Flow cytometry including sample injection needles and methods of use thereof

Abstract

The invention provides a flow cytometer with a sample injection needle. The flow cytometer includes a flow chamber for delivering particles in a central flow of the flow stream from a proximal end to a distal end, and a sample injection needle for delivering a sample fluid from a sample injection line at the proximal end to the flow chamber at the distal end to generate the central flow, the sample injection needle having a passage therethrough. The flow chamber of interest includes a flow chamber tapered structure at a proximal end, and the sample injection needle of interest is configured and placed to maintain a complete central flow under flow conditions that vary at one magnitude or more. The invention also provides a method of assembling the flow cytometer and a method of analyzing a sample using the flow cytometer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 388,406, filed on July 12, 2022; the disclosure of which is incorporated herein by reference in its entirety.

[0003] introduction

[0004] Characterization of analytes in biological fluids has become an important component of biological research, medical diagnostics, and assessment of a patient's overall health and well-being. Detection of analytes in biological fluids such as human blood or blood-derived products can provide results that can play a role in determining treatment regimens for patients suffering from a variety of disease states.

[0005] 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 other types of biological or chemical samples. 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. A flow cytometer delivers particles (including cells) in a fluid sample to a flow chamber as a cell stream, while also directing sheath fluid to the flow chamber. In order to characterize the components of the flow stream, the flow stream is illuminated with light. Changes in the material in the flow stream, such as morphology or the presence of fluorescent markers, may result in changes in the observed light, and these changes can be used for characterization and separation. In order to characterize the components in the flow stream, light must be directed onto the flow stream and collected. The light source of a flow cytometer can vary and can include one or more broad spectrum lamps, light emitting diodes, and single wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0006] Some flow cytometry systems are implemented using pressure-driven fluidics, where sample and sheath fluid are provided to a flow cell containing a detection region at a pressure above ambient pressure. Variation of the flow rate through the flow cell of a pressure-driven fluidics system can be achieved by varying the pressure in the sample tube and / or sheath fluid reservoir that feeds the flow cell. The ratio of sample fluid to sheath fluid flowing through the flow cell is controlled by the pressure levels in the sample tube and sheath fluid reservoir, as well as the ratio of the resistances of the sample fluid channel and the sheath fluid channel.

[0007] Alternatively, the flow cytometric system is implemented using vacuum-driven fluidics, wherein a vacuum pump draws a vacuum downstream of the flow chamber, and the sample fluid and sheath fluid are maintained at ambient pressure. By changing the degree of vacuum drawn by the vacuum pump, the flow rate through the flow chamber of the vacuum-driven fluidics system can be changed, and the ratio of the sample fluid and the sheath fluid flowing through the flow chamber is controlled by the ratio of the resistance of the sample fluid passage and the sheath fluid passage.

[0008] Some flow cytometers include an injection needle that plays an important role in introducing the sample into the flow chamber. The flow of sheath fluid within the flow chamber helps to establish the sample center and allows the sample cells to pass in a single file manner through the interrogation point of the cuvette. This technique is called hydrodynamic focusing. Summary of the invention

[0009] The inventors have recognized that imaging particles using a flow cytometer requires precise control of the speed of movement of cells within the detection point (i.e., where the sample is illuminated by one or more lasers). The inventors have found that failure to achieve such precise control may result in disruption of the flow stream and the core stream. In particular, the inventors have found that conventional sample injection mechanisms can produce "vortices" of the fluid that are associated with disturbances in the flow stream and the core stream. Therefore, the inventors have recognized that a flow cytometer with an improved sample injection needle is desired. Embodiments of the present invention meet this expectation.

[0010] Aspects of the present invention include flow cytometers. The flow cytometer includes a flow chamber for transporting particles in the center stream of the flow stream from the proximal end to the distal end, and a sample injection needle for transporting the sample fluid from the sample injection line at the proximal end to the flow chamber at the distal end to generate the center stream, the sample injection needle having a channel running through it. The flow chamber of interest includes a flow chamber cone at the proximal end, and the sample injection needle of interest is configured and placed to maintain a complete center stream under flow conditions that vary by one order of magnitude or more. In some embodiments, the flow chamber includes a sheath fluid introduction port for transporting sheath fluid to the flow chamber. In some such embodiments, the distal end of the sample injection needle is placed in the flow chamber cone and is separated from the sheath fluid introduction point by a longitudinal distance of 17 mm to 26 mm (e.g., 20 mm to 22 mm). In some cases, the sample injection needle is configured and placed to maintain a complete center stream under conditions where the flow velocity of the flow stream is 0.5 m / s to 10 m / s. In selected cases, the sample injection needle can be configured and placed to maintain a complete central flow under the condition that the flow rate of the sample fluid is 1 μL / min to 150 μL / min. In some cases, the sample injection needle includes an inner diameter of 0.2mm to 0.4mm (e.g., 0.25mm to 0.30mm). In some versions, the sample injection needle includes a tapered structure at the distal end. In some such versions, the tapered structure begins at a position where the distance separated from the distal end along the direction of the length of the sample injection needle is 3.5mm to 4.0mm (e.g., 3.8mm to 3.9mm). In other versions, the tapered structure begins at a position where the distance separated from the distal end along the direction of the length of the sample injection needle is 1.5mm to 2.0mm (e.g., 1.7mm to 1.8mm). In certain embodiments, the sample injection needle includes a tapered structure with a radius of 2.5mm to 12mm within a length span of 1.75mm to 4mm. In some embodiments, the sample injection needle includes a circular distal end. In selected cases, the flow cytometer includes multiple sheath fluid introduction ports, such as multiple sheath fluid introduction ports offset from each other so that the sheath fluid rotates through the conical structure of the flow chamber. The embodiment of the flow cytometer also includes a light source configured to illuminate the flow chamber at the detection point. In some cases, the distance between the far end of the flow chamber and the detection point is 13mm to 17mm (e.g., 14mm to 16mm). In an embodiment, the flow cytometer of the present invention includes one or more detectors configured to collect light from the flow chamber. In some versions, the flow cytometer includes a vacuum configured to draw fluid through the flow chamber.

[0011] Aspects of the present invention also include methods for analyzing samples. The method of interest includes introducing a particle sample into a flow cytometer including the flow chamber and a sample injection needle, and performing flow cytometry sorting on the particle sample. As described above, the flow cytometer includes a flow chamber for transporting particles in the central flow of the flow stream from the proximal end to the distal end, and a sample injection needle for transporting the sample fluid from the sample injection line at the proximal end to the flow chamber at the distal end to generate a central flow, the sample injection needle having a channel running through it. The flow chamber of interest includes a conical structure of the flow chamber at the proximal end, and the sample injection needle of interest is configured and placed to maintain a complete central flow under flow conditions that vary by one order of magnitude or more. In some cases, the particle sample is a biological sample (e.g., a cell).

[0012] Aspects of the present invention also include methods of assembling a flow cytometer. The method of interest includes placing a sample injection needle in the flow cytometer. As described above, the flow cytometer includes a flow chamber for transporting particles in a central stream of a flow stream from a proximal end to a distal end, and a sample injection needle for transporting a sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a central stream, the sample injection needle having a channel running through it. The flow chamber of interest includes a flow chamber tapered structure at a proximal end, and the sample injection needle of interest is configured and placed to maintain a complete central stream under flow conditions that vary by one order of magnitude or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present invention can be best understood when the following detailed description is read in conjunction with the accompanying drawings, which include the following:

[0014] Figures 1A to 1C A sample injection needle according to certain embodiments of the present invention is depicted.

[0015] FIG. 2A to FIG. 2C A diagram of the distal end of a sample injection needle is shown according to certain embodiments of the present invention.

[0016] FIG. 3A to FIG. 3B Depicted are flow chambers and sample injection needles according to certain embodiments of the present invention.

[0017] Figure 4 Depicted is a functional block diagram of a flow cytometry system according to certain embodiments.

[0018] Figure 5 A control system according to certain embodiments is depicted.

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

[0020] Figure 7A block diagram of a computing system is depicted in accordance with certain embodiments.

[0021] Figure 8 An image of the real-time central flow being detected by the laser is shown.

[0022] 9A to 9C Depicted is a sample injection needle used for needle performance analysis. DETAILED DESCRIPTION

[0023] A flow cytometer with a sample injection needle is provided. The flow cytometer includes a flow chamber for transporting particles in a central stream of a flow stream from a proximal end to a distal end, and a sample injection needle for transporting a sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a central stream, the sample injection needle having a channel running through it. The flow chamber of interest includes a flow chamber tapered structure at a proximal end, and the sample injection needle of interest is configured and positioned to maintain a complete central stream under flow conditions that vary by one order of magnitude or more. A method of assembling the flow cytometer and a method of analyzing a sample using the flow cytometer are also provided.

[0024] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as they can certainly vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0025] When providing a numerical range, it is understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value in the specified range are included in the present invention, wherein the precision of the intermediate value is one-tenth of the lower limit unit unless the context clearly stipulates otherwise. The upper and lower limits of these smaller ranges can be independently included in the smaller range and are also included in the present invention, but are subject to any explicitly excluded limits in the specified range. When the specified range includes one or two limits, the range excluding one or two of the included limits is also included in the present invention.

[0026] Certain ranges shown herein use the term "about" before a numerical value. As used herein, the term "about" provides written support for the exact number that follows it as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the unrecorded number that is close or approximately can be a number that is substantially equivalent to the specifically recited number when shown.

[0027] Unless otherwise defined, all technical terms and all scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and any 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 now described.

[0028] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for its disclosure content 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. In addition, the publication dates provided may be different from the actual publication dates and may need to be independently confirmed.

[0029] It is noted that, as used herein and in the appended claims, an element without a quantifier includes the singular and the plural unless the context clearly dictates otherwise. It is also noted that a claim may exclude any optional element. Therefore, this statement is intended to serve as an antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in connection with reciting claim elements, or for the use of a "negative" limitation.

[0030] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has independent components and features that can be easily separated or combined with the features in any other several embodiments without departing from the scope or spirit of the invention. Any recorded method can be carried out in the order of events recorded or in any other order that is logically possible.

[0031] Flow cytometry

[0032] As described above, aspects of the present invention include flow cytometers. The flow cytometer of interest includes a flow chamber for transporting particles in a central stream of a flow stream from a proximal end to a distal end, and a sample injection needle for transporting a sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a central stream, the sample injection needle having a channel running through it. The "central stream" described herein refers to a fluid stream described in its conventional sense, wherein the particles are focused by a sheath fluid flow (e.g., by hydrodynamic focusing) and transported through the flow chamber. Typically, the particles are transported through the central stream in a single-file manner. The size (e.g., diameter) of the central stream may vary as desired. In some cases, the diameter of the central stream may be approximately the same as the diameter of the particles being analyzed. In some cases, the diameter of the central stream is 5 μm to 25 μm, including 10 μm to 20 μm. The central stream diameter may be adjusted in proportion to the pressure (e.g., positive or negative pressure) applied when the particles are injected into the sheath fluid flow. In some cases, the flow rate of the sheath fluid remains unchanged. In this way, particles are injected into the sheath fluid and hydrodynamically focused so that laminar flow is generated and the particles move along the same axis at approximately the same rate.

