PARTICLE POPULATION

BACKGROUND

[0001] Microfluidic systems enable fluid-based experiments to be conducted using much smaller quantities of fluid as compared to microtiter plate-based experiments. These small volumes enable advantages such as a reduction in expensive chemicals used, a reduction in the amount of patient sample used which makes sample collection easier and less intrusive, a reduction in the amount of waste generated, and in some cases, a reduction in the time for processing.

BRIEF DESCRIPTION OF FIGURES

[0002] Various examples may be more completely understood in consideration of the following detailed description in connection with the accompanying drawings, in which:

[0003] FIG. 1 is a flow chart illustrating an example method of determining an error in detected passage of a target particle population, consistent with the present disclosure;

[0004] FIG. 2 is a diagram illustrating an example computing apparatus for determining an error in detected passage of a target particle population, consistent with the present disclosure;

[0005] FIG. 3 is a diagram illustrating an example apparatus for determining an error in detected passage of target particle populations, consistent with the present disclosure; [0006] FIG. 4 illustrates an example apparatus including multiple sensors, consistent with the present disclosure;

[0007] FIG. 5 is a diagram illustrating an example apparatus including multiple foyers for identifying errors in detected passage of a target particle population, consistent with the present disclosure;

[0008] FIG. 6 illustrates an example apparatus including an aspiration assembly, consistent with the present disclosure; and [0009] FIG. 7 illustrates an example apparatus including a secondary sensor, consistent with the present disclosure.

[0010] While various examples discussed herein are amenable to modifications and alternative forms, aspects thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the disclosure to the particular examples described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure including aspects defined in the claims. In addition, the term “example” as used throughout this application is by way of illustration, and not limitation.

DETAILED DESCRIPTION

[0011] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific examples in which the disclosure may be practiced. It is to be understood that other examples may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0012] The life sciences research and diagnostics industries are under pressure to reduce costs, increase throughput, and improve the utilization of patient samples. As a result, the instruments and tools used therein are moving from complex macrofluidic-based systems to simpler microfluidic-based technology, moving from pipetting-based technology to dispensing-based technology, and moving from performing a single test per sample to performing multiplexed tests per sample.

[0013] Inkjet-based systems can start with microliters of fluid and then dispense picoliters or nanoliters of fluid into specific locations on a substrate. These dispense locations can be specific target locations on the substrate surface, such as cavities, microwells, channels, or indentations into the substrate. As used herein, a microwell refers to or includes a column capable of storing a volume of fluid between a nanoliter and several milliliters of fluid. There may be tens, hundreds, or even thousands of dispense locations on the substrate, which may represent many tests on a small number of samples, a small number of tests on many samples, or a combination of the two. Additionally, multiple dispensing nozzles or printheads may dispense fluid on the substrate at a time to enable a high-throughput design.

[0014] In various life-science applications, it may be beneficial to isolate a target particle population in each of a plurality of target locations of the substrate. The particles may include cells, nucleic acids, amino acids, antibodies, chemical compounds, and beads (which may have a cell or antibody attached thereto), among other types of particles and combinations thereof. Different target particle populations or n-cells may be useful for different types of tests performed. As a specific example, it may be beneficial to isolate a particle, such as a cell or a single functionalized bead from a remainder of a sample (among other non-limiting examples), from a remainder of a sample. As a specific example, it may be beneficial to isolate a single cell. In some examples, the particle includes a single cell. Such samples may contain a viral or cellular material, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. Example samples may comprise mammalian and non-mammalian animal cells, plant cells, algae including blue- green algae, fungi, bacteria, protozoa, etc. Representative samples include whole blood and blood-derived products such as plasma, serum and buffy coat, urine, feces, cerebrospinal fluid or any other body fluids, tissues, cell cultures, cell suspensions, etc. Other example samples include fluids containing functionalized beads to which a portion of a biologic sample or other particles are attached. As an illustration, individual cells may be beneficial for cell line and monoclonal antibody development, as well as for Chimeric antigen receptor T- cell (CAR-T) treatments, among other examples. In such instances, cells may be chosen that have particular traits, and by isolating single cells, producers can ensure the highest purity and potency of the final product. For other example tests, a plurality of particles may be beneficial, such as for antibody tests and/or multiplexed analyses.

[0015] Detecting proper dispensing of a target particle population in a microfluidic device has a number of challenges. For instance, the firing of drops from an inkjet nozzle may create fluid flow through a microfluidic path. Particles are carried along from this fluid flow, but the number of fired drops to move a particle through the microfluidic path to the nozzle can vary depending on the path the particle takes. For example, particles that follow a path close to the center of the microfluidic path may take fewer drops to exit the nozzle than particles that follow a path near the walls.

[0016] Moreover, to enable a high throughput system, operating a microfluidic device at a high frequency may be beneficial. However, the faster the fluid flows through the microfluidic path, the higher the likelihood of obtaining weak sensor signals and incorrectly dispensing particles. Yet further, a system with a single sensor may assume a specific number of drops to flush a particle out of a nozzle. Making this assumed number of drops larger increases the likelihood of accidently dispensing more than the target particle population in a target location. Making this assumed number of drops smaller increases the likelihood of accidently dispensing less than the target particle population in the first target location and then accidently dispensing more than the target particle population in the subsequent target location.

[0017] It may be beneficial for a microfluidic device dispensing specific quantities of particles into each location of the substrate to have one-hundred percent occupancy, where every well in which a target particle population is desired is occupied by the target population, and every well has the correct number of particles. However, errors in particle dispensing can come from a variety of factors, such as clumping particles, variation in particle sizes, and variation in how the particles travel through the microfluidic path. Accurately and time-efficiently identifying errors in particle dispensing may increase test performance and improve viability of cells or other living matter.

[0018] Determining an error in detected passage of a target particle population, consistent with the present disclosure, involves use of a fluid dispensing apparatus that includes a sensor in each microfluidic path within a particle-dispensing printhead of the apparatus. Determining errors in the detected passage of the target particle population, consistent with the present disclosure, reduces the number of dispense reporting errors and errors caused by post-dispensing assessment, such as reduced risk of contamination and reduced risk of reduction in cell (or other living matter) viability. Detecting errors in the detected passage of the target particle population, consistent with the present disclosure, allows for an error correction mode of operation after the particle dispensing, allowing the apparatus to achieve high throughput and reduction in the overall number of errors.

