Removable circular nozzle for flow cytometer

Abstract

A circular nozzle assembly is disclosed. A cuvette nozzle subsystem (assembly) for a flow cytometry system is disclosed, comprising a cuvette assembly and a circular nozzle assembly selectively engaging the cuvette assembly. The cuvette assembly comprises a cuvette having a pocket and a flow channel, and a receptacle coupled to the cuvette within the pocket. The receptacle has a through-hole with a tapered conical portion and a cylindrical portion. The circular nozzle assembly comprises an O-ring gasket coupled to a nozzle body having a flow channel. The tapered conical portion of the nozzle body engages with the tapered conical portion of the through-hole to align the flow channels of the cuvette and the nozzle body with each other.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This patent application is a non-provisional patent application claiming the benefit of U.S. Provisional Patent Application No. 63 / 418,031, entitled "METHODS AND APPARATUS FOR REMOVABLE CIRCULAR NOZZLE IN FLOW CYTOMETERS," filed on October 20, 2022, by inventor Mikhail Blinkov, which is incorporated herein by reference for all intents and purposes.

[0002] This patent application incorporates by reference U.S. Patent Application No. 17 / 665,480, entitled "INTEGRATED COMPACT CELL SORTER," filed February 4, 2022 by inventors Glen Krueger et al., which is incorporated herein by reference for all intents and purposes. U.S. Patent Application No. 17 / 665,480 claims the benefit of U.S. Provisional Patent Application No. 63 / 172,072, entitled "INTEGRATED COMPACT CELL SORTER," filed April 7, 2021 by inventors Glen Krueger et al., which is incorporated herein by reference for all intents and purposes.

[0003] The disclosed embodiments relate generally to flow cytometer and cell sorter systems. [Background technology]

[0004] Flow cytometry and cell sorting involve the optical measurement of cells or particles of a test sample carried in a fluid stream. Cell sorting further sorts selected cells of interest into different containers (e.g., test tubes) for further use (e.g., testing) or enumeration. Laboratory instruments that accomplish these tasks are known as flow cytometers and cell sorters, respectively. Cell sorters may also be called sorting flow cytometers.

[0005] Cell sorters and flow cytometers are often configured with removable nozzles. This allows the nozzles to be periodically cleaned to avoid orifice clogging and cross-contamination between different samples being tested. Furthermore, different nozzles with different orifice diameters can be selected to accommodate different droplet sizes and drop delays between sample droplets of different biological samples. After periodic (e.g., daily) calibration of a flow cytometer with a selected nozzle, the nozzle may be removed multiple times during the same day. It is desirable for the removable nozzle to be reinserted in substantially the same position as when previously calibrated. This ensures that the droplet size and droplet delay (droplet quality) between sample droplets per sample run are substantially the same each time the removable nozzle is repositioned after cleaning.

[0006] Furthermore, for efficient droplet flow of sample liquid within the flow cytometer, it is desirable that the axes of the flow (fluid) channels and orifices within the nozzle be substantially aligned (concentric) with the axes of the flow channels within the cuvette. The channel alignment of these flow channels is desirably checked and set periodically (e.g., during initial assembly and reassembly). If the flow channels are misaligned, it is desirably to adjust the alignment of the nozzle and cuvette in the flow cytometer to substantially align the channels in order to improve droplet efficiency and droplet quality. Summary of the Invention

[0007] The embodiments are best summarized by the claims, however, a brief summary of some of the embodiments is provided here.

[0008] In one embodiment, a cuvette nozzle subsystem (assembly) for a flow cytometry system is disclosed. The cuvette nozzle subsystem includes a cuvette assembly and a circular nozzle assembly. The cuvette assembly includes a cuvette having a pocket and a flow channel, and a receptacle that is coupled to the cuvette within the pocket, the receptacle having a body with a through hole having a tapered conical portion and a cylindrical portion. A circular nozzle assembly selectively engages with the cuvette assembly. The circular nozzle assembly includes an O-ring gasket that is coupled to a nozzle body having a flow channel. The nozzle body has a tapered conical portion that engages with the tapered conical portion of the through hole in the receptacle to align the flow channels of the cuvette and the nozzle body with each other.

[0009] In another embodiment, a flow cytometer or cell sorter system is disclosed. The system includes a flow cell coupled in communication with a fluidics system to receive sheath fluid, wherein sample fluid carrying cells or particles flows through the flow cell and is surrounded by the sheath fluid. The flow cell includes: a flow cell body coupled around a droplet drive assembly to receive the sample fluid from a sample inlet tube; a cuvette coupled to a base of the flow cell body, the cuvette having a pocket and a cylindrical flow channel to receive the fluid flow of the sample fluid; a receptacle bonded within the pocket of the cuvette by adhesive, the receptacle having a body with a throughbore having a tapered conical portion; and a circular nozzle assembly selectively engageable with the receptacle and the cuvette. The circular nozzle assembly includes a nozzle body having a central flow channel and an O-ring gasket bonded to a top surface around the central flow channel. The nozzle body has an upper tapered conical portion that engages the tapered conical portion of the through-hole in the receptacle to align the cylindrical flow channel of the cuvette and the central flow channel of the nozzle body with one another.

[0010] In another embodiment, a method for circular nozzle assemblies in a flow cytometer or cell sorter system is disclosed, the method including: moving a first circular nozzle assembly upward into a pocket of a cuvette in a flow cytometer, further moving the first circular nozzle assembly upward to insert a tapered conical portion of the first circular nozzle assembly into a through-hole of a receptacle, and further moving the first circular nozzle assembly upward to engage the tapered conical portion of the first circular nozzle assembly with the tapered conical portion of the through-hole in the receptacle, bringing flow channels in the cuvette and the first circular nozzle assembly into alignment with one another.

[0011] In another embodiment, a method of joining a receptacle and a cuvette together for a subassembly of a flow cytometer or cell sorter is disclosed, the method including providing a pocket 1003 in a cuvette 406, applying a thin layer of adhesive to one or more portions of a top surface 1003T of the cuvette 406 within the pocket 1003, placing the receptacle in the pocket 1003 of the cuvette 406 such that a base of the receptacle engages one or more portions of the adhesive and the surface 1003T of the cuvette 406, with a through-hole in the receptacle surrounding a flow channel in the cuvette, and waiting a predetermined time (a waiting period or drying period) to allow the adhesive 1005 between the receptacle 1006 and the cuvette 406 to dry.

[0012] Various embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings. [Brief explanation of the drawings]

[0013] [Figure 1A] FIG. 1A is a basic conceptual diagram of a cell sorter system (sorting flow cytometer system) and a flow cytometer system. [Figure 1B] FIG. 1B is a front view of the compact cell sorter system with the various doors in a closed position. [Figure 1C] FIG. 1C is a front view of the compact cell sorter system with the various doors in an open position. [Figure 2A] FIG. 2A is a view of the droplet deposition unit (DDU) of the compact cell sorter system with the DDU door and sample entry door open. [Figure 2B] FIG. 2B is a view of the droplet deposition unit (DDU) of the compact cell sorter system with the DDU door and sample entry door open. [Figure 3A]FIG. 3A is a diagram of a fluidics bucket within the fluidics system of a compact cell sorter system. [Figure 3B] FIG. 3B is a diagram of a fluidics bucket within the fluidics system of the compact cell sorter system. [Figure 4A] FIG. 4A is a diagram of a flow cell within the fluidics system of a compact cell sorter system. [Figure 4B] FIG. 4B is a diagram of a flow cell within the fluidics system of the compact cell sorter system. [Figure 4C] FIG. 4C is a diagram of a flow cell within the fluidics system of a compact cell sorter system. [Figure 4D] FIG. 4D is a diagram of a flow cell within the fluidics system of a compact cell sorter system. [Figure 4E] FIG. 4E is a diagram of a flow cell within the fluidics system of the compact cell sorter system. [Figure 4F] FIG. 4F is a diagram of a flow cell within the fluidics system of a compact cell sorter system. [Figure 4G] FIG. 4G is a diagram of a flow cell within the fluidics system of a compact cell sorter system. [Figure 4H] FIG. 4H is an exploded view of the flow cell of the compact cell sorter system. [Figure 4I] FIG. 4I is an exploded view of the nozzle carriage assembly of the flow cell shown in FIG. 4H. [Figure 4J] FIG. 4J is a diagram of an example of left and right spring-loaded lever arms of a carriage linkage mechanism. [Figure 4K] FIG. 4K is a diagram of an example of left and right spring-loaded lever arms of a carriage linkage. [Figure 4L] FIG. 4L is a diagram of an example of left and right spring-loaded lever arms of a carriage linkage mechanism. [Figure 4M]FIG. 4M is a diagram of an example of left and right spring-loaded lever arms of a carriage linkage mechanism. [Figure 5A] FIG. 5A is a side view showing the movement of the flow cell carriage when the lever is raised. [Figure 5B] FIG. 5B is a side view showing the movement of the flow cell carriage as the lever descends. [Figure 6A] FIG. 6A is a cross-sectional view showing the movement of the flow cell carriage when the lever is raised. [Figure 6B] FIG. 6B is a cross-sectional view showing the movement of the flow cell carriage as the lever descends. [Figure 7A] FIG. 7A is a perspective view of a nozzle assembly within a flow cell of a compact cell sorter system. [Figure 7B] FIG. 7B is an exploded view of the nozzle assembly within the flow cell of the compact cell sorter system. [Figure 7C] FIG. 7C is an exploded view of the nozzle assembly within the flow cell of the compact cell sorter system. [Figure 7D] FIG. 7D is an exploded view of the nozzle assembly in the flow cell of the compact cell sorter system. [Figure 7E] FIG. 7E is a cross-sectional view of the nozzle assembly. [Figure 7F] FIG. 7F is an enlarged view of a portion of a cross-sectional view of the nozzle assembly. [Figure 8A] FIG. 8A is a diagram of the engagement / disengagement of the nozzle assembly with the flow cell of the compact cell sorter system. [Figure 8B] FIG. 8B is a perspective view of the flow cell showing the up and down movement of the carriage and nozzle assembly as the lever arm pivots. [Figure 8C] FIG. 8C is a perspective view of the flow cell showing the up and down movement of the carriage and nozzle assembly as the lever arm pivots. [Figure 9A] FIG. 9A is a perspective view of the engagement / disengagement of the nozzle assembly with the nozzle mount of the flow cell. [Figure 9B] FIG. 9B is a perspective view of the aligned nozzle assembly moving upward to engage the cuvette as the lever arm pivots. [Figure 9C] FIG. 9C is a perspective view of the aligned nozzle assembly moving upward to engage the cuvette as the lever arm pivots. [Figure 10A] FIG. 10A is an exploded side view of a cuvette nozzle assembly with a circular nozzle assembly. [Figure 10B] FIG. 10B is an inverted top perspective view of the cuvette nozzle assembly with the circular nozzle assembly disengaged from the tapered receptacle of the cuvette subassembly. [Figure 10C] FIG. 10C is an upside-down perspective view of the cuvette nozzle assembly with the circular nozzle assembly engaged in the tapered receptacle of the cuvette subassembly. [Figure 10D] FIG. 10D is an upside-down perspective cross-sectional view of the cuvette nozzle assembly. [Figure 10E] FIG. 10E is an inverted perspective view of the cuvette nozzle assembly from below. [Figure 11A] FIG. 11A is a perspective view of a circular nozzle assembly. [Figure 11B] FIG. 11B is a cross-sectional view of a circular nozzle assembly. [Figure 11C] FIG. 11C is a bottom view of the circular nozzle assembly. [Figure 11D] FIG. 11D is a top view of the circular nozzle assembly. [Figure 12A] FIG. 12A is a top perspective view of a tapered conical receptacle. [Figure 12B] FIG. 12B is a side view of a tapered conical receptacle. [Figure 12C] FIG. 12C is a bottom view of the tapered conical receptacle. [Figure 12D] FIG. 12D is a top view of a tapered cone receptacle. [Figure 13] FIG. 13 is an enlarged side view of the cuvette nozzle assembly in an upside-down position, with the circular nozzle assembly engaged with the tapered receptacle of the cuvette subassembly. [Figure 14A] FIG. 14A is a perspective view of an alignment fixture assembly that includes an alignment jig (fixture) that aligns and secures a tapered conical receptacle to a cuvette to form a cuvette subassembly. [Figure 14B] FIG. 14B is a perspective view of an alignment fixture assembly that includes an alignment jig (fixture) that aligns and secures a tapered conical receptacle to a cuvette to form a cuvette subassembly. [Figure 14C] FIG. 14C is an enlarged side view of the alignment fixture and cuvette subassembly. [Figure 15] FIG. 15 is a side view of the entire alignment system that aligns and secures the tapered cone receptacle to the cuvette to form the cuvette subassembly. [Figure 16A] FIG. 16A is an enlarged bottom view of the pocket of the cuvette subassembly showing the misalignment between the circular nozzle and the flow channel in the cuvette. [Figure 16B] FIG. 16B is an enlarged bottom view of the pocket of the cuvette subassembly showing proper alignment between the circular nozzle and the flow channel in the cuvette. [Figure 17A] FIG. 17A is a flow chart of the steps in the alignment process for aligning a circular nozzle and a flow channel in a cuvette. [Figure 17B] FIG. 17B is a flow chart of the steps in the alignment process for aligning the circular nozzle and the flow channel in the cuvette. [Figure 18A] FIG. 18A is an illustration of a poor quality droplet ejected from a nozzle. [Figure 18B] FIG. 18B is an illustration of a poor quality droplet ejected from a nozzle. [Figure 19A]FIG. 19A is an image of a high quality droplet ejected from the nozzle. [Figure 19B] FIG. 19B is an image of a high quality droplet ejected from the nozzle. DETAILED DESCRIPTION OF THE INVENTION