[0033] If a central flow associated with the present invention maintains a relatively constant shape throughout the length of the flow chamber, it can be described as a "complete" central flow. In some cases, the complete central flow of the present invention is defined by straight edges. In other words, when viewed in two dimensions, the boundaries of the complete flow stream appear straight. In selected versions, the flow stream and its component central streams are hydrodynamically focused and characterized by laminar flow. In some such versions, the straight edges of the central flow are substantially parallel to each other (e.g., such as the edges deviate from true parallel lines by five degrees or less, such as two degrees or less). In some cases, the complete central flow is characterized by no disturbances from the distal end of the sample injection needle to the distal end of the flow chamber. In some such cases, the complete central flow does not include vortices (e.g., vortices around the tip of the sample injection needle). In some cases, the flow cytometer and its component sample injection needles reduce flow stream disturbances and center flow disturbances 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, including a 100% reduction, compared to conventional instruments.

[0034] As described above, the sample injection needle is configured and positioned to maintain a complete center flow under flow conditions that vary by one order of magnitude or more. The term "order of magnitude" - also referred to as "order of magnitude" - is used in its conventional sense, referring to flow conditions that differ by a multiple of ten or more than ten times. In some cases, the sample injection needle is configured and positioned to maintain a complete center flow under flow conditions that vary by two orders of magnitude or more. For example, the flow conditions described herein may include sheath fluid flow rate, sample fluid flow rate, particle size (e.g., diameter), pressure (positive or negative pressure), etc. In some embodiments, the sample injection needle is configured and positioned to maintain a complete center flow under a sheath fluid flow rate that varies by one order of magnitude or more. For example, in some cases, the sample injection needle is configured and positioned to maintain a complete center flow under conditions where the flow rate of the flow stream is 0.5 m / s to 10 m / s. In selected cases, the sample injection needle is configured and positioned to maintain a complete center flow under a sample fluid flow rate that varies by one order of magnitude or more. For example, in some cases, the sample injection needle is configured and positioned to maintain full core flow at a sample fluid flow rate of 1 μL / min to 150 μL / min.

[0035] As described herein, "flow chamber" is described in its conventional sense and refers to a component containing a flow channel, which is configured to accommodate a liquid flow stream for transporting particles in a sheath fluid. In an embodiment, the flow chamber includes a colorimetric chamber. The colorimetric chamber of interest includes a container having a channel running through it. The flow chamber of interest includes a flow channel into which light can enter. In some cases, the flow chamber includes a transparent material (e.g., quartz) that allows light to pass through it. Any convenient flow chamber that transmits a fluid sample to a sample detection area can be used as the flow chamber described herein, wherein in some embodiments, the flow chamber includes a cylindrical flow chamber, a truncated conical flow chamber, or a flow chamber including a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminating in a plane, the plane having an orifice transverse to the longitudinal axis. The flow chamber of interest has a proximal end for receiving fluid from a sample fluid source (i.e., through a sample injection needle) and a sheath fluid source and a distal end for emitting fluid. Depending on the configuration of the flow cytometer, the fluid at the distal end of the flow chamber can flow to one or more different types of collection containers.

[0036] In some cases, the flow stream is configured to be illuminated by light from a light source at a detection point. The flow channel is configured for a flow stream, which may include a liquid sample injected from a sample tube through a sample injection line. In certain embodiments, the flow stream may include a narrow, fast-flowing liquid stream, which is arranged so that the particles separated along a straight line transported therein are separated from each other in a single column. The "detection point" described herein refers to an area in a flow chamber where the particles are illuminated by light from a light source, such as for analysis. The size of the detection point can vary as desired. For example, 0 μm represents the axis of the light emitted by the light source, and the detection point can be -100 μm to 100 μm, such as -50 μm to 50 μm, such as -25 μm to 40 μm, including -15 μm to 30 μm.

[0037] The flow chamber of interest includes a flow chamber taper at the proximal end. A "flow chamber taper" refers to a tapered (e.g., a right circular cone) recessed portion in a flow chamber that narrows toward the analysis region (i.e., detection region) of the flow chamber. The flow chamber taper can play a role in hydrodynamic focusing of the flow stream. The flow chamber taper described herein can be characterized in that the angle (i.e., the angle measured relative to an imaginary axis through the center of the taper to the generatrix of the taper) is 15° to 25°, e.g., 18° to 22°, e.g., 19° to 21°. In some cases, the flow chamber taper described herein can be characterized in that the angle is 20°.

[0038] In certain embodiments, flow cytometer includes sample fluid source.Sample fluid source can be any suitable reservoir or container (e.g., it has rigid wall or flexible wall) for accommodating sample fluid.The volume of sample fluid container can be 1mL to 100mL; For example, the volume of container can be 1mL to 90mL, 1mL to 80mL, 1mL to 70mL, 1mL to 60mL, 1mL to 50mL, 1mL to 40mL, 1mL to 30mL, 1mL to 20mL or 1mL to 10mL.In embodiments, sample fluid is provided to sample injection needle from sample fluid source by sample injection line (e.g., pipeline).

[0039] In some embodiments, the flow cytometer includes a sheath fluid reservoir. The sheath fluid reservoir can be any suitable reservoir or container for holding sheath fluid (e.g., it has rigid walls or flexible walls). In certain embodiments, the sheath fluid reservoir is fluidically coupled to the input end of the flow chamber. The volume of the sheath fluid container can be 1L to 100L; for example, the volume of the container can be 1L to 90L, 1L to 80L, 1L to 70L, 1L to 60L, 1L to 50L, 1L to 40L, 1L to 30L, 1L to 20L, or 1L to 10L. The sheath fluid reservoir can be fluidically coupled to a sheath fluid line configured to transport the sheath fluid from the reservoir to the flow chamber.

[0040] The flow cytometer of interest may also include one or more than one sheath fluid introduction port. The sheath fluid introduction port is fluidically connected to a sheath fluid source (i.e., a reservoir) and provides sheath fluid to the flow chamber at the proximal end. In an embodiment, the sheath fluid injection system is configured to provide a sheath fluid flow to the inner cavity of the flow chamber, such as a sheath fluid flow stream that produces a laminar flow around the sample flow stream together with the sample. Depending on the desired flow stream characteristics, the sheath fluid rate delivered to the flow chamber cavity can be 25 μL / s to 2500 μL / s, such as 50 μL / s to 1000 μL / s, including 75 μL / s or more than 75 μL / s to 750 μL / s.

[0041] In some embodiments, the sheath injection port is an orifice located on the lumen wall. The sheath injection port orifice can be any suitable shape, wherein the cross-sectional shape of interest includes but is not limited to a straight cross-sectional shape, such as a square, rectangle, irregular trapezoid, triangle, hexagon, etc., a curved cross-sectional shape, such as a circle, an oval, and an irregular shape, such as a parabola bottom connected to a plane top. The size of the sample injection port orifice can vary according to the shape, and in some cases, its opening is 0.1mm to 5.0mm, such as 0.2mm to 3.0mm, such as 0.5mm to 2.5mm, such as 0.75mm to 2.25mm, such as 1mm to 2mm, including 1.25mm to 1.75mm, such as 1.5mm.

[0042] In some cases, one or more than one sheath liquid introduction port is located in the flow chamber tapered structure (i.e., at the "base" of the tapered structure). In some cases, the flow cytometer includes a single sheath liquid introduction port. In other cases, the flow cytometer includes multiple sheath liquid introduction ports. The number of sheath liquid ports in multiple sheath liquid ports can vary, including, for example, 2 sheath liquid introduction ports, 3 sheath liquid introduction ports, 4 sheath liquid introduction ports, and 5 sheath liquid introduction ports. In a selected version, the flow cytometer includes 2 sheath liquid introduction ports. In some cases where the flow cytometers of the present invention include multiple sheath liquid introduction ports, multiple sheath liquid introduction ports are offset from each other so that the sheath liquid rotates through the flow chamber tapered structure (e.g., in a "toilet" manner). In some such cases, multiple sheath liquid introduction ports are offset from each other so that the sheath liquid rotates in a clockwise direction. In other cases, multiple sheath liquid introduction ports are offset from each other so that the sheath liquid rotates in a counterclockwise direction. In some cases, the rotation of the sheath liquid can also provide flow stream stability and center flow stability.

[0043] The sample injection needle of the present disclosure may include an elongated structure. "Elongated structure" refers to a sample injection needle whose length is greater than its width. In other words, the sample injection needle has a clearly distinguished proximal end and distal end. The proximal end is the end of the sample injection needle that receives the sample fluid (i.e., receives the sample fluid from a sample injection line connected to a sample fluid source fluid), and the distal end is the end of the sample injection needle that injects the sample into the flow chamber. The elongated structure can have any convenient cross-sectional shape, wherein the cross-sectional shapes of interest include, but are not limited to, straight cross-sectional shapes, such as squares, rectangles, irregular trapezoids, triangles, hexagons, etc., curved cross-sectional shapes, such as circles, ovals, and irregular shapes, such as the bottom of a parabola connected to a plane top. In an embodiment, the elongated structure has a substantially circular cross-sectional shape at a position along the length direction. A "substantially" circular cross section means that in an embodiment, the cross section at one or more than one position along the length direction of the outlet fitting may slightly deviate from a circular cross section, while the rest of the structure is characterized by a circular cross section. For example, in some versions, the elongated structure has a polygonal (e.g., hexagonal, pentagonal, etc.) cross section at one or more than one position along the length direction. In some cases, the width (e.g., cross-sectional diameter) of the elongated structure varies along the length direction of the outlet fitting. In other words, in these versions, the elongated structure is not a perfect cylinder, but a cross-sectional shape of certain regions having a diameter greater than or less than the diameter of the cross-sectional shape of other regions. In other cases, each portion of the sample injection needle has a circular cross section. In some such cases, different portions of the sample injection needle may be characterized by different diameters. In some cases, the outer diameter of at least part of the sample injection needle (i.e., the diameter measured from the geometric center of the sample injection needle to the outer edge) is 0.5 mm to 4 mm, such as 0.75 mm to 3 mm, such as 1 mm to 2 mm, such as 1.25 mm to 1.75 mm, including 1.5 mm to 1.6 mm. In some cases, the sample injection needle includes an outer diameter of 1.562 mm. The sample injection needle may have any convenient length, with the length of interest being 15 mm to 30 mm, such as 17 mm to 26 mm, including 20 mm to 22 mm. In some cases, the sample injection needle does not flatten at the distal end, such as to form a "duckbill" shape. In these cases, the sample injection needle has a substantially circular cross-section throughout.

