Flow cytometer with sample injection needle and method of using same

Abstract

Flow cytometers are provided that include a sample injection needle. The subject flow cytometers include a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at the proximal end to a flow cell at the distal end to generate a core stream. The subject flow cells have a flow cell cone at the proximal end, and the subject sample injection needles are constructed and arranged to maintain an intact core stream under flow conditions that vary by one or more orders of magnitude. Methods of assembling the subject flow cytometers and methods of analyzing samples using the subject flow cytometers are also provided.

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] Some flow cytometer systems are implemented using pressure-driven fluidics technology, in which sample and sheath fluids are delivered to a flow cell containing a detection region at pressures above ambient pressure. Varying the flow rate through the flow cell of a pressure-driven fluidics system is achieved by varying the pressure in the sample tube and / or sheath fluid reservoir that feed the flow cell. The ratio of sample fluid to sheath fluid flowing through the flow cell is controlled by both the pressure levels in the sample tube and sheath fluid reservoir and the ratio of the resistances of the sample fluid path and the sheath fluid path.

[0004] Alternatively, flow cytometer systems have been implemented using vacuum-driven fluidics technology, in which a vacuum pump applies a vacuum downstream of the flow cell while the sample and sheath fluids are held at ambient pressure. Varying the flow rate through the flow cell of a vacuum-driven fluidics system is achieved by varying the vacuum applied by the vacuum pump, and the ratio of sample and sheath fluids flowing through the flow cell is controlled by the ratio of the resistances of the sample fluid path and the sheath fluid path.

[0005] Some flow cytometers are equipped with an injection needle, which plays a key role in introducing the sample into the flow cell. Sheath flow within the flow cell helps establish the sample core, allowing the sample cells to pass the interrogation point of the cuvette in a single-file configuration. This technique is known as hydrodynamic focusing. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have recognized that imaging particles using a flow cytometer requires precise control of the velocity at which cells move through the interrogation point (i.e., the location where the sample is illuminated by one or more lasers). They have discovered that disruption of the flow stream and core stream can occur if such precise control is not possible. In particular, the present inventors have found that conventional sample injection mechanisms create fluid "vortices" associated with perturbations of the flow stream and core stream. Therefore, they have recognized that a flow cytometer with an improved sample injection needle is desirable. Embodiments of the present invention fulfill this need. [Means for solving the problem]

[0007] Aspects of the present invention include flow cytometers. The subject flow cytometers include a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at the proximal end to a flow cell at the distal end to generate the core stream. The flow cell of interest has a flow cell cone at its proximal end, and the sample injection needle of interest is configured and arranged to maintain the core stream intact under flow conditions that vary by one order of magnitude or more. In some embodiments, the flow cell has a sheath fluid injection port configured to deliver sheath fluid to the flow cell. In some such embodiments, the distal end of the sample injection needle is disposed within the flow cell cone and is separated from the sheath fluid introduction point by a longitudinal distance within a range of 17 mm to 26 mm (e.g., 20 mm to 22 mm). In some cases, the sample injection needle is configured and arranged to maintain the core stream intact under conditions in which the flow stream velocity is within a range of 0.5 m / s to 10 m / s. The sample injection needle may be constructed and arranged to maintain an intact core stream under conditions where the sample fluid flow rate is within a range of 1 μl / min to 150 μl / min. In some cases, the sample injection needle has an inner diameter within a range of 0.2 mm to 0.4 mm (e.g., 0.25 mm to 0.30 mm). In some variations, the sample injection needle has a tapered portion at the distal end. In some such variations, the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 3.5 mm to 4.0 mm (e.g., 3.8 mm to 3.9 mm). In other variations, the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 1.5 mm to 2.0 mm (e.g., 1.7 mm to 1.8 mm). In some embodiments, the sample injection needle has a taper range of 2.5 mm to 12 mm radius over a length of 1.75 mm to 4 mm. In some embodiments, the sample injection needle has a rounded distal end.In certain cases, the flow cytometer includes multiple sheath fluid introduction ports, e.g., the multiple sheath fluid introduction ports are offset from one another so that the sheath fluid swirls through the flow cell cone. Embodiments of the subject flow cytometers further include a light source configured to illuminate the flow cell at the interrogation point. In some cases, the distal end of the flow cell is spaced a distance within a range of 13 mm to 17 mm (e.g., 14 mm to 16 mm) from the interrogation point. In embodiments, flow cytometers of the present invention include one or more detectors configured to collect light from the flow cell. In some variations, the flow cytometer includes a vacuum to draw fluid through the flow cell.

[0008] Aspects of the present invention further include methods for analyzing samples. The featured methods involve introducing a particle sample into a flow cytometer equipped with a subject flow cell and a sample injection needle, and sorting the particle sample by flow cytometry. As described above, the subject flow cytometer includes a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passageway therethrough for directing sample fluid from a sample injection line at the proximal end to a flow cell at the distal end to generate the core stream. The subject flow cell includes a flow cell cone at its proximal end, and the subject sample injection needle is configured and arranged to maintain an intact core stream under flow conditions that vary by one or more orders of magnitude. In some cases, the particle sample is a biological sample (e.g., cells).

[0009] Aspects of the invention further include methods of assembling a flow cytometer. The methods of interest include placing a sample injection needle within the flow cytometer. As described above, the subject flow cytometers include a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at the proximal end to a flow cell at the distal end to generate the core stream. The flow cell of interest has a flow cell cone at its proximal end, and the sample injection needle of interest is constructed and arranged to maintain an intact core stream under flow conditions that vary by more than one order of magnitude. [Brief explanation of the drawings]

[0010] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which:

[0011] [Figure 1A] 1A and 1B illustrate a sample injection needle according to an embodiment of the present invention. [Figure 1B] 1A and 1B illustrate a sample injection needle according to an embodiment of the present invention. [Figure 1C] 1A and 1B illustrate a sample injection needle according to an embodiment of the present invention. [Figure 2A] 1A and 1B show examples of the distal end of a sample injection needle according to an embodiment of the present invention. [Figure 2B] 1A and 1B show examples of the distal end of a sample injection needle according to an embodiment of the present invention. [Figure 2C] 1A and 1B show examples of the distal end of a sample injection needle according to an embodiment of the present invention. [Figure 3A] FIG. 1 shows a flow cell and a sample injection needle according to an embodiment of the present invention. [Figure 3B] FIG. 1 shows a flow cell and a sample injection needle according to an embodiment of the present invention. [Figure 4] FIG. 1 is a functional block diagram illustrating a flow cytometry system according to an embodiment. [Figure 5]FIG. 1 illustrates 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] 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] FIG. 1 shows an image of an actual core stream being probed by a laser. [Figure 9A] FIG. 1 shows a sample injection needle used in needle performance analysis. [Figure 9B] FIG. 1 shows a sample injection needle used in needle performance analysis. [Figure 9C] FIG. 1 shows a sample injection needle used in needle performance analysis. DETAILED DESCRIPTION OF THE INVENTION

[0012] Flow cytometers are provided that include a sample injection needle. The subject flow cytometers include a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at the proximal end to a flow cell at the distal end to generate a core stream. The subject flow cells have a flow cell cone at the proximal end, and the subject sample injection needles are constructed and arranged to maintain an intact core stream under flow conditions that vary by one or more orders of magnitude. Methods of assembling the subject flow cytometers and methods of analyzing samples using the subject flow cytometers are also provided.

[0013] Before the present invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. The scope of the present invention 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.

[0014] When a range of values is given, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, 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 invention.

[0015] 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.

[0016] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described.

[0017] 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 invention 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.