[0014] It will be appreciated that some or all of the figures are for illustrative purposes and do not necessarily depict the actual relative sizes or locations 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.

[0015] 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 so as not to unnecessarily obscure aspects of the embodiments. It is important to note that directional terminology (e.g., "left," "right," "bottom," "top," "upper," "lower," etc.) is for illustrative purposes. Depending on the orientation defined for a given embodiment, other directional terminology may more accurately describe a given embodiment. Various sections of this specification are provided for organizational purposes; however, many details and advantages apply across multiple sections.

[0016] [System Overview] 1A is a basic conceptual diagram of a cell sorter system (sorting flow cytometer) 10. The five main subsystems of system 10 include an excitation optics system 12, a fluidics system 14, an emission optics system 16, an acquisition system 18, and an analysis system 20. Fluidics system 14 can include a sample loading system (not shown), an interrogation system 28, a cell sorting system 33, and a droplet receiving system 29. Generally, "system" and "subsystem" include hardware devices (electrical, mechanical, and electromechanical), software devices, or a combination thereof.

[0017] The excitation optics system 12 includes multiple (e.g., two to five) excitation channels 22A-22N, each having, for example, a different laser device 23A-23N and one or more optical elements 24-26 that direct the different laser light to spaced-apart optical interrogation regions 30A-30N along a line within a flow channel 27 of a flow cell 28. Exemplary optical elements of the one or more optical elements 24-26 include optical prisms and optical lenses. The excitation optics system 12 illuminates an optical interrogation region 30 within the flow cell 28. The fluidics system 14 conveys a fluid sample 32, surrounded by a sheath fluid, through each of the multiple optical interrogation regions 30A-30N within the flow cell / flow channel.

[0018] Emission optics system 16 includes multiple detector arrays 42A-42N, each of which includes, for example, one or more optical elements 40, such as optical fibers and one or more lenses that direct fluorescence and / or (forward, side, back) scattered light to various electro-optical detectors (transducers), including a side scatter channel (SSC) detector in each array and multiple (e.g., 16, 32, 48, 64) fluorescence wavelength range optical detectors, such as a first fluorescence optical detector (FL1) that receives fluorescence in a first wavelength range, a second fluorescence optical detector (FL2) that receives fluorescence in a second wavelength range, a third fluorescence optical detector (FL3) that receives fluorescence in a third wavelength range, a fourth fluorescence optical detector (FL4) that receives fluorescence in a fourth wavelength range, a fifth fluorescence optical detector (FL5) that receives fluorescence in a fifth wavelength range, and so on up to an Nth fluorescence optical detector (FLN) that receives fluorescence in an Nth wavelength range. Each detector array 42A-42N receives light corresponding to cells / particles and / or one or more fluorescent dyes attached thereto that are excited by different laser light impinging on them at interrogation regions / points 30A-30N along the flow channel 27 of the flow cell 28, respectively, via a corresponding one of the plurality of lasers 23A-23N. The emission optics system 16 collects photons emitted or scattered from the passing cells / particles and / or fluorescent dyes attached thereto. The emission optics system 16 directs and focuses these collected photons onto electro-optical detectors SSC, FL1, FL2, FL3, FL4, and FL5 in each detector array via fiber optic cables 39, one or more lenses 40, and one or more mirrors / filters 41, etc. The electro-optical detector SSC is a side scatter channel detector that detects light scattered from the cells / particles. Electro-optical detectors FL1, FL2, FL3, FL4, and FL5 are fluorescence detectors that may include bandpass or longpass filters to detect different specific fluorescence wavelength ranges from different fluorochromes excited by different lasers. Each electro-optical detector converts photons into electrical pulses and sends the electrical pulses to acquisition (electronics) system 18.

[0019] For each detector array 42A-42N, the acquisition (electronics) system 18 includes one or more analog-to-digital converters 47A-47N and one or more digital storage devices 48A-48N that can provide multiple detector channels (e.g., 16, 32, 48, or 64 channels) of spectral data signals. The spectral data signals can be signal processed (e.g., digitized by an A / D converter), time-stamped, and packetized together by a packetizer 52 into data packets corresponding to each cell / particle in the sample. These data packets for each cell / particle can be transmitted by the acquisition (electronics) system 18 to the analysis system 20 for further signal processing (e.g., time-domain to wavelength-domain conversion / transformation) and overall analysis. Alternatively, or in addition, the time-stamped digital spectral data signals from each detected channel can be transmitted directly to the analysis system 20 for signal processing.

[0020] The analysis system 20 includes a processor, memory, and data storage for storing data packets of time-stamped digital spectral data associated with detected cells / particles in the sample. The analysis system 20 further includes software having instructions executed by the processor to convert / transform data from the time domain to data in the wavelength / frequency domain and stitch / integrate the data together to provide an overall spectrum for cells / particles / dyes excited by different lasers and detected by the detector array. Detection of cell / particle types through one or more fluorescent dyes attached to the cells / particles allows for cell / particle counting in samples processed by a flow cytometer and / or cell sorter.

[0021] In some cases, it may be desirable to sort cells in a sample for further analysis by a cell sorter (sorting flow cytometer). Accordingly, the spectral data signals may be processed by a real-time sort controller 50 in the acquisition (electronics) system 18, and the spectral data signals may be used to control a sorting system 33 to sort cells or particles into one or more test tubes 34. In this case, the sorting system 33 communicates with and receives control signals from the real-time sort controller 50 in the acquisition (electronics) system 18. Instead of test tubes 34, the spectral data signals may be processed by the real-time sort controller 50 in the acquisition (electronics) system 18, and the spectral data signals may be used to control both the sorting system 33 and the droplet deposition system 29 to sort cells or particles into wells 35 of a moving capture tray / plate. In this case, both the droplet deposition system 29 and the sorting system 33 communicate with and receive control signals from the acquisition (electronics) system 18. In an alternative embodiment, the analysis system 20 can generate control signals from analysis of the spectral data signals to sort different cells / molecules and control the sorting system 33 and droplet deposition system 29 to capture droplets of sample with cells / particles into one or more of a plurality of wells 35 in a capture tray / plate.

[0022] U.S. patent application Ser. No. 15 / 817,277, now issued as U.S. Patent No. 10,871,438, entitled "FLOW CYTOMETERY SYSTEM WITH STEPPER FLOW CONTROL VALVE," filed Nov. 19, 2017, by David Vrane; U.S. patent application Ser. No. 15 / 659,610, entitled "COMPACT DETECTION MODULE FOR FLOW CYTOMETERS," filed July 25, 2017, by Ming Yan et al.; and U.S. patent application Ser. No. 15 / 942,430, entitled "COMPACT MULTI-COLOR FLOW CYTOMETER HAVING COMPACT DETECTION MODULE," filed March 30, 2018, by Ming Yan et al., each discloses exemplary flow cytometer systems and subsystems, all of which are incorporated herein by reference for all intents and purposes. U.S. Patent No. 9,934,511, entitled "Rapid Single Cell Based Parallel Biological Cell Sorter," issued June 19, 2016 to Wenbin Jiang, discloses a cell sorter system and is incorporated herein by reference for all intents and purposes.

[0023] [Compact cell sorter] FIG. 1B shows a front view of the integrated compact cell sorter system 100. The integrated compact cell sorter system 100 includes a chassis / frame 101 (see FIG. 1C) that supports the various systems and subsystems of the cell sorter. A fluidics panel / door 102, a flow cell door 111, a droplet deposition unit (DDU) door 112, and a sample entry door 113 are pivotally coupled to the chassis / frame 101 to cover and seal the various chambers of the cell sorter system. One or more side panels 114 are used to more securely cover other portions of the chassis / frame and internal subsystems. A fluidics input / output panel 104 connects the cell sorter system 100 to external fluid tanks and an external gas supply, such as a pressurized air supply.

[0024] 1C, a front view of the integrated, multiplexed cell sorter system 100 is shown with the doors open and the panels removed. The fluidics panel / door 102, flow cell door 111, DDU door 112, and sample entry door 113 of the integrated, compact cell sorter system 100 are pivoted to the open position about their hinges to allow visibility of the various systems and subsystems of the cell sorter system. The integrated, compact cell sorter system 100 includes a fluidics bucket 120 (part of the fluidics system), a deflection chamber 122, a flow cell 124, a sample pressure chamber 126, a droplet deposition unit (DDU) chamber or collection chamber 128, a sample entry station (SIS) 130, and a sorting and collection camera 132. The sample entry door 113 has a window 134 through which the sample tubes can be viewed when attached to the SIS 130. The DDU door 112 has a sorting and collection camera 132 that can watch droplets deflecting left and right from the slot in the deflection chamber 122 fall into the DDU chamber 128 and collect in test tubes or wells in a well plate.

[0025] The fluidics bucket 120 (part of the fluidics system) includes a bubble remover that eliminates air bubbles in the sheath fluid. The fluidics bucket 120 is further discussed with reference to Figures 3A and 3B. Pressure is applied to the fluidics system to encourage the flow of the sheath fluid and sample biological fluid.

[0026] A flow cell 124 is coupled in communication with the fluidics bucket 120 for receiving the sheath fluid. The sample biological fluid, along with cells or particles, passes through the flow cell 124 and is surrounded by the sheath fluid. The flow cell 124 is further discussed with reference to Figures 4A-4G.

[0027] The deflection chamber 122 is below the flow cell 124 and receives droplets of sample biological fluid and sheath fluid exiting the flow cell 124. The deflection chamber 122 selectively deflects one or more charged droplets away from the central flow path along one or more deflection paths. The deflection chamber 122 is further discussed with reference to Figures 16A and 16B.

[0028] A droplet deposition unit (DDU) chamber / system 128 communicates with the deflection chamber 122 for receiving selectively deflected droplets in the sample biological fluid stream carrying one or more biological cells or particles and depositing them into one or more containers. The DDU chamber 128 is further discussed with reference to Figures 2A and 2B.