[0044] In some cases, the sample injection needle includes a tapering structure at the distal end. In some such cases, the sample injection needle has a relatively constant outer diameter within most of its length (i.e., starting from the proximal end), but the outer diameter gradually (e.g., uniformly) decreases when approaching the distal end. In certain embodiments, the sample injection needle includes a tapering structure with a radius of 2.5mm to 12mm in a length span of 1.75mm to 4mm. For example, in some versions, the sample injection needle tapering structure has a radius of 2.79mm to 11.63mm in a length span of 1.78mm to 3.81mm. The tapering structure can start at different positions along the length direction of the sample injection needle. In some cases, the distance between the position along the length direction of the sample injection needle where the tapering structure starts and the distal end is 3mm to 5mm, such as 3.5mm to 4.0mm, including 3.8mm to 3.9mm. In some cases, such a distal end can be referred to as having a "super bullet" structure. In some embodiments with a super bullet structure, the tapering structure at the distal end is within a length span of 3.81 mm and has a radius of 11.63 mm. In other cases, the distance between the position along the length direction of the sample injection needle where the tapering structure begins and the distal end is 1 mm to 3 mm, such as 1.5 mm to 2 mm, including 1.7 mm to 1.8 mm. In some cases, such a distal end can be referred to as having a "bullet" structure. In some embodiments where the sample injection needle has a bullet structure, the tapering structure at the distal end is within a length span of 1.78 mm and has a radius of 2.79 mm. In other cases, the sample injection needle includes a rounded distal end.

[0045] The sample injection needle of the present invention also includes an opening at the distal end and a channel running through it, and the channel is used for sample fluid-optionally it contains particles-to be delivered to the flow chamber. In an embodiment, the opening is located at the geometric center of the cross section of the outlet fitting at the distal end. The opening can have any convenient cross-sectional shape, wherein the cross-sectional shape of interest includes but is not limited to a straight cross-sectional shape, such as a square, rectangle, irregular trapezoid, triangle, hexagon, etc., a curved cross-sectional shape, such as a circle, an oval, and an irregular shape, such as a parabola bottom connected to a plane top. In some cases, the opening has a circular cross-sectional shape. In other cases, the channel similarly includes a circular cross section. The opening can have any suitable diameter, such as a diameter of 0.1mm to 2mm, such as 0.1mm to 1mm, such as 0.2mm to 0.4mm, including 0.25mm to 0.30mm. Similarly, the channel can have any suitable diameter, such as a diameter of 0.1mm to 2mm, such as 0.1mm to 1mm, such as 0.2mm to 0.4mm, including 0.25mm to 0.30mm. In some cases, the opening and the channel have a circular cross-section with the same or similar diameters. In other cases, the diameter of the opening is different from (e.g., greater than) the diameter of the channel.

[0046] In some cases, the sample injection needle includes an adapter at the proximal end. In some cases, the adapter can be used to secure the sample injection needle within the flow cytometer. In some cases, the adapter includes one or more holes for receiving fasteners (e.g., pins, screws, etc.). In selected versions, the adapter includes multiple holes for receiving fasteners. In certain embodiments, the adapter includes three holes. In other cases, the adapter can be configured to be fluidically connected to the sample injection line to provide sample fluid (optionally containing particles) to the sample injection needle.

[0047] Figures 1A to 1C An alternative description of a sample injection needle according to certain embodiments of the present invention is provided. Figure 1A As shown, the sample injection needle 100a includes an elongated structure 101 and a tapered structure 102 at the distal end. Figure 1B An example of a sample injection needle with an adapter is provided. Figure 1B As shown, the sample injection needle 100b comprises an elongated structure 101, a tapered structure 102 at a distal end, and an adapter 103. The adapter 103 comprises a plurality of holes 105 for receiving fasteners (such as screws, not shown) for connecting the sample injection needle to the flow chamber. Figure 1C Cross-sectional views of sample injection needles according to some embodiments of the present invention are provided. As shown in section AA of sample injection needle 100c, a channel 104 running through the center of the needle is visible. Also shown is an elongated structure 101 and a tapered structure 102 at the distal end.

[0048] FIG. 2A to FIG. 2C An illustration of the distal end of a sample injection needle according to certain embodiments of the present invention is shown. FIG. 2A to FIG. 2C The exemplary dimensions shown are in inches. Figure 2A A sample injection needle distal end 202a having the above-described super bullet structure is shown. Figure 2B A sample injection needle distal end 202b is shown having a rounded distal end. Figure 2C A sample injection needle distal end 202c is described having the above-described bullet structure.

[0049] The sample injection needle of the present invention is placed in a specific manner relative to the flow chamber. The placement of the sample injection needle relative to the flow chamber can be described in a variety of ways. For example, in some cases, the placement of the sample injection needle is described as based on the position of the 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 placed within the tapered structure of the flow chamber, and the longitudinal distance separated from the sheath fluid introduction point is 15mm to 30mm, such as 17mm to 26mm, including 20mm to 22mm. The placement of the sample injection needle can also be described as based on the position of the distal end relative to the detection point (i.e., the point where the flow chamber is illuminated by one or more light sources). In some cases, the distance separating the distal end of the sample injection needle from the detection point is 10mm to 20mm, such as 13mm to 17mm, including 14mm to 16mm.

[0050] FIG. 3A to FIG. 3B Flow cells and sample injection needles according to certain embodiments of the present invention are described. Figure 3A As shown, the flow chamber 303 includes a flow chamber tapered structure 302 and a colorimetric chamber 304. A laser (not shown) illuminates the colorimetric chamber 304 at an illumination point 305. A sheath fluid introduction port 306 is configured to provide sheath fluid to the flow chamber tapered structure 302, and a sample injection needle 301 is configured to provide sample fluid to the flow chamber tapered structure 302, thereby maintaining a complete core flow (not shown) under flow conditions that vary by one order of magnitude or more. The sheath fluid and the sample fluid pass through the flow chamber along a direction d. Figure 3B Provided Figure 3A An alternative depiction of the same elements provided in FIG. Figure 3B As shown in the exemplary embodiment of , the separation distance between the distal end of the sample injection needle 301 and the sheath injection port 306 is 0.840 inches (21.3 mm). In addition, the separation distance between the distal end of the sample injection needle 301 and the irradiation point 305 is 0.5848 inches (14.85 mm).

[0051] After irradiating the particles in the flow chamber, light modulated by the particles can be observed. "Light modulated by the particles" refers to the light received from the particles in the flow stream after the light from the light source irradiates the particles. In some cases, the light modulated by the particles is side scattered light. As described herein, side scattered light refers to the light refracted and reflected from the surface and internal structure of the particles. In other embodiments, the light modulated by the particles includes forward scattered light (i.e., light that passes through or bypasses the particles mainly in a forward direction). In still other cases, the light modulated by the particles includes fluorescence (i.e., light emitted from fluorescein after irradiation with light of an excitation wavelength).

[0052] As described above, aspects of the present invention also include a light source configured to illuminate particles passing through the flow chamber at the detection point. Any convenient light source can be used as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser can be any convenient laser, such as a continuous wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near infrared diode laser. In other embodiments, the laser can 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 2 In some embodiments, the flow cytometer includes a 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 includes a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, the laser of interest includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and a combination thereof. In other cases, the flow cytometer includes a solid-state laser, such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO 4 Laser, Nd:YCa 4 O(BO 3 ) 3 Laser, Nd:YCOB laser, titanium sapphire laser, thulium YAG laser, ytterbium YAG laser, Yb 2 O 3 Laser or cerium-doped laser and combination thereof.

[0053] According to certain embodiments, the laser light source may also include one or more optical adjustment elements. In certain embodiments, the optical adjustment element is located between the light source and the flow chamber, and it may include any device capable of changing the spatial width of the illumination from the light source or some other characteristics of the illumination, such as illumination direction, wavelength, beam width, beam intensity, and focal spot. The optical adjustment scheme 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 schemes, and combinations thereof. In certain embodiments, the flow cytometer of interest includes one or more focusing lenses. In one example, the focusing lens can be a reduction lens. In other embodiments, the flow cytometer of interest includes an optical fiber.

[0054] When the optical adjustment element is configured to move, the optical adjustment element can be configured to move continuously or in discrete intervals, such as increments of 0.01μm or more, such as 0.05μm or more, such as 0.1μm or more, such as 0.1μm or more, such as 0.5μm or more, such as 1μm or more, such as 10μm or more, such as 100μm or more, such as 500μm or more, such as 1mm or more, such as 5mm or more, such as 10mm or more, including increments of 25mm or more.

[0055] Any displacement scheme can be used to move the optical adjustment element structure, such as connecting to a movable support table, or directly connecting to a motor-driven translation stage, a screw translation assembly, or a gear translation device, such as a stepper motor, a servo motor, a brushless motor, a brushed DC motor, a micro-step drive motor, a high-resolution stepper motor, or other types of motors.

[0056] The light source can be placed at any suitable distance from the flow chamber, such as a distance of 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, including 100 mm or more. In addition, the light source can be placed at any suitable angle relative to the flow chamber, such as an angle of 10 to 90 degrees, such as 15 to 85 degrees, such as 20 to 80 degrees, such as 25 to 75 degrees, including 30 to 60 degrees, such as an angle of 90 degrees.

[0057] In some embodiments, the light source of interest includes a plurality of lasers configured to provide lasers for discretely irradiating the flow stream, for example, 2 or more lasers, such as 3 or more lasers, such as 4 or more lasers, such as 5 or more lasers, such as 10 or more lasers, including 15 or more lasers configured to provide lasers for discretely irradiating the flow stream. Depending on the wavelength of light desired for irradiating the flow stream, the specific wavelength of each laser can vary from 200nm to 1500nm, such as from 250nm to 1250nm, such as from 300nm to 1000nm, such as from 350nm to 900nm, including from 400nm to 800nm. In certain embodiments, the lasers of interest may include one or more 405nm lasers, 488nm lasers, 561nm lasers, and 635nm lasers.

[0058] As described above, the flow cytometer of interest may also include one or more particle-modulated light detectors for detecting intensity data of particle-modulated light. In some embodiments, one or more particle-modulated light detectors include one or more forward-scattered light detectors configured to detect forward-scattered light. For example, the flow cytometer may include 1 forward-scattered light detector or multiple forward-scattered light detectors, such as 2 or more than 2, such as 3 or more than 3, such as 4 or more than 4, including 5 or more than 5. In certain embodiments, the flow cytometer includes 1 forward-scattered light detector. In other embodiments, the flow cytometer includes 2 forward-scattered light detectors.

[0059] The forward scattered light detector described herein can use any convenient detector for detecting the collected light. The detector of interest can include, but is not limited to, an optical sensor or optical detector, such as an active pixel sensor (APS), an avalanche photodiode, an image sensor, a charge coupled device (CCD), an enhanced charge coupled device (ICCD), a light emitting diode, a photon counter, a thermal radiation meter, a pyroelectric detector, a photoresistor, a photocell, a photodiode, a photomultiplier tube (PMT), a phototransistor, a quantum dot photoconductor or a quantum dot photodiode and a combination thereof, and other detectors. In certain 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 certain embodiments, the detector is a photomultiplier tube, such as an active detection surface area of ​​0.01 cm per region. 2 Up to 10cm 2 , such as 0.05cm2 Up to 9cm 2 , such as 0.1cm 2 Up to 8cm 2 , such as 0.5cm 2 Up to 7cm 2 And including 1cm 2 Up to 5cm 2 Photomultiplier tube.

