Flow cytometry system with stepper flow control valve

Abstract

Systems, methods, and devices for flow cytometry are provided. In one example, a flow cytometry system includes a dual laser device and dual scattering channels to measure the velocity of particles within a core stream of sample fluid. The total flow rate of the sample fluid and the sheath fluid surrounding the sample fluid is controlled and therefore kept constant by a feedback control system that controls a vacuum pump based on the differential pressure across the ends of the flow channels within the flow cell. A stepper flow control valve is disclosed that applies physical fluidic resistance to the flow of sheath fluid within the flow cytometer. The physical fluidic resistance regulates the flow rate of the sheath fluid, thereby regulating the flow rate of the sample fluid, in the flow cytometer.

Description

[Technical Field]

[0001] [Related Applications] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 424,464, filed November 19, 2016, by David Vrane et al., entitled "FLOW CYTOMETRY SYSTEM AND STEPPER MOTOR PINCH VALVE THEREFOR." This patent application also claims the benefit of U.S. Provisional Patent Application No. 62 / 517,147, filed June 8, 2017, by David Vrane, entitled "LINEAR RESISTANCE STEPPER VALVE FOR FLOW CYTOMETRY SYSTEM."

[0002] FIELD OF THE INVENTION Embodiments of the present invention relate generally to flow cytometry systems. [Background technology]

[0003] Flow cytometry involves the optical measurement of cells or particles of a test sample carried in a fluid stream, and the collection of instruments that accomplish this task is known as a flow cytometer.

[0004] Control of the flow fluid with the test sample in a flow cytometer is important for accurately analyzing the type and quantity of cells or particles in the test sample. If the velocity of the fluid flow is variable (e.g., too low or too high around a typical value), the identity of the cells or particles in the test sample may be inaccurately conveyed. Furthermore, overly complicated fluid control systems using additional control devices may be unreliable. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, it is desirable to improve the flow control system in a flow cytometer. [Means for solving the problem]

[0006] Embodiments are summarized by the claims. Briefly, however, systems, methods, and apparatus for flow cytometry fluidics in a flow cytometer are described. The flow cytometry system includes a dual laser device and dual scattering channels for measuring particle velocity within a core stream of a sample fluid. The first scattering channel detects first light scattering generated by particles passing through a first laser beam as they flow through the sample fluid. The second scattering channel detects second light scattering generated by particles passing through a second laser beam, the first and second laser beams being separated by a distance (L).

[0007] The flow cytometry system also includes a stepper motor-regulated valve (e.g., a linear resistance stepper motor-regulated valve) to control the ratio of sheath flow rate and sample flow rate in the flow path. The stepper motor-regulated valve applies a physical fluidic resistance to the flow of sheath fluid. The physical fluidic resistance regulates the flow rate of the sheath fluid, thereby regulating the flow rate of the sample fluid. The total flow rate of the sample fluid and the sheath fluid surrounding the sample fluid is controlled and consequently kept constant by a feedback control system that controls a vacuum pump based on the differential pressure across the flow path in the flow cell. Particle velocity in the flow path is a function of the total flow rate. [Brief explanation of the drawings]

[0008] Various embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings.

[0009] [Figure 1] Figure 1 is a basic conceptual diagram of a flow cytometer system. [Figure 2A] FIG. 2A is a schematic diagram of an exemplary flow cell such as the flow cell included in the fluidics system of FIG. [Figure 2B] FIG. 2B is a close-up view of fluid flow within the flow channel for the flow cell shown in FIG. 2A. [Figure 3A] FIG. 3A is a conceptual diagram of the interaction between a particle and a laser beam to form a light pulse. [Figure 3B] FIG. 3B is a graph illustrating the characteristics of a light pulse formed by the interaction between the particle shown in FIG. 3A and a laser beam. [Figure 4A] FIG. 4A is a conceptual diagram showing flow development in a channel. [Figure 4B] FIG. 4B is a diagram showing a cross-sectional view of the channel shown in FIG. 4A. [Figure 5] FIG. 5 is a basic conceptual diagram of the fluidics system of FIG. [Figure 6] FIG. 6 is a schematic diagram of a flow cytometry system with a dual laser device and dual scattering channels. [Figure 7] FIG. 7 is a schematic diagram of the fluidics system of FIG. [Figure 8] FIG. 8 is a diagram of an electrical circuit that models the main fluidic flows within the fluidic system. [Figure 9A] FIG. 9A is a side cutaway view of a stepper pinch valve. [Figure 9B] FIG. 9B is a front cutaway view of a stepper pinch valve. [Figure 9C] FIG. 9C is a perspective view of a stepper pinch valve. [Figure 10A] FIG. 10A is a block diagram showing further details of the fluidics system associated with the emission optics. [Figure 10B] FIG. 10B is a block diagram showing further details of the acquisition system associated with FIG. 10A. [Figure 10C] FIG. 10C is a block diagram of a peak sampling circuit that determines a timestamp at the peak of scattered light in the dual scattering channel. [Figure 10D] FIG. 10D is a waveform diagram illustrating digital sampling of the scattered light waveform to determine the peak amplitude and the timestamp associated with that peak amplitude. [Figure 11] FIG. 11 is a flow chart of an exemplary method for controlling vacuum relief in a fluidics system. [Figure 12] FIG. 12 is a flow chart of an exemplary method for controlling vacuum pump feedback in a fluidics system. [Figure 13] FIG. 13 is a diagram illustrating a fluidics system similar to that of FIG. [Figure 14] FIG. 14 is a schematic diagram of a fluidics system for a flow cytometer, including a linear resistance stepper valve. [Figure 15] FIG. 15 is a functional diagram of a linear resistance stepper valve. [Figure 16] FIG. 16 is an exemplary data spreadsheet for parameters of the linear resistance stepper valve of FIG. 15 for the fluidics system of FIG. [Figure 17A] FIG. 17A is an alternate view of one embodiment of a linear resistance stepper valve. [Figure 17B] FIG. 17B is an alternate view of an embodiment of a linear resistance stepper valve. [Figure 18] FIG. 18 is an exploded view of the linear resistance stepper valve shown in FIGS. 17A and 17B.

[0010] It will be appreciated that some or all of the figures are for illustrative purposes and do not necessarily depict the actual relative size or location of the elements shown. The figures are provided to illustrate one or more embodiments with the express understanding that they will not be used to limit the scope or meaning of the claims. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the following detailed description of the embodiments, numerous specific details are set forth. However, it will be apparent to those skilled in the art that the embodiments may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail as not to unnecessarily obscure aspects of the embodiments.

[0012] Systems, methods, and devices are provided for flow cytometry fluidics. The system includes a dual laser device and dual scattering channels to measure time differences and calculate particle velocities in a core stream of sample fluid. The system also includes stepper motor-regulated flow control valves to control the ratio of sheath flow rate and sample flow rate in the flow path. The total flow rate of the sample fluid and the sheath fluid surrounding the sample fluid is controlled and thus kept constant by a feedback control system that controls a vacuum pump based on the differential pressure across the flow path in the flow cell. Details of the system, method, and device are further described with reference to the figures.

[0013] The scattered light from the particles is not fluorescence emitted by markers attached to the particles. Dual side scatter channels (SSC) are described herein as receiving scattered light from particles flowing through a laser beam. However, scattered light can be received at many angles from the particles. The dual side scatter channels (SSC) described herein can alternatively be off-angle scatter channels, forward scatter channels, backscatter channels, or combinations thereof that receive scattered light from the particles at various angles. Thus, various devices for collecting scattered light at various angles are collectively and more broadly referred to herein as scatter channels.

[0014] 1 is a basic conceptual diagram of a flow cytometer system 100. The five major subsystems of the flow cytometer system 100 include an excitation optics system 102, a fluidics system 104, an emission optics system 106, an acquisition system 108, and an analysis system 110. Generally, a "system" includes hardware devices, software devices, or a combination thereof.

[0015] The excitation optics system 102 includes, for example, a laser device 112, an optical element 114, an optical element 116, and an optical element 118. Exemplary optical elements include an optical prism and an optical lens. The excitation optics system 102 illuminates an optical interrogation region 120. The fluidics system 104 transports a fluid sample 122 through the optical interrogation region 120. The emission optics system 106 includes, for example, an optical element 130 and optical detectors SSC, FL1, FL2, FL3, FL4, and FL5. The emission optics system 106 collects photons emitted or scattered from particles passing through it. The emission optics system 106 focuses these photons onto optical detectors SSC, FL1, FL2, FL3, FL4, and FL5. The optical detector SSC is a side scatter channel. Optical detectors FL1, FL2, FL3, FL4, and FL5 are fluorescence detectors and may include bandpass or longpass filters to detect particle fluorescence wavelengths. Each optical detector converts photons into electrical pulses and sends the electrical pulses to acquisition system 108. Acquisition system 108 processes and prepares these signals for analysis in analysis system 110. Various embodiments of flow cytometer 100 may be commercially available.

[0016] Figure 2A is a schematic diagram of an exemplary flow cell 206 included in the fluidics system 104 of Figure 1. Given the importance of the flow cell 206 to the flow cytometry system 100, an understanding of its intended function allows for practical consideration of how to improve flow cytometer performance.

[0017] Flow cell 206 provides the key fluid dynamic and optical conditions that allow for the collection of optical excitation and emission light from individual cells that sequentially pass through interrogation region 120. In a simplified representation shown in FIG. 2B, flow cell 206 has a transparent flow channel 216 through which sheath fluid 208 and sample fluid 210 pass. In a portion of transparent flow channel 216, a laser beam from a laser strikes particles within sample fluid 210. Scattered light from the particles and / or fluorescence from markers attached to the particles is collected by one or more optical sensors.

[0018] The ideal conditions for the flow channel 216 are: (a) the sample fluid 210 is surrounded by the sheath fluid 208, forming a core annular flow of the sample fluid 210, and (b) the stream of the sample fluid 210 is perfectly centered on the axis of the flow channel 204. If conditions (a) and (b) are met and the sheath-to-sample volume ratio is sufficiently high, particles within the core of the sample fluid 210 will (c) pass through the flow cell 206 in single file. For example, the core stream width 220 is narrow enough (e.g., 20 micrometers) to allow the particles to flow in single file. The sheath fluid 208 helps control the core stream velocity 222 of the sample fluid 210. The latter condition (c) ensures that each particle receives exclusive illumination within the optical interrogation region 120. The process of achieving these flow conditions is called hydrodynamic focusing of the core of the sample fluid 210.

[0019] Hydrodynamic focusing is typically achieved by injecting the sample fluid 210 into the center of the sheath flow 208 within a wide cross-section and then reducing the cross-section to convectively stretch the combined flow. Hydrodynamic focusing differs from acoustic focusing, which focuses particles into the center of a channel by a standing wave, as described in U.S. Patent Application Publication No. 2014 / 0147860, entitled "ACOUSTIC CYTOMETRY METHODS AND PROTOCOLS," filed June 27, 2012, by Kaduchak et al.

[0020] While hydrodynamic focusing can provide conditions that guarantee the interrogation of individual particles, it does not guarantee that measurements made on successive particles will be comparable. To produce comparable results, when considering two identical particles, the system must produce substantially identical optical outputs between the particles. These optical outputs are in the form of pulses.

[0021] 3A shows a conceptual diagram of the interaction between particle 304 and laser beam 302 to form optical pulse 306, as shown in the graph of FIG. 3B. In the graph shown in FIG. 3B, the amplitude (A) of optical pulse 306 depends on the excitation power and the emission characteristics of particle 304 (e.g., the size, shape, and number of dye molecules). However, the width or duration Δt of optical pulse 306 essentially depends on the diameter (D) of particle 304, the height (H) of excitation beam 302, and the velocity (V) of particle 304. This pulse-width relationship can be given by the following equation:

number

[0022] Therefore, two identical pulses traveling through the sample beam must have substantially the same velocity to produce the same pulse width. The particle velocity (V) at the interrogation region 120 (or interrogation point) is essentially determined by the flow behavior within the channel. As a result, the job of the fluidics 104 essentially comes down to ensuring that particle velocity remains as consistent as possible.

[0023] 2A and 2B, the fluidics system 104 is responsible for supplying the sheath fluid 208 and sample fluid 210 to the flow cell 206. The sheath flow rate is typically many times greater than the sample flow rate and therefore dominates the flow behavior in the flow cell 206. As a result, the sheath flow rate dominates the velocity behavior inside the channel and is the most important factor in determining particle velocity 222. The sample flow rate controls the core stream width 220, which is equally important because it controls the range of possible positions a given particle can assume along its velocity profile within the channel.

[0024] The velocity profile 212 inside the channel depends on several factors related to classical fluid mechanics. The convection region of the flow cell hydrodynamically focuses the core annular flow, stretching the sample stream until the particles (ideally) align with one another. Again, this is achieved by accelerating the sample flow 210 through a decreasing cross-sectional width 220. Beyond the convection region, the cross-sectional area of the flow remains constant. The flow enters this constant cross-section channel at the inlet 401 with a uniform velocity profile. The magnitude of the uniform velocity profile is equal to the flow rate divided by the cross-sectional area of the flow, as shown in the following equation:

number

[0025] FIG. 4A is a conceptual diagram illustrating flow development within a channel 216. The channel includes, without limitation, an inlet 401, an outlet 404, and a central axis 412. As the flow moves downstream through the channel 216, the viscous drag of the walls 214 decelerates adjacent water molecules. To maintain a constant flow rate (e.g., to keep mass constant), water molecules at the center of the channel 216 must accelerate to compensate for the decelerating particles near the walls 214. This communication of viscous drag through the fluid bulk continues until the viscous forces balance the drive shaft pressure gradient. Until this balance is reached, the sample core stream 210 continues to accelerate. For a rectangular channel with an aspect ratio near unity, as shown in FIG. 4B, the terminal velocity of the sample core stream 210 is approximately twice its initial uniform velocity at the channel inlet 401.