[0029] In one embodiment, the flow cell 124 comprises a flow cell body coupled in communication with a fluidics system to receive sheath fluid, the flow cell body having a charging port for charging droplets, the flow cell body having a chamber with a cylindrical portion and a funnel-shaped portion, the funnel-shaped portion forming a fluid stream of sample liquid surrounded by sheath fluid exiting a bottom opening; and a droplet drive assembly coupled to the flow cell body, the droplet drive assembly including a glass sample injection tube (SIT) inserted into the chamber of the flow cell body and having a first end located in the funnel-shaped portion of the chamber, the glass sample injection tube The present invention includes a droplet drive assembly having a second end coupled in communication with a fluidics system for receiving a sample liquid and injecting the sample liquid into a funnel-shaped portion of the chamber, and a cuvette coupled to the base of the flow cell body, the cuvette having a flow channel adjacent to a bottom opening of the flow cell body, the cuvette receiving a fluid stream of sample liquid surrounded by a sheath liquid exiting the bottom opening, the cuvette being transparent to light and allowing the sample liquid to be interrogated within the flow channel by a plurality of different lasers to determine a plurality of different types of cells or particles in the sample liquid.

[0030] In one embodiment, the flow cell 124 comprises a flow cell body coupled around the droplet actuation assembly to receive sample liquid from a sample inlet tube, the flow cell body coupled in communication with the fluidics system to receive sheath liquid, the flow cell body having a charging port for charging droplets, the flow cell body having a funnel-shaped portion that forms a fluid stream of sample liquid surrounded by sheath liquid flowing out of an opening, and a cuvette coupled to the base of the flow cell body, the cuvette having a channel that receives the fluid stream of sample liquid surrounded by sheath liquid flowing out of the opening, the cuvette being transparent to light and allowing the sample liquid to be interrogated within the channel by a plurality of different lasers to determine a plurality of different types of cells or particles present therein.

[0031] In one embodiment, the flow cell 124 further comprises a nozzle assembly that selectively engages the cuvette, the nozzle assembly having a nozzle and an O-ring around the nozzle that selectively presses against the face of the cuvette around the channel, the nozzle receiving a sample stream from the cuvette and forming sample droplets that flow out of the nozzle assembly; a carriage assembly that is slidably coupled to the flow cell body and slidably receives the nozzle assembly; and a linkage that is pivotally coupled to the carriage assembly and the flow cell body, the linkage comprising a lever arm for selectively engaging the nozzle with the cuvette to receive the fluid stream and selectively disengaging the nozzle from the cuvette for repair or replacement of the nozzle.

[0032] In one embodiment, the flow cell 124 further comprises a lever hinge formed to be statically coupled to the flow cell body, a carriage release lever rotatably coupled to the lever hinge, and two lever arms rotatably coupled to the carriage release lever and a carriage plate of the carriage assembly, wherein the two lever arms, carriage plate, carriage release lever, and lever hinge have a dynamic linkage mechanism that enables the carriage assembly to maintain vertical movement along the central axis.

[0033] In one embodiment, the flow cell 124 further comprises: a nozzle assembly having a nozzle handle with a body having a gripping end and a nozzle end, the body having a through-hole between its top and bottom surfaces near the nozzle end, a partial gland in the top surface extending around the through-hole, the partial gland having a slot extending from the through-hole to the nozzle end of the nozzle handle; a nozzle insert disposed within a portion of the through-hole in the body of the nozzle handle, the nozzle insert having a circular body with a central nozzle orifice concentric with the through-hole for flowing droplets of sample liquid, and a beveled ring extending from the circular body at its top surface; and a gasket disposed within the partial gland in contact with the beveled ring of the nozzle insert and extending partially over the top surface of the nozzle insert and the top surface of the nozzle handle, the gasket providing a seal around the central nozzle orifice, the slot extending from the partial gland to the nozzle end facilitating removal of the gasket.

[0034] In one embodiment, the DDU system 128 comprises a case or housing having an open face surrounded by an edge of the case, the case forming a portion of the containment chamber, the case having a top opening aligned with the deflection chamber for receiving selectively deflected droplets in the stream of sample biological fluid into one or more receptacles in the containment chamber; a seal attached around the edge of the case; one or more hinges coupled to the bottom of the case; and a door coupled to the one or more hinges, the door pivoting about the one or more hinges, the door pressing against the seal when closed to seal the containment chamber from the external environment.

[0035] In one embodiment, the DDU system 128 includes an electromagnetic lock comprising at least one electromagnet attached to the case and a metal latch coupled to the interior surface of the door, the metal latch being attracted to the at least one electromagnet when the door is closed and the at least one electromagnet is energized.

[0036] In one embodiment, the DDU system 128 includes a magnetic lock that includes at least one magnet attached to the case and a metal latch coupled to the inside surface of the door, the metal latch being attracted to the at least one magnet when the door is closed.

[0037] [DDU Chamber] 2A shows a portion of the deflection chamber 122 with door 222 open. The DDU chamber 128 of the cell sorter 100 can be seen with both doors 112 and 113 pivoted to the open position. An opening in the rear wall 208 of the DDU chamber 128 reveals the input air filter 204I and output air filter 204O mounted in a tunnel leading to the air conditioning chamber. Behind the wall 208 are one or more fans and at least one heating / air conditioning element to pump air through the air filters and maintain a desired temperature range for the samples in the SIS 130 and the sorted cells / molecules in the chamber 128.

[0038] At the base of the DDU chamber 128 is a separation plate 206, which separates the drive mechanism below the separation plate from the DDU chamber 128. Below the separation plate 206 is a magnetic control mechanism that controls the movement of the magnetic coupling puck 210, shown in FIG. 2B. The magnetic loading system for the DDU chamber and magnetic coupling puck 210 is disclosed in U.S. Provisional Patent Application No. 63 / 146,562, entitled "LOADING SYSTEM WITH MAGNETICALLY COUPLED SAMPLE MOVER FOR FLOW CYTOMETRY AND CELL SORTER SYSTEMS," filed February 5, 2021, by Babak Honaryar et al., which is incorporated herein by reference for all intents and purposes. The movement of the magnetic coupling puck 210 is controlled by the magnetic loading system below the separation plate 206.

[0039] FIG. 2B shows seals 212 attached along the edges of the DDU chamber 128 and sample loading station 130 to provide an air-resistant seal when the DDU door 112 and sample door 113 are closed. The DDU door 112 has a ledge 133 (shown in FIG. 1C ) that depresses the top seal portion 212T when closed. Other portions of the seal 212, such as the bottom 212B and sides 212S, 212L, are pressed by the doors 112 and 113 and compressed against the edges of the DDU chamber 128. When the doors are closed, the DDU chamber 128 is sealed off from the ambient air of the environment (e.g., laboratory) in which the cell sorter 100 is located. Additionally, the DDU chamber 128 and SIS 130 are subjected to a negative pressure via a vacuum to further help prevent cells / molecules / gases from leaking from the cell sorter into the ambient air of the environment, such as a laboratory.

[0040] The DDU door 112 and sample entry door 113 provide a good seal isolating the DDU chamber 128 from the rest of the flow cytometer / cell sorter 100 and the surrounding environment. Sample droplets sorted and captured within the DDU chamber 128 may require a temperature-controlled environment to maintain their integrity. Additionally, captured cells may be pathogens that are undesirable for aerosolization and escape into the environment. Therefore, with a magnetic loading system and sealed doors, the cell sorter can provide the DDU chamber 128 with an integrated filtration system and temperature-controlled environment.

[0041] [Fluidics Bucket] 3A and 3B show various views of the fluidics bucket 120, which is part of the fluidics system of the cell sorter system 100. In FIG. 3B, the fluidics bucket 120 includes a sample regulator 301 and a sheath regulator 302, which control the fluidic pressure of the sample fluid and the sheath fluid, respectively. The fluidics bucket 120 also includes a degasser switch 304 and a degasser pump 305 to provide air pressure so that a degasser 306 can remove air bubbles from the sheath fluid. The fluidics bucket also includes an aspirator pump 310 that draws waste out of the cell sorter system through a waste output port 334. A valve manifold 312 includes multiple valves that control the fluidics system, as well as a sample transducer 315 and a sheath transducer 316. The fluidics input / output panel 104 includes a supply air input 331, a sheath air output 332, a sheath fluid input 333, and a waste output 334. The sheath fluid 333 flows through a sheath filter 320 before entering the flow cytometer system. The fluidics bucket 120 includes a pressure switch that controls the opening pressure of the sample pressure chamber. An aspirator pump maintains a vacuum in a reservoir below the valve manifold 312.

[0042] [Flow cell assembly] Figures 4A-4G show various views and components of the flow cell assembly 124. In Figure 4A, the flow cell 124 has a ground connection 400 to a metal surface to protect the sample liquid from charges generated by the deflection unit and to remove any charges that may already be present.

[0043] 4B, flow cell 124 includes droplet actuation assembly 402, nozzle assembly 450, nozzle carriage assembly 442, and carriage release lever 441 of flow cell linkage 440. Flow cell 124 has multiple optical components including droplet camera 412, droplet strobe assembly 411, forward scatter assembly 413, and final focus lens 414. Final focus lens 414 can be focused by final focus adjustment 415. Droplet actuation assembly 402 has sample input port 408 that receives a hose or pipe carrying sample liquid.

[0044] Referring now to FIG. 4C, the fluid ports of the flow cell 124 are shown. The flow cell 124 receives sample fluid through the sample inlet port 408. The flow cell 124 receives sheath fluid through the sheath entry port 418. The flow cell 124 surrounds the flow of sample fluid with the sheath fluid. The flow cell 124 includes a conductive drain port fitting 419 that threads onto the drain port of the flow cell body 404 to expel fluid from the chamber within the flow cell and to impart an electrical charge to droplets of sample fluid with cells / particles. Both electrical wires and hoses are coupled to the conductive drain port fitting 419. The electrical wires communicate with the sort controller to receive signals synchronized with the droplets. Over time, the signal can be ground, one or more levels of positive charge voltage (e.g., +150, +300), or one or more levels of negative charge voltage (e.g., -150, -300) to keep the droplet uncharged, charge it positively, or charge it negatively, respectively.

[0045] 4D , there is shown a side cross-sectional view of flow cell 124. Flow cell 124 includes flow cell body 404, droplet drive assembly 402, cuvette 406, linkage assembly 440, carriage assembly 442, and nozzle assembly 450 including nozzle 704. Linkage assembly 440 includes carriage release lever 441 that pivots to move nozzle assembly up and down relative to cuvette 406. Droplet drive assembly 402 includes sample inlet tube 422.

[0046] The flow cell body 404 has a top surface, a bottom surface, a left surface, a right surface, a front surface, and a back surface. At the top surface, the flow cell body has a top chamber opening that leads into the chamber of the flow cell body 404. The droplet drive assembly (including the sample inlet tube) is mounted within the chamber through the top chamber opening. The flow cell body receives sample fluid from the sample inlet tube of the droplet drive assembly. At one side (e.g., the left side), the flow cell body has an input port that couples in communication with the fluidics system of the cytometer to receive sheath fluid. At the opposite side (e.g., the right side), the flow cell body has an output port aligned with the input port. The pressures of the sheath fluid and the sample fluid are independently controlled to achieve a desired flow rate at which the sample fluid, surrounded by the sheath fluid, leaves the chamber and enters the flow channel 906 of the cuvette 406.

[0047] The flow cell body 404 has an opening or pocket on the back. The pocket receives the cuvette 406 so that the flow channel 906 is aligned with the droplet stream from the bottom opening in the flow cell body chamber. Most of the cuvette 406 is obscured from the front by the opaque body of the flow cell 404 and the nozzle assembly attached to the carriage assembly and mount. The pocket is open on the left and right sides, allowing laser light from one or more lasers to pass through the sides of the cuvette and strike cells / particles flowing within the flow channel. Laser light can enter the cuvette from one side and be collected on the other side by a fiber optic or forward scatter detector.

[0048] The base or bottom of the flow cell body 404 also has a small cutout (an upwardly arching cutout) from the front to the back. Because the cuvette is highly concealed, the small cutout allows a microscope examination instrument to be inserted through the front of the flow cell body to view the flow channel within the cuvette 406 from the front of the flow assembly 124.

[0049] A large cutout in the base of the flow cell body allows the nozzle assembly 450 to fit in a mount 452 below the cuvette 406. The large cutout also allows the nozzle assembly 450 to move up and down by a linkage and carriage assembly to get under the cuvette.