[0060] In embodiments, the forward scattered light detector is configured to measure light continuously or to measure light at discrete intervals. In some cases, the detector of interest is configured to measure the collected light continuously. In other cases, the detector of interest is configured to measure at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or measuring light at some other intervals.

[0061] In other embodiments, one or more particle modulated light detectors may include one or more side scattered light detectors for detecting wavelengths of side scattered light (i.e., light refracted and reflected from the surface and internal structure of the particles). In some embodiments, the flow cytometer includes a single side scattered light detector. In other embodiments, the flow cytometer includes multiple side scattered light detectors, such as 2 or more than 2, such as 3 or more than 3, such as 4 or more than 4, including 5 or more than 5.

[0062] The side scattered light detector described herein can use any convenient detector for detecting the collected light. The detector of interest can include, but is not limited to, an optical sensor or optical detector, such as an active pixel sensor (APS), an avalanche photodiode, an image sensor, a charge coupled device (CCD), an enhanced charge coupled device (ICCD), a light emitting diode, a photon counter, a thermal radiation meter, a pyroelectric detector, a photoresistor, a photocell, a photodiode, a photomultiplier tube (PMT), a phototransistor, a quantum dot photoconductor or a quantum dot photodiode and a combination thereof, and other detectors. In certain 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 certain embodiments, the detector is a photomultiplier tube, for example, the active detection surface area of ​​each region is 0.01 cm 2 Up to 10cm 2 , such as 0.05cm 2 Up to 9cm 2 , such as 0.1cm 2 Up to 8cm2 , such as 0.5cm 2 Up to 7cm 2 And including 1cm 2 Up to 5cm 2 Photomultiplier tube.

[0063] In embodiments, the flow cytometer further comprises a fluorescence detector configured to detect light of one or more than one fluorescence wavelength. In other embodiments, the flow cytometer comprises a plurality of fluorescence detectors, such as 2 or more than 2, such as 3 or more than 3, such as 4 or more than 4, 5 or more than 5, 10 or more than 10, 15 or more than 15, including 20 or more than 20.

[0064] The fluorescence detectors described herein can use any convenient detector for detecting the collected light. Detectors of interest can include, but are not limited to, optical sensors or optical detectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, thermal radiation meters, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or quantum dot photodiodes and combinations thereof, and other detectors. In certain 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 certain embodiments, the detector is a photomultiplier tube, such as an active detection surface area of ​​0.01 cm per region. 2 Up to 10cm 2 , such as 0.05cm 2 Up to 9cm 2 , such as 0.1cm 2 Up to 8cm 2 , such as 0.5cm 2 Up to 7cm 2 And including 1cm 2 Up to 5cm 2 Photomultiplier tube.

[0065] When the flow cytometer includes multiple fluorescence detectors, each fluorescence detector can be the same, or the collection of fluorescence detectors can be a combination of different types of detectors. For example, when the flow cytometer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector (or imaging sensor) is a CMOS type device. In other embodiments, the first fluorescence detector and the second fluorescence detector are both CCD type devices. In other embodiments, the first fluorescence detector and the second fluorescence detector are both CMOS type devices. In other embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector is a photomultiplier tube (PMT). In other embodiments, the first fluorescence detector is a CMOS type device and the second fluorescence detector is a photomultiplier tube. In other embodiments, the first fluorescence detector and the second fluorescence detector are both photomultiplier tubes.

[0066] In embodiments of the present disclosure, the fluorescence detector of interest is configured to measure one or more wavelengths of light collected, such as 2 or more wavelengths of light, such as 5 or more than 5 different wavelengths of light, such as 10 or more than 10 different wavelengths of light, such as 25 or more than 25 different wavelengths of light, such as 50 or more than 50 different wavelengths of light, such as 100 or more than 100 different wavelengths of light, such as 200 or more than 200 different wavelengths of light, such as 300 or more than 300 different wavelengths of light, and including measuring 400 or more than 400 different wavelengths of light emitted by the sample in the flow stream. In some embodiments, 2 or more detectors in the flow cytometer described herein are configured to measure the collected light of the same wavelength or overlapping wavelengths.

[0067] In some embodiments, the fluorescence detector of interest is configured to measure the collected light within a certain wavelength range (e.g., 200nm to 1000nm). In certain embodiments, the detector of interest is configured to collect a spectrum of light within a certain wavelength range. For example, a flow cytometer may include one or more detectors configured to collect one or more spectra of light within a wavelength range of 200nm to 1000nm. In other embodiments, the detector of interest is configured to measure light of one or more specific wavelengths emitted by a sample in a flow stream. For example, a flow cytometer can 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, one or more detectors can be configured to pair with a specific fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0068] In some embodiments, flow cytometer includes one or more wavelength separators, and the wavelength separator is placed between the flow chamber and one or more detectors of the light modulated by particles. The term "wavelength separator" used herein refers to an optical element configured to separate the light collected from the sample into a predetermined spectral range in its conventional sense. In some embodiments, flow cytometer includes a single wavelength separator. In other embodiments, flow cytometer includes multiple wavelength separators, such as 2 or more than 2 wavelength separators, such as 3 or more than 3, such as 4 or more than 4, such as 5 or more than 5, such as 6 or more than 6, such as 7 or more than 7, such as 8 or more than 8, such as 9 or more than 9, such as 10 or more than 10, such as 15 or more than 15, such as 25 or more than 25, such as 50 or more than 50, such as 75 or more than 75, including 100 or more than 100 wavelength separators. In some embodiments, the wavelength separator is configured to pass light with a predetermined spectral range and reflect the remaining light of one or more spectral ranges, thereby separating the light collected from the sample into a predetermined spectral range. In other embodiments, the wavelength separator is configured to pass light with a predetermined spectral range and absorb the remaining light of one or more spectral ranges, thereby separating the light collected from the sample into a predetermined spectral range. In other embodiments, the wavelength separator is configured to spatially diffract the light collected from the sample to a predetermined spectral range. Each wavelength separator can be any convenient light separation scheme, 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 in the light detection system is a dichroic mirror.

[0069] Suitable flow cytometry systems may include, but are not limited to, 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 Throm 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. 24(3):203-255; the disclosure of which is incorporated herein by reference. In some cases, the flow cytometry system of interest includes the BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSR Fortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TMFlow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter, BD Biosciences FACSAria TM II cell sorter, BD Biosciences FACSAria TM III cell sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorter, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0070] In some embodiments, the system is a flow cytometric system, such as described in U.S. Pat. Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074. ; No. 10,302,545; No. 10,145,793; No. 10,113,967; No. 10,006,852; No. 9,952,076; No. 9,933,341; No. 9,726,527; No. 9,453,789; No. 9,200,334; No. 9,097,640; No. 9,095 ,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,8 No. 09,804; No. 6,372,506; No. 5,700,692; No. 5,643,796; No. 5,627,040; No. 5,620,842; No. 5,602,039; No. 4,987,086; No. 4,498,766; the disclosures of which are incorporated herein by reference in their entirety.

[0071] In some cases, the flow cytometric system of the present invention is configured to image particles in a flow stream by fluorescence imaging using radiofrequency tagged emission (FIRE), as described in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013) and in U.S. Pat. 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 , No. 408,758; No. 10,451,538; No. 10,620,111; and those described in U.S. 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.

[0072] Figure 4 A system 400 for flow cytometry according to an illustrative embodiment of the invention is shown. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a to 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 to 445g, one or more bandpass filters 450a to 450e, one or more long pass ("LP") filters 455a to 455b, and one or more fluorescence detectors 460a to 460f.

[0073] The excitation lasers 415a to 415c emit light in the form of laser beams. Figure 4 In the example system of , the wavelengths of the laser beams emitted from the excitation laser 415a to the excitation laser 415c are 488nm, 633nm and 325nm, respectively. The laser beams are first directed to pass through one or more beam splitters 445a and 445b. The beam splitter 445a transmits 488nm light and reflects 633nm light. The beam splitter 445b transmits UV light (light with a wavelength of 10nm to 400nm) and reflects 488nm light and 633nm light.

[0074] The laser beam is then directed to a focusing lens 420, which focuses the beam to the portion of the fluid stream where the particles of the sample are located in a flow chamber 425. A flow chamber is a portion of a fluidics system that directs particles in a stream, typically one particle at a time, to a focused laser beam for detection. A flow chamber may include a flow chamber in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0075] Light from one or more laser beams interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at several different wavelengths depending on the characteristics of the particles, such as size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present in the particles. The fluorescent emission, as well as the diffracted, refracted, reflected, and scattered light, can be sent through one or more beam splitters 445c to 445g, bandpass filters 450a to 450e, longpass filters 455a to 455b, and fluorescence collection lens 440 to one or more forward scatter detectors 430, side scatter detector 435, and one or more fluorescence detectors 460a to 460f.

[0076] The fluorescence collection lens 440 collects the light emitted by the particle-laser beam interaction and sends the light to one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a to bandpass filters 450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Therefore, a 510 / 20 filter extends 10nm on both sides of the center of the spectral band, or is 500nm to 520nm. Shortpass filters transmit light with a wavelength equal to or shorter than a specified wavelength. Longpass filters, such as longpass filters 455a to longpass filters 455b, transmit light with a wavelength equal to or longer than a specified wavelength of light. For example, longpass filter 455b is a 670nm longpass filter, which transmits light with a wavelength equal to or longer than 670nm. Filters are often chosen to optimize the specificity of the detector for a particular fluorescent dye. Filters can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0077] The forward scattering detector 430 is placed slightly off the axis of the direct beam through the flow chamber and is configured to detect diffracted light, i.e., excitation light that passes through or bypasses the particles mainly in the forward direction. The intensity of the light detected by the forward scattering detector depends on the overall size of the particles. The forward scattering detector may include a photodiode. The side scattering detector 435 is configured to detect refracted light and reflected light from the surface and internal structure of the particles, which tends to increase with the increase in the complexity of the particle structure. One or more fluorescence detectors 460a to 460f can detect fluorescence emission from fluorescent molecules associated with the particles. The side scattering detector 435 and the fluorescence detector may include a photomultiplier tube. The signals detected at the forward scattering detector 430, the side scattering detector 435, and the fluorescence detector may be converted into electronic signals (voltages) by the detectors. These data can provide information about the sample.

[0078] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to Figure 4 The depicted flow cytometer may also include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors, which may be of various wavelengths and have different configurations.