[0018] 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," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0019] 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 invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0020] flow cytometer As described above, embodiments of the present invention include a flow cytometer. Flow cytometers of interest include a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passageway therethrough for directing sample fluid from a sample injection line at the proximal end to the flow cell at the distal end to generate the core stream. The term "core stream" as used herein is used in its conventional sense to refer to a fluid stream that carries particles through the flow cell and is focused (e.g., hydrodynamically focused) by a sheath fluid stream. Generally, particles are transported through the core stream in a single file. The size (e.g., diameter) of the core stream may be varied as desired. In some cases, the diameter of the core stream may be approximately the same as the diameter of the particles being analyzed. In some cases, the diameter of the core stream is in the range of 5 μm to 25 μm, e.g., in the range of 10 μm to 20 μm. The diameter of the core stream may be adjusted proportionally to the pressure (e.g., positive or negative pressure) applied to the particles as they are injected into the sheath fluid stream. In some cases, the flow velocity of the sheath fluid remains constant. In this manner, particles are injected into the sheath fluid and hydrodynamically focused, creating a laminar flow in which the particles travel along the same axis at approximately the same velocity.

[0021] A core stream associated with the present invention may be described as "intact" if the core stream maintains a relatively constant shape throughout the entire length of the flow cell. In some cases, an intact core stream of the present invention is defined by straight edges. In other words, when viewed two-dimensionally, the boundaries of an intact flow stream appear straight. In certain variations, the flow stream and its constituent core streams are hydrodynamically focused and characterized by laminar flow. In some such variations, the straight edges of the core stream are substantially parallel to one another (e.g., the edges deviate from true parallel lines by no more than 5 degrees, e.g., no more than 2 degrees). In some cases, an intact core stream is not characterized by perturbations from the distal end of the sample injection needle to the distal end of the flow cell. In some such cases, an intact core stream does not include vortices (e.g., surrounding the tip of the sample injection needle). In some cases, the subject flow cytometers and their component sample injection needles reduce perturbations of the flow stream and core stream by 20% or more, 25% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, e.g., 100%, compared to conventional instruments.

[0022] As described above, the subject sample injection needles are configured and arranged to maintain an intact core stream under flow conditions that vary by one order of magnitude or more. The term "one order of magnitude" (also referred to as "one order of magnitude") is used in its conventional sense to refer to flow conditions that vary by a factor of ten or more. In some cases, the subject sample injection needles are configured and arranged to maintain an intact core stream under flow conditions that vary by two orders of magnitude or more. Flow conditions described herein include, for example, sheath fluid flow rate, sample fluid flow rate, particle size (e.g., diameter), pressure (positive or negative), etc. In some embodiments, the subject sample injection needles are configured and arranged to maintain an intact core stream under sheath fluid flow rates that vary by one order of magnitude or more. For example, in some cases, the sample injection needles are configured and arranged to maintain an intact core stream under conditions where the flow stream velocity is within a range of 0.5 m / s to 10 m / s. In certain cases, the subject sample injection needles are configured and arranged to maintain an intact core stream under sample fluid flow rates that vary by one order of magnitude or more. For example, in some cases, the sample injection needle is constructed and arranged to maintain an intact core stream under conditions where the sample fluid flow rate is in the range of 1 μl / min to 150 μl / min.

[0023] As used herein, the term "flow cell" is used in its conventional sense to refer to a component including a flow path configured to accommodate a liquid flow stream for carrying particles within a sheath fluid. In embodiments, flow cells of interest include cuvettes. Cuvettes of interest have a container with a passageway therethrough. Flow cells of interest include a flow path accessible to light. In some cases, the flow cell includes a transparent material (e.g., quartz) that allows light to pass through. Any convenient flow cell that delivers a fluid sample to a sample interrogation region may be used as a flow cell described herein; in some embodiments, the flow cell is a cylindrical flow cell, a frustoconical flow cell, or a flow cell including a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating in a flat surface with an orifice perpendicular to the longitudinal axis. Flow cells of interest receive fluid from a sample fluid source (i.e., via a sample injection needle) and have a proximal end for receiving fluid from a sheath fluid source and a distal end for discharging fluid. Depending on the configuration of the flow cytometer, the fluid at the distal end of the flow cell may be directed into one or more different types of collection vessels.

[0024] In some cases, the flow stream is configured to be illuminated with light from a light source at an investigation point. The flow stream, which defines the flow path, may include a liquid sample injected from a sample tube through a sample injection line. In some embodiments, the flow stream may include a narrow, rapidly flowing liquid stream such that linearly separated particles carried within the flow stream are aligned and separated from one another. The term "investigation point" as used herein refers to an area within a flow cell where particles are illuminated by light from a light source, e.g., for analysis. The size of the investigation point may vary as desired. For example, if 0 μm represents the axis of light emitted by the light source, the investigation point may be within a range of -100 μm to 100 μm, e.g., -50 μm to 50 μm, e.g., -25 μm to 40 μm, e.g., -15 μm to 30 μm.

[0025] The flow cells of interest have a flow cell cone at the proximal end. A "flow cell cone" refers to a conical (e.g., right-frustoconical) recess in the flow cell that narrows toward the analytical region (i.e., interrogation zone) of the flow cell. The flow cell cone may serve to hydrodynamically focus the flow stream. The flow cell cones described herein may be characterized by an angle (i.e., an angle measured with respect to an imaginary axis extending through the center of the flow cell cone to the generatrix of the flow cell cone) in the range of 15° to 25°, e.g., 18° to 22°, e.g., 19° to 21°. In some cases, the flow cell cones described herein may be characterized by an angle of 20°.

[0026] In certain embodiments, the flow cytometer includes a sample fluid source. The sample fluid source may be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding sample fluid. The volume of the sample fluid container may be in the range of 1 mL to 100 mL. For example, the volume of the container may be in the range of 1 mL to 90 mL, 1 mL to 80 mL, 1 mL to 70 mL, 1 mL to 60 mL, 1 mL to 50 mL, 1 mL to 40 mL, 1 mL to 30 mL, 1 mL to 20 mL, or 1 mL to 10 mL. In embodiments, the sample fluid is supplied from the sample fluid source to a sample injection needle via a sample injection line (e.g., tubing).

[0027] In some embodiments, the flow cytometer includes a sheath fluid reservoir. The sheath fluid reservoir may be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding sheath fluid. In some embodiments, the sheath fluid reservoir is fluidly coupled to the input of the flow cell. The volume of the sheath fluid container may be in the range of 1 L to 100 L. For example, the volume of the container may be in the range of 1 L to 90 L, 1 L to 80 L, 1 L to 70 L, 1 L to 60 L, 1 L to 50 L, 1 L to 40 L, 1 L to 30 L, 1 L to 20 L, or 1 L to 10 L. The sheath fluid reservoir may be fluidly connected to a sheath fluid line configured to carry the sheath fluid from the sheath fluid reservoir to the flow cell.

[0028] Flow cytometers of interest may further include one or more sheath fluid introduction ports. The sheath fluid introduction ports of interest are fluidly connected to a sheath fluid source (i.e., reservoir) and deliver sheath fluid to the proximal end of the flow cell. In embodiments, a sheath fluid injection system is configured to deliver a flow of sheath fluid, e.g., along with the sample, to the interior chamber of the flow cell to generate a laminated flow stream of sheath fluid surrounding the sample flow stream. The velocity of the sheath fluid delivered to the chamber of the flow cell may be between 25 μL / sec and 2500 μL / sec, e.g., between 50 μL / sec and 1000 μL / sec, e.g., between 75 μL / sec and 750 μL / sec, depending on the desired characteristics of the flow stream.