[0050] Laser light from one or more lasers is directed to one or more interrogation regions within the flow channel of the transparent cuvette, exciting cells / particles and / or one or more fluorescent dye markers attached thereto flowing through. On one side, the flow cell 124 further comprises one or more objective lenses 460A and 460B for capturing light (e.g., reflected light, scattered light, fluorescent light) from the cells / particles and / or one or more fluorescent dyes attached thereto. On the other side, the one or more objective lenses 460A and 460B can launch the captured light into a fiber optic cable.

[0051] To support movement of the linkage 440 and carriage assembly 442, the flow cell body 404 can include a plurality of threaded openings on its front surface. The threaded openings can receive threaded fasteners through holes in the linear slide rail 446 to attach the linear slide rail 446 to the front surface of the flow cell body. The linear slide rail 446 is used to slide the nozzle carriage assembly 442 up and down relative to the release lever 441 and linkage 440. The flow cell body can further include a shallow oval opening on its front surface to receive the spring 427 and detent 461 (a spring-loaded detent) to hold the position of the release lever 441, the linkage, the carriage assembly, and the nozzle assembly. The openings are oval so that the spring and detent can be moved up and down by adjusting the position of the hinge bracket 443.

[0052] 4E, a front cross-sectional view of flow cell 124 is shown. Flow cell 124 includes a flow cell body 404 that receives droplet drive assembly 402. Nozzle assembly 450 slides within a mount 452 that is coupled to the carriage assembly. Sample inlet tube 422 is preferably formed from glass to avoid surface etching in the presence of currents in the sheath liquid that charge the droplets and vibrations of the droplet drive that separate the droplets, which can cause leakage. Droplet drive assembly 402 includes a sample inlet 408 that receives sample liquid.

[0053] 4F, a cross-sectional view of flow cell 124 is shown through drain / charging port 419 with conductive hose fitting and sheath inlet port 418 with hose fitting. Sample inlet tube 422 is located in the center of the chamber within flow cell body 404. Flow cell 124 includes a back-focusing adjustment 463 for one or more objective lenses.

[0054] 4G shows a side view of flow cell 124. The central optical axes of objective lenses 460A and 460B are shown aligned to receive light from the cuvette. Objective lens mount 461 ensures that objective lenses 460A and 460B remain aligned. Flow cell body 404 is opaque to prevent light from other sources, such as the ambient light, from being captured by objective lenses 460A and 460B.

[0055] Nozzle assembly 450 slides in and out of mount 452 to service or repair nozzle assembly components or to replace nozzle openings with different diameters. The nozzle of the nozzle assembly receives a sample stream of fluid from the cuvette and forms droplets, preferably each with a single cell / particle to be sorted.

[0056] [Flow cell link mechanism and nozzle carriage] 4H-4L show various views and components of the flow cell linkage 440 and nozzle carriage assembly 442 of the flow cell assembly 124 of the cell sorter system 100. Figures 5A and 5B and 6A and 6B show side and cross-sectional views, respectively, of the flow cell assembly 124 to illustrate the operation of the flow cell linkage 440 and nozzle carriage assembly 442.

[0057] 4H, there is shown an exploded view of flow cell 124. Flow cell 124 comprises a flow cell linkage 440 and a nozzle carriage assembly 442. Nozzle assembly 450 slides in and out of mount 452. An exploded view of nozzle carriage assembly 442 is shown in FIG.

[0058] Flow cell linkage 440 has one or more links including carriage lever 441, left and right spring-loaded lever arms 444L and 444R, and nozzle carriage assembly 442, pivotally coupled together at pivot points by pivot shafts 445A-445C. Each of pivot shafts 445A-445C may include a washer along the shaft between the lever arm and pivot openings 447A, 447C, and 447F. Each of pivot shafts 445A-445C is retained within the pivot openings by circlips (retaining fasteners) 449.

[0059] The carriage lever 441 is pivotally mounted by a shaft 445C at a pivot point opening 447B in a projection extending from the lever to a pair of pivot point openings 447C in the arms of the lever hinge bracket 443.

[0060] A top pivot point opening 447D in each of the left and right lever arms 444L and 444R is pivotally coupled to the lever 441 at pivot point opening 447A by a shaft 445A. A bottom pivot point opening 447E in each of the left and right lever arms 444L and 444R is pivotally coupled to the nozzle carriage assembly 442 at pivot point opening 447F by a pivot shaft 445B. The nozzle carriage assembly 442 is slidingly coupled to a linear slide rail 446 that is attached to the flow cell body 404 by one or more fasteners (e.g., threaded screws or bolts).

[0061] In operation, carriage lever 441 pivots about pivot point opening 447B, causing the tops of lever arms 444L and 444R to raise or lower through shaft 445A at pivot point openings 447A, 447D. This translates to linear motion through the lever arms at bottom pivot point opening 447E. Linear motion in the lever arms is translated into linear motion in carriage assembly 442 by shaft 445B, which passes through bottom pivot openings 447E in lever arms 444L and 444R and pivot opening 447F in nozzle carriage assembly 442. When nozzle assembly 450 slides into mount 452, carriage assembly 442 can raise and lower the nozzle assembly to engage and disengage from cuvette 406.

[0062] The lever arms 444L and 444R are spring loaded between the upper and lower portions to ensure that the proper force is applied upward onto the nozzle assembly 450. This compresses the O-ring and ensures that it seals properly against the surface of the cuvette 406.

[0063] The flow cell linkage 440 is adjustable upward and downward by a hinge bracket 443. The hinge bracket 443 has a pair of elongated openings 460 on either side of a flange that attaches to the flow cell 404. A pair of screws or bolts (not shown) are inserted through the elongated openings 460 into threaded openings in the flow cell 404. The elongated openings 460 allow the bracket 443 to shift up or down around itself when the pair of screws or bolts are loosened. Moving the bracket 443 adjusts the flow cell linkage 440, including the carriage assembly 442, up or down.

[0064] Flow cell linkage 440 further comprises a spring-loaded lever detent 461, one end of which is inserted into an opening in flow cell 404, which can be coupled against spring 427 (see FIG. 4D). As shown in FIG. 4D, the opposite end of lever detent 461 rides up against rear cam 425 on lever hinge 441 to maintain flow cell linkage 440 in either the up or down position.

[0065] 4I, an exploded view of nozzle carriage assembly 442 is shown. Nozzle carriage assembly 442 includes carriage plate 465, linear bearing 464, nozzle mount 452, clamp plate 466, flat washer 467, lock washer 468, threaded bolt 469, and alignment tube 470, which are assembled together. Threaded bolt 469 is inserted through lock washer 468, flat washer 467, through hole 471C in clamp plate 466, hole 471D in nozzle mount 452, and the hollow cylinder inside alignment tube 470. The threads of bolt 469 thread into threaded hole 471E in the base of carriage plate 465, holding mount 452 coupled to the plate. Fasteners such as metal screws are inserted through a plurality of through holes 471A in the front face of carriage plate 465 and thread into threaded holes 471B in linear bearings 464 to join the plate and bearings together.

[0066] Linear bearing 464 includes a pair of guide rails 474 on its rear surface so that it slides along linear slide rails 446, shown in Figure 4H. The front surface of carriage plate 465 includes a pivot opening 447F that receives shaft 445B. A rectangular portion of the carriage plate extends from the front surface of the plate to form pivot opening 447F.

[0067] A ground wire lug 479 coupled to a ground wire is attached by fasteners near the center of the front of the carriage plate 465 to electrically ground the carriage assembly 442 .

[0068] 4J-4M, various views of lever arms 444L and 444R are shown. Each of lever arms 444L and 444R may include at least one small side cutout that allows the lever arm to pass over the end of shaft 445C. Each of the lever arms may also include a back cutout adjacent to the side cutout.

[0069] 4J shows rear notches 474 in each of lever arms 444L and 444R. The rear notches provide clearance for the heads of the mounting screws of bracket 443.

[0070] Figure 4K shows the spring-biased assembly of each lever arm. Figure 4K also shows through-holes 447D and 447E. Bolt 480 holds spring 476 and the upper and lower portions of each lever arm, which are spring-biased relative to one another. Bolt 480 has a shaft 477 with a smaller threaded portion 479 that threads into a threaded opening 478 in the upper portion until the larger shaft abuts the underside of the upper portion.

[0071] As shown in Figure 4L, the shaft of bolt 480 is inserted through a spring into an opening 481 at one end of the lower section. Bolt 480 can have a hex head, socket head, screw head, or other type of head that can be turned by inserting a tool into opening 481 at the end to reach the head deeper into the opening.

[0072] The spring 476 pushes up against the head of the bolt 480 at one end and against the bottom of the opening 481 in the lower section at the opposite end. The lower and upper sections of the lever arm are thus pulled slightly apart, allowing tension to build up until the spring is fully compressed. The spring provides tension to a cam to help hold the carriage release lever 441 and carriage assembly in place.

[0073] 5A and 5B, operation within the flow cell linkage 440 under the control of the carriage release lever 441 is shown. With the release lever 441 in its upward position, the nozzle carriage assembly 442 is at its highest position, such that the nozzle assembly 450 engages the cuvette 406. In this highest position, the lever arms 444L and 444R are substantially vertical. The cam on the release lever 441 is held in an upright position by friction from the detents. Depressing the release lever 441 causes the lever arms 444L and 444R to pivot together in parallel and away from the flow cell body 404, causing the nozzle carriage assembly 442 to slide downward within the guide rails. Lowering the nozzle carriage assembly 442 in this manner disengages the nozzle assembly 450 from the cuvette 406.

[0074] 5B, the carriage release lever 441 pivots about shaft 445C at the lever hinge / bracket 443. The lower ends of the lever arms 444L and 444R pivot about shaft 445B at the carriage plate of the carriage assembly 442. The upper ends of the lever arms pivot about shaft 445A at the release lever 441. The L-shaped configuration of the release levers forces the lever arms 444L and 444R outward and slightly downward relative to the flow cell body 404.

[0075] 6A and 6B better illustrate the disengagement of the nozzle assembly from the cuvette 406. In FIG. 6A, the release lever 441 is in the upward position. The lever arms 444L and 444R (see FIG. 5A) are in the upward vertical position. The nozzle assembly 450 is in the upward position and engages the cuvette 406. The O-ring seal of the nozzle assembly 450 pushes up against the cuvette 406, sealing around the nozzle and preventing fluid leakage.

[0076] 6B, the release lever 441 is in the down position, the lever arm is pivoted away from the flow cell body 404, and the carriage assembly is in the down position along with the nozzle assembly 450. The nozzle assembly 450 is therefore disengaged from the cuvette 406. A gap 602 is shown between the nozzle assembly 450 and the cuvette 406. In this down position, the nozzle assembly 450 can slide outward away from the mount 452 of the carriage assembly 442.

[0077] [Nozzle Assembly] 7A-7F, there are shown various views of nozzle assembly 450. Figures 7B-7D show various exploded views of nozzle assembly 450. Figure 7A as well as Figures 7E-7F show various assembled views of nozzle assembly 450.

[0078] The nozzle assembly 450 includes a three-dimensional nozzle body 702, a ceramic nozzle 704, a replaceable O-ring 706, and a partial gland opening 708. The partial gland opening 708 is a washer-shaped opening that includes a slot 710 at its rear end to allow for easy removal of the O-ring with a fingernail or small tool. Despite the slot 710, the O-ring 706 in the partial gland opening 708 provides a seal around the nozzle 704 that can withstand high pressures when pressed against a cuvette. The cross section of the three-dimensional body 702 generally includes a top portion, a middle portion below the top portion, and a base portion below the top and middle portions. The three-dimensional body 702 further includes a left rail 714L and a right rail 714R along the left and right sides of the base portion.

[0079] The three-dimensional body 702 is elongated and provides a handle at the front end via a left recess 712L and a right recess 712R at the top, middle, and bottom portions. At the rear end opposite the front end, the three-dimensional body 702 provides a nose or arched stop 716 at the base portion, providing two contact points. The nose or arched stop 716 extends from the base portion of the body through the middle portion to the top portion. The end 718 of the top portion extends slightly outward above the nose or arched stop 716 to ensure that the O-ring has sufficient support to seal the cuvette at the portion gland. Because the three-dimensional body 702 provides a handle, it may also be referred to herein as a nozzle handle 702.