[0079] In operation, the operation of the cytometer is controlled by the controller / processor 490, and the measurement data from the detectors may be stored in the memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is connected to the detectors to receive signals outputted therefrom, and the controller / processor 490 may be connected to the electrical and electromechanical components of the flow cytometer 410 to control lasers, fluid flow parameters, etc. Input / output (I / O) capabilities 497 may also be provided in the system. The memory 495, controller / processor 490, and I / O 497 may be provided integrally as an integral part of the flow cytometer 410. In such an embodiment, a display for displaying experimental data to a user of the cytometer 410 may also constitute a part of the I / O capabilities 497. Alternatively, part or all of the memory 495 and the controller / processor 490 and the I / O capabilities may be part of one or more external devices, such as a general purpose computer. In some embodiments, some or all of the memory 495 and controller / processor 490 may be in wireless or wired communication with the cytometer 410. The controller / processor 490, in conjunction with the memory 495 and I / O 497, may be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0080] Figure 4 The system shown includes six different detectors that detect fluorescence in six different bands (which may be referred to herein as "filter windows" for a given detector), which are defined by the configuration of filters and / or beam splitters in the beam path from the flow chamber 425 to each detector. Different fluorescent molecules in the fluorescein combination scheme used in the flow cytometer experiment emit light in their own characteristic bands. The specific fluorescent markers used in the experiment and their associated fluorescence emission bands can be selected to roughly match the filter windows of the detectors. I / O 497 can be configured to receive data about a flow cytometer experiment, which has a set of fluorescent markers and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O 497 can also be configured to receive biological data that assigns one or more markers to one or more cell populations, marker density data, emission spectrum data, data that assigns markers to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as marker spectral characteristics and flow cytometer configuration data can also be stored in memory 495. The controller / processor 490 may be configured to evaluate one or more assignments of a label to a marker.

[0081] In some embodiments, the system is a particle sorting system configured to sort particles with a closed particle sorting module, such as the particle sorting system described in U.S. Patent Publication No. 2017 / 0299493 filed on 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 decision module having multiple sorting decision units, such as the sorting decision module described in U.S. Patent Publication No. 2020 / 0256781 filed on December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting sample components includes a particle sorting module with a deflection plate, such as described in U.S. Patent Publication No. 2017 / 0299493 filed on March 28, 2017, the disclosure of which is incorporated herein by reference.

[0082] Figure 5 A functional block diagram is shown for one example of a control system for analyzing and displaying biological events, such as a processor 500. The processor 500 may be configured to perform various processes to control the graphical display of biological events.

[0083] The flow cytometer or sorting system 502 can be configured to acquire biological event data. For example, the flow cytometer can generate flow cytometric event data (e.g., data of light modulated by particles). The flow cytometer 502 can be configured to provide the biological event data to the processor 500. A data communication channel can be included between the flow cytometer 502 and the processor 500. The biological event data can be provided to the processor 500 via the data communication channel.

[0084] Processor 500 may be configured to receive biological event data from flow cytometer 502. The biological event data received from flow cytometer 502 may include flow cytometric event data. Processor 500 may be configured to provide a graphical display including a first graph of biological event data to display device 506. For example, processor 500 may also be configured to provide a region of interest in the form of a gate around a population of biological event data displayed by display device 506 and superimposed on the first graph. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest plotted on a single parameter histogram or a bivariate graph. In some embodiments, the display may be used to display particle parameters or saturated detector data.

[0085] Processor 500 may also be configured to display the bio-event data within the gate in a manner different from other events in the bio-event data outside the gate on display device 506. For example, processor 500 may be configured to distinguish the color of the bio-event data within the gate from the color of the bio-event data outside the gate. Display device 506 may be implemented in the form of a monitor, tablet computer, smart phone, or other electronic device configured to display a graphical interface.

[0086] The processor 500 may be configured to receive a gating selection signal identifying a gating from a first input device. For example, the first input device may be implemented in the form of a mouse 510. The mouse 510 may generate a gating selection signal to the processor 500 identifying a gating to be displayed on or operated by the display device 506 (e.g., by clicking when a cursor is positioned on or in a desired gating). In some embodiments, the first device may be implemented in the form of a keyboard 508 or other means for providing input signals to the processor 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, each of which may generate a trigger event.

[0087] A triggering event may cause processor 500 to change the manner in which data is displayed, change the portion of data actually displayed on display device 506, and / or provide input for further processing, such as selecting a population of interest for particle sorting.

[0088] In some embodiments, the processor 500 can be configured to detect when the mouse 510 initiates a gating selection. The processor 500 can also be configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on the specific distribution of the bio-event data received by the processor 500. In some embodiments, the processor 500 expands the first gate so that a second gate is generated (e.g., as described above).

[0089] Processor 500 may be connected to storage device 504. Storage device 504 may be configured to receive and store bio-event data from processor 500. Storage device 504 may also be configured to receive and store flow cytometric event data from processor 500. Storage device 504 may also be configured to allow processor 500 to retrieve bio-event data, such as flow cytometric event data.

[0090] The display device 506 can be configured to receive display data from the processor 500. The display data can include a graph of the biological event data and a gating that circles an outline of a portion of the graph. The display device 506 can also be configured to change the displayed information based on input received from the processor 500 and input from the flow cytometer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0091] In some embodiments, the processor 500 can generate a user interface to receive sample events for sorting. For example, the user interface can include a machine for receiving sample events or sample images. Sample events, images, or sample gating can be provided before collecting event data for the sample, or can be provided based on an initial set of events for a portion of the sample.

[0092] Fig. 6A is a schematic diagram of a particle sorting system 600 (e.g., flow cytometer 502) according to one embodiment described herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Fig. 6A As shown, a droplet formation transducer 602 (e.g., a piezoelectric oscillator) is connected to a fluid conduit 601, which can be connected 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 arranged in a single file to pass through a monitoring area 611 (e.g., a laser-stream intersection) and are illuminated by an illumination source 612 (e.g., a laser). The vibration of the droplet formation transducer 602 causes the moving fluid column 608 to break into a plurality of droplets 610, some of which contain particles 609.

[0093] In operation, a detection station 614 (e.g., an event detector) identifies a particle of interest (or a cell of interest) as it passes through the monitoring region 611. The detection station 614 inputs to a timing circuit 628, which in turn inputs to a flash charging circuit 630. At the droplet breakoff point, which is signaled by a timed droplet delay (Δt), the moving fluid column 608 can be flash charged to charge the droplet of interest. The droplet of interest can 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 container, such as a collection tube or a multi-well sample plate or a micro-well sample plate, in which a well or micro-well can be associated with a droplet of particular interest. As Fig. 6A As shown, the droplets can be collected in a drain container 638.

[0094] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal when a particle of interest passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 allows the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 can input an amplitude signal 620 and / or a phase 618 signal, which in turn is input (through an amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 in turn controls the droplet formation transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 can be included in the control system.

[0095] In some embodiments, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be connected to a memory configured to store detected events and sorting decisions based thereon. The event data for the particles can include sorting decisions. In some embodiments, the detection system 616 and the detection station 614 can be implemented as a single detection unit, or in a communication connection so that event measurements are collected by one of the detection system 616 or the detection station 614 and provided to a non-collecting element.

[0096] Figure 6B is a schematic diagram of a particle sorter system according to one embodiment presented herein. Figure 6B The particle sorter system 600 shown in FIG. 6 includes deflection plates 652 and 654. A charge can be applied via a stream charge wire in the barb. This forms a droplet stream 610 containing particles 609 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering information and fluorescence information. The particle information is analyzed by sorting electronics or other detection systems ( Figure 6B Deflector plates 652 and 654 may be independently controlled to attract or repel charged droplets, thereby directing the droplets toward a destination collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflector plates 652 and 654 can be controlled to direct particles along first path 662 toward container 674 or along second path 668 toward container 678. If the particle is not of interest (e.g., does not show scattering or illumination information within a specified sorting range), the deflector plates can allow the particle to continue along flow path 664. Such uncharged droplets can enter a waste container, such as through aspirator 670.

[0097] Sorting electronics may be included to initiate the collection of measurements, receive the fluorescent signals of the particles, and determine how to adjust the deflection plates to cause sorting of the particles. Figure 6B An exemplary implementation of the illustrated embodiment includes a BD FACSAria TM Series of flow cytometers.

[0098] Methods for analyzing samples

[0099] As described above, aspects of the present invention also include methods for analyzing samples. The method of interest includes introducing a particulate sample into the above-mentioned flow cytometer and performing flow cytometry sorting on the particulate sample. As described, the flow cytometer includes a flow chamber for transporting particles in the central stream of the flow stream from the proximal end to the distal end, and a sample injection needle for transporting the sample fluid from the sample injection line at the proximal end to the flow chamber at the distal end to generate the central stream, the sample injection needle having a channel running through it. The flow chamber of interest includes a flow chamber tapered structure at the proximal end, and the sample injection needle of interest is configured and placed to maintain a complete central stream under flow conditions that vary by one order of magnitude or more.

[0100] 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, plant, fungus, or a subset of animal tissue, animal cell or animal component, which can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood (amniotic cord blood), urine, vaginal fluid and semen in some cases. Therefore, "biological sample" refers to a subset of a natural organism or its tissue and refers to a homogenate, lysate or extract prepared from a subset of an organism or its tissue, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin slices, respiratory slices, gastrointestinal slices, cardiovascular slices and urogenital tract slices, tears, saliva, milk, blood cells, tumors, organs. A biological sample can be any type of organism's tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, such as plasma, tears, urine, semen, etc., wherein in some cases, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger stick (wherein the blood may or may not be combined with any reagents prior to assay, such as preservatives, anticoagulants, etc.).

[0101] In certain embodiments, the source of the sample is "mammal" or "mammals", wherein these terms are widely used to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method can be applied to samples obtained from human subjects of both sexes and at any stage of development (i.e., newborns, infants, teenagers, adolescents, adults), wherein in certain embodiments, the human subject is a teenager, adolescent, or adult. Although the present invention can be applied to samples from human subjects, it is understood that these methods can also be applied to samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0102] The cell of interest can be targeted for characterization according to a variety of parameters, such as phenotypic features identified by attaching a specific fluorescent marker to the cell of interest. In some embodiments, the system is configured to deflect the analyzed droplets determined to contain target cells. The method can be used to characterize a variety of cells. The 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 (such as HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells and erythroid cells. The target cells of interest include cells with convenient cell surface markers or antigens, which can be captured or labeled by their convenient affinity agents or conjugates. For example, the target cell may contain 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 α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cell is selected from HIV-containing cells from whole blood, bone marrow or umbilical cord blood, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+).

[0103] When practicing the method, a certain amount of initial fluid sample is injected into the flow cytometer. The amount of sample injected into the particle sorting module can vary, for example, 0.001 mL to 1000 mL, such as 0.005 mL to 900 mL, such as 0.01 mL to 800 mL, such as 0.05 mL to 700 mL, such as 0.1 mL to 600 mL, such as 0.5 mL to 500 mL, such as 1 mL to 400 mL, such as 2 mL to 300 mL and including 5 mL to 100 mL of sample.

