Modular flow cytometry system and sample processing method

Abstract

Described herein are modular flow cytometry systems and methods for processing samples. The system includes removable and replaceable automated or semi-automated modules. The sample passage module may be removed and installed in a microfluidic device cleaning module for cleaning, then reinstalled or stored for later use. The system further includes an optics module, an electronics module, and a mixing and collection module. The optics module includes a photo-scratching assembly and a detection laser assembly, which may be on the same side of the plane or surface of the flow cytometry device and on the opposite side of the detection assembly. The beam waist of the laser beam is wider in a direction perpendicular to the flow than in the direction of flow. The mixing and collection module can automatically mix samples being collected in sample tubes and can switch to another sample tube when the other tubes are full.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 230,568, filed August 6, 2021, U.S. Provisional Application No. 63 / 162,222, filed March 17, 2021, U.S. Provisional Application No. 63 / 119,769, filed December 1, 2020, and U.S. Provisional Application No. 63 / 117,104, filed November 23, 2020, which are incorporated by reference in their entireties.

[0002] This application is incorporated by reference in its entirety into U.S. Application No. 17 / 525,125, filed November 12, 2021, entitled MODULAR FLOW CYTOMETRY SYSTEMS AND METHODS OF PROCESSING SAMPLES (Klas et al.), Docket No. GS-38-2021-US2.

[0003] The present invention relates to automated and semi-automated systems and methods for processing samples, and more particularly to systems and methods in flow cytometer-based systems for analyzing, handling and processing fluids and components or particles in samples.

[0004] The present invention features a modular flow cytometry system and method for cleaning a sample passageway in a flow cytometer system.

[0005] The invention also features optical systems for use in particle detection and sorting in flow cytometry, including particle sorting using electromagnetic radiation and detection in microfluidic devices such as microfluidic chips.

[0006] The invention further features a mixing and collection system that mixes particles or components in a sample with a medium and maintains a desired suspension, such as a homogenous mixture, of the particles or components in the sample and the medium. [Background technology]

[0007] In the field of flow cytometry, it is often necessary to identify and sort particles, such as individual cells, after they have been properly aligned within the flow cytometer and either dispensed as a series of droplets, each containing an individual particle, or moved through the internal detection region of the flow cytometer. In some applications, it may be further necessary to remove certain particles from the flow stream, or to otherwise sort, inactivate, or deactivate particles in the flow stream.

[0008] In some systems, a microfluidic device, such as a microfluidic chip, is used to receive a particle sample and / or a sheath or directing fluid and focus, organize, or otherwise position individual particles from the particle sample within a fluid flow in one or more channels of the microfluidic device. The particles and fluid may flow through one or more interrogation and / or action sites in the channels of the microfluidic device where detection, sorting, and / or destruction processes are performed on the particles. The particles may then be released or otherwise removed from the microfluidic device, such as by fluid flow or pressure.

[0009] Existing systems and methods for processing particles or components in samples with flow cytometry-based systems include numerous steps, components, and elements, many of which rely on human intervention and manual processes. Extensive use of operator intervention, calibration, and operation in flow cytometry increases the likelihood of inaccurate system parameters or calibration, sample mishandling, contamination, and overall error. Existing systems and methods that rely primarily on human operators for all aspects of the process also result in extended downtime and lengthy adjustment and calibration steps based on the extensive dedicated manual intervention required. Manual processes may include preparing the sample, configuring sample and sheath fluid flow rates, configuring or calibrating the light path of an electromagnetic radiation emitter (e.g., a laser module), configuring laser module emission characteristics including power and duration, configuring detectors, configuring or programming sample processing characteristics, capturing or diverting samples, and mixing and storing the processed sample.

[0010] Existing systems, apparatus, and methods for processing samples, such as biofluid samples containing a plurality of cells, in flow cytometry devices, such as microfluidic systems, include using disposable microfluidic chips, cassettes, or cartridges. Other systems and methods include flow cytometry devices that may flow wash fluids or air through the sample processing passages. Still other systems and methods include separate, stand-alone devices that wash the microfluidic chips, cassettes, or cartridges.

[0011] However, none of the existing systems completely isolates the sample pathway used by the fluid sample from the flow cytometry or microfluidic analytical device. For example, in existing systems, while some of the sample pathways, such as the microfluidic chip or cassette, may be removed, other parts of the sample pathways that are contacted by the fluid sample are part of the flow cytometry device itself and cannot be removed from the system. These parts may include the pathways between the sample storage or mounting area and the microfluidic chip itself. Extensive cleaning and flushing of the flow cytometry device must be performed between sample runs. This results in extensive downtime between the processing of separate fluid samples, but is necessary to eliminate the possibility of biological or other contamination and to comply with the various rules and regulations related to the processing of biological samples. For example, after processing a first fluid sample, such as a semen sample from a first male animal, the system requires extensive cleaning to eliminate the possibility of cross-contamination with a second fluid sample, such as a semen sample from a second male animal. Elimination of cross-contamination is necessary when the samples contain different genetic material or traits and contamination would render the sample unsuitable for use.

[0012] An additional problem with existing systems and methods is that while parts of the system may be removed for cleaning, the cleaning process itself can be labor intensive and require extensive manual input and configuration. However, many systems treat components such as microfluidic chips, cartridges, and cassettes as disposable, single-use items, significantly increasing the amount of single-use plastic or glass waste by laboratories.

[0013] What is needed is a removable and replaceable sample passage module that may be removed from the flow cytometry device and cleaned with a separate cleaning device. The sample passage module should be reusable and easily removable to allow for exchange between sample passage modules on the flow cytometry device in order to operate the sample processing flow cytometry device nearly continuously.

[0014] Additionally, existing systems and methods include means for determining one or more properties of the particles based on the fluorescence of the particles detected by a detector such as an avalanche photodiode. The particles may be made to fluoresce by directed and focused irradiation of electromagnetic radiation such as a laser. The detected fluorescence may be used as a basis for a sorting or destroying decision.

[0015] In a sorting decision, certain particles that have certain fluorescent characteristics with certain properties or meet certain thresholds detected by one or more detectors are sorted based on those characteristics, which may include directing particles with certain characteristics into different flow streams and / or different collection vessels by means including magnetic fields, microvalves, or pneumatic or hydraulic pulses or jets.

[0016] There are advantages and disadvantages to using either sorting or destruction to process particles in flow cytometry. In the specific field of flow cytometry, sorting of sperm cells that contain either X or Y chromosome DNA for the purpose of sexing the sperm cells, it is important to preserve the viability or fertility of the sperm cells after processing in the flow cytometer system. A sorting process or method may cause undue stress to the selected or desired sperm cells, reducing the viability or fertility of the collected sample of sperm cells. A destruction process or method may leave sperm cells that were not completely inactivated in the collected sample, reducing the viability or fertility of the entire sample.

[0017] In a destruction decision that involves inactivating or damaging a particle, a particular particle that has a particular fluorescent characteristic, contains a particular property, or meets a particular threshold detected by one or more detectors may be damaged or inactivated. The destruction may include ablation or photodamage by directed and focused emission of electromagnetic radiation by a laser. A living cell, such as a spermatozoon, may be referred to as a "kill event" or "cell kill event" because the destruction or inactivation decision results in the death of the cell.

[0018] Additionally, with respect to the destruction decision process, aligning the laser or other electromagnetic radiation emitting device used in the destruction process is a particularly time-consuming process that requires extensive training and expertise to perform properly. This issue is particularly prevalent when changing between sample batches of a particular particle, such as when changing between unprocessed sperm samples from different male animals in semen processing. Existing systems and methods only adjust the height of the destruction or kill location within the interrogation or action location of the flow cytometer (e.g., the size of the kill beam or kill location in the longitudinal or flow direction), whose width is not controlled and which is a function of the laser divergence. Existing laser-based flow cytometer systems may also include multiple optical systems and paths to provide fluorescent and "kill" lasers and associated detector systems at single or sets of adjacent interrogation or action locations. These existing systems may require complex, time-consuming precise adjustments and configurations to function properly. If improperly adjusted, these existing systems may not function as desired, if at all. Multiple optical paths may also cause problems with crosstalk and interference between separate optical systems.

[0019] What is needed are improved systems and methods for particle detection and destruction at interrogation or action locations in flow cytometer systems that improve the rate, ratio, or rate of destruction for particles that have particular fluorescent characteristics, have particular properties, or meet particular thresholds detected by one or more detectors. What is needed is a laser-based flow cytometer that has adjustable, tunable, or improved width for the laser kill location.

[0020] Furthermore, sample mixing in existing systems requires a manual mixing process where the operator gently swirls the contents of the capture tube periodically, for example, every 5 minutes. This process is subject to several forms of error, ranging from interpretation of what is defined as gentle swirling, operator-to-operator variance in achieving mixing every 5 minutes, and removal of the tube from the path of the sample, e.g., cells, or causing the sample to splash out of the tube due to overmixing. Off-the-shelf orbital shakers can be automated, but have their own disadvantages. One is that they can take up space if they are too tall to fit under the pipetting equipment. Another disadvantage is that the orbital movement can move the capture tube out of or out of the path of the dripped sample. More compact mixers, such as vortexers, induce a quick vibrational motion to provide rapid mixing. However, these solutions can cause stress to the cells. What is needed are systems and methods that improve the sample collection and mixing process.

[0021] Additionally, there is a need to automate some or all of the processes required to process samples in a flow cytometry system, including automation or semi-automation of at least the manual processes described above, to improve efficiency, reduce downtime, and reduce operator-induced errors.

[0022] Existing cytometer systems are large, bulky, and non-modular. Their components may be shipped separately or disassembled from one another, requiring an expert or specialized training for adjustment and operation. Once assembled, the components are integrated into the instrument itself and cannot be individually removed or repaired without extensive disassembly of the entire device or the device's table. These systems are intricate and complex and do not lend themselves to on-site repair or calibration by relatively unskilled technicians. Furthermore, these systems require extensive downtime for maintenance, repair, or cleaning, resulting in interruptions of several hours or more if skilled technicians are not available. It would therefore be desirable to have a cytometer system with a modular assembly that can be easily adjusted, operated, and repaired by any operator without the need for specialized training.

[0023] The problems or issues described herein are exemplary and are not inclusive of all problems or issues that may be addressed, and one of ordinary skill in the art will appreciate that a system, apparatus, or method that provides a solution to or addresses the problems defined above also solves or addresses other similar, complementary, or undefined problems. [Prior art documents] [Patent documents]

[0024] [Patent Document 1] US8961904 [Patent Document 2] US9588100 [Patent Document 3] US10488320 [Patent Document 4] US10928298 [Patent Document 5] US10532357 [Patent Document 6] US16 / 279430 [Patent Document 7] US16 / 419756 [Patent Document 8] US16 / 597235 [Patent Document 9] US16 / 704175 [Patent Document 10] US16 / 741608 Summary of the Invention

[0025] Provided herein are automated and semi-automated systems, devices, and methods for processing samples in flow cytometry-based systems. In some aspects, the invention features a modular flow cytometry system for processing samples.

[0026] In some embodiments, the modular flow cytometry system may include a support structure, an electronics module, a sample path module, a fluidics module, an interrogation module, and a sample mixing and collection module. Each module may be configured and disposed on the support structure such that it can be independently removed and reinstalled from the support structure or replaced with another identical module. In some embodiments, the system may further include a microfluidic device cleaning module for cleaning the sample path module.

[0027] In some other embodiments, a modular flow cytometry system for processing samples may include a support structure, an electronics module with one or more computer processors and electronic ports, a sample path module with a microfluidic module having at least one microfluidic channel, a fluidics module fluidically coupled to the sample path module to circulate one or more fluids through the sample path module, an interrogation module including a detector and operably coupled to the electronics module, a detection laser and a kill laser for processing samples flowing through the microfluidic chip, and a sample mixing and collection module configured to collect and mix the processed samples exiting the microfluidic module. Each module may be configured and disposed on the support structure such that they can be separately removed and reinstalled from the support structure or replaced with another identical module for substantially continuous operation of the modular flow cytometry system. The kill laser and the detection laser may be disposed on a common side of the interrogation module. The sample mixing and collection modules may be rotated about a pivot to move in an arcuate path, allowing for mixing of the samples in a controlled and uniform motion. In some embodiments, the system may further comprise a microfluidic device cleaning module for cleaning the sample passage module.

[0028] In some embodiments, the invention features a modular flow cytometry system for gendering a sample. The system may include a chassis partitioned into multiple levels. A first level of the chassis includes an electronics module including one or more computer processors and a plurality of electronics ports. A second level of the chassis includes a sample path module including a microfluidic module for holding a microfluidic chip, and a fluidics module fluidly coupled to the sample path module to circulate one or more fluids through the sample path module. A third level of the chassis includes an interrogation module operably coupled to the electronics module, the interrogation module including a detection laser operably coupled to the detection assembly and a kill laser operably coupled to the detection assembly for processing samples flowing through the microfluidic chip, the kill laser and the detection laser being disposed on a common side of the interrogation module. Also disposed on the same second level of the chassis below the microfluidic module is a sample mixing and collection module configured to collect and automatically mix processed samples exiting the microfluidic chip. The sample mixing and collection module rotates about a pivot to move in an arcuate path, allowing for controlled, uniform motion to mix the samples. Each module can be configured and disposed on the support structure such that it can be independently removed from the support structure and reinstalled, or replaced with another identical module, for near continuous operation of the modular flow cytometry system.

[0029] In other embodiments, the sample passage module may further comprise a distribution block fluidically coupled to the microfluidic module. The distribution block may deliver a sample to the microfluidic module through a first fluidic line and a sheath fluid to the microfluidic module through a second fluidic line. In other embodiments, the system does not require a sheath fluid and the microfluidic module only needs to receive input from a single fluidic line that delivers the sample. In some embodiments, the distribution block comprises a fluidic manifold. In other embodiments, the distribution block further comprises a sample tube loader.

[0030] In some embodiments, the system may further comprise a microfluidic device cleaning module for cleaning the sample path module. The microfluidic device cleaning system is adapted to clean the sample path module circulating fluid through the sample path module. In some embodiments, the microfluidic device cleaning module may comprise a set of fluid reservoirs, a pump assembly, and a distribution block interface comprising a distribution block securing element and a distribution block adjustment element. The distribution block interface further comprises a set of fluid interfaces, at least one of which is in fluid communication with at least one of the set of fluid reservoirs, and a microfluidic device interface comprising a microfluidic device securing element and a microfluidic device alignment element. The microfluidic device interface further comprises a fluid interface in fluid communication with at least one of the set of fluid reservoirs. The distribution block interface may be adapted to secure a distribution block thereon, and the microfluidic device interface may be adapted to secure a microfluidic module thereon.

[0031] According to some embodiments, the interrogation module may further include a laser heat sink for the laser, which may draw air by natural convection to cool the optical module. In one embodiment, the kill laser and the detection laser are each disposed on a common side relative to a major surface of the microfluidic chip. In another embodiment, the kill laser and the detection laser are each adapted to emit a laser beam including an elliptical beam profile at a focal plane. In a preferred embodiment, the interrogation component of the interrogation module may be removed and replaced or reconnected.

[0032] In other embodiments, the sample mixing and collection module may include a rotating base, a set of collection tube holders disposed on the rotating base, a set of collection tubes each configured to be disposed in a collection tube holder of the set of collection tubes, and a fluid level sensor. One of the collection tubes may be positioned below the microfluidic chip such that a processed sample is collected in the collection tube. The sample mixing and collection module is configured to replace the collection tube with another collection tube to collect the processed sample when the fluid level sensor detects that the collection tube has reached a desired fill level. In some embodiments, the sample mixing and collection module may further include a mechanical diverter configured to prevent the processed sample exiting the microfluidic module from entering the sample mixing and collection module.

[0033] In other embodiments, the electronics module includes an electronics box with a removable lid for housing the computer processor and other electronic components. The electronic components can be removed from the electronics box and replaced or reconnected. In some embodiments, the electronics box is disposed on rails for easy movement. In other embodiments, the electronics module includes one or more fans for cooling the electronic components.

[0034] In some embodiments, one or more identification elements may be disposed on or etched into the surface of the microfluidic chip. Examples of the one or more identification elements include, but are not limited to, an alphanumeric string, a barcode, a QR code, a fiducial marker, or a combination thereof. In one embodiment, the one or more identification elements may be used to identify a chip currently mounted on a microfluidic module. In another embodiment, the one or more identification elements may be used to automatically align and position the microfluidic chip relative to a detection laser and a kill laser. For example, the microfluidic chip with the identification elements may be mounted on a multi-axis stage, and the multi-axis stage may be adjusted to reposition the microfluidic chip as needed based on reading of the identification elements by a detector, camera, scanner, or sensor.

[0035] In some embodiments, the system may further comprise an automatic sample level detector adapted to determine a sample level utilizing a sample flow rate. The sample flow rate is related to a cell rate. If the cell rate is lower or higher than a target cell rate or is not within a tolerance range, the automatic sample level detector may adjust the sample flow rate by adjusting the input sample pressure. If the input sample pressure exceeds a threshold pressure, the automatic sample level detector may provide a notification that the sample level is low or below a threshold level.

[0036] In one embodiment, the sample is a semen sample containing sperm cells from a non-human mammal, non-limiting examples of which include cows, pigs, horses, sheep, and goats.

[0037] According to another embodiment, the present invention provides a method for sexing sperm. In one embodiment, the method for sexing sperm includes classifying sperm cells as having a first characteristic, a second characteristic, or a third characteristic, and selectively wounding at least a portion of the sperm cells based on the classification. The first characteristic can be the presence of an X chromosome from one sperm cell, referred to as a singlet. The second characteristic can be the presence of a Y chromosome from one sperm cell. The third characteristic can be the presence of both an X and a Y chromosome, or the repetition of one of the chromosomes by multiple sperm cells, also referred to as polyploidy. By way of non-limiting example, the third characteristic can be an X and an X chromosome, an X and a Y chromosome, a Y and an X chromosome, or a Y and a Y chromosome by two sperm cells.

[0038] In another embodiment, a method for sexing sperm may include interrogating stained sperm cells with a first radiation source to produce fluorescent radiation, detecting the fluorescent radiation, classifying the sperm cells based on the detected fluorescent radiation, and selectively damaging the sperm cells with a second radiation source based on the classification.

[0039] In yet another embodiment, a method of sperm sexing includes delivering a fluid stream containing stained sperm cells to an interrogation location, interrogating the stained sperm cells with a first laser to generate fluorescent radiation, detecting the fluorescent radiation, classifying the sperm cells based on the detected fluorescent radiation, and selectively damaging one or more subpopulations of the classified sperm cells with a second laser based on the classification. In another embodiment, the method may further include staining the sperm cells to generate stained sperm cells. The sperm cells may be stained with a fluorescent DNA-binding dye.

[0040] In some embodiments, interrogating the stained sperm cells produces a fluorescent emission corresponding to a sperm cell or a fluorescent emission corresponding to multiple sperm cells. In one embodiment, damaging the sperm cells may cause DNA damage and / or membrane damage to the cells. In one embodiment, damaging the sperm cells causes the sperm cells to become infertile or dead sperm cells.

[0041] According to another embodiment, the present invention provides a method for producing a sexed semen product. In one embodiment, the method includes the steps of: providing a modular flow cytometry system comprising an electronics module, a sample passage module, a fluidics module, an interrogation module, and a sample mixing and collection module, each module configured to be separately removed and reinstalled or replaced with another of the same module and disposed within a support structure; flowing a semen sample having stained sperm cells through the fluidics module and the sample passage module; flowing a sheath fluid through the fluidics module and the sample passage module; causing the stained sperm cells to fluoresce using a detection laser in the interrogation module; detecting the fluorescence of the stained sperm cells; generating a sexed semen product by killing a subpopulation of the stained sperm cells using a kill laser in the interrogation module based on the detected fluorescence; collecting the sexed semen product flowing from the sample passage module into the sample mixing and collection module; and mixing the collected sexed semen product.

[0042] In other embodiments, the method may further include staining the semen sample containing the sperm cells to produce stained sperm cells. In yet other embodiments, the method may further include cleaning the sample passage module using a microfluidic device cleaning module. In some embodiments, the radiation from the detection laser and the kill laser may include an elliptical beam profile. In other embodiments, the sample mixing and collection module rotates about a pivot to move in an arcuate path, allowing mixing of the sexed semen product in a controlled, uniform motion.

[0043] In another embodiment, a method for producing a sexed semen product includes the steps of providing a modular flow cytometry system as described herein; staining a semen sample containing sperm cells to produce stained sperm cells; flowing the semen sample through a fluidics module and a sample passage module; flowing a sheath fluid through the fluidics module and the sample passage module, the sheath fluid, one or more channels in a microfluidic chip, or a combination thereof, that targets the stained sperm cells as the sheath fluid flows through the microfluidic chip; and causing the stained sperm cells to fluoresce with radiation from a detection laser having an elliptical beam profile. The method may include detecting the fluorescence of the sperm cells with a detection assembly, generating a sexed semen product by inactivating one or more subpopulations of the stained sperm cells with radiation from a kill laser based on the detected fluorescence, where the radiation from the kill laser includes an elliptical beam profile, and the detection laser and the kill laser are disposed on the same side of the microfluidic chip, collecting the sexed semen product flowing from the microfluidic chip in a sample mixing and collection module, and automatically mixing the collected sexed semen product based on a set of defined sample mixing parameters associated with the sample being processed. The sample mixing and collection module can rotate about a pivot to move in an arcuate path, allowing mixing of the sexed semen product with a controlled, uniform motion.

[0044] In some embodiments, the method may further include diverting the flow of sexed semen product exiting the microfluidic chip away from the sample mixing and collection module to a separate collector. In other embodiments, the method may further include automatically aligning and positioning the microfluidic chip with respect to the detection laser and the kill laser by using an identification element disposed on the microfluidic chip. In some other embodiments, the method may further include determining the sample level using an automatic sample level detector and indicating when the sample level is below a threshold level. In further embodiments, the method may include cleaning the sample passage module using a microfluidic device cleaning module.

[0045] In some embodiments, the method may further include diverting the flow of the sexed semen product away from the microfluidic chip when the collection tube of the sample mixing and collection module is filled to a desired level with the sexed semen product, rotating the rotating base of the sample mixing and collection module so that a second collection tube is positioned to collect the sexed semen product, and diverting the flow of the sexed semen product to the second collection tube.

[0046] According to another embodiment, the invention features a system, method, and apparatus for providing a removable and replaceable sample path module for use in a flow cytometry device, and a microfluidic device cleaning system for automatically or semi-automatically cleaning the removable and replaceable sample path module. The sample path module comprises a distribution block in fluid connection or communication with the microfluidic module. The distribution block of the removable and replaceable sample path module, the microfluidic module, and the fluid connections therebetween comprise an entire fluid path through which a fluid sample passes through the removable and replaceable sample path module and is processed by the flow cytometry device. The distribution block of the removable and replaceable sample path module and the microfluidic module interface with corresponding interfaces of either the flow cytometry device or the microfluidic device cleaning system to receive one or more fluids for cleaning or processing. The removable and replaceable sample passage module is releasably secured to a flow cytometry device or a microfluidic device cleaning module and can be used to process a fluid sample, removed, cleaned, and reused or replaced.

[0047] The microfluidic device cleaning system comprises an automated or semi-automated system for cleaning a sample path module, such as the removable and replaceable sample path module described above. The microfluidic device cleaning system comprises a distribution block of the sample path module and an interface for the microfluidic module, where one or more fluids can be processed through the sample path module to clean the sample path module. A set of one or more internal or external reservoirs contains one or more cleaning fluids used in the cleaning process. An internal or external control system, such as a computer, tablet, or dedicated microprocessor, is used to control the cleaning process. The control system is used to control the operation of other internal devices, including one or more internal valves, one or more internal pumps, and to process signals from one or more sensors, such as a proximity sensor, a camera or image sensor, and a flow sensor to control the operation of the valves or pumps.

[0048] Additionally, without wishing to be limited to any theory or mechanism of the invention, the methods and systems described herein are believed to be advantageous because the removable, replaceable, and washable modular sample path modules and microfluidic device cleaning system eliminate or substantially reduce the downtime typically consumed when replacing sample runs or microfluidic chips in flow cytometry devices used to process particles in fluid samples.

[0049] In a first embodiment, a removable and replaceable fluid path module for use in a flow cytometer system is provided, the fluid path comprising: a distribution block having a set of fluid inlets and a set of fluid outlets, at least one of the set of fluid inlets being fluidly connected to at least one of the set of fluid outlets to form a first sample path element; a microfluidic module having a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixation device, at least one of the set of fluid inlets being fluidly connected to at least one of the set of fluid outlets to form a second sample path element; and a fluid path providing fluid communication between the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, a portion of the fluid path joining and fluidly connecting the first sample path element and the second sample path element to form a sample path, wherein a sample being processed by the flow cytometer system through the removable and replaceable fluid path module is confined to the sample path during processing.

[0050] The microfluidic device fixture is adapted to fix one of the microfluidic chip or the microfluidic cassette in the microfluidic module. The microfluidic device fixture may comprise one or more adjustment means adapted to adjust the position of the microfluidic chip or the microfluidic cassette. The distribution block may comprise a fluidic manifold. The distribution block may further comprise a sample tube loader. The fluid path module may further comprise a distribution block, where another inlet of the set of fluid inlets is in fluid communication with another outlet of the set of fluid outlets to form a first buffer fluid path element, and another inlet of the set of fluid inlets of the microfluidic module is in fluid communication with another outlet of the set of fluid outlets to form a second buffer fluid path element. The fluid path further comprises a second portion that joins and fluidly communicates the first buffer fluid path element with the second buffer fluid path element to form a buffer fluid path, and the buffer fluid processed through the removable and replaceable fluid path module is confined to the buffer fluid path during processing.

[0051] The distribution block and the microfluidic module may each further comprise releasable fastening means adapted to fasten the removable and replaceable fluid path module to a flow cytometer system. The releasable fastening means may comprise at least one iron plate or at least one magnetic plate. The removable and replaceable fluid path module may be removable from the flow cytometer system by releasing the fastening means. The releasable fastening means of the removable and replaceable fluid path module may further be adapted to fasten the removable and replaceable fluid path module to a fluid path cleaning system.

[0052] The sample may be a mammalian semen sample. In one embodiment, the sample may include sperm cells, such as non-human sperm cells, including pig, cow, horse, and sheep. The sperm cells may be stained with a dye. A flow cytometer system may process the sample to identify subpopulations in the sample. The system may process the sample to perform a semen sexing operation.

[0053] In some embodiments, the fluid passage module may comprise an identification element. The identification element may comprise an optical identification element or a radio frequency identification element. The optical identification element may comprise a set of features or criteria on a microfluidic chip in the microfluidic module.

[0054] In another embodiment, a microfluidic device cleaning system is provided, the system comprising: a set of fluid reservoirs, a pump assembly, and a distribution block interface comprising a distribution block securing element and a distribution block adjustment element. The distribution block interface further comprises a set of fluid interfaces, at least one of which is in fluid communication with the fluid reservoirs, and a microfluidic device interface comprising a microfluidic device securing element and a microfluidic device alignment element. The microfluidic device interface further comprises a fluid interface in fluid communication with at least one of the set of fluid reservoirs, and the microfluidic device cleaning system is adapted to circulate fluid through a removable and replaceable fluid passage module secured to the distribution block interface and the microfluidic device interface.

[0055] The removable and replaceable fluid path module is adapted for use in a flow cytometer system and further comprises: a distribution block having a set of fluid inlets and a set of fluid outlets, at least one of the set of fluid inlets in fluid communication with at least one of the set of fluid outlets to form a first sample path element; a microfluidic module having a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixation device, at least one of the set of fluid inlets in fluid communication with at least one of the set of fluid outlets to form a second sample path element; and a fluid path providing fluid communication between the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, a portion of the fluid path joining and fluidly connecting the first sample path element and the second sample path element to form a sample path, wherein a sample processed by the flow cytometer system through the removable and replaceable fluid path module is confined to the sample path during processing.

[0056] The set of fluid reservoirs may include a wash fluid reservoir and a waste fluid reservoir. The set of fluid reservoirs may be disposed external to the housing of the system. The microfluidic device cleaning system may further include a plurality of releasable connections providing fluidic connection to the external set of fluid reservoirs. The pump assembly may include a liquid pump and an air pump. At least one of the pumps may include a peristaltic pump. The flow cytometer system may further include a set of valves for controlling internal fluid flow. The flow cytometer system may further include a set of fluid flow sensors adapted to provide a signal indicative of a flow rate in one or more elements of the flow cytometer system. The flow cytometer system may further include a set of proximity sensors adapted to provide a signal indicative of the presence or absence of a removable and replaceable fluid path module. The flow cytometer system may further include an imaging device for capturing a set of images of the removable and replaceable fluid path module, the distribution block interface, or the microfluidic device interface.