[0080] As shown in FIG. 7D, the three-dimensional body 702 includes a through-hole 720 that begins at the base of the partial gland 708, and the through-hole 720 has an upper receptacle portion 720U that receives the nozzle 704 and a lower droplet channel portion 720L that allows droplets to flow without interference from the sidewalls.

[0081] The three-dimensional body 702 is formed from a high-performance engineering thermoplastic polymer, such as polyetheretherketone (PEEK) from the polyaryletherketone (PAEK) family, to provide mechanical strength and high-temperature and chemical resistance. The three-dimensional body 702 is generally formed with low tolerances. This low tolerance allows the nozzle assembly to easily slide in and out of guides within the mount. This low tolerance also provides a somewhat loose friction fit with the mount, allowing for slight pivoting motion to clear debris from the two stops at the arched stops 716. Other thermoplastic polymers may be used to form the three-dimensional body 702 at low cost and with low tolerances.

[0082] The size (e.g., diameter, depth) and shape of the gland and nozzle are such that a low-cost standard rubber O-ring can be used as the replaceable O-ring 706. The O-ring can be made from ethylene propylene diene monomer (EPDM), a synthetic rubber that has excellent resistance to various environmental factors. In alternative embodiments, the O-ring can be made from silicone rubber or natural rubber.

[0083] The nozzle 704 is preferably a ceramic nozzle formed from an electrically insulating ceramic material to prevent electrical charges from being transferred to the droplets of sample liquid before they reach the deflection unit. As shown in FIGS. 7C-7F, the top of the nozzle 704 has a beveled ring 724 to properly receive and hold the circular cross-section of an O-ring at the depth of the partial gland. The top of the nozzle 704 has a droplet inlet 734, which leads to a somewhat larger diameter droplet channel 735 within the nozzle. When the nozzle 704 is friction-fitted into the upper receptacle portion 720U of the body 702, the nozzle channel 735 of the nozzle 704 communicates with a somewhat larger diameter lower droplet channel 720L of the through-hole 720, which extends the width of the body 702.

[0084] 7E and 7F show the nozzle 704 and O-ring 706 assembled into the through hole and partial gland opening in the nozzle assembly body 702. The O-ring 706 is held in the partial gland opening 708 by a beveled ring 724 on the top of the nozzle 704.

[0085] The nozzle assembly 450 is selectively slidably coupled and uncoupled relative to the nozzle mount 452. The tolerance between the nozzle body of the nozzle assembly 450 and the nozzle mount 452 is approximately 0.25 microns or greater for a loose fit. This is not an interference fit. This allows the nozzle assembly 450 to pivot about an axis that passes roughly through the nozzle orifice. This loose fit facilitates removal of debris between the nose and the nozzle mount receptacle for proper alignment of the nozzle orifice with the fluid flow channel in the cuvette 406.

[0086] The cuvette 406 can be formed from one or more pieces of optical grade quartz capable of receiving laser light and capturing reflected, scattered, and fluorescent light.

[0087] Sample droplets can be charged by a conductive host fitting mounted in the drain / charge port of the flow cell. Therefore, the nozzle assembly is formed from a non-conductive or insulating material to avoid charge loss through the ground path to the carriage assembly. The nozzle mount 452 and nozzle carriage assembly 442 are electrically grounded to shield the charged droplets from charges on the deflection plates below the nozzle mount.

[0088] 8A-8B and 9A-9B show the nozzle assembly 450 sliding in and out of the nozzle mount 452 of the carriage assembly 442 in the flow cell 124. This allows for maintenance of the nozzle assembly 450, including replacement of O-ring gaskets or seals.

[0089] Assume that the nozzle assembly 450 is to be pressed into the slot 910 in the mount 452. First, as better shown in Figures 9A and 9B, the bottom rails 714L and 714R of the nozzle assembly 450 are aligned with and inserted into the guide rail openings 914L and 914R, respectively, in the mount 452. The nozzle assembly 450 is pressed as far as possible into the mount's slot 910 so that the nose stop 716 of the body 702 engages the end wall of the mount at the slot 910.

[0090] During operation of the cell sorter (sorting flow cytometer), a stream of sample droplets carrying marked cells / particles flows from the SIT into the flow cell body and into the flow channel 906 of the cuvette 406 to be analyzed by a laser and detector. When the nozzle assembly 450 is properly aligned in the mount 452, the stream of droplets from the flow channel in the cuvette 406 is received by an opening in the nozzle of the nozzle assembly. The mount 452 has an opening 916 in the slot 910 that passes the stream of droplets received from the opening in the nozzle of the nozzle assembly 450. Therefore, it is desirable to achieve proper alignment of the nozzle assembly 450 in the mount 452.

[0091] Instead, imagine that nozzle assembly 450 is to be withdrawn for maintenance from slot 910 in mount 452. A user presses two fingers into left and right finger grips 712L and 712R of body 702 and slides nozzle assembly 450 out of slot 910, withdrawing it from mount 452.

[0092] 8B and 8C and 9B and 9C show how the nozzle assembly 450 in the mount 452 is raised and lowered by the flow cell linkage 440 and carriage assembly 442 to press and release the O-ring seal of the nozzle assembly 450 against the cuvette 406.

[0093] 8B and 9B, the release lever 441 and the nozzle assembly 450 engaged with the mount 452 of the carriage assembly 442 are in the lowered position. In the lowered position, a gap 602 exists between the cuvette 406 and the nozzle assembly 450, as shown in FIG. 9B. To engage the nozzle assembly 450 with the cuvette 406, the user raises the release lever 441 while pivoting it about shaft 445C. This causes the linkage assembly 440 to pivot about shaft 445B toward the flow cell body, raising the lever arms 444L and 444R and the carriage assembly 442. Because the nozzle assembly 450 is attached to the mount 452 of the carriage assembly 442, the nozzle assembly rises along with the carriage assembly.

[0094] 8C, 9C, and 10C, the release lever 441 and the nozzle assembly 450 engaged in the mount 452 are in a raised or upward position. The gap 602 between the nozzle assembly and the cuvette 406 is significantly reduced, and the O-ring seal of the nozzle assembly presses against the base of the cuvette 406 around the flow channel 906.

[0095] A spring-loaded detent slidably engages the rear cam 425 of the release lever 441 to maintain the selected position of the linkage, carriage assembly, and nozzle assembly. When the release lever 441 moves from the lower position to the upper position, the spring-loaded detent 461 rides up against the lower portion of the rear cam 425 and abuts against the upper portion of the rear cam 425, as shown in FIG. 4D. Between the lower and upper portions of the rear cam 425 is a ridge that is a greater radial distance from the shaft 445C than the lower portion of the cam. The upper portion of the cam can be a similar or smaller radial distance to the shaft than the ridge. Thus, when the release lever is in the upper position, the compression spring 427 behind the detent 461 is more compressed and applies a greater force to the cam 425. This spring force against the detent and the upper portion of the cam helps maintain the release lever in the upper position. The user depresses release lever 441 to overcome the force and friction exerted by the spring-loaded detent in the upper section, causing the cam to move to the lower section and actuating linkage 440 to lower carriage 442 and nozzle assembly 450.

[0096] To disengage the nozzle assembly 450 from the cuvette 406, the user depresses the release lever 441 while pivoting it about shaft 445C. This causes the linkage assembly 440 to pivot about shaft 445B away from the flow cell body, lowering the lever arms 444L and 444R and the carriage assembly 442. Because the nozzle assembly 450 is attached to a mount 452 on the carriage assembly 442, the nozzle assembly descends with the carriage assembly. Thus, in the lowered position, a large gap 602 is formed between the cuvette 406 and the nozzle assembly 450, allowing the nozzle assembly 450 to slide out from the mount 452 without damaging the cuvette 406. Once the nozzle assembly 450 has been slid out from the mount, a new nozzle assembly can be installed in its place and / or the used nozzle assembly can be subjected to maintenance and reinstalled after completion.

[0097] Although reference is made here to nozzle assembly 450, a test or alignment nozzle assembly can similarly be inserted and removed from nozzle mount 452. Additionally, the mount and carriage or elevator assembly and its linkages also allow the circular nozzle assembly to be engaged and disengaged from the cuvette or cuvette assembly.

[0098] [Cuvette Nozzle Subsystem] The nozzle assembly 450 described above can be engaged and disengaged from the flow cytometer by sliding it in and out of the nozzle mount 452. Furthermore, the nozzle assembly 450 is held in the nozzle mount 452 by fit and friction between engaging portions (e.g., side rails and rail slots, the bottom surface of the nozzle body, and the base of the slot). Unfortunately, nozzle assemblies are often removed (dismounted) from the mount, for example, to clear a clog in the nozzle orifice, or swapped for a different-sized flow channel to generate droplets of a different size fluid. When the nozzle assembly 450 is slid back into the mount, it may not stop or align in a substantially similar position within the mount. Thus, the flow channel in the nozzle may be slightly misaligned from the flow channel in the cuvette, which may result in a slightly altered droplet flow.

[0099] To avoid the need to recalibrate the droplet stream of a cell sorter or flow cytometer each time a nozzle assembly is replaced, it is desirable to have the nozzle assembly parked and aligned in substantially the same position within the mount so that the alignment between the flow channel within the nozzle and the cuvette remains substantially the same as previously calibrated. Advantageously, a cuvette nozzle subsystem 1000 comprising a circular nozzle assembly 1004 in a flow cytometry system (e.g., a flow cytometer or cell sorter) can implement such desirable functionality.

[0100] 10A-10E, a cuvette nozzle subsystem (assembly) 1000 of a flow cytometry system (e.g., a flow cytometer and cell sorter) will now be described. In FIG. 10A, an exploded view of the cuvette nozzle subassembly 1000 is shown. The cuvette nozzle subassembly 1000 includes a cuvette 406, a tapered receptacle 1006, and a circular nozzle assembly 1004. An adhesive or epoxy 1005 is used to bond the tapered receptacle 1006 and the cuvette 406 together to form a cuvette assembly 406'.

[0101] The cuvette 406 has a cuvette body 1002 with a pocket 1003 and a flow channel 906. The flow channel 906 in the body 1002 is between the top surface 1003T of the pocket 1003 and the top surface of the cuvette 406. The cuvette 406 is made of plastic, glass, quartz, or optical-grade quartz that is transmissive and transparent to the desired wavelength of laser light, light produced by a fluorescent dye for the detector to detect in the visible portion of the electromagnetic spectrum, and infrared light for the detector to detect in the invisible portion of the electromagnetic spectrum. Thus, the flow channel 906 allows fluid carrying particles (e.g., biological cells) to flow through it so that the particles can be analyzed using the laser light from the laser and infrared light with the detector. Within the pocket, the cuvette 406 has a left side 1003L, a back side 1003B, a right side 1003R, and a top side 1003T. The front and base of the pocket 1003 are open. A base opening in pocket 1003 can receive a tapered conical receptacle 1006 and a circular nozzle assembly 1004 .

[0102] Within the pocket 1003, the top surface of the tapered conical receptacle 1006 is bonded to the top side 1003T of the cuvette 406 by adhesive. The body of the tapered conical receptacle 1006 includes a tapered through-hole 1016. Adhesive 1005 can be thinly applied to the portion of the top surface 1003T within the pocket, avoiding the through-hole 1016 of the receptacle 1006. The through-hole 1016 includes a tapered conical surface 1016T that connects to a cylindrical surface (a relief with a ring-shaped opening) 1016C. The tapered conical surface 1016T can receive the top conical portion 1008T of the circular nozzle assembly 1004. The body of the receptacle 1006 can be made from a high-performance engineering thermoplastic polymer, such as polyetheretherketone (PEEK), or a ceramic material to provide mechanical strength, high-temperature resistance, and chemical resistance.