[0104] The method according to an embodiment of the present disclosure includes counting and optionally sorting the labeled particles (e.g., target cells) in the sample. When practicing the method, a fluid sample containing the particles is first introduced into the flow nozzle of the system. When leaving from the flow nozzle, the particles pass through the sample detection zone substantially one by one, each particle in the sample detection zone is illuminated by a light source, and the measurement of light scattering parameters and the measurement of fluorescence emission desired in some cases (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded separately for each particle. According to the properties of the flow stream being detected, a flow stream of 0.001 mm or more than 0.001 mm can be illuminated with light, such as 0.005 mm or more than 0.005 mm, such as 0.01 mm or more than 0.01 mm, such as 0.05 mm or more than 0.05 mm, such as 0.1 mm or more than 0.1 mm, such as 0.5 mm or more than 0.5 mm and including a flow stream of 1 mm or more than 1 mm. In certain embodiments, the method includes irradiating a planar cross-section of the flow stream in the sample detection zone, such as with a laser (as described above). In other embodiments, the method includes irradiating a predetermined length of the flow stream in the sample detection zone, which corresponds, for example, to the irradiation profile of a diffuse laser beam or lamp.

[0105] In certain embodiments, the method includes irradiating the flow stream at or near the flow chamber nozzle orifice. For example, the method can include irradiating the flow stream at a position about 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, and including 1 mm or more, from the nozzle orifice. In certain embodiments, the method includes irradiating the flow stream in close proximity to the flow chamber nozzle orifice.

[0106] In an embodiment of the method, as particles pass through a sensing region and are illuminated by an energy source, a detector, such as a photomultiplier tube (PMT), is used to record light passing through each particle (in some cases referred to as forward light scatter), light reflected in a direction orthogonal to the flow of particles through the sensing region (in some cases referred to as orthogonal light scatter or side light scatter), and if the particles are labeled with one or more than one fluorescent marker, the fluorescence emitted by the particles is also recorded. Each forward light scatter (FSC), side scatter (SSC), and fluorescence emission comprises an independent parameter for each particle (or each "event"). Thus, for example, two parameters, three parameters, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. As desired, the data recorded for each particle is analyzed in real time or stored in a data storage and analysis tool, such as a computer.

[0107] In certain embodiments, the detection and unique identification of particles is achieved by exposing the particles to excitation light and measuring the fluorescence of each particle in one or more detection channels, as desired. The fluorescence emitted in the detection channel used to identify the particle and its associated binding complex can be measured after excitation with a single light source, or can be measured after separate excitation with different light sources. If a separate excitation light source is used to excite the particle labels, the labels can be selected so that each excitation light source used can excite all labels.

[0108] The method in certain embodiments also includes data acquisition, analysis and recording, for example, using a computer to perform, wherein multiple data channels record the data of light scattering and fluorescence emitted by each particle from each detector when passing through the sample detection area of ​​the particle sorting module. In these embodiments, the analysis includes classifying and counting the particles so that each particle is displayed in the form of a set of digital parameter values. The system can be set to be triggered by a selected parameter to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for detecting a parameter, and can be used as a way to detect particles passing through a light source. When an event exceeding the selected parameter threshold is detected, the acquisition of light scattering data and fluorescence data of the particles is triggered. For particles or other components in the measured medium that cause a response below the threshold, data acquisition is not performed. The trigger parameter can be the detection value of the forward scattered light caused by the particle passing through the light beam. The flow cytometer then detects and collects the light scattering data and fluorescence data of the particles.

[0109] Subsequently, based on the data collected for the entire population, a specific subpopulation of interest is further analyzed by "gating". In order to select the appropriate gating, the data is plotted to obtain the best possible subpopulation separation. This process can be performed by plotting the side (i.e., orthogonal) light scatter (SSC) versus the forward light scatter (FSC) on a two-dimensional scatter plot. Subpopulations of particles (i.e., those cells within the gate) are then selected and particles that are not within the gate are excluded. If desired, a gating can be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Those particles within the gate are then further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis can be configured to obtain a count of particles of interest in the sample.

[0110] The method of interest may also include the use of particles in research, laboratory testing or treatment. In some embodiments, the method includes obtaining a single cell prepared from a target fluid biological sample or a tissue biological sample. For example, the method includes obtaining cells from a fluid sample or a tissue sample to be used as a research or diagnostic sample for a disease such as cancer. Similarly, the method includes obtaining cells from a fluid sample or a tissue sample for treatment. Cell therapy protocols are protocols that can prepare living cell materials including, for example, cells and tissues and introduce them into a subject as a therapeutic treatment. Conditions that can be treated by administering samples sorted by flow cytometry include, but are not limited to, blood diseases, immune system diseases, organ damage, etc.

[0111] A typical cell therapy protocol may include the following steps: sample collection, cell separation, genetic modification, culture, and in vitro amplification, cell collection, sample reduction and washing, biological preservation, storage, and introduction of cells into the subject. The protocol may begin with the collection of living cells and living tissues from the source tissue of the subject to produce cell samples and / or tissue samples. The sample may be collected by any suitable procedure, including, for example, administering a cell mobilization agent to the subject, drawing blood from the subject, removing bone marrow from the subject, etc. After collecting the sample, cell enrichment may be performed by several methods, including, for example, centrifugation-based methods, filtration-based methods, elutriation, magnetic separation methods, fluorescence-activated cell sorting (FACS), etc. 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 amplified in vitro. In some cases, the cells are preserved, such as cryopreserved, and stored for future use, and the cells are thawed during use and subsequently applied to the patient, such as cells may be infused into the patient.

[0112] Methods for assembling a flow cytometer

[0113] As described above, aspects of the present invention also include methods for assembling a flow cytometer. Methods of interest include placing a sample injection needle in a flow cytometer. A suitable flow cytometer includes a flow chamber for transporting particles in a central stream of a flow stream from a proximal end to a distal end. The flow chamber includes a flow chamber tapered structure at a proximal end. The method includes placing the sample injection needle in the flow chamber tapered structure so that a complete central stream is maintained under flow conditions that vary by one order of magnitude or more. As described, the sample injection needle has a channel running through it, which is used to transport a sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a central stream.

[0114] In an embodiment, the method includes placing the sample injection needle in the flow chamber tapered structure so that the distal end of the sample injection needle is separated from the sheath fluid introduction point by a longitudinal distance of 17 mm to 26 mm. For example, in some cases, the method includes placing the sample injection needle in the flow chamber tapered structure so that the distal end of the sample injection needle is separated from the sheath fluid introduction point by a longitudinal distance of 20 mm to 22 mm. In selected cases, the method includes placing the sample injection needle in the flow chamber tapered structure so that the distal end of the sample injection needle is separated from the detection point by a distance of 13 mm to 17 mm (e.g., 14 mm to 16 mm).

[0115] Computer control system

[0116] Aspects of the invention also include computer controlled systems, wherein the system includes one or more fully automated or partially automated computers. In some embodiments, the system includes a computer having a non-transitory computer-readable storage medium having a computer program stored thereon, wherein the computer program, when loaded onto the computer, includes instructions for receiving target or desired flow conditions (e.g., sheath fluid flow rate, sample fluid flow rate) and instructions for initiating a change in resistance state to achieve the target or desired flow conditions. Since the system described herein includes the flow chamber and sample injection needle of the present invention, the resulting core flow can remain intact.

[0117] 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 method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input-output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or it may be one of other available or soon-to-be-available processors. The processor executes an operating system, which interacts with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs, which can be written in various programming languages ​​known in the art, such as Java, Perl, C++, Python, other high-level languages ​​or low-level languages, and combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input-output control, file management and data management, memory management, and communication control and related services according to known techniques. In some embodiments, the processor includes an analog electronic device that provides feedback control, such as negative feedback control.

[0118] The system memory can 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 built-in hard disk or tape, optical media such as a compact optical disk, flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or unknown devices, including a compact optical disk drive, a tape drive, or a floppy disk drive. These types of memory storage devices typically read and / or write program storage media (not shown) such as compact optical disks. Any of these program storage media or others currently in use or that may be developed in the future can be considered a computer program product. It will be understood that 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 system memory and / or program storage devices used in conjunction with memory storage devices.

[0119] In some embodiments, a computer program product is described, which includes a computer usable medium having control logic (computer software program, including program code) stored therein. When the control logic is executed by a computer processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are mainly implemented in hardware using a hardware state machine. The implementation of a hardware state machine for performing the functions described herein will be apparent to those skilled in the relevant art.

[0120] The memory may be any suitable device in which the processor can store data and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including a disk or optical disk or tape or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor that is appropriately programmed from a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely through a communication channel, or pre-stored in a computer program product, such as a memory or some other portable or fixed computer-readable storage medium, any of which is connected to the memory for use. For example, a disk or optical disk may carry the programming and may be read by a disk writer / reader. The system of the present invention also includes programming, such as in the form of a computer program product, i.e., an algorithm for practicing the above-mentioned method. The programming according to the present invention may be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROMs; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0121] The processor may also access a communication channel to communicate with a user at a remote location. A remote location is one where the user is not directly interacting with the system and forwards 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 smart phone).

[0122] In some embodiments, the system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface may be configured for wired communication or wireless communication, including but not limited to radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), communication protocols, and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0123] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or physical interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port, to allow data communications between the system and other external devices, such as computer terminals (e.g., computer terminals in a physician's office or in a hospital environment), that are configured for similar complementary data communications.

[0124] In one embodiment, the communication interface is configured for infrared communication, communications or any other suitable wireless communication protocol to enable the system to communicate with other devices, such as computer terminals and / or networks, mobile phones with communication capabilities, personal digital assistants, or any other communication devices that a user may use in conjunction with.

[0125] In one embodiment, the communication interface is configured to provide a connection for data transmission using Internet Protocol (IP) through a cellular network, a short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0126] In one embodiment, the system is configured to communicate wirelessly with the server device via a communication interface, such as using 802.11 or The server device may be another portable device, such as a smartphone, a personal digital assistant (PDA), or a laptop computer; or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as a button, a keyboard, a mouse, or a touch screen.

[0127] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate with a network or server device using one or more of the above-mentioned communication protocols and / or communication mechanisms to communicate with data stored in the system, such as stored in an optional data storage unit.

[0128] The output controller may include a controller for any of various known display devices for presenting information to a user, whether a person or a machine, whether local or remote. If one of the display devices provides visual information, the information may generally be logically and / or physically organized as an array of pixels. A graphical user interface (GUI) controller may include any of various known or future software programs for providing a graphical input and output interface between the system and the user, and 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 accomplished in alternative embodiments using a network or other types of remote communications. The output manager may also provide information generated by the processing module to a user at a remote location, such as via the Internet, a telephone, or a satellite network, according to known techniques. The presentation of data by the output manager may be implemented in accordance with various known techniques. For example, the data may include an SQL document, an HTML document, or an XML document, an email or other file, or other forms of data. The data may include an Internet URL address so that a user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more than one platform present in the system may be any type of known computer platform or a type developed in the future, although they generally belong to a class of computers commonly referred to as servers. However, they may also be mainframe computers, workstations, or other types of computers. They may be connected by any known or future type of cable or other communication system, including wireless systems, whether networked or otherwise. They may be co-located or physically separated. Different operating systems may be used on any computer platform, which may depend on the type and / or model of computer platform chosen. Suitable operating systems include Android TM 、SGI Oracle and other operating systems.

