Fluid resistance unit, and flow cytometer and method involving the same

Abstract

A fluid resistance unit is provided. The fluid resistance unit of interest includes a plurality of valves and a plurality of resistors, and each resistor among the plurality of resistors is fluidly coupled to a different valve among the plurality of valves and is located at the same location as the valve. In one embodiment, the fluid resistance unit includes an inlet for receiving fluid, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet for discharging fluid. The valve line of interest includes a series of fluidly coupled valves, and the resistor line of interest includes a series of fluidly coupled resistors. Also provided are a method and a flow cytometer with the subject fluid resistance unit.

Description

Technical Field

[0001] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells in a blood sample or particles of interest within another type of biological or chemical sample.

[0002] A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing sheath fluid. The flow cytometer transfers particles (including cells) in the fluid sample as a cell stream to a flow cell while directing the sheath fluid to the flow cell. To evaluate the characteristics of the components of the flow stream, the flow stream is irradiated with light. Variations in the materials in the flow stream, such as the presence of morphological or fluorescent labels, can cause variations in the observed light, which enable characterization and separation. To evaluate the characteristics of the components in the flow stream, light must impinge on the flow stream and be collected. The light sources within a flow cytometer can vary and can include one or more broad-spectrum lamps, light-emitting diodes, as well as single-wavelength lasers. The light source is aligned with the flow stream such that the optical response from the illuminated particles is collected and quantified.

[0003] Some flow cytometry systems are implemented using pressure-driven fluidics, where the sample and sheath fluid are provided to a flow cell containing a detection region under a pressure greater than ambient pressure. Changes in the flow rate through the flow cell of a pressure-driven fluidics system are achieved by changing the pressure in the sample tube and / or sheath fluid reservoir that supplies the flow cell. The ratio of sample fluid to sheath fluid flowing through the flow cell is determined by both the pressure levels in the sample tube and sheath fluid reservoir and the ratio of the resistances of the sample fluid and sheath fluid paths.

[0004] Alternatively, the flow cytometer system is implemented using vacuum-driven fluidics where a vacuum pump pulls the downstream of the flow cell to a vacuum while the sample fluid and sheath fluid remain at ambient pressure. A change in the flow rate through the flow cell of the vacuum-driven fluidics system is achieved by changing the vacuum drawn by the vacuum pump, and the ratio of sample fluid to sheath fluid flowing through the flow cell is determined by the ratio of the resistance of the sample fluid and sheath fluid paths. Previous systems, such as those disclosed in U.S. Patent No. 8,528,427 by Vrane et al., employ fluid resistors and valves to provide a limited number of sheath fluid resistance states. SUMMARY OF THE INVENTION

[0005] The inventors have recognized that in order to further improve sample flow control in a vacuum-driven fluidics system, the resistance by which fluid is drawn into the flow cell through the fluidics system must be variable. Thus, systems and methods with increased resistance states are desirable. Embodiments of the fluid resistance units, flow cytometers, and methods described herein meet this need.

[0006] Aspects of the invention include a fluid resistance unit. The fluid resistance unit includes a plurality of valves and a plurality of resistors, each resistor of the plurality of resistors being fluidly coupled to a different valve in the plurality of valves and located at the same location as the valve. In embodiments, the subject fluid resistance unit includes an inlet for receiving fluid, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet for discharging fluid. The valve line of interest includes a series of fluidly coupled valves, and the resistor line of interest includes a series of fluidly coupled resistors. In some cases, the valve line comprises 2 to 6 valves (e.g., 4 valves). The resistor line may include 2 to 6 resistors (e.g., 4 resistors) in certain instances. In some embodiments, the fluid resistance unit comprises an equal number of resistors and valves. In such embodiments, the fluid resistance is x2 It may include x resistance states, where x is the number of valves. In the selected version, each valve in the valve line can be individually switched between an open position where the passage of fluid through the valve is not impeded and a closed position where the passage of fluid through the valve is impeded. In the closed position, the passage of fluid through the valve is partially or completely impeded. In some cases, each resistor in the resistor line includes an inner diameter in the range of 0.25 cm to 1.25 cm. In a particular case, the inner diameter of the resistors in the resistor line increases continuously or is constant. In the selected embodiment, each resistor in the resistor line has a length in the range of 1.5 cm to 90 cm. The length of the resistors in the resistor line may, in some cases, increase continuously. The number of connectors in the plurality of connectors can be in the range of, for example, 2 to 5 (e.g., 3). In the selected case, each connector in the plurality of connectors fluidly couples a portion of the valve line located between two valves in a series of valves to a portion of the resistor line located between two resistors in a series of resistors. The fluid resistance unit can be composed of, for example, a plastic tube or a metal tube.

[0007] Aspects of the present invention also include a flow cytometer. The flow cytometer of interest includes a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidly coupling to a sheath fluid reservoir, and a fluid resistance unit positioned between the sheath fluid line and the flow cell. The fluid resistance unit for use in the flow cytometer of interest has been described above and includes an inlet fluidly coupled to the sheath fluid line, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet fluidly coupling the fluid resistance unit to the flow cell. As discussed above, the valve line of interest includes a series of fluidly coupled valves, and the resistor line of interest includes a series of fluidly coupled resistors. In some instances, the flow cytometer includes a processor operably connected to each valve in the valve line. In such instances, the processor can be configured to change the resistance state of the fluid resistance unit by initiating a switch of a valve in the valve line from an open state to a closed state, or from a closed state to an open state. In a selected version, the flow cytometer includes a light source configured to irradiate the flow cell at an inspection point, a detector configured to collect particle-modulated light from the flow cell, and a sheath fluid reservoir.

[0008] Additional aspects of the present invention include a method of analyzing a sample. The method of interest includes: (a) introducing a particulate sample into a flow cytometer having a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidly coupling to a sheath fluid reservoir, and a fluid resistance unit positioned between the sheath fluid line and the flow cell; and (b) analyzing the sample by flow cytometry. The fluid resistance unit for use in the subject method includes an inlet fluidly coupled to the sheath fluid line, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet fluidly coupling the fluid resistance unit to the flow cell. As discussed above, the valve line of interest includes a series of fluidly coupled valves, and the resistor line of interest includes a series of fluidly coupled resistors. Embodiments of the method include changing the resistance state of the fluid resistance unit by switching the valves of the valve line from an open state to a closed state, or from a closed state to an open state. In certain instances, the particulate sample of interest includes a biological sample such as a cell.

[0009] The elements of the present invention further include a method of assembling a flow cytometer. The method of interest includes fluidly coupling a fluid resistance unit to a flow cell for transporting particles in a flow stream and a sheath fluid line for fluidly coupling to a sheath fluid reservoir. The fluid resistance unit for use in the subject method includes an inlet fluidly coupled to the sheath fluid line, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet fluidly coupling the fluid resistance unit to the flow cell. As discussed above, the valve line of interest includes a series of fluidly coupled valves, and the resistor line of interest includes a series of fluidly coupled resistors. The method according to some embodiments includes operably connecting a processor to each valve of the valve line. As discussed above, the processor of interest can be configured to change the resistance state of the fluid resistance unit by initiating a switch from an open state to a closed state, or from a closed state to an open state, of the valves in the valve line.

Brief Description of the Drawings

[0010] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures.

[0011]

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[0012] A fluid resistance unit is provided. The fluid resistance unit of interest includes a plurality of valves and a plurality of resistors, and each resistor in the plurality of resistors is fluidly coupled to a different valve in the plurality of valves and is located at the same location as the valve. In one embodiment, the fluid resistance unit includes an inlet for receiving fluid, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet for discharging fluid. The valve line of interest includes a series of fluidly coupled valves, and the resistor line of interest includes a series of fluidly coupled resistors. Also provided are methods and flow cytometers involving the subject fluid resistance unit.

[0013] Before the present invention is described in more detail, it is to be understood that the present invention is not limited to the particular embodiments described, and thus, of course, can vary. Also, since the scope of the present invention will be limited only by the appended claims, it is to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0014] When a range of values is provided, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of that range, down to one tenth of the unit of the lower limit, as well as any other stated value or intermediate value within the recited range, is understood to be encompassed by the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are likewise encompassed by the present invention, subject to any specifically excluded limits set forth in the recited range. When the recited range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present invention.

[0015] A particular range is presented herein with a numerical value preceded by the term "about." The term "about" is used herein to provide literal support for the exact number preceding it, as well as for a number that is close to, or approximates, the number preceding the term. In determining whether a number is close to, or approximates, a specifically recited number, a number that is close to, or approximates, an unrecited number may be a number that provides a substantial equivalent of the specifically recited number in the context in which it is presented.

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but representative illustrative methods and materials are described herein.

[0017] All publications and patents cited in this specification are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, to disclose and describe methods and / or materials, and publications are cited by reference in connection with those methods and / or materials. Any citation of a publication is for its disclosure prior to the filing date of this application, and this invention should not be construed as admitting that the invention has no right to antedate such publication on the ground of prior invention. Further, the provided publication dates may be different from the actual publication dates and may need to be individually verified.

[0018] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude optional elements. Accordingly, this description is intended to serve as a basis for using exclusive terms such as "solely", "only" in connection with the recitation of claim elements or the use of "negative" limitations.

[0019] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any of several other embodiments without departing from the scope or spirit of the invention. Any of the methods described can be performed in the order of the events described or any other order that is logically possible.

[0020] The systems and methods are described, or will be described, with functional descriptions for grammatical fluidity, but the claims should not be construed as necessarily limited by "means" or "step" limitation syntax unless expressly recited under 35 U.S.C. § 112, and should be given the full scope of the meaning of the definitions and equivalents provided by the claims under the doctrine of judicial equivalents. It should be clearly understood that when the claims are expressly recited under 35 U.S.C. § 112, full statutory equivalents under 35 U.S.C. § 112 should be given.

[0021] Fluid resistance unit As discussed above, aspects of the present invention include a fluid resistance unit. A "fluid resistance unit" means a device configured to provide a certain amount of resistance to a fluid being transported therethrough (e.g., using pressure or vacuum). For example, if the subject fluid resistance unit is employed in a flow cytometer (although other uses may be envisioned by those skilled in the art), the relative proportions of sheath fluid and sample fluid drawn through the flow cell can be adjusted by employing a particular resistance. When the total flow rate through the flow cell is held constant, adjusting the relative proportions of sheath fluid and sample fluid drawn through the flow cell also controls the sample fluid flow rate through the flow cell. In some cases, the subject fluid resistance unit increases the number of resistance states applicable to fluid flow, for example, compared to conventional devices for providing fluid resistance. A "resistance state" means a particular resistance level associated with the fluid resistance unit that is applied during a given time period during fluid flow. In some cases, the fluid resistance units of the present disclosure increase the number of resistance states to a number including two or more times, three or more times, four or more times, and five or more times. The increase in the number of resistance states increases the precision with which fluid flow can be controlled. The resistance states can be discrete or continuous. In some embodiments, the resistance states are discrete. In other embodiments, the resistance states are continuous.

[0022] The fluid resistance unit can be composed of any convenient material. In some cases, the fluid resistance unit includes a conduit (e.g., a tube). In such cases, the tube can consist of, for example, glass, plastic, or steel, as well as combinations thereof. The tube can have any convenient inner diameter ranging from 0.025 cm to 1.25 cm, for example, including 0.05 to 0.50 cm, such as in the range of 0.05 cm to 0.50 cm. The inner diameter of the conduit can be constant throughout the fluid resistance unit or can vary as needed. In an embodiment, the fluid resistance unit includes an inlet for receiving a liquid and an outlet for discharging the liquid. Any convenient inlet can be employed. For example, the inlet can be a portal within the conduit through which the fluid can enter. In a particular version, the inlet is, for example, a fluid coupling configured to couple to a sheath fluid line or includes the same. Similarly, a convenient outlet can be employed. In some embodiments, the outlet is configured to be fluidly coupled to a flow cell, for example, via a coupling.

[0023] Aspects of the fluid resistance unit include a plurality of valves. Any valve suitable for fluid applications can be employed, including, but not limited to, needle valves, globe valves, gate valves, plug valves, ball valves, butterfly valves, pinch valves, angle valves, cock valves (e.g., stopcock valves), etc. Each valve in the plurality of valves can be of the same type of valve or can be of different types of valves. In some cases, each valve in the plurality of valves is of the same type of valve. In other cases, one or more of the valves in the plurality of valves are of a different type of valve than another valve in the plurality of valves. The number of valves in the plurality of valves can vary as needed. In some embodiments, the number of valves in the plurality of valves is in the range including 2 to 10, for example, 2 to 8, for example, 2 to 6, and 2 to 4. In some embodiments, the plurality of valves includes 4 valves.