[0103] The circular nozzle assembly 1004 includes an O-ring gasket 1007 and a nozzle body 1008 that are bonded together. The nozzle body 1008 can be formed from a high-performance engineering thermoplastic polymer (e.g., PEEK) or a ceramic material for similar reasons as the receptacle body. The nozzle body has a ring groove or opening 1008R with a semicircular or U-shaped cross-section on its top surface to receive the O-ring gasket 1007. The nozzle body 1008 also has a central flow channel 1010 between the top surface and an open cylindrical chamber 1012, which allows for fluid flow and droplet formation in the flow. The O-ring gasket 1007 is positioned within the ring groove 1008R around the flow channel 1010.

[0104] The circular nozzle assembly 1004 is removably mounted (mounted and dismounted) on a movable circular mount 1099. An elevation device, such as the carriage linkage assembly (flow cell linkage) 440 shown in FIG. 4B, is coupled to the mount 1099 and moves the mount 1099 and circular nozzle assembly 1004 up and down together. This up and down movement, indicated by the double-headed arrow 1009, allows the circular nozzle assembly 1004 to engage (arrow 1009U in FIG. 10B) and disengage (arrow 1009D in FIG. 10B) from the tapered conical receptacle 1006. As the circular nozzle assembly moves upward into position, the top surface of the O-ring gasket 1007 engages the top surface 1003T of the cuvette, sealing around the flow channels 906, 1010 to prevent leakage. In one embodiment, O-ring gasket 1007 is a rubber gasket made by forming rubber into an O-ring shape. In another embodiment, O-ring gasket 1007 is a silicone gasket made by forming silicone into an O-ring shape. Other flexible materials can also be used to form O-ring gasket 1007. Nozzle body 1008 includes a cylindrical portion 1008C and a tapered conical portion 1008T.

[0105] The tapered cone receptacle 1006 is bonded to the cuvette 406 by adhesive 1005. In one embodiment, the adhesive 1005 is an epoxy resin. In another embodiment, the adhesive 1005 is a glue. The body of the tapered cone receptacle 1006 includes a through-hole 1016 having a hollow tapered cone portion 1016T that merges into a hollow cylindrical portion 1016C. The hollow cylindrical portion 1016C of the through-hole 1016 prevents adhesive from entering the through-hole and interfering with the engagement between the circular nozzle assembly and the cuvette assembly. As shown in FIG. 10B , the receptacle 1006 is located closer to the back side 1003B of the pocket 1003 than to the front side. As shown in FIG. 10D , the hollow tapered cone portion 1016T of the through-bore 1016 receives the tapered cone portion 1008T of the nozzle body 1008 of the circular nozzle assembly 1004. The surfaces of the hollow tapered cone portion 1016T and the tapered cone portion 1008T form the same angle when viewed from the same perspective. However, the surface area of ​​the tapered cone portion 1008T is larger than the surface area of ​​the hollow tapered cone portion 1016T, thereby allowing the circular nozzle assembly 1004 to slide deeper into the through-bore and press the O-ring gasket against the top surface of the cuvette. As shown in FIG. 10C , when the circular nozzle assembly 1004 engages the tapered cone receptacle 1006, the axes 1090, 1091 of the flow channel 906 and the flow channel 1010 are substantially aligned with one another within a tolerance range (e.g., 0 mm to 2 mm).

[0106] 10E, one or more portions of adhesive 1005 are placed on the surface 1003T of the cuvette 406 within the pocket 1003. The adhesive is carefully placed to avoid leakage from above / below the receptacle 1006 into the area within the through-hole 1016 of the surface 1003T. This is to avoid the adhesive interfering with the circular nozzle assembly 1004. After an alignment process that positions the receptacle around the flow channel in the cuvette, the adhesive 1005 is allowed to dry, bonding and holding the receptacle 1006 and cuvette 406 together as the cuvette assembly 406′.

[0107] 11A-11D, there are shown various views of a circular nozzle assembly 1004. The circular nozzle assembly 1004 comprises a nozzle body 1008 and an O-ring gasket 1007.

[0108] The nozzle body 1008 is a three-dimensional solid body comprising a flow channel 1010, an open cylindrical chamber 1012, and a ring-shaped opening 1008R. The exterior of the nozzle body 1008 has a cylindrical portion 1008C having a cylindrical shape and a tapered conical portion 1008T having a truncated conical (frusto-conical) shape. The exterior of the nozzle body 1008 further comprises a top surface 1008S and a bottom or base surface 1008B. The material of the nozzle body 1008 is carefully selected and manufactured (machined / lapped) to allow multiple circular nozzle assemblies 1004 to be interchangeable and to be mounted in each cuvette assembly 406′ with the same flow channel aligned. The diameter of the flow channel 1010 can be varied to allow for the generation of different droplet sizes from different circular nozzle assemblies.

[0109] The nozzle body 1008 further comprises a flow channel 1010 that begins at a tapered opening 1010T at the top surface 1008S and terminates in an open cylindrical chamber 1012. The open cylindrical chamber 1012 is substantially a hollow cylinder and comprises a beveled tapered ring portion 1012T, a central cylinder 1012C, and a beveled tapered ring portion 1012B at the base surface 1008B. The open cylindrical chamber 1012 has a larger diameter than the flow channel 1010, allowing the stream of droplets to fall from the circular nozzle assembly 1004 without interference.

[0110] Top surface 1008S has a U-shaped or semicircular ring 1008R that receives the lower portion of O-ring gasket 1007. The upper portion of O-ring gasket 1007 extends above top surface 1008S, as best seen in FIG. 11B. O-ring gasket 1007 can be a silicone gasket or a rubber gasket, where silicone or rubber is formed into the shape of an O-ring.

[0111] 12A-12D, various views of a tapered cone receptacle 1006 are shown. The tapered cone receptacle 1006 is a three-dimensional solid body with a through-hole 1016. The exterior of the receptacle 1006 has a rectangular or cuboid shape. The through-hole 1016 has a hollow tapered cone (hollow truncated cone) portion 1016T that merges into a hollow cylindrical portion 1016C. The hollow cylindrical portion 1016C of the through-hole 1016 allows the O-ring gasket 1007 in the nozzle to be pressed against the top surface 1003T of the cuvette 406. The hollow cylindrical portion 1016C further helps prevent adhesive from interfering with the engagement between the circular nozzle assembly and the cuvette assembly when the receptacle and cuvette are bonded together. The hollow tapered cone portion 1016T in the receptacle makes contact with the tapered cone portion 1008T of the circular nozzle assembly 1004, causing the flow channels to self-align with each other when the mount moves the circular nozzle assembly upward into the cuvette assembly 406'.

[0112] FIG. 13 shows an enlarged side view of the central portion of the cuvette nozzle assembly 1000. The size of the tapered cone receptacle 1006 is selected to leave gaps 1303R, 1303L between the side walls 1003R, 1003L within the pocket of the cuvette 406 and the left and right faces 1006S of the receptacle. This is to allow the receptacle 1006 to be adjusted left and right within the pocket 1003 to align the flow channels 906, 1006 with one another during initial calibration of the cuvette assembly. For similar reasons, as shown in FIGS. 16A and 16B, the size of the tapered cone receptacle 1006 is also selected to leave gap 1603B between the rear side (wall) 1003B of the pocket and the rear face 1006S of the receptacle. Gap 1603B allows receptacle 1006 to be adjusted back and forth within pocket 1003 to align the flow channels with one another during initial calibration of the cuvette assembly.

[0113] 14A and 14B show an alignment fixture 1402 that can be used to align the flow channels 906, 1006 with one another and mate the tapered cone receptacle 1006 with the cuvette 406 in the pocket. The cuvette nozzle subsystem 1000 mounted in the fixture 1402 forms the alignment fixture assembly 1400.

[0114] The alignment jig (fixture) 1402 includes a frame made up of a base 1403, a back 1404, and an arm 1405, and an XY-adjustable stage 1414. The alignment jig (fixture) 1402 further includes a Z-direction adjustment screw 1416Z that screws into the arm 1405. The XY-adjustable stage 1414 is attached to the base 1403 below the Z-direction adjustment screw 1416Z. The XY-adjustable stage 1414 includes an X-direction adjustment screw 1416X and a Y-direction adjustment screw 1416Y for moving the stage in the XY plane.

[0115] The Z-adjustment screw 1416Z has a top viewing hole 1420T with a viewing channel for viewing down into the top of the cuvette and its flow channel through the flow channel in the test circular nozzle. A light can shine upward into the flow channel of the cuvette 406 through the bottom viewing hole 1420B and viewing channel at the base and the XY-adjustable stage 1414. Optionally, a light can shine downward through the top viewing hole 1420T, the viewing channel, and the flow channel in the test circular nozzle, in which case the light will be viewed through the bottom viewing hole 1420B and its viewing channel. That is, the alignment between the flow channels at the interface between the cuvette and the circular nozzle can be viewed through either the top viewing hole 1420T or the bottom viewing hole 1420B.

[0116] 14C shows an enlarged side view of a portion of the alignment fixture assembly 1400 with the cuvette 406 attached to the stage 1414 by one or more clamps 1430. The alignment device 1408 on the Z-screw 1406Z makes contact with a circular test or alignment nozzle 1004'. The circular alignment nozzle 1004' is substantially similar to the circular nozzle 1004 but does not include an O-ring gasket or seal. The alignment nozzle 1004' can be used multiple times with the alignment fixture 1402 for each cuvette and receptacle that are mated together and aligned.

[0117] To view the alignment nozzle 1004', an upper viewing channel 1420U opens from the top viewing hole 1420T through the Z-screw 1406Z and the alignment device 1408. This allows for a view down into the alignment nozzle 1004' at the surface of the flow channel 1010 and the cuvette 406 and into its respective flow channel. To view the top of the cuvette 406, a lower viewing channel 1420L opens from the bottom viewing hole 1420B through the base 1403 and stage 1414. The transparency of the cuvette allows for a view through the cuvette to the top surface of the alignment nozzle 1004' and its respective flow channel.

[0118] By viewing the contact area from the bottom up or top down, one or both of the X- and Y-adjustment screws 1416X and 1416Y can be turned to move the XY-adjustable stage 1414 and align the cuvette and the flow channels in the circular nozzle with one another, if misaligned. A push rod 1406Y interfaces with the Y-adjustment screw 1416Y to move the XY-adjustable stage 1414 in the Y direction. A similar push rod interfaces with the X-adjustment screw 1416X to move the XY-adjustable stage 1414 in the X direction.

[0119] Before the adhesive 1005 between the receptacle 1006 and the cuvette 406 dries, the cuvette 406 is moved by the stage 1414 adjustable in the XY directions to readjust the positions of the test nozzle and the receptacle 1006 in the pocket 1003. This readjustment allows the flow channels to be aligned if there is any misalignment.

[0120] Referring now to FIG. 15 , one or more optical devices can be used to assist in viewing the contact portion of the cuvette nozzle assembly 1000 in the alignment fixture assembly 1400. A first optical device 1502T can be used to view the contact portion of the cuvette nozzle assembly down through the top viewing hole 1420T and the upper viewing channel 1420U. In one embodiment, the first optical device 1502T is an optical microscope or microscope camera to view the contact portion from above. Optionally, a second optical device 1502B can be used below the bottom viewing hole 1420B to assist the first optical device 1502T. In one embodiment, the optional second optical device 1502B is a lamp that shines light upward through the bottom viewing hole 1420B and the lower viewing channel 1420L. Alternatively, the lamp can be a laser that provides laser light. In another embodiment, the second optical device 1502B is an optical microscope or microscope camera for viewing the contact area from below, and the first optical device 1502T is a lamp that shines light downward through the top viewing hole 1420T and the upper viewing channel 1420U. Alternatively, the lamp can be a laser that provides laser light.