[0129] Figure 7 Depicted is a general architecture of an example computing device 600 in accordance with certain embodiments. Figure 7The general architecture of the computing device 700 described in the figure includes a layout of computer hardware components and computer software components. However, it is not necessary to display all of these generally conventional components in order to provide an achievable disclosure. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. The network interface 720 can provide a connection to one or more networks or computing systems. Therefore, the processing unit 710 can receive information and instructions from other computing systems or computing services via the network. The processing unit 710 can also communicate with and from the memory 770, and can also provide output information to the optional display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or program, such as data analysis software) stored as executable instructions in the non-temporary memory of the analysis system ) can display flow cytometry event data to a user. The input / output device interface 740 can also accept input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope or other input device.

[0130] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) that are executed by processing unit 710 to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in general management and general operations of computing device 700. Data may be stored in data storage device 790. Memory 770 may also contain computer program instructions and other information for implementing aspects of the present disclosure.

[0131] use

[0132] The sample injection needle, the flow cytometer and the method are suitable for various applications in which it is desired to analyze components in a sample in a fluid medium. The present invention is particularly suitable for improving the quality of the central flow in a fluid control system. For example, the sample injection needle, the flow cytometer and the method can be used to increase the degree to which the central flow remains intact. In some cases, the present invention can be used to reduce the formation of vortices in the tapered structure of the flow chamber.

[0133] Embodiments of the present invention are applicable to the application that may expect to use the cell prepared from biological sample for research, laboratory test or for treatment.In some embodiments, described method and described equipment can contribute to obtain and / or analyze the single cell prepared from target fluid biological sample or target tissue biological sample.For example, described method and described system contribute to obtain cell from fluid sample or tissue sample, for example, to be used as the research or diagnosis sample of the disease such as cancer.Equally, described method and described system can contribute to obtain cell from fluid sample or tissue sample for treatment.

[0134] Reagent test kit

[0135] Aspects of the present disclosure also include test kits. Test kits of interest include one or more than one sample injection needle. In one embodiment, the test kit includes a single sample injection needle. In other embodiments, the test kit includes multiple sample injection needles. If the test kit includes multiple sample injection needles, the sample injection needles can be the same or different. For example, in one case, the test kit includes a sample injection needle with a super bullet structure as described above, a sample injection needle with a bullet structure as described above, and a sample injection needle with a circular structure, or a combination of sample injection needles with these structures.

[0136] In addition to the above components, the kit (in some embodiments) may also include instructions, such as instructions for installing the sample injection needle of the present invention into a flow cytometer. These instructions may be present in the kit in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may exist is as printed information appearing on a suitable medium or substrate, such as one or more sheets of paper printed with information, in the packaging of the kit, in a package insert, etc. Another form of these instructions is a computer-readable medium, such as a floppy disk, a compact disc (CD), a portable flash drive, etc., on which information has been recorded. Another form in which these instructions may exist is a website, which can be used to access information on a remote website via the Internet.

[0137] The following examples are offered by way of illustration and not limitation.

[0138] experiment

[0139] introduction

[0140] From previous observations, it was found that the default injection needle (round) did not extend into the flow chamber and could not establish a stable sample center at the desired sample flow rate (1m / s sheath flow). The effect became more obvious as the sample flow rate increased. This was first discovered by running diluted food dye in the flow stream. This was later confirmed by observing the sample at the detection point with a microscope. There were 3 options for sample flow rates for evaluation: 8μL / min, 32μL / min, and 85μL / min. At 8μL / min, the sample center showed slight instability. At 32μL / min, the sample center moved all over the place. At 85μL / min, the sample center could not be established. Therefore, an experiment was wanted to study the effect of needle shape and length on sample center establishment.

[0141] method

[0142] Central flow analysis was performed on a Becton Dickinson flow cytometer using several sample injection needles at different extension positions within the flow chamber. Eight needles were compared in this study. Needles were grouped by tip shape and extension length and compared to the default needle. In particular, needles with rounded distal ends ( Fig. 9A ), having a bullet structure as described above ( Fig. 9B ) and having a super bullet structure as described above ( Fig. 9C ) sample injection needles. There are two round tip needles (0" extension needle and 1 / 8" extension needle), three bullet shaped needles (0" extension needle, 1 / 16" extension needle and 1 / 8" extension needle) and three super bullet shaped needles (0" extension needle, 1 / 16" extension needle and 1 / 8" extension needle).

[0143] For each needle, three sample flow rates (8 μL / min, 32 μL / min, and 85 μL / min) were applied. During each run, a camera was pointed at the colorimetric chamber to capture the sample center reflection. A flashlight was shone onto one of the fiber optic cables. This created a ring visible to the camera at the detection point. The physical center around the detection point was identified. The quality of the sample center was visually assessed from the video by determining whether the sample center was centered and whether the sample center was stable (minimal displacement).

[0144] result

[0145] Figure 8 An image of the injection needle for each sample taken from the camera is shown. Figure 8 The center portion of each image depicts the geometry of the central flow. The qualitative results of each sample injection needle at different extensions are tabulated as shown in Table 1 below.

[0146] Table 1: Comparison of needles at different sample flow rates (all in sheath flow of 1 m / s)

[0147]

[0148] discuss

[0149] The results show that the 1 / 8” extension had the best results for all tip shapes. The sample center could be established and centered. However, for shorter extensions (e.g., 1 / 16” extension), the more tapered the tip, the better the performance. The Bullet 1 / 16” sample center could not be centered at both 32 μL / min and 64 μL / min, while the Super Bullet passed both criteria at both sample flow rates.

[0150] in conclusion

[0151] This comparative study shows that longer needles and more tapered tips can produce centered and stable sample centers. From this comparative study, the 1 / 8" extended super bullet shape (with the highest tapering) is the best candidate for future injection needle development. This shows that sample injection needles can be configured and placed to maintain intact center flow under flow conditions that vary by an order of magnitude or more.

[0152] Notwithstanding the appended claims, the present invention may also be described by the following clauses:

[0153] 1. A flow cytometer, comprising:

[0154] a flow chamber for transporting particles in a center stream of the flow stream from a proximal end to a distal end, wherein the flow chamber includes a flow chamber taper at the proximal end; and

[0155] A sample injection needle for delivering sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a core flow, the sample injection needle having a channel running through it, wherein the sample injection needle is configured and positioned to maintain intact core flow under flow conditions that vary by one order of magnitude or more.

[0156] 2. The flow cytometer according to clause 1, further comprising a sheath fluid introduction port for delivering sheath fluid to the flow chamber.

[0157] 3. A flow cytometer according to clause 2, wherein the distal end of the sample injection needle is placed within the conical structure of the flow chamber and is separated from the sheath fluid introduction point by a longitudinal distance of 17 mm to 26 mm.

[0158] 4. A flow cytometer according to clause 3, wherein the longitudinal distance separating the distal end of the sample injection needle from the sheath fluid introduction point is 20 mm to 22 mm.

[0159] 5. A flow cytometer according to any of the preceding clauses, wherein the sample injection needle is configured and positioned to maintain an intact core flow under conditions where the flow velocity of the flow stream is between 0.5 m / s and 10 m / s.

[0160] 6. A flow cytometer according to any of the preceding clauses, wherein the sample injection needle is configured and positioned to maintain a complete core flow at a sample fluid flow rate of 1 μL / min to 150 μL / min.

[0161] 7. A flow cytometer according to any of the preceding clauses, wherein the sample injection needle comprises an inner diameter of 0.2 mm to 0.4 mm.

[0162] 8. A flow cytometer according to clause 7, wherein the sample injection needle comprises an inner diameter of 0.25 mm to 0.30 mm.

[0163] 9. A flow cytometer according to any of the preceding clauses, wherein the sample injection needle comprises a tapered structure at the distal end.

[0164] 10. The flow cytometer according to clause 9, wherein the tapered structure starts at a position separated from the distal end by a distance of 3.5 mm to 4.0 mm along the length direction of the sample injection needle.

[0165] 11. The flow cytometer according to clause 10, wherein the tapered structure starts at a position separated from the distal end by a distance of 3.8 mm to 3.9 mm along the length direction of the sample injection needle.

[0166] 12. The flow cytometer according to clause 9, wherein the tapered structure starts at a position separated from the distal end by a distance of 1.5 mm to 2.0 mm along the length direction of the sample injection needle.

[0167] 13. The flow cytometer according to clause 12, wherein the tapered structure starts at a position separated from the distal end by a distance of 1.7 mm to 1.8 mm along the length direction of the sample injection needle.

[0168] 14. A flow cytometer according to any one of clauses 9 to 13, wherein the tapered structure has a radius of 2.5 mm to 12 mm over a length span of 1.75 mm to 4 mm.

[0169] 15. A flow cytometer according to any one of clauses 1 to 8, wherein the sample injection needle comprises a rounded distal end.

[0170] 16. A flow cytometer according to clause 2, wherein the flow cytometer comprises a plurality of sheath fluid introduction ports.

[0171] 17. A flow cytometer according to clause 16, wherein the flow cytometer comprises two sheath fluid introduction ports.

[0172] 18. A flow cytometer according to clause 16 or 17, wherein the plurality of sheath fluid introduction ports are offset from one another so that the sheath fluid rotates through the tapered structure of the flow chamber.

[0173] 19. A flow cytometer according to any of the preceding clauses, further comprising a light source configured to illuminate the flow chamber at the detection point.

[0174] 20. The flow cytometer according to clause 19, wherein the distance separating the distal end of the sample injection needle from the detection point is 13 mm to 17 mm.

[0175] 21. The flow cytometer according to clause 20, wherein the distance separating the distal end of the sample injection needle from the detection point is 14 mm to 16 mm.

[0176] 22. A flow cytometer according to any of the preceding clauses, further comprising a detector configured to collect light from the flow chamber.

[0177] 23. A flow cytometer according to any of the preceding clauses, further comprising a vacuum configured to draw fluid through the flow chamber.

[0178] 24. A method for analyzing a sample, the method comprising:

[0179] (a) introducing a particle sample into a flow cytometer, the flow cytometer comprising:

[0180] a flow chamber for transporting particles in a center stream of the flow stream from a proximal end to a distal end, wherein the flow chamber includes a flow chamber taper at the proximal end; and

[0181] a sample injection needle for delivering sample fluid from a sample injection line at the proximal end to a flow chamber at the distal end to generate a core flow, the sample injection needle having a channel therethrough, wherein the sample injection needle is configured and positioned to maintain intact core flow under flow conditions that vary by an order of magnitude or more; and

[0182] (b) Flow cytometric analysis of particle samples.

[0183] 25. The method according to clause 24, wherein the flow cytometer further comprises a sheath fluid introduction port for delivering a sheath fluid to the flow chamber.