[0057] The microfluidic device cleaning system may further comprise a monitoring and interface system comprising a processor, a memory, and an input / output interface. The monitoring and interface system may be adapted to receive a set of inputs for selecting or configuring a cleaning protocol for the microfluidic device cleaning system. The set of inputs may include a set of user inputs. The set of inputs may be automatically determined based on the nature or characteristics of the removable and replaceable fluid path module. The monitoring and input system may be adapted to receive a set of inputs from at least one sensor and modify the cleaning protocol based thereon. The monitoring and input system may be adapted to transmit a set of control signals to one or more valves, actuators, or pumps based on the cleaning protocol. The microfluidic device cleaning system may further comprise a flow cytometry device. The flow cytometry device may comprise a fluorescence-based detection and laser ablation flow cytometry device.

[0058] In another embodiment, a method for replacing and cleaning a sample path module for a flow cytometry system is provided, the method comprising the steps of releasing a sample path module from a flow cytometry device, the sample path module comprising an entire fluid path for processing a sample by the flow cytometry system, removing the sample path module from the flow cytometry device, connecting the sample path module to a corresponding receptacle of a microfluidic device cleaning system, configuring a set of cleaning parameters for the microfluidic device cleaning system, and automatically cleaning the sample path module by the microfluidic device cleaning system based on the set of cleaning parameters.

[0059] In some embodiments, the sample path module is adapted for use in a flow cytometry system and further comprises a distribution block having a set of fluid inlets and a set of fluid outlets, where at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a first sample path element, a microfluidic module having a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixture, where at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a second sample path element, and a fluid passageway fluidly communicating between the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, where a portion of the fluid passageway joins and fluidly communicates the first sample path element with the second sample path element to form a fluid path, and where a sample processed by the flow cytometer system through the sample path module is confined to the fluid passageway during processing. The method may further comprise removing the sample path module from the microfluidic device cleaning system. The method may further include a set of cleaning parameters including an amount or volume of cleaning fluid, a fluid pressure, and a duration of the cleaning procedure. The duration of the cleaning procedure may include a number of paths to be cleaned, which is the number of sample passage volumes to be cleaned. The duration of the cleaning procedure may include at least one drying cycle.

[0060] In some embodiments, the method may further include detecting an obstruction in the sample passage module using an imaging sensor and stopping the automatic cleaning. Stopping the automatic cleaning may further include providing an error indication. The method may further include measuring a volume of fluid used for the automatic cleaning using at least one flow sensor. The measured volume may be used to determine a maintenance event for the microfluidic device cleaning system.

[0061] In another embodiment, the method may further include transmitting a signal from the at least one proximity sensor to a microfluidic device cleaning system indicating that the sample path module is properly connected, and initiating automatic cleaning based on the transmitted signal. The sample path module may be adapted for use in a flow cytometry system. The overall fluid path for processing the sample may further include a sample path with a fluid inlet and a fluid outlet in fluid communication, and a microfluidic chip with a set of fluid inlets and a set of fluid outlets, the set of fluid inlets being in fluid communication with the set of fluid outlets to form at least one microfluidic channel, the microfluidic chip being disposed between the fluid inlets and the fluid outlet of the sample path, the at least one microfluidic channel comprising an element of the sample path, and a sample being processed by the flow cytometry system through the sample path module is confined to the sample path during processing.

[0062] In another embodiment, there is provided a fluid passage module for use in processing a fluid sample comprising a plurality of particles, the fluid passage comprising: a sample passage having a fluid inlet and a fluid outlet in fluid communication; and a microfluidic chip having a set of fluid inlets and a set of fluid outlets, the set of fluid inlets being in fluid communication with the set of fluid outlets to form at least one microfluidic channel, the microfluidic chip being disposed between the fluid inlets and fluid outlets of the sample passage, the at least one microfluidic channel comprising elements of the sample passage, and a fluid sample passing through the fluid passage module and processed by a flow cytometry device is confined to the sample passage during processing.

[0063] The fluid path module of the above embodiment may further comprise a sheath fluid path comprising a fluid inlet and a fluid outlet in fluid communication. A microfluidic chip may be disposed between the fluid inlet and the fluid outlet of the sheath fluid path, and at least one microfluidic channel comprises an element of the sample path. The fluid path module may further comprise a distribution block comprising a set of fluid inlets and a set of fluid outlets, where at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a first sample path element, a microfluidic module comprising a microfluidic device fixation device adapted to fix the set of fluid inlets, the set of fluid outlets, and the microfluidic chip, where at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a second sample path element, and a fluid path fluidically connecting the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, where a portion of the fluid path joins and fluidically connects the first sample path element and the second sample path element to form the sample path.

[0064] The distribution block may comprise a fluidic manifold. The distribution block may further comprise a sample tube loader. The fluid path module may further comprise a releasable fastening means adapted to fasten the fluid path module to a flow cytometer system. The releasable fastening means may comprise at least one iron plate or at least one magnetic plate. The fluid path module may be removable from the flow cytometer system by releasing the fastening means. The releasable fastening means of the removable and replaceable fluid path module may further be adapted to fasten the removable and replaceable fluid path module to a fluid path cleaning system. The fluid sample may be a mammalian semen sample comprising a plurality of sperm cells. The flow cytometer system may process the sample to identify subpopulations in the fluid sample. The flow cytometer system may process the sample to perform a semen sexing operation. The fluid path module may comprise an identification element. The identification element may comprise an optical identification element or a radio frequency identification element. The optical discrimination element may comprise a set of features or criteria on a microfluidic chip in the microfluidic module.

[0065] According to another embodiment, the present invention provides a flow cytometer system with an improved optical path and combination of elements for delivering electromagnetic radiation to interrogation and / or action locations in a flow cytometer. The novel system of the present invention emits both a detection laser and a kill or ablation laser in the same direction as the sample fluid flow.

[0066] The present invention addresses existing problems in known systems and methods for optically detecting and destroying particles in flow cytometer systems. In existing systems, directing beams from two or more lasers through separate beam paths increases the chance of damage or degradation of components over time. In addition, emitting electromagnetic radiation from separate sides of the flow stream can require complex beam paths and complex orientations and arrangements of elements to focus and detect signals derived from the electromagnetic radiation. The present invention addresses these problems by emitting both a detection laser, for fluorescing particles such as those that have undergone a staining process, and a kill or ablation laser from the same direction as the sample fluid flow.

[0067] In addition, the shape of the kill beam spot in the ablation region of a flow cytometer system used in the sperm sexing process significantly impacts the photon dose delivered to a particular point in the sample flow, affecting the effectiveness of the kill laser on individual cells. The width of the kill laser kill beam or kill location provided by the present invention is equal to or greater than the width of the flow channel or droplet size in the flow cytometer system. The present invention provides for adjustment of the beam waist or width of the laser beam emission at the focal plane in the flow cytometer system, which is not a function of the laser divergence as determined by adjusting the height of the beam at the focal plane.

[0068] Additionally, without wishing to be limited to any theory or mechanism of the invention, it is believed that the methods and systems herein are advantageous because they provide faster kill alignment, more consistent and uniform shifting of sorted particles (i.e., more particles of the desired type are collected at the end of the sorting process), and more consistent performance of the flow cytometer sorting system. The present invention may also provide faster or easier training and setup compared to existing systems and methods based on a simplified optical path provided by positioning and firing of the detection laser and kill laser in the same direction as the sample fluid flow. The simple system of the present invention may also be applied or used in a wide range of microfluidic or flow cytometry systems, providing increased flexibility of implementation compared to existing systems and methods.

[0069] Additionally, without wishing to be limited to any theory or mechanism of the present invention, it is believed that the methods and systems herein are advantageous because the combination of adjustable beam expanders, cylindrical lenses and aspheric lenses with beam combiners such as polarizing beam splitters provides for positioning of the kill laser assembly and the detection laser assembly on a common side relative to the microfluidic chip, while providing an adjustable elliptical beam waist at the focal plane that can be specifically configured to the type or size of the microfluidic channel or chip.

[0070] In a first embodiment, the present invention provides a laser-based flow cytometry system for particle detection, the system comprising a laser assembly comprising a kill laser assembly and a detection laser assembly, the kill laser assembly and the detection laser assembly being disposed on a common side with respect to each other with respect to the flow cytometry system. The flow cytometry system may comprise a microfluidic device, such as a microfluidic chip or a microfluidic cassette. The kill laser assembly and the detection laser assembly are disposed on a common side with respect to a major surface of the microfluidic device. The major surface may be a top surface or a bottom surface. The laser assembly may further comprise a polarizing beam splitter. Each of the kill laser assembly and the detection laser assembly may further comprise an optical path adapted to direct electromagnetic radiation to a focal plane. The optical path of the kill laser assembly and the optical path of the detection laser assembly each comprise a fast axis beam expander and a slow axis beam expander. The optical path of the detection laser may comprise a pair of cylindrical lenses. The fast axis beam expander and the slow axis beam expander of the optical path for the kill laser assembly can be adapted to adjust the fast axis beam width and the slow axis beam width of the kill laser assembly. The fast axis beam expander and the slow axis beam expander of the optical path for the detection laser assembly can be adapted to adjust the fast axis beam width and the slow axis beam width of the detection laser assembly. The cylindrical lens of the detection laser optical path can be adapted to adjust and beam shape the fast axis beam width and the slow axis beam width of the detection laser assembly.

[0071] In some embodiments, the kill laser assembly and the detection laser assembly may be adapted to emit a laser beam including an elliptical beam profile at a focal plane. The elliptical beam profile of the kill laser assembly may have a fast axis beam width of 0.5-5 μm as measured at the focal plane. The elliptical beam profile of the kill laser assembly may have a slow axis beam width of 5-35 μm as measured at the focal plane. The elliptical beam profile of the kill laser assembly may have a fast axis beam width of 3.45-5 μm and a slow axis beam width of 5-35 μm as measured at the focal plane. The elliptical beam profile of the detection laser assembly may have a fast axis beam width of 3.5-20 μm and a slow axis beam width of 30-150 μm as measured at the focal plane. The kill laser assembly may operate at a pulse energy of 1-5 μJ per pulse. The kill laser assembly may comprise a pulse laser module having a pulse duration of 1-500 nanoseconds. The kill laser assembly may comprise a pulsed laser module having a pulse duration of 5 to 30 nanoseconds. The detection laser assembly may comprise a continuous wave laser module, a quasi-continuous wave laser module, or a pulsed laser module. The system may further comprise a detection assembly. The detection assembly may be adapted to detect one or more of fluorescence of the particle excited by radiation from the detection laser assembly, deactivation of the particle by radiation from the kill laser assembly, an image of the detection event, or an image of the kill event. The detection assembly may comprise at least one avalanche photodiode. The at least one avalanche photodiode may be adapted to detect fluorescence of the particle excited by radiation from the detection laser assembly. The at least one avalanche photodiode may be adapted to detect deactivation of the particle by radiation from the kill laser assembly. The detection assembly may comprise a CCD camera. The CCD camera may be adapted to capture an image of the detection event. The CCD camera may be adapted to capture an image of the kill event.

[0072] In another embodiment, the present invention provides a laser assembly comprising a laser module and an optical passage adapted to shape an electromagnetic radiation beam emitting from the laser module into an elliptical profile at a focal plane.

[0073] In some embodiments, the laser assembly is one of a detection laser assembly and a kill laser assembly. The optical path may include a fast axis beam expander and a slow axis beam expander. The optical path may include a pair of cylindrical lenses. The fast axis beam expander may be adapted to adjust a fast axis beam width for the beam emission. The slow axis beam expander may be adapted to adjust a slow axis beam width for the beam emission. The pair of cylindrical lenses may be adapted to adjust a beam profile at a focal plane of the beam emission. The elliptical beam profile of the laser assembly may have a fast axis beam width of 0.5 to 5 μm as measured at the focal plane. The elliptical beam profile of the laser assembly may have a slow axis beam width of 5 to 35 μm at the focal plane. The elliptical beam profile of the laser assembly may have a fast axis beam width of 3.45 to 5 μm and a slow axis beam width of 5 to 35 μm at the focal plane. The elliptical beam profile of the laser assembly may have a fast axis beam width of 3.5-20 μm and a slow axis beam width of 30-150 μm at the focal plane. The laser module may operate at a pulse energy of 1-5 μJ per pulse. The laser module may comprise a pulsed laser module having a pulse duration of 1-500 nanoseconds. The laser module may comprise a pulsed laser module having a pulse duration of 5-30 nanoseconds. The laser module may comprise a continuous wave laser module, a quasi-continuous wave laser module, or a pulsed laser module. The pair of cylindrical lenses may comprise an F25 cylindrical lens and an F150 cylindrical lens. The optical path may comprise an aspheric lens. The optical path may comprise a polarizing beam splitter. The optical path may comprise a harmonic separator. The optical path may comprise a wave plate.

[0074] In another embodiment, the present invention provides an optical path for a laser assembly for use in flow cytometry, the optical path comprising a beam expander, a wave plate, an aspheric lens, and a polarizing beam splitter, the beam expander adapted to shape an electromagnetic radiation beam emitting from a laser module into an elliptical beam profile at a focal plane.

[0075] In some embodiments, the beam expander comprises a fast axis beam expander and a slow axis beam expander. The fast axis beam expander can be adapted to adjust a fast axis beam width for the beam emission. The slow axis beam expander can be adapted to adjust a slow axis beam width for the beam emission. The elliptical beam profile can have a fast axis beam width of 0.5-5 μm and a slow axis beam width of 5-35 μm as measured at the focal plane. The laser module can operate with a pulse energy of 1-5 μJ per pulse. The laser module can comprise a pulsed laser module having a pulse duration of 1-500 nanoseconds. The laser module can comprise a pulsed laser having a duration of 5-30 nanoseconds. The optical path can further comprise a harmonic separator.

[0076] In another embodiment, the present invention provides a sexed non-human mammalian semen product, the product comprising a collection of sexed non-human mammalian sperm cells derived from a sample of unsexed non-human semen, the unsexed non-human semen comprising the steps of: staining the unsexed non-human semen sample; inserting the stained sample of unsexed non-human semen into a flow cytometry device; inserting a sheath fluid into the flow cytometry device, the sheath fluid and a channel of the flow cytometry device orienting and positioning the cells in the sample of unsexed non-human semen; and fluorescing a dye in the stained sample of unsexed non-human semen with radiation from a first laser module, the radiation from the first laser module comprising an elliptical beam profile. the dye fluorescing; detecting by a detector the fluorescence for each sperm cell in the stained sample of unsexed non-human semen; and inactivating a subset of the non-human sperm with radiation from a second laser module based on the detected fluorescence to create sexed non-human sperm, wherein the radiation from the second laser module comprises an elliptical beam profile, and the first laser module and the second laser module are disposed on a common side of the flow cytometry device opposite the detector.

[0077] In another embodiment, the present invention provides a method for sexing non-human mammalian semen, the method comprising the steps of staining a sample of unsexed non-human semen; inserting the stained sample of unsexed non-human semen into a flow cytometry device; inserting a sheath fluid into the flow cytometry device, the sheath fluid and a channel of the flow cytometry device orienting and positioning cells in the sample of unsexed non-human semen; and causing a dye in the stained sample of unsexed non-human semen to fluoresce with radiation from a first laser module, the first laser module emitting a first fluoresce signal to the dye in the sample of unsexed non-human semen. the radiation from the first laser module comprising an elliptical beam profile; detecting by a detector the fluorescence for each sperm cell in the stained sample of unsexed, non-human semen; inactivating a subset of the non-human sperm with radiation from a second laser module based on the detected fluorescence to create sexed, non-human semen, wherein the radiation from the second laser module comprises an elliptical beam profile, and the first laser module and the second laser module are disposed on a common side of the flow cytometry device opposite the detector; and collecting the sexed, non-human semen.

[0078] According to yet another embodiment, the present invention provides an automated or semi-automated system for routing or redirecting and mixing samples to be processed, for example in a microfluidic chip-based flow cytometry system. Providing an automated or semi-automated system for handling samples to be processed eliminates manual or operator-intensive steps from the processing of samples, improving the efficiency of the process and the quality of the samples processed. More uniform operation by means of automated sample routing and mixing reduces the chance of operator error by automated routine or repeatable steps in the sample processing operation.

[0079] Another object of the present invention is to provide a system and method that allows for automated mixing, more uniform mixing times, and a path to ensure that the inner diameter of the tube is always below the path of the cells. An embodiment of the present invention includes a mixing system that mixes gently along a pivot, allowing for automation of mixing for limited space applications. An embodiment of the present invention can reduce cell stress by controlling the mixing action, as well as reduce the time that the cells are in an overconcentrated solution of fluid.

[0080] In some aspects, the invention features a sample mixing system. The sample mixing system is used with a flow cytometry system for processing a sample containing constituent molecules or particles. For example, the sample mixing system may be used with a microfluidic chip-based flow cytometry system, where a sample, such as a biological or genetic sample containing a plurality of cells, is processed by a set of electromagnetic beam emitting devices, such as a laser. The cells in the sample may be processed in the microfluidic chip such that a subset of the sample is inactivated, destroyed, or removed following processing in the microfluidic chip, and the sample is released from the microfluidic chip by one or more outlets at the terminal end of the chip. The processed sample is allowed to flow into a sample capture tube disposed on the sample mixing system or diverted to a waste collection container based on meeting a set of criteria or parameters.

[0081] In some examples, a sample mixing system may include a rotating base, a plurality of collection tube holders disposed on the rotating base, and a plurality of collection tubes. Each collection tube is configured to be disposed in a collection tube holder. The collection tubes may contain a medium, such as a buffer medium, in which case mixing maintains buffering and controls the degree of buffering. The rotating base may be rotated such that the collection tube holders and collection tubes rotate or move in an arcuate path. This motion causes mixing of the contents of the collection tubes.

[0082] In some embodiments, the rotating base may be a disk disposed on a platform. In one embodiment, the rotating base is operatively coupled to a motor that rotates the rotating base about a central axis. The rotating base is rotated based on one or more of a blending arc defined in degrees, a blending arc defined in steps, and a rotational speed. In some embodiments, the rotating base may rotate back and forth. The rotating base rotates between approximately 10° and 180°.

[0083] In other embodiments, the rotating base may include magnets spaced apart from one another, and relatedly, the base of each collection tube holder may include a magnet, and each collection tube holder may be attached to the rotating base by magnetic attraction between the magnet of the collection tube holder and one of the magnets of the rotating base.

[0084] In some embodiments, the sample mixing system may further include one or more Hall effect sensors. For example, the south pole of one of the magnets in the rotating base may be oriented toward one or more Hall effect sensors, and the north pole of the other magnet is oriented toward the same one or more Hall effect sensors.

[0085] In other embodiments, the sample mixing system may include between about 2-6 collection tube holders. Each collection tube holder may include two or more arms extending upwardly from a base. The two or more arms are configured to hold a collection tube upright on the base. In some embodiments, the two or more arms are extendable to accommodate collection tubes of various sizes.

[0086] In some embodiments, the system may be positioned below the dispensing device such that the dispensing device dispenses a sample into one of the collection tubes. Preferably, the collection tube receiving the sample remains below the dispensing path of the dispensing device as the collection tube moves in an arcuate path to mix the sample. After mixing the sample in the collection tube for a period of time (e.g., 5-10 minutes), the system automatically places another collection tube below the dispensing device to dispense the sample into another collection tube.

[0087] In other embodiments, the sample mixing system may further comprise a sensor for detecting the liquid level in the collection tube. The sensor may be located at or near an exterior surface of the collection tube. In yet other embodiments, the sample mixing system may further comprise an auto dump configured to prevent flow from entering the collection tube.

[0088] In another aspect, the sample mixing system described herein may be used in a method of mixing sorted samples. The method may include collecting sorted samples in collection tubes of the sample mixing system, and automatically mixing the samples collected in the collection tubes by rotating a rotating base such that collection tubes disposed in the collection tube holders rotate or move uniformly in an arcuate path in a controlled motion to mix the collected samples. In a further embodiment, the method may include diverting the flow of sorted samples from the collection tubes to a separate collector when the collection tubes are filled to a desired level, rotating the rotating base such that a second collection tube is positioned to collect the sorted samples, and diverting the flow of sorted samples to the second collection tube.

[0089] One of the unique and inventive technical features of the present invention is the mixing induced automatically by the arcuate back and forth of the sample tube. Without wishing to limit the present invention to any theory or mechanism, it is believed that the technical features of the present invention advantageously enable more uniform automatic mixing of collection tubes under a dispensing device such as a microfluidic or droplet-based flow cytometer. The arcuate motion ensures a uniform concentration between the cell media and the cells in the sample tube while gradually dripping the cells into the tube and onto the media. The swapping capability allows the motor to quickly move from the "in use" tube to the "standby" tube after mixing of one tube is completed. The sample mixing system of the present invention is also small enough to fit into the allotted space inside the sexing device and requires less human intervention. No prior reference or work currently known has the unique and inventive technical features of the present invention.

[0090] According to another embodiment, the invention features a particle processing system including a flow cytometry assembly for processing particles or constituent molecules in a sample based on at least one characteristic, and an automated sample mixing system configured to automatically mix the sample processed by the flow cytometry assembly based on a defined set of parameters. In one embodiment, the automated sample mixing system can rotate about a pivot to move in an arcuate path, enabling mixing of the sample with a controlled uniform motion. The defined set of parameters includes one or more of a mixing arc defined in degrees, a mixing arc defined in steps, and a rotation speed.

[0091] In one embodiment, the automated sample mixing system further comprises a rotating base, a set of collection tube holders disposed on the rotating base, and a set of collection tubes, each configured to be disposed in a collection tube holder of the set of collection tubes. In another embodiment, the automated sample mixing system further comprises a fluid level sensor. The automated sample mixing system is configured to exchange a first tube with a second tube in the set of collection tubes when the fluid level sensor detects that the first tube has reached a desired fill level.

[0092] In some embodiments, the particle processing system further comprises a mechanical diverter configured to prevent an output stream containing the processed sample from the flow cytometer from entering the automated sample mixing system.

[0093] According to another embodiment, a particle processing system includes a flow cytometry assembly for processing particles or constituent molecules in a sample based on at least one characteristic, and a mechanical diverter configured to prevent an output stream containing the processed sample from the flow cytometer from entering a sample collection vessel based on a defined set of parameters. The mechanical diverter is configured to prevent an output stream containing the processed sample from the flow cytometer from entering an automated sample mixing system. The defined set of parameters for the mechanical diverter may include one or more of the purity of the sample, the number of particles in the sample, and the fill level of the collection vessel.

[0094] In other embodiments, the particle processing system may further comprise an automatic sample mixing system configured to automatically mix samples collected in the sample collection container and processed by the flow cytometry assembly based on a defined set of parameters. The defined set of parameters for the automatic sample mixing system may include one or more of a mixing arc defined in degrees, a mixing arc defined in steps, and a rotational speed. In a non-limiting embodiment, the automatic sample mixing system may comprise a rotating base, a set of collection tube holders disposed on the rotating base, and a set of collection tubes each configured to be disposed in a collection tube holder of the set of collection tubes. The automatic sample mixing system is configured to exchange a first tube with a second tube in the set of collection tubes when a fluid level sensor detects that the first tube has reached a desired fill level. In some embodiments, the automatic sample mixing system further comprises a fluid level sensor.

[0095] In a further embodiment, the present invention provides a method for producing a sexed non-human semen product. The method may include processing a semen sample in a flow cytometry device to select a first subpopulation of samples including at least one desired characteristic from a second subpopulation of samples including at least one undesirable characteristic, collecting a sample in a sample collection container that primarily includes the first subpopulation including the at least one desired characteristic, and automatically mixing the collected sample in the sample collection container using any of the sample mixing systems described herein. Mixing of the collected sample may be based on a set of defined sample mixing parameters associated with the sample being processed, such as one or more of a mixing arc defined in degrees, a mixing arc defined in steps, and a rotational speed. The sample mixing system may rotate about a pivot to move in an arcuate path, allowing mixing of the sample in a controlled, uniform motion.

[0096] In some embodiments, the method further includes mechanically diverting a portion of the processed sample that is collected in a sample collection container after the processed sample exits the flow cytometry device. Mechanically diverting the processed sample may be based on one or more defined parameters including purity of the sample, number of particles in the sample, and fill level of the collection container. In other embodiments, the method includes replacing a first tube with a second tube in the set of collection tubes when the fluid level sensor detects that the first tube has reached a desired fill level.

[0097] It will be apparent to those skilled in the art from the context, the specification, and the knowledge of those skilled in the art that any feature or combination of features described herein is included within the scope of the present invention, unless the features included in such combination are mutually inconsistent. Further advantages and aspects of the present invention are apparent in the following detailed description and claims.

