 mm.

[0341] Item 206. The method of item 205, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 2.02 mm to 2.04 mm.

[0342] Addendum 207. The method of any one of Addendums 196-206, further comprising positioning an objective lens in optical communication with the light source and the flow cell.

[0343] Item 208. The method of item 207, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5 mm to 5.2 mm.

[0344] Item 209. The method of item 208, wherein the surface of the cuvette is spaced from the flow path by a distance in the range of 5.05 mm to 5.1 mm.

[0345] Item 210. The method of item 209, wherein the surface of the cuvette is spaced from the flow channel by a distance in the range of 5.07 mm to 5.09 mm.

[0346] Addendum 211. The method of any one of Addendums 204-210, wherein the surface of the cuvette is provided with an anti-reflective coating.

[0347] Addendum 212. The method of any one of Addendums 174-211, wherein the cuvette has a height perpendicular to the rectangular cross section in the range of 5 mm to 9 mm.

[0348] Addendum 213. The method of Addendum 212, wherein the height of the cuvette is in the range of 6 mm to 8 mm.

[0349] Addendum 214. The method of Addendum 213, wherein the height of the cuvette is within the range of 7 mm to 7.2 mm.

[0350] Addendum 215. The method of any one of Addendums 174 to 214, wherein the cuvette has a length in the range of 8 mm to 12 mm.

[0351] Addendum 216. The method of Addendum 215, wherein the length of the cuvette is within the range of 9 mm to 11 mm.

[0352] Addendum 217. The method of Addendum 216, wherein the length of the cuvette is within the range of 10 mm to 10.2 mm.

[0353] Addendum 218. The method of any one of Addendums 174 to 217, wherein the width of the cuvette is within the range of 2 mm to 6 mm.

[0354] Addendum 219. The method of Addendum 218, wherein the width of the cuvette is in the range of 3 mm to 5 mm.

[0355] Addendum 220. The method of Addendum 219, wherein the width of the cuvette is within the range of 4 mm to 4.1 mm.

[0356] Addendum 221. The method described in Addendum 174, wherein the ratio of the width of the rectangular cross section of the channel to the width of the cuvette is within the range of 1:8.9 to 1:21.8.

[0357] Addendum 222. The method described in Addendum 221, wherein the ratio of the width of the rectangular cross section of the channel to the width of the cuvette is within the range of 1:12.5 to 1:19.2.

[0358] Addendum 223. The method according to Addendum 222, wherein the ratio of the width of the rectangular cross section of the channel to the width of the cuvette is within the range of 1:16.1 to 1:16.3.

[0359] Addendum 224. The method of Addendum 174, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:22.2 to 1:32.7.

[0360] Addendum 225. The method of Addendum 224, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:25 to 1:30.1.

[0361] Addendum 226. The method of Addendum 225, wherein the ratio of the width of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:28.2 to 1:28.6.

[0362] Addendum 227. The method described in Addendum 174, wherein the ratio of the length of the rectangular cross section of the channel to the length of the cuvette is within the range of 1:29.1 to 1:40.

[0363] Addendum 228. The method described in Addendum 227, wherein the ratio of the length of the rectangular cross section of the channel to the length of the cuvette is within the range of 1:31 to 1:35.6.

[0364] Addendum 229. The method described in Addendum 228, wherein the ratio of the length of the rectangular cross section of the channel to the length of the cuvette is within the range of 1:33.4 to 1:33.9.

[0365] Addendum 230. The method of Addendum 174, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:18.2 to 1:27.7.

[0366] Addendum 231. The method of Addendum 230, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:20.7 to 1:25.8.

[0367] Addendum 232. The method according to Addendum 231, wherein the ratio of the length of the rectangular cross section of the flow channel to the height perpendicular to the rectangular cross section is within the range of 1:23.6 to 1:23.7.

[0368] Addendum 233. The method of any one of Addendums 174 to 232, wherein the cuvette is made of fused silica.

[0369] Addendum 234. The method of any one of Addendums 174 to 233, wherein the flow cell has a collection efficiency in the range of 20% to 40%.

[0370] 235. The method of claim 234, wherein the flow cell has a collection efficiency in the range of 21% to 30%.

[0371] 236. The method of claim 235, wherein the flow cell has a collection efficiency in the range of 23% to 25%.

[0372] Addendum 237. The method of any one of Addendums 174-236, wherein the rectangular cross-section of the channel is symmetrical across the width, length, or both width and length of the cuvette.