[0029] In some embodiments, the sheath fluid injection port is an orifice in the wall of the internal chamber. The orifice of the sheath fluid injection port may be 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 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.

[0030] In some cases, the one or more sheath fluid introduction ports are located within the flow cell cone (i.e., at the "base" of the flow cell cone). In some cases, the flow cytometer includes one sheath fluid introduction port. In other cases, the flow cytometer includes multiple sheath fluid introduction ports. The number of sheath fluid introduction ports in the multiple sheath fluid introduction ports can vary, for example, two sheath fluid introduction ports, three sheath fluid introduction ports, four sheath fluid introduction ports, and five sheath fluid introduction ports. In certain variations, the flow cytometer includes two sheath fluid introduction ports. When a flow cytometer of the present invention includes multiple sheath fluid introduction ports, the multiple sheath fluid introduction ports are offset from one another to cause the sheath fluid to swirl (e.g., like a "toilet bowl") through the flow cell cone. In such cases, the multiple sheath fluid introduction ports are offset from one another to cause the sheath fluid to swirl in a clockwise direction. In other cases, the multiple sheath fluid introduction ports are offset from one another to cause the sheath fluid to swirl in a counterclockwise direction. In some cases, swirl of the sheath fluid provides additional stability to the flow stream and core stream.

[0031] The sample injection needle of the present disclosure may have an elongated structure. By "elongated structure" is meant that the sample injection needle has a length that is greater than its width. In other words, the sample injection needle has distinct proximal and distal ends. The proximal end is where the sample injection needle receives sample fluid (from a sample injection line fluidly connected to a sample fluid source), and the distal end is where the sample injection needle injects the sample into the flow cell. The elongated structure may have any convenient cross-sectional shape, and cross-sectional shapes of interest include, but are 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 embodiments, the elongated structure has a substantially circular cross-sectional shape along its length. By "substantially" circular cross-section, in embodiments, it is meant that one or more locations along the length of the outlet fitting may have a cross-section that deviates slightly from the circular cross-section that characterizes the remainder of the structure. For example, in some variations, the elongated structure has a polygonal (e.g., hexagonal, pentagonal, etc.) cross-section at one or more locations along its length. In some cases, the width (e.g., cross-sectional diameter) of the elongated structure varies along the length of the outlet fitting. In other words, in such variations, the elongated structure is not a perfect cylinder, but instead has some regions of its cross-sectional shape with a diameter that is larger or smaller than the diameter of other regions. In other cases, every portion of the sample injection needle has a circular cross-section. In some such cases, various portions of the sample injection needle may be characterized by different diameters. In some cases, at least a portion of the sample injection needle has an outer diameter (i.e., the outer diameter measured from the geometric center of the sample injection needle to its outer edge) in the range of 0.5 mm to 4 mm, e.g., 0.75 mm to 3 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm to 1.6 mm. In some cases, the outer diameter of the sample injection needle is 1.562 mm. The sample injection needle may have any convenient length, with lengths of interest being in the range of 15 mm to 30 mm, such as 17 mm to 26 mm, for example 20 mm to 22 mm. In some cases, the sample injection needle is not flat at the distal end, but has, for example, a "duck bill" shape.In these cases, the sample injection needle may have a substantially circular cross section throughout.

[0032] In some cases, the sample injection needle has a tapered portion at the distal end. In some such cases, the sample injection needle has a relatively constant outer diameter over most of its length (from the proximal end), but the outer diameter gradually (e.g., uniformly) decreases toward the distal end. In certain embodiments, the sample injection needle has a taper range of 2.5 mm to 12 mm radius over a length of 1.75 mm to 4 mm. For example, in one variation, the sample injection needle has a taper range of 2.79 mm to 11.63 mm radius over a length of 1.78 mm to 3.81 mm. The taper may begin at various locations along the length of the sample injection needle. In some cases, the taper begins at a distance along the length of the sample injection needle from the distal end within a range of 3 mm to 5 mm, e.g., 3.5 mm to 4.0 mm, e.g., 3.8 mm to 3.9 mm. Such a distal end may sometimes be referred to as having a "super bullet" configuration. In some embodiments having a super-bullet configuration, the distal end tapers to a radius of 11.63 mm over a length of 3.81 mm. In other cases, the taper begins at a distance along the length of the sample injection needle from the distal end within a range of 1 mm to 3 mm, e.g., 1.5 mm to 2 mm, e.g., 1.7 mm to 1.8 mm. Such distal ends may sometimes be referred to as having a "bullet" configuration. In some embodiments where the sample injection needle has a bullet configuration, the distal end tapers to a radius of 2.79 mm over a length of 1.78 mm. In other cases, the sample injection needle has a rounded distal end.

[0033] The sample injection needle of the present invention further comprises an opening at its distal end, and optionally a channel extending therethrough for conveying the particle-containing sample fluid to the flow cell. In embodiments, the opening is located at the geometric center of the cross-section of the outlet fitting at the distal end. The opening may have any convenient cross-sectional shape, and cross-sectional shapes of interest include, but are 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 bottom joined to a planar top. In some cases, the opening has a circular cross-sectional shape. In further cases, the channel similarly has a circular cross-sectional shape. The opening may have any suitable diameter, e.g., a diameter in the range of 0.1 mm to 2 mm, e.g., 0.1 mm to 1 mm, e.g., 0.2 mm to 0.4 mm, e.g., 0.25 mm to 0.30 mm. Similarly, the channel may have any suitable diameter, for example, the diameter is in the range of 0.1 mm to 2 mm, such as 0.1 mm to 1 mm, such as 0.2 mm to 0.4 mm, such as 0.25 mm to 0.30 mm. In some cases, the opening and the channel have circular cross-sectional shapes with the same or similar diameters. In other cases, the diameter of the opening is different (e.g., larger) than the diameter of the channel.

[0034] In some cases, the sample injection needle has an adapter at its proximal end. The adapter may also be used to secure the sample injection needle within a flow cytometer. In some such cases, the adapter has one or more holes configured to receive a mounting member (e.g., a pin, a screw, etc.). In certain variations, the adapter has multiple holes configured to receive a mounting member. In some embodiments, the adapter has three holes. In further cases, the adapter may be configured to fluidly connect to a sample injection line to supply sample fluid (optionally containing particles) to the sample injection needle.

[0035] 1A-1C are alternative views illustrating sample injection needles according to certain embodiments of the present invention. As shown in FIG. 1A, sample injection needle 100a has an elongated structure 101 and a tapered portion 102 at its distal end. FIG. 1B illustrates an example of a sample injection needle with an adapter. As shown in FIG. 1B, sample injection needle 100b has an elongated structure 101, a tapered portion 102 at its distal end, and an adapter 103. The adapter 103 has a plurality of holes 105 for receiving attachment members (e.g., screws, not shown) for attaching the sample injection needle to a flow cell. FIG. 1C is a cross-sectional view of a sample injection needle according to certain embodiments of the present invention. As shown in cross-section AA of sample injection needle 100c, a channel 104 is provided through the central portion of the sample injection needle. The elongated structure 101 and the tapered portion 102 at its distal end are also illustrated.

[0036] 2A-2C illustrate the distal end of a sample injection needle according to one embodiment of the present invention. Exemplary dimensions shown in FIGS. 2A-2C are in inches. FIG. 2A illustrates a sample injection needle distal end 202a having the super-bullet configuration described above. FIG. 2B illustrates a sample injection needle distal end 202b having a rounded distal end. FIG. 2C illustrates a sample injection needle distal end 202c having the bullet configuration described above.