[0019] In accordance with the present disclosure, a method of determining an error in detected passage of a target particle population into a target location includes receiving a sample on a die including a microfluidic chamber, the microfluidic chamber including a microfluidic path coupling a reservoir to a foyer, and moving the sample from the reservoir to the foyer by firing a nozzle fluidically coupled to the foyer. Different particle sizes and shapes can result in different quality signals at each sensor, such as different signal shapes and sizes. Furthermore, detection of a particle may be complicated by the relative size of the particle as compared to the volume of the sample. As non-limiting examples, example cell volumes for different types of cells are as follows: sperm cell 30 pm ³ ; red blood cell 100 pm ³ ; lymphocyte 130 pm ³ ; neutrophil 300 pm ³ ; beta cell 1 ,000 pm ³ ; enterocyte 1 ,400 pm ³ ; fibroblast 2,000 pm ³ ; HeLa, cervix 3,000 pm ³ ; hair cell (ear) 4,000 pm ³ ; osteoblast 4,000 pm ³ ; alveolar macrophage 5,000 pm ³ ; cardiomyocyte 15,000 pm ³ ; megakaryocyte 30,000 mhi ³ ; fat cell 600,000 miti ³ . Beads may be a variety of sizes, such as having a diameter of 10-400 miti or greater. As an illustration, detecting a sperm cell as opposed to a fat cell may be complicated, given the small volume of the sperm cell relative to other types of cells in the sample fluid. Failure to identify a single particle versus multiple particles may result in, among other things, incorrect dispensing of target particle populations within the target locations.

[0020] The method described herein, however, includes detecting passage of a target particle population of the sample to the foyer and into a target location of a substrate via a first sensor located proximate to the foyer, and determining an error in the detected passage of the target particle population into the target location of the substrate based on a signal received from the first sensor. The method further includes, in response to the determination, classifying the target location as erroneous. The classification provides an indication that the target location (e.g., wells) may or may not be relied upon to have the intended number of particles for research and development, and may therefore result in, among other things, an increased confidence in cell lines and/or test results.

[0021] As used herein, a sample refers to or includes a volume of fluid containing particles, such as a biologic sample or other fluid including functionalized beads. In some examples, beads may have a portion of the biologic sample attached thereto, such as cells, antibodies, proteins, nucleic acids, which may be scientifically-engineered or naturally occurring, etc. Although examples are not limited to beads functionalized with a biologic sample, and may include beads that are attached to scientifically-engineered particles, such as molecules, chemical compounds, and other functional groups. Example samples, such as biologic samples, contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. Non-limiting examples of a sample includes whole blood and blood-derived products such as plasma, serum and buffy coat, urine, feces, cerebrospinal fluid or any other bodily fluids, tissues, cell cultures, cell suspensions, etc. Non-limiting examples of particles contained in a sample include viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles, all types of mammalian and non-mammalian animal cells, plant cells, algae including blue-green algae, fungi, bacteria, and protozoa.

[0022] In some examples, a non-transitory computer-readable medium may store instructions which, when executed by a processor, may cause the processor to identify an error in detected passage of a target particle population into a target location. For instance, the non-transitory computer-readable medium may store instructions that cause the processor to detect passage of a target particle population of a sample along a microfluidic path into a target location of a substrate via a first sensor located proximate to a foyer, wherein the microfluidic path couples a reservoir storing the sample and the foyer, and a nozzle is fluidically coupled to the foyer to eject a first volume of the sample into the target location. The non-transitory computer-readable medium may store instructions to identify the error in the detected passage of the target particle population into the target location of the substrate based on a first signal received from the first sensor. The non-transitory computer-readable medium may store instructions to eject a second volume of the sample into the target location, via the nozzle, to correct the error.

[0023] In some examples, an apparatus for determining errors in detected passage of a target particle population includes a fluidic input to receive a sample, a die including a microfluidic chamber, and circuitry. In such examples, the microfluidic chamber includes a reservoir in fluidic contact with the fluidic input, a microfluidic path coupling the reservoir to a foyer, the foyer to contain a portion of the sample, and a first sensor located proximate to the foyer to detect passage of a target particle population of the sample into each of a plurality of target locations of a substrate. The microfluidic chamber further includes a nozzle to eject a volume of the portion of the sample into each of the plurality of target locations. The circuitry controls firing of the nozzle based on a plurality of signals received from the first sensor, detects passage of the target particle populations into the plurality of target locations via the first sensor, determines an error in the detected passage of the target particle population into a subset of the plurality of target locations based on the plurality of signals received from the first sensor, and in response to the determination, classifies the subset of the plurality of target locations as erroneous.

[0024] Turning now to the figures, FIG. 1 is a flowchart of an example method of determining an error in detected passage of a target particle population, consistent with the present disclosure. At 102, the method 100 includes receiving a sample on a die including a microfluidic chamber. As further described herein, the microfluidic chamber includes a microfluidic path coupling a reservoir to a foyer. At 104, the method 100 includes moving the sample from the reservoir to the foyer by firing a nozzle fluidically coupled to the foyer. At 106, the method 100 includes detecting passage of a target particle population of the sample to the foyer and into a target location of a substrate via a first sensor located proximate to the foyer. In some examples, an additional sensor or multiple sensors may be used to detect passage of the target particle population into the target location. For instance, the method 100 may include detecting the passage of the target particle population along the microfluidic path via the first sensor disposed within the microfluidic path, and detecting passage of the target particle population into the target location via a second sensor disposed between the first sensor and the nozzle.

[0025] A target particle population, as used herein, refers to or includes a defined number of particles, or n-particles, to be dispensed into a target location. A target particle population may include a single particle or multiple particles. In some specific examples, the target particle population includes a single particle, such as a single cell or a single bead. However, examples are not so limited. In various examples, different target locations of the substrate may have different target particle populations. The target location refers to or includes a particular location to which a target particle population is to be dispensed. The target location may be a particular well on a microwell plate or other types of substrates that a sample may be dispensed.