[0098] In order to facilitate a complete understanding of the present invention, reference will now be made to illustrative embodiments illustrated in the accompanying drawings, in which like elements are referenced with like numerals, and in which: These drawings should not be construed as limiting the present invention, but are intended to be illustrative and for reference purposes. [Brief description of the drawings]

[0099] [Figure 1] 1 illustrates a non-limiting example of a modular flow cytometry system of the present invention. [Diagram 2] Front view of three modular flow cytometry systems, each with a monitor, sheath chamber and scale assembly, mouse, collection tray, and scanner. [Figure 3A] FIG. 1 shows a front view of a modular flow cytometry system of the present invention. [Figure 3B] FIG. 2 shows a rear view of a modular flow cytometry system of the present invention. [Figure 4] FIG. 1 shows a block diagram of the instrument workflow of the modular flow cytometry system of the present invention. [Diagram 5] FIG. 1 is a front perspective view of an embodiment of a removable and replaceable sample passage module and a flow cytometry device for processing and analyzing samples. [Figure 6] FIG. 1 is a front perspective view of an embodiment of a removable and replaceable sample passage module and a microfluidic device cleaning system for cleaning the removable and replaceable sample passage module. [Figure 7] The paths between the dowel pins and balls from the distribution block and the microfluidic module to their respective dowel holes are shown. [Figure 8] FIG. 13 is another view of the paths between the dowel pins and balls from the distribution block and the microfluidic module to their respective dowel holes. [Figure 9A] 1 shows the internal components of the fluidics module. [Figure 9B]1 shows how the fluidics module is connected to the rear end fluidics ports of the system. [Figure 10] 1 is a flow chart illustrating an embodiment of a process and method for using a removable and replaceable sample passage in a flow cytometer system and for cleaning the removable and replaceable sample passage using a microfluidic device cleaning system. [Figure 11A] FIG. 1 is a block diagram illustrating an embodiment of a system and method for using a removable and replaceable sample passage in a flow cytometer system and for cleaning the removable and replaceable sample passage using a microfluidic device cleaning system. [Figure 11B] FIG. 1 is a block diagram illustrating an embodiment of a system and method for using a removable and replaceable sample passage in a flow cytometer system and for cleaning the removable and replaceable sample passage using a microfluidic device cleaning system. [Figure 12] FIG. 1 is a block diagram illustrating an embodiment of a microfluidic device cleaning system with an external fluid reservoir. [Figure 13] FIG. 1 is a block diagram illustrating an embodiment of a microfluidic device cleaning system with internal fluid reservoirs and a control system. [Figure 14] FIG. 1 is a block diagram illustrating an embodiment of a removable and replaceable sample path module comprising a microfluidic module and a distribution block. [Figure 15A] FIG. 13 shows another embodiment of a removable and replaceable sample path module comprising a microfluidic module and a distribution block. [Figure 15B] 15B shows a cross-sectional view of the distribution block of FIG. 15A. [Figure 16A] FIG. 1 is a block diagram illustrating a side view of a microfluidic modular embodiment of a removable and replaceable sample passage module. [Figure 16B]1 shows a non-limiting example of a microfluidic chip with an identification element. [Figure 16C] 1 shows a non-limiting arrangement for automatically aligning and positioning a microfluidic chip using the identification elements of the microfluidic chip. [Figure 17] FIG. 1 is a block diagram illustrating an embodiment of a system including a flow cytometry device, a microfluidic device cleaning system, a sample passage module, a local control system, and a remote server. [Figure 18] FIG. 1 is a block diagram showing an example of fluid flow within a microfluidic device cleaning system. [Figure 19A] 1 shows an embodiment of an optics module (interrogation module) of the present invention, which can float separately from the rest of the cytometry system and is isolated by rubber feet. [Figure 19B] 19B shows the internal layout of the optical system module of FIG. 19A. [Figure 20A] 1 shows an embodiment of an electronics module that may be disposed in a cytometry system of the present invention. [Figure 20B] Shows the electronics module covered with a lid. [Figure 20C] The internal components of the electronics module are shown. [Figure 21A] The control board located on the back of the cytometry system is shown, containing the electronic and fluidic ports. [Figure 21B] The control board located on the back of the cytometry system is shown, containing the electronic and fluidic ports. [Figure 22A] An electronic block diagram of the cytometry system is shown. [Figure 22B] An electronic block diagram of the cytometry system is shown. [Diagram 23] 1 is a diagram of a beam width for electromagnetic radiation emission of a kill laser in accordance with an embodiment of the present invention. [Figure 24A]11 is a chart showing the performance of a kill laser beam having an elliptical beam waist profile at the focal plane versus a kill laser beam having a circular beam waist profile at the focal plane in accordance with an embodiment of the present invention. [Figure 24B] 11 is a chart showing the performance of a kill laser beam having an elliptical beam waist profile at the focal plane versus a kill laser beam having a circular beam waist profile at the focal plane in accordance with an embodiment of the present invention. [Figure 25A] FIG. 1 is a block diagram of electrical, optical, and opto-mechanical elements of a laser-based particle processing system in accordance with an embodiment of the present invention. [Figure 25B] FIG. 1 is a block diagram of electrical, optical, and opto-mechanical elements of a laser-based particle processing system in accordance with an embodiment of the present invention. [Figure 26A] 2 is a schematic diagram of an optical path for an electromagnetic radiation beam emission from a detection laser module according to an embodiment of the present invention. [Figure 26B] 1 is a schematic diagram of an optical path for an electromagnetic radiation beam emission from a kill laser module according to an embodiment of the present invention. [Figure 27] 2 is a schematic diagram of optical and electrical elements of a detection assembly according to an embodiment of the present invention. [Figure 28] 1 is a schematic diagram of radiation at a kill spot and a detection spot in an action or interrogation area at a focal plane for detection by a detection assembly according to an embodiment of the present invention. [Figure 29A] 1 illustrates an exemplary graphical user interface representing an output of a measurement of a detected laser beam according to an embodiment of the present invention. [Figure 29B] 13 illustrates an exemplary graphical user interface representing a kill laser beam measurement output according to an embodiment of the present invention. [Diagram 30] A block diagram of how the sample stream is aligned to the detect and kill lasers to achieve the desired resolution and kill count is shown. [Diagram 31] 1 illustrates an alignment procedure that can be used in the present invention. [Diagram 32]A block diagram for automatically measuring sample levels and determining when the sample level is low is shown. [Diagram 33] A flow cytometry-based particle processing system including a sample mixing module and auto-dump components is shown. [Diagram 34] 1 shows a sample mixing system of the present invention with a magnetic collection tube disposed on a rotating base. [Figure 35A] FIG. 13 is a cross-sectional view showing the magnetic attachment of the collection tube holder. [Figure 35B] A top view of a Hall effect sensor installed in the motor of the sample mixing system. The Hall effect sensor is south pole actuated and used for position detection. A single magnet is oriented with its south pole towards the sensor and all other magnets are oriented with their north poles towards the sensor. [Figure 36A] 1 shows a sample mixing system integrated into a sample dispensing device. [Figure 36B] 1 shows a sample mixing system integrated into a sample dispensing device. [Figure 37A] 13 shows another embodiment of a sample mixing system with a larger collection tube. [Figure 37B] 13 shows another embodiment of a sample mixing system with a larger collection tube. [Figure 37C] 13 shows another embodiment of a sample mixing system with a larger collection tube. [Figure 38A] 1 illustrates an embodiment of a liquid level sensor being used in conjunction with a sample mixing system. [Figure 38B] FIG. 38A shows a close-up view of the liquid level sensor. [Figure 39A] 1 shows another embodiment of a liquid level sensor used in conjunction with a sample mixing system. The liquid level sensor is positioned approximately 10 mm away from the collection tube. [Figure 39B] The liquid level sensor is shown positioned approximately 6 mm away from the collection tube. [Figure 40A] A sample showing the auto-dump components of the mixing system. [Figure 40B] A sample showing the auto-dump components of the mixing system. [Diagram 41] 1 is a non-limiting flow chart for processing a sample according to an embodiment of the present invention. [Figure 42A] FIG. 1 is a block flow diagram of a method for collecting and mixing samples according to the present invention. [Figure 42B] FIG. 1 is a block flow diagram of a method for collecting and mixing samples according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0100] The present invention will now be described in more detail with reference to exemplary embodiments as shown in the accompanying drawings. Although the present invention is described herein with reference to exemplary embodiments, it should be understood that the present invention is not limited to such exemplary embodiments. Those possessing ordinary skill in the art and having the opportunity to utilize the teachings herein will recognize additional implementations, modifications and embodiments and other applications of use of the present invention, which are fully contemplated herein as being within the scope of the present invention as disclosed and claimed herein, and in which the present invention may be significantly beneficial.

[0101] In one aspect, the present disclosure relates to flow cytometry systems and methods that can separate particles or cellular material, such as sperm and other particles or cells, into various components and fractions. For example, various embodiments of the present invention provide for segregating components in a mixture, such as isolating viable and motile sperm from non-viable or non-motile sperm, segregating sperm by gender and other gender classification variations, segregating stem cells from cells in a population, segregating one or more labeled cells from unlabeled cells to distinguish desirable / undesirable characteristics, segregating genes in nuclear DNA according to specified characteristics, segregating cells based on surface markers, segregating cells based on membrane integrity (viability), potential or predicted reproductive state (fertility), ability to survive after freezing, etc., segregating cells from contaminants or debris, segregating healthy cells from damaged cells (i.e., cancer cells) (such as in a bone marrow harvest), segregating red blood cells from white blood cells and platelets in a plasma mixture, and segregating any cell from any other cellular component into its corresponding fraction.

[0102] As used herein, it is understood that a sperm sample should be provided from a mammal other than a human. The sperm sample may be a freshly collected sample from a source animal, or a thawed sample previously frozen and stored from a source animal. Examples of source animals include, but are not limited to, cattle, sheep, goats, horses, pigs, or other mammalian sources such as rabbits, alpacas, dogs, cats, ferrets, rats, mice, or buffalo. For example, bovine sperm cells may include sperm cells from domestic cattle, bison, African buffalo, water buffalo, or yaks. Furthermore, the sample may be a single semen, multiple semen collections from the same mammal, or multiple semen collections from two or more animals.

[0103] As used herein, the term "passage" refers to a fluid line, channel or tubing through which a fluid can flow. For example, a sheath fluid passageway refers to a line, channel or tubing through which a sheath fluid can flow, a sample passageway refers to a line, channel or tubing through which a sample fluid can flow, an air passageway refers to a line, channel or tubing through which air can flow, etc. In the sample passageway modules described herein, the module comprises an entire sample passageway through which the sample flows, thereby isolating the sample fluid from other modules during processing other than collection. This can prevent cross-contamination between different sample fluids and can also allow the sample passageway to be separated from other modules for cleaning. An entire sample passageway refers to all passageways through which a sample fluid flows, including paths that are separate and physically separated from each other by another component. In a similar manner, the term "total passage" may apply to a sheath fluid, air, or any other fluid, primarily in that the total passage comprises all of the passages through which a particular fluid flows, including passages that are separate and physically separated from each other by another component.

[0104] As used herein, the term "cleaned paths" refers to the number of sample passage volumes that are to be washed.

[0105] As used herein, the term "module" refers to multiple components that can be removed, reinstalled, or replaced as a unit. If an individual component may be removed, reinstalled, or replaced, the individual component is not a module.

[0106] According to some embodiments, the invention features a modular flow cytometry system having a chassis and a number of modules disposed therein, as shown in Figures 1-2. These modules include, but are not limited to, an electronics and control module, an interrogation (or optics) module, a fluidics module, a sample passage module, a microfluidic device cleaning module, and an automatic mixing and collection module. Vent holes or slots can be positioned at multiple locations along the chassis to allow proper and sufficient airflow and cooling to the electronics. Without wishing to limit the invention to a particular theory or mechanism, the modular flow cytometry system can have automation mechanisms including, but not limited to, automatic adjustment of tip position through the tip stage, automatic adjustment of the detection spot, automatic adjustment of the kill spot, automatic sample and sheath throughput, automatic collection and agitation, automatic dump (e.g., from failure conditions in sample collection, e.g., too high percent dead cells, too low resolution, contamination mistakes, alignment errors, etc.). The automation mechanisms can be controlled and operated by computer system components of the cytometer.

[0107] As shown in Figure 2, a sheath vessel may be fluidly coupled to the modular flow cytometry system. The sheath vessel may include a wheeled assembly for ease of transportation and a scale for monitoring the amount of sheath fluid in the vessel. Additional components that may be operatively coupled to the modular flow cytometry system include, but are not limited to, a mouse, a monitor, a touch screen, a keyboard, and an identification scanner, such as a barcode, QR code, or RFID scanner.

[0108] FIG. 3A shows the different components of the modular flow cytometry system in more detail. In some embodiments, the microfluidic module contains the chip cartridge and the microfluidic chip, which is magnetically held to the system. The sheath line is the plumbing between the fluids from the sheath inlet port and the microfluidic chip sheath channel. The sample line is the plumbing between the fluids from the sample tube and the microfluidic chip sample channel. The fluidic module directs the fluids from the sample inlet and sheath inlet, as well as the fluids from the fluidic assembly, to their respective ports in the microfluidic chip. The fluidic access screw allows for maintenance into the fluidic assembly. The liquid level sensor can be a capacitive sensor that detects the collection fill in the sample tube. The collection module includes a rotatable system that includes a collection tube holder that holds the collection tube, which switches between a "standby" position and a "collection" position. The visor is a swing-out cover that protects the chip module from environmental fluctuations during collection. The sample tubes are reservoirs that can hold samples, including semen samples or cleaning samples, and dispense them into the microfluidic sample channels. A storage tray can hold additional tubes and caps for convenience during operation.

[0109] Referring to FIG. 3B, the rear components of the modular flow cytometry system include a control box module containing the control panel and cover, fluidic ports containing quick disconnects to the sheath inlets, waste outputs, air inputs, air outputs to the sheath reservoirs, an electronics box module containing the power supply and main computer, an electronics power port for plugging into a cable to power the electronics box, and a status indicator to show overall system status.

[0110] The optical assembly is responsible for aligning and shaping the beam path from the kill laser and the detection laser to the microfluidic chip. The chip is viewed through a focusing lens into the camera. The detection laser is responsible for emitting light through each individual cell as it flows through the microfluidic chip and collecting the emission through the APD detection sensor to assess the sex of the cell. The kill laser is then responsible for sending a pulse of light at each ungated cell in the software, slicing and inactivating each of the ungated cells. From the software, the operator is able to optimize the cell sex detection resolution and kill slicing accuracy by controlling the movement of the chip stage and the detection beam.

[0111] In a non-limiting example of a sexing method implemented in the present invention, a sperm sample is loaded into a microchannel, surrounded by sheath fluid, and focused in all four directions. When the modular flow cytometry system is running, stained sperm cells in the sample are illuminated by a laser (either pulsed or continuous wave) to cause them to fluoresce. A decision process begins in a sensor in the system when light or photons from the cell's fluorescence strike an avalanche photodiode ("APD"). The signal from the APD is processed and sent to a field programmable gate array ("FPGA"). In the FPGA, if an appropriate amount of dead time has passed since the last detection event, then a new event state begins; otherwise, no event can occur at all.

[0112] At the start of the event state determination, if the signal value exceeds the threshold and the signal value exceeds the falling hysteresis value, then an event counter is incremented. It is then determined whether the event is a multi-peak event or a single-peak event. With respect to the signal or peak, the direction of the signal refraction change is determined and it is determined whether the signal is the first peak in a multi-peak event (doublet) or the second or subsequent peak in a multi-peak event. If the event is a multi-peak event, the peak count, peak voltage, and peak time are used to process the multi-peak event. Because singlets and doublets have approximately the same peak height, they appear in the same place on a 1-D histogram of peak heights ("intensity"). However, because the areas are significantly different, they appear in different places on a 2-D histogram of peak heights and areas.

[0113] If the event is a multi-peak event, the following data is updated for each data point in the event as the signal is processed until the end of the event is determined: peak voltage, peak time, integral area, and span value for the event. If the span value exceeds the minimum value for the event, the event is a valid event and the FPGA goes into "gating mode" to determine the gating result and use this result to decide whether to fire or not. Peaks that correspond to cells that are already dead result in a decision not to fire. The FPGA results, including peak voltage, area, and span, are sent to a digital signal processor ("DSP"). The DSP can make additional gating decisions or it can use the FPGA result to decide whether to fire or not. In DSP gating, the event data from the FPGA is processed to determine if the event data point is within a predefined gating polygon.

[0114] After the FPGA sends the signal to the DSP, it waits for the DSP gating result, and based on the DSP gating result, one or more peaks are added to the kill schedule based on the time stamp for the peaks in the event. For single peak, one event is added to the laser trigger schedule, and for multi-peak event, one or all peaks in the multi-peak event are added to the laser trigger schedule based on the current setting. For laser trigger, the FPGA constantly checks to know if a new trigger request is added to the schedule, and if a trigger request is added to the schedule, a delay is set based on the request. After the delay, the output from the FPGA causes the laser to fire at the sperm cell corresponding to the particular trigger request.

[0115] In some embodiments, the fluidics module is a liquid handling device that precisely controls the flow rate between the sheath and sample lines from the fluidics manifold to the microfluidic chip to allow optimal cell flow rates through the microfluidic chip and maximum allowable spacing between cells within the chip. The fluidics module is removable from the system.

[0116] In some embodiments, the fluidic module includes fluid regulators and fluid valves to control the flow rates for the sheath and sample fluids through the system. The fluidic module may also include a dual air regulator assembly near the rear of the system that can drop the housing air pressure to about 55 psi within the instrument and the air pressure to the sheath reservoir to less than 25 psi. The fluidic module houses the fluidic control PCB and controllable devices to precisely control the flow rate of fluids through the instrument. The fluidic module can be reached by triggering a release mechanism, such as by releasing a latch or twisting a knob until it is loose, and then sliding the module out. With reference to FIG. 9A, once the fluidic module has been pulled out of the instrument, maintenance staff can quickly reach the module by first unplugging the electrical port to the fluidic board, then holding the handle located at the top of the module and pulling the unit up. The fluidic module can be disconnected and removed from the system, for example for maintenance or diagnostics, and replaced with a backup fluidic module while the system remains operational. For example, the fluidics module may be removed to replace the sheath line filters once a month for preventive maintenance.

[0117] In order for any fluid to flow through the system, both the microfluidic and fluidic modules need to be properly seated in their respective spots. There are sensors to detect when the manifold is in place to reduce the chance of accidental spills. Both manifolds have handles attached to make them easier to grasp. In some embodiments, both the chip and fluidic manifolds are intended to be handled simultaneously. To load the fluidic and chip modules, the visor is lifted and then the module is moved to its intended location on the system. For the fluidic module, the two dowel pins from the module are aligned with holes in the fluidic module base and then pressed into place. For the chip module, the locating balls on the back of the module are aligned with locating holes in the chip module base and then pressed forward to slide into place on the system. Both modules should click into place when they are magnetically held. The sheath and sample lines are inserted into their respective slots on the clips just below the visor to hold the lines in place. Finally, the visor is closed. Removing the fluidics and chip modules from the system is the reverse of the loading procedure.

[0118] In some embodiments, a 5 mL tube sample with the desired fluid is loaded onto the fluidics module by placing the tube onto the sample tube loader and positioning the loaded sample tube loader perpendicular to the face of the instrument, directly underneath the fluidics module. Once the sample tube is centered and aligned, the sample tube loader is pushed up until the top face touches the fluidics module. The sample tube loader locks into place with the magnetic forces between the loader and the fluidics module. To remove the sample tube, twist the sample tube loader to break the magnetic forces with the fluidics module and pull it down to remove it from the fluidics manifold.

[0119] In other embodiments, the collection module can automate some parts of the collection process of the system. It has a sensor to detect when the collection tube is full. A liquid level sensor is calibrated to activate when the sample tube is full to a predefined level. The collection module can switch the full collection tube to a waiting empty collection tube and automatically mix the contents in the tube every 5 minutes. In some embodiments, the automated collection module includes a turntable base that holds two sample tubes. The collection base also includes a magnet to hold the sample tube holder in place in a specific orientation during collection. While collection is taking place, the collection module can frequently mix the contents. When the sample is finished being collected in the tube, the module switches the tube collecting the sample to the waiting tube. Finally, there is an automated system for redirecting the sample flow to a waste line without the need to stop the flow.

[0120] During monitoring of the sexing system, the operator's main interaction is with the collection module. When the software is started, the collection module returns to its designated location. Except when the collection module is moved, there is always one sample tube holder aligned with the sample collection zone, called the "collection" position. Directly below the sample collection zone and directly across from the sample tube holder is a second sample tube holder. The second sample tube holder is called the "standby" position.

[0121] During sample collection, the collection tubes are automatically mixed. The mixing protocol involves swiveling the collection tube back and forth for a few seconds and then returning it to the collection position. When a new collection tube is switched to the collection position, the finished tube is switched back to the waiting position and the full collection tube is removed and placed in the collection tray. A new collection tube is placed in the collection tube holder and loaded back to the waiting position.

[0122] Without wishing to be bound by any particular theory or mechanism, a novel and progressive feature of the modular flow cytometry system is the configuration and location of the modules relative to each other to allow efficient use of space. The system can be divided into three separate levels or floors. On the first floor (e.g., the bottom floor), the electronics module can slide in and out of the chassis. It includes a bulkhead plate or plates for protection from the fluids at the top level. Thus, the electronics module is physically isolated from the other modules. On the second floor (e.g., the middle floor), the fluidics module is at the front part to make it easier for the operator to reach. The third floor (e.g., the top floor) houses the optics module, which includes the laser, detector, and other optical components. The optics module is isolated from moving air so as not to disturb the optical components. Since the laser generates the most heat, the optics module is at the top level for optimal ventilation. Instead of using fans that can disturb the air around the optical components, a heat sink for the laser can draw air via natural convection to cool the components. The optics modules must be separated from the electronics modules because they both generate heat. Fans may be placed on the first floor and / or near the electronics modules to cool the electronic components. The layout of the modules further allows for reaching areas for frequent maintenance.

[0123] Another unique and inventive feature of the present invention is the modular nature of the entire system, which advantageously allows for easy assembly of the various modules and removal for maintenance and / or replacement, resulting in little downtime and near continuous operation. For example, the computer may be placed on rails for ease of movement and can be replaced with a similar electronics module. In the optics module, the laser can be removed, replaced, or reconnected. Other optical components, detectors, cameras, lenses, etc. can also be removed, replaced, or reconnected. The sample path module can be removed and cleaned using a cleaning module. The fluidics module can also be removed and cleaned. During maintenance, the fluidics module and the sample path module can be replaced with other cleaned modules. Replacement of the fluidics module or its components can allow for continuous operation of the flow cytometer. The system can be easily operated and maintained without the need for specialized technicians or specialized training. Furthermore, the modular nature also allows for easy shipping of the system with little or no assembly required after delivery.

[0124] Yet another unique and inventive feature of the present invention is the automated or semi-automated nature of the modular flow cytometry system, resulting in less system downtime, lower product contamination, and improved product uniformity and purity. For example, the physical location of the microfluidic chip can be adjusted to where it is needed using an automated chip stage adjustment procedure. Mixing of sample tubes containing processed samples is also automated by rotating the tubes as the samples are being collected, thereby reducing sample loss and reducing sperm cell damage. Further examples of the system's automation features include an automatic dump module that allows the redirection of processed sample streams to waste collection, and automated sample level detection that notifies the operator when the sample level is low.

[0125] The following sections describe in more detail the various modules of the modular flow cytometry system. Alternatives and equivalents are within the scope of the invention.

[0126] Sample passage module and microfluidic device cleaning module

[0127] In the field of commercial semen processing, for example in semen sexing operations, there is a requirement that microfluidic chips, cassettes, or cartridges should be washed to prevent contamination between sample runs.

[0128] In semen sexing, the process is a fluid sample change sub-process, e.g. a bull change sub-process, where the processing of one fluid sample (e.g. a semen sample) is completed and the system must prepare to process the subsequent sample. During bull change, the microfluidic chip used in the sexing process for the semen sample from the first bull must be cleaned to remove all the previous bull's semen in order to perform the next sexing process using the new bull's semen. Alternatively, a new or separate microfluidic chip may be used. Contamination between bulls, or more generally between animals, or between genetic fluid samples, is a possible consequence of microfluidic chips that were not cleaned thoroughly. Cross-contamination is a major problem if a contaminated product reaches the farmer, as it can result in the wrong breed or wrong sex of the offspring.

[0129] In existing systems and methods, reducing contamination between bulls is achieved by keeping the microfluidic chip fixed within the flow cytometry device or instrument. The chip cleaning procedure is performed on the flow cytometry device, where a cleaning solution or wash fluid, such as PARVOSOL, and sheath fluid or buffer are processed through the microfluidic chip within the flow cytometry device during a cleaning process of approximately 10 minutes within the period between bull changes. The procedure is a semi-autonomous process, where the operator sets the pressure for cleaning, configures the flow of the wash fluid, and sets the 10-minute cleaning timer.

[0130] However, in a commercial semen processing setup, the flow cytometry device loses valuable time that would otherwise be spent on semen processing if it is instead used to clean the chip. In a semi-autonomous cleaning procedure, it is also possible that the microfluidic chip is not completely cleaned. The semi-autonomous cleaning procedure performed on the flow cytometry device relies on the operator's judgment to set the cleaning flows correctly, to set the timers correctly, to monitor and ensure that the cleaning cycle does not end prematurely, and that enough cleaning solution, e.g., PARVOSOL, is in the sample tube for the entire cleaning cycle.

[0131] It is therefore an object of the present invention to provide a flow cytometry system including a replaceable or removable sample path module comprising a microfluidic module having a microfluidic chip, cassette or cartridge and further comprising a distribution block or fluidic manifold. In some embodiments, the flow cytometry system may include a microfluidic device cleaning system that provides automated cleaning of the sample path module using a pre-programmed or configured cleaning protocol.

[0132] Without wishing to limit the invention to any theory or mechanism, the methods and systems described herein are believed to be advantageous because the removable and replaceable fluid or sample passage module, comprising a microfluidic module holding a microfluidic chip and a distribution block or fluidic manifold, is portable and can be transported to a chip cleaning station, thereby providing a completely autonomous cleaning cycle and nearly continuous operation of the flow cytometry device.

[0133] In an exemplary embodiment, a removable and replaceable fluid or sample path module comprising a microfluidic module holding a microfluidic chip and a distribution block or manifold is releasably held (e.g., magnetically) in a flow cytometry system and can be releasably removed by an operator removing the sample path module from the flow cytometry system, such as by an outward force. The sample path module can then be carried to a chip cleaning station, which is a microfluidic device cleaning system, where a magnetic force can releasably secure the sample path module to the microfluidic device cleaning system, such that an operator can easily load the sample path module into the microfluidic device cleaning system.

[0134] Once on the microfluidic device cleaning system, the operator removes the sample tube loader used to push the sample tubes into the distribution block and replaces it with the sample reservoir on the microfluidic device cleaning system. The operator initiates the cleaning procedure by selecting a cleaning protocol or cleaning parameters through the user interface of the integrated or attached control system or computer. This initiates an automated cleaning procedure, which draws cleaning solution (e.g. PARVOSOL) from a container or reservoir connected to the back of the microfluidic device cleaning system and fills the sample reservoir to a level (e.g. 4.5 mL) pre-determined by the cleaning protocol. The microfluidic device cleaning system then primes deionized water or microbiological grade water through the sheath line or buffer line or flow path of the sample path module and flows it through the sample path module. Pressure is then applied to the sample reservoir to force the cleaning solution (e.g. PARVOSOL) through the sample line or flow path and thus through the sample path module. The procedure then involves flowing both the cleaning solution and deionized water through the sample passage module for an amount of time as determined by the cleaning protocol, e.g., 10 minutes, and finally, air is blown through the chip and through both the sample and sheath lines.

[0135] In some embodiments, the microfluidic device cleaning system includes a set of manifolds and valves within the microfluidic device cleaning system that allow switching between different liquids, which also allows the microfluidic device cleaning system to perform the same procedures for starting and stopping tip cleaning as on a flow cytometry system.

[0136] The removable and replaceable fluid or sample path module comprising the microfluidic chip and the microfluidic module holding the distribution block or manifold is now portable as opposed to being fixed. This allows an operator to remove the sample path module from the flow cytometry system after running a biological fluid sample such as a bull sample, carry the sample path module to the microfluidic device cleaning system (i.e., away from the flow cytometry device) to clean the instrument without unnecessary or excessive system processing downtime, and obtain a clean sample path module from storage and carry and place it on the flow cytometry device. The chip cleaning station is automated, where the cleaning protocol parameters, such as flow rates and timers, are pre-configured or programmed as part of the cleaning protocol. The microfluidic device cleaning system and cleaning protocol have built-in logic and algorithms to ensure that the flow rates are matched and the manifold is in place to achieve complete cleaning of the microfluidic chip in the sample path module.

[0137] The use of an automated system and removable and replaceable fluid or sample passage modules comprising a microfluidic module that holds a microfluidic chip and a distribution block or manifold provides results that are comparable to or better than existing systems and methods that require the cleaning process to be performed at or on the flow cytometry system while reducing the amount of downtime of the flow cytometry system. In some embodiments, it may take an operator approximately 13 to 16 minutes to perform a bull change cleaning process on the microfluidic device cleaning system. The duration of the cleaning process may be further optimized through future optimization and training plus.

[0138] Generally, the microfluidic device cleaning system and removable and replaceable fluid or sample passage modules comprising a microfluidic module that holds a microfluidic chip and a distribution block or manifold eliminates or substantially reduces the downtime of up to 10 minutes per fluid sample exchange for a flow cytometry system that was previously caused by the requirement to clean the microfluidic chip on the flow cytometry device.

[0139] Autonomous cleaning cycles provide thorough cleaning and require less oversight from the operator. Cleaning solutions, such as PARVOSOL, are shared in one or more larger reservoirs instead of requiring the operator to fill individual 5 mL tubes per cleaning routine. Additionally, removable and replaceable fluid or sample passage modules that comprise the microfluidic module holding the microfluidic chip and distribution block or manifold are easy to replace or replace if clogging appears, reducing downtime to replace microfluidic chips in existing systems.

[0140] With reference to FIG. 5, a front perspective view is provided illustrating one embodiment of a removable and replaceable sample passage module 100 and a flow cytometry system 10 for processing and analyzing samples. The flow cytometry system 10 may be any type of particle processing system capable of distinguishing between one or more particles based on observed or determined characteristics of the particles. For example, the flow cytometry system 10 may be a laser-based detection and inactivation system that uses one or more lasers and detectors to determine characteristics of particles, such as cells, in a sample stream and to inactivate a subset of the particles. Other methods of particle sorting, particle separation, or particle inactivation, such as fluid sorting, jet sorting, laser steering, holographic optical trapping, or electrophoresis, may also be used in the flow cytometry system 10.

[0141] The removable and replaceable sample path module 100 provides the entire fluid path for samples being processed by the flow cytometry system 10. The sample path module 100 includes a distribution block 110 and a microfluidic module 150. The distribution block 110 includes a body 112, a device face 111, a handle 114, a set of alignment pins or rods 116, and a sample straw 118. A set of releasable fittings, including a sample path fitting 103 and a sheath fluid or buffer fitting 105, are used to connect the sample fluid paths and the sheath fluid or buffer paths in the distribution block 110 to corresponding paths in the microfluidic module 150 via respective sample fluid tubing 102 (e.g., a first fluidic line) and sheath tubing or buffer tubing 104 (e.g., a second fluidic line). In an alternative embodiment, which does not require a sheath fluid, the sample fluid passages in the distribution block 110 may be coupled to corresponding passages in the microfluidic module 150 via the sample fluid lines 102, eliminating the need for the buffer lines 104. The sample fluid lines 102 and the sheath fluid lines or the buffer lines 104 may be secured to the microfluidic module 150 by a set of releasable fittings similar to those used on the distribution block 110. The set of releasable fittings used on the distribution block 110 and on the microfluidic module 150 may be fittings such as barbed fittings, locking collar fittings, threaded fittings, or other suitable fittings capable of securing the lines 102 and 104 under operational and pressurized conditions of the samples being processed by the flow cytometry system 10.