[0026] The sample core stream 210 approaches terminal velocity within the developing region 402. Within the developing region 402, the core stream accelerates. d ) is the physical distance from the inlet 401 required for the core stream 210 to achieve terminal velocity. As mentioned above, the development length (L d ) depends on viscosity, which in turn depends strongly on temperature. Higher temperatures result in longer growth lengths (L d ) results.

[0027] Developmental length (L d ), in the developed region 404, the core stream velocity in the channel 216 does not change with distance and the flow is considered "fully developed." Fully developed flow can also be temperature independent. However, this requires that the total flow rate through the channel 216 remains constant. The result of flow development is that the velocity profile changes from uniform (uniform velocity profile) at the channel inlet 401 to a constant velocity profile over the development length (L d ) until it develops into a fully parabolic shape (fully developed velocity profile).

[0028] With respect to flow cytometry, several important guidelines arise from knowledge of channel flow hydrodynamics. First, if the total flow rate through the channel 216 changes, the core stream velocity 222 will change. Thus, the particle pulse width (e.g., duration Δt) will change. Therefore, flow rate fluctuations should be minimized to ensure maximum system performance.

[0029] As a second guideline, maximum velocity occurs along the axis 412 of the channel. Therefore, any particle off axis 412 will have a slower velocity than a particle on or closer to axis 412. This means that the same particle will have a different pulse width (e.g., duration Δt) simply depending on its position within the core stream. Therefore, reducing the core stream width 220 (e.g., reducing the sample flow rate) minimizes pulse width variation due to the velocity profile.

[0030] As a third guideline, along the channel axis 412, the velocity increases by a factor of two in the developing region 402, and the length of the developing region 402 is temperature dependent. Therefore, if the interrogation region (or interrogation point) 120 is within the developing region 402, the pulse width (e.g., duration Δt) will vary with temperature. The development length (L d ), the core stream velocity does not change with distance along the channel.

[0031] FIG. 5 is a basic conceptual diagram of the fluidics system 104 of FIG. 1. The basic purpose of the fluidics system 104 is to provide sheath and sample to the flow cell 206 of FIG. 2. FIG. 5 shows the basic elements relevant to this purpose. A sheath straw 516 (e.g., tubing) is inserted into a plenum tank 504. The fluidics system 104 supplies sheath fluid 208 from a sheath tank 626. The fluidics system 104 typically draws sample fluid 210 from a standard laboratory vessel 506. A sample straw 518 (e.g., tubing) is inserted into the sample vessel 506. The fluidics system 104 injects the sample fluid 210 into the core 510 of the sheath flow 208 at the entrance to a contraction region 512 before the combined flow enters a flow path / observation orifice 520. Upon exiting flow path 216 at outlet 404, the combined flow is routed to waste 502 in waste container 508. Various commercial implementations attempt to meet these fluidics objectives.

[0032] Despite the variety of commercially available flow cytometry systems, there remains a need for a flow cytometry system that is flexible yet offers a relatively simple and reliable design, is capable of accessing a wide range of sample vessel types while providing undiminished stability over a wide range of operating conditions.

[0033] Thus, a flow cytometry system is provided that includes a dual laser device and dual scattering channels (e.g., dual side scattering channels—SSC) to measure particle velocity within a core stream of sample fluid. Typical systems measure pressure and then use the measured pressure to maintain a constant pressure and velocity in the system; unfortunately, this is an indirect method of controlling the velocity of sample particles. The system also includes a stepper motor-regulated flow control valve to control the ratio of sheath flow rate and sample flow rate in the flow path. The total flow rate of the sample fluid and the sheath fluid surrounding the sample fluid is controlled and consequently kept constant by a feedback control system that controls a vacuum pump based on the differential pressure across the flow path within the flow cell. Details of the system, method, and apparatus are further described with reference to the figures.

[0034] Referring now to Figure 6, a conceptual diagram of a flow cytometry system 600 is shown. Flow cytometry system 600 has dual laser devices (612, 614) and dual scattering channels (632, 634). Some aspects of flow cytometry system 600 are similar to system 100 of Figure 1. For example, flow cytometry system 600 has five major subsystems, including an excitation optics system 602, a fluidics system 604, an emission optics system 606, an acquisition system 608, and a host computer 610 with an analysis system. The host computer 610 includes a processor having digital mathematical logic and one or more storage devices that store instructions executable by the processor for using the digital mathematical logic to generate digital feedback control signals (e.g., mean particle flow velocity) that can be used to control one or more features of the flow cytometry system 600 (e.g., one or more valves / pumps / motors in the fluid control system 604 for controlling sample flow).

[0035] However, flow cytometry system 600 has important improvements over system 100 of Figure 1. For example, flow cytometry system 600 includes excitation optics 602 with dual laser devices (612, 614); an emission optics subsystem with dual optical components 130A and 130B, dual scatter channels (632, 634), and dual fluorescence channels FL1A-FL5A, FL1B-FL1B for acquisition; and an electronics subsystem 608 with dual analyzers 108A and 108B for parallel analysis. As further described with reference to other figures, the dual scatter channels (632, 634) play an important role in calculating the time delay or velocity of particles within the core stream of sample fluid so that the average time delay or average particle velocity can be determined.

[0036] Particles often do not fluoresce without a marker. Therefore, a fluorescence detector in the fluorescence channel will not detect particles that do not fluoresce. A scattering channel that detects scattered light can detect particles from scattered light whether or not the particles are marked with a marker. Thus, a series of dual lasers 612, 614 can excite particles in the sample stream, and a series of dual scattering channels can be used to detect particles at two points and, given the known distance and time delay between the lasers, determine the time delay or particle velocity between each particle.

[0037] Figure 7 shows a schematic diagram of the fluidics system 604 of Figure 6. The fluidics system 604 has a vacuum-based fluidics architecture with a time-based pressure regulation scheme for particle flow between laser beams (613, 615). The fluidics system 604 also provides continuous sample flow regulation in a vacuum-based system with a sample path that is completely free of transitions, discontinuities, or potentially cell-damaging peristaltic pumps. The fluidics system 604 seeks to maximize reliability and instrument uptime by avoiding the use of peristaltic pumps entirely.

[0038] Fluidics system 604 includes, without limitation, manifold assembly 701, isolation valves V1-V5, pinch valve V6, pressure transducers (e.g., probes) TR1 and TR2, accumulator vessel (vacuum chamber) 702, diaphragm vacuum pump 704, degasser pump 706, sheath fluid 208, sample fluid 210, sample vessel 506, output sensor 714, stepper mounting position 716, check valve 718, degasser 720, flow cell 206, sheath tank 726, plenum tank 504, plenum pump 728, stepper flow control valve 730, flush pump 732, sheath filter 734, waste tank 508, and plate loader 738, flow restrictors 740 and 742, and sheath float sensor 744.

[0039] The following provides an exemplary operating cycle for fluidics system 604 shown in FIG. 7. Upon startup, fluidics system 604 receives feedback from pressure transducers TR1 and TR2. The pressure difference sensed between pressure transducers TR1 and TR2 is continuous during operation, driving vacuum pump 704 to maintain a constant pressure difference between inlet 401 and outlet 404 of flow channel 216 in flow cell 206 shown in FIG. 4 until a minimum value is reached inside accumulator 702. Pressure transducers TR1 and TR2 may be differential pressure transducers DPTR that measure the differential pressure between inlet 401 and outlet 404 of flow channel 216 in flow cell 206. The minimum value is what fluidics system 604, at least initially, considers to be the differential pressure (e.g., setpoint pressure).

[0040] Fluidics system 604 is desirably bubble-free. Therefore, fluidics system 604 has a protocol for opening and closing valves V1-V5 to eliminate gas bubbles. For example, fluidics system 604 opens valve V4, which draws gas bubbles through sheath filter 734. Whenever sheath float sensor 744 in the plenum tank drops below a predetermined low level, plenum pump 728 draws fluid from sheath tank 726. The system is then ready for operation.

[0041] Once the sample vessel 506 is attached, the tube sensor 714 indicates that the fluidics system 604 is ready to lower the system's sample straw 518 into the sample vessel 506. The platter loader 738 includes a stepper motor that lowers the sample straw 518 into the sample vessel 506. When the sample straw 518 reaches the stepper sample injection tube position (SITD) 716, one or more sensors cause the fluidics system 604 to open valves V1 and V3. A regulation system within the fluidics system 604 begins to drive the differential pressure to meet the setpoint pressure.

[0042] In an advantageous embodiment of the fluidics system 604, a vacuum pump 704 evacuates an accumulator vessel 702. The accumulator vessel 702 serves as the driving vacuum source for the system, and a diaphragm maintains this source from a differential pressure reference derived from laser delay measurements made by the acquisition system 608. The accumulator vessel 702 also serves as a pulse attenuator and constant head reference for the outlet 404 of the flow cell 206, thereby isolating the flow cell 206 from head effects associated with the liquid level in the waste tank 508.

[0043] The fluidics system 604 supplies sheath fluid 208 to the flow cell 206 by means of a plenum tank 504. The liquid level in the plenum tank 504 is maintained at a precise level by periodic refilling by a plenum pump 728, which draws sheath from a sheath tank 726. In this way, the plenum tank 504 acts as a constant head reference for the inlet of the flow cell 206, thus isolating the flow cell 206 from head effects associated with the liquid level in the sheath tank 726.

[0044] Manifold assembly 701 contains a network of valves V1-V5 that control fluid flow within fluidics system 604. In the primary sample acquisition mode, valves V1 and V3 are open. In this mode, sheath fluid 208 and sample fluid 210 are simultaneously drawn into flow cell 206 by the vacuum within accumulator 702.

[0045] When valve V2 is closed, transducer TR1 measures the pressure inside flow cell 206 at inlet 401, and transducer TR2 measures the pressure at outlet 404 of flow cell 206, as shown in FIG. 4. Measuring the TR1 pressure through a static line between the flow cell drain port and closed valve V2 ensures that the differential pressure measurement does not include dynamic pressure drops associated with resistance in the sheath inlet port or sheath inlet line, which would otherwise distort the true differential pressure drop of the total flow between channel inlet 401 and channel outlet 404. When valves V1 and V3 are open, transducers TR1 and TR2 (or differential pressure transducer DPTR) measure the differential pressure across the end of channel 216 of flow cell 206. The pressure drop between outlet 404 and inlet 401, the differential pressure across the end of channel 216, is proportional to the total volumetric flow rate, which is proportional to the core stream velocity (assuming a near-constant temperature). See, for example, Equation 7. Operating temperatures are typically between about 15°C and 20°C, above which the viscosity of water can vary by as much as 20%. Volumetric flow rate (including sheath fluid velocity) is inversely proportional to viscosity.

[0046] When the fluidics system 604 is operating, it simultaneously draws the sheath fluid 208 and the sample fluid 210. The fluidics system 604 draws the sheath fluid 208 from the plenum tank 504, passes through the stepper flow control valve 730, through the flush pump 732, through the sheath filter 734, through valve V1, through the degasser 720, and to the flow cell 206. Simultaneously, the fluidics system 604 draws the sample fluid 210 from the sample vessel 506 and to the flow cell 206. Depending on the relative resistance between the sheath straw 516 and the sample straw 518, the fluidics system 604 regulates the relative flow rates between the sheath fluid 208 and the sample fluid 210. See, for example, Equation 3. The fluidics system 604 (e.g., stepper flow control valve 730 and vacuum pump 704) can regulate the flow rate of the sheath fluid 208 to be, for example, 13 milliliters / minute, while the fluidics system 604 regulates the flow rate of the sample fluid 210 to be, for example, 16 microliters / minute. Other flow rates are within the range of the system 600.

[0047] The stepper flow control valve 730 controls the continuously variable sample flow rate of fluid flow through the flow cell 206. The sheath straw 516 is inserted into the plenum tank 504. The sample straw 518 is inserted into the sample vessel 506. Compared to the sample straw 518, the sheath straw 516 has a relatively larger diameter to handle a relatively larger volumetric flow. Conversely, compared to the sheath straw 516, the sample straw 518 has a relatively smaller diameter to handle a relatively smaller volumetric flow. The diameter difference between the sheath straw 516 and the sample straw 518 controls the relative volumetric flow rates at which the sheath fluid 208 and the sample fluid 210 are aspirated from their respective vessels (504 and 506).

[0048] 4, 6, and 7, the vacuum pump 704 receives differential pressure feedback from the acquisition system 608 between the pressures sensed by transducers TR1 and TR2 in the fluidics control system 604. Based on the differential pressure feedback, the vacuum pump 704 is controlled to maintain a constant core stream velocity even as the stepper pinch valves are adjusted to change the sample flow rate. In this way, the total fluid flow rate through the flow cell 206 can remain substantially unchanged. The sheath flow rate changes very little with changes in the stepper pinch valves because the sheath flow rate is much greater than the sample flow rate. For example, to increase the sample flow rate from 10 μl / min to 30 μl / min, the sheath flow rate would only change from 15.99 ml / min to 15.97 ml / min. The stepper flow control valve allows the system to continuously vary the sample flow rate with little or no effect on the sheath flow rate.