[0026] In some examples, the method 100 may further include recording an indication of whether the target location includes the target particle population, or not, in a particle dispense map. A particle dispense map, as used herein, refers to or includes a map identifying detected particle populations of target locations e.g., wells, of a substrate. For example, the target location may be classified as including a target population location, being underpopulated, or being overpopulated based on signals measured by the first sensor. The circuitry may use signal parameters including peak height, peak width, time from last signal, and slope change to identify single particles versus clusters of particles, debris, and/or signal noise. Based on this information, the particle dispense map may be generated by indicating which location includes n- particles (e.g., the target number), more than n-particles, or less than n- particles. In particular examples, the particle dispense map may be generated in real time and/or on-the-fly while the apparatus is continuing to dispense fluid into further target locations. As used herein, real time refers to or includes processing of signals or other data within a threshold amount of time, e.g., seconds or milliseconds. On-the-fly, as used herein, refers to or includes processing that occurs while the fluid dispensing apparatus is in motion and/or another process is in progress.

[0027] At 108, the method 100 includes determining an error in the detected passage of the target particle population into the target location based on a signal received from the first sensor. In response to the determination, at 110, the method 100 includes classifying the target location as erroneous. Determining the error may include identifying the target location, which is previously classified as including the target particle population or n-particles, includes less than the target particle population or more than the target particle population. In such examples, the target location is reclassified as an underpopulated target location or an overpopulated target location. In other examples, determining the error may include identifying the target location, which is previously classified as including more or less than the target particle population, includes the target particle population. In such examples, the target location is reclassified as including the target particle population. In either examples, the circuitry may revise the particle dispense map by recording the reclassification for the target location.

[0028] As noted above, the circuitry may further analyze the signals from the first sensor, which may be associated with dispensing multiple target locations. In particular examples, the circuitry analyzes sensor signals to identify trends and/or variations in the signal parameters, such as the peak height, peak width, time from last signal, and slope change. Based on the analysis, the target location may be reclassified. In a number of examples, prior to dispensing the fluid, data may be received by the apparatus that identifies a type of substrate, a type of fluid and/or particle, a signal threshold for detecting passage of a particle, and/or an expected signal shape. The circuitry may adjust the signal threshold and/or detect errors based on analysis of the signals and signal shapes.

[0029] In various examples, the method 100 may include adjusting the signal threshold for detecting passage of a particle based on a plurality of signals from the first sensor. In such examples, the error is determined based on the adjusted signal threshold. The particles may be expected to be a particular size, which may result in an expected sensor signal of a particular height and/or width when the particle passes by the first sensor. Based on the expected particle size, a signal threshold may be set for detecting passage of a particle. Signals which are smaller or larger than the signal threshold may be determined to not include a particle, to be debris, to be signal noise and/or to include a clump of particles. By analyzing sensor signals, the method 100 may include identifying particles in the sample are of a different size than expected, and adjusting the signal threshold in response.

[0030] In further examples and/or in addition, the method 100 includes determining the error in the detected passage of the target particle population by analyzing the shape of the plurality of signals and determining the error based on the analyzed signal shapes. The signal shape may be analyzed to determine if the signal is representative of a particle, debris, a clump of particles, and/or sensor noise.

[0031] In some examples, the method 100 further includes verifying the classification of the target location using a secondary sensor. The secondary sensor may scan the substrate after dispensing is complete and the circuitry verifies the classification of the target location using data received from the secondary sensor. In specific examples, the secondary sensor may provide more accurate and/or detailed data as compared to the first sensor and the optional second sensor of the microfluidic device.

[0032] The method 100 may further include an error correction mode. For example, the method 100 may include correcting the error by ejecting a (second) volume of the sample into the target location via the nozzle. In specific examples, a target location that is classified as underpopulated may have the second volume of the sample ejected thereto by the fluid dispensing apparatus. In other examples and/or in addition, a target location that is classified as overpopulated may have fluid aspirated out, a solution fluid is ejected into the target location, followed by ejection of the second volume of the sample, as further illustrated by FIG. 6. In various examples, the substrate may be preloaded with the solution fluid in the target location and/or in each of a plurality of target locations.

[0033] In some examples, the method 100 includes terminating dispensing of the sample in the target location in response to detecting passage of the target particle population into the target location. After n-particles are dispensed into the target location, the fluid dispensing apparatus may move to the next target location and dispense fluid in the next target location. For example, although FIG. 1 illustrates a single target location, the method 100 may include detecting passage of the target particle population to the foyer and into a plurality of target locations of the substrate via the first sensor. In such examples, the method 100 further includes determining an error in the detected passage of the target particle population into a subset of the plurality of target locations based on a plurality of signals from the first sensor, the plurality of signals including the signal, and in response to the determination, classifying the subset of the plurality of target locations as erroneous.

[0034] Determining errors in detected passage of a target particle population may increase the accuracy of classification of the target location, which may allow for greater accuracy in a test performed on the sample. For example, test results of target locations which are identified as being overpopulated or underpopulated may be disregarded and/or the test may not be performed on such erroneous target locations. Detecting the errors using the apparatus in accordance with various examples may decrease the time consumed by the dispensing assessment and decrease errors as compared to manually assessing the substrate. As a specific example, a decrease in the time used for assessing the substrate may result in a decrease risk of loss of cell viability. Furthermore, the erroneous target locations may be corrected, thereby reducing the number of dispense errors of the apparatus.

[0035] FIG. 2 is a diagram illustrating an example computing apparatus for determining an error in detected passage of a target particle population, consistent with the present disclosure. In the example of FIG. 2, the computing apparatus 220 may include a processor 222 and a non-transitory computer- readable storage medium 226, and a memory 224. The non-transitory computer-readable storage medium 226 further includes instructions 228, 230, and 232 for determining an error in the detected passage of a target particle population into a target location. The computing apparatus 220 may be, for example, a dispensing instrument or a digital dispenser, a printer, a mobile device, multimedia device, a secure microprocessor, a notebook computer, a desktop computer, an all-in-one system, a server, a network device, a controller, a wireless device, or any other type of device capable of executing the instructions 228, 230, 232. In certain examples, the computing apparatus 220 may include or be connected to additional components such as memory, controllers, etc.