[0024] Aspects of the fluid resistance unit also include a plurality of resistors. The resistors described herein can be configured in any convenient form to restrict fluid flow. In some embodiments of the present invention, the fluid resistance unit is configured to provide a plurality of discrete resistance levels. This is achieved by providing alternative selectable fluid paths, each of which has a different fluid resistance. In such embodiments, each discrete path can comprise or consist of different resistors among the plurality of resistors. The fluid paths can be conduits (e.g., tubes having a certain length) with different lengths or inner diameters. Valves can be positioned within the fluid paths to enable selection of a desired fluid path from among the plurality of alternative fluid paths.

[0025] In certain cases, the fluid resistance unit includes a viscosity-dependent restrictor. In an embodiment, the viscosity-dependent restrictor is achieved by using a tube having a certain length and a substantially constant inner diameter, or a similar conduit. Such a resistor can be characterized as having discrete resistance levels. The resistance force for a fluid of a given viscosity depends on the length and internal cross-sectional area of the conduit, and the length is selected to provide the desired resistance. In contrast, simply pinching the tube at a single point or using a valve (such as a needle valve) to provide flow restriction results in convective acceleration. Due to the effect of temperature on viscosity, the temperature dependence of the resistance of a sheath fluid line incorporating a viscosity-dependent fluid resistor is the same as that of the resistance of a sample fluid line. Thus, the ratio of the resistance of the sample fluid line to that of the sheath fluid line remains the same as the temperature changes. In contrast, pinching is convectively dominated and the resistance is not a function of temperature, so the use of a pinching type of resistance will result in a change in the ratio of path resistance with a change in temperature.

[0026] In alternative embodiments of the present invention, the fluid resistance unit is continuously variable. In some such embodiments, at least one resistor includes a conduit made of a material that is at least partially compressible or deformable, such as a plastic tube having a length, and an adjustable pressure is applied to the outside of the tube. By adjusting the pressure applied to the outside of the tube, the diameter and / or shape of the tube changes, thereby changing the cross-sectional area of the fluid path and, accordingly, the fluid resistance of the tube. The continuously variable resistor can consist of a mechanically compressed tube having a length. For example, the tube can be wound around a cylindrical support and compressed between at least one movable plate having a surface perpendicular to the support. Alternatively, the continuously variable resistor can consist of a tube having a length that passes through the interior of a pressure chamber having an adjustable internal pressure. The internal pressure of the pressure chamber can be adjusted by connecting the chamber to an adjustable pressure source, such as a compressed air source, or by changing the size and / or shape of the pressure chamber by mechanical means or the like.

[0027] Each resistor in the plurality of resistors can be either the same type of resistor or different types of resistors. In some cases, each resistor in the plurality of resistors is the same type of resistor. In other cases, one or more of the resistors in the plurality of resistors are different types of resistors from another resistor in the plurality of resistors. In some such cases, one or more of the resistors in the plurality of resistors are continuously variable (i.e., can provide a range of resistances), while one or more other resistors are discrete. The number of resistors in the plurality of resistors can vary as needed. In some embodiments, the number of resistors in the plurality of resistors ranges from 2 to 10, for example, from 2 to 8, for example, from 2 to 6, and includes 2 to 4. In some embodiments, the plurality of resistors includes 4 resistors. Each resistor can have any convenient length. In some embodiments, each resistor has a length in the range including 1.5 cm to 90 cm, for example, 1.5 cm to 50 cm, and 1.5 cm to 45 cm. In some cases, at least one resistor has a length in the range of 35 cm to 45 cm, such as 40.64 cm. In some cases, at least one resistor has a length in the range of 15 cm to 25 cm, such as 20.32 cm. In some cases, at least one resistor has a length in the range of 5 cm to 15 cm, such as 10.16 cm. In some cases, at least one resistor has a length in the range of 1 cm to 10 cm, such as 5.08 cm. If the resistor includes a conduit (e.g., a tube) having a certain length, the tube can be arranged in any convenient manner. In some cases, the tube can be wound around a cylindrical support, for example, within a coil. In some such cases, the tube can be compressed between movable plates having a surface perpendicular to the support. However, any other suitable arrangement (e.g., without causing unintentional twisting within the tube) can be similarly employed.

[0028] Each resistor in the plurality of resistors can apply the same level of resistance or different levels of resistance. In some cases, all of the resistors apply the same level of resistance to each other. In some such cases, each resistor can be characterized by having the same inner diameter and length, i.e., the inner diameter and length are kept constant. In other embodiments, each resistor in the plurality of resistors applies a different level of resistance to each other. In a particular case, the resistance of the plurality of resistors increases continuously. In other words, the plurality of resistors includes a resistor characterized by the lowest resistance, a resistor characterized by a stepwise higher resistance compared to the lowest resistance, a resistor characterized by a stepwise higher resistance than the aforementioned resistance, and so on. In some such embodiments, the inner diameter and / or length of the resistor can vary. In a particular embodiment, the inner diameter of the resistor increases continuously. In an additional embodiment, the length of the resistance increases continuously. In a further embodiment, the inner diameter of the resistance is constant while the length of the resistance increases continuously.

[0029] In certain cases, each resistor in a plurality of resistors is fluidly coupled to a different valve in a plurality of valves and is located in the same location as the valve. As contemplated herein, two entities may be described as "fluidly coupled" if they have a conduit positioned therebetween configured to convey liquid from one entity to the other. Similarly, two entities may be described as "located in the same location" if they are adjacent and / or proximate to each other within a fluid resistance unit. For example, if entity A and entity B are fluidly coupled to each other via one or more conduits and there are no intervening entities within or along those conduits, entity A and B may be described as located in the same location. Thus, in certain instances, each resistor may be fluidly coupled to a valve and located in the same location as the valve such that the open or closed state of the valve affects whether and / or how fluid passes through the resistor located in the same location. In a selected version, the fluid resistance unit does not include valves that are not located in the same location as and / or not fluidly connected to a particular resistor. In these embodiments, the fluid resistance unit includes an equal number of resistors and valves.

[0030] When selected, each valve in a plurality of valves is individually switchable between an open position where passage of fluid through the valve is unobstructed and a closed position where passage of the valve's fluid is obstructed. Passage of fluid through each valve may be partially or completely obstructed in the closed position. When the valve is fully closed in the closed position, the fluid resistance unit is configured to provide discrete resistance levels. In these cases, the valve selects the resistance through which fluid passes, thereby achieving different resistance states. In some other cases where the valve is partially closed in the closed position, the fluid resistance unit is configured to provide continuous resistance levels. The valve may include any convenient mechanism for switching between the open and closed positions. In certain cases, the valve is under automatic control, for example, using a servo mechanism.

[0031] The components of the subject fluid resistance unit can be arranged in any suitable configuration. Exemplary configurations include a series configuration, a parallel configuration, and a configuration including both series and parallel elements. In some cases, the fluid resistance unit includes a series configuration. In some such embodiments, the fluid resistance unit includes an inlet for receiving fluid, a valve line fluidly coupled to the inlet, and a resistor line fluidly coupled to the inlet. Any convenient inlet can be employed. As discussed herein, a "valve line" refers to a plurality (i.e., a series) of valves fluidly coupled to each other, for example, via conduits. Similarly, a "resistor line" refers to a plurality (i.e., a series) of resistors fluidly coupled to each other, for example, via conduits. Embodiments of the fluid resistance unit also include a plurality of connectors fluidly coupling the valve line to the resistor line. A "connector" as described herein is a conduit that connects a portion of the valve line to a portion of the resistor line. In some cases, each connector in the plurality of connectors fluidly couples a portion of the valve line located between two valves in a series of valves to a portion of the resistor line located between two resistors in a series of resistors. Any convenient number of connectors can be employed. The number of connectors in the plurality of connectors can vary as needed. In some embodiments, the number of connectors in the plurality of connectors is in the range including 2 to 10, for example, 2 to 8, for example, 2 to 5, and 2 to 4. In some embodiments, the plurality of connectors includes three connectors. In a particular case, the plurality of connectors includes n - 1 connectors, where n is the number of resistors and / or valves.

[0032] In some embodiments with a series configuration, each valve in the valve line can be switched individually (e.g., via a servo mechanism) between an open position where the passage of fluid through the valve is unobstructed and a closed position where the passage of fluid through the valve is obstructed, e.g., a closed position where it can be completely or partially obstructed. In this way, the resistor through which the fluid passes can be adjusted. The resistance of the resistor in the resistor line can, in some embodiments, increase continuously. In some cases, each resistor in the resistor line has an inner diameter in the range including 0.025 - 1.25 cm, e.g., 0.050 - 0.50 cm, and 0.050 - 0.10 cm. In selected cases, the inner diameter of the resistor in the resistor line increases continuously. In other cases, the inner diameter of the resistance in the resistance line is constant. In a certain particular version, each resistor in the resistor line has a length in the range of 1.5 cm - 90 cm. If selected, the length of the resistor in the resistor line increases continuously. In some embodiments with a series configuration, the fluid resistance unit comprises an equal number of resistors and valves. In some such embodiments, the fluid resistance unit includes x 2 resistance states, where x is the number of valves. In an exemplary embodiment including four valves in series, the fluid resistance unit includes 16 resistance states.

[0033] In one example with a series configuration, the fluid enters through the inlet and can then branch into the resistor line and the valve line (e.g., via a Y - connector). When each valve in the valve line is closed, the fluid passes through each resistor in the resistor line before reaching the outlet. This scenario constitutes the highest resistance state. When the valve in the valve line closest to the inlet is open, an amount of fluid can bypass the first resistor. Since the next valve in the valve line is closed, the bypassed fluid enters the resistor line via the connector and passes through the remaining resistors. This will result in a resistance state that is step - by - step lower than the state where each valve is closed. In this way, any combination of open and closed valves produces different resistance states.

[0034] FIG. 1A depicts a fluid resistance unit 100a having a series configuration according to a particular embodiment of the present invention. The fluid resistance unit 100a includes an inlet A, a plurality of resistors R1 to R4 in a resistor line 101, valves V1 to V4 in a valve line 102, connectors 103a to 103c, and an outlet B. Since the fluid resistance unit 100a includes four valves, 4 2 = 16 resistance states are characterized. When all of V1 to V4 are closed, the fluid entering the inlet A must pass through each of R1 to R4, and thus the highest level of resistance is achieved. When valve V1 is open and valves V2 to V4 are closed, a certain amount of fluid that would otherwise have passed through R1 passes through V1 and connector 103a before passing through resistors R2 to R4. When valve V3 is open while valves V1 to V2 and V4 are closed, a certain amount of fluid that would otherwise have passed through R3 passes through V3 and returns to connector 103c. In this way, any of the 16 combinations of open and closed valves can be achieved, thereby resulting in 16 resistance states.

[0035] In some cases, the fluid resistance unit is characterized by a parallel configuration. In such an embodiment, the fluid resistance unit includes an inlet for receiving fluid, a plurality of fluid lines running in parallel, and an outlet for discharging the fluid. Each of the fluid lines includes both a valve and a resistor. A portion of each fluid line closer to the inlet includes a valve, while a portion of each fluid line closer to the outlet includes a resistor. In some embodiments, there are no parallel fluid lines that include a valve but not a resistor, or a resistor but not a valve. In other words, each parallel fluid line includes both a valve and a resistor. In some cases, the parallel fluid resistance units include an equal number of resistors and valves.

[0036] Figure 1B depicts a fluid resistance unit 100b having a parallel configuration according to a particular embodiment of the present invention. The fluid resistance unit 100b includes an inlet A, a plurality of valves V1-V4, a plurality of resistors R1-R4, parallel fluid lines 104a-104d, and an outlet B. Valve V1 is fluidly coupled to resistor R1 on fluid line 104a and is located at the same location as resistor R1, valve V2 is fluidly coupled to resistor R2 on fluid line 104b and is located at the same location as resistor R2, valve V3 is fluidly coupled to resistor R3 on fluid line 104c and is located at the same location as resistor R3, and valve V4 is fluidly coupled to resistor R4 on fluid line 104d and is located at the same location as resistor R4. In the embodiment of Figure 1B, each of the resistors R1-R4 is characterized by a different level of resistance (e.g., although not shown, they have continuously increasing lengths). Thus, different resistance states can be achieved depending on which of the valves V1-V4 are open or closed at a given point in time.