[0037] The sample injection needle of the present invention is positioned in a particular manner relative to the flow cell. The position of the sample injection needle relative to the flow cell may be described in multiple ways. For example, in some cases, the position of the sample injection needle is described in terms of the position of its distal end relative to one or more sheath fluid introduction ports. For example, in some cases, the distal end of the sample injection needle is positioned within the flow cell cone and is separated from the sheath fluid introduction point by a longitudinal distance ranging from 15 mm to 30 mm, e.g., 17 mm to 26 mm, e.g., 20 mm to 22 mm. The position of the sample injection needle may be further described in terms of the position of its distal end relative to an interrogation point (i.e., the point at which the flow cell is illuminated by one or more light sources). In some cases, the distal end of the sample injection needle is separated from the interrogation point by a distance ranging from 10 mm to 20 mm, e.g., 13 mm to 17 mm, e.g., 14 mm to 16 mm.

[0038] 3A-3B illustrate a flow cell and sample injection needle according to one embodiment of the present invention. As shown in FIG. 3A, flow cell 303 includes flow cell cone 302 and cuvette 304. A laser (not shown) illuminates cuvette 304 at illumination point 305. Sheath fluid introduction port 306 is configured to supply sheath fluid to flow cell cone 302, and sample injection needle 301 is configured to supply sample fluid to flow cell cone 302 so that a core stream (not shown) remains intact under flow conditions that vary by more than an order of magnitude. The sheath fluid and sample fluid travel through the flow cell in direction d. FIG. 3B is an alternative view of the same elements shown in FIG. 3A. As shown in the exemplary embodiment of FIG. 3B, the distal end of sample injection needle 301 is 0.840 inches (21.3 mm) from sheath fluid introduction port 306. Additionally, the distal end of the sample injection needle 301 is 0.5848 inches (14.85 mm) from the illumination point 305 .

[0039] Particle-modulated light may be observed after illuminating particles in a flow cell. "Particle-modulated light" refers to light received from particles in a flow stream after illuminating the particles with light from a light source. In some cases, the particle-modulated light is side-scattered light. As described herein, side-scattered light refers to light that is refracted and reflected off the surface and internal structure of a particle. In further embodiments, the particle-modulated light includes forward-scattered light (i.e., light that travels primarily forward through or around the particle). In still other cases, the particle-modulated light includes fluorescent light (i.e., light emitted from a fluorescent dye after illumination with excitation wavelength light).

[0040] As discussed above, aspects of the present invention further include a light source configured to illuminate particles passing through the flow cell at the interrogation point. Any convenient light source may be used as the light source described herein. 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 flow cytometer of interest 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, flow cytometers of interest include 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.

[0041] 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.

[0042] Where the optical adjustment component is configured to move, it may be configured to move continuously or at discrete intervals, for example in increments of 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 10 μm or more, for example 100 μm or more, for example 500 μm 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.

[0043] Any movement protocol may be used to move the optical adjustment component structure, for example, the optical adjustment component structure may be coupled to a movable support stage or directly coupled to a motor-driven parallel movement stage, a lead screw parallel movement assembly, a geared parallel movement device, for example, a device using stepper motors, servo motors, brushless electric motors, brushed DC motors, microstep drive motors, high resolution stepper motors, among other motor types.

[0044] 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, such as 5 mm or more, for example 10 mm or more, for example 25 mm or more, such as 100 mm or more. In addition, the light source may be positioned at any suitable angle relative to the flow cell, for example, at an angle between 10 degrees and 90 degrees, for example 15 degrees and 85 degrees, for example 20 degrees and 80 degrees, for example 25 degrees and 75 degrees, for example 30 degrees and 60 degrees, for example at an angle of 90 degrees.

[0045] 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.

[0046] As discussed above, flow cytometers of interest may further include one or more particle-modulated light detectors for detecting particle-modulated light intensity data. In some embodiments, the one or more particle-modulated light detectors include one or more forward scatter detectors configured to detect forward scatter. For example, flow cytometers of interest may include one forward scatter detector or multiple forward scatter detectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more forward scatter detectors. In some embodiments, the flow cytometer includes one forward scatter detector. In other embodiments, the flow cytometer includes two forward scatter detectors.

[0047] Any convenient detector for detecting collected light may be used in the forward scattered 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 quantum dot photodiodes, and combinations thereof, among other detectors. In some embodiments, the 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 ~9 cm 2 , e.g., 0.1 cm 2 ~8 cm 2 , e.g., 0.5 cm 2 ~7 cm 2 , e.g., 1 cm 2 ~5 cm 2The photomultiplier tube has an active detection surface area of each region within the range of .times. ...

[0048] In embodiments, the forward scattered light detector is configured to measure light continuously or at discrete intervals. In some cases, the detector of interest is configured to measure collected light continuously. In other cases, the detector of interest is configured to measure at discrete intervals, such as every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, e.g., every 1000 milliseconds, or other intervals.

[0049] In further embodiments, the one or more particle modulation light detectors may comprise one or more side scatter light detectors for detecting side scatter wavelengths of light (i.e., light refracted and reflected by the surface and internal structure of the particle). In some embodiments, the flow cytometer comprises one side scatter light detector. In other embodiments, the flow cytometer comprises multiple side scatter light detectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more side scatter light detectors.

[0050] 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 quantum dot photodiodes, and combinations thereof, among other detectors. In some embodiments, the 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 ~9 cm 2 , e.g., 0.1 cm 2 ~8 cm 2 , e.g., 0.5 cm 2 ~7 cm 2 , e.g., 1 cm 2 ~5 cm 2 The photomultiplier tube has an active detection surface area of each region within the range of .times. ...

[0051] In embodiments, the subject flow cytometers further comprise a fluorescence detector configured to detect light at one or more fluorescence wavelengths, hi other embodiments, the flow cytometers comprise a plurality of fluorescence detectors, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, 5 or more, 10 or more, 15 or more, e.g., 20 or more fluorescence detectors.

[0052] Any convenient detector for detecting collected light may be used with the fluorescence photodetectors 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 quantum dot photodiodes, and combinations thereof, among other detectors. In some embodiments, the 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 ~9 cm 2 , e.g., 0.1 cm 2 ~8 cm 2 , e.g., 0.5 cm 2 ~7 cm 2 , e.g., 1 cm 2 ~5 cm 2 The photomultiplier tube has an active detection surface area of each region within the range of .times. ...

[0053] 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 fluorescence detector and the second fluorescence detector are CCD-based devices. In still other embodiments, both the first fluorescence detector and the second fluorescence detector 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 fluorescence detector and the second fluorescence detector are photomultiplier tubes.

[0054] In embodiments of the present disclosure, the fluorescence light 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., two or more different wavelengths, e.g., three hundred or more different wavelengths, e.g., four hundred or more different wavelengths, e.g., two or more detectors of a flow cytometer as described herein are configured to measure the same or overlapping wavelengths of collected light.

[0055] In some embodiments, the fluorescence light 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 flow cytometer may include one or more detectors configured to measure light at one or more of the following wavelengths: 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.