[0036] The processor 222 may be a central processing unit (CPU), a semiconductor-based microprocessor, a graphics processing unit (GPU), a microcontroller, special purpose logic hardware controlled by microcode or other hardware devices suitable for retrieval and execution of instructions stored in the non-transitory computer-readable storage medium 226, or combinations thereof. The processor 222 may fetch, decode, and execute instructions 228, 230, 232 to detect passage of a target particle population into a target location and identify an error in the detected passage, as previously discussed. As an alternative or in addition to retrieving and executing instructions, the processor 222 may include at least one integrated circuit (IC), other control logic, other electronic circuits, or combinations thereof that include a number of electronic components for performing the functionality of instructions 228, 230, 232.

[0037] Non-transitory computer-readable storage medium 226 may be an electronic, magnetic, optical, or other physical storage device that contains or stores executable instructions. Thus, non-transitory computer-readable storage medium 226 may be, for example, Random Access Memory (RAM), an Electrically Erasable Programmable Read-Only Memory (EEPROM), a storage device, an optical disc, etc. In some examples, the computer-readable storage medium 226 may be a non-transitory storage medium, where the term ‘non- transitory’ does not encompass transitory propagating signals. As described in detail below, the non-transitory computer-readable storage medium 226 may be encoded with a series of executable instructions 228, 230, 232. In some examples, non-transitory computer-readable storage medium 226 may implement a memory 224 to store and/or execute instructions 228, 230, 232. Memory 224 may be any non-volatile memory, such as EEPROM, flash memory, etc.

[0038] In various examples, the non-transitory computer-readable storage medium 226 may store instructions 228, 230, 232 which, when executed by the processor 222, may cause the processor 222 to control a microfluidic chamber for detecting passage of a target particle population into a target location and identifying an error in the detected passage. For instance, the non-transitory computer-readable medium 226 may store detect passage of a target particle population instructions 228 that cause the processor 222 to detect passage of a target particle population of a sample along a microfluidic path into a target location of a substrate, via a first sensor located proximate to a foyer, wherein the microfluidic path couples a reservoir storing the sample and the foyer, and a nozzle is fluidically coupled to the foyer to eject a first volume of the sample into the target location.

[0039] Additionally, the medium 226 may store identify error instructions 230 that cause the processor 222 to identify an error in the detected passage of the target particle population into the target location of the substrate based on a first signal received from the first sensor. Yet further, the medium 226 may store eject a second volume instructions 232 that cause the processor 222 to eject a second volume of the sample into the target location, via the nozzle, to correct the error. The ejection of the second volume may be used to correct an underpopulated target location and/or an overpopulated target location via use of an aspiration assembly or subsystem, as further illustrated herein. In various examples, the medium 226 stores instructions that, when executed, cause the processor 222 to verify the target location includes the target particle population via the first sensor and during the ejection of the second volume. In a specific example, the medium 226 stores instructions that, when executed, cause the processor 222 to verify the target location includes the target particle population via the first sensor and during the ejection of the second volume, where the target particle population includes a single cell. In another specific example, the target particle population includes a single bead. The verification may include re-executing the detect passage of a target particle population instructions 228. [0040] In some examples, the medium 226 may include instructions that cause the processor 222 to record, in a particle dispense map, an indication of whether the target location includes the target particle population, is overpopulated, or is underpopulated based on the first signal or a plurality of signals measured by the first sensor. For example, the processor 222 may record the number of particles occupying each of a plurality of target locations. Each particle may generate a signal as it passes over the first sensor and the presence of a particle may be detected until the target particle population is reached. In examples in which the target particle population includes one particle, such as a single cell, each detected particle may be jetted into a separate well, and the map may be generated indicating which wells have a single particle, which wells have zero particles, and which wells have multiple particles. Although examples are not so limited, and the target particle population may include multiple particles and a map may be generated indicating which wells have the multiple particles or do not have the multiple particles based on a plurality of sensor signals. The identify error instructions 230 may cause the processor 222 to reclassify the target location (or a subset of target locations) as erroneous, as previously described. [0041] As noted above, and further illustrated by FIGs. 4 and 5, the microfluidic chamber may include a second sensor. In such examples, the medium 226 stores instructions that, when executed, cause the processor 222 to detect the passage of the target particle population along the microfluidic path via the first sensor disposed within the microfluidic path, detect passage of the target particle population into the target location via the second sensor disposed between the first sensor and the nozzle, and classify the target location as erroneous based the first signal from the first sensor and a second signal received from the second sensor.

[0042] In some examples, the ejection of a second volume may be used to correct an overpopulated target location. In such examples, the medium 226 stores instructions that, when executed, cause the processor 222 to classify the target location as being populated with greater than the target particle population (e.g., overpopulated) based on the first signal from the first sensor. In response, the medium 226 stores instructions that, when executed, cause the processor 222 to aspirate fluid out of the target location via an aspiration assembly, cause passage of a solution fluid along a second microfluidic path, and eject a volume of the solution fluid to the target location via a pipette of the aspiration assembly. The fluid dispensing apparatus, in such examples, further includes the aspiration assembly having the second microfluidic path that couples a solution supply reservoir storing the solution fluid to the pipette, as further illustrated by FIG. 6, although examples are not so limited.

[0043] In various examples, which may be used in combination with or alternatively to the aspiration assembly, the fluid dispensing apparatus may include a secondary sensor, which is separate from the fluid dispensing assembly, as further illustrated by FIG. 7. In such examples, the medium 226 stores instructions that, when executed, cause the processor 222 to, in response to the identified error, cause a stage coupled to the substrate to change positions and to locate the substrate proximate to the secondary sensor and to verify the target location is erroneous via data from the secondary sensor. [0044] FIG. 3 is a diagram illustrating an example apparatus for determining an error in detected passage of a target particle population, consistent with the present disclosure. The apparatus 340 may include a fluidic input 342 to receive a sample and a die including a microfluidic chamber 346. The microfluidic chamber 346 may be disposed beneath the fluidic input 342, such that fluid that flows through the fluidic input 342 is deposited onto the microfluidic chamber 346. Accordingly, the fluidic input 342 may include an aperture 344 to receive the sample. The location and general size of the microfluidic chamber 346 relative to the fluidic input 342 is illustrated in dashed lines, and an exploded view of microfluidic chamber 346 illustrates the various components of the of microfluidic chamber 346.

[0045] As an illustration, as the sample is received in fluidic input 342, the sample flows into aperture 344. The sample flows through the aperture 344 and onto the bottom side of fluidic input 342, where the microfluidic chamber 346 is disposed.