[0121] Referring now to FIG. 16A, a diagram of the interface between the receptacle 1006 / alignment nozzle 1004′ and the cuvette 406 in the alignment fixture assembly is shown. The flow channel 906 in the cuvette 406 is visually distinguishable from the flow channel 1010 in the alignment nozzle 1004′ using a microscope with a magnifying field of view. A standard research zoom microscope can be used to look up into the fixture and cuvette from below the fixture. In one embodiment, the flow channel 906 in the cuvette has a square or rectangular cross-section, while the flow channel 1010 in the circular nozzle has a circular or elliptical cross-section. The diameter or cross-sectional size of the flow channel 1010 in the nozzle is smaller than the side or cross-sectional size of the flow channel 906 in the cuvette. The side view of FIG. 13 better illustrates the difference in dimensions of the flow channels 906 and 1010. The transparency of the cuvette allows the nozzle / receptacle surface, flow channel 1010, and the outline of flow channel 906 relative to flow channel 1010 in nozzle 1004′ to be viewed through the cuvette from below the alignment fixture assembly. In this case, light from above and / or below can be used to assist in viewing the nozzle. If light (e.g., a narrow laser light) is shone through flow channel 1010 from above or through flow channel 906 from below, a microscope or some other optical sensor can be used to look into the other flow channel from below or above the fixture to detect the position of each flow channel and facilitate alignment. In either case, an optical alignment process is used to align the centers (axes) of flow channels 906, 1010 with respect to one another.

[0122] 16A and 16B show views from below the fixture through the cuvette 406 into the circular nozzle. As shown in FIG. 16A, the flow channel 906 in the cuvette 406 is misaligned with the flow channel 1010 in the circular alignment nozzle 1004'. The cuvette 406 is mounted on a stage that can be adjusted in the X direction 1310X and / or the Y direction 1310Y below the receptacle 1006 / alignment nozzle 1004' to align the flow channels 906, 1010 with each other. Gaps 1303R, 1303L, and 1303B between the side 1006S and the walls (sides) 1003R, 1003B, and 1003L of the cuvette 406 allow the receptacle 1006 / alignment nozzle 1004' to be adjusted in the X direction and / or the Y direction relative to the cuvette 406.

[0123] 16B shows another view of the interface between the receptacle 1006 / alignment nozzle 1004′ and the cuvette 406 in the alignment fixture assembly. After adjusting the position of the cuvette 406 relative to the receptacle 1006 / alignment nozzle 1004′, the flow channel 906 in the cuvette 406 is aligned with the flow channel 1010 in the alignment nozzle 1004′. This position can be maintained while the adhesive 1005 dries, holding the receptacle 1006 in a fixed position against the surface 1003T in the pocket 1003 of the cuvette 406.

[0124] 17A and 17B, there are shown steps in the alignment process for attaching and securing the tapered receptacle to the surface 1003T within the pocket 1003 of the cuvette 406. This alignment process aligns the flow channels within the cuvette and the circular nozzle with one another. The alignment process begins with step 1700 and then proceeds to step 1702.

[0125] In step 1702, the top surface of the cuvette is attached to the XY adjustable stage 1414 of the alignment fixture 1404 using one or more clamps 1430 shown in FIG. 14C.

[0126] In step 1704, one or more droplets of adhesive (e.g., four droplets placed near the corners of the receptacle) may be applied to one or more portions of the top surface 1003T of the pocket 1003 in the cuvette 406, ultimately forming a thin layer of adhesive 1005 between the cuvette and the receptacle. Care is taken to avoid the glue migrating beyond the surface 1003T into the area where the through-hole of the tapered conical receptacle 1006 is located. The thin layer of adhesive may be applied evenly to the surface 1003T of the cuvette, avoiding the circular area around the flow channel in the cuvette that opens through the through-hole of the receptacle.

[0127] In step 1706, the tapered conical receptacle 1006 is placed in the pocket 1003 with the base of the tapered conical receptacle 1006 resting on a thin layer of adhesive or epoxy resin 1005 on the top surface 1003T of the cuvette 406, so that the through hole with the tapered conical opening is around the cuvette flow channel.

[0128] In step 1708, the tapered portion 1008T of the circular alignment nozzle 1004' is placed into the hollow tapered cone portion of the through-hole 1016 of the tapered cone receptacle 1006. During the alignment process with the alignment nozzle 1004', the O-ring gasket does not contact the surface 1003T of the cuvette 406. During operation with the circular nozzle 1004, the top portion of the O-ring gasket 1007 may contact the surface 1003T of the cuvette 406.

[0129] In step 1710, the adjustable Z-screw 1416Z is threaded into the fixture arm 1405 so that it fits into the cylindrical bottom end of the circular alignment nozzle 1004'. In step 1712, the user views the position of the cuvette flow channel 906 in the cuvette relative to the nozzle flow channel 1010 in the circular alignment nozzle 1004' through the viewing holes 1420B, 1420U and their respective viewing channels. In step 1714, the position of the tapered conical receptacle 1006 and the circular alignment nozzle 1004' is adjusted by turning the X-screw 1416X and / or the Y-screw 1416Y in or out so that the cuvette flow channel is substantially centered (e.g., ±2 mm) relative to the nozzle flow channel. This step is repeated as necessary.

[0130] In step 1716, the Z-direction screw 1416Z is further threaded into the fixture arm to further press the circular alignment nozzle 1004′ and tapered conical receptacle 1006 down onto the surface 1003T of the cuvette 406 within the pocket 1003 in the cuvette 406. This allows pressure to be applied to clamp the tapered conical receptacle 1006 against the cuvette 406 and hold the position of the tapered conical receptacle 1006 relative to the cuvette 406, allowing the adhesive 1005 to dry and bonding the receptacle 1006 and cuvette 406 together. In step 1718, the tapered cone receptacle 1006 is held in place by a jig (fixture) for a predetermined time (waiting, curing, or drying time, e.g., at least 30 minutes) to allow the adhesive 1005 between the tapered cone receptacle 1006 and the cuvette 406 to dry.

[0131] In step 1720, after the drying period, the Z screw 1416Z is unscrewed to release the pressure clamping together the circular alignment nozzle 1004′, the tapered conical receptacle 1006, and the cuvette 406. Unscrewing the Z screw 1416Z lifts it away from the cuvette assembly, allowing it to be removed from the top surface of the XY adjustable stage 1414.

[0132] In step 1722, one or more clamps 1430 holding the cuvette 406 on top of the XY-adjustable stage 1414 are released. In step 1724, the cuvette assembly including the cuvette 406 and the tapered conical receptacle 1006 is removed from the XY-adjustable stage 1414. In step 1726, the cuvette assembly including the cuvette 406 and the tapered conical receptacle 1006 is assembled into a flow cytometer. In step 1728, the circular nozzle assembly 1004 having a nozzle body and an O-ring is assembled into the flow cytometer. In step 1730, referring to FIGS. 10A-10D , the circular nozzle assembly 1004 is engaged with the cuvette assembly including the tapered conical receptacle 1006 and the cuvette 406 in the flow cytometer. The nozzle assembly moves upward into the tapered conical receptacle so that the O-ring engages the top surface of the cuvette within the conical opening. This aligns the flow channels 906, 1010 in the cuvette and circular nozzle assembly 1004, respectively, allowing the O-ring 1007 to seal around the respective flow channels between the cuvette and circular nozzle assembly 1004, preventing fluid leakage. Fluid carrying particles can then pass through the flow cytometer and through the circular nozzle assembly and flow channels 906, 1010 in the cuvette. As the circular nozzle assembly moves upward (raises) into the tapered conical receptacle, the O-ring engages the top surface 1003T in the pocket 1003 of the cuvette 406, bringing the tapered conical surfaces 1016, 1008T of the receptacle 1006 and nozzle body 1008, respectively, face each other.

[0133] In step 1732, the circular nozzle assembly 1004 is disengaged from the cuvette assembly 406'. This includes disengaging the O-ring from surface 1003T and the tapered conical surface 1008T from the tapered conical surface 1016T of the receptacle 1006. This allows the circular nozzle assembly 1004 to be removed from the flow cytometer and replaced with a different circular nozzle assembly and / or its flow channel 1010 to be cleaned and unclogged. In step 1734, the flow channel 1010 in the circular nozzle assembly 1004 is cleaned and / or the O-ring 1007 is replaced in case the flow channel becomes clogged.

[0134] In step 1736, after cleaning, the circular nozzle assembly 1004 can be re-engaged with the flow cytometer. The circular nozzle assembly 1004 is moved upward into the cuvette assembly 406′, which includes the tapered conical receptacle 1006 and the cuvette 406. Once the flow channels are aligned with one another and the O-ring seals between the top surface of the nozzle body and the surface 1003T of the cuvette, the fluid with particles is passed through the flow cytometer for testing (analysis). After testing is complete, process steps 1732-1736 can be repeated for another sample fluid with particles, or the process can proceed to step 1799 and end.

[0135] [Droplet-Driven Assembly] The droplet drive assembly 402 of the flow cell 124 communicates with and receives sample liquid from the sample input station 130 of the fluidics system. The flow cell and the sample input station are communicatively coupled together by tubing. Generally, the droplet drive assembly 402 receives sample liquid under pressure at one end through a sample input port 408. At the other end, the droplet drive assembly 402 directs sample liquid from a sample injection tube (SIT) 422. As can be seen in Figures 4D-4G, the lower portion of the droplet drive assembly below the hub is inserted into a chamber of the flow cell body 404.

[0136] The droplet actuation assembly 402 includes, among other features, a sample entry port 408, a sample injection tube (SIT) 422, and a hollow cylindrical piezoelectric transducer. The upper end of the SIT 422 communicates with the sample entry port 408 and tubing to receive a flow of sample liquid. The sample injection tube (SIT) 422 injects the sample liquid into a funnel-shaped portion of a chamber within the flow cell body. The hollow cylindrical piezoelectric transducer is under user-selected software control (amplitude and frequency) to drive droplet formation.

[0137] The hollow cylindrical piezoelectric transducer, once assembled, is mounted around a portion of the SIT 422. The sample liquid containing cells / particles flows within the hollow central cylinder of the SIT 422. When energized by an alternating current (AC) signal (with selectable amplitude and frequency) from the electronics system, the hollow cylindrical piezoelectric transducer vibrates based on the frequency and amplitude of the AC signal. This vibration is coupled to an insulated cylindrical seal base, allowing the sample liquid to receive acoustic energy, which can help convert the sample liquid into a stream of small droplets that spread out in a single file from the nozzle. Ideally, each droplet contains a single cell / particle, but the size of the target cells / particles can vary. The size and frequency of droplet generation can vary depending on the nozzle opening diameter, sheath pressure, and fluid viscosity. At a given sheath liquid pressure, the frequency and amplitude of the AC signal can be set to a resonance point where droplet formation is stable and the desired droplet size is achieved. The nozzle assembly can be easily replaced to obtain nozzle openings with different diameters.

[0138] [Droplet quality] A nozzle in the nozzle assembly of the flow cell breaks the sample liquid into droplets. In a cell sorter, droplets with cells of interest in the central stream are sorted by deflecting the droplets away from the central stream. In the flow cell, the droplets are charged so that they can be deflected (sorted) away from the central stream by a charged deflection plate in a deflection chamber (unit) 122. The deflected droplets with cells of interest can be collected in a separate vessel (test tube, plate well) for further testing in a laboratory.

[0139] The stream of droplets containing biological cells exiting the nozzle should have an appropriate shape. The spacing between droplets should be appropriate so that each droplet can be deflected and collected in a separate vessel. Additionally, the break-off time point from the droplet stream should control droplet formation and be consistent between nozzle removal, cleaning, and reinsertion. Therefore, droplet shape and spacing between droplets can be important in a cell sorter system. The droplet yield and droplet shape quality of the droplet stream exiting the nozzle are related to how well the nozzle mates with the cuvette. The top surface of the nozzle and the bottom surface of the cuvette should be coplanar and the axis of the flow channel should be aligned, resulting in good droplet yield and good droplet shape quality.

[0140] Figures 18A and 18B show magnified and unmagnified views of low-quality droplets ejected from a nozzle in a nozzle assembly engaged with a cuvette in a flow cytometer / cell sorter under test, and Figures 19A and 19B show magnified and unmagnified views of high-quality droplets ejected from a nozzle in a nozzle assembly engaged with a cuvette in a flow cytometer / cell sorter under test.