[0142] Microfluidic module 150 includes a body 152, a handle 154, and a microfluidic device holder 156. Microfluidic device holder 156 is used to secure and position a microfluidic chip or cassette within microfluidic device holder 156. As shown in FIG. 14, a knob, handle, threaded rod, or other positioning device may be used to secure and position the microfluidic chip or cassette within microfluidic device holder 156 to align the chip or cassette with detection and / or inactivation optics within flow cytometry system 10.

[0143] The microfluidic device holder and distribution block 110 may each comprise one or more ferrous, ferrous, ferrimagnetic, or other magnetic plates or blocks that may be used to releasably secure the sample passage module 100 to the flow cytometry system 10. Additionally, or in alternative embodiments using a magnet-based fastening system, other latches, locks, or fasteners may be used to releasably secure the sample passage module 100 to the flow cytometry system 10. However, a fastening system that provides for quick and easy fastening of the sample passage module 100, as well as quick and easy release of the sample passage module 100 from the flow cytometry system 10, such as a magnet-based fastening system, may provide additional advantages related to ease of use and simplified operation.

[0144] When secured relative to the flow cytometry system 10, the sample passage module 100 provides the entire fluid pathway for samples being processed by the flow cytometry system 10. For example, samples contained within sample tubes 12 are pumped, pushed, or delivered through the sample passage module 110 via sample straws 118 by the action of fluid pressure, e.g., air or gas pressure within the sample tubes 12, which forces the sample through the sample straws 118 and into the distribution block 110 when the sample tubes 12 are mounted to or secured relative to the distribution block. Fluid pressure to the sample tubes 12 is provided from the flow cytometry system 10 via one or more fluid inlets, such as a set of fluid inlets on the device face 111 of the distribution block 110. A pump, such as a peristaltic pump within the flow cytometry system 10 or an external air pressure source provides air pressure to the sample tubes 12.

[0145] The fluid sample, which may contain particles such as sperm cells, passes through the distribution block 110 and via the sample fluid tubing 102 to the microfluidic module 150, where a microfluidic chip or cassette is used to process the fluid sample. After processing, the fluid sample may be collected in the automated fluid collection system 14 or automatically diverted into a waste collection tube, reservoir or container. During processing, the fluid sample from the sample tube 12 never comes into contact with any part of the flow cytometry system 10, except for the post-processing waste collection reservoir. In this manner, the sample passage module 100 may be removed from the flow cytometry system 10 after the fluid sample has been processed without any risk of contamination of other subsequently processed samples.

[0146] For example, when a first fluid sample, such as a semen sample having sperm cells, is processed by the flow cytometry system 10 via the sample path module 100, the sample path module 100 may be released or removed from the flow cytometry system 10 after processing and replaced with another similar or identical sample path module to process a second fluid sample without any risk of cross-contamination between the first and second fluid samples. This is particularly important when processing biological samples where cross-contamination is not only undesirable but may also be contrary to laws, regulations, or guidelines for processing and distributing biological samples. Cross-contamination may further create problems when genotyping or genetic sequencing is performed on the sample. The sample path module 100 provides an isolated, fully contained, removable sample path module for use in or with a flow cytometry device that effectively eliminates, or at least greatly reduces, the possibility of cross-contamination between individual samples.

[0147] 6, a front perspective view is provided illustrating one embodiment of a removable and replaceable sample path module 100 and a microfluidic device cleaning system 20 (or cleaning module) for cleaning the removable and replaceable sample path module 100. As shown in FIG 6, the sample path module 100 may be removed from the flow cytometry system 10 as shown in FIG 5 and aligned with and placed on, secured to, or fastened to the microfluidic device cleaning system 20.

[0148] The microfluidic device cleaning system 20 of Figure 6 (block diagram embodiments of which are shown in Figures 12 and 13) comprises a housing 22, a front section 24, a back section 26, a microfluidic module or chip interface 28, and a distribution block or fluidic manifold interface 30. A waste collection tube 32 directs waste from a microfluidic chip or cassette in the microfluidic module 150 through a grommet or opening 34 in the front section 24 of the housing 22 into a waste collection reservoir. Fluid, such as cleaning fluid or pressurized air, is provided to the cleaning fluid reservoir 38 from one or more internal or external fluid reservoirs via a connection through the opening or grommet 36.

[0149] In one embodiment, and with reference to the flow cytometry system 10 of FIG. 5 and the microfluidic device cleaning system 20 of FIG. 6, a set of valve fittings may be used at the interface of the distribution block 110 of the sample path module 100 or the microfluidic module 150. The valve fittings comprise fittings that open the valve when the fittings are connected and close the valve when the fittings are disconnected so that fluids in the flow cytometry system 10, the microfluidic device cleaning system 20 or the sample path module 100 are not allowed to leak or exit the system when a connection is made or disconnected. This prevents bio-contamination and further prevents exposure of the device or operator to hazardous waste or materials such as cleaning solvents. The valve or port assumes a closed position when removed or disconnected and opens to allow fluid flow only when a connection is made. In one embodiment, the valve fittings further comprise fittings that are actuated by an actuator or motor or by manual operation of the valve.

[0150] The operation of a flow cytometry system, such as flow cytometry system 10, and a microfluidic device cleaning system, such as microfluidic device cleaning system 20, that uses a removable and replaceable sample path module, such as sample path module 100, will now be described with reference to process 300 shown in the flow chart provided in Figure 10. Process 300 describes one example method for operating a flow cytometer using a removable and replaceable sample path module and removing the sample path module from the flow cytometer for cleaning with the microfluidic device cleaning system.

[0151] The process 300 begins at step 302 where the sample passage module is prepared to perform a process on a flow cytometry device. Preparing the sample passage module for a process may include installing or securing the sample passage module to the flow cytometry device. Securing the sample passage module to the flow cytometry device may include aligning the sample passage module to one or both of a distribution block interface and a microfluidic device or chip interface on the flow cytometry device. In one embodiment, aligning the microfluidic device may include using alignment pins or rods to precisely position the sample passage module relative to the flow cytometry device for installation. Other alignment devices may be used and may include a set of indexing marks on the sample passage module and the flow cytometry device, complementary geometrically corresponding faces or surfaces of the sample passage module and the flow cytometry device, or alignment channels, guides, or rails to align and position the sample passage module relative to the flow cytometry device for installation.

[0152] In some embodiments, the sample passage module may be secured to the flow cytometry device by ferrous, ferrous, or ferrimagnetic, or other magnetic material plates on a face of the sample passage module or the flow cytometry device and corresponding magnetic plates on an opposing surface. For example, in one embodiment, a set of four plates may be placed on a face of the distribution block and a corresponding set of four magnets may be placed on an interface surface of the flow cytometry device to align and releasably secure the distribution module of the sample passage module to the flow cytometry device. One or more magnets and corresponding plates may be used to secure either or both of the microfluidic module and the distribution block of the sample passage module. In another embodiment, the sample passage module may be secured to the flow cytometry device using a set of latches, a set of locking tabs, a set of fasteners such as a set of bolts, spring-loaded retaining pins, or retaining or locking lugs.

[0153] Additionally, the sample tube may be secured to a sample tube loader on the distribution block, either manually or automatically. For example, in one embodiment, the sample tube is removed or removed from the storage area, the lid or cover is removed, and the sample tube is positioned in the sample tube loader, pushed in, and secured by a spring-loaded mechanism, such as a set of locking lugs or tabs that are displaced by the action of the sample tube and return to their original position by the action of a biasing spring that secures the sample tube in place on the distribution block. In another embodiment, the sample tube is threaded into or against the distribution block via a corresponding threaded neck of the sample tube and an opening in the distribution block. In another embodiment, an actuator-driven loader moves the sample tube from the storage area and secures the sample tube to the distribution block, such as by a spring-loaded sample tube loader.

[0154] Once secured to the flow cytometry system, the sample path module may be primed to process a fluid sample. Priming may include flushing or priming the sample path module with a fluid, such as air, a buffer, or both, prior to processing the fluid sample. In step 304, a sample is processed through the sample path module by the flow cytometry device. A sample tube mounted in the distribution block is pressurized via a pressurized gas source within the flow cytometry device, such as a compressor, an air pump, or by an external pressurized air source. The pressurized gas is passed through the distribution block into the sample tube, and the pressurized gas in the sample tube forces the fluid sample through the sample straw of the distribution block. At the same time that the sample tube is being pressurized, a buffer or sheath fluid is pumped from an internal or external reservoir of the flow cytometry device by a pump or pressurized gas. The sheath fluid or buffer and the fluid sample from the sample tube travel through a distribution block via fluid connections, such as flexible hoses or tubes held in place by releasable fasteners, to the microfluidic module, where the fluid sample and sheath fluid or buffer pass or flow through a microfluidic chip, cartridge or cassette.

[0155] The particles in the fluid sample are analyzed by the flow cytometry device to determine one or more qualities or characteristics of the particles, such as by the methods described above, for the purpose of identifying one or more subgroups or subsets of the particles. After processing, the sheath fluid or buffer and the fluid sample may be collected in a sample collection tube and collected in a sample collection medium in the sample collection tube. If undesirable characteristics or conditions are identified, such as contamination, improper identification, instrument miscalibration, or other system or sample issues that create less than ideal sorting conditions, then some or all of the fluid sample or the sheath fluid or buffer may be diverted to a waste collection reservoir instead of being collected in the sample collection tube. After all of the fluid samples in the sample tubes loaded into the distribution block have been processed, processing of the samples by the flow cytometry device is complete.

[0156] Next, in step 306, the sample tubes are removed from the sample passage module and the sample passage module is removed from the flow cytometry system. The sample passage module may simply be pulled from the flow cytometry system if it is held in place by magnetic attraction, or a set of latches or fasteners may be removed or released to allow the sample passage module to be disconnected from the flow cytometry system. After the sample passage module is removed from the flow cytometry system, the sample tube loader, which may be a spring-loaded mechanism, may optionally be removed from the distribution block. This step may not be necessary if the wash fluid reservoir used by the microfluidic device cleaning system in the cleaning step is compatible with the sample tube loader.

[0157] In step 307, a replacement sample path module of similar or different configuration to the one just removed is installed or connected to the flow cytometry system in a similar manner as described above. No additional cleaning or sanitization of the flow cytometry system prior to processing of samples by the replaceable sample path module is required, since the fluid sample processed by the first sample path module does not contact any part of the flow cytometry system itself, thereby ensuring that no cross-contamination occurs between sample processing runs or between sample path modules. Once installed on the flow cytometry system, the replacement sample path module may be immediately used to process new fluid samples.

[0158] In step 308, the first sample path module removed from the flow cytometry system is installed or connected to a cleaning station, which in one embodiment is a microfluidic device cleaning module. The connection or installation of the sample path module on the microfluidic device cleaning module is similar to the installation of the sample path module on the flow cytometry system. However, instead of connecting the sample tubes to the distribution block, a wash fluid reservoir is connected to the distribution block. If compatible, a sample tube loader may be used to secure the wash fluid reservoir, otherwise the wash fluid reservoir may be secured by a set of fasteners or by magnetic attraction.

[0159] In step 310, a cleaning protocol is selected for cleaning the sample path module. In semi-automatic operation, the cleaning protocol is selected by an operator based on the configuration of the sample path module, including the type of microfluidic chip, cassette or cartridge in the microfluidic module of the sample path module, and further based on the fluid sample type to be processed by the sample path module. The cleaning protocol is selected using a user interface that provides selection of one of a plurality of cleaning protocols and configuration of one or more parameters of the cleaning protocol. For example, in one embodiment, a display and a set of user interface elements (e.g., switches, buttons, joysticks, knobs, etc.) are used to select the cleaning protocol. In another embodiment, a touch screen display and one or more graphical user interface elements are used to select the cleaning protocol. In another embodiment, the display and interface elements are on or integral with the microfluidic device cleaning system, whether presented in a graphical user interface or as a physical control device. In another embodiment, a separate control computer or system is provided that includes the display and interface elements. In one embodiment, a common control system is provided for the flow cytometry device and the microfluidic device cleaning system.

[0160] In fully automated operation, the cleaning protocol for the sample passage modules is determined automatically by the microfluidic device cleaning system. The identifier on the sample path module is used to identify the type of sample path module and may further be used to identify the type of fluid sample last processed by the sample path module. In one embodiment, an optically readable identifier is used on the sample path module. The optically readable identifier may be a bar code, a quick response ("QR") code, or other optically readable code or identifier capable of identifying an individual sample path module. In another embodiment, a radio wave identifier is used on the sample path module. The radio wave identifier may be a radio frequency identifier ("RFID") tag or chip, a near field communication ("NFC") tag or chip, a Bluetooth® wireless based device, or other radio wave identifier capable of identifying an individual sample path module.

[0161] For any type of identifier used with a sample path module, the type of sample path module may be determined locally on the microfluidic device cleaning system, or the identifier read from the sample path module may be transmitted or sent to a central system or server for processing or retrieval. For example, the identifier read from the sample path module may be compared to a database or table of known sample path modules to identify the unique sample path module. A set of characteristics may be read from at least one database entry, field, or record associated with the sample path module, where one of the database entries, fields, or records includes a cleaning protocol or a set of cleaning protocol configuration information.

[0162] A "cleaning protocol" includes a set of parameters that define the cleaning operation of the microfluidic device cleaning system for the sample passage module. A "cleaning protocol" includes the type of cleaning solution used (e.g., PARVOSOL, quaternary ammonium cleaning solution, biocidal (i.e. antiviral, antibacterial, antifungal) cleaning solution, bleach (e.g., sodium hypochlorite solution), alcohol (e.g., isopropyl alcohol), the volume of cleaning solution used, the type of rinsing solution used (e.g., deionized water, molecular biology grade water, biologically neutral solvents), duration of cleaning (e.g., 0-5 min, at least 5 min, 5 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69 min, 70 min, 71 min, 72 min, 73 , 5-10 min, at least 10 min, 10 min, 10-15 min, at least 15 min, 15 min, 15-20 min, at least 20 min, 20 min, 20-25 min, at least 25 min, 25 min, 25-30 min, at least 30 min, 30 min), the volume of the sample path in the sample path module, the number of sample path volumes to be cleaned ("paths to be cleaned"), the pressure used (e.g., 5 PSI, 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 the specified amount of time, at least 20 PSI, at least 30 PSI, at least 40 PSI, at least 50 PSI), the number of cleaning cycles (e.g., at least once, once, more than once, consecutively), the temperature to be used (e.g., at least 20°C, at least 30°C, at least 40°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, at least 90°C, at least 100°C), a clog detection procedure, a warning procedure, a drying procedure (e.g., drying the entire fluid path with pressurized gas for a specified amount of time, drying the entire fluid path until the detected moisture content drops below a specified level), a set of maintenance procedures or logging procedures (e.g., logging all cleaning protocol parameters and sensor data for each cleaning cycle or operation).

[0163] Additionally, a "cleaning protocol" may include one or more of a disinfection procedure or operation, a sanitization procedure or operation, or a cleaning procedure or operation. A cleaning procedure may include physical removal of contaminants or debris with or without reduction in the amount of "microbes", bacteria, fungi, or viruses. A sanitization procedure may include reduction in the amount of "microbes", bacteria, fungi, or viruses with or without physical removal of contaminants or debris. A disinfection procedure or sanitization procedure completely kills all "microbes", bacteria, fungi, or viruses with or without physical removal of contaminants or debris. A cleaning protocol may include specific parameters or configurations to accomplish one or more of a cleaning procedure or operation, a sanitization procedure or operation, or a disinfection procedure or operation. The set of parameters described above for a cleaning protocol may further include irradiation (e.g., with ultraviolet light) of one or more portions of the sample path module, or increasing the temperature of one or more portions of the sample path module, in addition to disinfection or sanitization with a fluid, and in addition to cleaning, to achieve disinfection or sanitization.

[0164] For example, in one embodiment, a cleaning protocol may include specifying a cleaning fluid (e.g., PARVOSOL), duration of cleaning (e.g., 10 minutes), number of cleaned paths to be performed (e.g., 20), temperature (e.g., 24° C.), pressure (e.g., 138 kPa), warning procedures (e.g., stopping cleaning and audio alarms, clogging detection procedures (e.g., increasing fluid pressure to a defined limit), rinsing or flushing fluids (e.g., deionized water or microbiology grade water), and drying procedures (e.g., flowing pressurized air through the sample path module for a defined period of time). One of skill in the art will appreciate that these are exemplary cleaning protocol parameters and that other configurable cleaning protocol parameters are considered within the scope of the present disclosure.

[0165] Determining the cleaning protocol to be used may be based on identifying the sample path module being cleaned. In one embodiment, the sample path module is identified based on one or more identification elements on the microfluidic chip. For example, the identification elements may be features or criteria on the microfluidic chip that are visually identified by processing a signal or image from an image sensor. In one embodiment, the sample path module is identified based on one or more identification elements on the sample path module. For example, an optical or radio frequency identifier on an exterior surface of the sample path module is scanned by a scanning device to identify the sample path module. In any embodiment, a cleaning protocol or a set of cleaning protocol parameters may be automatically identified by the system based on the identification of the sample path module. Alternatively, or in addition to the automatic identification, a set of information associated with the sample path module may be presented to a user in a graphical user interface. In one embodiment, a user may select from a set of cleaning protocols based in part on the information presented in the graphical user interface.

[0166] In one embodiment, an algorithm for clog detection monitors signal data from flow sensors in the fluid pathway to identify any flow resistance downstream of any device. For example, a flow sensor downstream of a component sending a signal indicating a lower than expected flow rate may be interpreted as indicating a clog, blockage, or other obstruction in a component upstream of the flow sensor. The system responds based on the particular flow sensor indicating a lower than expected flow rate. In one embodiment, a flow sensor downstream of a microfluidic module comprising a microfluidic chip sends a signal indicating a low flow rate. The system identifies the source of the low flow rate as likely to be the microfluidic chip based on the signal sent from the downstream flow sensor. In one embodiment, based on a determination that a clog is present in the microfluidic chip, the flow rate or pressure is increased to attempt to clear the clog. In another embodiment, a visual, audible, or audiovisual alarm, warning, or notification is provided to the user so that the user can take appropriate action to clear the clog. In another embodiment, operation is stopped to allow the clogged component to be removed or manually cleaned. In another embodiment, in addition to or instead of using a flow sensor, an image sensor may be used to visually identify a clog, such as in a transparent component such as a microfluidic chip. In another embodiment, when a clog or blockage is detected, a secondary operating mode is initiated. In another embodiment, when a clog or blockage is detected, a manual override mode is initiated.

[0167] After the cleaning protocol is determined and selected in step 312, the cleaning protocol is used by the microfluidic device cleaning system to perform a cleaning operation on the sample passage module according to the parameters of the cleaning protocol.

[0168] In some embodiments, alternative cleaning protocols may be used for samples used in a specific application or jurisdiction. For example, for samples to be manufactured, used, or sold in a jurisdiction, or for applications requiring higher cleaning or safety standards, system elements such as flow cytometry devices, microfluidic device cleaning systems, or sample path modules specific to that application or jurisdiction may be used. Jurisdiction or application specific system components are not used in or for the processing or cleaning of any other components used in any other jurisdiction or application. In one embodiment, a jurisdiction or application specific system may include one or more flow cytometry devices, microfluidic device cleaning systems, and sample path modules used to process fluid samples for a specific jurisdiction or application. In one embodiment, a jurisdiction may be a geographically, economically, or politically organized area. In one embodiment, an application may be for processing a specific fluid sample from a specific source. In one embodiment, an application may be for processing semen samples from a mammalian animal associated with a specific jurisdiction.

[0169] After the cleaning protocol is completed, the sample passage module is removed from the microfluidic device cleaning system in step 314 and either placed in a sanitized, safe storage location in step 316 or installed onto the same or another flow cytometry system in step 307.

[0170] 11A-11B, a block diagram 400 illustrating one embodiment of a system and method for using a removable and replaceable sample passage 100 in a flow cytometry system 10 and cleaning the removable and replaceable sample passage 100 using a microfluidic device cleaning system 20, and a block diagram 500 illustrating one embodiment of a system and method for using a removable and replaceable sample passage 502 in a flow cytometry system or device 10 and cleaning a removable and replaceable sample passage module 502 using a microfluidic device cleaning system 20, are provided, respectively.

[0171] In FIG. 11A, the sample path module 100 is shown positioned or installed on the flow cytometry system 10. The sample path module 100 may be installed and operated as described in the flow chart 300 and controlled by the control system 402. In one embodiment, the control systems 402 and 404 are a common system or are remote or "thin" clients connected to a central server or data center that stores, manages, and processes information. The sample tube 12 provides a fluid sample to the sample path module 100 for processing by the flow cytometry system 10. After processing of the fluid sample in the sample tube 12 by the flow cytometry system 10, the sample path module 100 may be removed from the flow cytometry system 10. The sample path module 100 may then be positioned, connected, or installed on the microfluidic device cleaning system 20. A wash fluid reservoir 38 is connected to the distribution block of the sample path module 100. The wash fluid reservoir 38 holds a small amount of wash fluid needed for a single cleaning operation of the sample passage module 100 and is filled from one or more larger internal or external fluid reservoirs.

[0172] In FIG. 11B, a sample path module 502 is shown positioned or installed on the flow cytometry system 10. The sample path module 502 may be installed and operated as described in the flow chart 300 and controlled by the control system 402. The sample path module 502 differs from the sample path module 100 shown in FIG. 11A in that the sample path module 502 combines a microfluidic module and a distribution block or manifold into a single element. While the sample path module 100 comprises a separate microfluidic module 150 and a distribution block 110 joined by one or more fluid connections, both of these components are combined into a single element in the sample path module 502. This configuration of the sample path module 502 provides for a simpler design and operation of the sample path module 502, where the configuration of the flow cytometry system 10 allows for a combined microfluidic module and distribution block to be used. The sample tube 12 provides a fluid sample to the sample path module 502 for processing by the flow cytometry system 10. After processing of the fluid sample in the sample tube 12 by the flow cytometry system 10, the sample path module 502 may be removed from the flow cytometry system 10. The sample path module 502 may then be positioned, connected, or installed on the microfluidic device cleaning system 20. A wash fluid reservoir 38 is connected to the distribution block of the sample path module 502. The wash fluid reservoir 38 holds a small amount of wash fluid needed for a single cleaning operation of the sample path module 502 and is filled from one or more larger internal or external fluid reservoirs.

[0173] 12, a block diagram illustrating one embodiment of a microfluidic device cleaning system 600 with external fluid reservoirs is provided. The microfluidic device cleaning system 600 comprises a primary housing or body 602 to which a set of fluid reservoirs (fluid reservoir 1 610a, fluid reservoir 2 610b, and fluid reservoir n 610n) and a waste reservoir 650 are releasably and fluidly connected. The fluid reservoirs 610a-610n hold one or more cleaning fluids or other fluids, which may include PARVOSOL, 7X, deionized water, microbiological grade water, isopropyl alcohol, ethanol, or other suitable cleaning, wetting, or rinsing solutions or solvents.

[0174] The computing or control system 690 is in electronic communication with one or more elements of the microfluidic device cleaning system 600 and includes a processor 692, which may be a commercially available microprocessor, FPGA, or other suitable processor (e.g., RISC, ARM, x86, x64, etc.), memory 693, a networking interface or transceiver 694, and an input / output ("I / O") interface 695. A display 691 may be integral to or in electronic communication with the computing system 690 and displays output from the computing system 690, including any user interface, graphical user interface, alerts, system information, or other output required by the system or desired by a user. The computing or control system 690 stores in memory 693 a set of cleaning protocols or procedures that control or manage the operation of the microfluidic device cleaning system 600. The protocol may include the parameters or mechanisms described above, but may also be modified or adjusted based on user input or based on sensor signals from flow, proximity or image sensors of the microfluidic device cleaning system 600. Additionally, the cleaning protocol may include algorithms or processes for controlling fault detection and response, such as when a clog or low fluid flow is detected. Fault responses may include a temporary increase in fluid flow or pressure, ceasing operation, or providing an audible, visual or audiovisual alarm, etc.

[0175] The fluid reservoirs 610a-610n are releasably connected to ports or openings in the body 602 of the microfluidic device cleaning system 600. A set of valves 612a, 612b, 612n control fluid flow from the respective reservoirs and are actuated based on control signals received from the computing system 690. A set of flow sensors 614a, 614b, 614n provide signals to the computing system 690 related to the volume or rate of flow from the respective fluid reservoirs to the microfluidic device cleaning system 600. The pump assembly 620 may be a single pump or multiple pumps, including liquid pumps and air pumps, one or more of which may be peristaltic pumps, to pump fluid from the reservoirs 610a-610n to the distribution interface 630. The fluid pumped by the pump assembly 620 may include pressurized gas, including pressurized air. Pump assembly 620 pumps fluid through one or more fluid passages and may include separate control valves 621 and 622 and flow sensors 623, 624 and 625. Valves 621 and 622 provide fluid flow control and may be used to redirect fluid flow, such as at valve 621, from between one or more fluid flow passages.

[0176] In one embodiment, the distribution interface 630 includes a set of magnets 632 for fastening the distribution block or distribution manifold of the sample path module. Other fastening devices such as latches, locking lugs, or fasteners may be used. The proximity sensor 634 may be an optical, ultrasonic, or magnetic proximity sensor configured to provide a signal to the computing system 690 indicating that the distribution block is properly aligned, positioned, or secured relative to the distribution interface 630. A set of fluid inlets from the pump side of the distribution interface 630 lead to a set of fluid outlets 631. The fluid outlets 631 allow fluids, such as compressed gas or cleaning fluid, to pass from the microfluidic device cleaning system 600 into the distribution block. Fluids pass through the distribution block of the sample path module to or through a microfluidic chip, cassette, or cartridge in the microfluidic module of the sample path module during a cleaning process as controlled by the cleaning procedure.

[0177] In one embodiment, the chip interface 640 includes a set of magnets 642 for fastening the microfluidic module to the sample passage module. Other fastening devices such as latches, locking lugs, or fasteners may be used. The proximity sensor 644 may be an optical, ultrasonic, or magnetic proximity sensor configured to provide a signal to the computing system 690 indicating that the microfluidic module is properly aligned, positioned, or secured to the chip interface 640. The image sensor 646 may be a charge-coupled device ("CCD") sensor or an active pixel sensor, such as a complementary metal oxide semiconductor ("CMOS") sensor, to capture image information that may be processed by the computing system 690 to determine whether a clog is present in the microfluidic chip, cassette, or cartridge in the microfluidic module. Simpler sensors, such as photoresistors or photodiodes, may also be used to determine whether a clog is present. For either sensor type, the image sensor 646 provides image data to the computing system 690 indicative of a clog in the fluid passage. Such image data may include reduced light levels, increased black levels, on signals, or off signals, which may indicate that some object or debris is blocking the fluid flow path. The computing system 690 may process this input data to initiate a fault or unclog process. The image sensor 646 may also be used to capture images of identification markings on the microfluidic chip, such as features, fiducial markers, reference images, or reference markings, which may be processed by the computing system 690 to identify the unique microfluidic chip and provide calibration or adjustment information for the optical or sensor components. Optically identifying the microfluidic chip may be used to automatically determine a cleaning protocol or set of cleaning parameters for the microfluidic chip or sample passage module.

[0178] Valves 652 and flow sensors 654 may be used to respectively control and monitor the flow of fluids, including gases, cleaning solutions, and rinsing or other solutions, as they exit the microfluidic chip, cassette, or cartridge and flow into a waste reservoir 650. In one embodiment, an ultraviolet ("UV") light emitting sterilization device 648, which may be a UV light emitting diode ("LED"), is used in combination with the fluid-based cleaning and sterilization to further sterilize and clean the microfluidic chip.

[0179] The set of valves, image sensors and proximity sensors provide signal outputs that are processed by the computing system 690 to determine one or more states or conditions of the microfluidic device cleaning system 600 and compare the determined or detected state to an ideal or desired system state. For example, a flow sensor may be used to determine the amount of fluid that has flowed from one or more reservoirs, which may then be used to determine the amount of fluid remaining in the reservoir and signal an operator that the reservoir is low on fluid and needs replacing. The flow sensor may also be used to determine the amount of fluid that has flowed through a particular component, tube or piping run, which should be used to determine a maintenance event for that component, such as replacing the pump or tube at the end of a maintenance life or cycle. For example, a peristaltic pump may have a predetermined life and needs to be replaced at the end of its operating life. By measuring the fluid flow before or after the pump, an accurate determination can be made as to when the pump should be replaced.