[0037] In certain cases, the fluid resistance unit is characterized by a configuration involving both parallel and series arrangements. In these cases, at least two sets of valves and resistors are positioned within parallel fluid lines (e.g., as depicted and described with respect to Figure 1B). Additionally, at least one resistor-valve pair is located upstream and / or downstream of the parallel fluid lines, where "upstream" and "downstream" are defined with respect to the direction of fluid flow. In other words, the resistor-valve pair is "in series" with the resistor-valve pairs that are "in parallel".

[0038] Figure 1C depicts a fluid resistance unit 100c characterized by a configuration involving both parallel and series arrangements according to a particular embodiment of the present invention. The fluid resistance unit 100c includes an inlet A, valves V1 to V3, resistors R1 to R3, parallel fluid lines 105a to 105b, and an outlet B. Valve V2 is fluidly coupled to resistor R2 on fluid line 105a and is located at the same location as resistor R2. Valve V3 is fluidly coupled to resistor R3 on fluid line 105b and is located at the same location as resistor R3. Valve V1 and resistor R1 are located at the same location as each other and are fluidly coupled, and are positioned upstream of fluid lines 105a to 105b. Although not shown in Figure 1C, valve V1 may optionally be fluidly coupled to a conduit that bypasses resistor R1. Valve V1 may be configured to divert fluid to such a conduit in the event of a desired event having a lower resistance state.

[0039] Flow cytometer Aspects of the present invention also include a flow cytometer. The flow cytometer of interest includes a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidly coupling to a sheath fluid reservoir, and a fluid resistance unit positioned between the sheath fluid line and the flow cell. As discussed above, the fluid resistance unit of the present invention includes a plurality of valves and a plurality of resistors, each resistor in the plurality of resistors being fluidly coupled to a different valve in the plurality of valves and being located at the same location as the valve. In some cases, the fluid resistance unit of the present invention includes an inlet fluidly coupled to the sheath fluid line, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet fluidly coupling the fluid resistance unit to the flow cell.

[0040] As contemplated herein, a "flow cell" refers to a component, as explained in its conventional meaning, that includes a flow channel having a liquid flow stream for transporting particles in a sheath fluid. In embodiments, the flow cell of the subject matter includes a cuvette. The cuvette of interest includes a container passing therethrough. The flow stream may include a liquid sample injected from a sample tube. The flow cell of interest includes a flow channel that is optically accessible. In some instances, the flow cell includes a transparent material (e.g., quartz) that permits the passage of light. Any convenient flow cell that propagates a fluid sample to a sample inspection region may be employed as the flow cell described herein, and in some embodiments, the flow cell is a flow cell that includes a cylindrical flow cell, a frustoconical flow cell, or a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating in a flat surface having an orifice that is transverse to the longitudinal axis.

[0041] In certain embodiments, a flow cytometer includes a sample fluid source. The sample fluid source can be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding the sample fluid. The sample fluid container can have a volume in the range of 1 mL to 100 mL, and for example, the volume of the container can be in the range of 1 mL to 90 mL, 1 mL to 80 mL, 1 mL to 70 mL, 1 mL to 60 mL, 1 mL to 50 mL, 1 mL to 40 mL, 1 mL to 30 mL, 1 mL to 20 mL, or 1 mL to 10 mL.

[0042] In some embodiments, the flow cytometer includes a sheath fluid reservoir. This sheath fluid reservoir can be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding the sheath fluid. In certain embodiments, the sheath fluid reservoir is fluidly coupled to the input of the flow cell. The sheath fluid container can have a volume in the range of 1 L to 100 L. For example, the volume of the container can be in the range of 1 L to 90 L, 1 L to 80 L, 1 L to 70 L, 1 L to 60 L, 1 L to 50 L, 1 L to 40 L, 1 L to 30 L, 1 L to 20 L, or 1 L to 10 L. The sheath fluid reservoir can be fluidly connected to a sheath fluid line configured to carry the sheath fluid from the reservoir to the flow cell.

[0043] In some embodiments, the flow cell includes a sample injection port configured to provide a sample from a sample fluid source to the flow cell. This sample injection port can be an orifice positioned in the wall of the internal chamber or a conduit positioned at the proximal end of the internal chamber. When the sample injection port is an orifice positioned in the wall of the internal chamber, the sample injection port orifice can be of any suitable shape, and examples of cross-sectional shapes of interest include, but are not limited to, cross-sectional shapes composed of straight lines such as square, rectangular, trapezoidal, triangular, hexagonal, etc., cross-sectional shapes composed of curves such as circular, elliptical, etc., and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on the shape. In specific cases, it can have an opening in the range of 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, 0.1 mm to 5.0 mm, including 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0044] In certain cases, the sample injection port is a conduit positioned at the proximal end of the flow cell internal chamber. For example, the sample injection port can be a conduit positioned to have an orifice of the sample injection port along the flow cell orifice. When the sample injection port is a conduit positioned along the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, and examples of cross-sectional shapes of interest include, but are not limited to, cross-sectional shapes composed of straight lines such as squares, rectangles, trapezoids, triangles, hexagons, etc., cross-sectional shapes composed of curves such as circles, ellipses, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. The orifice of the conduit can vary depending on the shape, and in certain cases, it has an opening in the range of 0.2 to 3.0 mm, such as including 1.25 mm to 1.75 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, 0.1 mm to 5.0 mm, for example, 1.5 mm. The shape of the tip of the sample injection port can be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port can include a bevel tip having a bevel angle in the range of 1 to 10 degrees, such as including 2 to 9 degrees, 3 to 8 degrees, 4 to 7 degrees, including a 5-degree bevel angle.

[0045] In some embodiments, the flow cell also includes a sheath fluid injection port configured to provide a sheath fluid from a sheath fluid source to the flow cell. In an embodiment, the sheath fluid injection system is configured to provide a flow of the sheath fluid to the flow cell internal chamber, for example, together with the sample, to generate a laminar flow stream of the sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid carried to the flow cell chamber can be in the range of 50 μL / second to 1000 μL / second, such as including 75 μL / second or more to 750 μL / second, 25 μL / second to 2500 μL / second.

[0046] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the internal chamber. The sheath fluid injection port orifice can be of any suitable shape, and examples of cross-sectional shapes of interest include, but are not limited to, cross-sectional shapes composed of straight lines such as square, rectangular, trapezoidal, triangular, hexagonal, etc., cross-sectional shapes composed of curves such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. The size of the sample injection port orifice can vary depending on the shape, and in certain cases, it includes ranges such as 1.25 mm to 1.75 mm, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, etc., such as 1 mm to 2 mm, etc., 0.1 mm to 5.0 mm, and has an opening, for example, of 1.5 mm.

[0047] The flow cytometer of the present invention includes a fluid resistance unit (e.g., the sheath fluid injection port of the flow cell) positioned between the sheath fluid reservoir and the flow cell. For example, if the sheath fluid reservoir includes a sheath fluid line fluidly coupled thereto, the inlet of the fluid resistance unit can be fluidly coupled to the sheath fluid line (e.g., via a joint). Similarly, the outlet of the fluid resistance unit can be coupled to the sheath fluid injection port of the flow cell (e.g., via a joint). Any fluid resistance unit such as those described above can be employed in the flow cytometer. In some embodiments, the fluid resistance units are in series. In other embodiments, the fluid resistance units are in parallel. In still other embodiments, the fluid resistance unit includes both parallel and series elements.

[0048] In some embodiments, the system further includes a pump (e.g., a vacuum pump) in fluid communication with the flow cell for propagating a flow stream through the flow cell. Any convenient fluid pump protocol may be employed to control the flow of the flow stream through the flow cell. In certain instances, the system includes a peristaltic pump, such as a peristaltic pump having a pulse damper. The pump in the subject system is configured to carry fluid through the flow cell at a rate suitable for multi-photon counting of light from the sample in the flow stream. For example, the system may include a pump configured to flow a sample through the flow cell at a rate in the range of 1 nL / min to 250 nL / min, such as 1 nL / min to 100 nL / min, 2 nL / min to 90 nL / min, 3 nL / min to 80 nL / min, 4 nL / min to 70 nL / min, 5 nL / min to 60 nL / min, including 10 nL / min to 50 nL / min. In certain embodiments, the flow rate of the flow stream is 5 nL / min to 6 nL / min.

[0049] FIG. 2 depicts a schematic view of the elements of a flow cytometry system of the present invention. System vacuum is caused by a vacuum pump 211, which draws sheath fluid from a sheath reservoir 202 and sample fluid containing particles to be analyzed through a flow cell 200 from a sample tube 201, where optical analysis is performed (the optical system is not shown). Waste effluent, which is a mixture of sample and sheath fluid exiting the flow cell, is discharged to a waste reservoir 203.

[0050] Pulses in the vacuum caused by a vacuum pump 211, typically a diaphragm type pump, are attenuated by an accumulator 255, also referred to as a pulse damper. This accumulator may be a sealed canister having an internal volume many times (e.g., 10 to 1000 times) the stroke volume of the vacuum pump.

[0051] The transducer 231 measures the pressure drop caused by the vacuum pump 211 with respect to atmospheric pressure. This pressure drop is referred to herein as the "static pressure drop". This static pressure drop can be measured from inside the accumulator 255 so that a stable measured value can be obtained. The transducer 231 is typically connected to the accumulator 255 by a short tube such that the pressure in the tube equals the pressure in the accumulator. It is desirable to include an air bleed (e.g., a small orifice connecting the inside of the tube to the outside air) in the tube connecting the transducer 231 and the accumulator 255 positioned near the transducer so that a small amount of air can be drawn by the vacuum in the accumulator and drawn through the tube and through the tube. The air bleed must be small enough so that the air flow through the tube does not significantly affect the measurement of the static pressure drop. A slight air flow through the tube in the direction from the orifice (near the transducer) towards the accumulator prevents any fluid or bubbles that may be present in the accumulator from entering the transducer from the tube and may affect the accuracy of the measurement.

[0052] The transducer 232 measures the pressure drop across the entire cuvette 102 (measured from upstream of the flow cell to the accumulator 255). This pressure drop is referred to herein as the "dynamic pressure drop". For a given total flow rate through the cuvette, the dynamic pressure drop is constant. Thus, by adjusting the power of the vacuum pump 211 to provide a constant dynamic pressure drop, a constant total flow rate through the cuvette can be maintained.

[0053] The sample fluid is drawn into the flow cell 200 through the sample line 220 and through the sample inlet port 208. The entire sample path has a fluid resistance R0. The sheath fluid is drawn into the flow cell 200 through the fluid resistance unit 222 and through the sheath inlet port 210. In one embodiment, the fluid resistance of the fluid resistance unit is adjustable to a plurality of discrete fluid resistance set values. In another embodiment, the fluid resistance of the fluid resistance unit is continuously adjustable over a predetermined range.

[0054] The relative proportions of the sample fluid and the sheath fluid drawn into the flow cell 200 depend on the ratio of the sample line fluid resistance R0 to the fluid resistance of the fluid resistance unit 222. Thus, by adjusting the fluid resistance of the fluid resistance unit 222, the ratio of the sample fluid to the sheath fluid can be controlled. If the total flow rate through the flow cell is held constant by maintaining a constant dynamic pressure drop, each decrease in the effective fluid resistance of the fluid resistance unit 222 results in a decrease in the sample fluid flow rate and an increase in the sheath fluid flow rate. Thus, the desired sample flow rate can be selected by appropriate adjustment of the total fluid resistance of the fluid resistance unit 222 while maintaining a constant flow rate through the flow cell.

[0055] Valve 253 enables complete shut-off of the fluid flow through the flow cell. The flow can be paused, for example, to enable a change to a new sample source after each sample analysis. In this system, the fluid flow can be paused by closing a valve located in the fluid path between the flow cell and the pump. When this valve is closed, the dynamic pressure drop becomes zero, the first feedback loop between the dynamic pressure drop and the pump stops, and the second feedback loop between the static pressure drop and the pump activates. The second feedback loop enables maintaining the static pressure drop at a constant level during the pause state, such as maintaining the static pressure drop of the system that existed while in the running state before pausing the system (while under the control of the first feedback loop). When the fluid flow through the flow cell is restarted, the control of the pump is switched back to the first feedback loop, which enables maintaining a constant flow in the flow cell even after a change in the sample flow rate. This switching between the feedback control circuits eliminates large fluctuations in the vacuum during the transition between the running and pause states while maintaining a constant flow rate through the flow cell for all samples.