[0046] The microfluidic chamber 346 may include a reservoir 348 in fluidic contact with the fluidic input 342. The microfluidic chamber 346 may further include a microfluidic path 357 coupling the reservoir 348 to a foyer 350, the foyer 350 to contain a portion of the sample. As illustrated, a first sensor 356 may be disposed proximate to the foyer 350 to detect passage of a target particle population within the sample into a plurality of target locations of a substrate. A nozzle 360 may eject a volume of the portion of the sample into each of the plurality of target locations of the substrate (not illustrated in FIG. 3). Circuitry 352 may control firing of the nozzle 360 based on a plurality of signals received from the first sensor 356 by sending signals to the nozzle 360 via electrical connects 354, and may detect passage of the target particle population into each of the plurality of target locations via the first sensor 356. For example, the circuitry 352 may classify and record in a particle dispense map, an indication of whether each of the target locations include the target particle population, an overpopulation, and/or an underpopulation based on signals measured by the first sensor 356. [0047] The sample may flow from the aperture 344 to the reservoir 348 of the microfluidic chamber 346. As the sample, including particles, flows from the reservoir 348 to the foyer 350, the first sensor 356 may detect passage of the target particle population, such as n-particles, of the sample into the foyer 350, which is interpreted by the circuitry 352 as the target particle population dispensing into the respective target location. Although FIG. 3 illustrates the first sensor 356 as being disposed within the microfluidic path 357 coupling the reservoir 348 to the foyer 350, examples are not so limited and the first sensor 356 may be disposed proximate to the nozzle 360. In such examples, the first sensor 356 may detect passage of the target particle population from the foyer 350 into each of the plurality of target locations.

[0048] The first sensor 356 may be an impedance-based sensor, a capacitance-based sensor or another type of sensor, such as an optical sensor, a thermal sensor, a voltammetric sensor, an amperometric/coulometric sensor, a transistor, such as a field-effect transistor, a magnetic sensor, among others. An impedance-based sensor or a capacitance-based sensor may include a pair of electrodes that measure the impedance or capacitance of the fluid containing the sample, with the capacitance and/or impedance being measured between the electrodes. For example, the impedance or capacitance may be measured for a current or voltage path between the two electrodes. More specifically, a high-frequency alternating (e.g., sine-wave) current or voltage may be applied to one electrode and the interaction of the alternating electrical field with the fluid is monitored at the other electrode, which may be in the form of an alternating current signal. The two electrodes may be separated from the fluid by a dielectric layer. Changes in impedance and/or capacitance between the electrodes may indicate the presence of a particle. The impedance measurements may be processed by the circuitry 352 to determine the presence of a particle. Impedance-based and capacitive-based sensors may not contact the particles, which may increase cell viability, and may be used to sense particles without the use of a label and/or imaging. Additionally, impedance-based sensors and capacitive-based sensors may be inexpensive, small in size, and may provide sensor signals at high speeds, as compared to other types of sensors.

[0049] The microfluidic chamber 346 may include a nozzle 360 to eject a volume of the portion of the sample into each of the plurality of target locations. The nozzle 360 may include a fluid ejector, such as a thermal inkjet resistor, to eject the sample onto the respective target location. As such, the microfluidic chamber 346 may include the circuitry 352 to control firing of the nozzle 360. [0050] In various examples, the circuitry 352 may determine an error in the detected passage of the respective target particle population into a subset of a plurality of target locations based on the plurality of signals received from the first sensor. In response to the determination, the circuitry 352 may classify the subset of the plurality of target locations as erroneous. For example, the circuitry 352 may reclassify each of the subset of the plurality of target locations as being one of unpopulated and overpopulated, and may revise the particle dispense map that identifies each of the plurality of target locations as being one of containing the target particle population, being unpopulated, and being overpopulated based on the classification. The classification may additionally or alternatively include reclassifying a portion of the subset of the plurality of target locations as containing the target particle population, which were previously classified as one of unpopulated and overpopulated.

[0051] In some examples, the apparatus 340 may operate in an error correction mode. In the error correction mode, the circuitry 352 may control firing of the nozzle 360 to eject a second volume of the sample into a portion (or all) of the subset of plurality of target locations to correct the respective errors. The portion may include target locations that are underpopulated and the circuitry 352 may eject the second volume to increase the particle population in the portion of the subset of the target locations to the target particle population.

In other examples and/or in addition, the portion (or a subset of the portion) may include target locations that are overpopulated and the circuitry 352 may control an aspiration assembly to aspirate fluid out the portion (or subset) of the subset of the target locations and eject solution fluid into the portion of the subset of target locations, and control the nozzle 360 to eject the second volume of the sample into the portion.

[0052] FIG. 4 illustrates an example apparatus including multiple sensors, consistent with the present disclosure. Similar to apparatus 340 illustrated by FIG. 3, the apparatus 440 of FIG. 4 includes a fluid input 442, an aperture 444, and a reservoir 448 which receives a sample on the microfluidic chamber 446. A microfluidic path 457 couples the reservoir 448 and the foyer 450, and a first sensor 456 is located proximate to the foyer 350. As illustrated by FIG. 4, the microfluidic chamber 446 may further include a second sensor 458 disposed between the first sensor 456 and the nozzle 460 to detect passage of the target particle population past the foyer 450.

[0053] In some examples, the first sensor 456 may be disposed within the microfluidic path 457, and the second sensor 458 may be disposed within a threshold distance of the nozzle 460 to detect passage of the particle or the target particle population of the sample into the nozzle 460. As used herein, the threshold distance may be a distance close enough to the nozzle 460 such that passage of a particle into the nozzle 460 may be detected. An example range of the threshold distance of the first sensor 456, as measured from an edge of the nozzle 460, may be 5-100 micrometers (urn), although examples are not so limited.

[0054] The first sensor 456 and the second sensor 458 may be the same type of sensor, or different types of sensors. For instance, the first sensor 456 may be an impedance-based sensor or a capacitance-based sensor. Similarly, the second sensor 458 may be an impedance-based sensor or a capacitance- based sensor. Examples of the present disclosure are not limited to impedance- based sensors or capacitance-based sensors, and additional and/or different types of sensors may be used.