[0056] In some embodiments, the flow cytometer includes one or more wavelength separators disposed between the flow cell and the one or more particle modulation light detectors. The term "wavelength separator" is used herein in its conventional sense to refer to an optical component configured to separate light collected from a sample into predetermined spectral regions. In some embodiments, the flow cytometer includes one wavelength separator. In other embodiments, the flow cytometer includes multiple wavelength separators, e.g., two or more wavelength separators, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., fifteen or more, e.g., twenty-five or more, e.g., fifty or more, e.g., seventy-five or more, e.g., one hundred or more wavelength separators. In some embodiments, the wavelength separators are configured to separate light collected from a sample into predetermined spectral regions by passing light having predetermined spectral regions and reflecting light in one or more remaining spectral regions. In other embodiments, the wavelength separators are configured to separate light collected from a sample into predetermined spectral regions by passing light having predetermined spectral regions and absorbing light in one or more remaining spectral regions. In yet other embodiments, the wavelength separator is configured to spatially diffract light collected from the sample into predetermined spectral regions. Each wavelength separator may be any convenient light separation protocol, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In certain embodiments, the wavelength separator of a subject optical detection system is a dichroic mirror.

[0057] 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(pt 1):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 ...Verse™ flow cytometer, BD Biosciences FACSSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFor These include the BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, BD Biosciences FACSMelody™ cell sorter, and BD Biosciences FACSymphony™ S6 cell sorter.

[0058] 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.

[0059] In some cases, the flow cytometry system of the present invention may be implemented using the techniques described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, U.S. Patent Application Publication No. 200900222, and the like. and US Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, the disclosures of which are incorporated herein by reference, for imaging particles in a flowstream by Fluorescence Imaging Using Radio Frequency Tag Emission (FIRE).

[0060] 4 shows a system 400 for flow cytometry according to an exemplary embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. Flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more longpass (LP) filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0061] Pump lasers 415a-415c emit light in the form of laser beams. The wavelengths of the laser beams emitted from pump lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system of FIG. 4. The laser beams are first directed through one or more of beam splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits UV light (light having a wavelength in the range of 10-400 nm) and reflects 488 nm and 633 nm light.

[0062] The laser beam is then directed to a focusing lens 420, which focuses the laser beam onto the portion of the fluid stream where the sample particles reside in a flow chamber 425. The flow chamber is the part of the fluid system that directs particles in the stream, typically one at a time, into the focused laser beam for investigation. The flow chamber can comprise a flow cell in a benchtop flow cytometer or a nozzle tip in a stream-in air cytometer.

[0063] Light from one or more laser beams interacts with particles in the sample by 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 through one or more of beam splitters 445c-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collection lens 440 to one or more of forward scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a-460f.

[0064] The fluorescence collection lens 440 collects light emitted by particle-laser beam interactions and directs it toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the bandpass filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number represents the extent 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. Shortpass filters transmit light with wavelengths below a specified wavelength. Longpass filters, such as longpass filters 455a-455b, transmit light with wavelengths above a specified wavelength. For example, longpass filter 455b, a 670 nm longpass filter, transmits light above 670 nm. Filters are often selected to optimize the specificity of the detector for a particular fluorochrome. The filter may be configured so that the spectral band of light transmitted to the detector approximates the emission peak of the fluorescent dye.

[0065] The forward scatter detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell 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 light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect light refracted and reflected from the particle's surface and internal structure, which tends to increase as the particle's structural complexity increases. Fluorescent emission from fluorescent molecules bound to the particle may be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected by the forward scatter detector 430, side scatter detector 435, and fluorescence detector may be converted to electronic signals (voltages) by the detectors. This data may provide information about the sample.

[0066] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to the flow cytometer shown in Figure 4, 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 at various wavelengths and in a variety of different configurations.

[0067] During operation, the operation of the flow cytometer is controlled by the controller / processor 490, and measurement data from the detectors may be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 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 410 to control lasers, fluid flow parameters, etc. An input / output (I / O) function 497 may also be provided in the system. The memory 495, controller / processor 490, and I / O function 497 may be provided entirely as an integral part of the flow cytometer 410. In such an embodiment, a display may also form part of the I / O function 497 to present experimental data to a user of the flow cytometer 410. Alternatively, some or all of the memory 495 and the controller / processor 490 and I / O function 497 may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of memory 495 and controller / processor 490 may be in wireless or wired communication with flow cytometer 410. Controller / processor 490 in conjunction with memory 495 and I / O functionality 497 may be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0068] The system illustrated in FIG. 4 includes six different detectors that detect fluorescence within six different wavelength bands (which may be referred to herein as "filter windows" for a given detector) as determined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Various fluorescent molecules in a panel of fluorescent dyes used in a flow cytometer experiment emit light in their own unique wavelength bands. The particular fluorescent labels used in the experiment and their associated fluorescence emission bands may be selected to generally coincide with the filter windows of the detectors. The I / O function 497 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. The I / O function 497 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 also be stored in memory 495. The controller / processor 490 may be configured to evaluate one or more assignments of labels to markers.

[0069] In some embodiments, the subject system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as 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 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 described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0070] 5 is a functional block diagram of an example of a control system for analyzing and displaying biological events, such as a processor 800. The processor 800 can be configured to perform various processes for controlling the graphical display of biological events.

[0071] A flow cytometer or sorting system 802 may be configured to acquire bio-event data. For example, a flow cytometer may generate flow cytometry event data (e.g., particle-modulated light data). The flow cytometer 802 may be configured to provide the bio-event data to the processor 800. A data communication channel may be provided between the flow cytometer 802 and the processor 800. The bio-event data may be provided to the processor 800 via the data communication channel.

[0072] The processor 800 may be configured to receive bio-event data from the flow cytometer 802. The bio-event data received from the flow cytometer 802 may include flow cytometry event data. The processor 800 may be configured to provide a graphical representation including a first plot of the bio-event data to the display device 806. The processor 800 may be further configured to render a region of interest as a gate around a population of the bio-event data displayed by the display device 806, 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 one parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0073] The processor 800 may be further configured to display the bioevent data within the gate on the display device 806 differently from other events within the bioevent data outside the gate. For example, the processor 800 may be configured to render the color of the bioevent data included within the gate differently from the color of the bioevent data outside the gate. The display device 806 may be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to provide a graphical interface.

[0074] The processor 800 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 810. The mouse 810 may be capable of sending a gate selection signal to the processor 800 identifying a gate to be displayed on or manipulated via the display device 806 (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 808 or other means for providing input signals to the processor 800, 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 810 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.

[0075] A trigger event can cause the processor 800 to change how the data is displayed, what portions of the data are actually displayed on the display device 806, and / or provide input to further processing, such as selecting a population of interest for particle sorting.

[0076] In some embodiments, the processor 800 may be configured to detect when a gate selection is initiated by the mouse 810. The processor 800 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 processor 800. In some embodiments, the processor 800 extends the first gate such that a second gate is generated (e.g., as described above).

[0077] The processor 800 may be connected to a storage device 804. The storage device 804 may be configured to receive and store biological event data from the processor 800. The storage device 804 may be further configured to receive and store flow cytometry event data from the processor 800. The storage device 804 may be further configured to enable retrieval of biological event data, such as flow cytometry event data, by the processor 800.

[0078] The display device 806 may be configured to receive display data from the processor 800. The display data may include plots of biological event data and gates outlining sections of the plots. The display device 806 may be further configured to modify the information displayed in response to input received from the processor 800 in conjunction with input from the flow cytometer 802, the storage device 804, the keyboard 808, and / or the mouse 810.

[0079] In some embodiments, the processor 800 can generate a user interface for receiving example events for filtering. For example, the user interface can include a mechanism 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 based on an initial set of events for a portion of the sample.

[0080] FIG. 6A is a schematic diagram illustrating a particle sorting system 600 (e.g., a flow cytometer 802) 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., a laser-stream intersection) 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 .

[0081] 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.