[0056] Valve 251 enables complete shut-off of the sheath fluid flow. Valve 251 is used to temporarily stop the sheath fluid flow following connection of the sample tube 201 to the sample line 220 and temporarily increase (“boost”) the sample fluid flow rate to shorten the time taken for the sample fluid to be drawn into the flow cell 200. When the sample fluid reaches the flow cell, valve 251 is opened and the sheath fluid flow establishes a hydrodynamically focused stream, and the flow rates of the sample and sheath fluids return to the desired flow rates for analysis. Valves 251 and 253 can be automatically controlled in a coordinated manner such that valve 253 is opened for a predetermined time before opening valve 251 to enable a vacuum to occur within the flow cell before opening said valve 251.

[0057] The controller 261 having the switching mechanism 263 controls the switching between the adjustment of the power of the vacuum pump 211 to provide a constant dynamic pressure drop and the adjustment of the power of the vacuum pump 211 to provide a constant static pressure drop. To maintain a constant dynamic pressure drop, the controller compares the dynamic pressure drop measured by the transducer 232 with the stored desired dynamic pressure drop P D This desired dynamic pressure drop P D is determined during the setup of the device as the dynamic pressure drop that provides the desired flow rate through the flow cell. To maintain a constant static pressure drop, the controller compares the static pressure drop measured by the transducer 231 with the stored desired static pressure drop P S The desired static pressure drop P S is the measured static pressure drop corresponding to the device to be operated at the desired flow rate through the flow cell, depending on the selected setting of the fluid resistance unit. A plurality of values for the desired static pressure drop P S can be stored, each corresponding to a predetermined resistance setting value of the fluid resistance unit. Alternatively, the desired static pressure drop P S can be saved immediately before the system is paused, and the system can be restarted with the same resistance setting value of the fluid resistance unit that was active immediately before the system was paused. In some embodiments, the pressure drop feedback circuit (via the controller 261), as well as the automatic control of the valves 251 and 253, will be provided in a coordinated manner.

[0058] The flow sensor 235 is positioned on the sample line 220 to provide a direct measurement of the sample fluid flow rate. Suitable high-precision liquid flow sensors and liquid flow meters with a measurement range up to nanoliters per minute are commercially available, for example, from Sensirion (Westlake Village, CA). The flow sensor 235 is optional but facilitates the setup of the flow system. During the setup of the instrument (system calibration), the fluid resistance of the fluid resistance unit 222 is adjusted to provide the desired ratio of sheath to sample fluid, and the flow sensor provides an independent measurement of the resulting sample fluid flow rate. Alternatively, the flow rate of the sample fluid can be measured by other means such as analyzing a sample containing test particles of a known concentration. By measuring the detection rate of the test particles, the flow rate within the sample line 220 can be inferred.

[0059] In some embodiments, the subject flow cytometer may be operated in conjunction with programmable logic implemented in hardware, software, firmware, or any combination thereof to vary the provided state of the fluid resistance unit. For example, when the programmable logic is implemented in software, the resistance state adjustment may be at least partially realized by a computer-readable data storage medium containing program code that includes instructions configured to initiate switching of a valve in a valve line from an open state to a closed state at runtime. The subject programmable logic may be implemented in any of a variety of devices such as a specially programmed event processing computer, a wireless communication device, or an integrated circuit device. In some embodiments, the programmable logic may be executed by a specially programmed processor that may include one or more processors such as one or more digital signal processors (DSPs), configurable microprocessors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or other equivalent integrated circuits or discrete logic circuits. Combinations of computing devices, such as a combination of a DSP and a microprocessor, multiple microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration in at least partial data connection state, may implement one or more of the described features.

[0060] Embodiments of the subject flow site meter include a processor. The processor of interest is operably connected to each valve of the valve line and is configured to change the resistance state of the fluid resistance unit by initiating a change in the valve of the valve line from an open state to a closed state or from a closed state to an open state. In some cases, the processor is configured to compare the actual flow rate with a target flow rate and adjust the resistance state of the fluid resistance unit accordingly to achieve the target flow rate. In the selected version, the processor operates automatically, for example, with an automatic feedback control circuit. In some cases, the processor can receive input from a user. In such cases, the processor can receive an input regarding the target flow rate, and the processor changes the resistance state of the fluid resistance unit to achieve the target flow rate.

[0061] Figure 3 depicts an automatic feedback control circuit for automatically adjusting the sample fluid flow rate to obtain a preselected sample fluid flow rate during system calibration. The fluid resistance unit 322 is configured to enable adjustment of the fluid resistance using a servo mechanism. The processor 361 compares the flow rate measured by the flow sensor 235 with the stored desired flow rate FR and adjusts the fluid resistance of the variable resistance fluid resistor 322 until the measured flow rate matches the desired flow rate FR. The processor 361 is configured to open or close any of the valves within the fluid resistance unit 322, for example, via a servo mechanism.

[0062] As mentioned above, the flow cytometer of the present invention includes a flow cell for transporting particles in a flow stream. The flow stream can be configured such that light from a light source irradiates at an inspection point. The flow stream in which a flow channel is configured can include a liquid sample injected from a sample tube. In certain embodiments, the flow stream can include a narrow, rapidly flowing liquid stream in which linearly separated particles transported therein are separated from each other in a single file manner. The "inspection point" discussed herein refers to, for example, a region within the flow cell where particles are irradiated by light from a light source for analysis. The size of the inspection point can vary as needed. For example, when 0 μm represents the axis of the light emitted by the light source, the inspection point can range from -15 μm to 30 μm, including -50 μm to 50 μm, such as -25 μm to 40 μm, such as -100 μm to 100 μm.

[0063] After the particles are irradiated in the flow cell, particle-modulated light can be observed. "Particle-modulated light" means the light received from the particles in the flow stream after irradiating the particles with light from a light source. In some cases, the particle-modulated light is side-scattered light. As discussed herein, side-scattered light refers to the light diffracted and reflected from the surface and internal structure of the particles. In additional embodiments, the particle-modulated light includes forward-scattered light (i.e., light that travels mostly in the forward direction through or around the particles). In still other cases, the particle-modulated light includes fluorescence (i.e., light emitted from a fluorescent dye after irradiation with excitation wavelength light).

[0064] Aspects of the present invention also include a light source configured to irradiate particles passing through a flow cell at an inspection point. Any convenient light source may be employed 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 CO2 laser, a CO laser, an argon-fluoride (ArF) excimer laser, a krypton-fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluoride (XeF) excimer laser, or a combination thereof. In other cases, the flow cytometer of interest 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 combinations thereof. In still other cases, the flow cytometer of interest includes a solid laser such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a slim YAG laser, a ytterbium YAG laser, a ytterbium2O3 laser, or a cerium-doped laser, and combinations thereof.

[0065] A laser light source according to certain embodiments may also include one or more optical adjustment components. In certain embodiments, the optical adjustment component is located between the light source and the flow cell and can be any device that can change the spatial width of the irradiation or some other characteristics of the irradiation from the light source, such as, for example, the irradiation direction, wavelength, beam width, beam intensity, and focus. The optical adjustment protocol can 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, collimating protocols, and combinations thereof. In certain embodiments, the flow cytometer of interest includes one or more focusing lenses. The focusing lens can be, in one example, a non-magnifying lens. In still other embodiments, the flow cytometer of interest includes an optical fiber.

[0066] If the optical adjustment component is configured to move, the optical adjustment component can be moved continuously or at discrete intervals, such as increments of 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 10 μm or more, 100 μm or more, 500 μm or more, 1 mm or more, 5 mm or more, 10 mm or more, including increments of 25 mm or more, for example, increments of 0.01 μm or more.

[0067] Any displacement protocol for moving the optical adjustment component structure can be employed, such as being coupled to a movable support stage or being directly coupled to a motor-operated translation stage, a lead screw translation assembly, a gear-type translation device, among others, including, for example, a stepping motor, a servo motor, a brushless electric motor, a brushed DC motor, a microstepping drive motor, a high-resolution stepping motor.

[0068] The light source can be positioned at any suitable distance from the flow cell. For example, here, the light source and the flow cell are separated by a distance of 0.01 mm or more, such as 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, 0.005 mm or more, including distances of 100 mm or more. Further, the light source can be positioned at any suitable angle with respect to the flow cell, for example, at an angle in the range of 10 degrees to 90 degrees, including 15 degrees to 85 degrees, such as 20 degrees to 80 degrees, 25 degrees to 75 degrees, 30 degrees to 60 degrees, for example, at an angle of 90 degrees.

[0069] In some embodiments, the light source of interest includes a plurality of lasers configured to provide laser light for discrete illumination of the flow stream, including 2 or more lasers, such as 3 or more lasers, 4 or more lasers, 5 or more lasers, 10 or more lasers, 15 or more lasers configured to provide laser light for discrete illumination of the flow stream. Depending on the desired wavelength of the light for irradiating the flow stream, each laser can have a specific wavelength that varies from 250 nm to 1250 nm, such as 300 nm to 1000 nm, 350 nm to 900 nm, 200 nm to 1500 nm, including 400 nm to 800 nm. In certain embodiments, the lasers of interest can include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0070] The flow cytometer of interest can further include one or more particle-modulated light detectors for detecting particle-modulated light intensity data. In some embodiments, the particle-modulated light detector includes one or more forward-scattered light detectors (plural possible) configured to detect forward-scattered light. For example, the particle analyzer of the subject matter can include one forward-scattered light detector, or a plurality of (e.g., 2 or more, such as 3 or more, 4 or more, and 5 or more included) forward-scattered light detectors. In certain embodiments, the particle analyzer includes one forward-scattered light detector. In other embodiments, the particle analyzer includes two forward-scattered light detectors.

[0071] Any convenient detector for detecting the collected light can be used in the forward scattered light detector described herein. Detectors of interest can include, among other detectors, active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), intensified charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, but are not limited thereto. In certain embodiments, the collected light is measured with a charge coupled device (CCD), semiconductor charge coupled device (CCD), active pixel sensor (APS), complementary metal oxide semiconductor (CMOS) image sensor, or N-type metal oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector is 0.01 cm 2 ~10 cm 2 , for example, 0.05 cm 2 ~9 cm 2 , for example, 0.1 cm 2 ~8 cm 2 , for example, 0.5 cm 2 ~7 cm 2 , and 1 cm 2 ~5 cm 2 and is a photomultiplier tube such as a photomultiplier tube having an active detection surface area for each region in the range including.

[0072] In embodiments, the forward scattered light detector is configured to measure light continuously or at discrete intervals. In some cases, the detector of interest is configured to continuously measure the collected light. In other cases, the detector of interest is configured to measure light at discrete intervals, such as at 0.001 milliseconds per, 0.01 milliseconds per, 0.1 milliseconds per, 1 millisecond per, 10 milliseconds per, 100 milliseconds per, or some other interval, including every 1000 milliseconds.

[0073] In additional embodiments, one or more particle-modulated light detectors may include one or more side-scattered light detectors for detecting the side-scattered wavelength of light (i.e., the light refracted and reflected from the surface and internal structure of the particle). In some embodiments, the particle analyzer includes a single side-scattered light detector. In other embodiments, the particle analyzer includes multiple (e.g., two or more, e.g., three or more, e.g., four or more, and including five or more) side-scattered light detectors.

[0074] Any convenient detector for detecting the collected light may be used for the side-scattered light detectors described herein. Detectors of interest may include, among others, active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors, or photodiodes, and optical sensors or detectors such as combinations thereof, but are not limited thereto. 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 has an active detection surface area in each region in the range including 0.01 cm 2 ~10 cm 2 e.g., 0.05 cm 2 ~9 cm 2 e.g., 0.1 2 ~8 cm 2 e.g., 0.5 cm 2 ~7 cm 2 and 1 cm 2 ~5 cm 2 and is a photomultiplier tube such as a photomultiplier tube having an active detection surface area in each region of the range.