[0055] The circuitry 452 may detect passage of the target particle population into the plurality of target locations via signals from the first sensor 456 and the second sensor 458. The particle may be expected to take a certain amount of time between the first sensor 456 and the second sensor 458, t ₀ ± 6t depending on the size of the particle. If the particle is a clump, it may traverse the path longer, and may not fit within the bounds t ₀ ± 6t and therefore may not be classified as a particle of interest. The bound may be soft, with particles taking longer time than t ₀ being assigned a lower probability of being the particle of interest. The circuitry 452 may determine an error in the detected passage of the target particle population via a post-dispense analysis, such as by analyzing the signal sizes and shapes, and revising the classification as previously described. The use of the signals from the first sensor 456 and the second sensor 458 may provide a more accurate classification as compared to use of the first sensor 456 without the second sensor 458.

[0056] While FIG. 4 illustrates the second sensor 458 being disposed at an end of the foyer 450 near the nozzle 460, examples are not so limited. For instance, in various examples the second sensor 458 may be disposed within the nozzle 460. Including the second sensor 458 in the nozzle 460 has the advantage of measuring the signal immediately before the particle exits the microfluidic path (i.e. is ejected out nozzle 460).

[0057] Although not illustrated, in some examples, additional sensors may be disposed within foyer 450. A second and third (or more) sensor in the fluid path enables progress of the particle through the microfluidic path to be more closely tracked. Signals for the three (or more) sensors can be aggregated to decrease the likelihood of false positives or negatives. In the case when a second particle enters the foyer 450 before the first particle has exited; a third sensor increases the likelihood that these two particles can be distinguished from one another and dispensed in a particular target location or to a different location, such that the target location is occupied by the target particle population. Regardless of the number of sensors, the circuitry 452 may control a firing rate of the nozzle 460, by sending a signal to the nozzle 460 via electrical connects 454.

[0058] While particle concentration is intended to be low enough that one particle is in the foyer at a time, there may be instances in which a second particle has entered the foyer before the first particle has left the foyer. In a system with a single sensor, this means that there is a high probability of a double dispense, with more particles being dispensed than intended. By including a plurality of sensors as described herein, with a sensor or a plurality of sensors within the foyer region, the progress of the particle through the foyer may be tracked and may provide for an improved probability that a target particle population is dispensed into a location even when multiple particles reside in the foyer at once. As an example, particles may pass the first sensor 456, and subsequently flow backwards toward the first sensor 456 rather than toward nozzle 460 due to back pressure from firing the nozzle 460. Without an additional sensor disposed between the first sensor 456 and nozzle 460, dispensing errors may occur because the backflow prevents the particle from being dispensed in the target location and which may, instead, be dispensed in the next target location. The end result of such backflow, when a single sensor is used, is that one well may have no particles in it while the next well may have two particles in it when both wells are intended to have a single particle.

[0059] Another example of errors that may occur with a single sensor, is dependent on the size/volume of the particle and the path that the particle takes within the foyer. For example, a particle with a smaller volume, such as a red blood cell, may travel down a center of the foyer 450 or down an edge of foyer 450. If the red blood cell travels down the edge of the foyer 450, it may take longer (e.g., more firing of the nozzle 460) for the red blood cell to reach the nozzle 460 and dispense into the target location. Similarly, if the red blood cell travels down the center of the foyer 450, the red blood cell may reach the nozzle 460 faster (e.g., with less firing of the nozzle 460). Without an additional sensor or sensors disposed between the first sensor 456 and nozzle 460, the nozzle 460 may fire a specified number of times after particle is detected at the first sensor 456 with the assumption that the particle has exited the nozzle 460 and into the target location. However, such assumption may be incorrect based on the volume of the particle and the route that the particle has taken within the foyer 450. By adding the additional sensor, such as the second sensor 458, the movement of the particle within the foyer 450 may be detected and confidence increased that the target particle population is dispensed in each target location. [0060] FIG. 5 is a diagram illustrating an example apparatus including multiple foyers for determining an error in detected passage of a target particle population, consistent with the present disclosure. In the example illustrated in FIG. 5, each foyer 550-1 , 550-2, 550-3, and 550-4 is coupled to a respective nozzle 560-1 , 560-2, 560-3, and 560-4. Each nozzle 560-1 , 560-2, 560-3, and 560-4 may dispense a target particle population into a different respective target location. The target locations may be within a high-density micro-titer plate, such as a 1536 well plate, where the spacing between the nozzles 560-1 , 560-2, 560- 3, and 560-4 match the spacing between different wells without moving the substrate. These paths may be similar and redundant, with the redundancy used to increase throughput and/or to increase system robustness. These paths may be operated separately, concurrently, or in a round-robin fashion (cycling between which path is the active path). As discussed with regards to FIG. 1 , the apparatus 540 may include a die including a microfluidic chamber 546, and circuitry 552 to control firing of the plurality of target nozzles 560-1 , 560-2, 560- 3, 560-4.

[0061] Each foyer 550-1 , 550-2, 550-3, and 550-4 is coupled to a different respective microfluidic path. Similarly, each foyer 550-1 , 550-2, 550-3, and 550- 4 includes a first sensor 556-1 , 556-2, 556-3, and 556-4, respectively. The first sensors 556-1 , 556-2, 556-3, and 556-4 detect passage of particles from the reservoir 548 into the respective foyer 550-1 , 550-2, 550-3, 550-4. In various examples, although examples are not so limited, a second sensor 558-1 , 558-2, 5258-3, and 558-4 in each respective foyer 550-1 , 550-2, 550-3, 550-4 detects passage of the target particle population passing into a respective nozzle, e.g., 560-1 , 560-2, 560-3, 560-4. The circuitry 552 may control firing of each respective nozzle based on the signals received from the associated sensors, detect passages of the target particle population into the plurality of target locations via the signals, and determines errors in a subset of the detected passages via the signals.

[0062] As illustrated in FIG. 5, the microfluidic chamber 546 may include a plurality of foyers 550-1 , 550-2, 550-3, and 550-4, each coupled to a common reservoir 548. Each respective foyer 550-1 , 550-2, 550-3, and 550-4 may be coupled to the reservoir by a different respective microfluidic path. For each respective foyer, passage of particles within the foyer may be monitored using the associated sensors.

[0063] In cases where the multiple fluidic paths are addressing what is effectively the same target location within a low-density micro-titer plate, such as a 384, 96, 48, 24 or lower-density well plate, the microfluidic paths can be operated concurrently or in a round-robin method to increase throughput or to enable slower operation of each microfluidic path as a way to decrease errors.