[0180] Similar measurements and feedback may be provided for any system components where measured values ​​may be compared to stored values ​​such as maintenance intervals or useful life to provide an indication as to when a component or device needs to be replaced. In another example, an image sensor may be used alone or in combination with one or more flow sensors to detect or determine when a component of the microfluidic device cleaning system 600 becomes clogged. Based on the type of clog as determined by the measured system conditions, a signal may be sent to an operator or a fault condition action may be triggered, such as an abort action or increasing flow rate or pressure to break up the clog. Sensors, including flow sensors, may also be used to track the volume of the sample path module cleaned (e.g., number of paths cleaned) to accurately determine when the sample path module is clean enough to terminate the cleaning operation.

[0181] Valves in the microfluidic device cleaning system may be used to control the flow of fluids in the microfluidic device cleaning system 600, for example, to divert or stop the flow of fluids to or from particular components of the microfluidic device cleaning system 600. The valves may be solenoid valves, actuator controlled valves, or pneumatically or hydraulically controlled valves operated by control signals from the computing system 690.

[0182] FIG. 13 provides a block diagram illustrating one embodiment of a microfluidic device cleaning system 700 with internal fluid reservoirs and a control system. The microfluidic device cleaning system 700 is substantially similar to the microfluidic device cleaning system 600, except that all system elements, including the computing system 690, the display 691, the waste reservoir 650, and the fluid reservoirs 610a, 610b, through 610n, are located within a body or housing 702. In both the microfluidic device cleaning systems 600 and 700, the number and location of the reservoirs and the configuration of the computing system 690 may be modified (e.g., included or omitted, or internal or external) based on the needs of a particular implementation or process. For example, in one embodiment, the microfluidic device cleaning system may include a single external fluid reservoir 660 used for both wash fluids and waste fluids, in which the fluids are filtered or recycled. In another embodiment, the microfluidic device cleaning system may include an internal waste reservoir and an external fluid reservoir. In another embodiment, the microfluidic device cleaning system may include two internal fluid reservoirs and an external waste reservoir. In any of the embodiments, the microfluidic device cleaning system may include an internal gas compressor or pump, or a connection to an external pressurized gas source.

[0183] 14, a block diagram illustrating one embodiment of a removable and replaceable sample path module 800 including a microfluidic module 850 and a distribution block 810 is provided. The sample path module 800 includes a sample fluid path 830 and a sheath fluid path 832 that run from the distribution block 810 to the microfluidic module 850. The sample path module 800 may be used with a flow cytometry device or may be used with a microfluidic device cleaning system. It should be understood that the sample path module 800, as depicted in the embodiment in FIG. 14, is a configuration that is common to either a flow cytometry device or a microfluidic device cleaning system.

[0184] The distribution block 810 comprises a body or manifold 811 comprising sample passage elements 814, gas path elements 815, sheath fluid or buffer path elements 817, and connecting path elements 816. A spring loaded sample tube loader 813 clamps and seals a fluid reservoir 822 against a surface, such as a bottom surface, of the distribution block body 811. Alternatively, such as in the case of a microfluidic device cleaning system, the sample tube loader 813 may be removed and the fluid reservoir 822 may be held in place relative to the distribution block 810 by other suitable means. In one embodiment, the fluid reservoir 822 is a wash fluid reservoir that is clamped against the distribution block 810 by corresponding threads or lugs and a set of magnets. In one embodiment, the fluid reservoir 822 is a sample fluid reservoir that is fully retained and clamped within the sample tube loader 813, which is releasably secured to the distribution block 110 by a set of magnets. In another embodiment, the sample tube loader 813 further comprises a spring or spring biasing means for applying pressure to a fluid reservoir 822, such as a sample tube, to ensure that the seal against the sample straw 812 extends into the fluid reservoir 822 and into the fluid 824 contained therein. In a flow cytometry system, when processing a biological fluid sample, the fluid 824 may be a semen sample and the fluid reservoir 822 may be a sample tube. In a microfluidic device cleaning system, the fluid 824 in the fluid reservoir 822 may be a cleaning solution, solution or solvent, or water, such as deionized water or microbiological grade water.

[0185] A pressurized fluid, such as pressurized gas, enters the sample tube through a port or opening 820. Pressure from the pressurized gas forces or flows fluid 824 through the sample straw 812 and into the sample path element 814. A sheath fluid or buffer, or a flushing or rinsing liquid or solvent, is flowed or pumped through the sheath fluid or buffer path element 817. Pressurized gas may also be diverted, such as by an internal or external valve, from the gas path 815 through the connecting path element 816 to the sheath fluid or buffer path element 816 to provide pressurized gas into the sheath fluid path 832, such as for flushing or drying. Similarly, pressurized gas may be forced through the sample path element 814 to wash or dry the sample fluid path 830. Hoses, tubes or pipes 831 and 833 are releasably held in place by fitting sets 819 and 852, and 818 and 853, joining the distribution block 810 and the microfluidic module 850 in fluid connections and forming part of the sample fluid passage 830 and the sheath fluid or buffer passage 832, respectively.

[0186] The microfluidic module 850 comprises a body 851 in which a microfluidic chip (which may be a microfluidic cassette or cartridge) 880 is positioned and held by a microfluidic device holder 856. The device holder 856 is used to secure and position the microfluidic chip 880 in the body 851. The device holder 856 is used to adjust the relative position and angle of the microfluidic chip 880, for example by thumb screws, threaded rods or other suitable positioning means, to align the microfluidic chip 880 with a detection optical system or a laser kill optical system in a flow cytometry device or with an image sensor in a microfluidic device cleaning system. In another embodiment, the position of the microfluidic chip 880 is fixed within the microfluidic module 850 by pins or by corresponding geometric features of the microfluidic module 850, and the position of the kill laser, detection laser or other optical device is adjusted in the X, Y or Z directions relative to the microfluidic chip 880 within the flow cytometer or microfluidic device cleaning system.

[0187] In the illustrated embodiment, the microfluidic chip 880 comprises a body 881 and a set of internal fluidic channels. Channels 855 in the microfluidic module body 851 direct sheath fluid, wash fluid or pressurized gas into a sheath input 882, through a sheath channel 883, into a common channel 886, through a detection region 887 and out through one or more outlets 888 and 889 to be collected in a waste reservoir 890 as waste fluid 892. Channels 854 in the microfluidic module body 851 direct sample fluid, wash fluid or pressurized gas into a sample input 884, through a sample channel 885, into a common channel 886, through a detection region 887 and out through one or more outlets 888 and 889 to be collected in a waste reservoir 890 as waste fluid 892.

[0188] The sample path module 800 is a fully modular device that can be removed from a flow cytometry device and placed on a microfluidic device cleaning system for a fully automated cleaning process. Components of the sample path module 800, such as the distribution block 810 and the microfluidic module 850, may be removed, separated, and replaced separately for cleaning or maintenance purposes. The sample fluid path 830 of the sample path module 800 provides the entire sample fluid path for the sample fluid when the sample path module 800 is used with a flow cytometry device. The sample fluid processed by the sample path module 800 in the flow cytometry device does not contact any part of the flow cytometry device except when the sample is collected in a sample collection tube or diverted to a waste collection container. This allows for easy and quick replacement of the sample path module 800 between sample runs, for example to change the source of the semen sample. The replaceable, removable, reusable and modular nature of the sample passage module 800 reduces or eliminates downtime of commercial flow cytometry instruments and further reduces waste that can be generated by the use of disposable or single-use microfluidic devices.

[0189] 16A, a block diagram illustrating a side view of one embodiment of a removable and replaceable sample passage module microfluidic module 950 is provided. Microfluidic chip 880 may be a microfluidic chip with internal sample fluid and buffer or sheath fluid flow channels as depicted, or may have any configuration of sample and buffer or sheath fluid flow channels that will be apparent to one of skill in the art, and is disposed within chip fixture 955 of body 951 of microfluidic module 950. Sample lines are connected to connection 952 and fed to microfluidic chip 880 through internal passage 954, and sheath fluid or buffer lines are connected to connection 953 and fed to microfluidic chip 880 through internal passage 955. Connections 952 and 953 may be releasable connections as described above, and the properties and functions of microfluidic chip 880 may be as described above, or may be subject to modifications known to one of skill in the art.

[0190] Additional details and examples of microfluidic chips that may be used in accordance with the systems and methods of the present invention can be found in the following commonly owned patents and patent applications: US8961904, US9588100, US10488320, US10928298, US10532357, US16 / 279430, US16 / 419756, US16 / 597235, US16 / 704175 and US16 / 741608, the contents and specifications of which are incorporated herein by reference.

[0191] The microfluidic chip 880 is secured within the chip fixture 955 by a plate and fasteners 959 on the face of the chip fixture 955, by a set of retaining pins 960, by a set of corresponding geometric features 961, or by any combination of these elements. The front, front or front plate element 956 of the microfluidic module 950 is generally orthogonal to the chip fixture 955. The front 956 includes one or more magnets 956 that are used to releasably secure the microfluidic module 950 to a flow cytometer or microfluidic device cleaning system.

[0192] 17, a block diagram is provided illustrating an embodiment of a modular flow cytometry system 1000 including a flow cytometry device 1010, a microfluidic device cleaning system 1020, a sample passage module 1100, a local control system 1400, and a remote server 1500. In the system 1000, a removable and replaceable sample passage module 1100 is used in conjunction with the flow cytometry device 1010 and the microfluidic device cleaning system 1020. In one embodiment, the flow cytometry device 1010 includes a distribution block interface 1018, a microfluidic module interface 1016, a set of unprocessed sample tubes 1012 secured in a sample tube holder, and an automated processed sample mixing station 1014. In one embodiment, the microfluidic device cleaning system 1020 includes a distribution block interface 1630, a microfluidic device interface 1640, and a wash fluid reservoir 1038. Wash fluid may be supplied to the reservoir 1038 from one or more internal or external fluid reservoirs, and waste wash fluid may be returned to the microfluidic device cleaning system 1020 for internal or external storage.

[0193] The microfluidic module interface 1016 and the microfluidic device interface 1640 each comprise adjustable elements for positioning an optical imaging device (e.g., a camera, a microscope imaging device), a detector (e.g., a photomultiplier tube, an avalanche photodiode), or an illumination device (e.g., a kill laser or a detection laser) relative to a microfluidic chip in the microfluidic module of the sample passage module 1100. The position and angle of the adjustable elements may be adjusted manually (e.g., by a set of screws, etc.) or by an element of the flow cytometry system 1010 or the microfluidic device cleaning system 1020 (e.g., an actuator, a stepper motor). This provides for accurate positioning of any optical or detection element of the flow cytometry device 1010 or the microfluidic device cleaning system 1020 relative to a portion of the microfluidic chip in the sample passage module 1100 (e.g., an operating area, a detection area, or a calibration marking). The adjustable elements may be moved in the X, Y or Z directions, or any combination of these directions, or may be tilted relative to the microfluidic chip to properly align the adjustable elements.

[0194] The sample passage module 1100 is held and secured, such as by a set of magnets, in a holding or storage station 1700 that is physically adjacent to one or both of the flow cytometry device 1010 and the microfluidic device cleaning system 1020. A scanning device 1720, which is a device capable of reading or scanning optical or radio frequency identification tags (e.g., a bar code scanner or RFID reader), is used to read or scan the identification element 1956 on the sample passage module 1100 before it is connected to or placed in the flow cytometry device 1010 for use in processing a fluid sample. This information is transmitted to the local control system 1400. The local control system 1400 may be one or more computing devices, such as a desktop computer, laptop computer, tablet computer, thin client computer or other suitable computer, and may be external to the flow cytometry device 1010 and the microfluidic device cleaning system 1020, may be integral to one or both of the flow cytometry device 1010 and the microfluidic device cleaning system 1020, may be shared by the flow cytometry system 1010 and the microfluidic device cleaning system 1020, or may comprise a separate station or terminal for each of the flow cytometry system 1010 and the microfluidic device cleaning system 1020.

[0195] The local control system 1400 receives and processes the identification information scanned by the scanning device 1720 from the sample passage module 1100. The graphical user interface 1410 displays information associated with the sample passage module 1100, such as the configuration, the type of microfluidic chip, the appropriate flow cytometry or cleaning protocols or parameters, maintenance information, or warning information, to inform the user about how the sample passage module 1100 is being used and configured. Additionally, this information may be used by the flow cytometry system 1010 and the microfluidic device cleaning system 1020, respectively, to automatically configure flow cytometry or cleaning protocols based on the configuration of the sample passage module 1100. Additionally or alternatively, scanning may be used to track the location or status of the sample passage module 1100, and similar identification elements may be scanned on the flow cytometry device 1010 and the microfluidic device cleaning system 1020 to specifically track how and when the sample passage module 1100 has been used and to identify where the sample passage module 1100 is at any given time.

[0196] Information used by the local control system 1400 may be stored locally or in a remote server 1500 that includes one or more databases. The databases stored in the remote server 1500 include information about the specific sample passage module 1100, including usage logs, maintenance information, configuration information, flow cytometry protocols, and cleaning protocols. This information may alternatively be stored in the local control system 1400. In one embodiment, information stored in the local control system 1400 or the remote server 1500 may be shared between multiple systems, i.e., between two or more systems. For example, in one embodiment, configuration and usage information from one flow cytometry device 1010 may be shared with other flow cytometry systems, configuration and usage information from one microfluidic device cleaning system 1020 may be shared with other microfluidic device cleaning systems, and information about the sample passage module 1100 may be communicated and shared between any connected devices or systems.

[0197] The user interface 1410 provides a user with a GUI and a set of user operable or controllable elements, e.g., graphical user interface elements, to view and control parameters or information associated with any aspect of the system 1100. For example, in one embodiment, a user is provided with a set of user interface elements in the user interface 1410 to configure a flow cytometry protocol for a particular sample passage module 1100 and for a particular sample being processed, such as a semen sample. In another embodiment, a user is provided with a set of user interface elements in the user interface 1410 to configure a cleaning protocol for a particular sample passage module 1100. The user interface 1410 is further configured to provide alerts or information regarding the current operating status of the flow cytometry device 1010 or the microfluidic device.

[0198] In one embodiment, any flow cytometry system (e.g., flow cytometry system 10), microfluidic device cleaning system (e.g., microfluidic device cleaning system 20) and sample path module (e.g., sample path module 100) and any fluid path, i.e., any components that come into physical contact with fluid, should be chemically inert. For example, silicate glass, polyvinylidene fluoride ("PVDF"), polytetrafluoroethylene ("PTFE"), perfluoroalkoxyalkane ("PFA"), polyetheretherketone ("PEEK"), or other suitable materials may be used to prevent damage, corrosion, or other undesirable reactions in components that may be exposed to corrosive substances (e.g., acids, oxidizers, bases) or other hazardous substances that may be used in the cleaning process or procedure. In particular, the selection of materials for components in any fluid path should prioritize materials that may be safely placed in contact with any chemicals used in the cleaning or sample processing operation or process, and should use materials that are substantially chemically inert.

[0199] Referring now to FIG. 18, a block diagram 2000 is provided illustrating one embodiment of fluid flow within a microfluidic device cleaning system, such as the microfluidic device cleaning system 20, 600, 700, or 1020. The diagram 2000 illustrates one embodiment of the movement and control of fluids into, through, and out of the microfluidic device cleaning system. A set of fluid sources, including a sheath line source 2010, a sample line source 2012, and a gas source 2014, each of which may be an internal or external source, may be provided from a fluid reservoir (e.g., bag, container, tank), a compressed gas source or tank, and may include a cleaning solution, a solvent such as water, and compressed air, comprises a set of inputs for the fluidic system. Overall, three fluidic pathways are provided: a sheath fluid line or pathway 2002, a gas line or pathway 2006, and a sample line or pathway 2004. In the sheath fluid line 2002, the flow of fluid from one of the sources 2010 is controlled by a corresponding valve 2020 to allow or restrict flow to the sheath line manifold 2030. In the sample fluid line 2004, the flow of fluid from one of the sources 2012 is controlled by a corresponding valve 2022 to allow or restrict flow to the sample line manifold 2032. One or more fluid inputs, for example a particular wash or solution, a solvent such as water or other fluids, may be shared or provided to both the sheath fluid line 2002 and the sample line 2004. Control of the fluid path for the shared fluid is achieved by operation of the appropriate corresponding valve in the set of valves 2020 or 2022. With respect to the gas line 2006, the gas pressure from the gas or air input 2014 may be a pressurized external or internal gas source and is regulated by an air regulator 2040 to allow or regulate the pressurized gas in the fluid system.

[0200] In the sheath line 2002, a sheath line pump 2050, which may be a peristaltic pump or other suitable pump, draws or pumps one or more fluids from a source 2010 to a main fluidic manifold 2080. The flow of fluids as pumped by the sheath line pump 2050 is measured by a flow sensor 2060 downstream of the pump and controlled by a valve 2070. In the sample line 2004, a sample line pump 2052, which may be a peristaltic pump or other suitable pump, draws or pumps one or more fluids from a source 2010 or 2012 to a main fluidic manifold 2080. The flow of fluids as pumped by the sample line pump 2052 is measured by a flow sensor 2060 downstream of the pump and controlled by the operation of a valve downstream of the flow sensor. The flow sensor 2060 is used to measure the amount of fluid moved through the flow line or the velocity of the fluid flow through the flow line and may be used to detect or identify a lower than desired fluid flow rate, which may indicate a clog or obstruction in the fluid flow line. Compressed gas from the air input 2014 is regulated by an air regulator 2040 and the flow of compressed gas is controlled by valves 2072 and 2074 to allow gas to flow into the fluidic manifold 2080 or into the sheath fluid line 2002. The gas is used to clear the sample line 2004 or the sheath fluid line 2002 and to dry the fluid lines after a cleaning operation.

[0201] Fluids, including gases, flow into the fluidic manifold 2080 and through the sample pathway module before returning at the chip manifold 2090. Waste fluids, including cleaning fluids and rinsing agents, flow to and are received at the waste output 2100, which directs the waste fluids to an internal or external waste reservoir or drain line. The waste valve 2102 is used to divert any fluid from the sample line 2004, sheath fluid line 2002, or gas line 2006 directly into the waste output 2100 without passing through the sample pathway module. Fluid diversion may be performed manually or automatically as a result of an error or fault condition, including based on a detected clog or obstruction.

[0202] On-chip identification

[0203] 16A and 16B, the microfluidic chip may include an identification element 957 for on-system object identification and referencing for auto-focusing the microfluidic chip. For example, one or more identification elements 957 may be located on a front portion 956 of the chip. The identification element 957 may be a visual or optical identification element, such as a QR code or barcode, or may be a wireless identification element, such as an RFID or NFC tag, or may be a combination of an optical or visual element and a wireless identification element.

[0204] Although each microfluidic chip is intended to be geometrically identical, variations during manufacturing may cause the chips to differ slightly. The identification element can allow comparison of chips against instrument performance, which is useful for eliminating poor performing chips and identifying high performing chips. The identification element also allows for automatic tracking of how long the chip has been in use to inform the user when the chip needs to be removed for cleaning.

[0205] In some embodiments, the identification element 957 is used to identify a particular microfluidic module 950 and the microfluidic chip 880 installed or disposed within the module 950. This allows for easy identification of the elements for cleaning, storage and removal to avoid cross contamination, and for configuration of flow cytometry or cleaning parameters or protocols based on the type and configuration of the microfluidic chip 880 and microfluidic module 950 as identified by the identification element 957.

[0206] In other embodiments, the identification element can also be used to automatically align and position the microfluidic chip within the flow cytometry device. This can save the operator time by eliminating the need to manually focus and align the microfluidic chip. As shown in FIG. 16C, in one embodiment, the microfluidic chip with the identification element is placed on a 3-axis stage, which is a stage that can move on three axes. A camera and a light source, such as a bright field LED light, may be used to image the microfluidic chip and provide feedback to the 3-axis stage to properly align the chip. The camera and light source may be fixed in place and alternative levels of illumination may be required. The dimensions of the identification element should be small enough to be completely visible within the field of view of the camera. The 3-axis stage allows for repositioning to move the chip in focus by moving the chip, and thus the etch pattern, instead of refocusing and repositioning the camera.

[0207] In some embodiments, the chip may have one or more identification elements 957. For example, multiple identification elements 957 may be required to achieve accurate focusing and positioning. In some embodiments, the identification elements 957 may be laser etched into each chip. The identification elements may be any pattern that can be deciphered through image analysis. Patterns include, but are not limited to, alphanumeric sequences, bar codes, QR codes, and the like, which are applicable and equivalent.

[0208] In another application, the identification element 957 may be used to indicate which chip is currently mounted on the microfluidic module. Since the etched pattern can store information, the flow cytometry device may be able to read the etched pattern on the chip to indicate which chip is on the instrument.

[0209] Optical module and electronic module

[0210] Without wishing to limit the invention to a particular application or embodiment, the systems, devices and methods described herein, such as the removable and replaceable sample passage module 100, may be used with particle processing systems, such as a semen sorting system (e.g., flow cytometry system 10). In one embodiment, the semen sorting system may comprise a flow cytometry apparatus or a microfluidic chip or device, comprising a flow chamber configured to direct a fluid flow containing sample particles through a particle interrogation location. The sample particles may be cells, such as sperm, that have been stained or otherwise treated prior to entering the flow cytometry apparatus or microfluidic device. The flow cytometry apparatus or microfluidic device may further comprise a laser configured to emit electromagnetic radiation along a beam path to the particle interrogation location, and a detector configured to detect the radiation from the particles. After detection, the particles may be sorted or inactivated, for example by ablation with electromagnetic radiation, before or after exiting the flow cytometry apparatus or microfluidic device. Additional detectors may be used to determine the effectiveness of particle inactivation, e.g., to determine or detect the amount or quantity of electromagnetic radiation to which individual particles have been exposed, and to determine whether individual particles have been sufficiently inactivated or damaged. The particles may be X- or Y-chromosome sorted or sexed semen, e.g., X-chromosome sexed semen, which is collected in one or more collection vessels.

[0211] The removable and replaceable sample passage module 100 may also be used in or with a method for analyzing particles, such as sperm cells, contained in a fluid stream as the particles flow through an interrogation location in a flow cytometry apparatus or in a microfluidic chip or device. Such a method includes illuminating the fluid stream and the particles contained therein using electromagnetic radiation emitted from a laser. A detector detects electromagnetic radiation emitted from the interrogation location by the particles, and a processor determines a characteristic of the particles in the fluid stream based in part on the signal from the detector. After detection, the particles may be sorted or inactivated, such as by a physical sorting and removal process, or by inactivation by ablation or photodamage with electromagnetic radiation, before or after exiting the flow cytometry apparatus or microfluidic device. The particles may be X- or Y-chromosome sorted or sexed semen, for example sexed semen having an X or Y chromosome, and may then be collected in one or more collection vessels.

[0212] The removable and replaceable sample passage module 100 may also be used in or with a method for assessing the total amount of DNA in a sperm cell's nucleus using a flow cytometry apparatus or a microfluidic chip or device. The method may include staining the DNA in the sperm cell's nucleus and then illuminating the stained DNA in the sperm cell's nucleus with an electromagnetic emission device, such as a laser, as the sperm cell passes through the laser in a corresponding region of the flow cytometry apparatus or microfluidic device. A detector, such as an avalanche photodiode, detects the fluorescent light emitted from the illuminated and stained DNA in the sperm cell's nucleus at a detection or interrogation location in the flow cytometry apparatus or microfluidic device. The method may further include distinguishing between X chromosome bearing sperm cells and Y chromosome bearing sperm cells by first determining the sex of the sperm cell using the detected total amount of DNA in the sperm cell's nucleus and then distinguishing between the sperm cells based on the sex determination. The sperm cell characteristic may include the corresponding total amount of DNA in the sperm cell's nucleus. The method may further include inactivating a given sperm cell based on the determined total DNA count in the nucleus of the given sperm cell. The inactivating may include photodamaging or ablating the given sperm cell by emitting electromagnetic radiation. The method may further include forming a plurality of droplets having one of the sperm cells entrapped therein, differently charging each of the droplets based on the sex differentiation characteristic of the sperm cell entrapped therein, deflecting one of the droplets, and differentially collecting each of the droplets based on the sex differentiation characteristic of the sperm cell entrapped therein. The method may further include collecting the sperm cells after determination, sorting, or inactivation as the sperm cells exit the microfluidic device. The particles may be X- or Y-chromosome sorted or sexed semen, e.g., sexed semen having an X or Y chromosome, and are collected in one or more collection vessels.

[0213] In some embodiments, the invention provides an electromagnetic radiation kill beam having an adjustable elliptical beam waist profile at a focal plane, the electromagnetic radiation kill beam being independently adjustable in height and width and not a factor of or derived from each other, and optical and optomechanical modules for both positioning and emitting detection lasers and kill lasers from the same direction relative to the sample fluid flow or from a common side with respect to the flow cytometry device.

[0214] FIG. 19 shows one embodiment of an interrogation or optics module 7020 that includes the detection optics and the detection and kill lasers. The optics module floats separately from the rest of the module system and may be isolated with rubber feet, i.e., removable from the chassis. FIG. 20 shows the internal layout of the optics module. Both the detection and kill come from one side in combination with the use of a beam cube. The optics module combines "S" and "P" polarized beams of the same wavelength. A filter in front of the target protects it from damage. An aspheric lens focuses the kill laser instead of the target. Both lasers have shaped lenses to control the spot size at the tip.

[0215] Figures 20A-20B and 21A-21B show an electronics module 7010 that comprises an electronics box that houses the electrical components and a control panel. The box includes a removable lid that protects the interior of the box. The electronics module can be removed from the chassis as well. Figure 22 is a schematic diagram of the electronic components of a modular flow cytometry system.

[0216] Referring now to FIG. 23, a diagram illustrating the beam width of the electromagnetic radiation emission of a kill laser having an elliptical profile at the focal plane according to one embodiment of the present invention is provided. The width of the beam is configured, calibrated, or adjusted to be 5 μm to 35 μm wide in the X direction, where the Y direction is longitudinal along the length of the flow path or channel in a microfluidic device through which a sample containing a set of particles and / or sheath fluid flows. The width of the kill beam as shown is 20 μm, which is wider than the exemplary core width shown. The 5-20 μm core width is the width of a fluid, such as a sample fluid, that is bounded by other focusing fluids, not necessarily by physical channel walls. The physical channel in a flow cytometer may be over 100 μm wide, but the core, which is a constrained fluid and in which the particles or sample are placed, is 5-20 μm wide. For example, a single sheath designed microfluidic chip ("SSMC") channel may have a core width of 5-20 μm, and a dual sheath designed microfluidic chip ("DSMC") may have a core width of 5-15 μm. The width of the kill beam is configured by adjustment of one or more optical or opto-mechanical components, for example as provided in Figures 25A, 25B, and 26B, to provide a beam width equal to or greater than the core width in the working or interrogation region of the microfluidic channel.

[0217] The kill beam has an elliptical profile at the focal plane and has a Gaussian intensity profile, in this case the highest concentration of electromagnetic radiation emission, e.g. photons, is at the center of the kill beam. A factor in the effectiveness of the kill beam in inactivating or destroying particles passing through the focal plane in the action or interrogation region of a microfluidic channel or in a droplet in a droplet-based flow cytometer is the amount of electromagnetic radiation emission, e.g. photons, in a unit of space that strikes a particle, e.g. a sperm cell, passing through that space. The kill beam needs to have sufficient intensity over the surface area of ​​the particle to ensure that the particle has been sufficiently inactivated, e.g. by photodamage or ablation. The laser module used to generate the kill beam is a pulsed laser module, e.g. a pulsed laser module having a pulse duration of 1-500 nanoseconds and an available pulse energy of 1-5 μJ of available energy per pulse. In another embodiment, the pulsed laser module has a pulse duration of 5-30 nanoseconds. Increasing the pulse energy increases the size of the kill beam. In existing systems and methods, this may result in a circularly shaped beam having an increased beam waist, such as by increasing the radius and circumference of the beam at the focal plane. However, it may be more effective to utilize an elliptical profile kill beam, which may output the same amount of electromagnetic radiation per unit area at a lower pulse energy and over a wider area at the focal plane than existing circular profile beams. Providing an elliptical profile by expanding the width, rather than the height, of the kill beam provides an increased kill width or effective area at the same pulse energy level of the laser module, as compared to a circular kill beam of the same area.