[0075] In an embodiment, the subject particle analyzer also includes a fluorescence detector configured to detect one or more fluorescence wavelengths of light. In other embodiments, the particle analyzer includes a plurality (e.g., two or more, e.g., three or more, e.g., four or more, five or more, ten or more, fifteen or more, and twenty or more) of fluorescence detectors.

[0076] Any convenient detector for detecting the collected light can be used with the fluorescence detectors described herein. Detectors of interest include, among others, active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors, or photodiodes, and optical sensors or detectors such as combinations thereof, but are not limited thereto. In certain embodiments, the collected light is measured by 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 has an active detection surface area in each region in the range including 0.01 cm 2 ~10 cm 2 , e.g., 0.05 cm 2 ~9 cm 2 , e.g., 0.1 cm 2 ~8 cm 2 , e.g., 0.5 cm 2 ~7 cm 2 , and 1 cm 2 ~5 cm 2 and is a photomultiplier tube such as a photomultiplier tube having an active detection surface area in each region of the range.

[0077] When the particle analyzer of the subject includes a plurality of 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 particle analyzer of the subject includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-type device, and the second fluorescence detector (or image sensor) is a CMOS-type device. In other embodiments, both the first fluorescence detector and the second fluorescence detector are CCD-type devices. In still other embodiments, both the first fluorescence detector and the second fluorescence detector are CMOS-type devices. In still other embodiments, the first fluorescence detector is a CCD-type device, and the second fluorescence detector is a photomultiplier tube (PMT). In still other embodiments, the first fluorescence detector is a CMOS-type device, and the second fluorescence detector is a photomultiplier tube. In still other embodiments, both the first fluorescence detector and the second fluorescence detector are photomultiplier tubes.

[0078] In embodiments of the present disclosure, the fluorescence detector of interest is configured to measure light collected at one or more wavelengths, including, for example, measuring light emitted by a sample in the flow stream at two or more wavelengths, such as five or more different wavelengths, such as ten or more different wavelengths, such as twenty-five or more different wavelengths, such as fifty or more different wavelengths, such as one hundred or more different wavelengths, such as two hundred or more different wavelengths, such as three hundred or more different wavelengths, and four hundred or more different wavelengths. In some embodiments, two or more detectors within the particle analyzer described herein are configured to measure the same, or overlapping, wavelengths of the collected light.

[0079] In some embodiments, the fluorescence detector of interest is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect a spectrum of light over a range of wavelengths. For example, a particle analyzer can include one or more detectors configured to collect a spectrum of light over one or more of the wavelength ranges from 200 nm to 1000 nm. In still other embodiments, the detector of interest is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a particle analyzer can include one or more detectors configured to measure light at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, one or more detectors can be configured to pair with a specific fluorophore, such as those used with a sample in a fluorescence assay.

[0080] In some embodiments, the particle analyzer includes one or more wavelength separators disposed between the flow cell and the particle modulation light detector(s). The term "wavelength separator" as used herein refers, in its conventional meaning, to an optical component configured to separate light collected from a sample into a predetermined spectral range. In some embodiments, the particle analyzer includes a single wavelength separator. In other embodiments, the particle analyzer includes a plurality of wavelength separators, such as two or more wavelength separators, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty-five or more, such as fifty or more, such as seventy-five or more, and one hundred or more wavelength separators. In other embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by passing light having a predetermined spectral range and reflecting light in one or more remaining spectral ranges. In other embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by passing light having a predetermined spectral range and absorbing light in one or more remaining spectral ranges. In still other embodiments, the wavelength separator is configured to spatially diffract light collected from a sample into a predetermined spectral range. Each wavelength separator can be any convenient light separation protocol, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In other embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In certain embodiments, the wavelength separator within the optical detection system of the subject is a dichroic mirror.

[0081] Suitable flow cytometry systems include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt1):17-28; Linden, et.al., Semin 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 disclosures of which are incorporated herein by reference.In certain instances, the flow cytometry system of interest includes BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and BD Biosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, and the like.

[0082] In some embodiments, the subject system is a flow cytometry system such as those described in U.S. Patent Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 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; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766, the disclosures of which are hereby incorporated by reference in their entirety.

[0083] Figure 4 shows a system 400 for flow cytometry, according to an illustrative embodiment of the present invention. 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 - 415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a - 445g, one or more bandpass filters 450a - 450e, one or more long pass ("LP") filters 455a - 455b, and one or more fluorescence detectors 460a - 460f.

[0084] The excitation lasers 415a to 415c emit light in the form of laser beams. The wavelengths of the laser beams emitted from the excitation lasers 415a to 415c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system of FIG. 4. The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. The beam splitter 445b transmits UV light (light having wavelengths in the range of 10 to 400 nm) and reflects light at 488 nm and 633 nm.

[0085] The laser beams are then directed to a focusing lens 420, which focuses the beam onto a portion of the fluid stream in which the particles of the sample are located within the flow chamber 425. The flow chamber is part of a fluidics system that directs the particles in the stream, typically one at a time, to the focused laser beam for inspection. The flow chamber can comprise a flow cell within a benchtop flow cytometer or a nozzle tip within a stream-in air cytometer.

[0086] The light from the laser beams interacts with the particles in the sample by diffraction, refraction, reflection, scattering, and absorption with re-emission at various different wavelengths depending on particle characteristics such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally occurring on or in the particles. Fluorescent emission, as well as diffracted light, refracted light, reflected light, and scattered light, can be routed through one or more of the beam splitters 445c to 445g, bandpass filters 450a to 450e, longpass filters 455a to 455b, and fluorescence collection lens 440 to one or more of the forward scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a to 460f.

[0087] The fluorescence collection lens 440 collects the light emitted from the particle laser beam interaction and routes the light towards one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a - 450e, enable a narrow wavelength range 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. Thus, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short - pass filters transmit the wavelengths of light below a specified wavelength. Long - pass filters, such as long - pass filters 455a - 455b, transmit the wavelengths of light above a specified wavelength. For example, long - pass filter 455b, which is a 670 nm long - pass filter, transmits light of 670 nm and above. Filters are often selected to optimize the specificity of the detector for a particular fluorescent dye. Those filters can be configured such that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0088] The forward scatter detector 430 is positioned slightly off - axis from the direct beam passing through the flow cell and is configured to detect diffracted light, excitation light that travels mostly in the forward direction through or around the particles. The intensity of the light detected by the forward scatter detector depends on the overall size of the particles. The forward scatter detector can include a photodiode. The side scatter detector 435 is configured to detect diffracted and reflected light from the surface and internal structure of the particles, which tends to increase as the particle structure becomes more complex. Fluorescent emission from fluorescent molecules associated with the particles can be detected by one or more fluorescence detectors 460a - 460f. The side scatter detector 435 and the fluorescence detectors can include photomultiplier tubes. The signals detected by the forward scatter detector 430, the side scatter detector 435, and the fluorescence detectors can be converted by the detectors into electronic signals (voltages). This data can provide information about the sample.

[0089] One of ordinary skill in the art will recognize that the flow cytometer according to one embodiment of the present invention is not limited to the flow cytometer depicted in FIG. 4, but may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in various different configurations.

[0090] During operation, the operation of the flow cytometer is controlled by a controller / processor 490, and measurement data from the detector can be stored in a memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive an output signal therefrom and is also coupled to the electrical and electromechanical components of the flow cytometer 410 to control lasers, fluid flow parameters, etc. An input / output (I / O) function unit 497 may also be provided within the system. The memory 495, the controller / processor 490, and the I / O 497 may be provided as an integral part of the flow cytometer 410. In such an embodiment, the display may also form part of the I / O function unit 497 for presenting experimental data to the user of the flow cytometer 410. Alternatively, some or all of the memory 495, the controller / processor 490, and the I / O function unit 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 the controller / processor 490 can communicate with the flow cytometer 410 wirelessly or by wire. Together with the memory 495 and the I / O 497, the controller / processor 490 may be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0091] The system illustrated in FIG. 4 includes six different detectors that detect fluorescence within six different wavelength bands (which may be referred to herein as "filter windows" for a given detector), as defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules within the fluorescent panel used for flow cytometry experiments will emit light in their own characteristic wavelength bands. The specific fluorescent labels used in the experiment, and their associated fluorescence emission bands, can be selected to generally match the filter windows of the detectors. I / O 497 can be configured to receive data related to flow cytometry experiments with a panel of fluorescent labels and multiple cell parent populations having multiple markers, where each cell parent population has a subset of the multiple markers. I / O 497 can also be configured to receive biological data that assigns one or more markers to one or more cell parent populations, marker concentration data, emission spectrum data, data that assigns labels to one or more markers, and cytometer configuration data. Flow cytometry experiment data such as label spectral characteristics and flow cytometer configuration data can also be stored in memory 495. The controller / processor 490 can be configured to evaluate one or more assignments of labels to markers.

[0092] FIG. 5 shows a functional block diagram of an example of a processing system for analyzing and displaying biological events. The processor 500 can be configured to perform various processes for controlling a graphic display of biological events.

[0093] The flow cytometer 502 can be configured to acquire biological event data. For example, the flow cytometer can generate flow cytometry event data (e.g., particle modulated light data). 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.

[0094] Processor 500 can be configured to receive biological event data from flow cytometer 502. The biological event data received from flow cytometer 502 can include flow cytometry event data. Processor 500 can be configured to provide a graphical display of the biological event data to display device 506. Processor 500 can be further configured to render a region of interest as a gate around a population of biological event data indicated by display device 506. In some embodiments, the gate can be a logical combination of one or more graphical regions of interest drawn on a single parameter histogram or bivariate plot. In some embodiments, the display can be used to display particle parameters or saturated detector data.

[0095] Processor 500 can be further configured to display the biological event data on display device 506 within the gate differently from other events within the biological event data outside the gate. For example, processor 500 can be configured to render the color of the biological event data included within the gate to be different from the color of the biological event data outside the gate. Display device 506 can be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0096] The processor 500 can be configured to receive a gate selection signal that identifies a gate from a first input device. For example, the first input device can be implemented as a mouse 510. This mouse 510 can initiate a gate selection signal to the processor 500 that identifies a gate to be displayed or manipulated via the display device 506 (e.g., by clicking on the desired gate when positioning a cursor there). In some embodiments, the first device can be implemented as a keyboard 508, or other means for providing an input signal to the processor 500, such as a touch screen, input pen, light detector, or voice recognition system. Some input devices can include multiple input functions. In such embodiments, each input function can be considered an input device. For example, as shown in FIG. 5, the mouse 510 can include a right mouse button and a left mouse button, each of which can generate an activation event.

[0097] This activation event can cause the processor 500 to change how data is displayed, which portions of the data are actually displayed on the display device 506, and / or provide input to further processing, such as the selection of a population of interest for particle sorting.

[0098] In some embodiments, the processor 500 can be configured to detect when a gate selection is initiated by the mouse 510. The processor 500 can be further configured to automatically modify the visualization of the plot to facilitate the gate control process. This modification can be based on a particular distribution of the biological event data received by the processor 500. In some embodiments, the processor 500 expands the first gate such that a second gate is generated (e.g., as discussed above).

[0099] Processor 500 may be connected to memory device 504. This memory device 504 may be configured to receive and store biometric event data from processor 500. Memory device 504 may also be configured to receive and store flow cytometry event data from processor 500. Memory device 504 may be further configured by processor 500 to enable retrieval of biometric event data such as flow cytometry event data.

[0100] Display device 506 may be configured to receive display data from processor 500. The display data may include a plot of biometric event data and gates that delineate the contours of the plot segments. Display device 506 may be further configured to modify the information presented in accordance with input received from processor 500 in conjunction with input from flow cytometer 502, memory device 504, keyboard 508, and / or mouse 510.

[0101] Method for Analyzing a Sample Aspects of the present invention also include a method for analyzing a sample. The method of interest includes (a) introducing a particulate sample into a flow cytometer having a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidly coupling to a sheath fluid reservoir, and a fluid resistance unit positioned between the sheath fluid line and the flow cell, and (b) analyzing the sample with the flow cytometer. The fluid resistance unit for use in the subject method is described above and includes an inlet fluidly coupled to the sheath fluid line, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet fluidly coupling the fluid resistance unit to the flow cell.

[0102] In some embodiments, the method also includes changing the resistance state of the fluid resistance unit by switching a valve in the valve line from an open state to a closed state, or from a closed state to an open state. In such embodiments, the practitioner of the method can initiate the switching of one or more valves, for example, by entering a desired flow rate into the processor of the flow cytometer.