In the round-robin operation, the circuitry 552 cycles between operating the various paths. For instance, nozzle 560-1 may fire to draw a particle through foyer 550-1 and out of nozzle 560-1 , then nozzle 560-2 may fire to draw a particle through foyer 560-2 and out of nozzle 560-2, then nozzle 560-3 may fire to draw a particle through foyer 560-3 and out of nozzle 560-3, and nozzle 560- 4 may fire to draw a particle through foyer 560-4 and out of nozzle 560-4. Additionally and/or alternatively, various nozzles may be fired concurrently. As an illustration, nozzles 560-1 and 560-2 may fire at a same time, and nozzles 560-3 and 560-4 may fire at a same time. Operation of the nozzles illustrated in FIG. 5 is not limited to those examples described, and the plurality of nozzles 560-1 , 560-2, 560-3, 560-4 may fire in any sequence and/or combination contemplated.

[0064] Although the circuitry 352, 452, 552 of the example apparatuses of FIGs. 3-5 are illustrated as being disposed separate from the respective dies, sometimes referred to as “off-die”, examples are not so limited. In some examples, the circuitry 352, 452, 552 may be disposed on the dies, which may be referred to as “on-die”.

[0065] FIG. 6 illustrates an example apparatus including an aspiration assembly, consistent with the present disclosure. The fluid dispensing apparatus 670 may deposit fluid, such as a sample, onto a substrate 678.

[0066] The fluid dispensing apparatus 670 includes a fluid dispensing assembly (e.g., a particle-dispensing printhead) 674, a fluid supply assembly 682, a substrate transport assembly 680, and circuitry 672. The fluid dispensing apparatus 670 may include additionally non-illustrated components, such as a mounting assembly and a power supply that provides power to the various electrical components of the fluid dispensing apparatus 670 and the aspiration assembly 684. The fluid dispensing assembly 674 includes a microfluidic device having a plurality of dies 675 that eject drops of fluid through a plurality of nozzles 676 toward the substrate 678 to dispense target particle populations of the sample on different target locations of the substrate 678. Each die 675 includes a sensor located proximate to a foyer, as previously described and illustrated by FIGs. 3-5. The nozzles 676 may be arranged in columns or arrays such that properly sequenced ejection of fluid from nozzles 676 causes a volume of the sample to be dispensed to different target locations of the substrate 678 as the fluid dispensing assembly 674 and substrate 678 are moved relative to each other.

[0067] The fluid supply assembly 682 supplies sample to the fluid dispensing assembly 674 and includes a first reservoir 681 for storing the sample. The fluid flows from the first reservoir 681 to the fluid dispensing assembly 674.

[0068] Although not illustrated, the apparatus 670 may include a mounting assembly that positions the fluid dispensing assembly 674 relative to the substrate transport assembly 680, and the substrate transport assembly 680 positions the substrate 678 relative to the fluid dispensing assembly 674. A dispensing zone 677 is defined adjacent to the nozzles 676 in an area between the fluid dispensing assembly 674 and the substrate 678. In various examples, the fluid dispensing assembly 674 is a scanning type fluid dispensing assembly. In a scanning type fluid dispensing assembly, the mounting assembly includes a carriage for moving the fluid dispensing assembly 674 relative to substrate transport assembly 680 to scan the substrate 678. In other examples, the fluid dispensing assembly 674 is a non-scanning type fluid dispensing assembly. In a non-scanning fluid dispensing assembly, mounting assembly fixes the fluid dispensing assembly 674 at a prescribed position relative to substrate transport assembly 680 and the substrate transport assembly 680 may include a stage for moving and positioning the substrate 678 relative to the fluid dispensing assembly 674. [0069] The circuitry 672 may include a processor, machine readable instructions, and other printer electronics for communicating with and controlling the fluid dispensing assembly 674, the substrate transport assembly 680, and other components of the apparatus 670. In various examples, the circuitry 672 may include the computing apparatus 220 as previously illustrated by FIG. 2. The circuitry 672 may receive data from a host system, such as a computer, and includes memory for temporarily storing data. The data may be sent to the fluid dispensing apparatus 670 along an electronic, infrared, optical, or other information transfer path. The data may represent, for example, identification of a type of substrate, a target particle population, and/or positions of the target locations on the substrate. The data may additionally represent an initial signal threshold for identifying passage of a particle and/or an expected signal shape. Using the data, the circuitry 672 controls the fluid dispensing assembly 674 to eject fluid drops from the nozzles 676.

[0070] As previously described, the circuitry 672 may detect passage of the target particle population into each of a plurality of target locations via sensor signals received from sensors of the dies 675. The circuitry 672 may further determine errors in the detected passage of a subset of the target locations. [0071] In various examples, the circuitry 672 may control correction of the errors of overpopulated target locations and/or underpopulated target locations. For correcting underpopulated locations, the circuitry 672 may control the fluid dispensing assembly 674 to eject a second volume of the sample to increase the particle population in underpopulated target location(s), thereby correcting target location(s) which were previously underpopulated.

[0072] For correcting overpopulations, the apparatus 670 may include an aspiration assembly 684. The aspiration assembly 684 may include a first pipette 685 and a second pipette 686. The first pipette 685 is coupled to a suction source, such as a vacuum pump or other pneumatic tools, and aspirates out 687 any fluid in the identified overpopulated target location(s). The second pipette 686 is coupled to the solution supply assembly 688 that supplies solution fluid along a second microfluidic path to the second pipette and the second pipette 686 is to eject 683 a volume of the solution fluid to the overpopulated target location(s) after the previous fluid is removed. The solution fluid may include suspension fluid, buffer fluid, or cell growth media, among other fluids. The volume of solution fluid ejected may be larger than the volume of the sample ejected, such as a milliliter range.

[0073] However examples are not so limited and in a number of examples, similar to the fluid dispensing assembly 674, the aspiration assembly 684 may include a second microfluidic device having a pipette and suction source that aspirates fluid out from the target location(s) and having a plurality of dies that eject drops of solution fluid through a nozzle toward the substrate so as to dispense volumes of the solution fluid on different target location(s) of the substrate 678. Each die includes a nozzle or a plurality of nozzles for ejection of solution fluid from the nozzle(s) and that causes a volume of the solution fluid to be dispensed to different target locations of the substrate 678 as the aspiration assembly 684 and substrate 678 are moved relative to each other. Each die may include a second foyer and the second microfluidic path that couples a second reservoir 689 storing the solution fluid to the respective second foyer which is coupled to the respective nozzle.