[0218] Referring now to FIG. 24A, the diagram illustrates how the pulse energy in μJ increases for a beam that increases the beam width in μm for a particular beam shape according to one embodiment of the present invention. The kill width, i.e., effective area at the focal plane where there is a sufficient amount of electromagnetic radiation to deactivate or destroy a particle, for the expanded or elliptical beam shape of the present invention is increased as the laser pulse energy increases, but is higher at any given pulse energy level when compared to a standard circular beam. The expanded, elliptical beam has a larger kill width in μm than a standard circular beam where there is sufficient electromagnetic radiation at the focal plane to completely deactivate a particle. In the exemplary embodiment provided in the diagram in FIG. 24A, this difference between the expanded and standard beam is approximately 2.5 μm at lower pulse energies, e.g., 1.8 μJ, but can be as much as 7.5 μm at higher pulse energies, e.g., 3 μJ. Expanded beams have a larger kill beam width in μm than standard beams where there is enough electromagnetic radiation at the focal plane to partially deactivate or slice a particle. This difference between expanded and standard beams is roughly 2.5 μm at lower pulse energies, e.g. 1.8 μJ, but can be as much as 5 μm at higher pulse energies, e.g. 3 μJ. Similar results are achieved at lower pulse energies, e.g. 1 μJ, and at higher pulse energies, e.g. 5 μJ.

[0219] If the beam width of an elliptical kill beam, such as the beam shown in Figure 23, is increased to 30 μm, the difference between the elliptical kill beam and the standard circular kill beam may be 1.33 + / - 0.06% in sliced ​​particle width and 1.29 + / - 0.15% in killed or fully inactivated particle width. This difference exists for laser radiation pulse energies of 1 μJ to 5 μJ and can become even larger as the laser radiation pulse energy increases.

[0220] Without wishing to limit the present invention to a particular theory or mechanism, the optical and opto-mechanical systems of the present invention increase the kill width of the beam emitted by the kill laser to provide similar kill performance or effectiveness with respect to the kill volume in either the X dimension or the Z dimension (where the Y dimension is the flow direction dimension in a flow cytometer system).

[0221] For example, referring to the chart shown in FIG. 24B, in one non-limiting exemplary embodiment, a 30 μm wide kill beam with an elliptical profile at the focal plane was used at a radiant energy of 2.4 μJ. However, the radiated pulse energy may be 1-5 μJ per pulse and the pulse duration may be 1-500 nanoseconds, or in one embodiment, 5-30 nanoseconds. In this exemplary embodiment, the 30 μm wide kill beam demonstrated improved kill counts as the percentage of particles ablated or photodamaged by the electromagnetic radiation of the kill laser. The 30 μm wide kill beam with an elliptical profile at the focal plane provided a differential improvement in relative location of + / - 10 μm from the ideal or determined kill or action location. Whereas a typical kill beam or kill location less than 18μm wide with a circular profile in the focal plane was unable to effectively kill, inactivate, significantly photodamage or ablate particles more than + / - 10μm from the ideal or determined kill location, the improved 30μm wide kill beam with an elliptical profile in the focal plane achieves inactivation, killing, destruction or significant photodamage or ablation of at least 90% of particles subjected to the kill laser. When comparing particles such as sperm cells killed, killed or sliced ​​by the narrower circular beam, the improvement of the wider elliptical beam is readily apparent in terms of killing effectiveness or slicing.

[0222] Without wishing to limit the invention to a particular embodiment, the wider kill beams described above may be used with existing flow cytometry systems and in existing flow cytometry optics configurations. However, the wider kill beams described herein provide additional advantages when used with flow cytometry systems having a detection laser and a kill or inactivation laser positioned on a common side relative to a major or primary face of a flow cytometry device, such as a microfluidic chip. These advantages include additional adjustability and configurability, as well as the ease of doing so with respect to beam size, shape and positioning, as provided by having optical components or paths positioned on a common side of the system.

[0223] 25A, a block diagram of an optical-mechanical system 3400 for positioning a set of laser assemblies 3410, including a detection laser assembly 3430 and a kill laser assembly 3420, on the same side of a major (e.g., top or bottom) surface of a microfluidic chip, and a detection assembly 3450 on the opposite side, according to one embodiment of the present invention. The optical-mechanical system 3400 is used to detect certain characteristics of particles and to inactivate, kill, photodamage, or ablate particles, such as sperm cells, passing through an action or interrogation region 3404 in a flow path 3402 of a flow cytometer system, such as within a channel of a microfluidic chip. The optical-mechanical system 3400 may further be used to detect the outcome (e.g., success, failure, partial success) of a kill action or photodamage ablation event. The kill laser assembly 3420 includes a kill laser 3422, which may be a pulsed laser module with a pulse duration of 1-500 nanoseconds, a set of one or more beam expander optics 3424, and a cylindrical lens pair 3426. The profile or shape and configuration of the lenses of the beam expander 3424 and the cylindrical lens pair 3426 provide compensation for misalignment up to + / - 5°. This provides a simpler alignment and calibration process and further provides some margin for operator error in alignment and calibration. The detection laser assembly 3430 includes a detection laser 3432, which may be a coherent continuous wave ("CW") laser, a quasi-CW laser, or a pulsed laser, a set of one or more beam expander optics 3434, and a cylindrical lens pair 3436. Mirror 3428 directs the electromagnetic radiation emission ("beam") of kill laser 3422 towards beam combiner 3412 to combine the beam emission of kill laser 3422 with the beam emission of detection laser 3432 for further directing both beam emissions towards focal plane 3452 within interrogation or interrogation region 3404 of flow path 3402. Here, "combining" does not only refer to a physical combination or overlap of beam emissions, but may also refer to a reorientation of one or more of the beams to be directed in the same direction and positioned in close physical proximity where the beam emissions do not coincide.

[0224] On the detector side of the optomechanical system 3400, a detection assembly 3450 includes one or more filters, such as a short or long pass filter 3454, a mirror 3456, a near ultraviolet ("NUV") objective lens 3458, a mirror 3460, a mirror 3462, a kill sensor 3464, a camera 3468, and a detection sensor 3470. The kill sensor 3464 and the detection sensor 3470 may be avalanche photodiode type sensors, and the camera 3468 may be a charge-coupled device ("CCD") camera. In another embodiment, the kill sensor 3464 and the detection sensor 3470 may each include a photomultiplier tube ("PMT"), a complementary metal oxide semiconductor ("CMOS") sensor, or an electron multiplying charge-coupled device ("EMCCD") type sensor. In another embodiment, the camera 3468 may comprise a complementary metal oxide semiconductor ("CMOS") or electron multiplying charge coupled device ("EMCCD") type sensor.

[0225] Without wishing to limit the invention to a particular application or embodiment, the systems, devices and methods of the invention, e.g., the opto-mechanical system 3400, may be used with particle processing systems, such as semen sorting systems. In one embodiment, the semen sorting system may comprise a flow cytometry apparatus or a microfluidic chip or device comprising a flow chamber configured to direct a fluid flow containing sample particles through a particle interrogation location (e.g., action or interrogation location 3404). The sample particles may be cells, such as sperm, that have been stained or otherwise processed prior to entering the flow cytometry apparatus or microfluidic device. The flow cytometry apparatus or microfluidic device may further comprise a laser (e.g., detection laser assembly 3430) configured to emit electromagnetic radiation along a beam path to the particle interrogation location, and a detector (e.g., detection assembly 3450) configured to detect radiation from the particles. After detection, the particles may be sorted or inactivated, such as by ablation with electromagnetic radiation (e.g., by kill laser assembly 3420) before or after exiting the flow cytometry apparatus or microfluidic device. Additional detectors may be used to determine the effectiveness of the particle inactivation, such as to determine or detect the total amount or quantity of electromagnetic radiation to which individual particles were exposed, and to determine whether individual particles have been sufficiently inactivated or damaged. The particles may be X- or Y-chromosome sorted or sexed semen, such as X-chromosome sexed semen, and are then collected in one or more collection vessels.

[0226] The opto-mechanical system 3400 may also be used with or in a method for analyzing particles, such as sperm cells, contained in a fluid stream as the particles flow through an interrogation location (e.g., working or interrogation location 3404) in a flow cytometry apparatus or microfluidic chip or device. Such a method includes illuminating the fluid stream and particles contained therein using electromagnetic radiation emitted from a laser (e.g., detection laser assembly 3430). A detector (e.g., detection assembly 3450) detects the electromagnetic radiation emitted from the interrogation location by the particle, and a processor determines a characteristic of the particle in the fluid stream based in part on the signal from the detector. After detection, the particle may be sorted or inactivated, such as by a physical sorting and removal process, or by ablation or photodamage with electromagnetic radiation (e.g., by kill laser assembly 3420), before or after it exits the flow cytometry apparatus or microfluidic device. The particles may be X- or Y-chromosome sorted or sexed semen, for example sexed semen having an X or Y chromosome, which is then collected in one or more collection vessels.

[0227] The opto-mechanical system 3400 may also be used with or in a method for assessing the amount of DNA in the nucleus of a sperm cell using a flow cytometry apparatus or a microfluidic chip or device. The method may include staining the DNA in the nucleus of a sperm cell and then emitting the stained DNA in the nucleus of the sperm cell by an electromagnetic emission device such as a laser (e.g., detection laser assembly 3430) when the sperm cell passes the laser in a corresponding region (e.g., action or interrogation location 3404) of the flow cytometry apparatus or microfluidic device. A detector (e.g., detection assembly 3450), such as an avalanche photodiode, detects the fluorescent light emitted from the emitted stained DNA in the nucleus of the sperm cell at the detection or interrogation location (e.g., action or interrogation 3404) in the flow cytometry apparatus or microfluidic device. The method may further include distinguishing between sperm cells having an X chromosome and sperm cells having a Y chromosome by first determining the sex of the sperm cells using the detected amount of DNA in the sperm cell's nucleus, and then distinguishing between the plurality of sperm cells based on the sex determination. The sperm cell characteristics may include a corresponding amount of DNA in the sperm cell's nucleus. The method may further include inactivating a given sperm cell based on the determined amount of DNA in the given sperm cell's nucleus. The inactivating may include photodamaging or ablating the given sperm cell by emission of electromagnetic radiation (e.g., by a kill laser assembly 3420). The method may further include forming a plurality of droplets having one of the sperm cells entrapped therein, separately charging each of the droplets based on the sex differentiation characteristic of the sperm cell entrapped therein, deflecting one of the droplets, and differentially collecting each of the droplets based on the sex differentiation characteristic of the sperm cell entrapped therein. The method may further include collecting the sperm cells after determination, sorting or inactivation as they exit the microfluidic device.The particles may be X- or Y-chromosome sorted or sexed semen, for example sexed semen having an X or Y chromosome, and are collected in one or more collection vessels.

[0228] Without wishing to limit the invention to a particular application or embodiment, the opto-mechanical system 3400 provides an improvement over existing systems and methods for interrogating, detecting, and deactivating particles by positioning the kill laser assembly 3420 and the detection laser assembly 3430 of a set of laser assemblies 3410 on a common side of a flow cytometry device, such as a microfluidic chip. For example, the kill laser assembly 3420 and the detection laser assembly 3430 may be positioned or located on the same or common side relative to the top or front side of the microfluidic chip, or relative to the bottom or back side of the microfluidic chip, and the detection assembly 3450 may be positioned or located on the opposite side relative to the laser assembly 3410. This configuration provides an advantage over configurations in which the kill laser assembly and the detection laser assembly are positioned on opposite sides of the flow plane of the flow cytometry device.

[0229] For example, in configurations involving positioning the kill laser assembly and the detection laser assembly on opposite sides of the flow plane of a flow cytometry device, the radiation beam of the kill laser assembly may need to pass through the objective lens of the detection assembly, which may cause damage or degradation to one or more optical components of the detection assembly. By positioning the kill laser assembly 3420 on a common side with the detection laser assembly 3430, the radiation beam of the kill laser assembly 3420 does not need to first pass through any optical components of the detection assembly 3450, which may cause unnecessary or undesirable degradation, damage or wear.

[0230] Additionally, positioning the kill laser assembly 3420 and the detection laser assembly 3430 on a common side of the flow cytometry device simplifies the setup, alignment and assembly of the optical and mechanical elements of the system 3400. This provides greater stability of the system 3400, and also improves detection resolution and reduces the signal to noise ratio. Commonly positioning the kill laser assembly 3420 and the detection laser assembly 3430 provides greater ability to eliminate undesired wavelengths on the detection assembly 3450 side of the system 3400. Furthermore, when the kill laser assembly 3420 and the detection laser assembly 3430 are commonly positioned, the set of laser assemblies 3410 may share optical components, reducing system complexity and providing inherent compensation for physical vibration or movement of the system when the assemblies share a subset of components in the optical path. If the kill laser assembly 3420 and the detection laser assembly 3430 do not share optical components and are not commonly positioned, vibrations of the system 3400 can cause the radiation beams from both assemblies 3420 and 3430 to move or oscillate independently, potentially creating problems with the accuracy and effectiveness of detection and deactivation. However, if commonly positioned, vibrations can cause the radiation beams of the kill laser assembly 3420 and the detection laser assembly 3430 to oscillate in common, reducing the loss in the effectiveness and accuracy of detection and deactivation.

[0231] Continuing with the optomechanical system 3400 in FIG. 25A, the configuration of the kill laser assembly 3420 alone does not provide the advantages described above, but may be used with wider kill beams as shown and described in FIGS. 1-3. For example, an electromagnetic radiation emission or laser beam is emitted from a laser head, which may be a kill laser module 3422 (e.g., a pulsed laser module with a pulse duration of 1-500 nanoseconds and a pulse energy of 1-5 μJ). The beam passes through a series of optical components, namely a beam expander 3424, which shapes the beam into a desired shape and size, which is typically circular. The beam continues to pass through a pair of cylindrical lenses 3426, which continue to shape the beam from a circular profile to an elliptical profile. The beam is then directed by a mirror 3428 towards a beam combiner 3412, where the beams from the kill laser module 3422 and the detection laser module 3432 are combined and projected towards a focal plane.

[0232] Additionally, the positions of the kill laser assembly 3420 and the detection laser assembly 3430 may be swapped or reversed so that the beam or radiation from the kill laser assembly 3420 passes directly through the polarizing beam splitter or combiner 3412 and the beam or radiation from the detection laser assembly 3430 is redirected 90 degrees toward the focal plane. The radiation from the kill laser assembly 3420 and the detection laser assembly 3430 may or may not be perfectly combined, i.e., the radiation may not be perfectly coincident at the focal plane 3452, depending on the flow cytometer and particle processing task in which the assemblies are used.

[0233] 25B, a block diagram of an opto-mechanical system 3500 for positioning a set of laser assemblies 3510, including a detection laser assembly 3530 and a kill laser assembly 3520, on the same side of a major (e.g., top or bottom) surface of a microfluidic chip and opposite a detection assembly 3550, is provided according to one embodiment of the present invention. The opto-mechanical system 3500 is used to detect certain characteristics of particles and to inactivate, kill, photodamage, or ablate particles, such as sperm cells, passing through an action or interrogation region 3504 in a flow path 3502 of a flow cytometer system, such as within a channel of a microfluidic chip. The opto-mechanical system 3500 may further be used to detect the outcome (e.g., success, failure, partial success) of a kill action or photodamage ablation event. The kill laser assembly 3520 comprises a kill laser 3522, which may be a pulsed laser module having a pulse duration of 1-500 nanoseconds, a set of one or more beam expander optics 3524, a wave plate 3526, and an aspheric lens 3528. The detection laser assembly 3530 comprises a detection laser 3532, which may be a coherent continuous wave ("CW") laser, a quasi-CW laser, or a pulsed laser, and a cylindrical lens 3534. A mirror 3512 directs the electromagnetic radiation emission ("beam") of the detection laser 3532 towards a beam combiner 3514, which is a polarizing beam splitter, to combine the beam emission of the kill laser 3522 with the beam emission of the detection laser 3532 and further direct both beam emissions towards a focal plane 3552 within an interrogation or working region 3504 of the flow path 3502. Here, "combining" may refer not only to the physical joining or overlapping of beam radiation, but also to the redirection of one or more of the beams to be directed in the same direction and positioned in close physical proximity.

[0234] On the detector side of the optomechanical system 3500, the detection assembly 3550 includes a filter, such as a high pass filter 3554, a NUV objective lens 3556, a dichroic mirror 3558, a kill sensor 3564, a camera 3560, a beam splitter 3562, and a detection sensor 3566. The kill sensor 3564 and the detection sensor 3566 may be avalanche photodiode type sensors, and the camera 3560 may be a CCD camera. In another embodiment, the kill sensor 3564 and the detection sensor 3566 may each comprise a PMT, a CMOS sensor, or an EMCCD type sensor. In another embodiment, the camera 3560 may comprise a CMOS or EMCCD type sensor.

[0235] Without wishing to limit the invention to a particular application or embodiment, the systems, apparatus and methods of the present invention, such as the opto-mechanical system 3500, may be used in conjunction with particle processing systems such as the semen sorting system described above with respect to the system 3400 shown in FIG. 25A.

[0236] The optical-mechanical system 3500 may also be used in conjunction with or in methods for analyzing particles, such as sperm cells, contained in a fluid stream as the particles flow through an interrogation location (e.g., action or interrogation location 3504) in a flow cytometry apparatus or in a microfluidic chip or device, such as the methods described above with respect to system 3400 shown in FIG. 25A.

[0237] The opto-mechanical system 3500 may also be used in conjunction with or in a method for assessing the amount of DNA in a sperm cell nucleus using a flow cytometry apparatus or microfluidic chip or device, such as the method described above with respect to system 3400 shown in FIG. 25A.

[0238] Without wishing to limit the invention to a particular application or embodiment, the opto-mechanical system 3500 provides an improvement over existing systems and methods for interrogating, detecting, and deactivating particles by positioning the kill laser assembly 3520 and the detection laser assembly 3530 of a set of laser assemblies 3510 on a common side of a flow cytometry device, such as a microfluidic chip. For example, the kill laser assembly 3520 and the detection laser assembly 3530 may be positioned or located on the same or common side relative to a top or front surface of the microfluidic chip, or relative to a bottom or back surface of the microfluidic chip, and the detection assembly 3550 may be positioned or located on the opposite side relative to the laser assembly 3510. This configuration provides an advantage over configurations in which the kill laser assembly and the detection laser assembly are positioned on opposite sides of the flow plane of the flow cytometry device.

[0239] For example, in configurations involving positioning the kill laser assembly and the detection laser assembly on opposite sides of the flow plane of a flow cytometry device, the radiation beam of the kill laser assembly may need to pass through an objective lens, which may cause damage or degradation to one or more optical components of the detection assembly. By positioning the kill laser assembly 3520 on a common side with the detection laser assembly 3530, the radiation beam of the kill laser assembly 3520 does not need to first pass through any optical components of the detection assembly 3550, which may cause unnecessary or undesirable degradation, damage or wear.

[0240] Additionally, positioning the kill laser assembly 3520 and the detection laser assembly 3530 on a common side of the flow cytometry device simplifies the setup, alignment and assembly of the optical and mechanical elements of the system 3500. This provides better stability of the system 3500 and also provides improved detection resolution and reduced signal to noise ratio. Commonly positioning the kill laser assembly 3520 and the detection laser assembly 3530 provides a better ability to eliminate undesired wavelengths on the detection assembly 3550 side of the system 3500. Furthermore, when the kill laser assembly 3520 and the detection laser assembly 3530 are commonly positioned, the set of laser assemblies 3510 may share optical components, reducing the complexity of the system and providing inherent compensation for physical vibration or movement of the system. If the kill laser assembly 3520 and the detection laser assembly 3530 do not share optical components and are not commonly positioned, vibrations of the system 3500 can cause the radiation beams from both assemblies 3520 and 3530 to move or oscillate independently, potentially creating problems with the accuracy and effectiveness of detection and deactivation. However, if commonly positioned, vibrations can cause the radiation beams of the kill laser assembly 3520 and the detection laser assembly 3530 to oscillate in common, reducing loss in the effectiveness and accuracy of detection and deactivation.

[0241] Continuing with the optomechanical system 3500 in FIG. 25B, the configuration of the kill laser assembly 3520 alone does not provide the advantages described above, but may be used with wider kill beams as shown and described in FIGS. 1-3. For example, electromagnetic radiation radiation or laser beam is emitted from a laser head, which may be a kill laser module 3522 (e.g., a pulsed laser module having a pulse duration of 1-500 nanoseconds). The beam passes through a series of optical components, namely a beam expander 3524, to shape the beam into the desired shape and size, which is typically circular. The beam subsequently passes through a wave plate 3526, which changes the polarization state of the beam as it passes through the plate, and then through an aspheric lens 3528, changing the profile from circular to elliptical. These components may be adjusted to independently adjust the height and width of the elliptical profile of the beam at the focal plane 3552. The beams are then directed through a beam combiner 3514 where the beams from the kill laser module 3522 and the detection laser module 3532 are combined and projected towards a focal plane. The radiation from the kill laser assembly 3520 and the detection laser assembly 3530 may or may not be perfectly combined, i.e., the radiation may not be perfectly coincident at the focal plane 3552, depending on the flow cytometer and particle processing task in which the assemblies are used.

[0242] Optical paths for a detection laser assembly (e.g., detection laser assemblies 3430 and 3530) and a kill laser assembly (e.g., kill laser assemblies 3420 and 3520) may be used with systems 3500 and 3400, and are provided in the exemplary embodiment shown in Figures 26A and 26B, respectively. In the exemplary embodiment, the laser-based particle detection and deactivation system includes a detection laser assembly having an optical path 3600, shown in Figure 26A, and a kill laser assembly having an optical path 3700, shown in Figure 26B. The optical path 3600 for the detection laser includes a detection laser module 3602, a first harmonic separator 3604, a second harmonic separator 3606, an F150 cylindrical lens 3608, an F25 cylindrical lens 3610, a polarizing beam splitter 3612, and terminates at a focal plane or a microfluidic chip.

[0243] The optical path 3700 for the kill laser assembly includes a kill laser module 3702, a first harmonic separator 3704, an F50 cylindrical lens 3706, an F25 cylindrical lens 3708, a wave plate 3710, a second harmonic separator 3712, an F-25 cylindrical lens 3714, an F150 cylindrical lens 3716, a third harmonic separator 3718, an aspheric lens 3720, a polarizing beam splitter 3722, and terminates at a focal plane or a microfluidic chip.

[0244] Without wishing to limit the invention to a particular application or embodiment, the improved systems and optical paths of the present invention, including opto-mechanical system 3400 shown in FIG. 25A, opto-mechanical system 3500 shown in FIG. 25B, and optical paths 3600 and 3700 shown in FIG. 26A and FIG. 26B, respectively, improve upon existing systems through the incorporation of spherical lenses in at least the kill laser beam path, and through the use of polarizing beam splitters to combine radiation from the kill laser module and the detection laser module. Specifically, the use of aspheric lenses in configurations such as those shown in the exemplary embodiments provides for shaping the beam radiation from higher pulse energy lasers without risk of damage to optical elements, including the aspheric lenses. The aspheric lenses further provide for precise adjustment and / or calibration of the beam radiation to obtain optimal beam shape and size at the focal point. The use of such optical components further provides for independent adjustment of beam waist height and width for beam radiation at the focal plane. Additionally, in example embodiments, a polarizing beam splitter may be used to combine the beam radiation from the kill laser module and the detection laser module, where both beam radiations are at or near a common wavelength and the radiation of both modules is polarized. Other methods for combining beam radiation, such as dichroic mirrors, may not be suitable or optimal in a flow cytometer system having the kill laser module and the detection laser module positioned on a common side of the flow cytometer and on opposite sides of the detection module or assembly. This type of example configuration, with the unusual use of a polarizing beam splitter, achieves superior results compared to other beam combining systems or methods.

[0245] 28, a diagram of radiation from a detection spot 4001 and a kill spot 4003 is provided. Detection radiation 4014 is generated at detection spot 4001 at an action or interrogation location in focal plane 4002, and kill radiation 4012 is generated at kill spot 4003 in focal plane 4002. Optical elements of an optical path, such as those shown in FIG. 27, shape and direct both detection radiation 4014 and kill radiation 4012 so that both can be separately analyzed, detected, and / or captured by one or more sensors or devices, such as system 4400 provided in FIG. 27.

[0246] 27, a diagram of the optical, opto-mechanical and electrical components of a detection assembly 4400 according to one embodiment of the present invention is provided. The detection assembly 4400 includes a filter 4404, which may be a high pass filter, an objective lens 4406, which may be a NUV 20x objective lens, a tube lens 4408, which may be a 100 mm or 200 mm tube lens, and one or more filters or splitters, such as a dichroic mirror 4420 and a splitter 4422, to filter, split and / or redirect electromagnetic radiation emissions either from detection laser excitation of a particle (detection radiation 4414) or from kill laser excitation of a particle (kill radiation 4412). The detection radiation 4414 occurs at a detection spot at an action or interrogation location of the focal plane 4402, and the kill radiation 4412 occurs at a kill spot of the focal plane 4402. At least the filter 4404, objective lens 4406 and tube lens 4408 shape and direct both the detection radiation 4414 and the kill radiation 4412 so that both can be separately analyzed, detected and / or captured by one or more sensors or devices such as a CCD camera 4430, a kill avalanche photodiode ("APD") or a kill detector 4432 and a detector 4434. The CCD camera 4430 is used to capture images of the radiation 4412 and 4414, which may be used in determining the type or quality of the radiation, such as determining the amount of fluorescence of a particle caused by the electromagnetic radiation emission of the kill laser or the detection laser. The dichroic mirror 4420 is used to reflect a portion or a very small amount of the radiation 4412 and 4414 towards the CCD camera 4430 and allow the remainder of the radiation to pass through towards the kill detector 4432 and the detector 4434. Kill detector 4432 is used to detect whether a kill event by a kill laser to inactivate a particle is successful. Detector 4434 is used to determine the type and quality of the particle's emission, such as in particle emission 4414, and to determine the quality or characteristics of the particle, such as the DNA content of a sperm cell, based on the determined level of cell fluorescence.

[0247] "Example 1"

[0248] While not wishing to limit the invention to any particular application or embodiment, the following illustrative embodiment is described.

[0249] The flow cytometry particle sorting system includes a detection laser assembly, a microfluidic chip, a detection sensor, a set of signal processing electronics, a graphical user interface, a CCD camera, a kill laser module, and a kill sensor. The detection laser assembly is configured to excite a dye, such as a Hoechst dye, within the particles, which may be individual sperm cells. The beam from the detection laser is focused to a specified size, such as an ellipse having a fast axis of 3.5-20 μm and a slow axis of 30-150 μm. The microfluidic chip accepts a particle sample, such as an ejaculated sample containing sperm, and focuses or orients the particles using a sheath fluid within one or more microchannels in the chip. The detection sensor collects radiation at the detection laser location. The signal processing electronics may include digital electronic components including a processor and memory, or may include analog electronic components, or a combination of digital and analog processing components. The signal processing electronics receives one or more signals from one or more sensors or cameras, converts or interprets the signals, and makes a decision based on the input signals. The determination may be a sex or gender determination of the sperm cells based on the fluorescence detected based on the amount of DNA in the cells. A graphical user interface ("GUI") displays information regarding the status of the system including processing rate, laser pulse energy, kill rate, detection rate, fluid levels, sample collection levels, and further provides one or more user interface elements for configuring system elements such as laser pulse energy, flow rate, temperature, pressure, and desired sample distortion. A CCD camera captures images of one or both of the kill and detection locations and provides feedback via the GUI for user alignment of the optical and opto-mechanical elements of the system. The kill laser kills or inactivates the undesired particles. The kill laser radiation is focused to a small location or spot to effectively slice, ablate, photodamage, or otherwise kill particles such as sperm cells. The focused laser radiation may be an elliptical profile shape with a fast axis of 3.5-5 μm and a slow axis of 5-35 μm.

[0250] The detection laser assembly includes a CW laser module, a laser shutter, a set of one or more harmonic separators, a horizontally oriented F150 cylindrical lens, a vertically oriented F25 cylindrical lens, and a polarizing beam splitter. The CW laser module emits a CW laser beam that is used to cause the particles to fluoresce. The laser shutter prevents the laser from reaching the user's access point when the safety switch is activated. The harmonic separator is used to reflect 355 nm light while transmitting other wavelengths of light, removing the 352 nm and 1064 nm carrier wavelengths from the laser beam. The F150 cylindrical lens shapes the horizontal dimension of the detection laser at the tip or focal plane, and is disposed on an adjustable base or stage that allows the beam position to be adjusted horizontally. The F25 cylindrical lens shapes the vertical dimension of the detection laser at the tip or focal plane, and is disposed on a base or stage that allows the beam position to be adjusted vertically. The polarizing beam splitter reflects S-polarized light while transmitting P-polarized laser light, allowing the detection laser beam to be transmitted through the beam splitter onto the chip or focal plane.