[0103] In some cases, the sample analyzed by the method is a biological sample. The term "biological sample" is used in its conventional meaning to refer to a whole organism, plant, fungus, or, in certain cases, a subset of animal tissues, cells, or components that can be found in, for example, blood, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to both a natural organism or a subset of its tissues, as well as, without limitation, for example, a homogenate, lysate, or extract prepared from a subset of a biological organism or its tissues, including plasma, serum, cerebrospinal fluid, lymph fluid, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, and urinary organs, tears, saliva, milk, blood cells, tumors, and organs. A biological sample can be any type of biological tissue, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or a derivative thereof, for example, plasma, tears, urine, semen, etc., and in some cases, the sample is a blood sample including whole blood, such as blood obtained by venipuncture or finger stick (the blood may or may not be combined with any reagents such as preservatives, anticoagulants, etc. prior to the assay).

[0104] In certain embodiments, the sample source is a "mammal" or "mammalian animal", terms that are widely used to describe organisms within the class of mammals, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. These methods can be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is a juvenile, adolescent, or adult. While the present invention can be applied to samples from human subjects, it is to be understood that it can also be practiced with samples from other animal subjects (i.e., "non-human subjects") such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0105] The cells of interest can be targeted in order to be characterized according to various parameters, such as phenotypic characteristics that are identified by attaching a specific fluorescent label to the cells of interest. In some embodiments, the system is configured to deflect analyzed droplets determined to contain the target cells. A variety of cells can be characterized using the methods of the subject matter. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythrocyte cells. Target cells of interest include cells having a convenient cell surface marker or cell surface antigen that can be captured or labeled by a convenient affinity agent or complex thereof. For example, the target cells can include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0106] When practicing the methods of the subject matter, an amount of an initial fluid sample is injected into a flow cytometer. The amount of sample injected into the particle sorting module can vary in the range of 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, including samples of 5 mL to 100 mL.

[0107] The method according to embodiments of the present disclosure includes counting and optionally sorting labeled particles (e.g., target cells) in a sample. When implementing the method of the subject matter, a fluid sample containing the particles is first introduced into the flow nozzle of the system. When exiting the flow nozzle, the particles pass substantially one by one through the sample inspection region, where each of the particles is irradiated with respect to a light source, and, if necessary, measured values of light scattering parameters and, in some cases, fluorescence emission (e.g., measurement values of two or more light scattering parameters and one or more fluorescence emissions) are recorded separately for each particle. Depending on the characteristics of the flow stream being inspected, from 0.001 mm or more of the flow stream, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, can be irradiated with light, including that 1 mm or more of the flow stream can be irradiated with light. In certain embodiments, the method includes irradiating a planar cross-section of the flow stream within the sample inspection region, for example, with a laser (as described above). In other embodiments, the method includes irradiating a predetermined length of the flow stream within the sample inspection region, such as corresponding to the irradiation profile of a diffused laser beam or a lamp.

[0108] In certain embodiments, the method includes irradiating the flow stream at or near the flow cell nozzle orifice. For example, the method can include irradiating the flow stream at a position of about 0.001 mm or more from the nozzle orifice, including 1 mm or more from the nozzle orifice, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more. In certain embodiments, the method includes irradiating the flow stream directly adjacent to the flow cell nozzle orifice.

[0109] In an embodiment of the method, a detector such as a photomultiplier tube (or "PMT") is used to record the light passing through each particle (in certain cases, referred to as forward light scattering), the light reflected orthogonally to the direction of the flow of particles passing through the sensing region (in some cases, referred to as orthogonal or side light scattering), and the fluorescence emitted from the particles when labeled with a fluorescent marker, when the particles pass through the sensing region and are illuminated by an energy source. Each of forward light scattering (or FSC), side scatter (SSC), and fluorescence emission includes distinct parameters for each particle (or "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers.

[0110] The data recorded for each particle is analyzed in real time or stored in a data storage and analysis means such as a computer, if necessary. U.S. Patent No. 4,284,412 describes the configuration and use of a flow cytometer of interest equipped with a single light source, while U.S. Patent No. 4,727,020 describes the configuration and use of a flow cytometer equipped with two light sources.

[0111] In certain embodiments, the particles are detected and uniquely identified by exposing the particles to excitation light and measuring the fluorescence of each particle in one or more detection channels, if necessary. The fluorescence emitted in the detection channels used to identify the particles and the binding complexes associated with the particles can be measured following excitation with a single light source or separately following excitation with distinct light sources. When separate excitation light sources are used to excite the particle labels, the labels can be selected such that all labels are excitable by each of the excitation light sources used.

[0112] The method in certain embodiments also includes obtaining, analyzing, and recording data by a computer or the like, where multiple data channels record data from each detector for light scattering and fluorescence emitted by each particle as each particle passes through the sample inspection region of the particle sorting module. In these embodiments, the analysis includes classifying and counting the particles such that each particle exists as a set of digitized parameter values. The system of the subject can be set to activate with selected parameters to distinguish particles of interest from background and noise. "Activation" refers to a preset threshold for detecting a parameter and can be used as a means for detecting the passage of particles through the light source. Detection of an event exceeding the threshold for the selected parameter activates the acquisition of light scattering and fluorescence data for the particle. For particles or other components in the medium being analyzed that cause a response below the threshold, no data is acquired. The activation parameter can be the detection of forward scattered light caused by a particle passing through the light beam. The flow cytometer then detects and collects the light scattering and fluorescence data of the particles.

[0113] A particular subpopulation of interest is then further analyzed by "gating" based on the data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best separation possible of the subpopulation. This procedure can be performed by plotting forward light scatter (FSC) versus side (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot plot. A subpopulation of particles (i.e., those cells within the gate) is then selected and particles not within the gate are excluded. If desired, the gate can be selected by using a cursor on the computer screen to draw a line around the desired subpopulation. Those particles within the gate are then further analyzed, for example, by plotting other parameters for these particles, such as fluorescence. If desired, the above analysis can be configured to result in a count of the particles of interest in the sample.

[0114] The method of interest may further include employing particles in research, laboratory tests, or therapies. In some embodiments, the method of the subject includes obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the method of the subject includes obtaining cells from a fluid or tissue sample used as a specimen for research or diagnosis of a disease such as cancer. Similarly, the method of the subject includes obtaining cells from a fluid or tissue sample used in therapy. A cell therapy protocol is, for example, a protocol that can prepare viable cell materials including cells and tissues and introduce them into a subject as a therapeutic treatment. Conditions that can be treated by administration of a sample sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, organ damage, and the like.

[0115] A typical cell therapy protocol may include steps of sample collection, cell isolation, genetic recombination, culture, and in vitro proliferation, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of cells into a subject. The protocol can start with collecting viable cells and tissues from a source tissue of a subject to generate a sample of cells and / or tissues. The sample can be collected via any suitable procedure including, for example, administering a cell mobilizing agent to the subject, collecting blood from the subject, removing bone marrow from the subject, and the like. After collecting the sample, cell concentration can occur via several methods including, for example, centrifugation-based methods, filter-based methods, elution, magnetic separation methods, fluorescence-activated cell sorting (FACS), and the like. In some cases, the concentrated cells can be genetically recombined by any convenient method, for example, nuclease-mediated gene editing. The genetically recombined cells can be cultured, activated, and proliferated in vitro. In some cases, the cells are retained and, for example, cryopreserved and stored for future use. For future use, the cells are thawed and then administered to a patient. For example, the cells can be injected into the patient.

[0116] Method for assembling a flow cytometer As discussed above, aspects of the present invention also include a method of assembling a flow cytometer. The method of interest includes fluidly coupling a fluid resistance unit to a flow cell for transporting particles in a flow stream and a sheath fluid line for fluidly coupling to a sheath fluid reservoir. The fluid resistance unit for use in the subject method includes an inlet fluidly coupled to the sheath fluid line, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet fluidly coupling the fluid resistance unit to the flow cell. As discussed above, the valve line of interest includes a series of fluidly coupled valves and the resistor line of interest includes a series of fluidly coupled resistors.

[0117] Embodiments of the method of interest include operably connecting a processor to each valve of the valve line. As discussed above, the processor of interest is configured to change the resistance state of the fluid resistance unit by initiating a switch of a valve in the valve line from an open state to a closed state or from a closed state to an open state. The processor can be operably connected, for example, to a servo mechanism configured to initiate a switch from an open state to a closed state or from a closed state to an open state. The method of the present invention can also include connecting a sheath fluid reservoir to the sheath fluid line. As discussed above, the sheath fluid reservoir can be any suitable reservoir or container (e.g., having a rigid or flexible wall) for holding the sheath fluid. In certain embodiments, the method includes fluidly coupling the sheath fluid reservoir to the input of the flow cell.

[0118] Computer control system Aspects of the present disclosure further include computer control systems, which include one or more computers for full or partial automation. In some embodiments, the system includes a computer having a non-transitory computer-readable storage medium storing a computer program, which, when loaded onto the computer, receives a target or desired flow rate and includes instructions for initiating a change in the resistance state within a fluid resistance unit to achieve the target or desired flow rate. The system may include a display and an operator input device. The operator input device can be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses a memory having instructions stored for performing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, a memory storage device, and an input / output controller, cache memory, a data backup unit, and many other devices. The processor can be a commercially available processor or one of other processors that are available or will become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to cooperate and execute the functions of various computer programs written in various programming languages such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as known in the art. 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 and data management, memory management, and communication control and related services, all according to well-known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0119] 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 resident hard disks or tapes, optical media such as read / write compact disks, flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including compact disk drives, tape drives, or disk drives. Memory storage devices of this type typically read from and / or write to a program storage medium (not shown), such as a compact disk. Any of these program storage media, or others currently in use or that may be developed later, can be considered a computer program product. As will be understood, these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or a program storage device used in conjunction with the memory storage device.

[0120] In some embodiments, the computer program product is described as comprising a computer-usable medium having control logic (a computer software program including program code) stored therein. The control logic, when executed by a processor, causes the computer or processor to perform the functions described herein. In other embodiments, some of the functions are implemented primarily in hardware, using, for example, a hardware state machine. Embodiments of a hardware state machine for performing the functions described herein will be apparent to those of ordinary skill in the relevant art.

[0121] The memory can be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including magnetic or optical disks, or tapes, or RAM, or any other suitable device, either fixed or portable). The processor can include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming can be provided remotely to the processor via a communication channel, or can be pre-stored in a computer program product, such as a memory or any other portable or fixed computer-readable storage medium, using any of those devices together with the memory. For example, a magnetic or optical disk can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product, and algorithms for use in implementing the above methods. The programming according to the present invention can 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 magnetic tapes, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0122] The processor can also have access to a communication channel for communicating with a user at a remote location. A remote location means a location where the user does not have direct contact with the system and where input information is relayed to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0123] In some embodiments, the system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface may include a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface may be configured for wired 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), Bluetooth® communication protocol, and cellular communication such as code division multiple access (CDMA) or global system for mobile communications (GSM) for mobile communication).

[0124] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the subject system and other external devices, such as a computer terminal configured for similar complementary data communication (e.g., in a clinic or hospital environment).

[0125] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, to enable the subject system to communicate with other devices, such as a computer terminal and / or a network, a communicable mobile phone, a personal digital assistant, or any other communication device that the user may use in combination.

[0126] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet protocol (IP) via a mobile phone network, short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0127] In one embodiment, the subject system is configured to wirelessly communicate with a server device via a communication interface using a common standard protocol such as, for example, the 802.11 or Bluetooth® RF protocol, or the IrDA infrared protocol. The server device can be another portable device such as a smartphone, a personal digital assistant (PDA) or a notebook 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 input devices such as buttons, a keyboard, a mouse, or a touch screen.

[0128] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically, with a network or a server device, data stored within the subject system, e.g., within an optional data storage unit, using one or more of the communication protocols and / or mechanisms described above.

[0129] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, locally or remotely. When one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input 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 the system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to the user at a remote location, according to known techniques, e.g., via the Internet, telephone, or satellite network. The presentation of data by the output manager may be implemented according to a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include an Internet URL address so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. One or more platforms present within the system of the subject matter may typically be of any type of known computer platform or type to be developed in the future, but they will generally be of the class of computers referred to as servers. However, they may be mainframe computers, workstations, or other computer types. They may be connected via other communication systems, including any known or future type of cable wiring, or a wireless system, whether networked or not. They may be located in the same place or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems may be adopted on any of the computer platforms. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBMi®, Android™, SGI IRIX®, Oracle Solaris®, etc.