[0082] 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 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. Detection system 616 enables the instrument to accurately calculate the position of each detected particle within the droplet. Detection system 616 can provide amplitude signal 620 and / or phase signal 618, which are then provided (via amplifier 622) to amplitude control circuit 626 and / or frequency control circuit 624. Amplitude control circuit 626 and / or frequency control circuit 624 then control droplet forming transducer 602. Amplitude control circuit 626 and / or frequency control circuit 624 can be provided within the control system.

[0083] 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.

[0084] 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. A 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.

[0085] 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. Exemplary implementations of the embodiment shown in Figure 6B include the BD FACSAria™ line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0086] How to analyze the sample As discussed above, aspects of the invention further include methods for analyzing samples. The featured methods include introducing a particle sample into the flow cytometer described above and sorting the particle sample by flow cytometry. As discussed above, the subject flow cytometers include a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at the proximal end to the flow cell at the distal end to generate the core stream. The subject flow cells include a flow cell cone at the proximal end, and the subject sample injection needles are configured and arranged to maintain an intact core stream under flow conditions that vary by more than one order of magnitude.

[0087] 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).

[0088] 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 invention 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.

[0089] 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.

[0090] 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.

[0091] 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, such that the illumination profile corresponds to that of a diffuse laser beam or lamp.

[0092] 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.

[0093] 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 light scatter), the light reflected perpendicular to the direction of particle flow through the detection region (sometimes referred to as orthogonal or side light scatter), 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 light scatter (FSC), side light scatter (SSC), and fluorescent emission each have separate parameters per 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.

[0094] 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.

[0095] 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.

[0096] 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 light scatter (FSC) versus side (i.e., orthogonal) light 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.

[0097] 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.

[0098] 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.

[0099] How to Assemble a Flow Cytometer As described above, aspects of the present invention further include methods for assembling a flow cytometer. In the methods of interest, a sample injection needle is positioned within the flow cytometer. A suitable flow cytometer includes a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end. The flow cell of interest has a flow cell cone at its proximal end. In the methods, the sample injection needle is positioned within the flow cell cone such that an intact core stream is maintained under flow conditions that vary by more than one order of magnitude. As described above, the sample injection needle has a passageway therethrough for delivering sample fluid from a sample injection line at its proximal end to a flow cell at its distal end to generate the core stream.

[0100] In embodiments, the method positions the distal end of the sample injection needle within the flow cell cone such that the distal end is a longitudinal distance within a range of 17 mm to 26 mm from the sheath fluid introduction point. For example, in some cases, the method positions the distal end of the sample injection needle within the flow cell cone such that the distal end is a longitudinal distance within a range of 20 mm to 22 mm from the sheath fluid introduction point. In particular cases, the method positions the sample injection needle within the flow cell cone such that the distal end is a distance within a range of 13 mm to 17 mm (e.g., 14 mm to 16 mm) from the interrogation point.

[0101] Computer Control System Aspects of the invention further include computer control systems, 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 having instructions, when loaded into the computer, to receive target or desired flow conditions (e.g., sheath fluid flow rate, sample fluid flow rate) and initiate a change in resistance state to achieve the target or desired flow conditions. Because the systems described herein comprise the flow cells and sample injection needles of the invention, the resulting core stream may remain intact.

[0102] 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 elements 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.

[0103] 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.

[0104] 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.

[0105] 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 communications 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 invention further comprises 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 of the present invention 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.

[0106] 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).

[0107] 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).

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] FIG. 7 illustrates a general configuration of an exemplary computing device 900 according to one embodiment. The general configuration of the computing device 900 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, the computing device 900 includes a processing unit 910, a network interface 920, a computer-readable medium drive 930, an input / output device interface 940, a display 950, and input devices 960, all of which may communicate with each other via a communications bus. The network interface 920 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 910 may receive information and instructions from other computing systems or services via a network. The processing unit 910 may further communicate with a memory 970 and may further provide output information for an optional display 950 via the input / output device interface 940. 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 940 may further accept input from any input device 960, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0115] The memory 970 may include computer program instructions (grouped in some embodiments as modules or components) that the processing unit 910 executes to implement one or more embodiments. The memory 970 generally includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. The memory 970 may store an operating system 972 that provides computer program instructions for use by the processing unit 910 in the general management and operation of the computing device 900. Data may be stored in the data storage device 990. The memory 970 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0116] usefulness The subject sample injection needles, flow cytometers, and methods are used in a variety of applications where it is desirable to analyze components in a sample within a fluid medium. The invention is particularly useful for improving the quality of a core stream in a fluid system. For example, the sample injection needles, flow cytometers, and methods may be used to increase the extent to which the core stream remains intact. In some cases, the invention may be used to reduce the formation of vortices within a flow cell cone.

[0117] Embodiments of the present invention further 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 target 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.

[0118] kit Aspects of the present disclosure further include kits. Kits of interest include one or more sample injection needles. In one embodiment, the kit includes one sample injection needle. In other embodiments, the kit includes multiple sample injection needles. When the kit includes multiple sample injection needles, the sample injection needles may be the same or different. For example, in one example, the kit includes sample injection needles having a super-bullet configuration as described above, a bullet configuration as described above, and a rounded configuration, or some combination of sample injection needles having these configurations.

[0119] In addition to the above components, the subject kits may (in some embodiments) further include instructions for use, for example, for attaching a sample injection needle of the present invention to 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 present 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.

[0120] The following examples are offered by way of illustration and not by way of limitation.

[0121] experiment introduction Previous observations have shown that the default injection needle (blunt) that does not extend into the flow cell is unable to establish a stable sample core at the desired sample flow rate (1 m / s sheath flow). This effect became more pronounced as the sample flow rate increased. This effect was first observed by injecting diluted food dye into the flow stream and later confirmed by observing the sample at the investigation point using a microscope. Three sample flow rate options were evaluated (8 μl / min, 32 μl / min, and 85 μl / min). At 8 μl / min, the sample core showed slight instability. At 32 μl / min, the sample core detached. At 85 μl / min, the sample core could not be established. As a result, experiments were required to study the effect of needle shape and length on sample core establishment.

[0122] method Core stream analysis was performed on a Becton Dickinson flow cytometer using multiple sample injection needles at various extension positions within the flow cell. Eight needles were compared in this study. Needles were grouped by tip shape and extension length relative to the default needle. Specifically, sample injection needles with a rounded distal end (Figure 9A), a bullet configuration as described above (Figure 9B), and a super-bullet configuration as described above (Figure 9C) were compared. There were two rounded tip needles (0-inch extension and 1 / 8-inch extension), three bullet-shaped needles (0-inch extension, 1 / 16-inch extension, and 1 / 8-inch extension), and three super-bullet-shaped needles (0-inch extension, 1 / 16-inch extension, and 1 / 8-inch extension).

[0123] Three sample flow rates (8 μl / min, 32 μl / min, and 85 μl / min) were applied per needle. During each run, a camera was aimed at the cuvette to capture the reflection of the sample core. A flashlight shone a light on one of the fiber cables. This created a ring visible to the camera at the interrogation point. A physical center around the detection point was identified. The quality of the sample core was visually assessed from the footage by determining whether the sample core was centered and whether it was stable (minimal deviation).

[0124] result The images captured by the camera for each sample injection needle are shown in Figure 8. As shown in Figure 8, the center of each image represents the shape of the core stream. The qualitative results for each sample injection needle of various extensions are shown in Table 1 below.

[0125] [Table 1]

[0126] Discussion The results showed that the 1 / 8 inch extension gave the best results for all tip geometries. The sample core could be established and was centered. However, for shorter extensions (e.g., 1 / 16 inch extensions), the more tapered the tip, the better the performance. The bullet 1 / 16 inch sample core was off-center and failed at 32 μl / min and 64 μl / min, while the super bullet met both criteria at both sample flow rates.