[0251] The kill laser assembly includes a kill laser module, one or more sets of UV mirrors, a vertically oriented F-25 cylindrical lens, a vertically oriented F+150 cylindrical lens, an F25 aspheric lens, and a polarizing beam splitter. The kill laser module is a pulsed laser module that emits a pulsed laser beam at 1-5 μJ. The one or more sets of UV lenses each reflect the laser beam 90 degrees and are mounted on a tilt / tilt mount for steering or directing the kill laser beam. The F-25 cylindrical lens is used in conjunction with the F+150 cylindrical lens to expand the kill laser beam vertically. The height of the kill laser at the tip or focal plane can be controlled by adjusting the separation of the F-25 cylindrical lens and the F+150 cylindrical lens. F-25 and F+150 cylindrical lenses can expand the kill laser beam from 1mm to 6mm. F25 aspheric lenses may be used to focus the kill laser beam on all axes to a small elliptical spot on the focal plane of the microfluidic chip. Aspheric lenses reduce spherical aberration compared to standard or common spherical lenses.

[0252] The cell detection assembly includes an objective lens, a 390 long pass filter, a first F100 spherical tube lens, a 90:10 beam splitter, a detection sensor, a kill sensor, a second F100 spherical tube lens, and a CCD camera. The objective lens collects dye emission from both the detection and kill locations at the focal plane of a fluid path in a flow cytometry device (e.g., a microchannel in a microfluidic chip), and further collects a portion of the laser wavelengths from the kill laser and the detection laser to measure the size of the laser spot. The 390 long pass filter transmits wavelengths longer than 390 nm and reflects wavelengths less than 390 nm, reflecting a portion of the light toward the CCD camera and allowing the remaining light to transmit to the kill sensor and the detection sensor. The 90:10 beam splitter transmits 90% of all light, reflects 10%, transmits 90% of the light that passes through the 390 long pass filter to the detect sensor, and reflects 10% of the light to the kill sensor. The detect sensor is an avalanche photodiode that collects the light emitted at the detect location. The kill sensor is an avalanche photodiode that collects the light emitted at the kill location. A first F100 spherical lens focuses the light collected from the objective onto the detect sensor and kill sensor, and a second F100 spherical lens focuses a portion of the light collected from the objective as reflected by the 390 long pass filter onto the CCD camera. The CCD camera images the light collected by the objective and is a source of user feedback for system alignment as well as measurement of the size of the laser spot.

[0253] "Example 2"

[0254] While not wishing to limit the invention to any particular application or embodiment, the following illustrative embodiment is described.

[0255] Typically, pulsed lasers are used for fluorescent dyes in particles in flow cytometry systems. For example, existing systems for sperm sexing use pulsed lasers to fluoresce Hoechst dyes, such as Hoechst 33324, in sperm cells to determine the amount and therefore type of DNA in the sperm cells in the sexing process. However, the use of CW lasers offers advantages in efficiency, calibration, and adjustment compared to conventional pulsed lasers. The use of CW lasers in the present invention offers the above improvements over existing pulsed detection lasers and detection laser assemblies while maintaining the same fertility or viability of sperm cells sorted in this manner within a statistically significant margin of error. The fertility rate, as a measure of fertility, of cells irradiated by CW lasers was at least as good, if not better in some circumstances, than cells irradiated with a pulsed laser. In particular, the difference in fertility was about 1%, within the margin of error of measurement. Thus, the use of CW lasers in particle detection does not measurably affect the viability or fertility of sperm cells treated using CW lasers compared to the control.

[0256] "Example 3"

[0257] While not wishing to limit the invention to any particular application or embodiment, the following illustrative embodiment is described.

[0258] The laser-based particle detection and inactivation system comprises a kill laser assembly and a detection laser assembly. The kill laser assembly may operate at a power of 350mW to 2W. The power of the detection laser assembly may be set from 10mW to 50mW. The power output by the detection laser assembly may be subject to approximately 10% power loss and a measured power drift of less than + / - 1% over a 24-hour period. Accurate and stable detection laser power may be achieved by increasing the laser power for the first minute of operation and then decreasing the laser power to provide stable detection power at an elevated temperature of approximately 4 degrees Celsius.

[0259] 29A and 29B, there are shown respective diagrams of representative graphical user interface outputs measuring the size and intensity of the detection laser beam at the focal plane (FIG. 29A) and the size and intensity of the kill laser beam at the focal plane (FIG. 29B). The outputs shown in FIG. 29A and FIG. 29B may be achieved using the laser module power settings described in Example 3 herein above. The major axis of the detection laser ellipse is between 95 and 110 μm, and the minor axis of the ellipse is between 12 and 15 μm. Specifically, as shown in FIG. 29A, the major or slow axis is 104.2 μm, and the minor or fast axis is 11.2 μm. The major axis of the kill laser ellipse is between 20 and 25 μm, and the minor axis of the ellipse is between 2 and 4 μm. Specifically, as shown in FIG. 29B, the major or slow axis is 20.9 μm, and the minor or fast axis is 3.45 μm. However, the placement and positioning of the optical elements may be adjusted or calibrated, and the pulse energy for the laser module may be adjusted to shape the beam emission as desired.

[0260] "Example 4"

[0261] While not wishing to limit the invention to any particular application or embodiment, the following illustrative embodiment is described.

[0262] The laser-based particle detection and inactivation system comprises a kill laser assembly and a detection laser assembly. The detection laser assembly comprises an annular light emitting diode ("LED") and a power sensor. The annular LED has an inner diameter of 6 mm and is positioned between a set of cylindrical lenses in the optical path of the detection laser. The power sensor has TO-5 packaging and is disposed on a printed circuit board. The power sensor is positioned in or adjacent to the optical path of the detection laser, such as by a polarizing beam splitter, and detects the power of the detection laser beam. This is used to continuously monitor the output power and provide feedback to an operator. The detected power may also be used in a feedback loop to automatically or semi-automatically adjust the output power.

[0263] "Example 5"

[0264] The following are non-limiting examples of how to determine particle velocity in a sample stream within a microfluidic chip and can be used for kill-trigger delay timing, flow rate adjustment, etc.

[0265] For cell velocity calibration, the cell velocity is calculated as follows: Speed ​​= Detection Spot Y - Kill Spot Y / Trigger Delay

[0266] If the current speed deviates significantly from the target speed, a new flow rate is calculated using the following equation: (Flow1 / Velocity1)=(Flow2 / Velocity2) In this formula, Flow1 and Rate1 are the current values, Rate2 is the target rate, and Flow2 is the new flow rate.

[0267] The cell rate (cells / sec), which is equal to events / sec, is a fluidic parameter that is desired to be kept constant. The flow cytometer tracks the number of cells or events reported by the detector in the last second. This fluidic parameter is controlled through a pressure regulator in the fluidics module, so if the cells / sec is too low, the pressure increases, and if the cells / sec is too high, the pressure decreases. Pressure is applied to the sample, so the higher the pressure, the more of the sample is forced through the flow cytometer. The sample is mixed with a sheath fluid, which is kept at a constant flow rate by a PID controller operating in the fluidics module. In some embodiments, the sheath flow is controlled by the opening and closing of a valve, such as a pressure regulator.

[0268] Alignment Procedure

[0269] Existing systems require an operator to align the kill laser to the stream of cell cores to effectively kill the targeted cells. The operator aligns the stream of cores to the laser in three dimensions using a two-camera system, with one camera monitoring the cell location and another monitoring the kill laser. Each spot is given a centroid location in the image. The positional relationship between the spots is then maintained during collection. Alignment is achieved using the image, a two-axis stage, and a time delay. The two-axis stage allows alignment at the center of the laser, and the time delay allows the laser to target the cells as they pass through the microfluidic channel. Focusing the stream of cores at a focal point concentrates most of the laser energy on the cells, resulting in a high purity product. After alignment, the operator gates on the appropriate population, and cells outside of that population are targeted by the kill laser. Problems that arise during operation include laser drift and cell velocity drift that cause misalignment of the system where targeted cells are not effectively killed or missed. The operator must identify and correct the misalignment.

[0270] However, operators cannot be trusted to identify and correct misalignments with the required frequency to maintain product purity. Additionally, operators cannot correct for drift between the cameras, i.e., both measurement devices themselves drift, and measurement accuracy was not precise enough to maintain alignment within tolerances. Automating the alignment process reduces labor costs, improves product consistency, and increases product quality by eliminating operator dependency.

[0271] Thus, in some embodiments, the invention features automatic alignment correction by measuring the misalignment and incrementally correcting the stage position or delay until the misalignment is within an acceptable range.

[0272] Referring to FIG. 30, a procedure for aligning the microfluidic chip stage with the detection laser and kill laser to achieve a desired resolution is shown. First, the microfluidic chip stage is aligned with the detection laser to achieve the desired detection resolution. Next, the position of the microfluidic chip stage is adjusted so that the kill laser is aligned with the center of the sample stream, thereby achieving the desired resolution. If the kill count is below the kill count threshold, the microfluidic chip stage is repositioned relative to the kill laser until the kill count threshold is reached or exceeded.

[0273] Figure 31 shows the procedure for aligning the instrument by adjusting the position of the core stream relative to the kill laser and measuring the properties of the fluorescence produced by the kill laser and the cell core stream. During initial alignment, the operator optimizes the position of the core stream relative to the core laser. This spot in the 2D image space is marked with crosshairs superimposed on the image. The orange crosshairs are fixed and used as a position to maintain during collection. The red crosshairs are a measurement of the activity of the spot on the image. If the spot moves, the red crosshairs follow but the orange crosshairs do not.

[0274] In some embodiments, a left-right adjustment may be required if the core flow has deviated left or right from its original position. The distance between the crosshairs is measured and the tip / core flow is moved left or right until the distance between the crosshairs is zero. In other embodiments, the focus may be optimized by sweeping in one direction from one extreme until a zero slope condition is met in the focus optimization metric.

[0275] In some embodiments, delay adjustments may be required if the cell speed or the distance between the detection laser and the kill laser changes. After left-right and focus optimization, the location of the spot on the image may be marked as the target location. The cell fires and the current location of said cell in the image is measured relative to the target location. After measuring the distance between the crosshairs, the delay is adjusted until the current location relative to the target location is zero. For example, the delay is decreased or increased accordingly until the distance between the crosshairs is zero.

[0276] While not wishing to limit the invention to any particular theory or mechanism, the alignment procedure had the advantage that no additional hardware was required to measure the laser position. Furthermore, the system was optically stable and the laser only moved 0-1 μm during collection. This allowed the initial alignment to be maintained even after validation without the need to measure the location of the moving kill laser or to frequently realign to maintain product quality.

[0277] Detection of depleted samples

[0278] Semen processing requires making several samples over the time of operation to maintain the vitality of the semen. Thus, a volume of 3-5 mL is processed at a time, and a newly made sample is replaced when the previous sample runs out. By visually checking the level of the sample tube, the operator identified when the sample was low and determined when it should be replaced. This could lead to complete sample depletion, resulting in extended downtime.

[0279] The present invention therefore solves this problem by providing an indication of when the sample is depleted before it is completely depleted, helping to reduce operator burden and instrument downtime. The uniqueness of this solution is in monitoring the controller output as a means of detecting known change patterns.

[0280] Referring to Figure 32, a sample is forced from a 5 mL container through a small diameter tubing by applying upward pressure on the fluid. Since the sample in the tubing is assumed to be of uniform concentration, the rate at which cells leave the container (or cell rate) is proportional to the applied pressure. As pressure increases, the cell rate increases. Ideally, the cell rate is held constant to maintain constant instrument performance. Due to local variations in concentration as well as downstream pressure, the pressure may need to be adjusted slightly to keep the cell rate on target.

[0281] In reality, the concentration does not remain uniform throughout the duration of the sample. Sedimentation occurs and cells fall towards the bottom, leaving the lower part of the sample more concentrated than the top. As the fluid level decreases, the local concentration near the tube outlet decreases continuously. To maintain a constant throughput, an increasing pressure is required. This pressure increases exponentially as the sample begins to deplete. The method of the present invention requires monitoring this change in pressure during collection. The operator is then notified when the change in pressure exceeds a threshold value.

[0282] As shown in Figure 32, the sample vessel contains sperm cells in solution. A pressure regulator receives a constant pressure from a source. The pressure set point is received from the PC / GUI. Pressure is output to the sample tube containing the sperm cells and applied above the fluid level. Small diameter tubing is placed inside the sample vessel with the end of the tubing near the bottom of the vessel. The other end of the tubing is inserted into a microfluidic chip that is used to focus the cells in a flow cytometer for measurement of cell rate. The cells are driven out of the vessel through the small diameter tubing as pressure is applied. The flow cytometer measures the cell rate and sex. The cell rate is input into a feedback loop to maintain a constant cell rate.

[0283] The feedback controller may be a PID-based controller that uses cell rate as an input and pressure as an output. The objective of the PID controller is to maintain a constant cell rate, which may be user specified. If the constant cell rate deviates from the baseline, e.g., the cell rate decreases, the feedback controller increases the pressure output from the pressure regulator into the sample vessel to maintain a constant cell rate. If the pressure exceeds the pressure set point, this indicates that the amount of sample in the vessel is low, and the operator is notified of the low level.

[0284] The above solution has the advantage that no additional hardware is required to determine the sample fluid level. However, in alternative embodiments, the present invention may use one or more additional components, such as a liquid level sensor, to measure the level directly. Examples of liquid level sensors are described herein.

[0285] Particle Processing

[0286] 33, a sample, which may be a semen sample containing sperm cells, enters the microfluidic chip 850 through a sample passage, and sheath fluid enters the chip through one or more sheath fluid passages. The sample and sheath fluid may be pumped into the chip by a fluid pump, a syringe pump, or other suitable pumping means at a rate and pressure suitable for the individual sample.

[0287] In some embodiments, one or more electromagnetic radiation emitters are directed at or to the chip using an optical path. The laser detection module may be based on a continuous wave laser and may be configured to cause fluorescence emission in a dye injected into particles or components in the sample. The laser kill module may be based on a pulsed or micro-pulsed laser and may be configured to inactivate, such as by photoablation, one or more particles or components in the sample based on a characteristic of the sample detected by the laser detection module. The detection module collects or detects radiation from the particles or components in the sample to identify one or more characteristics of the particles or components, such as the amount or type of DNA, and provides feedback to the system and / or operator. The detection module may comprise one or more of a camera, a photomultiplier tube, or an avalanche photodiode-based detector. Based on the detected characteristic, the kill laser in the emission module emits a kill laser beam to inactivate or destroy a subset of particles or components in the sample.

[0288] The processed sample exits the tip 850 through one or more openings at the end of the tip. The auto-dump assembly 5160 comprises an actuator and a mechanical diverter. The actuator may be a two-position linear actuator or other suitable mechanical or electromechanical actuation means. The actuator operates to move the diverter in or out of the path of the processed sample exiting the tip to either allow the sample to pass into the collection tube 5130 or to divert the sample into a waste collection container or area. The determination is based on one or more qualities of the sample, such as purity, or based on operator input. The determination may be based on whether a sample tube is present in the sample collection path.

[0289] In some embodiments, the sample mixing assembly or module 5100 comprises one or more sample collection containers or tubes 5130 held in one or more collection tube holders 5120 that are integral to or secured to a rotating base 5110. The rotating base 5110 is magnetically secured to a platform 5360 or held by a bolt or threaded cap. The rotating base 5110 is rotated, oscillated, or moved by an actuator or motor 5340, which may be a stepper motor, linear actuator, brushless motor, or other suitable drive means capable of rotating the rotating base 5110. The rotating base 5110 rotates in an arcuate path to mix samples collected in the sample collection tubes 5130 or to rotate a sample collection tube on the base into a sample collection path.

[0290] Although optical means of detecting the sample have been described, it will be understood that other detection means may be used in the modular flow cytometry system of the present invention. Alternatively, or in addition to the above embodiments of detecting the sample, the sample may be detected using electrodes, a camera, a magnet, electroporation, impedance, or conductivity.

[0291] Although a kill laser has been described for damaging the sample, it will be understood that other means for damaging cells may be used in the modular flow cytometry system of the present invention. Alternatively or in conjunction with the embodiments relating to damaging cells, cells may be inactivated or killed using physical deformation or electrical means such as the use of electrodes or electroporation.

[0292] In other alternative embodiments, the sexed sperm cells may be separated into two or more subpopulations using separation means including, but not limited to, physical barriers, droplet separation, acoustic means, laser steering, electrodes, or combinations thereof.

[0293] Automated Mixing and Collection Module

[0294] 34, the present invention features a sample mixing system 5100 that includes a rotating base 5110, a plurality of collection tube holders 5120 disposed on top of the rotating base, and a plurality of collection tubes 5130. Each tube is configured to fit and be disposed in the collection tube holders of the plurality of collection tubes. Without wishing to limit the present invention to a particular theory or mechanism, the rotating base 5110 can be rotated such that the collection tube holders 5120 and collection tubes 5130 disposed therein rotate or move in an arcuate path, and the contents in the collection tubes 5130 are mixed by said motion.

[0295] In some embodiments, the rotating base 5110 can rotate back and forth on the platform 5360 on which it is disposed. The rotating base 5110 can be operatively coupled to a motor 5340 that rotates said rotating base 5110 about its central axis. The rotating base 5110 can rotate about its central axis in a first direction, a second direction, or both directions. For example, the rotating base 5110 can rotate in a first direction, a second direction, or both directions to change from an "in use" collection tube to the next or adjacent "standby" collection tube. For switching purposes, in some embodiments, the rotating base 5110 can rotate from about 45° to 360°.

[0296] In some embodiments, the motor is a stepper motor that is used to automatically induce mixing of the sample tube back and forth in an arc. From other mixing solutions, this is a pivot-based motion. The motor moves along an arc, which allows the collection tube 5130 to remain in the drip path below the microfluidic module. In some embodiments, for mixing purposes, the rotating base 5110 can rotate approximately 10° to 40°.

[0297] In other embodiments, the rotating base 5110 can move in an orbital path to induce mixing of the sample tubes in a pivoting motion similar to the motion of an orbital shaker, i.e., orbital movement. Alternatively or in conjunction with axial rotation, the axis of the rotating base can move along a circular path having a minor axis such that the collection tube 5130 also moves in a circular path while remaining below the drip path of the microfluidic module.

[0298] In some preferred embodiments, the mixing technique allows for gentle agitation or mixing of the collection tube. In some embodiments, the mixing technique is performed periodically. For example, the collection tube is mixed periodically for a duration. For example, the collection tube is mixed about every 3-8 minutes for a duration ranging from about 10-120 seconds. In some embodiments, the system 5100 allows for automatic switching from an "in use" collection tube to an "on standby" collection tube while focusing on the primary task of mixing the contents therein. In some embodiments, the design is optimized such that the mixing device is configured to fit within the space provided within the modular flow cytometry system.

[0299] In one embodiment, a mixing profile for the stepper motor 5340 may include + / - 50 steps of left, back to center, and right motion out of a possible 1600 steps. In a non-limiting example, for example, a mixing profile with + / - 50 steps for each of the left, back to center, and right motion may include an arc of + / - 11.25 degrees for a total arc of 22.5 degrees. However, this range may be increased or decreased based on the size of the sample output stream, the size of the sample collection vessel or tube, and the degree of mixing required. For example, a stronger mixing profile may be obtained using smaller steps that cause shorter, more rapid movements. This causes an impulse type motion to the stirred or mixed sample instead of a rocking motion. In another example, a mixing profile having more than 50 steps of motion in each direction and more than 22.5 degrees of arc motion may be desired or required for either larger sample vessels, more viscous samples, samples that require more thorough or uniform mixing, or larger sample sizes. Additionally, other means for rotating the rotating base 5110 may be used to rotate or move the base, such as a linear actuator that rotates the base or moves the base in a reciprocating motion, a brushed or brushless electric motor that rotates or moves the base, a fluid actuator such as a pneumatic actuator that rotates or moves the base, or a piezoelectric actuator that agitates or moves the base.

[0300] The specified configuration of the mixing profile may be configured manually by a user and be unique for each individual sample being processed, or the specified configuration of the mixing profile may be determined automatically based on the type of sample. For example, a code read from an information storage means, such as a barcode, QR code, or RFID tag, may be read from the sample container by a machine and used to configure, retrieve, or otherwise identify a set of configuration information used to define a mixing profile for the sample. The information storage means may contain the mixing profile information or may include a location, pointer, or address where such information is found. The mixing profile entered into or read by the system of the present invention is used to automatically mix, stir, or agitate samples processed in the sample mixer according to the mixing profile with or without minimal additional input or action from a user or operator.

[0301] In some embodiments, the rotating base 5110 is a disk. Referring to FIG. 35A, the rotating base 5110 may include magnets 5112 that are positioned separately from one another. Each collection tube holder's base 5122 also includes a magnet 5124. The collection tube holder 5120 may be attached to the rotating base 5110 by magnetic attraction between the collection tube holder's magnet 5124 and one of the rotating base's magnets 5112. In another embodiment, instead of or in addition to the use of the magnet 5124, a threaded nut or knob may be used to secure the rotating base 5110 to a corresponding threaded rod or bolt.

[0302] In some embodiments, the system may include between about two and six collection tube holders 5120. The number and location of the rotating base magnets 5112 may be determined by the number of collection tube holders. For example, if the system includes two collection tube holders 5120, the tube holders are diametrically opposed or 180° apart. With four collection tube holders 5120, the arrangement may be 90° apart, and with six collection tube holders 5120, the arrangement may be 60° apart.

[0303] In other embodiments, the collection tube holder may include two or more arms 5126 projecting upward from the base 5122. The two or more arms 5126 are configured to hold the collection tube 5130 upright. In some embodiments, the tube holder base may be circular or square. In a non-limiting example, FIG. 34 shows a collection tube holder with two arms 5126 disposed on either side of the tube holder base 5122 and projecting upward therefrom. Another embodiment may include four arms 5126 equidistant from each other, for example 90° apart, or at the corners of a square base. In one embodiment, the arms 5126 may be connected to an edge of the tube holder base 5122, or the arms 5126 may be attached to the top surface of the tube holder base 5122. In another embodiment, the arms 5126 may be curved when viewed from above and straight when viewed from the side. Alternatively, the arm 5126 can be straight when viewed from above and from the side. In yet other embodiments, the collection tube holder 5120 may comprise a round or square tube with fully enclosed sides. In another embodiment, the collection tube holder may comprise at least one arm or protrusion having a fastening means for fastening a collection tube or container to the collection tube holder. The fastening means may be a friction fit opening, a set of magnets, a magnet and an element of magnetic receptive material, a metal or plastic clip or fastener, a hook and loop fastener, or a retaining ring or collar.

[0304] In a preferred embodiment, the two or more arms 5126 are expandable to accommodate collection tubes 5130 of various sizes. In one embodiment, the volume capacity of the collection tubes ranges from about 50 ml to about 300 ml. For example, the collection tubes may have a volume of 50 ml or 250 ml. In another embodiment, the diameter of the collection tubes may range from about 100 mm to about 200 mm. In another embodiment, the diameter of the collection tubes may range from about 50 mm to about 150 mm. The height of the collection tubes may range from about 150 mm to about 250 mm. In other embodiments, the height of the tube holder may be shorter than the collection tubes. The height of the tube holder may range from 50 mm to 100 mm. However, any suitable collection tube or container may be used, secured to a rotatable or movable substrate by any suitable means as described herein.

[0305] In some embodiments, the sample mixing system 5100 may further comprise one or more Hall effect sensors. In a non-limiting example, as shown in FIG. 35B, one of the magnets 5112 in the rotating base 5110 may be oriented with its south pole oriented toward one or more Hall effect sensors, and the other magnets 5112 may be oriented with their north poles directed toward one or more Hall effect sensors. The north ends or north poles of the magnets 5112 at the top and bottom of the base 5110 are configured to hold or clip a sample tube loader in place on the base 5110. The sample tube loader holds and positions the sample collection tube or container at the magnet's position. In another embodiment, one or more sample tube holders are integrated into the base 5110. In another embodiment, the sample tube holders are clipped by a press fit into a corresponding opening on the base, one or more plastic clips, a spring-loaded clamp, or one or more fasteners such as screws, bolts, or rivets. In one embodiment, a magnet 5112 having a south or south pole disposed on the top of the base 5110 is used to track or return to a return position or position 0. A Hall effect sensor may be used to detect or identify this magnet to determine position 0, after which the base 5110 may be rotated or turned to a collection position. Other sensors such as an optical sensor and corresponding markings or physical features on the base 5110, an electrical switch, a conductive area on the base 5110 and a corresponding conductive area at a fixed position, a mechanical switch, a hard stop such as a physical lip, edge or engagement tab on the base 5110, or an absolute encoder may be used in place of a Hall effect sensor and corresponding magnet to track or return to a return position or position 0. Any of these sensor types may be used to identify the return position or position 0 and move the base 5110 to a specified or set position.

[0306] According to some embodiments, the sample mixing system 5100 may be implemented in a method for mixing a sample. For example, the present invention may be used in the processing of semen samples, e.g., mixing sperm cells. When used in such a sample processing system, the sample mixing system 5100 may be a component of a flow cytometry system, such as one that employs a microfluidic chip and one or more optical components in the processing of components or particles in the sample. This may be, for example, the determination of a characteristic of the particle, such as the amount or type of DNA in a sperm cell. The sample thus processed by the flow cytometry system may exit the processing area and be collected in a sample collection container or tube, and may be collected with additional media or fluids required to keep the sample in suspension, such as a homogenously mixed suspension.

[0307] As shown in FIG. 36A and FIG. 36B, in one embodiment, the system 5100 can be placed under a dispensing device. The dispensing device can dispense a sample into one of the collection tubes 5130 as the rotating base moves through the arcuate path. In a preferred embodiment, the collection tube 5130 that receives the sample remains under the dispensing path of the dispensing device as said collection tube 5130 moves through the arcuate path, thereby mixing the sample in the collection tube 5130. In a non-limiting example, the sample can include sperm cells and the collection tube 5130 can include a medium. While not wishing to limit the invention to a particular theory or mechanism, the method can reduce the time that the cells are in an overconcentrated solution of the medium. For example, the medium can be a buffered medium that is in the tube before adding the sample or that is added with the sample. The medium is mixed or agitated to maintain buffering and control the degree of buffering.

[0308] In some embodiments, the sample may be dropped into a collection tube. In other embodiments, the sample may be continuously dispensed into the collection tube. A fluid pump, such as a syringe pump or other suitable hydraulic or pneumatic pump, may be used to force the sample through a flow cytometer device, such as a microfluidic chip. The sample is inserted into the flow cytometer, processed by one or more systems or elements of the flow cytometer, and ejected from one or more outlets at the end of the flow cytometer. For example, in one embodiment, the sample and at least one sheath fluid are inserted into a microfluidic chip, the sample is processed in an action or interrogation region of the microfluidic chip, such as a set of electromagnetic radiation emitting devices (e.g., a laser module and associated optics), and the sample is ejected from one or more outlets at the end of the microfluidic chip. The processed sample is then dripped, dropped, or drained into a sample collection tube through one or more channels or openings by the action of fluid pressure and / or gravity.

[0309] After mixing the sample in one collection tube 5130, e.g., an "in use" tube, for a period of time, the system 5100 may automatically place another collection tube 5130, e.g., an "in use" tube, under the dispenser so that the sample may be dispensed into the collection tube 5130. In some embodiments, the time for mixing is about 3-8 minutes.

[0310] Liquid Level Sensor

[0311] In some embodiments, the sample mixing system includes a sensor that detects the liquid level inside the collection tube. The sensor may be located at or near the surface of the collection tube. For example, the sensor is located 1-10 mm from the surface. In one embodiment, FIGS. 38A and 38B show an example of a square capacitance sensor for liquid level detection. In another embodiment, FIGS. 39A and 39B show an example of a tubular capacitance sensor for liquid level detection. In another embodiment, the liquid level sensor is an infrared sensor.