[0130] FIG. 6 depicts a general architecture of an exemplary computing device 600 according to a particular embodiment. The general architecture of the computing device 600 depicted in FIG. 6 includes the arrangement of computer hardware and software components. However, not all of these generally traditional elements necessarily need to be shown in order to provide an effective disclosure. As illustrated, the computing device 600 includes a processing unit 610, a network interface 620, a computer-readable media drive 630, an input / output device interface 640, a display 650, and an input device 660, all of which may communicate with each other via a communication bus. The network interface 620 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 610 may receive information and instructions from other computing systems or services via the network. The processing unit 610 may also communicate with a memory 670 and may further provide output information for an optional display 650 via the input / output device interface 640. For example, analysis software (e.g., data analysis software or programs such as FlowJo®) stored as executable instructions in the non-transitory memory of an analysis system may be capable of displaying flow cytometry event data to a user. The input / output device interface 640 may also receive input from an optional input device 660 such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device.

[0131] Memory 670 may contain computer program instructions that the processing unit 610 executes in sequence to implement one or more embodiments (in some embodiments, grouped as modules or components). Memory 670 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 670 may store an operating system 672 that provides computer program instructions for use by the processing unit 610 in the general management and operation of computing device 600. Data may be stored in data storage device 690. Memory 670 may further contain computer program instructions and other information for implementing aspects of the present disclosure.

[0132] Usefulness The subject fluid resistance units, flow cytometers, and methods find use in a variety of applications where it is desirable to analyze components in a sample within a fluid medium. The present invention finds use particularly in applications where it is desirable to improve sample flow rate control in a vacuum-driven fluidics system. For example, the fluid resistance units, flow cytometers, and methods of the present invention can be employed to increase the number of resistance states as compared to previously employed mechanisms for generating resistance in vacuum-driven fluidics.

[0133] Embodiments of the present invention find use in applications where cells prepared from a biological sample may be desired for use in research, laboratory testing, or therapy. In some embodiments, the subject methods and devices may facilitate obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods and systems facilitate obtaining cells from a fluid or tissue sample that is used as a specimen for research or diagnosis of a disease such as cancer. Similarly, the subject methods and systems may facilitate obtaining cells from a fluid or tissue sample used in therapy.

[0134] Kit Aspects of the present disclosure further include a kit. The kit of interest includes a fluid resistance unit. The fluid resistance unit for use in the subject kit is as described above and includes a plurality of valves and a plurality of resistors, each resistor in the plurality of resistors being fluidly coupled to a different valve in the plurality of valves and located at the same location as the valve. In one embodiment, the fluid resistance unit includes an inlet for receiving fluid, a valve line fluidly coupled to the inlet, a resistor line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet for discharging fluid.

[0135] In addition to the above components, the subject kit may further include, for example, instructions (in some embodiments) for implementing the fluid resistance unit of the present invention. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as a piece of paper on which the information is printed, within the package of the kit, within an attached document, etc. Yet another form of these instructions is a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Yet another form of these instructions that may be present is a website address that may be used via the Internet to access the information at a remote site.

[0136] The following examples are presented by way of illustration and not by way of limitation.

[0137] Experiment A fluid resistance unit was constructed according to a series of embodiments of FIG. 1A. FIG. 7B depicts the specifications of the constructed fluid resistance unit. As shown in FIG. 7B, the resistors in the resistor line have a constant inner diameter of 0.030 inches (0.0762 cm) and continuously increasing lengths. Resistor R1 has a length of 2 inches (5.08 cm), resistor R2 has a length of 4 inches (10.16 cm), resistor R3 has a length of 8 inches (20.32 cm), and resistor R4 has a length of 16 inches (40.64 cm). The fluid resistance unit includes four resistor-valve pairs and thus contains 2 = 16 resistance states. The fluid was subsequently driven through the fluid resistance unit in each of the 16 resistance states, in order from the highest level of resistance to the lowest level of resistance. At the highest level of resistance, all valves are closed. At the second highest level of resistance, all valves except V1 are closed. At the third highest level of resistance, all valves except V2 are closed, and thus 16 resistance states are realized. Subsequently, the sample fluid flow rate (μL / min) was measured at each of the 16 resistance states. As shown in FIG. 7A, the highest level of resistance produced the highest flow rate. The flow rate decreased as the resistance decreased. As shown on the right side of FIG. 7A, the flow rates achieved using previous mechanisms for generating fluid resistance are added for comparison. The conventional mechanisms shown allow only three sample flow rates, while the fluid resistance unit of the present invention allows 16 sample flow rates.

[0138] Notwithstanding the appended claims, the present disclosure also is defined by the following clauses. 1. A flow cytometer comprising a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidly coupling to a sheath fluid reservoir, and a fluid resistance unit positioned between the sheath fluid line and the flow cell, the fluid resistance unit comprising an inlet fluidly coupled to the sheath fluid line, A valve line fluidly coupled to an inlet and comprising a series of fluidly coupled valves, A resistor line fluidly coupled to the inlet and comprising a series of fluidly coupled resistors, A plurality of connectors fluidly coupling the valve line to the resistor line, A flow cytometer comprising an outlet fluidly coupling a fluid resistance unit to a flow cell.

[0139] 2. The flow cytometer according to clause 1, wherein the valve line comprises 2 to 6 valves.

[0140] 3. The flow cytometer according to clause 1 or 2, wherein the resistor line comprises 2 to 6 resistors.

[0141] 4. The flow cytometer according to any one of the preceding clauses, wherein the fluid resistance unit comprises an equal number of resistors and valves.

[0142] 5. The flow cytometer according to clause 4, wherein the fluid resistance unit comprises x 2 resistive states, where x is the number of valves.

[0143] 6. The flow cytometer according to any one of the preceding clauses, wherein each valve of the valve line is individually switchable between an open position where passage of fluid through the valve is unimpeded and a closed position where passage of fluid through the valve is impeded.

[0144] 7. The flow cytometer according to clause 6, wherein passage of fluid through the valve is partially impeded in the closed position.

[0145] 8. The flow cytometer according to clause 6, wherein passage of fluid through the valve is completely impeded in the closed position.

[0146] 9. The flow cytometer according to any one of clauses 6 to 8, further comprising a processor operably connected to each valve of the valve line, the processor being configured to change the resistance state of the fluid resistance unit by initiating a switch of a valve in the valve line from an open state to a closed state or from a closed state to an open state.

[0147] 10. The flow cytometer according to any one of the preceding clauses, wherein each resistor in the resistor line has an inner diameter in the range of 0.025 cm to 1.25 cm.

[0148] 11. The flow cytometer according to clause 10, wherein the inner diameter of the resistor in the resistor line increases continuously.

[0149] 12. The flow cytometer according to clause 10, wherein the inner diameter of the resistor in the resistor line is constant.

[0150] 13. The flow cytometer according to any one of the preceding clauses, wherein each resistor in the resistor line has a length in the range of 1.5 cm to 90 cm.

[0151] 14. The flow cytometer according to clause 13, wherein the length of the resistor in the resistor line increases continuously.

[0152] 15. The flow cytometer according to any one of the preceding clauses, wherein the number of connectors in the plurality of connectors is in the range of 2 to 5.

[0153] 16. The flow cytometer according to any one of the preceding clauses, wherein each connector in the plurality of connectors fluidly couples a portion of the valve line located between two valves in a series of valves to a portion of the resistor line located between two resistors in a series of resistors.

[0154] 17. The flow cytometer according to any one of the preceding clauses, wherein the fluid resistance unit consists of a plastic tube.

[0155] 18. The flow resistance unit is the flow cytometer according to any one of the preceding clauses, which consists of a metal tube.

[0156] 19. The flow cytometer according to any one of the preceding clauses, further comprising a light source configured to irradiate the flow cell at an inspection point.

[0157] 20. The flow cytometer according to any one of the preceding clauses, further comprising a detector configured to collect particle-modulated light from the flow cell.

[0158] 21. The flow cytometer according to any one of the preceding clauses, further comprising a sheath fluid reservoir.

[0159] 22. A method for analyzing a sample, comprising: (a) introducing a particulate sample into a flow cytometer; and (b) analyzing the sample by flow cytometry, wherein the flow cytometer comprises: a flow cell for transporting particles in a flow stream; a sheath fluid line for fluidly coupling to a sheath fluid reservoir; a flow resistance unit positioned between the sheath fluid line and the flow cell, wherein the flow resistance unit comprises: an inlet fluidly coupled to the sheath fluid line; a valve line fluidly coupled to the inlet and comprising a series of fluidly coupled valves; a resistor line fluidly coupled to the inlet and comprising a series of fluidly coupled resistors; a plurality of connectors fluidly coupling the valve line to the resistor line; and an outlet fluidly coupling the flow resistance unit to the flow cell.

[0160] 23. The method according to clause 22, wherein the valve line comprises 2 to 6 valves.

[0161] 24. The resistor line is the method according to clause 22 or 23, comprising 2 to 6 resistors.

[0162] 25. The fluid resistance unit is the method according to clause 22 or 24, comprising an equal number of resistors and valves.

[0163] 26. The fluid resistance unit includes x 2 resistive states, where x is the number of valves, according to the method of clause 25.

[0164] 27. Each valve of the valve line is individually switchable between an open position where the passage of fluid through the valve is not impeded and a closed position where the passage of fluid through the valve is impeded, according to the method of any one of clauses 22 to 26.

[0165] 28. The passage of fluid through the valve is partially impeded in the closed position, according to the method of clause 27.

[0166] 29. The passage of fluid through the valve is completely impeded in the closed position, according to the method of clause 27.

[0167] 30. The method further includes changing the resistance state of the fluid resistance unit by switching the valve of the valve line from the open state to the closed state, or from the closed state to the open state, according to any one of clauses 27 to 29.

[0168] 31. Each resistor of the resistor line has an inner diameter in the range of 0.025 cm to 1.25 cm, according to the method of any one of clauses 22 to 30.

[0169] 32. The inner diameter of the resistors of the resistor line increases continuously, according to the method of clause 31.

[0170] 33. The inner diameter of the resistors of the resistor line is constant, according to the method of clause 31.

[0171] 34. Each resistor of the resistor line has a length in the range of 1.5 cm to 90 cm, and is made by the method described in any one of clauses 22 to 33.

[0172] 35. The length of the resistor in the resistor line increases continuously, and is made by the method described in clause 34.

[0173] 36. The number of connectors in the plurality of connectors is in the range of 2 to 5, and is made by the method described in any one of clauses 22 to 35.

[0174] 37. Each connector in the plurality of connectors fluidly couples a portion of the valve line located between two valves in a series of valves to a portion of the resistor line located between two resistors in a series of resistors, and is made by the method described in any one of clauses 22 to 36.

[0175] 38. The fluid resistance unit is made of a plastic tube, and is made by the method described in any one of clauses 22 to 37.

[0176] 39. The fluid resistance unit is made of a metal tube, and is made by the method described in any one of clauses 22 to 38.

[0177] 40. The sample is a biological sample, and is made by the method described in any one of clauses 22 to 39.

[0178] 41. The sample contains cells, and is made by the method described in any one of clauses 22 to 40.

[0179] 42. The flow cytometer further includes a sheath fluid reservoir fluidly coupled to the sheath fluid line, and is made by the method described in any one of clauses 22 to 41.

[0180] 43. A fluid resistance unit, an inlet for receiving fluid, a valve line fluidly coupled to the inlet and having a series of fluidly coupled valves, a resistor line fluidly coupled to the inlet and having a series of fluidly coupled resistors, A plurality of connectors fluidly coupling a valve line to a resistor line, and an outlet for discharging fluid, a fluid resistance unit.

[0181] 44. The valve line includes 2 to 6 valves, the fluid resistance unit according to clause 43.

[0182] 45. The valve line includes 4 valves, the fluid resistance unit according to clause 44.

[0183] 46. The resistor line includes 2 to 6 resistors, the fluid resistance unit according to any one of clauses 43 to 45.

[0184] 47. The resistor line includes 4 resistors, the fluid resistance unit according to clause 46.

[0185] 48. The fluid resistance unit includes an equal number of resistors and valves, the fluid resistance unit according to any one of clauses 43 to 47.