[0127] conclusion This comparative study showed that longer needles and more tapered tips can produce a stable, central sample core. This comparative study found that the 1 / 8-inch extended super-bullet shape (most tapered) is the best candidate for future injection needles. Therefore, it was shown that sample injection needles can be constructed and arranged to maintain an intact core stream under flow conditions that vary by more than an order of magnitude.

[0128] Regardless of the scope of the appended claims, the present invention may be further defined by the following notes.

[0129] Clause 1. A flow cell for transporting particles from a proximal end to a distal end within a core stream of a flow stream, the flow cell having a flow cell cone at the proximal end; a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at a proximal end to a flow cell at a distal end to generate a core stream; It is equipped with The sample injection needle is configured and positioned in a flow cytometer to maintain an intact core stream under flow conditions that vary by more than one order of magnitude.

[0130] Appendix 2. The flow cytometer of Appendix 1, further comprising a sheath fluid introduction port for delivering sheath fluid to the flow cell.

[0131] Appendix 3. The flow cytometer of Appendix 2, wherein the distal end of the sample injection needle is positioned within the flow cell cone and is a longitudinal distance from the sheath fluid introduction point within the range of 17 mm to 26 mm.

[0132] Appendix 4. The flow cytometer of Appendix 3, wherein the distal end of the sample injection needle is a longitudinal distance in the range of 20 mm to 22 mm from the sheath fluid introduction point.

[0133] Appendix 5. The flow cytometer of any one of Appendixes 1 to 4, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow stream flow velocity is within a range of 0.5 m / s to 10 m / s.

[0134] Appendix 6. The flow cytometer of any one of Appendixes 1 to 5, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow rate of the sample fluid is within the range of 1 μl / min to 150 μl / min.

[0135] Appendix 7. The flow cytometer according to any one of Appendixes 1 to 6, wherein the sample injection needle has an inner diameter in the range of 0.2 mm to 0.4 mm.

[0136] Appendix 8. The flow cytometer according to Appendix 7, wherein the sample injection needle has an inner diameter in the range of 0.25 mm to 0.30 mm.

[0137] Appendix 9. A flow cytometer according to any one of appendices 1 to 8, wherein the sample injection needle has a tapered portion at the distal end.

[0138] Clause 10: The flow cytometer of clause 9, wherein the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 3.5 mm to 4.0 mm.

[0139] Clause 11. The flow cytometer of clause 10, wherein the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 3.8 mm to 3.9 mm.

[0140] Clause 12. The flow cytometer of clause 9, wherein the tapered portion begins at a distance along the length of the sample injection needle that is within a range of 1.5 mm to 2.0 mm from the distal end.

[0141] 13. The flow cytometer of claim 12, wherein the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 1.7 mm to 1.8 mm.

[0142] Appendix 14. A flow cytometer according to any one of appendices 9 to 13, wherein the tapered section has a tapered range of 2.5 mm to 12 mm radius over a length of 1.75 mm to 4 mm.

[0143] Appendix 15. A flow cytometer according to any one of appendices 1 to 8, wherein the sample injection needle has a rounded distal end.

[0144] Clause 16. The flow cytometer of clause 2, comprising a plurality of sheath fluid introduction ports.

[0145] Clause 17. The flow cytometer of clause 16, comprising two sheath fluid introduction ports.

[0146] Item 18. A flow cytometer according to item 16 or 17, wherein the multiple sheath fluid introduction ports are offset from one another so that the sheath fluid swirls through the flow cell cone.

[0147] Clause 19. The flow cytometer of any one of clauses 1 to 18, further comprising a light source configured to illuminate the flow cell at the interrogation point.

[0148] 20. The flow cytometer of claim 19, wherein the distal end of the sample injection needle is spaced from the interrogation point by a distance in the range of 13 mm to 17 mm.

[0149] Item 21. The flow cytometer of item 20, wherein the distal end of the sample injection needle is spaced from the investigation point by a distance in the range of 14 mm to 16 mm.

[0150] Clause 22. The flow cytometer of any one of clauses 1 to 21, further comprising a detector configured to collect light from the flow cell.

[0151] Clause 23. The flow cytometer of any one of clauses 1 to 22, further comprising a vacuum to draw fluid through the flow cell.

[0152] Appendix 24. A method for analyzing a sample, comprising: (a) a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, the flow cell having a flow cell cone at the proximal end; a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at a proximal end to a flow cell at a distal end to generate a core stream, the sample injection needle constructed and arranged to maintain an intact core stream under flow conditions that vary by one or more orders of magnitude; introducing the particle sample into a flow cytometer comprising: (b) A method in which the particle sample is analyzed by flow cytometry.

[0153] 25. The method of claim 24, wherein the flow cytometer further comprises a sheath fluid introduction port for delivering sheath fluid to the flow cell.

[0154] Item 26. The method of item 25, wherein the distal end of the sample injection needle is positioned within the flow cell cone and is a longitudinal distance within the range of 17 mm to 26 mm from the sheath fluid introduction point.

[0155] Item 27. The method of item 26, wherein the distal end of the sample injection needle is a longitudinal distance in the range of 20 mm to 22 mm from the sheath fluid introduction point.

[0156] Item 28. The method of any one of Items 24-27, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow velocity of the flow stream is within the range of 0.5 m / s to 10 m / s.

[0157] Addendum 29. The method of any one of Addendums 24 to 28, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow rate of the sample fluid is within the range of 1 μl / min to 150 μl / min.

[0158] Appendix 30. The method of any one of Appendixes 24 to 29, wherein the sample injection needle has an inner diameter in the range of 0.2 mm to 0.4 mm.

[0159] Appendix 31. The method of Appendix 30, wherein the sample injection needle has an inner diameter in the range of 0.25 mm to 0.30 mm.

[0160] Appendix 32. The method of any one of Appendixes 24 to 31, wherein the sample injection needle has a tapered portion at the distal end.

[0161] Item 33. The method of item 32, wherein the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 3.5 mm to 4.0 mm.

[0162] Item 34. The method of item 33, wherein the tapered portion begins at a distance within a range of 3.8 mm to 3.9 mm from the distal end along the length of the sample injection needle.

[0163] Item 35. The method of item 32, wherein the tapered portion begins at a distance within a range of 1.5 mm to 2.0 mm from the distal end along the length of the sample injection needle.

[0164] Item 36. The method of item 35, wherein the tapered portion begins at a distance within a range of 1.7 mm to 1.8 mm from the distal end along the length of the sample injection needle.

[0165] Clause 37. The method of any one of clauses 32-36, wherein the tapered portion has a taper range of 2.5 mm to 12 mm radius over a length of 1.75 mm to 4 mm.

[0166] Addendum 38. The method of any one of Addendums 24 to 31, wherein the sample injection needle has a rounded distal end.

[0167] Item 39. The method of item 25, wherein the flow cytometer is equipped with multiple sheath fluid introduction ports.

[0168] Item 40. The method of item 39, wherein the flow cytometer is equipped with two sheath fluid introduction ports.

[0169] Clause 41. The method of clause 39 or 40, wherein the multiple sheath fluid introduction ports are offset from one another so that the sheath fluid swirls through the flow cell cone.

[0170] Addendum 42. The method of any one of Addendums 24 to 41, wherein the flow cytometer further comprises a light source configured to illuminate the flow cell at the interrogation point.