[0141] Figures 18A and 18B show diagrams of a low-quality droplet stream 1801 from a nozzle orifice (flow channel). In Figure 18B, a cross section 1810 of the stream 1801 is enlarged to measure droplet quality. Lines 1811, 1812, and 1813 measure droplet delay, droplet spacing, droplet center, etc., and can be used to obtain an overall view of the flow conditions. Droplets 1815A-1815D are asymmetrical without rounded droplet shapes. Poor engagement between the nozzle and the cuvette can result in such poor droplet quality. A nozzle with good separation would exhibit symmetrical, well-rounded droplet shapes.

[0142] 19A and 19B show diagrams of a high-quality droplet stream 1901 from a nozzle orifice (flow channel). In FIG. 19B, a cross section 1910 of the stream 1901 is enlarged to measure droplet quality. Lines 1911, 1912, and 1913 measure droplet delay, droplet spacing, droplet center, etc., and can be used to get an overall view of the flow conditions. Droplets 1915A-1915D are symmetrical. The droplets have a well-rounded elliptical shape. This indicates that good droplet quality results from a nozzle with good separation and good engagement between the nozzle and the cuvette.

[0143] Some flow control adjustments can be made to improve poor droplet quality to an acceptable level even when engagement between the nozzle and cuvette is poor. However, the settings that may need to be adjusted (e.g., increasing the amplitude and / or frequency of the vibrations provided by the cylindrical piezoelectric transducer in the SIT) can overload components within the flow cytometer / cell sorter, leading to premature failure that requires replacement or repair. To avoid component overload and reduce maintenance costs for the flow cytometer / cell sorter, it is desirable to provide good mechanical engagement between the nozzle and cuvette to provide good droplet quality at nominal settings.

[0144] [advantage] Flow Cytometers and Sorting Having a circular nozzle assembly in a flow cytometer (e.g., cell sorter 100) has several advantages: After calibration to the nozzle, the circular nozzle assembly can be removed, cleaned, and repositioned in a substantially similar position to provide substantially similar droplet quality from run to run.

[0145] Allowing the flow channels in the cuvette and nozzle to be adjustable so that they are aligned has several advantages. Better alignment between flow channels can lead to better droplet quality. Improved droplet quality allows droplet settings to be closer to nominal within a range. In the long term, improved droplet quality can result in lower maintenance costs for the flow cytometer or cell sorter system.

[0146] The present disclosure contemplates other embodiments or objectives. It will be recognized that embodiments 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 have been described, it is evident that many alternatives, modifications, permutations, and variations will become apparent in light of the above description. For example, threaded openings may have different dimensions, in which case the dimensions of various threaded fasteners will differ from those set forth herein. It is therefore intended that the claimed invention encompass all such alternatives, modifications, and variations that fall within the scope of the appended claims. The mere fact that a product, process, or method exhibits differences from one or more of the described exemplary embodiments does not mean that the product or process is outside the scope (literal and / or other legally permitted) of the appended claims.

Claims

1. 1. A cuvette nozzle subsystem of a flow cytometry system, the cuvette nozzle subsystem comprising:

1. A cuvette assembly comprising: a cuvette having a pocket and a flow channel; a receptacle coupled to the cuvette within the pocket, the receptacle having a body with a throughbore having a tapered conical portion and a cylindrical portion to form a flow channel; a cuvette assembly including: a circular nozzle assembly selectively engaging with the cuvette assembly, the circular nozzle assembly having an O-ring gasket coupled to a nozzle body having a flow channel, the nozzle body having a tapered conical portion that engages with the tapered conical portion of the through-hole in the receptacle to align the flow channels of the cuvette and the nozzle body with each other; 1. A cuvette nozzle subsystem comprising:

2. the O-ring gasket fits within a semicircular ring portion on the top surface of the nozzle body such that a portion of the O-ring gasket extends above the top surface; the extended portions of the O-ring gasket engage surfaces within the pockets of the cuvette around each of the flow channels when the circular nozzle assembly is engaged with the cuvette assembly to prevent fluid leakage. The cuvette nozzle subsystem of claim 1 .

3. the nozzle body further includes a cylindrical portion removably coupled to a mount, the mount allowing the circular nozzle assembly to move vertically upward toward and away from the cuvette assembly; the vertical movement upward toward the cuvette positions the portion of the O-ring gasket against the surface within the pocket of the cuvette to provide a seal around each of the flow channels between the cuvette and the nozzle body.

3. The cuvette nozzle subsystem of claim 2.

4. 4. The cuvette nozzle subsystem of claim 3, wherein the vertical movement away from the cuvette enables the circular nozzle assembly to be disengaged from the mount, thereby allowing the circular nozzle assembly to be replaced.

5. The cuvette nozzle subsystem of claim 3 , wherein the vertical movement away from the cuvette enables the circular nozzle assembly to be disengaged from the mount.

6. The cuvette nozzle subsystem of claim 5 , wherein the flow channel within the circular nozzle assembly can be cleaned by dismounting the circular nozzle assembly from the mount.

7. The cuvette nozzle subsystem of claim 5 , wherein the O-ring gasket can be replaced by dismounting the circular nozzle assembly from the mount.

8. The cuvette nozzle subsystem of claim 5 , wherein the circular nozzle assembly can be replaced by disengaging the circular nozzle assembly from the mount.

9. The cuvette nozzle subsystem of claim 5 , wherein the flow channels in the nozzle body can have different diameters to form droplets of different droplet sizes.

10. The cuvette nozzle subsystem of claim 3 further comprising a flow cell linkage including at least one link coupled to the mount.

11. 1. A flow cytometer or cell sorter system, the system comprising: a flow cell coupled in communication with a fluidics system to receive a sheath fluid, wherein sample fluid with cells or particles flows through the flow cell and is surrounded by the sheath fluid, the flow cell comprising: a flow cell body coupled around the droplet actuation assembly for receiving a fluid stream of the sample liquid from a sample inlet tube; a cuvette coupled to a base of the flow cell body, the cuvette having a pocket and a cylindrical flow channel for receiving the fluid stream of the sample liquid; a receptacle coupled within the pocket of the cuvette by an adhesive, the receptacle having a body with a through hole having a tapered conical portion; a circular nozzle assembly selectively engageable with the receptacle and the cuvette, the circular nozzle assembly including a nozzle body having a central flow channel and an O-ring gasket bonded to a top surface around the central flow channel, the nozzle body having an upper tapered conical portion that engages with the tapered conical portion of the through-hole in the receptacle to align the cylindrical flow channel of the cuvette and the central flow channel of the nozzle body with one another; A flow cytometer or cell sorter system comprising:

12. 12. The flow cytometer or cell sorter system of claim 11, wherein the through-hole of the receptacle further comprises a cylindrical portion connected to the tapered conical portion to prevent adhesive from entering the through-hole.

13. the O-ring gasket fits within a ring opening in the top surface of the nozzle body such that a portion of the O-ring gasket extends above the top surface; 12. The flow cytometer or cell sorter system of claim 11, wherein the extended portion of the O-ring gasket engages with a surface within the pocket of the cuvette around the cylindrical flow channel when the circular nozzle assembly engages with the receptacle and the cuvette to prevent fluid leakage.

14. 12. The flow cytometer or cell sorter system of claim 11, wherein the cuvette is transparent to light across the electromagnetic spectrum, including laser light, fluorescence in the visible portion of the electromagnetic spectrum, and infrared light in the invisible portion of the electromagnetic spectrum, such that particles in the cylindrical flow channel can be excited by the laser light from a laser and the infrared light and fluorescence can be detected by one or more detectors.

15. 12. The flow cytometer or cell sorter system of claim 11, wherein the nozzle body further comprises a lower cylindrical portion connected to the upper tapered conical portion, and the system further comprises a movable circular mount capable of attaching and detaching the circular nozzle assembly.

16. the movable circular mount allows the circular nozzle assembly to move vertically upward to engage the cuvette and the receptacle at the tapered conical portion of the through-bore; 16. The flow cytometer or cell sorter system of claim 15, wherein the movable circular mount further allows the circular nozzle assembly to move vertically downward to disengage from the cuvette and the receptacle.

17. 1. A method for a circular nozzle assembly of a flow cytometer or cell sorter system, comprising: moving a first circular nozzle assembly upward into a pocket of a cuvette in a flow cytometer; further moving the first circular nozzle assembly upward so that a tapered conical portion of the first circular nozzle assembly is inserted into the through hole of the receptacle; further moving the first circular nozzle assembly upward to engage the tapered conical portion of the first circular nozzle assembly with the tapered conical portion of the through-hole in the receptacle and bring flow channels in the cuvette and the first circular nozzle assembly into alignment with one another; A method comprising:

18. moving the first circular nozzle assembly upwardly to engage the tapered conical portion comprises:

18. The method of claim 17, further comprising engaging an O-ring gasket of the first circular nozzle assembly within the pocket with the surface of the cuvette at the through hole around the cuvette and the flow channel in the circular nozzle assembly to seal and prevent leakage of fluid outside the O-ring gasket in the circular nozzle assembly.

19. 20. The method of claim 18, further comprising moving the first circular nozzle assembly downward to disengage from the cuvette and the receptacle.

20. 20. The method of claim 19, wherein the upward and downward movement of the first circular nozzle assembly is vertical movement.

21. 20. The method of claim 19, further comprising replacing the first circular nozzle assembly with a second circular nozzle assembly.

22. The method of claim 19 further comprising cleaning the flow channel in the first circular nozzle assembly.

23. 20. The method of claim 19, further comprising replacing the O-ring gasket in the first circular nozzle assembly.

24. 1. A method for joining together a receptacle and a cuvette for a subassembly of a flow cytometer or cell sorter, comprising: Providing a pocket (1003) in the cuvette (406); applying a thin layer of adhesive (1005) to one or more portions of the top surface (1003T) within the pocket (1003) of the cuvette (406); placing the receptacle (1006) in the pocket (1003) of the cuvette such that a base of the receptacle (1006) engages the thin layer of adhesive and the surface (1003T) of the cuvette (406), the through-hole (1016) of the receptacle being around a cuvette flow channel (906) in the cuvette (406); waiting a predetermined time to allow the thin layer of adhesive (1005) between the receptacle (1006) and the cuvette (406) to dry; A method comprising:

25. attaching the top surface of the cuvette (406) to the top surface of an XY adjustable stage (1414) of an alignment fixture (1404) using one or more clamps (1430) before applying the thin layer of adhesive (1005); After positioning the receptacle, positioning a tapered portion (1008T) of a circular alignment nozzle (1004') within the through-hole (1006) of the receptacle so as to engage with a hollow tapered cone portion of the through-hole (1016); Before the waiting, turning an adjustable Z-screw (1416Z) on the jig arm (1405) of said alignment fixture (1404) to engage the cylindrical bottom end of said circular alignment nozzle (1004'); looking down through viewing holes (1420B, 1420U) into viewing channels in the adjustable Z-screws (1416Z) to view the position of the cuvette flow channel (906) in the cuvette (406) relative to the nozzle flow channel (1010) in the circular alignment nozzle (1004'); turning the X-screw (1416X) and / or the Y-screw (1416Y) of the XY adjustable stage (1414) to adjust the position of the tapered conical receptacle (1006) and the circular alignment nozzle (1004') so that the cuvette flow channel (906) is centered relative to the nozzle flow channel (1010); 25. The method of claim 24, further comprising:

26. Before the waiting, 26. The method of claim 25, further comprising rotating the Z-direction screw (1416Z) further downward onto the jig arm (1405) to further press the circular alignment nozzle (1004') and the tapered conical receptacle (1006) down into the thin layer of adhesive (1005) on the surface (1003T) of the cuvette (406).

27. Before the waiting, 27. The method of claim 26, further comprising maintaining the position of the tapered conical receptacle (1006) relative to the cuvette (406).

28. After the drying time, unscrewing the Z-screw (1416Z) to release the pressure clamping the circular alignment nozzle (1004'), the tapered conical receptacle (1006), and the cuvette (406) together; Raising the circular alignment nozzle (1004') away from the tapered cone receptacle (1006) and moving the circular alignment nozzle (1004') away from the alignment fixture (1404); 28. The method of claim 27, further comprising:

29. 29. The method of claim 28, further comprising releasing the one or more clamps (1430) holding the cuvette (406) to the top surface of the XY adjustable stage (1414) after unscrewing the Z screw (1416Z).

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