[0049] 7 , the stepper flow control valve 730 includes a stepper motor 731 that remotely adjusts the flow resistance of the sheath straw 516, which is typically less than the flow resistance of the sample straw 518. The stepper flow control valve 730 can increase or decrease the flow resistance of the sheath straw 516 while the fluidics system 604 applies approximately the same vacuum pressure to the fluid. Increasing the flow resistance of the sheath straw 516 causes the fluidics system 604 to increase the amount of sample fluid 210 (relative to the sheath fluid 208) drawn from the sample reservoir 506. In contrast, decreasing the flow resistance of the sheath straw 516 causes the fluidics system 604 to decrease the amount of sample fluid 210 (relative to the sheath fluid 208) drawn from the sample reservoir 506. Thus, control of the sheath straw 516 by the stepper flow control valve 730 enables the fluidics system 604 to have continuously variable flow rates (e.g., variable sample flow rate and variable sheath flow rate) while maintaining a constant overall flow and therefore a constant (e.g., stable) particle velocity.

[0050] Controlling the flow rate of the sheath fluid 208 controls the flow rate of the sample fluid 210, including the sample particles therein. Thus, the system 600 can control the sheath volumetric flow rate, and thereby the sample volumetric flow rate and particle velocity. The system 600 performs these controls of pressure and velocity in a closed loop (as opposed to an open loop). Note that typical commercial systems perform open loop corrections, which require stopping undesired system pressurization.

[0051] The stepper flow control valve 730 regulates the flow rate through, for example, flush pump P2, valve V1, degasser 720, and flow cell 206. Valve V2 allows fluid flow to flush the system. Valve V2 is then closed to set up sampling. During the cleaning operation before the next sample is sampled, flush pump P2 pushes sheath fluid through valve V1, degasser 720, and flow cell 206, with valves V2 and V3 closed, to allow backflushing of the sample straw 518. Valve V2 then opens after backflushing is complete to relieve pressure in the flow cell.

[0052] Advantageously, the stepper flow control valve 730 and its stepper motor 731 allow for precise control of the fluid, taking into account that the viscosity of the sample fluid 210 can be highly variable. For example, blood as the sample fluid 210 can have a viscosity that is twice the viscosity of the sheath fluid 208. Note that the volumetric flow rate is inversely proportional to the viscosity.

[0053] With valves V1 and V3 open, fluidics system 604 draws sheath fluid 208 and sample fluid 210 through the top of flow cell 206, through valve V3, and into accumulator 702. Accumulator 702 acts as a waste bucket for the fluidics system. Vacuum pump 704 maintains (e.g., regulates) the vacuum pressure within fluidics system 604. Vacuum pump 704 also pumps waste from accumulator 702 to waste pump tank 508.

[0054] When the sample fluid 210 is flowing at a high flow rate (e.g., the flow resistance of the sheath straw 516 is substantially high), the vacuum of the fluidics system 604 is higher to maintain a differential pressure across the flow cell 206. If the user desires a lower flow rate for the sample fluid 210, the flow resistance of the sheath straw 516 is appropriately reduced. At this point, the vacuum pump 704 is operating at a relatively high vacuum, which forces the fluidics system 604 to flow the sheath fluid 208 through the system at a much higher velocity, thereby flowing particles at a steady velocity in the sample fluid 210. Thus, the fluidics system 604 detects that the differential pressure is too high and opens the pressure relief valve V5, pulsing the valve V5 until the differential setpoint is met. Thus, the valve V5 maintains (e.g., regulates) the pressure within the system 600.

[0055] Restrictors 740 and 742 are always open to fluidics system 604 to allow a partial bleed of fluid into accumulator 702. Therefore, vacuum pump 704 is always pumping at least a small amount, which keeps accumulator 702 free of excess liquid and ensures that accumulator 702 does not fill up.

[0056] The degasser 720 removes gas from the fluidics system 604. Generally, gas is generated whenever a vacuum is applied to a fluid. Therefore, the degasser 720 removes gas from the fluidics system 604 before it reaches the flow cell 206. The degasser pump 706 also serves as the source for the sample injection tube (SIT) flush aspiration vacuum.

[0057] Whenever the fluid 210 to be sampled (e.g., blood) flows through the fluidics system 604, the sample fluid 210 leaves residue in the SIT line. Residue is highly undesirable because it can contaminate subsequent sample fluids. Therefore, the fluidics system 604 achieves SIT flushing via flush pump 732. When the system is finished flowing the sample fluid, it opens valve V1 but does not open valves V2 or V3. The system then activates flush pump P2 so that the fluidics system 604 pressurizes the entire sheath fluid 208 path. Because the sheath fluid 208 has nowhere to go (because valves V2 and V3 are closed), the fluidics system 604 pushes the sheath fluid 208 back through the sample fluid 210 path, thereby rinsing the interior of the SIT path. The degasser pump 706 siphons backflush from the SIT path before it drips onto surfaces below the SIT. This action of the degasser 720 is enabled by the opening of valve V6, which allows fluid to flow from the flow cell 206, along the path of valve V6, through the degasser pump 706, and into the waste tank 508. Such flushing cleans both the inner and outer diameters of the SIT (e.g., blowing into a straw).

[0058] The degasser pump 706 is activated (e.g., turned on) whenever a vacuum switch on the degasser 720 is triggered because the vacuum falls below a predetermined value (e.g., −9 pounds per square inch). The fluidics system 604 draws gas through the check valve 718. In this case, the degasser 720 draws a relatively small amount of gas from the sheath fluid 208. The degasser 720 remains isolated. The check valve prevents the vacuum pressure inside the degasser 720 from being lost while a SIT flush is occurring.

[0059] The degasser 720 degasses the fluid whenever the fluidics system 604 reduces the gas pressure on the fluid. For example, gas forms within the fluid, similar to the gas that forms within a pressurized soda can when it is opened. In this system, the sheath fluid 208 within the plenum tank 504 is at approximately atmospheric pressure, while the pressure within the accumulator 702 is below atmospheric pressure. As the sheath fluid 208 flows along the sheath path, the pressure on the sheath fluid 208 continually decreases. Because of the continually lower pressure, air bubbles tend to form within the sheath fluid 208. This problem is exacerbated if the sheath fluid is pre-aerated. Air bubbles tend to be approximately the same size as particles within the sample fluid 210. These air bubbles introduce background optical noise when the fluidics system 604 attempts to detect and analyze particles within the sample fluid 210. Note that for this reason, many manufacturers do not build flow cytometry systems that operate using a vacuum. To combat the formation of air bubbles within the sheath fluid 208 , the degasser 720 removes air bubbles (eg, gas) from the fluid solution before they can enter the flow cell 206 .

[0060] 8 is a diagram of an electrical circuit 800 that models the primary fluidic flows within fluidic system 604. In FIG. 8, electrical circuit 800 includes, without limitation, a flow cell resistor R FC , variable sheath resistor R SH , sample resistor R SA, pressure transducers TR1 and TR2, and accumulator voltage V.

[0061] Variable sheath resistor R SH and sample resistor R SA is the flow cell resistor R FC The variable sheath resistor R SH and sample resistor R SA models the ratio of sheath fluid to sample fluid entering flow cell 206 at inlet 401. Using the hydrodynamic equivalent of Ohm's law and keeping in mind conservation of mass, the governing equation for flow in this circuit 800 is approximated by the following equation:

number

number

[0062] Equation 3 is analogous to stating that for the vacuum fluidics architecture of the preferred embodiment, the pressure drops across the sheath and sample legs of the primary circuit are equal, apart from the hydrostatic head difference between the liquid levels in the sheath plenum and the sample vessel. Equation 4 is analogous to stating that the total flow rate through the flow cell is equal to the sum of the sheath flow rate and the sample flow rate.

[0063] The sheath flow rate dominates the sample flow rate in terms of the velocity profile for lower sample flow rates, but as the sample flow rate increases, it is technically more accurate to describe things in terms of the total flow rate through the flow cell 206. Substituting equation (4) into equation (3) to eliminate the sheath flow rate yields the following expression for the sample flow rate:

number

[0064] An examination of Equation 5 shows that, for a fixed total flow rate, the sample flow rate can be easily manipulated by changing the sheath path resistance. In the present invention, this is accomplished through the use of a stepper motor-regulated flow control valve, which allows for a continuously variable flow rate in the following range:

number

[0065] Equation 6 shows that if the sheath resistance were infinite, ideally all of the flow would be through the sample path. And if the sheath resistance were zero, ideally any flow through the sample path would be due to the pressure head between the plenum and the sample vessel. In reality, however, the sheath path resistance will always have a non-zero value, and this value will affect the lower limit of the achievable sample flow rate.

[0066] Hydrostatic term ΔP for system performance h It is equally important to recognize the effect of the hydrostatic term ΔP when the sample level is below the plenum level. h The sign of is positive. This means that the flow range is SH This means that for some choice of , the sheath path resistance R can be reduced to allow for a low sample flow rate, ensuring a sufficiently low flow rate to minimize the core stream width. SH can be used to offset a finite value of

[0067] However, if the sample level exceeds the plenum level, the hydrostatic term ΔP h The sign of is negative. In this case, the sample flow rate has a lower positive limit, which is determined by the non-zero sheath path resistance R SHcombined with , it may be too high to achieve a core stream width as narrow as the particle diameter. This case should be avoided because the resolution performance will be limited by the fluidics system due to the higher CV (coefficient of variation).

[0068] 9A-9C show various orientations of a stepper pinch valve 900, which is one embodiment of the stepper flow control valve 730 of FIG. 7. FIG. 9A shows a side cutaway view of the stepper pinch valve 900. FIG. 9B shows a front cutaway view of the stepper pinch valve 900. FIG. 9C shows a perspective view of the stepper pinch valve 900. The stepper pinch valve 900 includes, without limitation, a head 901, a pinch anvil 902, a pinch hammer 903, a hammer guide 904, a nut 905, a lead screw 906, a stepper motor 907, sensors 908 and 909, a tubing passage 910, a home switch 911, a limit switch 912, and a mounting plate 913. Because the stepper flow control valve 730 includes a stepper motor that adjusts the valve and the flow rate, it can also be referred to as a stepper motor-regulated flow control valve. Thus, the stepper pinch valve 900 may also be referred to as a stepper motor adjusted flow pinch valve 900 since it includes a stepper motor 907 that adjusts the valve and the flow rate.

[0069] A stepper motor 907 rotatably drives and couples to a lead screw 906. A nut 905 threads onto and couples to the lead screw 906. The nut 905 is in mechanical communication with a pinch hammer 903, which is in mechanical communication with a pinch anvil 902. Rotational movement of the lead screw 906 generates translational movement of the nut 905. The translational movement of the nut 905 generates translational movement of the pinch hammer 903 into / out of the pinch anvil 902. The translational movement of the pinch hammer 903 changes (e.g., decreases / increases) the spacing (e.g., diameter) of a tubing passage 910. The tubing passage 910 is a concave cavity formed between the pinch hammer 903 and the pinch anvil 902. A sheath straw 516 passes through the tubing passage 910 and can contact the pinch hammer 903 and the pinch anvil 902. Thus, the diameter of the sheath straw 516 varies (e.g., decreases / increases) according to the spacing of the tubing passage 910. The hammer guide 904 prevents the pinch hammer 903 from spinning relative to the motor shaft. Thus, the pinch hammer 903 is moved vertically by the translation of the rotation of the lead screw 906.

[0070] Sensors 908 and 909 are in mechanical communication with the pinch hammer 903 and / or pinch anvil 902. The sensors 908 and 909 detect when the pinch hammer 903 or pinch anvil 902 are at their limits. Upon start-up of the system 600, the system 600 determines the position of the pinch hammer 903 within its range of motion via the sensors 908 and 909. For example, an algorithm in the firmware of the system 600 automatically cycles the pinch hammer 903 in translation to trigger the sensors 908 and 909. The system 600 calibrates the position of the pinch hammer 903. Once the system calibrates the position of the pinch hammer 903, the system 600 is configured to set a value for the pinch hammer 903 (e.g., the diameter of the tubing passage 910) between the translation limits to achieve a specific flow rate.

[0071] The following is an example of open-loop calibration of the pinch hammer 903. The system finds the position of the stepper motor 907 (and therefore the pinch hammer 903) when the sheath fluid 208 flow rate is zero. In open-loop mode, a titration is performed for the stepper value that yields a particular flow rate. For example, assume a stepper motor range of 1 to 1000 steps. The system 600 can determine that step #540 corresponds to a flow rate of 60 milliliters / minute, while step #800 corresponds to a flow rate of 120 milliliters / minute. These stepper values and corresponding flow rates are stored for the system to interpolate other subsequent settings of the stepper motor for desired flow rates. In this example, the flow rate is linearly proportional to the stepper number. This open-loop calibration technique is quite robust and accurate.

[0072] An alternative to using open-loop calibration is to use a feedback device (e.g., a thermal pulse flowmeter) that measures volumetric flow rate. For example, system 600 can receive an input requesting a sample fluid flow rate of 60 microliters / minute. A feedback circuit coupled to the feedback device drives stepper motor 907 to a predetermined position until the flow rate reaches approximately a predetermined rate, such as 60 microliters / minute. Such flow rate calibration can be referred to as closed-loop feedback using a thermal pulse flowmeter.