[0312] Without wishing to be bound by any particular theory or mechanism, the liquid level sensor can detect when a desired level of fluid is reached in the collection tube. Once the liquid level is filled, the system 5100 automatically places another collection tube 5130 under the dispensing device, and the liquid level sensor then detects the liquid level inside this collection tube. This may be done, for example, by rotating the base 110 to place another collection tube under the sample collection location or sample dispense path. Once the first tube is determined to be filled to the desired level, or the sensor detects a certain level of fluid, e.g., fluid is splashed or raised to a certain point by mixing the sample fluid in the first collection tube, the first collection tube may be rotated out of the sample dispense path and the second tube rotated into the path. When there is no collection tube under the sample dispense path or at the sample dispense location, the sample being processed may be dumped or diverted into a waste collection location as described below.

[0313] Auto Dump Module

[0314] With reference to FIGS. 40A and 40B, in some embodiments, an auto-dump subsystem may be coupled with a sample mixing system. An "auto-dump" system includes a mechanical diverter configured to route a sample processed by a flow cytometer, such as a microfluidic chip-based system. The auto-dump mechanical diverter is used to automatically divert, redirect, or change the path of a processed sample based on a determined set of parameters. For example, the auto-dump mechanical diverter may be configured to automatically divert or redirect a processed sample based on a determination that the sample contains components or particles (e.g., cells, such as sperm cells) that were not properly processed by the flow cytometry system. In one embodiment, this may be one or more sperm cells in a semen sample that were not fully or properly inactivated by a laser-based "laser kill" system of the flow cytometer. If cells that should have been inactivated are present in the processed sample, the sample may be diverted into a waste collection location.

[0315] Without wishing to limit the invention, the mechanical diverter or auto dump is configured to prevent flow through the autonomous system into the collection tube at the discretion of the software. In some embodiments, the auto dump may include a cap that disperses sheath fluid away from the collection chute. The auto dump cap may be coated with a fluoropolymer such as CYTOP™. In other embodiments, fluid droplets cannot roll under the cap. In one embodiment, the cap may include an undercut to further ensure that fluid does not enter under the cap.

[0316] In one embodiment, a flow cytometry system, such as a microfluidic chip-based system, processes samples continuously. The entire processed sample is collected in a sample collection tube, and the sample is not diverted within the microfluidic chip to another collection location, such as a waste collection location. However, it may be desirable for the processed sample to be diverted after being released from an outlet in the microfluidic chip under certain circumstances. For example, if the processing components, such as the laser optical components, are not calibrated or misaligned, the sample being processed may contain undesired elements that should have been removed or inactivated by the processing system. This may be a subset of the sample that should have been removed or inactivated, such as cells in the sample that should have been removed or inactivated by the processing system. When this occurs, the improperly processed sample is mechanically diverted so that it is not collected in the sample collection tube. This prevents the processed sample containing the undesired characteristic from mixing with or contaminating the sample containing the desired characteristic.

[0317] This may be referred to as "filtering" or "not filtering". For example, the default position may be a normally closed mechanical diverter that "filters" all samples from the microfluidic chip unless a set of desired conditions are met. This may be when the sample only or mainly contains cells or particles with a particular characteristic, such as DNA moieties, or when a suitable sample collection tube is positioned to collect the processed sample. Once the desired conditions are met, the mechanical diverter is opened so as not to filter the processed sample. When the diverter is opened, the processed sample may be collected by a sample collection tube or the like. The filtered sample may be dumped or diverted into a waste collection location, such as a waste collection bin or a sample collection tube designated for waste collection.

[0318] The mechanical diverter may be operated by a linear actuator such as a two-position linear actuator, a solenoid, or a fluid-actuated rotation system such as a pneumatically actuated rotation system that includes a rotating cap with at least one opening through which the processed sample can be collected into a sample collection tube.

[0319] In some embodiments, the auto dump subsystem can accept all chemicals used in the process. The subsystem can also determine if the collection chute is open or closed. In other embodiments, the subsystem can be disassembled and reassembled for maintenance inside the equipment. For example, two screws, circled areas in FIG. 40A, can be removed to remove the actuator of the auto dump subsystem 5160.

[0320] While not wishing to limit the invention to a particular application or embodiment, the systems, devices, and methods of the present invention may be used in conjunction with particle processing systems, such as the sperm sexing system demonstrated in FIG. 41. In one embodiment, the sperm sexing system may comprise a flow cytometry apparatus or a microfluidic chip or device comprising a flow chamber configured to direct a flow of fluid containing sample particles through a particle interrogation location. The sample particles may be cells, such as sperm, that have been stained or otherwise treated prior to entering the flow cytometry apparatus or microfluidic device. The flow cytometry device may further comprise a laser configured to emit electromagnetic radiation along a beam path to the particle interrogation location, and a detector configured to detect the radiation from the particles. Following detection, the particles may be sorted or destroyed, such as by ablation with electromagnetic radiation, before or after exiting the flow cytometry apparatus or microfluidic device. The sample mixing system of the present invention may be used to mix the collected particle and fluid samples following the interrogation, determination, and sorting or destruction processes.

[0321] The sample mixing system of the present invention may be used in conjunction with or in a method of analyzing particles, such as sperm cells, contained in a fluid stream as the particles flow through an interrogation location in a flow cytometry apparatus or microfluidic chip or device. Such a method includes illuminating the fluid stream and the particles contained therein using electromagnetic radiation emitted from a laser. A detector, such as an avalanche photodiode, detects the electromagnetic radiation emitted by the particles from the interrogation location, and a processor determines a characteristic of the particles in the fluid stream based in part on the signal from the detector. Following detection, the particles may be sorted or destroyed, such as by ablation or photodamage with electromagnetic radiation, before or after exiting the flow cytometry apparatus or microfluidic device. The sample mixing system of the present invention may be used to mix the particles and fluid samples collected following the interrogation, determination, and sorting or destruction processes.

[0322] The sample mixing system of the present invention may be used in conjunction with or in a method of assessing the amount of DNA in the nucleus of a sperm cell using a flow cytometry apparatus or a microfluidic chip or device. The method may include staining the DNA in the nucleus of the sperm cell and then illuminating the stained DNA in the nucleus of the sperm cell with an electromagnetic radiation device, such as a laser, as the sperm cell passes the laser in a corresponding region of the flow cytometry apparatus or microfluidic device. A detector, such as an avalanche photodiode, detects the fluorescence emanating from the illuminated and stained DNA in the nucleus of the sperm cell at a detection or interrogation location in the flow cytometry apparatus or microfluidic device. The method may further include distinguishing between X chromosome-bearing sperm cells and Y chromosome-bearing sperm cells by first determining the sex of the sperm cell using the detected amount of DNA in the nucleus of the sperm cell and then distinguishing between the plurality of sperm cells based on the sex determination. The sperm cell characteristic may include a corresponding amount of DNA in the nucleus of the sperm cell. The method may further include inactivating a given sperm cell based on the determined amount of DNA in the nucleus of the given sperm cell. The inactivation may involve photodamage or ablation of given sperm cells by emission of electromagnetic radiation.

[0323] In other embodiments, the method may further include forming a plurality of droplets having one of the carried sperm cells, differentially charging each of the droplets based on the gender-distinguishing characteristic of the sperm cell carried in the droplet, deflecting each of the droplets, and differentially collecting each of the droplets based on the gender-distinguishing characteristic of the sperm cell carried in the droplet. The method may further include collecting the sperm cells after interrogation, sorting, or inactivation as the sperm cells exit the microfluidic device. The sample mixing system of the present invention may be used to mix the particle and fluid samples collected following the interrogation, interrogation, and sorting or destruction processes.

[0324] Additional Automation Features

[0325] In some embodiments, in addition to automating sample routing using mechanical diverters or auto dumps, and automating the sample mixing process using a sample mixing system, additional elements of the flow cytometry-based particle processing system may be automated. For example, parameters related to the fluid pressure used with the sample, the amount, volume or velocity of the sheath fluid used, the power or duration of the laser module, or the desired characteristics to be identified in the particles, such as cells, may be automatically determined based on the sample type detected by the flow cytometry system or entered by the operator. In one embodiment, this may include scanning a tag or identifier on the sample by the operator. The tag or identifier may be a barcode, QR code, RFID tag, or similar information-encoding tag on the sample, but may also include a set of configuration information for the flow cytometry system, or may include an address, pointer, or link to a location where such information is stored. Based on the information scanned in the tag or identifier on the sample, the flow cytometry system may be automatically configured with the optimal parameters for the individual sample or sample type.

[0326] For example, semen samples from individual boss Taurus bulls may differ in consistency, sperm cell density, pH (e.g., acidity), fertility, or fragility and may require specific parameters for successful processing of the semen sample, e.g., identifying and inactivating sperm cells from within the sample that contain the amount or type of DNA, such as X chromosome containing sperm cells or Y chromosome containing sperm cells. The specific processing requirements may be stored in a tag or identifier on the sample or in a database record or location identified by the tag or identifier, and those processing requirements may include configuration requirements for the flow cytometry-based system.

[0327] The flow rates and / or pressures of the sample and sheath fluids, the chromosome types to be detected, the laser power and / or duration, the pH of the medium, the amount or type of additives to be automatically introduced by the system, and other parameters may be set and automatically performed by the system for that specified sample. In this manner, the sample may be processed under conditions that are ideal for the specified sample without requiring a human operator to piecemeal use this information to find, configure, or calibrate the flow cytometry system.

[0328] Working Example

[0329] The following examples are intended to be illustrative only and not limiting.

[0330] Example Set A:

[0331] "Example A1" 1. A modular flow cytometry system for processing samples, comprising: a support structure; - an electronics module; - a sample passage module; a fluidics module; a query module; - Sample mixing and collection module In a system comprising: - each module is arranged on the support structure and configured to be separately removed and reinstalled or replaced by another of the same module; system.

[0332] "Example A2" The system of Example A1, further comprising a microfluidic device cleaning module for cleaning the sample passage module.

[0333] "Example A3" 1. A modular flow cytometry system for processing samples, comprising: a support structure; - an electronics module comprising one or more computer processors and electronic ports; - a sample passage module comprising a microfluidic module having at least one microfluidic channel; - a fluidics module fluidly coupled to the sample path module to circulate one or more fluids through the sample path module; - an interrogation module operatively coupled to the electronics module, an interrogation module including a detector, as well as a detection laser and a kill laser for processing the sample flowing through the microfluidic chip; a sample mixing and collection module configured to collect and mix the processed sample exiting the microfluidic module; In a system comprising: - each of the modules is configured to be separately removed and reinstalled or replaced with another of the same module and disposed on the support structure for substantially continuous operation of the modular flow cytometry system; system.

[0334] "Example A4" The system according to embodiment A3, wherein the kill laser and the detection laser are disposed on a common side of the interrogation module.

[0335] "Example A5" The system of example A3 or example A4, wherein the sample mixing and collection module rotates about a pivot to move in an arcuate path, enabling mixing of the sample in a controlled, uniform motion.

[0336] "Example A6" 1. A modular flow cytometry system for sexing a sample, comprising: - A chassis divided into multiple levels; - an electronics module disposed on a first, lowest level of a plurality of levels of a chassis, the electronics module including one or more computer processors and a plurality of electronics ports; - a sample passage module disposed on a second level of the chassis, the sample passage module including a microfluidic module for holding a microfluidic chip; - a fluidics module disposed on a second level of the chassis, the fluidics module fluidly coupled to the sample pathway module to circulate one or more fluids through the sample pathway module; - an interrogation module disposed on a third level of the chassis and operably coupled to the electronics module, the interrogation module including a detection laser and a kill laser operably coupled to the detection assembly for processing a sample flowing through the microfluidic chip, the kill laser and the detection laser being disposed on a common side of the interrogation module with respect to each other; - a sample mixing and collection module configured to collect and automatically mix processed samples emerging from the microfluidic chip and disposed below the microfluidic module on the same second level of the chassis, the sample mixing and collection module rotating about a pivot to move in an arcuate path to enable mixing of the samples in a controlled and uniform motion; In a system comprising: - each module is configured to be independently removed and reinstalled or replaced with another of the same module for near continuous operation of the modular flow cytometry system; system.

[0337] "Example A7" A system described in any one of Examples A3 to A6, wherein the sample passage module further comprises a distribution block fluidly coupled to the microfluidic module, the distribution block delivering a sample to the microfluidic module through a first fluidic line and delivering a sheath fluid to the microfluidic module through a second fluidic line.

[0338] "Example A8" The system of example A7, wherein the distribution block comprises a fluidic manifold.

[0339] "Example A9" The system of example A7 or example A8, wherein the distribution block further comprises a sample tube loader.

[0340] "Example A10" The system of any one of Examples A3 to A9, further comprising a microfluidic device cleaning module.

[0341] "Example A11" Microfluidic Device Cleaning Module - a set of fluid reservoirs; a pump assembly; a distribution block interface comprising a distribution block fixing element and a distribution block adjustment element, the distribution block interface further comprising a set of fluid interfaces, at least one of the set of fluid interfaces being in fluid communication with at least one of the set of fluid reservoirs; - a microfluidic device interface comprising a microfluidic device fixing element and a microfluidic device alignment element, the microfluidic device interface further comprising a fluid interface in fluid communication with at least one of the set of fluid reservoirs; a microfluidic device comprising a cleaning module; - a distribution block interface adapted to secure a distribution block thereon, a microfluidic device interface adapted to secure a microfluidic module thereon, and a microfluidic device cleaning system adapted to clean the sample passage module circulating fluid through the sample passage module; The system described in Example A10.

[0342] "Example A12" The system of any one of embodiments A6 to A11, wherein the kill laser and the detection laser are each disposed on a common side relative to a main surface of the microfluidic chip.

[0343] "Example A13" The system of any one of embodiments A3-A12, wherein the kill laser and the detection laser are each adapted to emit a laser beam including an elliptical beam profile at a focal plane.

[0344] "Example A14" A sample mixing and collection module, - a rotating base; - a set of collection tube holders arranged on a rotating base; - a set of collection tubes, each configured to be disposed in a collection tube holder of the set of collection tubes; - Fluid level sensors and A system comprising: - one of the collection tubes is positioned under the microfluidic chip such that the processed sample is collected in said collection tube; - the sample mixing and collection module is configured to replace the collection tube with another collection tube to collect the processed sample when the fluid level sensor detects that the collection tube has reached a desired fill level; The system according to any one of the embodiments A1 to A13.

[0345] "Example A15" A system described in any one of Examples A1 to A14, wherein the sample mixing and collection module further comprises a mechanical diverter configured to prevent processed sample exiting the microfluidic module from entering the sample mixing and collection module.

[0346] "Example A16" A system described in any one of embodiments A1 to A15, wherein the interrogation module comprises a laser heat sink for the laser, and the laser heat sink is capable of drawing in air by natural convection to cool the interrogation module.

[0347] "Example A17" The system according to any one of Examples A1 to A16, wherein the sample is a semen sample comprising sperm cells.

[0348] "Example A18" A system described in any one of Examples A1 to A17, wherein the electronics module includes an electronics box with a removable lid for housing a computer processor and other electronic components, and the electronic components are removable and can be replaced or reconnected.

[0349] "Example A19" A system as described in Example A18, wherein the electronics box is arranged on rails for easy movement.

[0350] "Example A20" The system of any one of embodiments A1-A19, wherein the electronics module includes one or more fans for cooling the electronic components.

[0351] "Example A21" The system of any one of embodiments A1-A20, wherein the interrogation component of the interrogation module is removable and can be replaced or reconnected.

[0352] "Example A22" The system of any one of Examples A6-A21, wherein the one or more identification elements are disposed on or etched into a surface of the microfluidic chip.

[0353] "Example A23" The system described in Example A22, wherein the one or more identification elements include an alphanumeric string, a barcode, a QR code (registered trademark), a fiducial marker, or a combination thereof.

[0354] "Example A24" The system of example A22 or example A23, wherein the one or more identification elements are used to identify a chip currently mounted in the microfluidic module.

[0355] "Example A25" The system of any one of Examples A22-A24, wherein the identification element is used to automatically align and position the microfluidic chip with respect to the detection laser and the kill laser.

[0356] "Example A26" The system of any one of Examples A22-A25, wherein the microfluidic chip with the identification element is mounted on a multi-axis stage that repositions the microfluidic chip.

[0357] "Example A27" A system described in any one of embodiments A1 to A26, further comprising an automatic sample level detector adapted to determine a sample level using the sample flow rate and to notify when the sample level falls below a threshold level.

[0358] "Example A28" 1. A method for sexing sperm, comprising: - classifying the sperm cells as having a first characteristic, a second characteristic, or a third characteristic; - selectively damaging at least a portion of the sperm cells based on these classifications; The method includes:

[0359] "Example A29" The method of example A28, wherein the first characteristic is the presence of an X chromosome from one sperm cell, the second characteristic is the presence of a Y chromosome from one sperm cell, and the third characteristic is the presence of both an X and a Y chromosome or repetition of one of the chromosomes by multiple sperm cells.

[0360] "Example A30" 1. A method for sexing sperm, comprising: - interrogating the stained sperm cells with a first radiation source to produce fluorescent radiation; - detecting said fluorescent radiation; - classifying the sperm cells based on the detected fluorescent emission; selectively damaging the sperm cells based on these classifications with a second radiation source. The method includes:

[0361] "Example A31" 1. A method for sexing sperm, comprising: - delivering a fluid stream containing stained sperm cells to an interrogation location; - interrogating the stained sperm cells with a first laser to cause them to produce fluorescent radiation; - detecting said fluorescent radiation; - classifying the sperm cells based on the detected fluorescent emission; - selectively damaging one or more subpopulations of the classified sperm cells based on these classifications using a second laser. The method includes:

[0362] "Example A32" The method of any one of Examples A28 to A31, further comprising staining the sperm cells to produce stained sperm cells.

[0363] "Example A33" The method according to any one of Examples A28 to A32, wherein the sperm cells are stained with a fluorescent DNA-binding dye.

[0364] "Example A34" The method of any one of Examples A30-A33, wherein the stained sperm cells are interrogated to generate a fluorescent emission corresponding to a single sperm cell or a fluorescent emission corresponding to a plurality of sperm cells.

[0365] "Example A35" The method according to any one of Examples A28 to A34, wherein the sperm cells are injured, thereby causing DNA damage and / or membrane damage to the cells.

[0366] "Example A36" The method according to any one of Examples A28 to A35, wherein the sperm cells are rendered infertile or dead by damaging said sperm cells.

[0367] "Example A37" 1. A method for producing a sexed semen product, comprising: - providing a modular flow cytometry system comprising an electronics module, a sample passage module, a fluidics module, an interrogation module, and a sample mixing and collection module, each module disposed within a support structure and configured to be separately removed and reinstalled or replaced by another of the same module; - flowing the semen sample having the stained sperm cells through a fluidics module and a sample passage module; - flowing a sheath fluid through the fluidic system module and the sample passage module; - causing the stained sperm cells to fluoresce using a detection laser in an interrogation module; - detecting the fluorescence of the stained sperm cells; - generating a sexed semen product by killing a subpopulation of stained sperm cells using a kill laser in an interrogation module based on the detected fluorescence; - collecting the sexed semen product flowing from the sample passage module in a sample mixing and collection module; - mixing the collected sexed semen products; The method includes:

[0368] "Example A38" The method of embodiment A37, wherein the radiation from the detection laser and the kill laser comprises an elliptical beam profile.

[0369] "Example A39" The method of example A37 or example A38, further comprising staining the semen sample containing the sperm cells to produce stained sperm cells.

[0370] "Example A40" The method of any one of Examples A37-A39, wherein the sample mixing and collection module rotates about a pivot to move in an arcuate path to enable mixing of the sexed semen product in a controlled, uniform motion.

[0371] "Example A41" The method of any one of Examples A37-A40, further comprising cleaning the sample passage module using a microfluidic device cleaning module.

[0372] "Example A42" 1. A method for producing a sexed semen product, comprising: - providing a modular flow cytometry system according to any one of Examples A6 to A27; - staining a semen sample containing sperm cells to produce stained sperm cells; - flowing the semen sample through a fluidics module and a sample passage module; - flowing a sheath fluid through the fluidics module and the sample passage module, where the sheath fluid, one or more channels in the microfluidic chip, or a combination thereof, targets the stained sperm cells as the sheath fluid flows through the microfluidic chip; - causing the stained sperm cells to fluoresce with radiation from a detection laser having an elliptical beam profile; - detecting fluorescence of the sperm cells by a detection assembly; - generating a sexed semen product by inactivating one or more subpopulations of the stained sperm cells with radiation from a kill laser based on the detected fluorescence, the radiation from the kill laser comprising an elliptical beam profile, and the detection laser and the kill laser are disposed on the same side of the microfluidic chip; - collecting the sexed semen product flowing from the microfluidic chip in a sample mixing and collection module; - automatically mixing the collected sexed semen product based on a set of defined sample mixing parameters associated with the sample being processed, wherein the sample mixing and collection module rotates about a pivot to move in an arcuate path to enable mixing of the sexed semen product in a controlled, uniform motion; The method includes:

[0373] "Example A43" The method of example A42, further comprising diverting the flow of sexed semen product exiting the microfluidic chip to a separate collector away from the sample mixing and collection module.

[0374] "Example A44" - redirecting the flow of sexed semen product away from the microfluidic chip when the collection tube of the sample mixing and collection module is filled with the sexed semen product to a desired level; - rotating a rotating base of the sample mixing and collection module such that a second collection tube is positioned to collect the sexed semen product; - redirecting the flow of sexed semen product to a second collection tube; The method of example A42 or example A43, further comprising:

[0375] "Example A45" The method of any one of Examples A42 to A44, further comprising automatically aligning and positioning the microfluidic chip relative to the detection laser and the kill laser by using an identification element disposed on the microfluidic chip.

[0376] "Example A46" The method of any one of embodiments A42-A45, further comprising using an automatic sample level detector to determine the sample level and notifying when the sample level falls below a threshold level.

[0377] "Example A47" The method of any one of Examples A42-A46, further comprising cleaning the sample passage module using a microfluidic device cleaning module.

[0378] Example Set B:

[0379] "Example B1" A removable and replaceable fluid path module for use in a flow cytometer system, the fluid path comprising: - a distribution block comprising a set of fluid inlets and a set of fluid outlets, at least one of the set of fluid inlets in fluid communication with at least one of the set of fluid outlets to form a first sample path element; - a microfluidic module comprising a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixture, wherein at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a second sample path element; - a fluid passageway in fluid communication between a set of fluid outlets of the distribution block and a set of fluid inlets of the microfluidic module, a portion of the fluid passageway joining and in fluid communication with a first sample passage element and a second sample passage element to form a sample passage; 1. A fluid passage module comprising: - samples processed by the flow cytometer system through a removable and replaceable fluid path module are confined to the sample path during processing; Fluid passage module.

[0380] "Example B2" The fluid passage module of embodiment B1, wherein the microfluidic device fixing device is adapted to fix one of a microfluidic chip or a microfluidic cassette in the microfluidic module.

[0381] "Example B3" The fluid passage module of embodiment B2, wherein the microfluidic device fixing device comprises one or more adjustment means adapted to adjust the position of the microfluidic chip or the microfluidic cassette.

[0382] "Example B4" The fluid passage module of any one of embodiments B1-B3, wherein the distribution block comprises a fluidic manifold.

[0383] "Example B5" The fluid passage module of any one of embodiments B1-B4, wherein the distribution block further comprises a sample tube loader.

[0384] "Example B6" the distribution block further comprising a set of separate fluid inlets in fluid communication with a set of separate fluid outlets to form a first buffer fluid path element; - the microfluidic module further comprises another set of fluid inlets in fluid communication with another set of fluid outlets to form a second buffer fluid path element; - the fluid passage further comprises a second portion joining and in fluid communication with the first buffer fluid path element and the second buffer fluid path element to form the buffer fluid passage; 1. A fluid passage module comprising: - the buffer fluid processed through the removable and replaceable fluid passage module is confined to the buffer fluid passage during processing; The fluid passage module according to any one of the embodiments B1 to B5.

[0385] "Example B7" A fluid passage module described in any one of embodiments B1 to B6, wherein each of the distribution block and the microfluidic module further comprises releasable fixing means adapted to fix the removable and replaceable fluid passage module to a flow cytometer system.

[0386] "Example B8" The fluid passage module of embodiment B7, wherein the releasable fixing means comprises at least one iron plate or at least one magnetic plate.

[0387] "Example B9" A fluid path module as described in embodiment B7 or embodiment B8, wherein the removable and replaceable fluid path module can be removed from the flow cytometer system by releasing the fixing means.

[0388] "Example B10" A fluid passage module described in any one of embodiments B7 to B9, wherein the releasable fixing means of the removable and replaceable fluid passage module is further adapted to fix the removable and replaceable fluid passage module to a fluid passage cleaning system.

[0389] "Example B11" The fluid passage module of any one of embodiments B1 to B10, wherein the sample is a mammalian semen sample.

[0390] "Example B12" The fluid path module of any one of embodiments B1-B11, wherein a flow cytometer system processes the sample to identify subpopulations in the sample.

[0391] "Example B13" The fluid passage module of any one of embodiments B1-B12, wherein a flow cytometer system processes the sample to perform a semen sexing operation.

[0392] "Example B14" The fluid passage module of any one of embodiments B1-B13, wherein the fluid passage module comprises an identification element.

[0393] "Example B15" The fluid passage module of embodiment B14, wherein the identification element comprises an optical iden...

Claims

1. 1. A modular flow cytometry system for processing samples, comprising: a. a multi-level chassis; b. an electronics module disposed in a first, lowest level of the chassis, the electronics module including one or more computer processors and a plurality of electronics ports; c. a sample path module, the sample path module being disposed on a second level of the chassis and including a microfluidic module for holding a microfluidic chip; d. a fluidic system module disposed in the second level of the chassis and fluidically coupled to the sample path module to circulate one or more fluids through the sample path module; e. an interrogation module disposed on a third level of the chassis and operably coupled to the electronics module, the interrogation module including a detection laser operably coupled to a detection assembly and a kill laser operably coupled to the detection assembly for processing a sample flowing through the microfluidic chip, the kill laser and the detection laser being disposed on a common side of the interrogation module; f. a sample mixing and collection module configured to collect and automatically mix the processed samples exiting the microfluidic chip, the sample mixing and collection module disposed below the microfluidic module on the same second level of the chassis, and rotating about a pivot to move in an arcuate path to enable mixing of the samples in a controlled, uniform motion; 1. A modular flow cytometry system comprising: A modular flow cytometry system, wherein each module is configured to be separately removed and reinstalled or replaced with another of the same module for substantially continuous operation of the modular flow cytometry system.

2. 2. The system of claim 1, wherein the sample passage module further comprises a distribution block fluidically coupled to the microfluidic module, the distribution block delivering the sample to the microfluidic module through a first fluidic line and delivering sheath fluid to the microfluidic module through a second fluidic line.

3. 11. The system further comprising a microfluidic device cleaning module for cleaning the sample passage module, the microfluidic device cleaning module comprising: a. a set of fluid reservoirs; b. a pump assembly; c. a distribution block interface comprising a distribution block securing element and a distribution block adjusting element, the distribution block interface further comprising a set of fluid interfaces, at least one of the set of fluid interfaces in fluid communication with at least one of the set of fluid reservoirs; d. a microfluidic device interface comprising a microfluidic device securing element and a microfluidic device alignment element, the microfluidic device interface further comprising a fluid interface in fluid communication with at least one of the set of fluid reservoirs; Equipped with the distribution block interface is adapted to secure the distribution block thereon, the microfluidic device interface is adapted to secure the microfluidic module thereon, and a microfluidic device cleaning system is adapted to clean the sample path module which circulates fluid through the sample path module. The system of claim 2.

4. 2. The system of claim 1, wherein one or more identification elements are disposed on or etched into a surface of the microfluidic chip and are used to identify a chip currently mounted in the microfluidic module and to automatically align and position the microfluidic chip relative to the detection laser and the kill laser.

5. The system of claim 4 , wherein the microfluidic chip with the identification element is mounted on a multi-axis stage that uses the identification element to reposition the microfluidic chip.

6. 2. The system of claim 1, wherein the sample passage module is configured to process particles or constituent molecules in a sample based on at least one characteristic, and the sample mixing and collection module is configured to automatically mix the sample processed by the sample passage module based on a defined set of parameters.

7. the sample mixing and collection module comprising: a. a rotating base; b. a set of collection tube holders disposed on the rotating base; c. a set of collection tubes, each configured to be disposed in a collection tube holder of the set of collection tubes; The system of claim 1 further comprising:

8. 8. The system of claim 7, wherein the sample mixing and collection module further comprises a fluid level sensor and is configured to replace the first tube with a second tube in the set of collection tubes when the fluid level sensor detects that a first tube has reached a desired fill level.

Images