[0373] Addendum 238. The method of any one of Addendums 174 to 237, wherein the flow path extends the entire height of the cuvette.

[0374] Although the foregoing disclosure has been described in some detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art, in view of the teachings of the present disclosure, that certain changes and modifications may be made without departing from the spirit and scope of the appended claims.

[0375] Thus, the foregoing merely illustrates the essence of the present disclosure. It is clear that those skilled in the art will be able to devise various configurations that embody the essence of the present disclosure and are within the spirit and scope of the present disclosure, even though not explicitly described or shown herein. Furthermore, all examples and conditional language set forth herein are intended essentially to aid the reader in understanding the essence of the disclosure and the concepts provided by the inventors to advance the art, and should not be construed as limiting the examples and conditions specifically set forth. Furthermore, all statements herein that describe the essence, aspects, and embodiments of the present disclosure, as well as specific examples of the present disclosure, are intended to encompass both structural and functional equivalents of the present disclosure. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., all elements developed that perform the same function, regardless of structure. Furthermore, the descriptions disclosed herein are not intended to be publicly disclosed, regardless of whether such disclosure is explicitly recited in the claims.

[0376] Accordingly, it is not intended that the scope of the present disclosure be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present disclosure are embodied by the appended claims. With respect to claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly provided to be invoked with respect to a limitation in a claim only when the precise phrase "means for" or "step for" appears at the beginning of such limitation in the claim; if such precise phrase is not used in a claim limitation, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0377] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Patent Application No. 63 / 642,501, filed May 3, 2024, the entire disclosure of which is incorporated herein by reference.

Claims

1. a flow cell comprising a cuvette configured to carry particles in a flow stream, the cuvette having a flow path with a rectangular cross section and an aspect ratio in the range of 1.0 to 1.4; a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell; a detector configured to collect light emitted from the illuminated particles; A flow cytometer comprising:

2. 2. The flow cytometer of claim 1, wherein the aspect ratio is in the range of 1.1 to 1.

3.

3. 3. The flow cytometer of claim 1, wherein the rectangular cross section of the flow channel has a length within a range of 275 μm to 325 μm, 290 μm to 310 μm, or 295 μm to 305 μm.

4. 4. The flow cytometer of claim 1, wherein the rectangular cross section of the flow channel has a width within a range of 225 μm to 275 μm, 240 μm to 260 μm, or 245 μm to 255 μm.

5. 5. The flow cytometer according to claim 1, configured as an imaging flow cytometer.

6. 6. The flow cytometer of claim 5, wherein the light source comprises a light beam generator configured to generate at least a first beam of frequency-shifted light and a second beam of frequency-shifted light.

7. 7. The flow cytometer of claim 1, further comprising a sample fluid line configured to supply particles to the flow cell.

8. (c) an imaging mode in which the flow cytometer is configured to deliver particles in a flow stream to the flow cell at a first velocity; and (d) a non-imaging mode, wherein the flow cytometer is configured to deliver particles in a flow stream to the flow cell at a second velocity greater than the first velocity.

8. The flow cytometer of claim 7, configured to operate at

9. 9. The flow cytometer of claim 1, further comprising a collection lens in optical communication with the flow cell and the detector.

10. 10. The flow cytometer according to claim 1, wherein the cuvette has a height perpendicular to the rectangular cross section within the range of 5 mm to 9 mm.

11. 11. The flow cytometer of claim 1, wherein the cuvette is made of fused silica.

12. 12. The flow cytometer of claim 1, wherein the rectangular cross section of the flow channel is symmetrical across the width, length, or both the width and length of the cuvette.

13. 13. A flow cytometer according to claim 1, wherein the flow path extends over the entire height of the cuvette.

14. 1. A method for analyzing a sample fluid, comprising: (a) a flow cell comprising a cuvette configured to carry particles in a flow stream, the cuvette having a flow passage with a rectangular cross section and an aspect ratio in the range of 1.0 to 1.4; a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell; a detector configured to collect light emitted from the illuminated particles; introducing a sample fluid into a flow cytometer comprising: (b) analyzing the sample fluid by irradiating particles within the flowstream.

15. a cuvette configured to carry particles in a flow stream; The cuvette is a flow cell having a flow channel with a rectangular cross section and an aspect ratio in the range of 1.0 to 1.4.

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