[0073] 7, the stepper motor-regulated flow control valve 730 represents a significant improvement over discrete resistance flow paths or coiled tubing compression mechanisms (e.g., as found in U.S. Patent No. 8,528,427, filed by Vrane and Norton and entitled "DUAL FEEDBACK VACUUM FLUIDICS FOR A FLOW-TYPE PARTICLE ANALYZER") while maintaining an obstruction-free sample path. Furthermore, this simpler fluidics system 604 is suitable for continuous feedback control of the sample flow rate.

[0074] In another embodiment, a non-invasive flow meter (e.g., a SENSIRION SLI flow meter) in the sample path provides feedback to the stepper position of the adjustable flow control valve. This allows the system to provide true sample flow control without the complexity, expense, or reliability issues associated with syringe drives. Stepper pinch valve 900 is one embodiment of a stepper flow control valve 730 that may be used in the system. Other types and embodiments of flow control valves, such as the linear flow control valves described herein, may be used in the system to control fluid flow.

[0075] 10A and 10B show further details of the fluidics system 604, emission optics 606, and acquisition system 608 shown in FIG. 6. Direct control of the core stream velocity is achieved by utilizing dual laser delay measurements made by the acquisition system 608 based on particle flow within the fluidics system 604. The time delay measurements ΔT between particles passing through the dual lasers can be used as a proxy for particle velocity to generate a feedback control signal for the average time delay that controls the flow rate within the flow channel. Alternatively, with a known distance L between the lasers, the particle velocity can be calculated and used to generate the average particle velocity as a feedback control signal that controls the flow rate within the flow channel. To maintain the average particle velocity at a desired constant value, the vacuum pump can be controlled to control the core stream velocity and maintain the average time delay or average particle velocity constant.

[0076] To utilize the widest bandwidth of emitted light to identify biological particles (e.g., cell lines), fluidics system 604 uses a dual laser device (laser 612, laser 614) that emits two laser beams (laser beam 613, laser beam 615), typically with two excitation laser wavelengths. Dual serial scattering channels (scattering channel 632 and scattering channel 634) are used to detect scattered light from the two laser beams (laser beam 613, laser beam 615) of known separation at different times within the flow channel.

[0077] System 600 utilizes the concept that particles passing through a laser beam (613 or 615) always generate scattered light, but do not always fluoresce. System 600 detects the scattered light from the particles using dual scattering channels (scattering channel 632, scattering channel 634) to calculate particle velocity within the sample fluid. The laser beams (613, 615) are spatially separated by a known, predetermined distance L, as shown in FIG. 10A. The laser separation distance L, as shown in FIG. 10A, can be minimized to the extent possible while limiting crosstalk between the dual lasers 612 and 614 that generate laser beams 613, 615.

[0078] Particle 1006 first flows (e.g., passes) through a first laser beam 613, causing emission optics 606 to generate pulses 1012 that measure the light scattered off the particle by the first laser beam. Particle 1006 continues its flow in the flow channel and then flows (e.g., passes) through a second laser beam 615. Emission optics 606 generates pulses 1014 that measure the light scattered off the particle by the second laser beam.

[0079] The scattering channel 632 detects the pulse 1012 with an optical detector (e.g., a photodetector) and sends pulse information to the acquisition system 608. The pulse information from the first scattering channel 632 can include, among other information, for example, a particle identifier (e.g., particle 1006), a laser identifier (e.g., laser device 612 or laser beam 613), and a first timestamp (e.g., timestamp #1). The second scattering channel 634 detects the second pulse 1014 with an optical detector (e.g., a photodetector) and sends pulse information about the second pulse to the acquisition system 608. The pulse information from the scattering channel 634 can include, among other information, for example, a particle identifier (e.g., particle 1006), a laser identifier (e.g., laser device 614 or laser beam 615), and a timestamp (e.g., timestamp #2).

[0080] 10C, there is shown a block diagram of a peak sampling circuit 1060 that determines a timestamp from two peaks in pulse signals 1012, 1014 indicative of scattered light detected by the first scattering channel 632 and the second scattering channel 634. The first scattering channel 632 includes a photodetector 1062A having an output coupled to a low noise gain amplifier 1063A to generate the pulse signal 1012 shown in FIG. 10B. The second scattering channel 634 includes a photodetector 1062B having an output coupled to an input to a low noise gain amplifier 1063B to generate the pulse signal 1014 shown in FIG. 10B.

[0081] The circuit 1060 further includes a clock or timer 1061 which generates a clock signal CLK and a timestamp signal TS. The clock signal CLK is used to synchronize both the circuit and the device. The timestamp signal TS is used to timestamp the digital samples of the scattering pulses 1012, 1014 to obtain the time difference between particles passing through the first scattering channel 632 and the second scattering channel 634.

[0082] FIG. 10D shows pulse signals 1012, 1014. It is desirable to periodically sample the pulse signals 1012, 1014 to generate multiple digital signals with respective timestamps. A threshold value TH is set that enables digital sampling of the pulse. Amplitude values of the pulse signal below the threshold are considered to be noise, which disables or terminates digital sampling of the pulse. When the pulse amplitude exceeds the threshold, digital sampling begins and the first sample is taken at timestamp TS1. N samples can be taken at timestamps separated by the sample period. The final sample is taken at timestamp TSN, after which the amplitude of the pulse signal falls below threshold TH, thereby indicating the end of digital acquisition of the pulse.

[0083] Referring back to FIG. 10C , the threshold value T is coupled to one input of comparators 1064A and 1064B for each scattering channel. The analog outputs A1, A2 of the gain amplifiers 1063A and 1063B are coupled to second inputs of the comparators 1064A and 1064B. The comparators 1064A and 1064B compare the amplitude of the pulse signal to a threshold value. If the comparator determines that the amplitude of the pulse signal exceeds the threshold value T, the comparator generates an enable signal EN1, EN2 at its output terminal to initiate digital sampling. If the comparator detects that the amplitude of the pulse signal is below the threshold value T, the comparator shuts off the enable signal EN1, EN2 to stop further digital sampling of the pulse because the amplitude may be considered noise.

[0084] Analog outputs A1, A2 of gain amplifiers 1063A and 1063B are similarly coupled to analog input terminals of analog-to-digital converters (ADCs) 1065A and 1065B. A clock signal is coupled to the clock terminals of analog-to-digital converters (ADCs) 1065A and 1065B. Enable outputs EN1, EN2 from comparators 1064A and 1064B are coupled to the enable input terminals of analog-to-digital converters (ADCs) 1065A and 1065B. Digital outputs D1, D2 of analog-to-digital converters (ADCs) 1065A and 1065B are coupled to first data inputs of dual-port storage devices 1066A and 1066B for storing digital data samples. A timestamp TS output from clock circuit 1061 is coupled to a second data input of dual-port storage devices 1066A and 1066B for storing a timestamp associated with each digital data sample.

[0085] The clock signal is coupled to clock terminals of analog-to-digital converters (ADCs) 1065A and 1065B and to clock terminals of dual port storage devices 1066A and 1066B to simultaneously store the digital samples and timestamps. Enable outputs EN1, EN2 from comparators 1064A and 1064B are further coupled to enable terminals of dual port storage devices 1066A and 1066B to enable storage of multiple digital samples D1, D2 representing pulses 1012, 1014 in dual port storage devices 1066A and 1066B.

[0086] Dual port storage devices 1066A and 1066B are coupled to TS1 and TS2 register / flip-flops 1051 and 1052 to transfer respective outputs TSpeak1, TSpeak2. The outputs of dual port storage devices 1066A and 1066B are coupled to the data inputs of the TS1 and TS2 register / flip-flops.

[0087] Dual-port storage devices 1066A and 1066B store respective digital samples associated with respective timestamps. Preferably, peak amplitudes Peak1 and Peak2 are searched for in each pulse signal and the associated timestamps are selected as peak timestamps TSpeak1 and TSpeak2. As can be seen in FIG. 10D , the largest digital sample values indicate the peak amplitudes Peak1 and Peak2 of pulses 1012 and 1014. The timestamps associated with the peak amplitudes Peak1 and Peak2 are the peak timestamps TSpeak1 and TSpeak2 of peaks Peak1 and Peak2 of pulses 1012 and 1014, respectively.

[0088] To determine the peak amplitudes Peak1, Peak2 and select the respective timestamps TSpeak1, TSpeak2, the dual-ported storage devices 1066A and 1066B can be sortable tables. In this case, the dual-ported storage devices 1066A and 1066B are sorted such that the sample values for the peak amplitudes Peak1, Peak2 are determined to have the largest digital value and its associated timestamp selected as the peak timestamps TSpeak1, TSpeak2. Alternatively, the dual-ported storage devices 1066A and 1066B can include logical comparison capabilities to indicate the peak amplitudes Peak1, Peak2 and associated peak timestamps TSpeak1, TSpeak2 for each pulse 1012, 1014.

[0089] The peak timestamp TSpeak (e.g., TSpeak1 and / or TSpeak2) for the peak amplitude within each pulse for a particle is stored in registers 1051, 1052. Once the peak timestamp TSpeak for each pulse is stored in a register, a subsequent digital subtraction can be performed to determine the time difference, as described further below.

[0090] 10B , the acquisition system 608 may further include a data acquisition chip DAC 1002 that includes, among other devices, a field programmable gate array (FPGA) particle calculation logic 1050. The particle calculation logic 1050 is coupled to the scattering channels 632, 634 to receive the pulse information and generate a digital timestamp. The logic 1050 includes a timekeeping device, such as a clock or timer 1061, to generate the digital timestamp in response to the sampling of the pulse information by the ADCs 1065A and 1065B. The particle calculation logic 1050 includes registers / flip-flops 1051, 1052 that store digital values for a first peak timestamp TSpeak1 and a second peak timestamp TSpeak2. A digital mathematical logic device (adder or subtractor) 1053 is coupled to the registers 1051, 1052 to receive the digital timestamps and calculate the time difference between the first peak timestamp TSpeak1 and the second peak timestamp TSpeak2.

[0091] The packetizer 1004 is coupled to the particle velocity calculation logic 1050 and the scattering channels 632, 634. The packetizer 1004 collects information and organizes it together into event data packets for particles 1006 flowing in a channel that intersects the two laser beams 613, 615. An event data packet is generated for each particle in the flow and sent to a host computer that executes acquisition software instructions as part of the acquisition system. The event data packet contains pulse information for each scattering channel as well as the time difference between peak timestamps.

[0092] For each particle of the plurality of particles in the stream, the acquisition system 608 has at least the following information: the distance L between the laser devices (612, 614), the particle identifier, the laser identifier, the corresponding timestamp, and the time difference (time delay) between the peak timestamps for each particle. The time delay for each particle may be stored in a storage device and accumulated so that an average time delay may be determined. The average time delay may be determined by adding the accumulation of time delays together and dividing by the total number of particles associated with that time delay. The average time delay may be used to generate a control signal that maintains a constant flow rate or velocity of the particles and a constant average time delay.

[0093] The acquisition system 608 having a digital mathematical logic device (e.g., device 1053 or a host computer 610 having processor logic, memory, and instructions) can further calculate the particle velocity of each particle according to Equation 8 (e.g., particle velocity equals distance L divided by the time difference between timestamps) and include that value as part of the event data packet for the particle. With multiple particles flowing through the flow path for a known period of time, the system (e.g., device 1053 or a host computer 610 having processor mathematical logic, memory, and instructions) can accurately calculate the average particle velocity for multiple particles passing through over a period of time.

[0094] The particle velocity for each particle may be stored in a storage device and accumulated so that an average particle velocity may be determined by adding the accumulations of particle velocities together and dividing by the total number of particles associated with that particle velocity.

[0095] To identify each particle, the density of particles in the sample fluid is relatively low, so that particles do not pass by the laser devices (612, 614) too frequently. For example, the fluidics system 604 can operate at a sample flow rate and particle density such that particles flow through the laser devices (612, 614) at less than 10,000 particles / second (e.g., events / second) and an average particle velocity of about 5 meters / second. In this example, the 10,000 particles / second (e.g., events / second) can be referred to as the "event rate" or particle frequency. With such an event rate and average particle velocity, the separation between particles is typically between about 150 and 200 micrometers, which is sufficient separation to have a sufficiently high signal-to-noise ratio.

[0096] In contrast, if particles pass by the laser devices (612, 614) at a high event rate, for example, 50,000 particles / second, at the same flow rate, the fluidics system 604 may generate an excessively large amount of particle coincidence between the laser devices (612, 614). In such a case, the system 600 may erroneously interpret two different particles as the same particle due to the reduced particle separation required for the higher event rate. Such coincidence may result in an excessively low signal-to-noise ratio, making the calculations of the acquisition system 608 unreliable.

[0097] Fortunately, it is easy for system 600 to maintain a large enough separation between particles so that coincidences are not a problem. Furthermore, system 600 calculates the average particle velocity of multiple particles over a period of time. The average particle velocity can be used as a feedback control signal to adjust the flow rate as needed to maintain a high signal-to-noise ratio in each of the two scattering channels. Therefore, any coincidences that may occur can be treated as outliers, as long as the separation between particles is maintained high enough.