[0171] Item 43. The method of item 42, wherein the distal end of the sample injection needle is spaced from the investigation point by a distance in the range of 13 mm to 17 mm.

[0172] Item 44. The method of item 43, wherein the distal end of the sample injection needle is spaced from the investigation point by a distance in the range of 14 mm to 16 mm.

[0173] Appendix 45. The method of any one of appendices 24 to 44, wherein the sample is a biological sample.

[0174] Item 46. The method of item 45, wherein the sample comprises cells.

[0175] Appendix 47. The method of any one of appendices 24 to 46, wherein the sample is sorted by flow cytometry.

[0176] Appendix 48. A method for assembling a flow cytometer, comprising: a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at the proximal end to the flow cell at the distal end to generate the core stream; A method in which the sample injection needle is positioned within the flow cell cone such that an intact core stream is maintained under flow conditions that vary by more than one order of magnitude.

[0177] Item 49. The method of item 48, wherein the flow cytometer further comprises a sheath fluid introduction port for delivering sheath fluid to the flow cell.

[0178] Item 50. The method of item 49, wherein the distal end of the sample injection needle is positioned within the flow cell cone such that the distal end of the sample injection needle is a longitudinal distance within the range of 17 mm to 26 mm from the sheath fluid introduction point.

[0179] Item 51. The method of item 50, wherein the distal end of the sample injection needle is positioned within the flow cell cone such that the distal end of the sample injection needle is a longitudinal distance within the range of 20 mm to 22 mm from the sheath fluid introduction point.

[0180] Addendum 52. The method of any one of Addendums 48 to 51, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow velocity of the flow stream is within the range of 0.5 m / s to 10 m / s.

[0181] Addendum 53. The method of any one of Addendums 48 to 52, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow rate of the sample fluid is within the range of 1 μl / min to 150 μl / min.

[0182] Addendum 54. The method of any one of Addendums 48 to 53, wherein the sample injection needle has an inner diameter in the range of 0.2 mm to 0.4 mm.

[0183] Appendix 55. The method of Appendix 54, wherein the sample injection needle has an inner diameter in the range of 0.25 mm to 0.30 mm.

[0184] Addendum 56. The method of any one of Addendums 48 to 55, wherein the sample injection needle has a tapered portion at the distal end.

[0185] Item 57. The method of item 56, wherein the tapered portion begins at a distance within a range of 3.5 mm to 4.0 mm from the distal end along the length of the sample injection needle.

[0186] Item 58. The method of item 57, wherein the tapered portion begins at a distance within a range of 3.8 mm to 3.9 mm from the distal end along the length of the sample injection needle.

[0187] Item 59. The method of item 56, wherein the tapered portion begins at a distance within a range of 1.5 mm to 2.0 mm from the distal end along the length of the sample injection needle.

[0188] Item 60. The method of item 59, wherein the tapered portion begins at a distance within a range of 1.7 mm to 1.8 mm from the distal end along the length of the sample injection needle.

[0189] Addendum 61. The method of any one of Addendums 56-60, wherein the tapered portion has a taper range of 2.5 mm to 12 mm radius over a length of 1.75 mm to 4 mm.

[0190] Addendum 62. The method of any one of Addendums 48 to 55, wherein the sample injection needle has a rounded distal end.

[0191] Addendum 63. The method of Addendum 49, wherein the flow cytometer is equipped with multiple sheath fluid introduction ports.

[0192] Item 64. The method of item 63, wherein the flow cytometer is equipped with two sheath fluid introduction ports.

[0193] Item 65. The method of any one of items 63 to 64, wherein the multiple sheath fluid introduction ports are offset from one another so that the sheath fluid swirls through the flow cell cone.

[0194] Addendum 66. The method of any one of Addendums 48 to 65, wherein the flow cytometer comprises a light source configured to illuminate the flow cell at the interrogation point.

[0195] Item 67. The method of item 66, wherein the sample injection needle is positioned within the flow cell cone such that the distal end of the sample injection needle is a distance within the range of 13 mm to 17 mm from the interrogation point.

[0196] Item 68. The method of item 67, wherein the distal end of the sample injection needle is spaced from the investigation point by a distance in the range of 14 mm to 16 mm.

[0197] Although the foregoing invention 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 that, in view of the teachings of the present invention, certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

[0198] Accordingly, the foregoing merely illustrates the essence of the present invention. It is clear that those skilled in the art will be able to devise various configurations that embody the essence of the present invention and are within the spirit and scope of the present invention, although 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 present invention and the concepts provided by the inventors to advance the art, and should not be construed as limiting the scope of the present invention to the specifically set forth examples and conditions. Furthermore, all statements herein that describe the essence, aspects, and embodiments of the present invention, as well as specific examples of the present invention, are intended to encompass both structural and functional equivalents of the present invention. 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.

[0199] 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 / 388,406, filed July 12, 2022, the entire disclosure of which is incorporated herein by reference.

Claims

1. a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, the flow cell having a flow cell cone at the proximal end; a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at a proximal end to the flow cell at a distal end to generate a core stream; It is equipped with A flow cytometer wherein the sample injection needle is constructed and arranged to maintain an intact core stream under flow conditions that vary by more than one order of magnitude.

2. 10. The flow cytometer of claim 1, further comprising a sheath fluid introduction port for delivering sheath fluid to the flow cell.

3. 3. The flow cytometer of claim 2, wherein the distal end of the sample injection needle is disposed within the flow cell cone and is a longitudinal distance in the range of 17 mm to 26 mm from the sheath fluid introduction point.

4. 4. The flow cytometer of claim 1, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow velocity of the flow stream is within a range of 0.5 m / s to 10 m / s.

5. 5. A flow cytometer as described in any one of claims 1 to 4, wherein the sample injection needle is configured and arranged to maintain an intact core stream under conditions where the flow rate of the sample fluid is within the range of 1 μl / min to 150 μl / min.

6. 6. The flow cytometer according to claim 1, wherein the sample injection needle has an inner diameter in the range of 0.2 mm to 0.4 mm.

7. 7. The flow cytometer according to claim 1, wherein the sample injection needle has a tapered portion at the distal end.

8. 8. The flow cytometer of claim 7, wherein the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 3.5 mm to 4.0 mm.

9. 8. The flow cytometer of claim 7, wherein the tapered portion begins at a distance along the length of the sample injection needle from the distal end within a range of 1.5 mm to 2.0 mm.

10. 7. The flow cytometer of claim 1, wherein the sample injection needle has a rounded distal end.

11. 3. The flow cytometer of claim 2, comprising a plurality of sheath fluid introduction ports.

12. 12. The flow cytometer of claim 11, wherein the plurality of sheath fluid introduction ports are offset from one another so that sheath fluid swirls through the flow cell cone.

13. 1. A method for analyzing a sample, comprising: (a) a flow cell for transporting particles within a core stream of a flow stream from a proximal end to a distal end, the flow cell having a flow cell cone at the proximal end; a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at a proximal end to the flow cell at a distal end to generate a core stream, the sample injection needle constructed and arranged to maintain an intact core stream under flow conditions that vary by one or more orders of magnitude; introducing the particle sample into a flow cytometer comprising: (b) analyzing the particle sample by flow cytometry.

14. 14. The method of claim 13, wherein the sample is sorted by flow cytometry.

15. 1. A method of assembling a flow cytometer, comprising: a sample injection needle having a passageway therethrough for delivering sample fluid from a sample injection line at a proximal end to the flow cell at a distal end to generate a core stream; The method of claim 1, wherein the sample injection needle is positioned within the flow cell cone such that an intact core stream is maintained under flow conditions that vary by more than one order of magnitude.

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