[0184] 26. The method according to clause 25, wherein the distal end of the sample injection needle is positioned within the flow chamber tapered structure and is separated from the sheath fluid introduction point by a longitudinal distance of 17 mm to 26 mm.

[0185] 27. The method according to clause 26, wherein the distal end of the sample injection needle is separated from the sheath fluid introduction point by a longitudinal distance of 20 mm to 22 mm.

[0186] 28. The method according to any one of clauses 24 to 27, wherein the sample injection needle is configured and positioned to maintain an intact core flow at a flow rate of the flowing stream of 0.5 m / s to 10 m / s.

[0187] 29. The method according to any one of clauses 24 to 28, wherein the sample injection needle is configured and positioned to maintain full core flow at a sample fluid flow rate of 1 μL / min to 150 μL / min.

[0188] 30. The method according to clause 24 or 29, wherein the sample injection needle comprises an inner diameter of 0.2 mm to 0.4 mm.

[0189] 31. The method according to clause 30, wherein the sample injection needle comprises an inner diameter of 0.25 mm to 0.30 mm.

[0190] 32. The method according to any one of clauses 24 to 31, wherein the sample injection needle comprises a tapered structure at the distal end.

[0191] 33. The method according to clause 32, wherein the tapered structure starts at a position separated from the distal end by a distance of 3.5 mm to 4.0 mm along the length of the sample injection needle.

[0192] 34. The method according to clause 33, wherein the tapered structure starts at a position separated from the distal end by a distance of 3.8 mm to 3.9 mm along the length of the sample injection needle.

[0193] 35. The method according to clause 32, wherein the tapered structure starts at a position separated from the distal end by a distance of 1.5 mm to 2.0 mm along the length of the sample injection needle.

[0194] 36. A flow cytometer according to clause 35, wherein the tapered structure starts at a position separated from the distal end by a distance of 1.7 mm to 1.8 mm along the length of the sample injection needle.

[0195] 37. A method according to any of clauses 32 to 36, wherein the tapered structure has a radius of 2.5 mm to 12 mm over a length span of 1.75 mm to 4 mm.

[0196] 38. The method according to any one of clauses 24 to 31, wherein the sample injection needle comprises a rounded distal end.

[0197] 39. The method of clause 25, wherein the flow cytometer comprises a plurality of sheath fluid introduction ports.

[0198] 40. The method according to clause 39, wherein the flow cytometer comprises two sheath fluid introduction ports.

[0199] 41. The method according to clause 39 or 40, wherein the plurality of sheath fluid introduction ports are offset from one another so that the sheath fluid rotates through the tapered structure of the flow chamber.

[0200] 42. A method according to any one of clauses 24 to 41, wherein the flow cytometer further comprises a light source configured to illuminate the flow chamber at the detection point.

[0201] 43. The method according to clause 42, wherein the distal end of the sample injection needle is separated from the detection point by a distance of 13 mm to 17 mm.

[0202] 44. The method according to clause 43, wherein the distal end of the sample injection needle is separated from the detection point by a distance of 14 mm to 16 mm.

[0203] 45. The method according to any one of clauses 24 to 44, wherein the sample is a biological sample.

[0204] 46. ​​A method according to clause 45, wherein the sample comprises cells.

[0205] 47. A method according to any one of clauses 24 to 46, wherein the method further comprises flow cytometry sorting of the sample.

[0206] 48. A method of assembling a flow cytometer, the method comprising placing a sample injection needle in the flow cytometer, the flow cytometer comprising a flow chamber for transporting particles in a central stream of a flow stream from a proximal end to a distal end, wherein the flow chamber comprises a flow chamber taper at the proximal end, and wherein:

[0207] The sample injection needle has a channel therethrough for conveying sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a core flow, and

[0208] The method includes placing the sample injection needle within a flow chamber tapered structure such that intact core flow is maintained under flow conditions that vary by an order of magnitude or more.

[0209] 49. The method according to clause 48, wherein the flow cytometer further comprises a sheath fluid introduction port for delivering a sheath fluid to the flow chamber.

[0210] 50. The method of clause 49, further comprising placing the distal end of the sample injection needle within the flow chamber tapered structure such that the distal end of the sample injection needle is separated from a sheath fluid introduction point by a longitudinal distance of 17 mm to 26 mm.

[0211] 51. The method of clause 50, further comprising placing the distal end of the sample injection needle within the flow chamber tapered structure such that the distal end of the sample injection needle is separated from a sheath fluid introduction point by a longitudinal distance of 20 mm to 22 mm.

[0212] 52. A method according to any one of clauses 48 to 51, wherein the sample injection needle is configured and positioned to maintain an intact core flow at a flow rate of the flowing stream of 0.5 m / s to 10 m / s.

[0213] 53. A method according to any one of clauses 48 to 52, wherein the sample injection needle is configured and positioned to maintain full core flow at a sample fluid flow rate of 1 μL / min to 150 μL / min.

[0214] 54. The method according to any one of clauses 48 to 53, wherein the sample injection needle comprises an inner diameter of 0.2 mm to 0.4 mm.

[0215] 55. The method according to clause 54, wherein the sample injection needle comprises an inner diameter of 0.25 mm to 0.30 mm.

[0216] 56. The method according to any one of clauses 48 to 55, wherein the sample injection needle comprises a tapered structure at the distal end.

[0217] 57. The method according to clause 56, wherein the tapered structure begins at a position separated from the distal end by a distance of 3.5 mm to 4.0 mm along the length of the sample injection needle.

[0218] 58. The method according to clause 57, wherein the tapered structure begins at a position separated from the distal end by a distance of 3.8 mm to 3.9 mm along the length of the sample injection needle.

[0219] 59. The method according to clause 56, wherein the tapered structure starts at a position separated from the distal end by a distance of 1.5 mm to 2.0 mm along the length of the sample injection needle.

[0220] 60. The method according to clause 59, wherein the tapered structure begins at a position separated from the distal end by a distance of 1.7 mm to 1.8 mm along the length of the sample injection needle.

[0221] 61. A method according to any of clauses 56 to 60, wherein the tapered structure has a radius of 2.5 mm to 12 mm over a length span of 1.75 mm to 4 mm.

[0222] 62. The method of any one of clauses 48 to 55, wherein the sample injection needle comprises a rounded distal end.

[0223] 63. The method of clause 49, wherein the flow cytometer comprises a plurality of sheath fluid introduction ports.

[0224] 64. A method according to clause 63, wherein the flow cytometer comprises two sheath fluid introduction ports.

[0225] 65. The method of clause 63 or 64, wherein the plurality of sheath fluid introduction ports are offset from one another such that the sheath fluid rotates through the tapered structure of the flow chamber.

[0226] 66. A method according to any one of clauses 48 to 65, wherein the flow cytometer comprises a light source configured to illuminate the flow chamber at the detection point.

[0227] 67. The method according to clause 66, wherein the method comprises placing the sample injection needle in the tapered structure of the flow chamber so that the distal end of the sample injection needle is separated from the detection point by a distance of 13 mm to 17 mm.

[0228] 68. The method according to clause 67, wherein the distal end of the sample injection needle is separated from the detection point by a distance of 14 mm to 16 mm.

[0229] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that, in light of the teachings of the invention, certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0230] Therefore, the above only illustrates the principle of the present invention.It will be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language recorded herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to advance this area, and should be interpreted as not being limited to these specifically recorded examples and conditions.In addition, all statements of the principles, aspects and embodiments of the present invention and its specific embodiments recorded herein are intended to include equivalents of their structure and function.In addition, it is intended that such equivalents include currently known equivalents and equivalents developed in the future, i.e., any element developed to perform the same function, regardless of its structure.In addition, any content disclosed herein is not intended to be dedicated to the public, regardless of whether such disclosure is clearly recorded in the claims.

Claims

1. A flow cytometer, comprising: a flow chamber for transporting particles in a central stream of the flow stream from a proximal end to a distal end, wherein the flow chamber comprises a flow chamber tapered structure at the proximal end; and A sample injection needle for delivering sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a core flow, the sample injection needle having a channel running through it, wherein the sample injection needle is configured and positioned to maintain an intact core flow under flow conditions that vary by one order of magnitude or more.

2. The flow cytometer according to claim 1, further comprising a sheath fluid introduction port for delivering sheath fluid to the flow chamber.

3. The flow cytometer according to claim 2, wherein the distal end of the sample injection needle is placed within the conical structure of the flow chamber and is separated from the sheath fluid introduction point by a longitudinal distance of 17 mm to 26 mm.

4. The flow cytometer according to any one of the preceding claims, wherein the sample injection needle is configured and positioned to maintain an intact core flow under the condition that the flow velocity of the flow stream is 0.5 m / s to 10 m / s.

5. The flow cytometer according to any one of the preceding claims, wherein the sample injection needle is configured and positioned to maintain an intact core flow at a flow rate of the sample fluid of 1 μL / min to 150 μL / min.

6. The flow cytometer according to any one of the preceding claims, wherein the sample injection needle comprises an inner diameter of 0.2 mm to 0.4 mm.

7. The flow cytometer according to any one of the preceding claims, wherein the sample injection needle comprises a tapered structure at the distal end. 8 . The flow cytometer according to claim 7 , wherein the tapered structure starts at a position separated from the distal end by a distance of 3.5 mm to 4.0 mm along the length direction of the sample injection needle.

9. The flow cytometer according to claim 7, wherein the tapered structure starts at a position separated from the distal end by a distance of 1.5 mm to 2.0 mm along the length direction of the sample injection needle.

10. The flow cytometer according to any one of claims 1 to 6, wherein the sample injection needle comprises a rounded distal end.

11. The flow cytometer according to claim 2, wherein the flow cytometer comprises a plurality of sheath fluid introduction ports.

12. The flow cytometer according to claim 11, wherein the plurality of sheath fluid introduction ports are offset from each other so that the sheath fluid rotates through the tapered structure of the flow chamber.

13. A method for analyzing a sample, the method comprising: (a) introducing a particle sample into a flow cytometer, the flow cytometer comprising: a flow chamber for transporting particles in a central stream of the flow stream from the proximal end to the distal end, wherein the flow chamber comprises a flow chamber tapered structure at the proximal end; and a sample injection needle for delivering a sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a core flow, the sample injection needle having a channel therethrough, wherein the sample injection needle is configured and positioned to maintain an intact core flow under flow conditions that vary by one order of magnitude or more; and (b) performing flow cytometric analysis on the particle sample.

14. The method of claim 13, further comprising subjecting the sample to flow cytometric sorting.

15. A method of assembling a flow cytometer, the method comprising placing a sample injection needle in the flow cytometer, the flow cytometer comprising a flow chamber for transporting particles in a central stream of a flow stream from a proximal end to a distal end, wherein the flow chamber comprises a flow chamber tapered structure at the proximal end, and wherein: The sample injection needle has a channel therethrough for conveying sample fluid from a sample injection line at a proximal end to a flow chamber at a distal end to generate a core flow, and The method includes placing the sample injection needle within a flow chamber tapered structure such that intact core flow is maintained under flow conditions that vary by an order of magnitude or more.

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