[0098] Typical commercial flow cytometer systems are fundamentally different. While they may have dual lasers, the dual lasers are not used to measure particle velocity. They may only have one side-scatter channel, which detects scattered light from particles at one location via the first laser. The second laser typically has only a fluorescence detector, which only detects fluorescence from markers attached to particles. Particles are not always marked with fluorescent markers, and even if they are, they do not always fluoresce due to the wrong wavelength of excitation laser light. A second laser associated with the fluorescence detector, rather than the scatter channel, can detect only the fluorescence from markers attached to particles. Typical commercial systems cannot calculate particle velocity using fluorescence alone. This is because particles cannot be labeled with fluorescent markers that fluoresce under both lasers, which excite at different wavelengths. For example, consider a particle in a sample that is chemically colored red by a fluorescent marker that fluoresces when excited by a red-wavelength laser but not a blue-wavelength laser. Typically, dual lasers have different wavelengths—one laser to excite and detect red-dyed particles and another laser to excite and detect blue-dyed particles, but not both. Thus, particle velocity cannot be measured by two fluorescence detectors, with only one of the two lasers exciting each fluorescing particle in the sample stream. Two scattering channels can detect particles as they flow by their associated lasers, regardless of their fluorescent dye markers or laser excitation wavelengths.

[0099] In contrast to typical commercial systems, system 600 takes advantage of the fact that particles passing through laser beams (613 and 615) consistently generate scattered light. Therefore, system 600 is configured with dual scatter detector channels (632, 634) to detect scattered light from particles twice, once struck by laser beam 613 and once by laser beam 615.

[0100] The core stream velocity is proportional to the volumetric flow rate in the flow passage 216. In a technically precise sense, the core stream velocity is proportional to the total flow rate in the flow passage. Furthermore, the total volumetric flow rate is proportional to the differential pressure across the ends of the flow passage. For completeness, these relationships may be expressed formally as the following equations:

number

[0101] The laser delay describes the particle flow time between two spatially separated laser beams (613, 615), as shown in Figure 10A. Referring to Figures 10A and 10B, the laser delay can be determined from the difference between the timestamps associated with signal pulses detected by the in-line detector channels (632, 634) for the same particle 1006. In one embodiment, the laser beams (613, 615) can provide a laser delay that is precise to within about ±0.1 microseconds. The laser delay is related to the particle velocity by the following equation:

number

[0102] Laser delay is a critical parameter for data acquisition in flow cytometers with spatially separated laser beams (613, 615). Accurate knowledge of its value allows the acquisition system to associate pulses from different laser beams (613, 615) with the same particle 1006. Still referring to FIG. 10B , pulse 1012 is generated when a particle passes through laser beam 613. Shortly thereafter, the particle will generate pulse 1014 when it passes through laser beam 615. If the acquisition system 608 knows the correct laser delay, it can correctly associate pulse 1012 and pulse 1014 with the same particle. Laser delay calculations are typically performed as part of the daily quality control procedure for the cytometer and are assumed to remain constant for the remainder of the day. In this example, the acquisition system 608 includes electronics such as a DAC (digital-to-analog converter) 1002 integrated with an FPGA (field-programmable gate array).

[0103] In reality, however, there is some uncertainty in the laser delay, primarily due to core stream velocity variations. These variations can arise from many sources, such as changes in liquid level, temperature shifts, particle position within the core stream, and laser position drift. To compensate for the uncertainty in the laser delay, the acquisition system uses the concept of window extension. Window extension adds a tolerance band to the pulse width measured in the laser beam 613 and applies this increased window as the expected interval to collect pulse 1012. The increased window is essentially a fluidics system tolerance factor that allows the acquisition system 608 to correlate pulses over a range of laser delays. A larger window extension allows for greater velocity variations, but at the expense of a lower signal-to-noise ratio because more background light is integrated into pulse 1014 (the second pulse) during portions of the collection interval where no particle signal is present.

[0104] An advantageous embodiment of system 600 uses continuous monitoring of laser delay by acquisition system 608. A moving average of the laser delay value is fed to fluidics system 604 and used to periodically correct the differential pressure setpoint value to ensure that the laser delay remains within very tight tolerances. Such feedback (e.g., correction) ensures that a window widening approach is the least likely. The basic algorithm for differential setpoint correction is given by the following equation:

number

number

[0105] Equation 10 shows the possible range of flow regimes inside a channel. The lower limit represents the flow resistance due to purely inviscid (Bernoulli) flow. The upper limit represents the flow resistance due to purely viscous (Poiseuille) flow.

[0106] 7 and 8, the laser delay corrected differential pressure set point is used in a feedback loop, where the feedback parameter is the differential pressure measured across the end of the flow cell 206. The system gain G is calculated using a PID (proportional-integral-derivative) controller and the vacuum pump 704, accumulator 702, and primary flow path resistance network (R FC , R SH , R SA ) and the physical characteristics of the pressure transducers (TR1, TR2).

[0107] Typical flow cytometers can use differential feedback. However, they are hampered by cumbersome sample flow schemes that require switching between differential and static feedback modes between running and stopped states. Also, flow cytometers require a temperature input to compensate for viscosity changes.

[0108] Advantageously, embodiments of the present invention eliminate the need for any of these commercially available schemes. Utilizing laser delay provides direct measurement and control of core stream velocity. Temperature compensation is unnecessary because thermal effects contribute to laser delay shift. Given stable laser spacing, using laser delay as a feedback parameter essentially reduces to using core stream velocity as a feedback parameter to maintain itself. A rather important nuance is that laser delay acts as the outer loop of the combined feedback system as a correction to the differential setpoint. Differential pressure acts as the inner loop. This allows the acquisition system 808 to calculate an accurate running average based on thousands of laser delay measurements. In the absence of an event (e.g., a particle), the system maintains the pressure differential based on the last updated setpoint correction. This prevents the system from entering an "open loop" and ensures robust control.

[0109] In typical fluidics systems, the aforementioned awkwardness of sample flow rate management stems from the limitations of the vacuum feedback scheme. Such systems do not incorporate any pressure feedback mechanism that can release the vacuum in the accumulator. The vacuum pump can only increase or maintain the vacuum level in the accumulator. This vacuum pump limitation is particularly troublesome because, to reduce the sample flow rate while maintaining a constant total flow rate, the system must reduce the accumulator pressure in response to lower sheath path resistance. Such systems only achieve this reduction by topping off the accumulator with sheath fluid directly from the plenum (compromising the waste head level) as a special operating case. During this special operating case, which consumes significant workflow time, the acquisition system must be disabled.

[0110] Advantageously, system 600 completely solves this problem of typical systems by incorporating a dedicated vacuum air bleed valve (e.g., valve V5 in FIG. 7) controlled by a pressure relief algorithm that is part of the vacuum feedback control loop.

[0111] 11 is a block diagram of a feedback control system and method 1100 for controlling vacuum relief in the fluidics system 604 of a flow cytometry system. The vacuum pump cannot operate in reverse. Therefore, a vacuum relief valve, such as valve V5 in FIG. 7, can be used to provide a faster supply of air to the accumulator 702 to resolve an over-vacuum condition.

[0112] 7 , which controls valve V5 coupled between accumulator 702 and flow restrictor 740 to atmospheric pressure. Acquisition system 608 may include, for example, firmware layer 1108A that executes the control process and analog interface circuitry 1108B that interfaces to peripheral hardware 1110. Analysis system 610 may include, for example, peripheral hardware 1110 with which analysis system 610 interfaces to sense and control vacuum within the flow cytometry system. Peripheral hardware 1110 may include pressure transducers TR2 and TR1 (or differential pressure transducer DPTR) that sense the differential pressure across the ends of flow channel 216 of flow cell 206 and control controllable valve V5 to release the vacuum within accumulator 702 to atmosphere, thereby increasing the pressure within accumulator 702 and altering the differential pressure.

[0113] Using system and method 1100, valve V5 acts as a pressure (vacuum) relief mechanism to compensate for the pressure (e.g., pounds per square inch or PSI) in accumulator 702 shown in FIG. 7. System and method 1100 allows the fluidics system in a flow cytometry system to be continuous and much more responsive than typical flow cytometry systems. Method 1100 also eliminates the need for the so-called dual feedback loops (static and dynamic) used in other flow cytometry systems (such as those described in U.S. Patent No. 8,528,427, entitled "DUAL FEEDBACK VACUUM FLUIDICS FOR A FLOW-TYPE PARTICLE ANALYZER," issued September 10, 2013 to David Vrane et al.).

[0114] System parameters for the feedback control system and method 1100 include, without limitation, input system parameters of offset 1101, gain 1102, pressure set point 1104, pressure set point tolerance 1105, vacuum pump status 1106, and valve 5 status 1107, and output system parameter of pressure readout 1103.

[0115] Valve V5 is opened to release the vacuum in the fluidics system's accumulator 702 more quickly than it can be bled out to atmosphere by flow restrictor 742. Generally, during normal operation of vacuum pump 704, valve V5 is opened periodically for short periods of time. There are unusual cases where valve V5 is opened periodically.

[0116] When there is a need for a full vacuum release, such as during a system cleaning cycle, the state of valve 5 1107 is set to true, bypassing much of the firmware logic through OR gate 1142 and opening valve V5 for a period of time until the state of valve 5 1107 is set to false.

[0117] If the vacuum pump 704 is not being used, there may be little vacuum in the accumulator, and there is no need to control valve V5 to open and dump the vacuum in the accumulator 702. In this case, the vacuum pump state 1106 may be set to false, indicating an off state. The vacuum pump state is one of two inputs to the AND gate 1140. If the vacuum pump state is set to false, the process that generates the bit value ζ is ignored, and the output of the AND gate 1140 is false. In this case, unless valve V5 is in state 1107, valve V5 is not controlled to open and dump the vacuum in the accumulator 702.

[0118] The firmware layer 1108A contains logic that interfaces to certain actions or processes and system parameters.

[0119] At action 1120, the system receives a pressure reading 1101, a gain 1102, and a digital feedback error signal e from analog circuitry 1108B. The system calculates a pressure reading 1103 based on the error signal e using a gain coefficient K2 1102 and an offset OS 1101.

[0120] In action 1121 , the system reads the pressure reading 1103 and / or the delay parameter 1133 before performing the comparison in action 1122 .

[0121] At action 1122, the system determines whether the pressure reading 1103 minus the pressure setpoint 1104 is greater than the pressure setpoint tolerance 1105. If it is not (no), the system proceeds to action 1124, where the system sets a counter N to zero (0) and jumps to action 1132. However, if it is greater (yes), the system proceeds to action 1126, whereupon the system increments the current counter value N and proceeds to action 1128.

[0122] At action 1128, the system determines whether the counter value N is greater than or equal to a predetermined number (e.g., 10). If yes at action 1128, the system sets the bit value ζ equal to true (logic 1). However, if no at either action 1128 or action 1122, the system sets the bit value ζ equal to false (logic 0) at action 1132. In either case, the bit value ζ is stored with its output coupled to an input of a two-input logic gate 1140. After setting the bit at either action 1130 or 1132, the process continues with action 1133. At action 1133, a predetermined wait time (e.g., 0.1 seconds) elapses before the process loops back to action 1121 to repeat one or more of actions 1121, 1122, 1124, 1126, 1128, 1130, and 1132.

[0123] With wait 1133 set to 0.1 seconds and the predetermined number of comparisons set to 10, action 1128 generally results in a 1 second delay before bit value ζ is set to true and valve V5 can be actuated to release the vacuum in the accumulator during normal control and operation of vacuum pump 704.

[0124] Logic gates 1140, 1142 perform logic functions before valve V5 is opened by valve driver circuit 1138. The logical AND gate causes the system to perform a logical AND operation 1140 on bit value ζ with vacuum pump state 1106. If bit value ζ and vacuum pump state 1106 are both true, the resulting output of logical AND operation 1140 is true. Otherwise, if either bit value ζ or vacuum pump state 1106 is false, the resulting output of logical AND operation 1140 is false. The system then performs a logical OR operation 1142 on the result of logical AND operation 1140 with state 1107 of valve V5. If either the result of logical AND operation 1140 or state 1107 of valve V5 is true, the resulting output of logical OR operation 1142 is true. The resulting output of logical OR operation 1142 is false only if both logical AND operation 1140 and state 1107 of valve V5 are false.

[0125] Valve driver circuit 1138 is coupled to the output of the OR gate to receive the result of the logical OR operation 1142 and control valve V5.

[0126] At action 1136, a pressure transducer regulation and PI (proportional-integral) feedback circuit generates an analog feedback error signal e in response to the differential pressure sensed by pressure transducers TR2 and TR1 (or differential pressure transducer DPTR) across the flow path of the flow cell. At action 1134, an ADC (analog-to-digital converter) receives the analog feedback error signal e and converts it to a digital feedback signal e that firmware layer 1108A can digitally process. The digital feedback signal e is input to action 1120 to complete the feedback loop, continuing the cycle of actions. Other actions, processes, and / or details are discussed with reference to the figures and may be part of feedback control system and method 1100, depending on the implementation.

[0127] 12, a block diagram of an exemplary method and feedback control system 1200 for controlling the vacuum pump 704 in the fluidics system 604 shown in FIG. 7 is shown. In one embodiment, the acquisition system 608 includes a firmware layer 1208A and analog circuitry 1208B that implement and provide the method and feedback control system 1200 for controlling the vacuum pump 704. The analysis system 610 interfaces with peripheral hardware 1210 to sense and control the vacuum pump motor, thereby generating vacuum within the flow cytometry system. The peripheral hardware 1210 includes at least one pressure transducer (e.g., pressure transducer TR2 shown in FIG. 7) and the vacuum pump motor M704.

[0128] Using the method and feedback control system 1200, the system generally drives the vacuum pump 704 to a pressure setpoint and controls the core velocity flow within the flow cell 206 when changes are present. A pressure correction factor can be generated in response to the measured average time delay of particle flow between the lasers (e.g., laser delay 1202) sensed by a pair of scattering channels. Particle velocity is inversely proportional to the time delay between the lasers. If the particle time delay between the lasers along the flow path increases, indicating slower fluid and particle velocities, the vacuum can be increased to increase the core velocity flow and decrease the particle time delay between the lasers. If the particle time delay between the lasers along the flow path decreases, indicating faster fluid and particle velocities, the vacuum can be decreased to decrease the core velocity flow and increase the particle time delay between the lasers. The pressure setpoint 1204 is corrected in response to the measured time delay between the lasers before driving the vacuum pump 704.

[0129] Input system parameters include, but are not limited to, offset 1201, gain 1202, pressure set point 1204, reference set point 1205, correction constant 1206, and measured laser delay 1207. Output system parameters include pressure readout 1203.

[0130] The firmware layer 1208A contains mathematical logic that controls certain actions or processes and system parameters and interfaces to the analog circuitry 1208B. The analog circuitry 1208B contains certain actions or processes and devices that interface to the firmware layer 1208A.

[0131] Analog circuit element 1208B includes a pressure transducer regulation and PI (proportional integral) feedback circuit that receives the differential pressure across transducers TR2 and TR1 (or differential pressure transducer DPTR). At action 1228, the pressure transducer regulation and PI (proportional integral) feedback circuit generates an analog feedback signal -e based on the voltage received from the differential pressure sensed by transducers TR2 and TR1.

[0132] At action 1224, an analog-to-digital converter (ADC) receives the analog feedback signal-e and converts it to a digital feedback signal-e for use by the digital logic and processes of firmware layer 1208A.

[0133] At action 1220, the pressure calculation device receives the offset 1201, the gain 1202, and the digital feedback signal -e from the analog circuitry 1208B. The pressure calculation device calculates a pressure reading 1203 based on the offset 1201, the gain 1202, and the digital feedback signal -e.

[0134] At action 1222, a pressure correction calculator receives a reference laser delay 1205, a correction constant 1206, and a measured laser delay 1207. The pressure correction calculator calculates a differential pressure ΔP, a correction factor, which is based on the reference laser delay 1205, the correction constant 1206, and the measured laser delay 1207.

[0135] At action 1221, a summer or adder adds the pressure set point P to the differential pressure ΔP to form a desired pressure value P. The output from the summer or adder, a digital value, is coupled to an analog-to-digital converter (ADC) 1226.

[0136] In action 1226, the ADC receives the digital value for the desired pressure value P from the summer or adder and converts it to an analog value for the desired pressure P.

[0137] At action 1227, the system calculates a commanded pressure (Pe) by adding or summing together the desired pressure P from the DAC and a negative feedback signal -e from the pressure transducer regulation and PI (proportional integral) feedback circuit 1228. The commanded pressure (Pe) is coupled to the pump driver circuit.

[0138] At action 1230, the pump driver circuit receives the specified pressure (Pe) from the summer or adder 1227. The pump driver circuit is coupled to the vacuum pump motor 704. If the specified pressure begins to increase (reducing vacuum), the pump driver circuit can drive the vacuum pump motor 704 with a higher voltage to increase the vacuum in the accumulator 702. If the specified pressure begins to decrease (increasing vacuum), the pump driver circuit can drive the vacuum pump motor 704 with a lower voltage to decrease the vacuum in the accumulator 702.

[0139] The actions of firmware layer 1208A and analog circuitry 1208B continuously loop in a cycle of actions. Other actions, processes, and / or details are discussed with reference to the figures and may be part of method and system 1200, depending on the implementation.

[0140] Figure 13 shows a fluidics system 1304 that is similar to the fluidics system of Figure 7. However, in the fluidics system 1304 of Figure 13, the positioning of the degasser 1320 differs from the positioning of the degasser 720 of Figure 7.

[0141] The fluidics system 1304 of Figure 13 has a vacuum-based fluidics architecture with a time-based pressure regulation scheme for particle flow between the laser beams (613, 615) shown in Figure 6. The fluidics system 1304 similarly provides continuous sample flow regulation in a vacuum-based system with a sample path that is completely free of transitions, discontinuities, or potentially cell-damaging peristaltic pumps. The fluidics system 1304 seeks to maximize reliability and instrument uptime by avoiding the use of peristaltic pumps entirely.

[0142] Fluidics system 1304 includes, without limitation, manifold assembly 701, isolation valves V1-V5, pinch valve V6, pressure transducers (e.g., probes) TR1 and TR2, accumulator vessel (vacuum chamber) 702, diaphragm vacuum pump 704, degasser pump 706, plenum fluid 208, sample fluid 210, sample vessel 506, output sensor 714, stepper mounting position 716, check valve 718, degasser 1320, flow cell 206, sheath tank 726, plenum tank 504, plenum pump 728, stepper pinch valve 730, flush pump 732, sheath filter 734, waste tank 508, and plate loader 738, flow restrictors 740 and 742, and sheath float sensor 744.

[0143] 14, an alternative embodiment of a fluidics system 1404 for a flow cytometer is shown. The fluidics system 1404 includes a linear resistance stepper valve V7. The linear resistance stepper valve V7 includes a stepper motor that adjusts the flow rate of the sheath fluid 208 to control the flow rate of the sample fluid 210. The fluidics system 1404 does not include the stepper pinch valve 730 and flush pump 732 included in the fluidics system 604 of FIG. 7 and the fluidics system 1304 of FIG. 13.

[0144] The fluidics system 1404 of Figure 14 has a vacuum-based fluidics architecture with a time-based pressure regulation scheme for particle flow between laser beams (613, 615) as described with reference to Figure 6. The fluidics system 1404 similarly provides continuous sample flow regulation in a vacuum-based system with a sample path that is completely free of transitions, discontinuities, or potentially cell-damaging peristaltic pumps. The fluidics system 1404 seeks to maximize reliability and instrument uptime by avoiding the use of peristaltic pumps entirely.

[0145] Fluidics system 1404 includes, without limitation, manifold assembly 701, isolation valves V1-V5, pinch valve V6, pressure transducers (e.g., probes) TR1 and TR2, accumulator vessel (vacuum chamber) 702, diaphragm vacuum pump 704, degasser pump 706, plenum fluid 208, sample fluid 210, sample vessel 506, output sensor 714, stepper mounting position 716, check valve 718, linear resistance stepper valve V7, flow cell 206, sheath tank 726, plenum tank 504, plenum pump 728, sheath filter 734, waste tank 508, plate loader 738, flow restrictors 740 and 742, and sheath float sensor 744.

[0146] The following provides an exemplary operating cycle for the fluidics system 1404 shown in FIG. 14. Upon startup, the fluidics system 1404 receives feedback from pressure transducers TR1 and TR2. The pressure difference sensed between transducers TR1 and TR2 is continuous during operation, driving the vacuum pump 704 to maintain a constant pressure difference between the inlet 401 and outlet 404 of the flow channel 216 in the flow cell 206 shown in FIG. 4 until a minimum value is reached inside the accumulator 702. The pressure transducers TR1 and TR2 may be differential pressure transducers DPTR that measure the differential pressure between the inlet 401 and outlet 404 of the flow channel 216 in the flow cell 206. The minimum value is what the fluidics system 1404 considers to be, at least initially, a differential pressure (e.g., a setpoint pressure).

[0147] Fluidics system 1404 is desirably bubble-free. Therefore, fluidics system 1404 has a protocol for opening and closing valves V1-V5 to eliminate gas bubbles. For example, fluidics system 1404 opens valve V4, which draws gas bubbles through sheath filter 734. If at any time sheath float sensor 744 in the plenum tank drops below a predetermined low level, plenum pump 728 draws fluid from sheath tank 726. The system is then ready for operation.

[0148] Once the sample vessel 506 is attached, the tube sensor 714 indicates that the fluidics system 1404 is ready to lower the system's sample straw 518 into the sample vessel 506. In an alternative sampling scenario, the platter loader 738 includes x-stage and y-stage stepper motors that allow the sample straw 518 to draw fluid from a well plate, such as a 96-well plate or a 384-well plate, instead of the sample vessel 506. When the sample straw 518 reaches the stepper sample injection tube position (SITD) 716, one or more sensors cause the fluidics system 1404 to open valves V1 and V3. A regulation system within the fluidics system 1404 begins to drive a differential pressure to meet the setpoint pressure.

[0149] In one embodiment of the fluidics system 1404, a vacuum pump 704 evacuates an accumulator vessel 702. The accumulator vessel 702 serves as the driving vacuum source for the system, and the vacuum pump 704 maintains this source from a differential pressure reference derived from laser delay measurements made by the acquisition system 608. The accumulator vessel 702 also serves as a pulse attenuator and constant head reference for the outlet 404 of the flow cell 206, thereby isolating the flow cell 206 from head effects related to the liquid level in the waste tank 508.

[0150] The fluidics system 1404 supplies sheath fluid 208 to the flow cell 206 by means of a plenum tank 504. The liquid level in the plenum tank 504 is maintained at a precise level by periodic refilling by a plenum pump 728, which draws sheath from a sheath tank 726. The plenum tank 504 thus acts as a constant head reference for the inlet of the flow cell 206, thus isolating the flow cell 206 from head effects associated with the liquid level in the sheath tank 726.

[0151] Manifold assembly 701 contains a network of valves V1-V5 that control fluid flow within fluidics system 1404. In the primary sample acquisition mode, valves V1 and V3 are open. In this mode, sheath fluid 208 and sample fluid 210 are simultaneously drawn into flow cell 206 by the vacuum within accumulator 702.

[0152] When valve V2 is closed, transducer TR1 measures the pressure inside flow cell 206 at inlet 401, and transducer TR2 measures the pressure at outlet 404 of flow cell 206, as shown in FIG. 4. When valves V1 and V3 are open, transducers TR1 and TR2 (or differential pressure transducer DPTR) measure the differential pressure across the end of flow channel 216 of flow cell 206. The pressure drop between outlet 404 and inlet 401, the differential pressure across the end of flow channel 216, is proportional to the total volumetric flow rate, which is proportional to the core stream velocity (assuming a nearly constant temperature). See, for example, Equation 7. Operating temperatures are typically between about 15°C and 20°C, above which the viscosity of water can vary by as much as 20%. Volumetric flow rate (including sheath fluid velocity) is inversely proportional to viscosity.

[0153] When fluidics system 1404 is operating, it simultaneously removes sheath fluid 208 and sample fluid 210. Fluidics system 1404 removes sheath fluid 208 from plenum tank 504, passes through sheath filter 734, through degasser 1320, through valve V1, through linear resistance stepper valve V7, and to flow cell 206. Simultaneously, fluidics system 1404 removes sample fluid 210 from sample vessel 506 and delivers it to flow cell 206.

[0154] The relative physical (fluidic) resistance between the sheath straw 516 and the linear resistance stepper valve V7 causes the fluidics system 1404 to adjust the relative flow rates between the sheath fluid 208 and the sample fluid 210. The fluidics system 1404 (e.g., through the use of the linear resistance stepper valve V7) can adjust the flow rate of the sheath fluid 208 to be, for example, 10.50 milliliters / minute, while the fluidics system 1404 adjusts the flow rate of the sample fluid 210 to be, for example, 10.00 microliters / minute. Other flow rates are also within the scope of the fluidics system 1404.

[0155] The linear resistance stepper valve V7 includes a drive motor with a variable piston valve to provide variable physical fluid resistance to regulate the flow rate of the sheath fluid 208 within the flow cell 206. Regulating and controlling the flow rate of the sheath fluid 208 allows the system to continuously control the variable flow rate of the sample fluid 210 within the flow cell 206.

[0156] The sheath straw 516 is inserted into the plenum tank 504. The sample straw 518 is inserted into the sample vessel 506. Compared to the sample straw 518, the sheath straw 516 has a relatively larger diameter to handle a relatively larger volumetric flow. Conversely, compared to the sheath straw 516, the sample straw 518 has a relatively smaller diameter to handle a relatively smaller volumetric flow. The diameter difference between the sheath straw 516 and the sample straw 518 affects the relative solution rates at which the sheath fluid 208 and the sample fluid 210 are aspirated from their respective vessels (504 and 506).

[0157] 4, 6, and 14, the vacuum pump 704 receives differential pressure feedback from the acquisition system 608 between the pressures sensed by transducers TR1 and TR2 in the fluidics system 1404. Based on the differential pressure feedback, the vacuum pump 704 is controlled to maintain a constant core stream velocity even as the linear resistance stepper valve V7 adjusts to vary the sheath flow rate. In this way, the total fluid flow rate through the flow cell 206 can remain substantially unchanged. The linear resistance stepper valve V7 allows the system to continuously vary the sample flow rate by directly varying the sheath flow rate. Controlling the flow rate of the sheath fluid 208 with the linear resistance stepper valve V7 allows the fluidics system 1404 to have continuously variable flow rates (e.g., variable sample flow rate and variable sheath flow rate) while maintaining a constant overall flow and, therefore, a constant (e.g., stable) particle velocity.

[0158] Adjusting (e.g., controlling) the flow rate of the sheath fluid 208 adjusts (e.g., controls) the flow rate of the sample fluid 210, including the sample particles therein. Thus, the system 600 can control the sheath volumetric flow rate, and thereby the sample volumetric flow rate and particle velocity. The system 600 performs these controls of pressure and velocity in a closed loop (previously open loop). Note that typical commercial systems perform open loop corrections, which require stopping undesired system pressurization.

[0159] Linear resistance stepper valve V7, for example, regulates the flow rate through valve V1, degasser 1320, and flow cell 206. Valve V2 turns on the fluid flow to flush the system, after which valve V2 is closed to set up sampling.

[0160] Advantageously, the linear resistance stepper valve V7 allows for precise control of the fluid, taking into account that the viscosity of the sample fluid 210 can be highly variable. For example, blood as the sample fluid 210 can have a viscosity that is twice the viscosity of the sheath fluid 208. Note that the volumetric flow rate is inversely proportional to the viscosity.

[0161] With valves V1 and V3 open, fluidics system 1404 draws sheath fluid 208 and sample fluid 210 through the top of flow cell 206, through valve V3, and into accumulator 702. Accumulator 702 serves as a waste bucket for the fluidics system. Vacuum pump 704 maintains (e.g., regulates) vacuum pressure within fluidics system 1404. Vacuum pump 704 also pumps waste from accumulator 702 to waste pump tank 508.

[0162] When the sample fluid is flowing at a high rate (e.g., the flow rate of the sheath fluid through the linear resistance stepper valve V7 is substantially reduced), the vacuum in the fluidics system 1404 is higher to maintain the differential pressure across the flow cell 206. If the user desires a lower flow rate for the sample fluid 210, the flow rate of the sheath fluid through the linear resistance stepper valve V7 is increased accordingly. At this point, the vacuum pump 704 is operating at a relatively high vacuum, which forces the fluidics system 1404 to flow the sheath fluid 208 through the system at a much higher velocity, thereby flowing particles at a steady velocity in the sample fluid 210. Thus, the fluidics system 1404 detects that the differential pressure is too high and opens the pressure relief valve V5, pulsing the valve V5 until the differential setpoint is met. Thus, the valve V5 maintains (e.g., regulates) the pressure within the system 600.

[0163] Restrictors 740 and 742 are substantially always open to fluidics system 1404 to allow partial bleeding of fluid into accumulator 702. As such, vacuum pump 704 is substantially always pumping at least a small amount, which ensures that accumulator 702 remains free of excess liquid and therefore does not fill up.

[0164] The degasser 1320 removes gas from the fluidics system 1404. Generally, gas is generated whenever a vacuum is applied to a fluid. Therefore, the degasser 1320 removes gas from the fluidics system 1404 before it reaches the flow cell 206. The degasser pump 706 also serves as the source for the sample injection tube (SIT) flush aspiration vacuum.

[0165] The degasser pump 706 is activated (e.g., turned on) whenever the vacuum switch on the degasser 1320 is triggered because the vacuum falls below a predetermined value (e.g., −9 pounds per square inch). The fluidics system 1404 draws gas through the check valve 718. In this case, the degasser 1320 draws a relatively small amount of gas from the sheath fluid 208. The degasser 1320 remains isolated. Check valve V6 prevents the vacuum pressure inside the degasser 1320 from being lost while a SIT flush is occurring.

[0166] The degasser 1320 degasses the fluid whenever the fluidics system 1404 reduces the gas pressure on the fluid. For example, gas forms within the fluid, similar to the gas that forms within a pressurized soda can when it is opened. In this system, the sheath fluid 208 within the plenum tank 504 is at approximately atmospheric pressure, while the pressure within the accumulator 702 is below atmospheric pressure. As the sheath fluid 208 flows along the sheath path, the pressure on the sheath fluid 208 continually decreases. Because of the continually lower pressure, air bubbles tend to form within the sheath fluid 208. This problem is exacerbated if the sheath fluid is pre-aerated. Air bubbles tend to be approximately the same size as particles within the sample fluid 210. These air bubbles introduce background optical noise when the fluidics system 1404 attempts to detect and analyze particles within the sample fluid 210. Note that for this reason, many manufacturers do not build flow cytometry systems that operate using a vacuum. To combat the formation of air bubbles within the sheath fluid 208 , the degasser 1320 removes air bubbles (eg, gas) from the fluid solution before they can enter the flow cell 206 .

[0167] 15, a schematic diagram of a linear resistance stepper valve V7 is shown. The linear resistance stepper valve V7 includes, but is not limited to, an inlet port 1502, an outlet port 1504, a piston 1508, a piston bore (hollow cylinder) 1506, and a stepper motor 1512. Sheath fluid 208 enters the inlet port 1502, enters the piston bore 1506, flows past the piston 1508 and through the piston bore 1506, exits the piston bore 1506 via the outlet port 1504, and flows to the flow cell 206 (shown in FIG. 14).

[0168] Piston bore 1506 is a substantially cylindrical space having a central axis. Piston bore 1506 is formed by the inner wall of linear resistance stepper valve V7. Piston 1508 is a substantially cylindrical device having a central axis. The central axis of piston 1508 and the central axis of piston bore 1506 are substantially collinear. Diameter D0 is the diameter (e.g., inner diameter) across the hollow circular cylinder of piston bore 1506. Diameter D i is the diameter across the circular cylinder of the piston 1508. As the piston 1508 extends into the piston bore 1506, the piston 1508 decreases the volume of the space (chamber) inside the piston bore 1506. Conversely, as the piston 1508 is withdrawn from the cylinder, the volume of the space (chamber) inside the piston bore 1506 increases.

[0169] The piston 1508 and piston bore 1506 each have spaced apart but overlapping cylindrical sidewalls. The piston 1508 and piston bore 1506 are separated from one another by a variable height ring-shaped space or gap, also referred to herein as an annular region. The diameter D of the piston bore 1506 and the diameter D of the piston 1508 are i The radial distance between helps establish a linear physical fluid resistance for a desired fluid viscosity to flow through the variable height annular space or gap.

[0170] The length L is the distance that the piston 1508 extends along a collinear axis into the annular region of the piston bore 1506 (sidewall overlap distance). The length L is also the distance between the diameter D of the piston bore 1506 and the diameter D of the piston 1508. i The height of the annular region of the ring-shaped space or gap between the

[0171] Piston bore diameter D0 and piston diameter D ishould be designed and machined to precisely apply the desired amount of physical fluid resistance (drag) to the sheath fluid 208 flowing through the linear resistance stepper valve V7. In one embodiment, Equation 11 below describes the physical fluid resistance R within the variable height annular region that the piston 1508 and piston bore 1506 apply to the sheath fluid 208 flowing through the linear resistance stepper valve V7: SH This stipulates:

number

[0172] Equation 11 includes the following parameters: R SH = the resistance that the linear resistance stepper V7 applies to the sheath fluid 208, D0 = diameter of piston bore 1506, D i = diameter of piston 1508, μ=dynamic viscosity of the flowing sheath fluid 208, L = diameter D0 and D i The length (height) of the annular region (gap) between

[0173] Advantageously, the physical fluid resistance R of the linear resistance stepper valve V7 is determined in accordance with Equation 11: SH is the diameter D0 of the piston bore 1506 and the diameter D of the piston 1508 i As the piston 1508 is pushed further into the piston bore 1506, a higher annular region (gap) exists between the sidewall of the piston and the sidewall of the piston bore, thereby increasing the height or length L of the annular region (gap). As the piston 1508 is withdrawn from the piston bore 1506, the annular region (gap) between the sidewall of the piston and the sidewall of the piston bore becomes shorter, thereby decreasing the length L of the annular region (gap).

[0174] The linearity between length L and physical fluid resistance makes it easy to adjust the sheath flow rate (and therefore the sample rate) with sufficiently high precision within the fluidics system 1404. A linear stepper motor 1512 can easily push and pull the piston 1508 to adjust the length L (height) of the annular region (gap).

[0175] 7 regulates the sample flow rate by controlling the sheath flow rate by changing the diameter of the stepper pinch valve 730. Unfortunately, the resistance of the stepper pinch valve 730 tends to be proportional to the diameter of the tubing of the stepper pinch valve 730 raised to the fourth power (e.g., resistance ∝ diameter 4 ). Nonlinearity makes it difficult to regulate the sheath flow rate (and therefore the sample flow rate) with sufficient precision. Another challenge with the system 604 of FIG. 7 is that the sample flow rate can be more difficult to control because the materials typically used for the tubing of the stepper pinch valve 730 tend not to provide reliable and repeatable resistance.

[0176] 14 and 15, the fluidics system 1404 may operate in a conventional manner without the flush pump 732 of FIG. 7. For example, the fluidics system 1404 may close valve V1, open valve V7, push piston 1508, and backflush fluid downstream of the sample path 210 to remove (e.g., flush) carryover fluid (e.g., from a previous run). Alternatively, the fluidics system 1404 may close valve V1, open valve V7, and pull piston 1508 to remove (e.g., boost) sample fluid to prime the sheath path 208. The fluidics system 1404 may then open valves V1 and V7. The linear resistance stepper valve V7 may then act like an electronic resistance to the electric current, providing a physical fluidic resistance to the sheath fluid flowing through the valve.

[0177] In one embodiment, the total volume of sample tube 210 is approximately 13 microliters (μl), while the total volume of valve V7 varies anywhere between approximately 500 μl and 5000 μl, depending on the piston and bore diameter selection. Thus, only a relatively small displacement of piston 1508 within bore 1506 is required to clear sample path 210. Such small displacement facilitates coordination between the flushing position and the normal operating home position of valve V7.

[0178] The time when valve V7 is used as a flush pump or boost pump is during the transition stage (e.g., start / stop of operation). One stroke of the piston in the bore of valve V7 is typically required to flush or boost the fluidics system 1404. Thus, the linear resistance stepper valve V7 effectively operates as a flush pump, a boost pump, and / or a linear fluidic resistor. In contrast, the fluidics system 604 of FIG. 7 performs backflushing by pressurizing the fluidics system 604, turning on the flush pump 732, and applying pressure to force the sample fluid 210 back down the sample path.

[0179] 16, an exemplary data spreadsheet 1600 for parameters 1650 of the linear resistance stepper valve V7 is shown. The data spreadsheet 1600 includes four different setups (1601, 1602, 1603, 1604) having different piston positions of the linear resistance stepper valve V7 for the fluidics system 1404. Each setup (1601, 1602, 1603, 1604) is defined by parameters 1650 that conform to Equation 11 discussed herein.

[0180] The parameters 1650 of the Linear Resistance Stepper Valve V7 include, but are not limited to, temperature (Celsius), viscosity (Pascal-seconds), SIT diameter (mm), SIT length (mm), SIT resistance (Pascal-seconds / cubic millimeter), piston bore diameter D0 (mm), piston diameter D i(mm), piston bore length (mm), sheath valve resistance (Pascal-seconds / cubic millimeter), sample flow rate (microliters / minute), sheath flow rate (microliters / minute), head difference (mm), head pressure (Pascals), required sheath resistance (Pascal-seconds / cubic millimeter), required valve resistance (Pascal-seconds / cubic millimeter), and required length L (mm).

[0181] Changes to any of these parameters affect the others in a predictable manner. For comparison, all of the exemplary setups (1601, 1602, 1603, 1604) are configured with, among other parameters, temperature, viscosity, SIT diameter, SIT length, SIT resistance, piston bore diameter D0, piston diameter D1, and so on. i , and has the same value for piston bore length.

[0182] The data spreadsheet 1600 responds to a variable length L of overlap between the sidewall of the piston and the sidewall of the bore for, among other parameters, a constant sheath flow rate, a constant piston diameter D i , and a constant piston bore diameter D0, exemplary calculations are provided for an exemplary design of a linear stepper valve to provide a variable sample flow rate in a flow cytometer. For example, in data spreadsheet 1600, a given sheath flow rate is 10.50 ml / min, and a given piston diameter D i is 6.38 mm and the given piston bore diameter D0 is 6.47 mm.

[0183] Note that the linear resistance stepper valve V7 has a predictable amount of base sheath valve resistance. In the example of Figure 16, the base sheath valve resistance is 14.68 Pa·s / mm 3 Also, the linear resistance stepper valve V7 linearly adds a predictable amount of valve resistance whenever the length L is greater than zero. For example, in setup 1601, which has a length of zero, the valve resistance is 0.00 Pa·s / mm 3In setup 1602 with a length of 0.42 mm, the valve resistance is calculated to be 2.26 Pa·s / mm 3 For setup 1603 with a length of 4.16 mm, the required valve resistance is calculated to be 22.63 Pa·s / mm 3 For setup 1604 with a length of 10.41 mm, the required valve resistance is calculated to be 56.58 Pa·s / mm 3 Each resistor for each setup is approximately 5 times the length, thereby demonstrating linearity.

[0184] Thus, with this exemplary design, in setup 1601, for a desired sample flow rate of 10.00 μl / min, the required length L is 0.00 mm. In setup 1602, for a desired sample flow rate of 12.00 ml / min, the required length L is 0.42 mm. In setup 1603, for a desired sample flow rate of 30.00 ml / min, the required length L is 4.16 mm. In setup 1604, for a desired sample flow rate of 60.00 ml / min, the required length L is 10.41 mm.

[0185] During the machining (manufacturing) of the linear resistance stepper valve V7, the piston bore diameter D0 and piston diameter D i are two important parameters that can be changed as needed. The piston bore diameter D0 is i 16, there is a maximum available travel distance for piston 1508 into piston bore 1506 of about 12.70 mm. For greater granularity in controlling sheath resistance, piston bore diameter D0 and piston diameter D1 are adjusted to have piston 1508 use most (e.g., as much) of the maximum available travel distance. iIt is desirable to design (e.g., size) the linear resistance stepper valve V7 such that a longer length L tends to provide more accurate repeatability for the parameters of the linear resistance stepper valve V7.

[0186] Figures 17A and 17B show two different views of one embodiment of a linear resistance stepper valve V7. Figure 17A shows a cross-sectional view of the linear resistance stepper valve V7. Figure 17B shows a three-dimensional perspective view 1751 of the linear resistance stepper valve V7. Figure 18 shows an exploded three-dimensional view of the linear resistance stepper valve V7.

[0187] 17A and 17B and 18, the linear resistance stepper valve V7 includes, without limitation, a piston bore 1506, a seal 1704, a packing ring 1706, a piston 1508, a lead screw 1708, a home switch 1710, an isolation valve 1720, a valve head 1702, an outlet port 1504, an inlet port 1502, a homing pin 1712, a stepper motor 1512, a guide body 1714, a guide pin 1716, and a home switch 1718.

[0188] A stepper motor 1512 rotatably drives and is coupled to a lead screw 1708. The lead screw 1708 is threaded into and coupled to the piston 1508. The rotational motion of the lead screw 1708 generates translational motion of the piston 1508. The translational motion of the piston 1508 moves the piston 1508 a variable length L within the annular region of the piston bore 1506, as described with reference to FIG. 15. The translational motion of the piston 1508 can also be used to flush or boost fluid through components coupled to the valve V7, as described with reference to FIGS. 14 and 15.

[0189] Seal 1704 is in physical contact with piston 1508 and piston bore 1506. Seal 1704 physically prevents fluid entering inlet port 1502 from seeping out of piston bore 1506. Seal 1704 may also wash / wipe away salt that accumulates on piston 1508.

[0190] FIG. 18 shows the separate components that form linear resistance stepper valve V7, including, but not limited to, piston bore 1506, seal 1704, packing ring 1706, piston 1508, lead screw 1708, home switch 1710, home switch screw 1802, isolation valve 1720, isolation valve screw 1804, valve head 1702, outlet port 1504, inlet port 1502, homing pin 1712, stepper motor 1512, guide body 1714, guide pin 1716, and home switch 1718.

[0191] 14, the temperature of the components within the flow cytometer, including valve V7, and the fluids used within fluidics system 1404 can both change, such as from a change in room temperature. As the temperature changes, the viscosity changes for the fluids change depending on the piston bore diameter D0 or piston diameter D of linear resistance stepper valve V7. i The change in temperature is substantially more significant than the change in flow resistance of the linear resistance stepper valve V7. On the other hand, both the linear resistance stepper valve V7 and the sample path 210 are viscosity dominated (e.g., viscosity is important). Fortunately, both the valve V7 and the sample path 210 are affected approximately equally (or proportionally) by temperature. For example, the fluid resistances in the valve V7 and the sample path 210 change approximately equally (or proportionally) with temperature. Therefore, the effect of temperature change on both the valve V7 and the sample path 210 is self-compensating.

[0192] The SIT pathways and associated components can be made of plastic because their expansion / contraction (with temperature) is approximately equal or proportional. Therefore, the SIT pathways and associated components are all governed by similar thermal and resistance behavior. The SIT pathways and associated components are thereby thermally stable.

[0193] In contrast, the fluidics system 604 of Figure 7 is not viscosity-dominated. A substantial convective pressure drop can occur across the stepper pinch valve 730. The convective pressure drop is both viscosity-independent and viscosity-dominant. This independence can be problematic.

[0194] Advantageously, the fluidics system 1404 of FIG. 14 is more thermally stable than the fluidics system 604 of FIG. 7. Therefore, materials for the linear resistance stepper valve V7 can be selected to target resistance performance and repeatability during operation (e.g., targeting temperature / pressure effects a priori). The guide body 1714 and the valve head 1702 can include, for example, stainless steel and / or other materials. The material forming the piston 1508 can include, for example, ceramic, polytetrafluoroethylene (PTFE), and / or other materials. The material of the piston 1508 should be sufficiently slippery to allow salt to be removed, as salt accumulates on the piston 1508. For example, the fluidics system 1404 can flush / wipe away salt buildup by flushing the piston 1508 with a back seal 1704 applied to the piston 1508. Generally, materials for physically dependent components are selected to expand or contract at nominally similar rates (or nominally proportional rates) with changes in temperature and / or pressure.

[0195] Systems, methods, and devices for flow cytometry fluidics in a flow cytometer are described. In particular, a system is provided that includes a dual laser device and dual scattering channels to measure the velocity of particles within a core stream of a sample fluid. The first scattering channel detects first light scattering generated by particles passing through a first laser beam as they flow through the sample fluid. The second scattering channel detects second light scattering generated by particles passing through a second laser beam, the first and second laser beams being separated by a distance (L). The system also includes a stepper motor-adjusted flow control valve for controlling the ratio of sheath flow rate and sample flow rate in a flow path within the flow cell. The total flow rate of the sample fluid and the sheath fluid surrounding the sample fluid is controlled and thus kept constant by a feedback control system that controls a vacuum pump based on the differential pressure across the ends of the flow path within the flow cell.

[0196] According to some embodiments, the stepper motor-regulated flow control valve is a stepper motor-regulated pinch valve. According to other embodiments, the stepper motor-regulated flow control valve is a linear resistance stepper valve that applies a physical fluidic resistance to the flow of sheath fluid. In this case, the physical fluidic resistance regulates the flow rate of the sheath fluid, thereby regulating the flow rate of the sample fluid.

[0197] When implemented in software, the elements of embodiments of the present invention are essentially programs, code segments, or instructions that perform the necessary tasks. The programs, code segments, or instructions can be stored in a processor-readable medium or storage device that can be read and executed by a processor. The processor-readable medium can include any medium that can store information. Examples of processor-readable media include, but are not limited to, electronic circuits, semiconductor memory devices, read-only memories (ROMs), flash memories, erasable programmable read-only memories (EPROMs), floppy disks, CD-ROMs, optical disks, and magnetic disks. The programs or code segments can be downloaded via a computer network such as the Internet, an intranet, etc., and stored in the processor-readable medium or storage device.

[0198] Some portions of the preceding detailed descriptions may be presented in terms of algorithms and symbolic representations that perform operations on data bits within a computer memory. These algorithmic descriptions and representations are the tools used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art. An algorithm is herein generally conceived to be a self-consistent sequence of operations leading to a desired result. The operations are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities may take the form of electrical (e.g., current or voltage) or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, levels, elements, symbols, characters, terms, numbers, or the like.

[0199] However, all of these and similar terms are to be associated with, and are merely convenient labels applied to, the appropriate physical quantities. Unless otherwise specifically stated, and as is clear from the above discussion, it will be recognized that throughout the description, discussions utilizing terms such as "processing" or "computing" or "calculating" or "determining" or "displaying" refer to the actions and processes of a computer system, processing logic, or similar electronic computing device, which actions and processes automatically or semi-automatically manipulate and convert data represented as physical (electronic) quantities in the computer system's registers and memory into other data that are similarly represented as physical quantities in the computer system's memory or registers or other such information storage, transmission, or display device.

[0200] Additionally, embodiments of the present invention are not described with reference to any particular programming language, although it will be appreciated that a variety of programming languages can be used to implement the teachings of embodiments of the present invention as described herein.

[0201] The present disclosure contemplates other embodiments or objectives. It will be recognized that embodiments of the invention may be practiced by means other than those of the described embodiments, which are presented herein for purposes of illustration and not limitation. The specification and drawings are not intended to limit the exclusive scope of this patent document. It is noted that various equivalents to the specific embodiments discussed herein may also be practiced in accordance with the claimed invention. That is, while specific embodiments of the invention have been described, it is evident that many alternatives, modifications, substitutions, and variations will become apparent in light of the above description. Accordingly, it is intended that the present invention embrace all such alternatives, modifications, and variations that fall within the scope of the appended claims. The mere mention of a product, process, or method departing from one or more of the described exemplary embodiments does not mean that the product or process falls outside the scope (literal and / or otherwise legally permitted) of the claims.

Claims

1. 1. A device for controlling the flow of particles in a flow channel of a flow cytometer, comprising: a linear resistance stepper valve that applies a physical fluid resistance to a flow of a sheath fluid surrounding a flow of a sample fluid, the physical fluid resistance adjusting a flow rate of the sheath fluid and thereby adjusting a flow rate of the sample fluid, the linear resistance stepper valve comprising: an inner wall defining a piston bore; an inlet port for receiving the sheath fluid into the piston bore; an outlet port for delivering the sheath fluid from the piston bore; and a piston configured to enter the piston bore and move a length; a timekeeping device that generates a clock signal and a digital timestamp signal; a first register in communication with the timekeeping device, the first register storing a first peak digital timestamp based on the first scattering channel detecting a first peak of light scattered by a particle flowing through the first laser beam; a second register in communication with the timekeeping device, the second register storing a second peak digital timestamp based on a second scattering channel detecting a second peak in light scattered by the particle passing through a second laser beam; a digital mathematical logic device coupled to the first register and the second register that receives the first peak digital timestamp and the second peak digital timestamp associated with the particle, the digital mathematical logic device configured to determine a time difference between the first peak digital timestamp and the second peak digital timestamp; a storage device in communication with the digital mathematical logic device for storing and accumulating a plurality of particle velocities over a period of time; a second digital mathematical logic device coupled to the storage device and configured to determine an average particle velocity by adding the plurality of particle velocities together and dividing by a total number of particles; Equipped with the time difference is inversely proportional to particle velocity and contributes to generating a feedback control signal that controls core fluid flow rate in the channel; The mean particle velocity is a feedback control signal for the flow cytometer system to control the flow rate and ensure a high signal-to-noise ratio in particle detection through each of the two side scatter channels.

2. 10. The apparatus of claim 1, further comprising: a packetization device coupled to the digital mathematical logic device that receives the time differences and organizes the time differences together into data packets having information about the particles.

3. 10. The apparatus of claim 1, wherein the time difference contributes to generating the feedback control signal that controls a vacuum pump that varies a fluid flow rate in the flow path to maintain a constant average time difference and a constant average particle velocity.

4. 2. The apparatus of claim 1, wherein the digital mathematical logic device is further configured to determine a particle velocity of the particle by dividing the distance of the flow path between the position of the first laser and the position of the second laser by the time difference.

5. 10. The apparatus of claim 1, wherein the digital mathematical logic device is further configured to store and accumulate in the storage device a plurality of time differences for a plurality of particles over a period of time, and determine an average time difference by adding the plurality of time differences together and dividing by a total number of particles.

6. a storage device in communication with the digital mathematical logic device for storing and accumulating a plurality of timestamp differences over a period of time; a second digital mathematical logic device coupled to the storage device configured to determine an average timestamp difference by adding together the plurality of timestamp differences and dividing by a total number of particles; 2. The apparatus of claim 1, wherein the average timestamp difference is the feedback control signal of the flow cytometer system for controlling flow rates and ensuring a high signal-to-noise ratio in particle detection through each of two side scatter channels.

7. a first scattering channel including a first photodetector adjacent to the flow path for receiving light scattered by the particles flowing through the first laser beam, and a first gain amplifier coupled to the first photodetector for amplifying a signal from the first photodetector and generating a first pulse signal having the first peak; a first comparator coupled to the first gain amplifier for receiving the first pulse signal, the first comparator configured to compare an amplitude of the first pulse signal with a threshold and generate a first sample enable signal; a first analog-to-digital converter (ADC) coupled to the first gain amplifier to receive the first pulse signal and coupled to the first comparator to receive the first sample enable signal, the first analog-to-digital converter (ADC) configured, when enabled by the first sample enable signal, to periodically obtain digital samples of the amplitude of the first pulse signal in accordance with a clock signal; a second scattering channel including a second photodetector adjacent to the flow path for receiving light scattered by the particles flowing through the second laser beam, and a second gain amplifier coupled to the second photodetector for amplifying a signal from the second photodetector to generate a second pulse signal having the second peak; a second comparator coupled to the second gain amplifier for receiving the second pulse signal, the second comparator configured to compare an amplitude of the second pulse signal with the threshold and generate a second sample enable signal; a second analog-to-digital converter (ADC) coupled to the second gain amplifier to receive the second pulse signal and coupled to the second comparator to receive the second sample enable signal, the second analog-to-digital converter (ADC) configured, when enabled by the second sample enable signal, to periodically obtain digital samples of the amplitude of the second pulse signal in accordance with the clock signal; Furthermore, one digital sample of the digital samples of amplitude periodically obtained by the first ADC is a first peak amplitude associated with the first peak digital timestamp; 2. The apparatus of claim 1, wherein one of the digital samples of amplitude periodically obtained by the second ADC is a second peak amplitude associated with the second peak digital timestamp.

8. a first storage device coupled to the timekeeping device, the first ADC, and the first register, configured to receive and store a plurality of digital timestamps respectively associated with a plurality of digital amplitude samples of the first pulse signal associated with the particle passing through the first laser beam, the first dual-port storage device further configured to determine a first peak amplitude within the plurality of digital amplitude samples and select the associated timestamp as the first peak digital timestamp; a second storage device coupled to the timekeeping device, the second ADC, and the second register, configured to receive and store a plurality of digital timestamps respectively associated with a plurality of digital amplitude samples of the second pulse signal associated with the particle passing through the second laser beam, the second storage device being further configured to determine a second peak amplitude within the plurality of digital amplitude samples and select the associated timestamp as the second peak digital timestamp; The apparatus of claim 7 further comprising:

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