[0186] 49. The fluid resistance unit includes x 2 resistive states, where x is the number of valves, the fluid resistance unit according to clause 48.

[0187] 50. Each valve of the valve line is individually switchable between an open position where the passage of fluid through the valve is unobstructed and a closed position where the passage of fluid through the valve is obstructed, the fluid resistance unit according to any one of clauses 43 to 49.

[0188] 51. The passage of fluid through the valve is partially obstructed in the closed position, the fluid resistance unit according to clause 50.

[0189] 52. The passage of fluid through the valve is completely obstructed in the closed position, the fluid resistance unit according to clause 50.

[0190] 53. Each resistor in the resistor line is a fluid resistance unit according to any one of clauses 43 to 52, having an inner diameter in the range of 0.025 cm to 1.25 cm.

[0191] 54. The inner diameter of the resistor in the resistor line is continuously increasing, and it is a fluid resistance unit according to clause 53.

[0192] 55. The inner diameter of the resistor in the resistor line is constant, and it is a fluid resistance unit according to clause 53.

[0193] 56. Each resistor in the resistor line has a length in the range of 1.5 cm to 90 cm, and it is a fluid resistance unit according to any one of clauses 43 to 55.

[0194] 57. The length of the resistor in the resistor line is continuously increasing, and it is a fluid resistance unit according to clause 56.

[0195] 58. The number of connectors in the plurality of connectors is in the range of 2 to 5, and it is a fluid resistance unit according to any one of clauses 43 to 57.

[0196] 59. The fluid resistance unit includes three connectors, and it is a fluid resistance unit according to clause 58.

[0197] 60. Each connector in the plurality of connectors fluidly couples a portion of the valve line located between two valves in a series of valves to a portion of the resistor line located between two resistors in a series of resistors, and it is a fluid resistance unit according to any one of clauses 43 to 59.

[0198] 61. The fluid resistance unit is made of a plastic tube, and it is a fluid resistance unit according to any one of clauses 43 to 60.

[0199] 62. The fluid resistance unit is made of a metal tube, and it is a fluid resistance unit according to any one of clauses 43 to 61.

[0200] 63. A method of assembling a flow cytometer, comprising fluidly coupling a fluid resistance unit to a flow cell for transporting particles in a flow stream, and a sheath fluid line for fluidly coupling to a sheath fluid reservoir wherein the fluid resistance unit comprises an inlet for receiving fluid, a valve line fluidly coupled to the inlet and comprising a series of fluidly coupled valves, a resistor line fluidly coupled to the inlet and comprising a series of fluidly coupled resistors, a plurality of connectors fluidly coupling the valve line to the resistor line, and an outlet for discharging fluid. A method.

[0201] 64. The method according to clause 63, wherein the valve line comprises 2 to 6 valves.

[0202] 65. The method according to clause 63 or 64, wherein the resistor line comprises 2 to 6 resistors.

[0203] 66. The method according to clause 63 or 65, wherein the fluid resistance unit comprises an equal number of resistors and valves.

[0204] 67. The method according to clause 66, wherein the fluid resistance unit comprises x 2 resistance states, where x is the number of valves.

[0205] 68. The method according to any one of clauses 63 to 67, wherein each valve in the valve line is individually switchable between an open position where passage of fluid through the valve is unobstructed and a closed position where passage of fluid through the valve is obstructed.

[0206] 69. The method according to clause 68, wherein passage of fluid through the valve is partially obstructed in the closed position.

[0207] 70. The method according to clause 68, wherein the passage of fluid through the valve is completely blocked in the closed position.

[0208] 71. The method according to any one of clauses 68 to 70, further comprising operably connecting a processor to each valve of the valve line, the processor being configured to change the resistance state of the fluid resistance unit by initiating a switch from the open state to the closed state or from the closed state to the open state of the valve in the valve line.

[0209] 72. The method according to any one of clauses 63 to 71, wherein each resistor of the resistor line has an inner diameter in the range of 0.025 cm to 1.25 cm.

[0210] 73. The method according to clause 72, wherein the inner diameter of the resistor in the resistor line increases continuously.

[0211] 74. The method according to clause 72, wherein the inner diameter of the resistor in the resistor line is constant.

[0212] 75. The method according to any one of clauses 63 to 74, wherein each resistor of the resistor line has a length in the range of 1.5 cm to 90 cm.

[0213] 76. The method according to clause 75, wherein the length of the resistor in the resistor line increases continuously.

[0214] 77. The method according to any one of clauses 63 to 76, wherein the number of connectors in the plurality of connectors is in the range of 2 to 5.

[0215] 78. The method according to any one of clauses 63 to 77, wherein each connector in the plurality of connectors fluidly couples a portion of the valve line located between two valves in a series of valves to a portion of the resistor line located between two resistors in a series of resistors.

[0216] 79. The fluid resistance unit is a method according to any one of clauses 63 to 78, which consists of a plastic tube.

[0217] 80. The fluid resistance unit is a method according to any one of clauses 63 to 79, which consists of a metal tube.

[0218] 81. The method according to any one of clauses 63 to 80, further comprising fluidly connecting a sheath fluid reservoir to a sheath fluid line.

[0219] 82. A fluid resistance unit, comprising a plurality of valves and, a plurality of resistors, wherein each resistor among the plurality of resistors is fluidly coupled to a different valve among the plurality of valves and is located at the same location as the valve.

[0220] 83. The fluid resistance unit according to clause 82, comprising 2 to 6 valves.

[0221] 84. The fluid resistance unit according to clause 83, comprising 4 valves.

[0222] 85. The fluid resistance unit according to any one of clauses 82 to 84, comprising 2 to 6 resistors.

[0223] 86. The fluid resistance unit according to clause 85, comprising 4 resistors.

[0224] 87. The fluid resistance unit according to any one of clauses 82 to 86, comprising an equal number of resistors and valves.

[0225] 88. The fluid resistance unit according to clause 87, comprising x 2 resistive states, where x is the number of valves.

[0226] 89. Each valve in the plurality of valves is individually switchable between an open position where the passage of fluid through the valve is not obstructed and a closed position where the passage of fluid through the valve is obstructed, according to any one of clauses 82 to 88. The fluid resistance unit according to any one of clauses 82 to 88.

[0227] 90. The passage of fluid through the valve is partially obstructed in the closed position, according to the fluid resistance unit of clause 89.

[0228] 91. The passage of fluid through the valve is completely obstructed in the closed position, according to the fluid resistance unit of clause 89.

[0229] 92. Each resistor in the resistor line has an inner diameter in the range of 0.025 cm to 1.25 cm, according to any one of clauses 82 to 91. The fluid resistance unit according to any one of clauses 82 to 91.

[0230] 93. The inner diameter of the resistor in the resistor line is constant, according to the fluid resistance unit of clause 92.

[0231] 94. Each resistor in the resistor line has a length in the range of 1.5 cm to 90 cm, according to any one of clauses 82 to 93. The fluid resistance unit according to any one of clauses 82 to 93.

[0232] 95. The fluid resistance unit is made of a plastic tube, according to any one of clauses 82 to 94. The fluid resistance unit according to any one of clauses 82 to 94.

[0233] 96. The fluid resistance unit is made of a metal tube, according to any one of clauses 82 to 94. The fluid resistance unit according to any one of clauses 82 to 94.

[0234] The above invention has been described in some detail by way of illustration and example for the purpose of clear understanding. However, it will be readily apparent to those skilled in the art that, in light of the teachings of the present invention, without departing from the spirit or scope of the appended claims, some changes and modifications can be made to those inventions.

[0235] Accordingly, the foregoing merely illustrates the principles of the present invention. Those skilled in the art will appreciate that, although not explicitly described or illustrated herein, various modifications that embody the principles of the present invention and fall within the spirit and scope of the present invention can be devised. Further, all of the examples and conditional terms listed herein are primarily intended to assist the reader in understanding the principles of the present invention and the concepts provided by the inventors to advance the art, and are not to be construed as being limited to such specifically listed examples and conditions. Additionally, all descriptions in this specification, including those listing the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Further, nothing disclosed herein is intended to be dedicated to the public whether or not such disclosure is explicitly recited in the claims.

[0236] Accordingly, it is not intended that the scope of the present invention be limited to the exemplary embodiments illustrated and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is expressly defined as being cited for a limitation in a claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim, and 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is not cited when such exact phrase is not used in a claim limitation.

[0237] Cross - Reference to Related Applications Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 349,367, filed on June 6, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

Claims

1. A flow cytometer, comprising: a flow cell for transporting particles in a flow stream; a sheath fluid line for fluidly coupling to a sheath fluid reservoir; a fluid resistance unit positioned between the sheath fluid line and the flow cell, wherein the fluid resistance unit comprises: an inlet fluidly coupled to the sheath fluid line; a valve line fluidly coupled to the inlet and comprising a series of fluidly coupled valves; a resistor line fluidly coupled to the inlet and comprising a series of fluidly coupled resistors; a plurality of connectors fluidly coupling the valve line to the resistor line; and an outlet fluidly coupling the fluid resistance unit to the flow cell.

2. The flow cytometer according to claim 1, wherein the valve line comprises 2 to 6 valves.

3. The flow cytometer according to claim 1 or 2, wherein the resistor line comprises 2 to 6 resistors.

4. The flow cytometer according to any one of the preceding claims, wherein the fluid resistance unit comprises an equal number of resistors and valves.

5. The fluid resistance unit includes x ２ resistance states, where x is the number of the valves, the flow cytometer according to claim 4.

6. The flow cytometer according to any one of the preceding claims, wherein each valve in the valve line is individually switchable between an open position where passage of fluid through the valve is unobstructed and a closed position where passage of fluid through the valve is obstructed.

7. The flow cytometer according to claim 6, further comprising a processor operably connected to each valve in the valve line, the processor configured to change the resistance state of the fluid resistance unit by initiating the switching of a valve in the valve line from an open state to a closed state or from a closed state to an open state.

8. The flow cytometer according to any one of the preceding claims, wherein each connector in the plurality of connectors fluidly couples a portion of the valve line located between two valves in the series of valves to a portion of the resistor line located between two resistors in the series of resistors.

9. The flow cytometer according to any one of the preceding claims, further comprising a light source configured to irradiate the flow cell at an inspection point.

10. The flow cytometer according to any one of the preceding claims, further comprising a detector configured to collect particle-modulated light from the flow cell. **Claim 11** The flow cytometer according to any one of the preceding claims, further comprising a sheath fluid reservoir. **Claim 12** A method for analyzing a sample, comprising: (a) introducing a particulate sample into a flow cytometer; (b) analyzing the sample by flow cytometry, wherein the flow cytometer comprises: a flow cell for transporting particles in a flow stream; a sheath fluid line for fluidly coupling to a sheath fluid reservoir; a fluid resistance unit positioned between the sheath fluid line and the flow cell, wherein the fluid resistance unit comprises: an inlet fluidly coupled to the sheath fluid line; a valve line fluidly coupled to the inlet and comprising a series of fluidly coupled valves; a resistor line fluidly coupled to the inlet and comprising a series of fluidly coupled resistors; a plurality of connectors fluidly coupling the valve line to the resistor line; and an outlet fluidly coupling the fluid resistance unit to the flow cell. **Claim 13** A fluid resistance unit comprising: an inlet for receiving fluid; a valve line fluidly coupled to the inlet and comprising a series of fluidly coupled valves; a resistor line fluidly coupled to the inlet and comprising a series of fluidly coupled resistors; a plurality of connectors fluidly coupling the valve line to the resistor line; and an outlet for discharging fluid. **Claim 14** A method of assembling a flow cytometer, comprising fluidly coupling a fluid resistance unit to a flow cell for transporting particles in a flow stream and a sheath fluid line for fluidly coupling to a sheath fluid reservoir, wherein the fluid resistance unit comprises: an inlet for receiving fluid; a valve line fluidly coupled to the inlet and comprising a series of fluidly coupled valves; a resistor line fluidly coupled to the inlet and comprising a series of fluidly coupled resistors; a plurality of connectors fluidly coupling the valve line to the resistor line; and an outlet for discharging fluid. **Claim 15** A fluid resistance unit comprising a plurality of valves and a plurality of resistors. **Claim 16** A fluid resistance unit comprising: a plurality of valves; and a plurality of resistors. Each resistor in the plurality of resistors is a fluid resistance unit that is fluidly coupled to different valves in the plurality of valves and is located at the same location as the valve.

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