[0074] The solution supply assembly 688 supplies solution fluid to the aspiration assembly 684 and includes a second reservoir 689 that stores the solution fluid, sometimes interchangeably referred to as “a solution supply reservoir”. The solution fluid flows from the second reservoir 689 to the aspiration assembly 684. The apparatus 670 may further include a waste reservoir 690 for storing aspirated fluid, which may be coupled to the first pipette 685. Although examples are not so limited, and the apparatus 670 may include a spittoon location on the substrate 678 and/or the substrate transport assembly 680 to which the waste is aspirated out of the target location and dispensed on the spittoon location.

[0075] Although not illustrated, the apparatus 670 may include a second mounting assembly that positions the aspiration assembly 684 relative to the substrate transport assembly 680, and the substrate transport assembly 680 positions the substrate 678 relative to the aspiration assembly 684. As noted above, the substrate transport assembly 680 may include a stage that positions the substrate 678 relative to the fluid dispensing assembly 674, moves the substrate 678 toward the aspiration assembly 684, and positions the substrate 678 relative to the aspiration assembly 684, as illustrated by the arrow 691. [0076] After dispensing the solution fluid to the identified target location(s), the substrate transport assembly 680 may move the substrate 678 toward the fluid dispensing assembly 674 and position the substrate 678 relative to the fluid dispensing assembly 674. The fluid dispensing assembly 674 may eject drops of fluid through respective nozzles 676 toward the substrate 678 to dispense target particle populations into the identified target locations of the substrate 678, thereby correcting the respective target location(s) which were previously overpopulated.

[0077] FIG. 7 illustrates an example apparatus including a secondary sensor, consistent with the present disclosure. Similar to apparatus 670 illustrated in FIG. 6, the fluid dispensing apparatus 771 includes a fluid dispensing assembly 774, a fluid supply assembly 782, a substrate transport assembly 780, and circuitry 772. The fluid dispensing assembly 774 includes a microfluidic device having a plurality of dies 775 that ejects drops of fluid through a plurality of nozzles 776 toward the substrate 778 to dispense target particle populations on different target locations of the substrate 778. Each die 775 includes a sensor located proximate to a foyer, as previously described and illustrated by FIGs. 3-5. The fluid supply assembly 782 supplies a sample to the fluid dispensing assembly 774 and includes a first reservoir 781 for storing the sample. The substrate transport assembly 780 may include a stage that positions the substrate 778 relative to the fluid dispensing assembly 774.

[0078] The circuitry 772 controls the fluid dispensing assembly 774 to eject fluid drops from the nozzles 776 and to detect passage of the target particle population into each of a plurality of target locations via sensor signals received from sensors of the dies 775. The circuitry 772 may detect errors in the detected passage of a subset of the plurality of target locations and may classify the subset of plurality of target locations as erroneous.

[0079] In some specific examples, the apparatus 771 further includes a secondary sensor 794 that may be used to verify the classification of the plurality of target locations. After dispensing each of the target locations, the circuitry 772 may position the substrate 778 relative to the secondary sensor 794. As noted above, the substrate transport assembly 780 may include a stage that positions the substrate 778 relative to the fluid dispensing assembly 774, moves the substrate 778 toward the secondary sensor 794, and positions the substrate 778 relative to the secondary sensor 794, as illustrated by the arrow 793.

[0080] The secondary sensor 794 may scan the substrate 778 and the circuitry 772 verifies the classification of each of the target locations using data received from the secondary sensor 794. Example secondary sensors include an optical camera, a microscope, and a thermal camera, among other types of sensors.

[0081] As with FIG. 6, the circuitry 772 may additionally control correction of the errors of underpopulated target location(s) and, optionally, overpopulated target location(s) by controlling the fluid dispensing assembly 774 to eject a second volume of the sample to increase the particle population in the respective location(s). For correcting overpopulated location(s), the apparatus 771 may further include an aspiration assembly, such as the aspiration assembly 684 of FIG. 6.

[0082] Terms to exemplify orientation, such as left/right, and top/bottom, may be used herein to refer to relative positions of elements as shown in the figures. It should be understood that the terminology is used for notational convenience and that in actual use the disclosed structures may be oriented different from the orientation shown in the figures. Thus, the terms should not be construed in a limiting manner.

[0083] Various terminology as used in the Specification, including the claims, connote a plain meaning in the art unless otherwise indicated. As examples, the Specification describes and/or illustrates aspects useful for implementing the claimed disclosure by way of various structure, such as circuits or circuitry selected or designed to carry out specific acts or functions, as may be recognized in the figures or the related discussion as depicted by or using terms such as blocks, device, and system, and/or other examples. It will also be appreciated that certain of these blocks may also be used in combination to exemplify how operational aspects have been designed and/or arranged. Whether alone or in combination with other such blocks or circuitry including discrete circuit elements such as transistors, resistors, these above- characterized blocks may be circuits coded by fixed design and/or by configurable circuitry and/or circuit elements for carrying out such operational aspects. In certain examples, such a programmable circuit refers to or includes computer circuits, including memory circuitry for storing and accessing a set of program code to be accessed/executed as instructions and/or configuration data to perform the related operation. Depending on the data-processing application, such instructions and/or data may be for implementation in logic circuitry, with the instructions as may be stored in and accessible from a memory circuit. Such instructions may be stored in and accessible from a memory via a fixed circuitry, a limited group of configuration code, or instructions characterized by way of object code.

[0084] Where the Specification may make reference to a “first [type of structure]”, a “second [type of structure]”, etc., the adjectives “first” and “second” are not used to connote any description of the structure or to provide any substantive meaning; rather, such adjectives are merely used for English- language antecedence to differentiate one such similarly-named structure from another similarly-named structure designed or coded to perform or carry out the operation associated with the structure.

[0085] Although specific examples have been illustrated and described herein, a variety of alternate and/or equivalent implementations may be substituted for the specific examples shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific examples discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof.

What is Claimed is: