Cell selection device and method

Abstract

In some examples, a method is provided for sorting cells within a microfluidic flow by adjusting an offset delay and / or a selection period for sorting cells within the microfluidic flow. In one aspect, the method includes: detecting cell events using waveforms in cell emission signals received from the microfluidic flow and associated with cell events within the microfluidic flow; classifying the cell events into selected cell events using the waveforms; and sorting one or more cells from the selected cell events within a selection period following the selected cell events after an offset delay; wherein the selection period and / or offset delay are adjusted based on the waveform width of the waveform associated with the selected cell events.

Description

1. Technical Field

[0001] This disclosure relates to cell selection and sorting within microfluidic systems. These methods provide enhanced sorting of biological cells such as sperm cells. 2. Background Technology

[0002] The classification of biological cells with different characteristics is useful for many subsequent processes. For example, classifying sperm cells into X and Y populations allows for downstream separation or sorting of these two populations. One type of sperm cell may be better suited for certain types of animal husbandry. For instance, bovine X sperm cells are better suited for inseminating dairy cows to produce a milk-producing herd dominated by female offspring.

[0003] The classification and sorting of bovine sperm cells presents several challenges, including low sorting efficiency, low enrichment, and slow sorting speed. Low sorting efficiency results in a low percentage of desired cells (e.g., X sperm cells) collected compared to the total number of cells required to be incorporated into the classification and sorting system. Low sorting efficiency can be caused by a number of factors, including poor orientation of cells used for classification, inaccurate classification techniques, inefficient sorting techniques, and associated processes negatively impacting cell motility. Low enrichment means that the enrichment of cells with desired characteristics is lower than expected or desired levels. Slow sorting speed prolongs the time that biological cells are outside of optimal storage conditions, and therefore may also affect cell motility and viability.

[0004] References to patent specifications, other external documents, or other sources of information in this specification are generally made to provide context for discussing the features of the invention disclosed herein. Unless otherwise specifically stated, references to such external documents should not be construed as an admission that such documents or sources of information are prior art or constitute part of common general knowledge in the art within any jurisdiction.

[0005] The object of this invention is to provide an improved method for processing cells within a microfluidic flow, or at least to provide the public with a useful option for cell classification and / or sorting methods. 3. Summary of the Invention

[0006] In some examples, a method for timing the sorting of cells within a microfluidic flow is provided. The method includes: detecting cell events using waveforms in cell emission signals received from and associated with cell events within the microfluidic flow; classifying the cell events into selected cell events using the waveforms; and sorting one or more cells from the selected cell events within a selected time period, the selected time period depending on the waveform width of the waveform associated with the selected cell events.

[0007] The effectiveness and efficiency of cell sorting can be improved by sorting cells based on the waveform width of the waveform associated with a cell event. Cell events can be associated with a single cell or multiple tightly clustered cells. An example implements improved timing associated with the sorting of cells or other particles.

[0008] In some examples, the selected time period and / or the offset between the end of the selected cell event and the start of the corresponding selected time period can be adjusted based on the waveform width of the cell event.

[0009] In some examples, the waveform width can be determined by detecting the rising edge, followed by the falling edge, of a waveform in a cell emission signal received from the microfluidic flow and associated with a cellular event. In other examples, the waveform width can be determined by detecting a peak in the waveform and using a predetermined duration before and after the peak. In yet another example, the waveform width can be determined by detecting either a preceding rising edge or a subsequent falling edge of the waveform and using the duration between the peak and the preceding rising edge or subsequent falling edge.

[0010] In some examples, sorting of one or more cells in a selected cellular event within a selected time period uses a pulse sorting arrangement that generates regular pulses, and sorting of cells associated with the selected cellular event is achieved by controlling gating opening, during which one or more of these regular pulses are directed into the microfluidic flow. The gating opening time period is controlled based on the detection of waveforms associated with the cellular event and the timing of one or more of these regular pulses.

[0011] In some examples, selected cells can be sorted from unselected or unwanted cells by means of pressure applied by applying electromagnetic radiation to selected and / or unselected cells. In other examples, electromagnetic radiation can be used to inactivate selected or unselected cells.

[0012] In some examples, the cells can be bovine sperm cells, and sorting is used to separate X and Y cells.

[0013] In some examples, the method can be implemented using a pulse sorting arrangement, such as a pulsed laser, to sort one or more cells. In some examples, sorting includes controlling one or more gated opening periods during a selection period, wherein during the gated opening period, one or more regular pulses are directed to the microfluidic flow, and the gated opening period is controlled based on the detection of cellular events and the timing of one or more of these regular pulses.

[0014] In some examples, the gating opening period includes a switching delay, where the gating opening period is controlled to prevent regular pulses from occurring during the switching delay. The switching delay corresponds to the time required for the switching device to switch from fully disconnected to fully closed, or vice versa. For example, this could correspond to the switching time required from one or more regular pulses being directed into the microfluidic flow to one or more regular pulses being directed out of the microfluidic flow, or vice versa.

[0015] In some examples, the gating opening period is controlled in response to the detection of a regular pulse after the detection of a cell event, or the gating opening period is controlled based on the detection of a regular pulse before the detection of a cell event.

[0016] In some examples, the gating start-up period is initiated after a certain start-up delay following the detection of a cell event or the classification of a cell event into a selected cell event. This start-up delay includes a predetermined delay and a variable delay depending on the timing of one or more of these regular pulses. The predetermined delay may depend on the transit time of a cell in the microfluidic flow between a detection position and a sorting position, where the detection position corresponds to the detection of a cell event and the sorting position corresponds to the sorting of cells associated with the cell event. The variable delay may depend on the detection of a regular pulse after the detection of a cell event. In some examples, the variable delay can be calculated using the time difference between classifying a cell event into a selected cell event and the detection of the next regular pulse.

[0017] In some examples, a method is provided for determining the z-axis orientation of cells within a microfluidic flow. The method includes: detecting cells using waveforms in cell emission signals received from and associated with cells within the microfluidic flow; determining the z-axis orientation of the cells by identifying a maximum value in the waveform corresponding to a first portion of the cell and identifying a maximum value in the waveform corresponding to a second portion of the cell; wherein the z-axis orientation of the cells is determined based on the order of the two maximum values ​​in the waveform.

[0018] In some examples, a method is provided for adjusting an offset delay and / or a selection period for sorting cells within a microfluidic flow. The method includes: detecting cell events using waveforms in cell emission signals received from and associated with cell events within the microfluidic flow; classifying the cell events into selected cell events using the waveforms; and sorting one or more cells from the selected cell events within a selection period following the selected cell events with an offset delay. The selection period and / or offset delay are adjusted based on the waveform width of the waveform associated with the selected cell events.

[0019] In some examples, a method for processing cells within a microfluidic flow is provided. The method includes: detecting a cell event by detecting a rising edge, followed by a falling edge, of a waveform in a cell emission signal received from and associated with a cell event within the microfluidic flow; classifying the cell event into selected cell events using the waveform; and sorting one or more cells from the selected cell events using an offset delay from the detected falling edge.

[0020] In some examples, a method for processing cells within a microfluidic flow is provided. The method includes: detecting cellular events within the microfluidic flow using received emission signals associated with the microfluidic flow; classifying the cellular events as unselected or selected cellular events; and sorting one or more cells from the selected cellular events within a selected time period, the selection time period depending on the end of the selected cellular event and the duration of the selected cellular event.

[0021] A computer program is also provided, comprising processor instructions that, when executed by a processor, cause the processor to perform any of the methods described above. In some examples, a non-transitory medium is provided on which the computer program is stored.

[0022] In some examples, an apparatus is provided for timing the sorting of cells within a microfluidic flow. The apparatus includes a processor and a memory configured to: detect cell events using waveforms in cell emission signals received from and associated with cell events within the microfluidic flow; classify the cell events into selected cell events using the waveforms; and classify one or more cells from the selected cell events within a selected time period, the selected time period depending on the waveform width of the waveform associated with the selected cell events.

[0023] In some examples, an apparatus for processing cells within a microfluidic flow is provided. The apparatus includes a processor and a memory configured to: detect cellular events within the microfluidic flow using received emission signals associated with the microfluidic flow; classify the cellular events as unselected or selected cellular events; and classify one or more cells from the selected cellular events within a selected time period, the selected time period depending on the end of the selected cellular event and the duration of the selected cellular event.

[0024] In some examples, an apparatus for processing cells within a microfluidic flow is provided. The apparatus includes: components for detecting cellular events using waveforms in cellular emission signals received from and associated with cellular events within the microfluidic flow; components for directing interrogating electromagnetic radiation to cells within the microfluidic flow to facilitate responsive emission signals from the cells; components for directing sorting electromagnetic radiation to selected cells within the microfluidic flow; and optical components for directing the interrogating and / or sorting electromagnetic radiation. The optical components are adjustable according to the characteristics of the waveforms.

[0025] In some examples, an apparatus for processing cells within a microfluidic flow is provided. The apparatus includes: a component for delivering the microfluidic flow; a component for detecting cellular events using waveforms in cellular emission signals received from and associated with cellular events within the microfluidic flow; a component for directing interrogating electromagnetic radiation to cells within the microfluidic flow to facilitate responsive emission signals from the cells; and a component for directing sorting electromagnetic radiation to selected cells within the microfluidic flow. The component for delivering the microfluidic flow is adjustable to change the path of the microfluidic flow according to the characteristics of the waveform.

[0026] In some examples, a method is provided for sorting cells within a microfluidic flow using a pulse sorting arrangement that generates regular pulses. The method includes: detecting cellular events within the microfluidic flow; classifying the cellular events into selected cellular events; and sorting cells associated with the selected cellular events by controlling a gating opening period during which one or more of these regular pulses are directed into the microfluidic flow. The gating opening period is controlled based on the detection of the cellular events and the timing of one or more of these regular pulses.

[0027] In some examples, a method for timing the sorting of cells within a microfluidic flow is provided, wherein the cell sorting includes using a pulse sorting arrangement structure that generates regular pulses. The method includes: detecting cell events using waveforms in cell emission signals received from the microfluidic flow and associated with cell events within the microfluidic flow; classifying the cell events into selected cell events using the waveforms; and sorting one or more cells from the selected cell events within a selected time period, the selected time period depending on the waveform width of the waveform associated with the selected cell events, wherein the sorting of one or more cells from the selected cell events is achieved by controlling a gating opening time period during which one or more regular pulses are directed into the microfluidic flow. The gating opening time period is controlled based on the detection of cell events and the timing of one or more regular pulses.

[0028] In some examples, sorting of one or more cells within a selected cell event during a selected time period depends on the end of the selected cell event and the duration of the selected cell event. In some examples, the selected time period may include one or more gating open time periods.

[0029] By controlling the application of some regular pulses to the microfluidic flow based on the detection of cellular events and one or more of these regular pulses, cells associated with cellular events are more accurately targeted. This increases the likelihood of sorting cells associated with cellular events (such as unwanted Y sperm cells) while not sorting cells unrelated to cellular events (such as desired X sperm cells). This, in turn, improves sorting efficiency and reduces collateral damage to other cells unrelated to cellular events.

[0030] In some examples, the gating opening period may include a switching delay corresponding to the time required for the switching device to switch from being guided into the microfluidic flow by one or more regular pulses to being guided out of the microfluidic flow by one or more regular pulses, or vice versa; and the gating opening period may be controlled to prevent regular pulses from occurring during the switching delay.

[0031] In some examples, the gating opening period can be controlled in response to the detection of a regular pulse after the detection of a cellular event. Alternatively, the gating opening period can be controlled based on the detection of a regular pulse before the detection of a cellular event. The detection of regular pulses can be implemented in a laser pulse-based system using a photodetector.

[0032] In some examples, the gating start-up period can be initiated after a certain start-up delay following the detection of a cell event or the classification of a cell event into a selected cell event. This start-up delay includes a predetermined delay and a variable delay depending on the timing of one or more of these regular pulses. The predetermined delay may depend on the transit time of a cell in the microfluidic flow between a detection position and a sorting position, where the detection position corresponds to the detection of a cell event and the sorting position corresponds to the sorting of cells associated with the cell event. The variable delay may depend on the detection of a regular pulse after the detection of a cell event. The variable delay can be calculated using the time difference between classifying a cell event into a selected cell event and the detection of the next regular pulse.

[0033] In some examples, the gating opening period can be one or more of the following: equal to or less than the inter-pulse period between regular pulses; 30%-70% of the inter-pulse period; 46%-60% of the inter-pulse period, or about 50%.

[0034] In some examples, the gating opening period is controlled to overlap with a single pulse. The single pulse can be timed within the central portion of the gating opening period, which includes one of the following: the middle 80% of the gating period; the middle 50% of the gating period; or the middle of the gating period.

[0035] In some examples, the gating opening period can be controlled to overlap with two or more pulses in response to the detection of a cellular event associated with a single cell. In some examples, the gating opening period can be controlled to overlap with two or more pulses in response to the detection of cellular events associated with multiple cells. The gating opening period can end after the detection of the last cell in the cellular event or after classifying the cellular event as a selected cellular event, wherein the end delay period depends on the transit time of the cell in the microfluidic flow between a detection position and a sorting position, the detection position corresponding to the detection of the cellular event and the sorting position corresponding to the sorting of the cell associated with the cellular event. The end delay period can include the transit time minus a variable end delay, which depends on the switching delay from when one or more regular pulses are directed into the microfluidic flow to when one or more regular pulses are directed out of the microfluidic flow, or vice versa. The variable end delay can be calculated in response to determining that the next pulse will coincide with the switching delay associated with the end gating period.

[0036] In some examples, the pulse sorting arrangement may include a pulsed laser that generates regular laser pulses. Sorting may include using the laser pulses to nudge, inactivate, or ablate cells associated with selected cellular events. Gating opening periods may be associated with an optical switch that is controlled to switch the laser pulse into and out of the microfluidic flow. The optical switch may include one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector; or an electro-optic modulator.

[0037] In some examples, the cells are sperm cells.

[0038] In some examples, a method is provided for sorting cells within multiple microfluidic flows using a pulsed laser that generates regular laser pulses. The method includes: splitting the laser pulses into multiple beams, each beam associated with a corresponding microfluidic flow; detecting cellular events within each microfluidic flow; classifying the cellular events in each microfluidic flow into selected cellular events; and sorting cells associated with the selected cellular events in each microfluidic flow using the regular laser pulses of the corresponding beams.

[0039] By sharing a common source of laser pulses used to sort cells in multiple microfluidic flows, cost and complexity can be reduced, while also making the method / system more robust. This approach allows for miniaturization and parallel processing, which improves both throughput and speed, as well as portability and / or processing power.

[0040] In some examples, one or more beam splitters can be used to split a regular laser pulse into multiple beams. The beam splitter can be a polarization beam splitter, and the beam splitting ratio is adjusted by adjusting the ratio of light polarized in a first plane to light polarized in a second plane. The amount of light in the first plane relative to the second plane can be adjusted by a polarization adjuster.

[0041] In some examples, the method uses N or N-1 beamsplitters or polarization beamsplitters to sort cells within N microfluidic flows. The method may include adjusting the power transfer properties of at least some of the beamsplitters in response to at least one of cell event properties (such as detection, classification, and / or sorting properties) and beam states (such as deactivation of detection, classification, and / or sorting for one or more microfluidic flows in the microfluidic flow). In some examples, the method uses N or N-1 polarization beamsplitters to sort cells within N microfluidic flows. The method may include adjusting the polarization of at least some of the N or N-1 polarization beamsplitters in response to at least one of cell event properties (such as detection, classification, and / or sorting properties) or beam states (such as deactivation of detection, classification, and / or sorting for one or more microfluidic flows in the microfluidic flow).

[0042] In some examples, sorting cells associated with selected cellular events may include controlling a corresponding gating period during which one or more regular laser pulses from the beam are directed to a corresponding microfluidic flow; wherein the corresponding gating period is controlled based on the detection of cellular events in the corresponding microfluidic flow and the timing of one or more regular laser pulses. The corresponding gating period may include a switching delay from when one or more regular laser pulses from the corresponding beam are directed to the corresponding microfluidic flow to when one or more regular laser pulses from the corresponding beam are directed away from the corresponding microfluidic flow, or vice versa. The corresponding gating period may be controlled to prevent regular laser pulses from the corresponding beam from occurring during the switching delay.

[0043] In some examples, the corresponding gating opening period can be controlled in response to the detection of a regular laser pulse after the detection of a cellular event in the corresponding microfluidic flow. Alternatively, the corresponding gating opening period can be controlled based on the detection of a regular laser pulse before the detection of a cellular event.

[0044] In some examples, the corresponding gating start-up period can be initiated after a certain start-up delay following the detection of a cellular event in the corresponding microfluidic flow or the classification of a cellular event into a selected cellular event for the corresponding microfluidic flow. This start-up delay includes a predetermined delay and a variable delay depending on the timing of one or more of these regular laser pulses. The predetermined delay may depend on the transit time of a cell in the corresponding microfluidic flow between a detection position and a sorting position, where the detection position corresponds to the detection of a cellular event in the corresponding microfluidic flow, and the sorting position corresponds to the sorting of cells associated with the cellular event in the corresponding microfluidic flow. The variable delay may depend on the detection of a regular laser pulse after the detection of a cellular event. The variable delay can be calculated using the time difference between classifying a cellular event into a selected cellular event for the corresponding microfluidic flow and the detection of the next regular laser pulse.

[0045] In some examples, the corresponding gating opening period can be one or more of the following: equal to or less than the inter-pulse period between regular laser pulses; 30%-70% of the inter-pulse period; 40%-60% of the inter-pulse period, or about 50%.

[0046] In some examples, the corresponding gating opening period can be controlled to overlap with a single laser pulse. The single laser pulse can be timed within the central portion of the corresponding gating opening period, which includes one of the following: the middle 80% of the corresponding gating period; the middle 50% of the corresponding gating period; or the middle of the corresponding gating period.

[0047] In some examples, the corresponding gating opening period can be controlled to overlap with two or more laser pulses in response to the detection of cellular events associated with multiple cells in the corresponding microfluidic flow. The corresponding gating opening period can end after the detection of the last cell in the cellular event or after classifying the cellular event as a selected cellular event in the corresponding microfluidic flow, wherein the end delay depends on the transit time of the cells in the corresponding microfluidic flow traveling between a detection position and a sorting position, where the detection position corresponds to detecting cellular events in the corresponding microfluidic flow and the sorting position corresponds to sorting cells associated with cellular events in the corresponding microfluidic flow. The end delay can include the transit time minus a variable end delay, which depends on the switching delay from when one or more regular laser pulses of the corresponding beam are guided into the corresponding microfluidic flow to when one or more regular laser pulses are guided away from the corresponding microfluidic flow, or vice versa. The variable end delay can be calculated in response to determining that the next laser pulse will coincide with the switching delay associated with ending the corresponding gating period.

[0048] In some examples, sorting may include using the laser pulse to nudge, inactivate, or ablate cells associated with selected cellular events.

[0049] In some examples, the corresponding gating opening period can be associated with an optical switch that is controlled to switch laser pulses into and out of the corresponding microfluidic flow. The optical switch may include one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector; or an electro-optic modulator.

[0050] In some examples, a sorting apparatus is provided for sorting cells within a microfluidic flow using a pulse sorting arrangement that generates regular pulses. The apparatus includes: a pulse sorting arrangement that generates regular pulses; a detection component for detecting cellular events within the microfluidic flow; a classification component for classifying cellular events into selected cellular events; and a sorting component for sorting cells associated with selected cellular events by controlling a gating opening period during which one or more of these regular pulses are directed into the microfluidic flow. The gating opening period is controlled based on the detection of cellular events and the timing of one or more of these regular pulses.

[0051] In some examples, a sorting apparatus is provided for sorting cells within multiple microfluidic flows using a pulsed laser that generates regular laser pulses. The apparatus includes: a pulsed laser that generates regular laser pulses; a beam splitter for splitting the laser pulses into multiple beams, each beam associated with a corresponding microfluidic flow; one or more detection components for detecting cellular events within the corresponding microfluidic flow; one or more classification components for classifying cellular events in the corresponding microfluidic flow into selected cellular events; and corresponding sorting components for sorting the selected cellular events in the corresponding microfluidic flow using regular laser pulses.

[0052] In some examples, a method for timing the sorting of cells within a microfluidic flow is provided. The method includes: detecting cell events using waveforms in cell emission signals received from and associated with cell events within the microfluidic flow; classifying the cell events into selected cell events using the waveforms; and sorting one or more cells from the selected cell events within a selected time period, the selected time period depending on the waveform width of the waveform associated with the selected cell events.

[0053] Aspects of the invention may also be broadly described as the parts, elements, and features individually or jointly mentioned or indicated in the description of this application, any or all combinations of two or more of said parts, elements, or features, and wherein a particular technical element mentioned herein has a known equivalent in the field to which the invention pertains, such known equivalents being considered incorporated herein as if set forth separately. 4. Description of the attached drawings

[0054] The invention will now be described by way of example only and with reference to the accompanying drawings, in which:

[0055] Figure 1 These are schematic diagrams of systems for processing cells, based on some examples;

[0056] Figure 2a An evaluation of single-cell event waveforms for receiving transmitted signals is illustrated based on several examples;

[0057] Figure 2b An evaluation of the waveform of a multicellular event receiving a transmitted signal is illustrated based on several examples;

[0058] Figures 3a to 3d The waveforms, peak detection, and cell inactivation of single-cell and multi-cell events received from transmitted signals are illustrated.

[0059] Figure 4 The following examples illustrate cell event waveforms, falling edge detection, and cell inactivation based on received transmitted signals;

[0060] Figure 5a The following are examples of single-cell event waveforms for receiving transmitted signals and laser pulses for sorting single-cell events.

[0061] Figure 5b The following are examples of multicellular event waveforms for receiving transmitted signals and laser pulses for sorting cells in multicellular events;

[0062] Figure 6a The following are examples of single-cell event waveforms for receiving transmitted signals and laser pulses for sorting single-cell events.

[0063] Figure 6b The following are examples of multicellular event waveforms for receiving transmitted signals and laser pulses for sorting cells in multicellular events;

[0064] Figure 7 The methods for sorting selected cells are illustrated based on some examples;

[0065] Figure 8This is a graph illustrating the relationship between the selected time period of a cell event and the waveform width, based on some examples.

[0066] Figure 9 Examples of controllers for processing cells are shown;

[0067] Figure 10 Sperm cells are illustrated;

[0068] Figure 11 The following are examples of waveforms related to the z-axis orientation of sperm cells;

[0069] Figure 12a and Figure 12b The waveforms of sperm cells in head-first z-axis orientation and tail-first z-axis orientation are illustrated respectively, based on some examples;

[0070] Figures 13a to 13d illustrate z-axis orientation-related waveforms of sperm cells based on fluorescence emission determined from different angles, according to some examples;

[0071] Figure 14 These are schematic diagrams of systems for processing cells, based on some examples;

[0072] Figures 15a to 15c The waveforms of single-cell and multi-cell events, peak detection, laser pulses, and cell inactivation are illustrated in the received transmitted signals.

[0073] Figure 16 An example of a control method for a single-cell event is illustrated, including the timing of the laser pulse, the ablation amplitude, and control signaling including the use of different control periods;

[0074] Figure 17 An example of a control method for a single-cell event is illustrated, including the timing of the laser pulse, the ablation amplitude, and control signaling including the use of different control periods;

[0075] Figure 18 An example of a control method for multi-cell events is illustrated, including the timing of laser pulses, ablation amplitude, and control signaling including the use of different control periods;

[0076] Figure 19 An example of a control method for multi-cell events is illustrated, including the timing of laser pulses, ablation amplitude, and control signaling including the use of different control periods;

[0077] Figure 20 Methods for sorting cells within a microfluidic flow are illustrated according to some examples;

[0078] Figures 21 to 23 Systems for processing cells are illustrated according to some examples;

[0079] Figure 24 Examples of controllers for processing cells are shown;

[0080] Figure 25 The pulse sorting arrangement structure is illustrated according to some examples. 5. Detailed Implementation

[0081] In the claims and the foregoing description, all transitional phrases such as “comprising,” “including,” “with,” “having,” “containing,” “involving,” “holding,” “constituting,” etc., should be understood as open-ended, that is, referring to including but not limited to. Only the transitional phrases “constituting of” and “constituting substantially of” should be closed or semi-closed transitional phrases, respectively.

[0082] As used herein, the term “about” refers to a reasonable amount of deviation that modifies a term so that the final result will not be significantly altered. For example, when applied to a value, the term should be understood as including a deviation of + / - 5% of that value.

[0083] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions incorporated by reference in other documents, and / or the general meaning of the defined terms.

[0084] Unless otherwise expressly indicated, the indefinite articles “a” and “an” as used in this specification and claims shall be understood to mean “at least one / a kind”.

[0085] The terms “may” and “can” are used interchangeably in this disclosure and indicate that the mentioned element, component, structure, function, functionality, objective, advantage, operation, step, process, apparatus, system, device, result or description can be used, included or produced, or otherwise represent the proposition indicated in a statement in which the term is used (or mentioned) for a particular example.

[0086] The phrase “and / or” as used in this specification and claims should be understood to mean “any one or both” of the elements so combined—that is, elements that exist together in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, that is, “one or more” of the elements so combined. In addition to the elements specifically indicated by the “and / or” clause, other elements may optionally be present, whether related to or unrelated to those specifically indicated. Thus, as a non-limiting example, the reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising / including,” may in one example refer only to A (optionally including elements other than B); in another example, only to B (optionally including elements other than A); in yet another example, to both A and B (optionally including other elements); and so on.

[0087] As used in this specification and claims, when referring to a list of one or more elements, the phrase "at least one / at least one of" should be understood to mean at least one element selected from any one or more elements in the list, but not necessarily including each element specifically listed in the list and at least one element from each element, and does not exclude any combination of elements in the list. In addition to the elements specifically indicated in the list referred to by the phrase "at least one / at least one of," this definition also allows for elements that may be optionally present, whether related to or unrelated to those specifically indicated elements. Therefore, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B"), in one example, may refer to at least one A, optionally including more than one A, where B is absent (and optionally including elements other than B); in another example, may refer to at least one B, optionally including more than one B, where A is absent (and optionally including elements other than A); in yet another example, refers to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements); and so on.

[0088] The intent is to refer to the range of numbers disclosed herein (e.g., 1 to 10) as well as to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any range of any rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus all subranges of all ranges explicitly disclosed herein are explicitly disclosed. These are merely examples of specific intents, and all possible combinations of numerical values ​​between the listed minimum and maximum values ​​should be considered as expressly stated in a similar manner in this application.

[0089] Whenever a range is given in the specification (e.g., temperature range, time range, or composition range), all intermediate ranges and sub-ranges, as well as all individual values ​​included in the given range, are intended to be included in this disclosure.

[0090] Specific details, such as particular examples or examples used for illustrative purposes rather than limiting, are set forth below. Those skilled in the art will understand that other examples may be employed in addition to these specific details. In some cases, detailed descriptions of well-known methods, nodes, interfaces, circuits, and devices have been omitted to avoid obscuring the description with unnecessary detail. Those skilled in the art will understand that the described functionality can be implemented in one or more nodes using hardware circuitry (e.g., analog and / or discrete logic gates, ASICs, PLAs, etc., interconnected to perform specific functions) and / or using software programs and data in conjunction with one or more digital microprocessors or general-purpose computers. Nodes communicating using an air interface also have suitable radio communication circuitry. Furthermore, where appropriate, this technology can be additionally considered to be fully embodied in any form of computer-readable storage, such as solid-state memory, disk, or optical disk containing a suitable set of computer instructions that will cause a processor to execute the technology described herein.

[0091] The hardware implementation may include, but is not limited to, digital signal processor (DSP) hardware, reduced instruction set processors, hardware (e.g., digital or analog) circuitry, including but not limited to one or more application-specific integrated circuits (ASICs) and / or one or more field-programmable gate arrays (FPGAs), and (where appropriate) state machines capable of performing such functions. Memory may be used to store temporary variables, maintain and transfer data between processes, non-volatile configuration settings, standard message formats, etc. Any suitable form of volatile and non-volatile memory device may be used, including random access memory (RAM) implemented as metal-oxide-semiconductor (MOS) or integrated circuits (ICs), and storage devices implemented as hard disk drives and flash memory.

[0092] Some or all of the described devices or functionalities may be instantiated in cloud environments such as Docker, Kubernetes, or Spark. The cloud functionality may be instantiated at the network edge, device edge, local location, or on a remote server coupled via a network such as 4G or 5G. Alternatively, the functionality may be implemented in dedicated hardware.

[0093] The term "constraint" as used in this article refers to a constraint on the cross-sectional shape and size of a cellular flow in a fluid flow. For example, it can constrain the diameter of a circular cross-section of the flow, or constrain the dimensions of the major and minor axes of an elliptical cross-section flow, thereby producing a single narrow trajectory in which any polar axis of the cell deviates minimally from the longitudinal axis of the flow's defined center.

[0094] The term "rotational orientation" for asymmetric cells (including sperm cells) refers to the principal angle of a representative sample of the cell relative to an axis substantially perpendicular to the cell's flow axis. Without any feature applying directional torque to the cell, the rotational orientation of the face is expected to be randomly distributed and oriented at any angle of approximately 360°. Cell samples for which directional torque has been applied by a directional force or feature will have a non-random angular rotational orientation, which preferably oriented the cell face at a specific angle, making the principal angle determinable or observable.

[0095] The term "z-axis orientation" refers to the direction of travel of elongated cells within a microfluidic flow. The z-axis is aligned with the direction of the flow. In these examples, the elongated cells are defined by a longitudinal axis, which facilitates function or movement within a specific environment. This longitudinal axis extends longitudinally from one end of the cell to the other, thus defining the primary direction of the cell's extension and generally influencing its movement and interaction with its surrounding environment. In cases where such elongated cells have different structures at each end of the longitudinal axis, they can be said to exhibit z-axis orientation because one structure or form guides the direction of travel. For elongated cells containing a head and tail, such as sperm cells, z-axis orientation indicates whether the cell within the flow travels head-first or tail-first.

[0096] "Cell" and "X cell" are referred to herein as examples of specific types of particles that may be desired to be retained within a microfluidic sorting arrangement. The use of the term "cell" herein does not require that the cell be a living cell. It will be readily understood by those skilled in the art that reference to X cell is intended to indicate any other particle or cell having properties suitable for inquiry and sorting according to the invention. Specifically, X cell herein can be replaced by any type of cell, including substantially symmetrical and asymmetrical cells, neurons, erythrocytes, tagged cells, viruses, or microbiomes, as will be known to those skilled in the art.

[0097] While some examples are descriptions of cells, this disclosure is not limited to the handling or sorting of cells, but is equally applicable to other types of particles. Therefore, the term "cell" as used herein includes other types of particles suitable for detection and sorting according to the methods and apparatus described herein. As used in this context, a particle refers to any discrete unit that can be manipulated and sorted within a microfluidic system, primarily fluorescently labeled particles. This includes, but is not limited to: chemical entities, such as labeled compounds; biological entities, such as cells, specifically sperm cells, DNA or RNA carriers or molecules, oligomers; proteins, such as enzymes or antibodies, viruses, liposomes, plasmids, hormones, microvesicles, and exogenous bodies; beads, such as beads labeled with specific molecules for identification or separation purposes; and nanoparticles, including nanoparticles used for drug delivery or imaging, such as metal nanoparticles, quantum dots, carbon-based nanoparticles, polymer nanoparticles, silica nanoparticles, and magnetic nanoparticles.

[0098] As used herein, the term "microfluidic flow" refers to a liquid flow having at least one dimension in which surface forces dominate volume forces. In examples, this could include liquid flows having sub-millimeter diameters or other cross-sectional dimensions. In examples, microfluidic flow could be a continuous phase flow of a liquid, such as an uninterrupted flow of one or more aqueous solutions. This could be a laminar flow having a sample flow comprising cells and a sheath flow surrounding the sample flow. Microfluidic flow may alternatively or additionally include dispersed flows of droplets.

[0099] As used herein, the term "flow environment" refers to an environment through which a microfluidic flow can flow. Examples include microchannels, which may comprise a material, such as glass, forming an elongated lumen or path through which the microfluidic flow passes. The path may be completely surrounded by material between each end of the path. The path may have at least one boundary that exposes the microfluidic flow to the fluid environment, wherein the material is in contact with another boundary of the microfluidic flow. In another example, the flow environment may be a fluid environment or volume, which may be substantially static or itself fluid. In this example, the microfluidic flow may not be in contact with a material substrate but may be completely surrounded by a fluid environment. The fluid environment may be a liquid (such as an aqueous solution) or a gas (such as air).

[0100] The term "sorting" is intended to refer to any process that separates or is capable of separating a population of cells with a first characteristic from a population of cells with a different characteristic. Those skilled in the art will understand that various methods can be employed within the sorting arrangement described herein to achieve the collection of cellular components rich in the desired characteristics. For example, electrostatic sorting methods or microbubble-induced particle sorting are known to those skilled in the art, and these methods are designed to accomplish the same task. Here, microbubbles or droplets containing gently pushed cells may move laterally relative to an axis corresponding to the flow direction and are not necessarily retained within the separated fluid flow. While increasing the power of the radiation source or similar alternative modifications to the sorting arrangement may increase the initial separation distance between the particle flows, it may be desirable to have the ability to do so without affecting cell viability. Therefore, when using such a sorting method, the radiation source needs to be carefully controlled to ensure that selected cells are displaced into different flow paths compared to unselected cells, and that the selected cells do not become immobile or inactive due to radiation. This displacement effect is preferably achieved by applying electromagnetic radiation (e.g., by a laser) to change the orientation of the cells from the first flow path to a different flow path. The particle flow path containing selected or unselected cells can then be directed to the first collection container, while the particle flow containing other populations of unwanted cells can be discarded or collected in a second, different collection container.

[0101] The term "cellular emission signal" is intended to refer to any signal detectable from a microfluidic flow, and it indicates the presence of one or more cells within the microfluidic flow at a location corresponding to the detection or interrogation area. In one example, a UV beam is directed into the microfluidic flow, and the presence of one or more cells results in fluorescence emission detected as a cellular emission signal.

[0102] The term "selected cell event" is intended to refer to the identification of a cell or group of cells within a microfluidic flow that is intended for use with respect to a treatment or disposal that may be provided with regard to other cells that do not correspond to the selected cell event. In one example, a selected cell event may correspond to an unwanted cell, such as a Y sperm cell that may be intended for inactivation; this is compared to other cells that do not correspond to the selected cell event and are not intended for inactivation. Selected cell events can be detected using cell emission signals received from the microfluidic flow.

[0103] The term "selection period" is intended to refer to the duration for which one or more cells corresponding to a selected cellular event are processed. In one example, this could be the time period during which an inactivating laser is applied to one or more cells. In another example, the selection period could refer to the time period during which a pulse sorting arrangement that generates regular pulses is directed to one or more cells or a microfluidic flow containing said one or more cells. In some examples, this can be controlled by a gating opening period that depends on the timing of detecting cellular events and regular pulses (e.g., laser pulses). The selection period can include one or more gating opening periods. In yet another example, the selection period could refer to the duration during which a non-inactivating laser (which can be pulsed or continuous) is applied to one or more cells to push them from one laminar flow path within a microfluidic flow into another laminar flow path. This, in turn, allows cells in different laminar flow paths to be separated or sorted from each other.

[0104] The term "gated opening period" is intended to refer to a controllable period of time in a device or apparatus that is switchable or controllable to direct a regular pulse into or away from the microfluidic flow. A gated opening period indicates when a regular pulse is directed into the microfluidic flow. A gated opening period may correspond to a "selection period" as referred to herein, or, in some examples where a selection period comprises two or more gated opening periods, to a portion of a selection period. For example, an optical switch or gating may be controlled to allow an incident laser pulse to be directed into the microfluidic flow during a gated opening period and to block or direct the laser pulse away from the microfluidic flow outside of the gated opening period. Some controllable devices may be associated with a switching delay, during which a portion of the power of the regular pulse may be directed into the microfluidic flow. Depending on the implementation, this switching delay may or may not be considered when controlling the gated opening period.

[0105] The term "regular pulse" is intended to refer to a pulse used in an applied physical phenomenon to interact with cells in order to sort one group of cells flowing in a microfluidic flow from another. Pulses are regular because they share a common inter-pulse distance or time period between adjacent pulses. An example applied physical phenomenon is laser, where a regular pulse refers to a brief, controlled emission of laser energy that occurs at regular intervals, followed by a period of no emission before the next pulse. These emissions can last for any duration, from several microseconds to several femtoseconds, depending on the laser's design. Each pulse delivers concentrated energy in a short time, which can produce high peak power during those intervals, making pulsed lasers suitable for applications requiring precise and high-intensity light in short, controlled phases. These pulses can be directed in the form of beams, which can be used to sort cells in a microfluidic flow. These regular pulses can be generated as regular pulses using controlled devices or by switching continuous energy beams. Example regular pulses include laser pulses as well as electrostatic or electromagnetic pulses. Pulses can be separated from adjacent pulses by the inter-pulse time period.

[0106] The term "transit time" refers to the time it takes for a cell in a microfluidic flow to travel from an interrogation zone or cell detection zone exposed to detection radiation to a sorting zone or region. The sorting zone or region may include a sorting arrangement configured to expose cells to at least one regular pulse to sort cells based on detection.

[0107] The term "nudge" refers to applying at least one of force and torque to cells within a population to induce at least one of displacement and orientation of those cells relative to an axis defined by the direction of microfluidic flow. This alters the trajectory of a population of cells compared to different populations, allowing them to be collected individually or otherwise further processed. In some examples, this displacement or orientation can be achieved using pressure applied to selected or unselected cells via a cell-directed impingement laser or bubble.

[0108] The term "pulsed laser" is intended to refer to any arrangement configured to generate regular laser pulses. Examples may include a laser that generates regular laser pulses at a predetermined rate when powered on, or a continuous laser paired with a switching or blocking arrangement configured to deliver regular laser pulses at a predetermined rate.

[0109] The term "designated pulse" is intended to refer to a controlled number of regular pulses used to interact with a number of cells in order to enable sorting. In one example, one or more designated laser pulses may be used to interact with a number of cells.

[0110] Figure 1An illustrative cell processing system 100 is shown, comprising a preparation station 105 that delivers prepared cells to an input arrangement 110 that delivers the cells into a microfluidic flow 115 for downstream processing. The microfluidic flow 115 may be a laminar flow having a predetermined cross-sectional size and transported within a flow environment. In this example, the flow environment may include a volume of gas (such as air) or microchannels that completely or partially surround the microfluidic flow. One or more irradiators 120 generate an interrogation beam, such as an infrared (IR) or ultraviolet (UV) irradiator or other radiation device. The interrogation beam irradiates cells within the microfluidic flow at an interrogation region 125. The radiation to the cells induces emission signals, such as scattered light or fluorescence detected by one or more detectors 130. Measurement characteristics of the detected emission signals generate one or more signals, which are then forwarded to a controller 135.

[0111] In one example, the interrogation beam includes at least one laser source configured to deliver light to a cell to induce bond vibrations in the cell's DNA. In this example, an emission signal is detected and provides characteristics of the bond vibrations used to calculate the amount of DNA carried by the sperm cell. This technique can be used to identify whether a sperm cell carries an X chromosome or a Y chromosome. In one example, the emission signal includes at least one of resonant mid-infrared absorption, non-resonant mid-infrared absorption, and cell scattering. Any of these emission signals can be used to determine cell properties. Another example involves using a quantum cascade laser (QCL) to deliver light to a cell within a microfluidic flow to induce resonant mid-infrared absorption of DNA, one or more analytes from the cell, or another cellular component. After interrogation, a suitable detector, such as a mid-infrared detector, can be used to detect characteristic features of the transmitted mid-infrared wavelength light. This characteristic provides an indication of the quantity or identity of intracellular DNA, analytes, or other cellular components (collectively, "cellular components"), which can be used to identify one or more cellular properties.

[0112] Controller 135 may include a processor and memory and is configured to interpret received emitted signals to control sorting arrangement 140. In one example, the emitted signals include a fluorescence signal. Controller 135 is configured to detect single-cell events, each single-cell event comprising a single cell within a microfluidic flow passing through interrogation region 125. This results in light being received at detector 130, which generates a signal that will be used by the controller to detect the single-cell event, as detailed below. Controller 135 may also be configured to detect multi-cell events, each multi-cell event comprising a densely clustered plurality of cells within a microfluidic flow passing through interrogation region 125. The densely clustered cells of a multi-cell event may not be individually resolved into a single-cell event.

[0113] The controller can also be configured to classify cellular events into selected cellular events and unselected cellular events. This could correspond to classifying cells within at least some of these cellular events into different populations P1 and P2 based on analysis of the emission signals associated with these cells. An example controller 135 configured to classify cells into different populations is described in International Patent Publication WO2022139597A1, which is incorporated herein by reference.

[0114] In some aspects, sorting selected cells or cellular events can include: laser targeting of cells, using fluid pressure to alter the trajectory or orientation of cells, mechanical sorting, piezoelectric actuation, dielectric electrophoresis of droplets, electrolysis or electroporation, optical manipulation, optical trapping, holographic steering, acoustic-assisted hydrodynamic focusing, application of photon pressure, acoustic deflection, laser inactivation, or laser ablation. Sorting can affect at least one of forces and torques on cells in a population to induce at least one of displacement and orientation of these cells relative to an axis defined by the flow direction of the microfluidic flow. When sorting involves the inactivation of cells associated with selected cellular events, this includes imparting energy to the cells associated with the selected cellular events, such energy being sufficient to damage the cells, thereby causing them to become non-viable.

[0115] In certain examples, the sorting arrangement may include: microbubble-based sorting, such as using lasers, sparks, or thermal vapor; pneumatic and solenoid valve-based cell sorting, such as using polydimethylsiloxane (PDMS); or piezoelectric actuation, such as using PDMS valves. In another example, the sorting arrangement may include a radiation source configured to irradiate a microfluidic flow at a target region 145 of the sorting beam. The radiation source is directed to or near cells associated with selected cellular events to cause changes in the orientation, positioning, or direction of travel of the cells, or to achieve inactivation, including ablation or damage to selected cells therein. In this example, the radiation emitted from the radiation source that affects the cells is referred to as the “sorting beam.” In any of the examples provided herein, the sorting beam may include an elongated beam profile. For example, the elongated beam profile may include a line, an ellipse, a rectangle, or a rounded rectangle. In some examples, the radiation source of the sorting arrangement includes a laser and may be configured for pulsed operation. The laser may include a nanosecond, picosecond, or femtosecond laser. The specific characteristics of the sorting beam may vary depending on the desired frequency, power, and wavelength. Furthermore, different cells may require different sorting beams for sorting. Those skilled in the art will be able to determine the required frequency, power, and pulse duration to adapt the sorting beam to the cell type and flow rate. However, in some examples, the sorting beams include frequencies between 100 kHz and 3000 kHz.

[0116] A switch is used to control the emission of a sorting beam toward a microfluidic flow. In one example, the switch includes an acousto-optic modulator or a Pockels cell or an electro-optic deflector or electro-optic modulator. The switch is used to rapidly control the emission of laser toward a cell at an appropriate time and for a sustained desired period of time, referred to herein as a selection period. In some examples, the laser may be pulsed, wherein the selection period includes one or more gated-on periods during which one or more laser pulses are directed toward the microfluidic flow. As detailed below, the one or more gated-on periods are controlled depending on the detection of cellular events for sorting and the timing of the laser pulses. In some examples, the system also includes at least one of free-space optics, optical fibers, and other waveguides configured to direct and focus radiation from a radiation source toward the microfluidic flow.

[0117] This arrangement is particularly useful for removing unwanted cells from a population that includes both desired and unwanted cells. For example, during the generation of a cell population for CAR T-cell therapy, certain types of cells may not exhibit the desired phenotype. Cells in this first population (P1) are disrupted, degenerated, or rendered immobile by the sorting arrangement. An alternative population (P2) of cells not exhibiting the desired characteristics (e.g., desired cells not yet selected by the controller) remains undisturbed. The sorted or processed cells in the microfluidic flow 115 can then be collected in one or more collection containers 160 for further use. The sorting arrangement 140 thus provides a cell population (P2) rich in the desired characteristics. When using sperm cells, this desired population may include motile X cells.

[0118] In one example, the sorting beam includes a laser configured to inactivate cells. Inactivation is the process of transferring energy to a cell that is sufficient to permanently deprive it of vitality for its normal function or purpose. For example, sperm cells can be inactivated to rapidly induce permanent immobility, or sperm cells can be inactivated to “prime” them so that they cannot survive downstream processes such as freezing and thawing. In the former, inactivation may involve complete ablation, which means the cell surface membrane ruptures, thus disrupting cell integrity. During “priming,” the cell surface membrane remains substantially intact, even if motility may be reduced or stopped. Inactivation may also involve damaging intracellular DNA, such as through UV irradiation, or impairing the integrity of extracellular structures essential for survival, such as destroying or removing the sperm cell’s flagella (tail), resulting in a lack of vitality.

[0119] Downstream of the sorting arrangement 140, cells may be collected in one or more collection containers 160p1, 160p2. When separating selected or unselected cells into different flow paths using a force or torque applied to at least one of the two populations, microfluidic channels or other mechanisms may be used to separate desired and unwanted cells, such that the two cell populations are guided to their respective collection containers 160p1, 160p2. In other examples, where selected cells are inactivated but remain within the same flow path as unselected cells, all cells (or cell remnants) may be guided to a single collection container 160p1, which has a higher concentration of live unselected cells (e.g., desired intact cells) to selected cells (e.g., unwanted inactivated cells) compared to the concentration of live desired cells to unwanted cells upstream of the sorting arrangement 140.

[0120] refer to Figure 2a The diagram illustrates a waveform or pulse corresponding to a single event. This waveform extends along the time axis t, and its height corresponds to the amplitude of the signal emitted by the receiving cell associated with the microfluidic flow carrying the cell. Waveform 800a includes a single peak 842a, initially rising from the baseline 822 before falling back to the baseline. The peak in the waveform corresponds to the detection of the cell head and / or body in a single-cell event. In the example of a sperm cell, the cell comprises a large head, a smaller middle portion, and an elongated tail; in the case of a sperm cell, the peak primarily corresponds to the head portion of the cell.

[0121] The rising edge threshold 824 is the waveform height or amplitude used to detect the rising edge of a waveform. A rising edge 860 is detected at time tr when the received transmitted signal rises above the rising edge threshold from the baseline 822. The baseline 822 can be zero, signal noise floor, or any other suitable data. The rising edge threshold 824 can be determined experimentally or through trial and error. In one example, the rising edge threshold or falling edge threshold (described below) is set as follows:

[0122] a. Determine the maximum amplitude of the background noise level;

[0123] b. Determine the minimum amplitude of the waveform generated by the cell;

[0124] c. Set the threshold level to be greater than a but less than b.

[0125] A falling edge is detected at time tf 865, where the intensity decreases from the peak value 842a and drops below the falling edge threshold 864 at falling edge (tf) 865. The falling edge threshold can depend on the waveform 800a, for example, and may be set as a percentage of the average height 852 of the waveform or the maximum height of the waveform. In one example, the falling edge threshold 865 is set below the rising edge threshold 860. This is to reduce the possibility of transient error triggering due to high-frequency noise that may fluctuate rapidly above and then below the threshold.

[0126] The time or duration between the detected rising edge 860 (tr) and the detected falling edge 865 (tf) is the waveform (or pulse) width 854. It should be understood that the waveform width for single-cell events will be similar, but the waveform width for multi-cell events will vary depending on the number of densely aggregated cells and their spatial distribution within the microfluidic flow. Multi-cell events can be detected by assessing whether the waveform width exceeds a predetermined time, where this time is specific to single-cell events.

[0127] refer to Figure 2b The diagram illustrates a waveform or pulse corresponding to a multicellular event. This waveform extends along the time axis t, and its height corresponds to the amplitude of the received and transmitted signals associated with the microfluidic flow carrying the cells. Peaks in the waveform correspond to cellular events, which in this multicellular event represent multiple densely clustered cells. Depending on how the cells in the multicellular event are positioned along the X, Y, and Z axes of the microfluidic flow and how they are oriented within it, not all cells are capable of producing peaks. For example, in cases where the cells are substantially transparent, the beam can diffract around the cells, and when two signals are close together or overlap, this causes the two signals to merge into a single large peak. Waveform 800b includes three peaks 842b, 844b, and 846b, and initially rises from the baseline 822 before falling back to the baseline.

[0128] As described above, the rising edge threshold 824 is the waveform height or amplitude used to detect the rising edge of the waveform. When the received transmitted signal rises from the baseline 822 to above the rising edge threshold, a rising edge 860 is detected at time tr.

[0129] A falling edge is detected at time tf (865), and a falling edge threshold of 866 is used to determine whether one of the intensity drop slopes following peaks 842, 844, and 846 exceeds this threshold. The falling edge threshold can be set in several ways to trigger falling edge detection and thus indicate cellular events.

[0130] a. In one example, the fall edge threshold 866 is defined by a measure of the emission intensity at a predetermined height above the calibration baseline 822. For example, this could be a predetermined percentage of the maximum or average waveform height relative to a representative number of the measured waveforms. In this example, a fall edge 865 is detected when the intensity drops below this absolute value.

[0131] b. In another example, the falling edge threshold 866 is defined by a relative intensity decrease of 858b from the peak maximum value 846b.

[0132] c. In another example, the falling edge threshold depends on the average height of the waveform corresponding to a similar cell, for example, a predetermined percentage (e.g., 50%) of the average peak height maximum of 852 of a representative number of previously measured waveforms.

[0133] Any method for defining a falling edge threshold (and thus a falling edge event trigger) may be used in the methods of the invention described herein.

[0134] The falling edge threshold 866 for multi-cell events may differ from the falling edge threshold for single-cell events, for example, to accommodate adjacent peaks of similar or different heights within the waveform. The falling edge ramp following the second peak 844b extends downwards by a distance or height 856, which in this example is insufficient to exceed the falling edge threshold 866 and therefore does not trigger the detection of a falling edge. The falling edge ramp following the third peak 846b extends beyond the falling edge threshold and thus triggers the detection of a falling edge 865. In one example, the falling edge threshold 866 is set below the rising edge threshold 824. This is to reduce the possibility of momentary false triggering due to high-frequency noise that may fluctuate rapidly above and then below the threshold.

[0135] The time or duration between the detected rising edge 860 (tr) and the detected falling edge 865 (tf) is the waveform (or pulse) width 854. It should be understood that the waveform width for single-cell events will be similar, but the waveform width for multi-cell events will vary depending on the number of densely clustered cells and their spatial distribution within the microfluidic flow.

[0136] Figures 3a to 3d The waveforms of cellular events, peak detection, and cell inactivation upon receiving the emitted signal are illustrated. The emitted signal described herein may include fluorescence emission signals. Figure 3a A single-cell event waveform of 210s is illustrated, and Figures 3b to 3dA multi-cell event waveform 210m is illustrated. Single-cell events are associated with single cells 205s passing through the cell processing system, and multi-cell events are associated with multiple cells 205m densely clustered through the system. The densely clustered cells 205m may overlap, making it difficult or impossible to resolve these cells individually, for example, using the corresponding single-cell event waveform.

[0137] For single-cell events, a peak detection signal was detected in the waveform at 215 s. The peak detection signal can be triggered in various ways. For example, it can be detected in the following ways:

[0138] a. Determine the maximum height or amplitude of the received transmitted signal, for example by determining when the transmitted signal rises above the rising edge threshold and then the gradient reaches 0;

[0139] b. Determine the time point at which the transmitted signal drops below the falling edge threshold after first exceeding the rising edge threshold; or

[0140] c. A peak detection signal is generated for 215 seconds whenever the current amplitude drops below the moving average value by generating a moving average value of the received transmitted signal within a short window.

[0141] exist Figure 3a In one example shown, a falling edge threshold 212 is illustrated. In one example, this could be a predetermined percentage of the maximum or average waveform height or intensity relative to the representative number of measured waveforms within a local window.

[0142] For this peak detection method, it can be seen that the actual peak maximum value precedes the detection of the falling edge, and this difference defines the "detection delay time". If needed, this detection delay time can be considered using an estimate of the detection delay time between peak detections. This detection delay time can be estimated or determined experimentally or in any other suitable manner. The cell transit duration is defined by the time between cell detection and the effect of the cell transfer sorting arrangement structure. This duration can be determined experimentally, for example, by selecting all cells for sorting and maximizing the total number of sorted cells. For example, using an inactivating laser as the target for sorting all cells, the actual number of inactivated cells can be measured while varying the duration to achieve the maximum number of inactivated cells. For a given cell velocity and distance between the interrogation beam in interrogation region 125 and the sorting arrangement structure 140, the predetermined cell transit duration (PCTD) can then be used to set the selection period for a specific cell event or the end of the on-time (when the cell is no longer affected by the sorting arrangement structure). Various other peak detection and sorting methods can also be used alternatively.

[0143] A trigger signal is activated at 220 s for a selection period, pulse, or on-time, which begins after a determined offset delay or time following the peak detected at 215 s, so as to coincide with the cells selected for sorting at 205 s. In this example, both the selection period and the offset delay are predetermined, for example, a selection period of 1 μs, and the offset delay has a predetermined duration, for example, 61 μs. The end of the selection period can be set at a predetermined cell transit time after the detection of a cellular event. This type of triggering is referred to herein as single-cell static timing selection triggering; however, an adaptive timing selection triggering mode in which the offset delay and / or selection period can be adjusted may alternatively be used, as detailed below.

[0144] In the static timing selection trigger example of the present invention, the predetermined offset delay may correspond to the cell passing through interrogation region 125 and... Figure 1 The predetermined offset delay is the duration between the sorting arrangements 140 (predetermined cell transit time (PCTD)) minus the predetermined selection period or on-time. In one example including an electrostatic cell sorting arrangement, the predetermined offset delay may correspond to a droplet delay value, which is the time between a cell passing through the interrogation region 125 and reaching the droplet detachment point, at which charge is applied to the droplet containing the target cell for downstream sorting. In another example where selection leads to activation of the inactivation laser, the predetermined offset delay may correspond to the time taken for a cell to move from the interrogation beam to the sorting region or for the inactivation beam to be activated.

[0145] refer to Figure 3b In one example of a multi-cell event including cells A, B, and C, the peak detection signal 215m indicates the time at which a falling edge is detected in the waveform. This falling edge is detected when the emission intensity decreases beyond a predetermined falling edge threshold, shown as 212m. In this case, because the falling edge threshold is crossed only once, only one peak can be resolved, corresponding to cell A. This results in cells B and C not being able to be evaluated for sorting.

[0146] exist Figure 3c In the alternative detection method shown, the falling edge thresholds 212-1 and 212-2 are variable and can be set by decreasing the intensity from the peak maximum. The peak maximum can be detected in any way, such as when the gradient is 0. In this case, two peaks corresponding to cells A and B are detected. In this case, it can be seen that there are two well-resolved peaks that are detected, although a third undetected peak can also be seen.

[0147] The laser trigger signal 220m for the falling edge or local maximum of the detected multicellular event includes one or more pulses 221 and 222 with a predetermined offset delay or time following the peak detected in 215m, so as to coincide with cells 205m selected for sorting (e.g., via inactivation). The predetermined offset delay may correspond to the detected cells passing through interrogation region 125 with... Figure 1 The duration between the sorting arrangement structures 140. Each selection period or laser activation period can be a predetermined duration. The sorting action applied to the cells is represented by a star above the cells in the diagram. Figure 3c As can be seen in the diagram, the predetermined offset delay after cell A in the laser trigger signal selection period or laser on-time on the right side 221 is consistent. The detected cells and the laser trigger signal selection period or laser on-time on the left side 222 are consistent with the predetermined offset delay after cell B is detected. Since cell C, which was not detected, is outside the selection period, it is not selected for sorting or not laser-processed during the laser on-time. The waveform corresponding to peak C is not evaluated or processed. This can lead to inaccurate cell count measurements and may result in contamination of the collected sample if cell C is one that should be selected for sorting. This illustrates a problem that can occur in multi-cell events where densely clustered cells cannot be resolved well and not all cells can be processed. Specifically, as Figure 3b As shown, if only one falling edge is to be triggered during a multi-cell event, the cells in the multi-cell event may not be selected and therefore will not be processed to improve the purity of the collected cell sample.

[0148] refer to Figure 3d In one example of a multicellular event, a single selection period can be triggered after the end of the multicellular event is detected. The duration of the single selection period can be predetermined to align with the expected start and end times of multiple overlapping waveforms. For example, by determining the average time from the rising edge to the falling edge. However, as... Figure 3d As shown, a falling edge threshold of 212m results in the detection of two cells at 215m, even though three cells are actually present. The triggering of a single selection period or on-pulse with a predetermined offset delay after the end of the multi-cell event detection is referred to herein as multi-cell static timing selection triggering.

[0149] The proximity of two cells in time can trigger a multi-cell mode. Although two cells were detected, and the selection period or laser on-time 223 was extended to select cells A and B for sorting, cell C was not selected.

[0150] In one example, a predetermined offset delay such as 61 μs and a predetermined selection period or opening period such as 1 μs can be used.

[0151] Figure 4 The diagram illustrates cellular event waveforms of received transmission signals, peak detection, and cell inactivation, based on several examples. A single-cell event waveform 310s is illustrated on the left, and a multi-cell event waveform 310m is illustrated on the right. Single-cell events are associated with a single cell 305s passing through the cell processing system, while multi-cell events are associated with multiple closely grouped cells 305m passing through the system.

[0152] It should be noted that, for example, sorting via inactivation is only associated with selected cell events, and cell events not classified as selected may not undergo sorting actions such as inactivation. Various factors can be used to configure whether a cell event is classified as selected or unselected, such as whether the cell event is a single-cell event or a multi-cell event and / or whether one or more cells associated with the cell event are desired or unwanted cells. In one example, cell X could be unselected (i.e., desired), and cell Y could be selected (i.e., unwanted).

[0153] For a selected single-cell event, the detected falling edge 315s of the received transmitted signal is determined at the end of waveform 310s, as described below. The laser trigger signal 320s includes a pulse, an on-time period, or a selection period 325s, which begins after an offset delay of 330s following the detected falling edge 315s. For a selected multi-cell event, the detected falling edge 315m of the received transmitted signal is determined at the end of waveform 310m. The laser trigger signal 320m includes a pulse, an on-time period, or a selection period 325m, which begins after an offset delay of 330m following the detected falling edge 315m. Example selection trigger modes for single-cell and multi-cell events are described in more detail below.

[0154] It can be seen that the selection period of 325m for multi-cell events is longer than the selection period of 325s for single-cell events. This allows for sorting of multiple cells within a multi-cell event, for example, via inactivation. The end of the selection period can be a predetermined time or duration after the end of waveforms 310s and 310m detected using the falling edge of the generated waveform. In one example, this could correspond to the last (or only) cell passing through in a cell event. Figure 1 The time difference between the system's inspection area 125 and the cell flowing along the microfluidic flow through the sorting beam 140. The offset period 330m for multi-cell events is correspondingly shorter than the offset period 330s for single-cell events because the selection period or on-pulse is longer in multi-cell events.

[0155] Waveforms associated with cellular events can be used to classify the waveform and / or one or more cells associated with it. Characteristics of the waveform can be used to classify associated cellular events as unselected or selected.

[0156] The waveform has a waveform width (or pulse width PW) corresponding to the time difference between the detected rising edge and falling edge.

[0157] In one example, the integral of the waveform can be determined and used to classify cells into, for example, unselected X cells and selected Y cells. The integral represents the total amount of light collected during the time the cell crosses the interrogation beam. This measurement is more stable and less susceptible to noise compared to the peak maximum. The integral of the waveform is calculated between two time points, which can be labeled a and b. In one example, a and b are equidistant before and after the peak maximum. In another example, a and b are chosen to intersect with a threshold that is selected to be higher than the background noise level detected by the detector.

[0158] In one example, the peak value, or height, is used to classify cells. This corresponds to the maximum amplitude of the signal generated when a cell passes through an interrogation beam. It is proportional to the intensity of light scattering or fluorescence emitted from the cell.

[0159] Cell events can be classified as selected or unselected based on whether they include unwanted or desired cells. In cases where the classification of cells within a cell event cannot be determined, the cell event can be classified as selected. This may occur when cells in a multi-cell event are too tightly clustered to be individually resolved and classified. The sorting arrangement can then perform sorting of one or more unwanted or desired cells from the cell event based on the classification of the cells or cell event.

[0160] All 305m cells fell within the selection period and were therefore all selected for sorting. In some examples, this might be desirable. For instance, when selecting cells with unwanted properties for inactivation, it might be preferable to inactivate any cells that cannot be resolved, rather than allowing those cells to potentially contaminate unresolved desired cells and reduce the enrichment of the desired properties. However, this strategy of inactivating all unresolved cells could waste cells that would be considered desired if resolved.

[0161] The method described in this article can provide one or more advantages, including:

[0162] 1) Enhance the enrichment of desired cellular properties by improving cell sorting. In one example, the sorting action prevents unwanted cell progression (especially during multicellular events).

[0163] 2) Increase cell throughput while keeping cell selection metrics constant. That is, allow fewer unwanted cell types to pass through, meaning that a sorting arrangement can be used to collect higher cell concentrations. This has the effect of increasing the speed at which the sample passes through the system. This advantage can be particularly important when cells degrade based on the time spent flowing through the cell processing system. Therefore, increased cell throughput can ultimately lead to more viable collected cells due to the reduced sample flow time through the system.

[0164] 3) Real-time monitoring of cell and culture medium quality / characteristics. For example...

[0165] a) Sample quality,

[0166] b) Cell quality

[0167] c) Mass of the sheath fluid or buffer fluid.

[0168] In some examples, the offset delay from the detection of a single-cell or multi-cell event to the selection period that triggers the event can be predetermined, i.e., static, such as 61 μs and 5 μs. As mentioned herein, this form of triggering is referred to as static timing selection triggering, with single-cell events as an example. Figure 3a As shown, an example of a multicellular event is as follows: Figures 3b to 3d As shown. The actual time will depend on the settings of the cell processing system used, for example... Figure 1 The velocity of the microfluidic flow and the distance between the detection area 125 and the sorting arrangement structure 140.

[0169] In the following example, the offset delay or timing used to trigger the selected time period is adaptive. Figure 5a An example of applying sorting action to a single-cell event is illustrated, and graphs of the received emission signal, sorting trigger signal, laser pulse, and cell are shown from top to bottom; when queried to generate a waveform in the received emission signal (e.g., at detection area 125), the cell appears gray, while it appears black when sorted via inactivation (e.g., at sorting arrangement structure 140).

[0170] The received transmitted signal includes a waveform corresponding to the detection of a single-cell event—a gray cell. This waveform includes a detected rising edge and a detected falling edge. The waveform has a waveform width (or pulse width PW) corresponding to the time difference between the detected rising and falling edges. The trigger signal includes a selection period or on-time, which begins after an offset delay following the detected falling edge. Example times for waveform width (15 μs), offset delay (51 μs), and selection period or on-time (15 μs) are shown; however, it should be understood that other durations of these parameters may be used.

[0171] In this example, an adaptive timing selection trigger mode is used. In this trigger mode, the selected period or on-time (e.g., 15 μs) is set to be the same as or similar to the waveform width (15 μs). Figure 5a In the example, the selected time period corresponds to three laser pulses as indicated, resulting in three interactions with the cell—illustrated as a star shape. Figure 3a Compared to the shorter selection period in the example, Figure 5a The extended selection period can be used in different ways. For example, an additional laser pulse can be used to apply additional energy to selected cells to increase their likelihood of inactivation. Similarly, using a longer selection period can increase the likelihood of the laser pulse striking a specific part of the cell (e.g., the cell body), which can improve, for example, inactivation. On the other hand, a laser with lower power can be used, where the energy delivered to the cell diffuses over a longer selection period, which can still inactivate the target cell but may reduce collateral damage to other cells—for example, unselected or desired cells (not shown) can be oriented such that their tails overlap with a portion of the cell being inactivated. In this scenario, reducing the laser power can reduce its effect on the tail of the desired cell while still being sufficient to inactivate the cell selected for inactivation.

[0172] In other examples, the offset delay and / or on-time or selection period can be adjusted based on factors such as the number or type of cells, cell size, rotational orientation, z-axis orientation, or the sorting type used (e.g., inactivation via ablation, or a method that causes less damage but is still sufficient to inactivate cells). For example, a longer on-time or selection period, such as 35 μs, can be used. This allows more energy input to the cells, potentially leading to complete ablation (i.e., cell membrane rupture); in contrast, less energy input may only result in cell damage, ultimately leading to loss of activity (i.e., cell pretreatment). In this case, a shorter offset delay is used, which is the previously predetermined offset minus the increase in on-time.

[0173] Alternatively, a predetermined offset delay corresponding to the end of the selection period (when the cells pass through the sorting process) can be used to subtract the selection period, for example, 66-15=51μs. In one example, the on-time can be modified to minimize the possibility of collateral damage to neighboring cells. For example, the selection period or on-time can be reduced to avoid affecting cells outside the target cell event. For instance, the tail of a desired sperm cell may be oriented in a way that extends into the (non-target) cell event, and if the selection period is not shortened, this tail would otherwise suffer unwanted damage from the laser pulse. Similarly, heating the fluid around the target cell may affect nearby desired cells, and therefore shortening the selection period can mitigate this effect.

[0174] In another example, the on-time can be modified to compensate for variations in cell positioning. For instance, if the detected cell is outside the focal point of the sorting beam, the on-time can be increased to allow more energy to be supplied to the cell, thus compensating for the lower intensity. In yet another example, the offset delay is altered to account for the cell's z-axis orientation (head-to-tail, tail-to-head, off-axis, etc.).

[0175] In some examples, the selected time period can correspond to the gating opening period and can be timed to span multiple laser pulses from a regular pulse laser sorting arrangement. (As per...) Figure 16 and Figure 17 In a further example, the gating opening period within a selected time period is timed to ensure that the full laser pulse is directed to the cell or microfluidic flow. This concept of timing and selecting pulses during a selected time period can be further extended to, for example, regarding… Figure 18 and Figure 19 The description includes multiple cellular events and multiple pulses. In these examples, an optical switch can be used and controlled to be on only during a predetermined gating period around each laser pulse within a selected time period.

[0176] In some examples, the sorting beam comprises an inactivating laser, which includes pulsed lasers such as nanosecond, picosecond, or femtosecond lasers. In one example, the pulse duration is between approximately 10 femtoseconds and 100 nanoseconds. Those skilled in the art will understand that different pulse durations will affect laser properties (such as energy and power output) and will influence the physical behavior of the interaction between the cell and the laser beam. In some examples, the pulse duration may also affect other aspects such as repetition rate and beam quality.

[0177] In one example, the sorting beam comprises an inactivating laser having a wavelength in the IR, UV, or visible wavelength range. In another example, the sorting beam has an X-ray or gamma-ray wavelength. In one example, the sorting beam has a wavelength between approximately 100 nm and 400 nm, 380 nm and 780 nm, or 760 nm and 10 μm. In some examples, a sorting beam with a UV wavelength is used. This has the advantage of exciting certain DNA staining agents commonly used for cell interrogation (e.g., Hoechst-33342). Therefore, stained cells will absorb the stain better, which can provide the advantage of being able to use a lower power beam compared to other non-UV wavelengths.

[0178] During a selected time period, on-time, or gated on-time, the sorted beam can be controlled to guide it to the microfluidic flow. The number of laser pulses applied to the microfluidic flow (and any cells overlapping with the laser) will depend on the selected time period, timing, and inter-pulse interval. Figure 6a In the example shown, one laser pulse is applied to the (black) cell; however, in other configurations, when the selected time period is long and / or its start time is the same as or close to the laser pulse, multiple laser pulses can be applied to the cell, such that two or more laser pulses can be included in the selected time period - this also depends on configuration parameters such as the laser pulse frequency and the duration of the selected time period.

[0179] In one example, the z-axis length of the cell is measured, and a laser with a sufficient repetition rate is turned on before or simultaneously with the cell entering the sorting beam target region 145, and the laser is turned off after the cell (or a portion of the cell) leaves the sorting beam target region. The duration between laser on and off (also referred to herein as a selection period or gated on period) or the length of the target region can vary with the cell length to ensure that the cell receives a predetermined number of pulses. Optionally, the sorting beam target region can be larger than the cell length by one cell length to compensate for variations in cell velocity. Those skilled in the art will understand that references to turning the laser on or off herein are intended to indicate allowing the laser to interact with the cell or microfluidic flow, rather than indicating the power state of the laser itself. Switching from on to off and from off to on can be achieved through a switching arrangement such as a pulse pickup, wherein the laser can be guided away or blocked during the off period and guided toward the cell or microfluidic flow during the on period or gated on period.

[0180] The selection of time periods can be achieved using an acousto-optic modulator or other switches to control the emission of laser pulses toward the microfluidic flow during the selected time period—indicated, for example, by the black laser pulses within the “on time” delimitation line in Figures 5 and 6. At other times, the laser pulses can be directed elsewhere or absorbed / blocked—indicated, for example, by the gray laser pulses outside the “on time” delimitation line in Figures 5 and 6.

[0181] In some examples, when inactivation-based sorting is employed, the laser can be tuned to have low absorption in water (or other carrier media) and high absorption in cells.

[0182] In some examples, high-repetition-rate pulsed lasers can be used, thereby shortening the selection period or the on-time. In this case, high repetition rate means that the z-axis length of each cell head is greater than one pulse. For example, for selection periods >10 μs, lasers of 200 kHz to 500 kHz can be used to achieve multiple-emission cell targeting. In another example, for selection periods of 0.5 μs to 5 μs, lasers of 500 kHz to 5000 kHz can be used to achieve multiple-emission cell targeting.

[0183] Other examples may employ one or more of the following laser characteristics: a small focal spot on the z-axis (travel direction); high beam quality, such as low M... 2 Uniform illumination across the flow (line profile); energy density sufficient to ablate cells.

[0184] Figure 5b An example of sorting multi-cell events using an adaptive timing-based selective triggering process is illustrated. It shows graphs from top to bottom of the received signal, trigger, pulsed laser, and cells; when interrogated to generate a waveform in the received signal (e.g., at detection area 125), the cells appear gray, while during sorting they appear black (e.g., at sorting arrangement structure 140).

[0185] As previously noted, the predetermined cell transition duration refers to the time required from the moment a cell is detected in the interrogation area or detection location until it becomes susceptible to the sorting process (such as the application of a laser or the action of an electrostatically charged sorting plate) in the sorting area or sorting location. This will depend on the configuration of the cell processing system used, for example... Figure 1 The velocity of the microfluidic flow and the distance between the detection area 125 and the sorting arrangement structure 140.

[0186] The received transmission signal includes a waveform corresponding to the detection of a multicellular event—gray cells. This waveform includes a detected rising edge and a detected falling edge exceeding a falling edge threshold as previously described. The waveform has a waveform width (or pulse width PW) corresponding to the time difference between the detected rising and falling edges. The trigger signal includes a selection period or on-time that begins after an offset delay following the detected falling edge. The end of the selection period may correspond to cell detection (e.g., using the previously described falling edge thresholding technique) plus a predetermined cell transition duration. The offset delay can be calculated by subtracting the waveform width from the predetermined cell transition duration. Example times for the waveform width (34 μs), offset delay (32 μs), and selection period or on-time (34.5 μs) are shown; however, it should be understood that other durations for these parameters may be used. For example, the waveform width may be less than 50 μs, for example, in the range of 0.1 μs to 50 μs. In some examples, the offset delay may be in the range of 0.5 μs to 50 μs, and the selection period or on-time may be in the range of 0.5 μs to 100 μs. These values ​​depend on a range of factors, such as the beam distance between the interrogation beam and the sorting beam, the speed at which cells pass through the system, the processor speed at which it resolves and determines whether a cell is selected, the actuation time of any sorting arrangement, pump capacity, microfluidic pressure limitations, or other factors. Those skilled in the art will understand that adjustments are made based on these parameters and appropriate experimental procedures to achieve effective cell targeting. The impact of these parameter variations on cell targeting outcomes can be assessed by methods including visual inspection or cellular fluorescence detection during sorting, or by downstream measurements of the purity percentage of selected or unselected cells according to known methods.

[0187] In this example, with Figure 5a Similar to single-cell events, an adaptive timing selection trigger mode is used. In this trigger mode, the selected time period or on-time (e.g., 15 μs) is set to be the same as or similar to the waveform width (15 μs). A predetermined duration is used to calculate the offset time period, which corresponds to the time it takes for a cell to traverse from the detected examination area to the end of the sorting or laser-activated area. As previously noted, the actual time will depend on the settings of the cell processing system used, such as... Figure 1 The velocity of the microfluidic flow and the distance between the detection area 125 and the sorting arrangement structure 140 are considered. In this example, the predetermined duration is 66 μs, after which the cells are no longer affected by the sorting beam. The offset delay is calculated by subtracting the waveform width of 34 μs from the predetermined duration—in this example, 66 μs - 34 μs = 32 μs. This allows the sorting beam to act on all cells within a multicellular event.

[0188] exist Figure 5b In the example, the selected time period corresponds to seven laser pulses as indicated, resulting in seven interactions with the cell—illustrated as a star shape. Figures 3b to 3d Compared to the shorter selection period in either of the examples, Figure 5b The use of extended selection time in the process allows all cells within a multicellular event to be targeted by the laser pulse.

[0189] exist Figure 5b In the example, the offset delay and / or on-time or selected period are adjusted based on the waveform width. As noted above, in this example, the selected period or on-time is the waveform width of 34 μs, and the offset delay is the predetermined cell transition duration (PCTD) minus the selected period (or waveform width, if kept constant), for example, 66 (predetermined) - 34 (width) = 32 μs. This is described by the following equation:

[0190] Equation 1: OD = PCTD - WW

[0191] Where OD is the offset delay, PCTD is the predetermined cell transition duration, and WW is the waveform width or duration. This method allows the duration of the sorting process to correspond to the period during which cells can be affected by the sorting process—in other words, the duration of the waveform width corresponds to the time when cells with multi-cell events are detected, and the sorting process is applied for that same time (selection period) but delayed to allow the detected cells to reach the sorting process. This arrangement ensures that all cell heads of the detected sperm cells pass through the focus of the inactivation laser.

[0192] The selection of the time interval can be adjusted based on factors such as cell type, size, z-axis orientation, or the sorting method used (e.g., ablation, pretreatment, or other methods). For example, extending the on-time allows more energy to be delivered to the cells, resulting in complete ablation, while shortening the on-time reduces the likelihood of collateral damage to nearby cells in need. The selection of the time interval can correspond to, for example, the selection of the time interval related to, cell type, size, z-axis orientation, or sorting method used. Figures 16 to 19 The described method utilizes a gated opening period with a conventional pulsed laser, where an optical switch directs selected pulses to cells or microfluidic flows at specific intervals. This enhances sorting and reduces collateral damage to the desired cells.

[0193] In one example, the waveform width is greater than a predetermined delay offset, for instance, where a cluster of cells provides a large number of overlapping emission events. In this case, the calculation above for the delay offset period may result in a negative time, or the calculation may not be completed before the cell passes the sorting beam position. To avoid this, the system can be configured to detect long multi-cell events and initiate the selection period once the waveform width exceeds a threshold waveform width (e.g., a predetermined delay).

[0194] In another example, the offset delay can be based on a proportion of the waveform width. As mentioned in this article, this process is referred to as adaptive selection triggering for adaptive timing. It can be described as follows:

[0195] Equation 2: OD=PCTD-aWW-b

[0196] Where PCTD is the predetermined cell transition duration, a is a coefficient multiplied by the waveform width WW, and b is a constant.

[0197] Figure 6a An example of applying sorting action to a single-cell event is illustrated, and graphs of the received emission signal, sorting trigger signal, laser pulse, and cell are shown from top to bottom; when queried to generate a waveform in the received emission signal (e.g., at the detection or inspection area 125), the cell appears gray, while it appears black when sorted via inactivation (e.g., at the sorting location or arrangement structure 140).

[0198] As previously described, the received transmitted signal includes a waveform corresponding to the detection of a single-cell event—a gray cell. This waveform includes a detected rising edge and a detected falling edge. The waveform has a waveform width (or pulse width PW) corresponding to the time difference between the detected rising and falling edges. The trigger signal includes a selection period or on-time, which begins after an offset delay following the detected falling edge. In this example, an adaptive selection trigger mode with adaptive timing is used. In this trigger mode, the selection period or on-time (e.g., 4 μs) is calculated based on a proportion of the waveform width (e.g., 15 μs). The selection period can be calculated by multiplying the waveform width by a coefficient a and adding a constant b. This is described by the following equation:

[0199] Equation 3: SP (selected time period) = aWW (coefficient × waveform width) + c (constant)

[0200] Similarly, the offset delay can be calculated by multiplying the waveform width by a coefficient and subtracting that coefficient and a constant from the predetermined cell transition delay. This is described by the equation shown above for OD. See below for reference. Figure 8The coefficients a and b are described in more detail.

[0201] exist Figure 6a In the example, the selected time period corresponds to a single laser pulse as indicated, resulting in a single interaction with the cell—illustrated as a star shape. Figure 5a Compared to the longer selection period in the example, Figure 6a The shorter selection period can be used in different ways. For example, reducing the number of laser pulses can avoid collateral damage to nearby desired cells, including those that may partially overlap with the target cells. A reduced number of laser pulses can be used to avoid complete cell ablation while still inducing their eventual inactivation (as described in the previous pretreatment).

[0202] Figure 6b An example of applying sorting action to a multi-cell event is illustrated, and graphs of the received emission signal, sorting trigger signal, laser pulse, and cells are shown from top to bottom; when asked to generate a waveform in the received emission signal (e.g., at detection area 125), the cells appear gray, while when sorted via inactivation, they appear black (e.g., at sorting arrangement structure 140).

[0203] As previously described, the received transmitted signal includes a waveform corresponding to the detection of a multicellular event—gray cells. This waveform includes a detected rising edge and a detected falling edge. The waveform has a waveform width (or pulse width PW) corresponding to the time difference between the detected rising and falling edges. The trigger signal includes a selection period or on-time, which begins after an offset delay following the detected falling edge. In this example, an adaptive selection trigger mode with adaptive timing is used. In this trigger mode, the selection period or on-time (e.g., 34 μs) is calculated based on a proportion of the waveform width. The selection period can be calculated by multiplying the waveform width by a coefficient a and adding a constant b. This is described by Equation 3 above.

[0204] Similarly, the offset delay can be calculated by multiplying the waveform width by a coefficient and subtracting that coefficient and a constant from the predetermined cell transition delay. This is described by Equation 2 above.

[0205] exist Figure 6b In the example, the selected time period corresponds to six laser pulses as indicated, resulting in six interactions with the cell—illustrated as a star shape. Figure 5b Compared to the longer selection period in the example, Figure 6bThe shorter selection period can be used in different ways. For example, reducing the number of laser pulses can avoid collateral damage to nearby desired cells, including those that may partially overlap with the target cells in a multicellular event. A reduced number of laser pulses can be used to avoid complete cell ablation while still inducing their eventual inactivation (as described in the previous pretreatment).

[0206] Such as about Figure 5a , Figure 5b , Figure 6a , Figure 6b As described, during a selected time period or on-time, the sorting beam can be controlled to be guided to the microfluidic flow. The number of laser pulses applied to the microfluidic flow (and any cells overlapping with the laser) will depend on the selected time period and timing. Figure 5a and Figure 5b In the example shown (selective triggering of adaptive timing), and Figure 6a and Figure 6b Compared to the example (adaptive selection triggering with adaptive timing), more laser pulses are applied to (multiple) (black) cells. In other configurations, different numbers of laser pulses can be applied for the same (or different) waveform widths. These laser pulses are sufficient to sort all cells in a multi-cell event and inactivate them.

[0207] In some examples, according to about Figures 16 to 19 The method described is for selecting laser pulses.

[0208] The selection of the time period can be achieved using an acousto-optic modulator or other switches to control the emission of the laser pulse toward the microfluidic flow during the selected period. At other times, the laser pulse can be redirected or absorbed / blocked—indicated by the gray laser pulse.

[0209] While some examples determine the waveform width of a cell event by using the rising and falling edges of the waveform, others may use different methods. In one example, a peak or maximum value may be determined for the waveform, and predetermined durations or "widths" may be added before and after the peak to provide an estimated waveform width that can be used in the previously described adjustments to the selected time period and / or delay offset. These additional predetermined durations may be determined based on experimental and / or analytical analysis of waveforms from multiple cell events. In some examples, the predetermined durations applied before and after the peak may differ.

[0210] In some examples, where a cell event comprises multiple peaks, a predetermined time interval can be added after the last peak and before the first peak to determine the total waveform width of the multi-cell event.

[0211] In other examples, the falling edge of the waveform, as previously described, can be used to determine the duration or period following the peak. This duration or period can then be additionally added before (and after) the detected peak to determine the estimated waveform width. In the case of multi-celled events with multiple peaks, the duration between the last peak and the falling edge can then also be added before the first peak and any intermediate peaks to determine the waveform width.

[0212] like Figure 10 As shown, a sperm cell includes a head 1007, a midpiece 1008, and a tail 1011. The tail can be divided into a principal component 1009 and a terminal component 1010. The head includes a plasma membrane 1001, an acrosome 1002, and a nucleus 1003. The nucleus contains genomic DNA. The head is connected to the midpiece via a connector 1004. The midpiece contains mitochondria 1005 within a mitochondrial sheath that spirally wraps around the midpiece of the tail and provides the energy required for tail movement. The tail moves back and forth in a whip-like motion to propel the sperm toward the egg. Mitochondria contain their own genome (mitochondrial DNA, “mtDNA”), which encodes a limited number of genes. There are 5 to 10 copies of mtDNA in a mitochondrial cell, and in the case of somatic cells, there are 1000 to 5000 copies in a single cell. Sperm cells have approximately 50 to 75 mitochondria, and each mitochondrial contains an average of one copy of mtDNA.

[0213] The inventors have surprisingly discovered that mtDNA can be used to enhance the sorting and targeting of sperm cells stained with DNA-specific staining agents. This technique involves detecting waveforms with sufficient fidelity to identify genomic DNA (gDNA) from the cell nucleus, which produces gDNA peaks, and mitochondrial DNA (mtDNA) from the midsection. The relationship between the mtDNA peaks and gDNA allows the user to determine at least one property of the cell, selected from: z-axis orientation (i.e., head-first or tail-first); and the alignment of the cell along its longitudinal axis (head-to-tail) relative to the z-axis flow direction.

[0214] The emission characteristics of mtDNA are significantly smaller than those of gDNA because the amount of DNA present in the midsection of the sperm cell is correspondingly lower than that in the head. Therefore, the detected cell emission will produce a significantly smaller peak compared to de novo gDNA peaks (such as those from semi-overlapping, overlapping, or adjacent cell heads). Cell emission values ​​can be determined by any suitable method (such as peak integration or maxima). The distinction between gDNA and mtDNA peaks can be made by any appropriate method. In some examples, the peaks are distinguished as follows:

[0215] 1. Calculate the centroid of the waveform and compare it with the maximum value. If the maximum value is before the centroid, the cell is moving head-to-head, and if the maximum value is after the centroid, the cell is moving tail-to-head.

[0216] 2. Similar to about Figure 3d The described method sets a high fall-edge threshold of 212m, which occurs after the first peak (mtDNA or gDNA) and before the second peak (mtDNA or gDNA). Another fall-edge threshold is set much lower, which is triggered only when the descent approaches the baseline, for example... Figure 3b The figure shows 212m. For a single sperm cell, the time between the first and second descending edges will be within a well-defined range, where the peaks correspond to gDNA and mtDNA, and can be referred to as the head-to-mid transition period, “HMTP”. This HTMP should be predictable and within a constant range for a given flow rate, and the sperm cell should be traveling head-to-tail or tail-to-head, and substantially aligned with the Z-axis of the flow. To distinguish between a single-cell event with two peaks (corresponding to mtDNA and gDNA) and a multi-cell event, the time between the descending edges (or peak maxima) can be measured and evaluated against a predetermined HMTP. If the time is outside the HMTP range, this indicates a multi-cell event. If it is within the range, it indicates with high probability that the peaks correspond to mtDNA and gDNA. Therefore, this provides a mechanism to avoid false positives in multi-cell events when attempting to determine the cell's Z-axis orientation.

[0217] In order to detect the emission in the midstream and distinguish it from the emission of gDNA in the cell nucleus, both the beam and the emission must provide sufficient fidelity. This fidelity can be achieved in a variety of ways.

[0218] In one example, when measured along the longitudinal head-to-tail axis, the interrogation beam width is less than or equal to half the head length. The head of a bovine sperm cell is approximately 8 μm to 11 μm long. Therefore, in one example, the interrogation beam width is less than approximately 5 μm. Higher fidelity can typically be achieved with a narrower beam, such as less than one-third of the head length, for example, 4 μm or less than 3 μm.

[0219] In another example, the emission signal from mtDNA is separated from the emission signal from gDNA.

[0220] In another example, the properties of the mtDNA emission signal differ from those of the gDNA emission signal. For instance, an mtDNA-specific staining agent is used, which provides a different emission signal compared to the gDNA emission signal.

[0221] In one example, a mitochondrial-specific stain is used to induce emission signals. This dye may or may not stain mtDNA; it can also stain other mitochondrial-specific cellular components. In another example, a mitochondrial (non-mtDNA) stain can be used in combination with an mtDNA emission stain to enhance emission intensity, thereby effectively identifying at least one cellular property, such as those described above.

[0222] Those skilled in the art will understand that the concept of detecting DNA or other cellular components from multiple longitudinal cellular locations to understand cellular properties (such as Z-axis orientation) can also be applied to other cells with components such as DNA that are found at multiple longitudinal locations using similar techniques.

[0223] In some cases, the cell head may overlap with the sperm tail, and the intention is to hit the sperm head. This could result in the overlapping sperm tail also being hit, such as... Figure 5b As shown in Figure 510. In one example, the present invention provides a method for selecting cells, the method comprising:

[0224] a. Determine the z-axis orientation of the cell based on the shape of the waveform (i.e., whether the head flows forward or the tail flows forward).

[0225] b. Determine the selection period, wherein the selection period is timed to begin based on the determination of the z-axis orientation; and

[0226] c. Select the cell.

[0227] Cell selection can be performed according to any selection or inactivation method described herein. The start of the selection period is adjusted to select preferred portions of the cells. For example, the selection period can be timed to select only the head and not the tail, or only the tail and not the head. For example, selecting the head of a cell by inactivation laser is more likely to cause cell membrane rupture. This may result in the release of cellular components such as DNA into the medium. Free DNA or other cellular components may be undesirable, as these components may impair the health of the remaining cells or interfere with the analysis of the remaining cells or cell medium. Therefore, this technique has the potential to improve cell sorting and analysis techniques. Based on the determination of the z-axis orientation, the actions associated with the selection period can be adjusted. For example, the laser or inactivation laser designed to change the orientation, position, or direction of travel of the cells can be adjusted to achieve one or more of the following: reducing laser power, increasing laser power, or changing the laser direction.

[0228] In another example, the method of selecting cells by determining z-axis orientation can be combined with the techniques described herein to determine multicellular events. In this example, a first cell in the multicellular event can be determined to be in a first z-axis orientation, and a second cell or other cells in the multicellular event can be determined to be in a second z-axis orientation. Based on the corresponding z-axis orientation, the actions associated with the selection period can be adjusted. For example, as shown in 510, the tail of a first cell traveling with a head-forward z-axis orientation may overlap with the head of a second cell traveling with a tail-forward z-axis orientation. Selecting the first cell (e.g., via an inactivation laser) may damage the second cell due to the possibility of damage to the tail of the second cell. Therefore, in this case, it may be preferable to reduce the inactivation power or omit the excitation inactivation to allow both cells to pass through. Alternatively, it may be decided to extend the selection period to cover both cells (i.e., inactivate them).

[0229] In an alternative example, a first cell traveling with its tail-forward z-axis orientation can be selected based on selection criteria. This cell may be close to or overlap with an unselected second cell traveling with its head-forward z-axis orientation. In this example, the selection period can be shortened to minimize the possibility of selecting (e.g., damaging) the second cell. In this example, only the first portion of the head of the first cell is acted upon, while the overlapping portion remains unacted, to reduce the possibility of damaging the overlapping second cell.

[0230] refer to Figure 11Two waveforms are shown, in which representative cells are also illustrated. Each cell 1105f and 1105g includes a cell head 1106f and 1106g, a mid-section 1107f and 1107g, and a tail 1108f and 1108g. These cellular portions are described in more detail below. Each cell 1105f and 1105g is associated with high-resolution waveforms 1110f and 1110g, respectively. In these high-resolution waveforms, it can be seen that each waveform includes two peaks or maxima, corresponding to the higher maxima or peaks 1112f and 1112g of the cell head 1106f and 1106g, respectively, and the lower maxima peaks 1114f and 1114g of the mid-section 1107f and 1107g, respectively. A waveform can be considered as a series of cell-emitted signal values, all of which are above (or deviate from) a signal value baseline (e.g., zero), where a first maximum and a second maximum correspond to respective local maximum signal values ​​or amplitudes within the waveform, which corresponds to DNA in the cell head or cell segment—the maximum value associated with the cell head has a higher signal value or amplitude than the maximum value associated with the cell segment. In this context, the term "above" is intended to convey deviation from the baseline and can therefore refer to a negative signal value, depending on the specific implementation of the signal value determination circuitry. In some examples, the waveform can be further defined by a series of signal values ​​that are 10% or 50% above the first maximum signal value after it. In other examples, the waveform can be defined by a maximum duration after the first maximum and / or when a signal value in the series returns to within a predetermined offset from the baseline. By identifying different height maximums within the same waveform, the order of different parts of the cell can be determined. For example, if the first maximum value (cell head) is higher than the second maximum value (cell segment), the cell is oriented head-first in the direction of travel. On the other hand, if the first maximum value (the middle of the cell) is lower than the second maximum value (the head of the cell), the cell is oriented with the tail in front.

[0231] from Figure 11 It can also be seen that the cell's z-axis orientation in the z-direction, or the direction of cell movement, affects the shape of the corresponding waveform. Cell 1105f on the left is oriented with its tail in front, resulting in waveform 1110f, where the lower peak 1112f appears before the higher peak 1114f. Conversely, cell 1105g on the right is oriented with its head in front, resulting in waveform 1110g, where the lower peak 1114g occurs after the higher peak 1112g. Therefore, by examining the waveform of a single-cell event, the z-axis orientation of the corresponding cell can be determined.

[0232] exist Figure 11As can be observed in the depicted figures, the cell travels from right to left. When the cell moves past the stationary detector (e.g., on the left side of the figure), the head of cell 1105f will emit a signal at 1106f, which is recorded as a peak reading 1110f before the middle segment 1107f, and the middle segment is recorded as a smaller peak 1114f after the larger peak.

[0233] Figure 12a and Figure 12b The actual waveforms detected from the cells are shown. The mid-range emission signal can be observed as secondary peaks 1201a and 1201b adjacent to the main peaks associated with heads 1202a and 1202b. The background noise threshold 1203 can also be observed. Figure 12a and Figure 12b The waveforms shown were obtained using an interrogation beam of approximately 3 μm. To achieve accurate resolution of the primary and secondary peaks, it is useful to use a beam width smaller than the width of the sperm head. Therefore, in some examples, the beam width is smaller than the length of the sperm head being analyzed. In some examples, the beam width is less than approximately 10 μm. Figure 12a The position of the secondary peak 1201a, located to the left of the main peak 1202a, indicates the z-axis orientation of the cell, which is related to the superimposed depiction of cell 1204 and the corresponding mid-segment 1205.

[0234] Figures 13a to 13d show further waveform examples that sequentially detect the head and midsection of sperm cells. Sperm cells have been overlaid on the waveforms to indicate the observed z-axis orientation of the sperm. The two waveforms in each figure are overlapping waveforms detected by orthogonal detectors. In one example, information from both detectors is combined to determine the z-axis orientation with greater precision than data from a single detector. The upper (higher maximum) waveform 1305 in each figure corresponds to the first detector, and the lower waveform 1310 corresponds to fluorescence detected by the second detector.

[0235] Figure 13a illustrates the difference between the first detector waveform shape 1305 and the second detector waveform shape 1310. In contrast, Figure 13b shows the first detector waveform shape 1305, which is substantially identical to the second detector waveform shape 1310. The similarity of waveform shapes is considered to be due to the x-axis or y-axis orientation of the cells. X-axis orientation refers to the dominant orientation of the cell head-tail direction in the x-axis (across the microfluidic flow), while y-axis orientation refers to the dominant orientation of the cell head in the y-axis (the up-down direction in the flow). Therefore, a method for determining the x-axis or y-axis orientation is provided by comparing the similarity between the first waveform 1305 and the second waveform 1310 to determine the x-axis or y-axis orientation relative to the first and / or second detectors. Based on the x-axis or y-axis orientation, at least one cell sorting parameter can be adjusted to improve cell selection, targeting, or sorting. In one example, a correlation parameter between the two waveforms can be determined, and if the correlation parameter exceeds a threshold, it can indicate one orientation; while if the correlation parameter is below the threshold or another lower threshold, it can indicate another orientation. The correlation parameter can be the cross-correlation coefficient of the waveform, although other measures may be used alternatively.

[0236] This additional information regarding cell orientation along the x, y, or z axes can be used to adjust the selective processing of cell events. For example, if it is determined that a desired cell with its tail in the forward orientation is within a predetermined distance of a certain cell event (single-cell event or multi-cell event), it indicates that the desired cell overlaps with one or more unwanted cells or cells to be targeted. This determination of overlap can then be used to adjust the selective processing of the aforementioned unwanted cell events, for example, to avoid affecting the tail of subsequently desired cells.

[0237] In one example, the modified treatment could be to prevent selective processing of previously unwanted cellular events. While this would allow cells in that unwanted event to pass through unselected, it also avoids potential damage to the tails of subsequently desired cells, thus protecting their viability.

[0238] In another example, when it is determined that there are overlapping cells requiring selection, adaptive triggering can be selected from adaptive timing used for selection processing of previous cell events. Figure 5a or Figure 5b Switch to adaptive timing with adaptive selection triggering. Figure 6a or Figure 6b This effectively shortens the selection period as previously described, thus preventing damage to the tails of cells that are subsequently needed.

[0239] Figure 7 Methods for processing cells according to some examples are illustrated. Method 600 can be used in any suitable device (such as...) Figure 1This method is implemented in the controller 135. It analyzes cellular events and determines whether to classify the cells within those events.

[0240] At 605, the method detects the rising edge and subsequent falling edge of the waveform, and determines the waveform width or duration. This can be used with respect to... Figure 2a and Figure 2b The method described is used to achieve this, however other methods may be chosen instead.

[0241] At 610, the method determines whether the waveform corresponds to a multi-cell event comprising multiple cells tightly clustered together. In this case, it may be impossible to fully resolve each cell. In contrast, a single-cell event may include a single cell with a defined waveform and waveform width and / or height range that leads to the expected pattern. Cell events that do not meet these expectations can be classified as multi-cell events. Alternatively, multi-cell events can be determined based on one or more characteristics of the waveform, such as a waveform width greater than a multi-cell event threshold, more than one detected peak, or other characteristics.

[0242] In this method, multicellular events are considered selected cellular events; however, in other examples, further analysis of the waveform can be performed to classify multicellular events as unselected or selected. For example, in some multicellular events, sufficient information about the cells within the multicellular event can be determined to classify the event as desired, and thus not select those cells for inactivation, or classify them into different containers for the desired cells. An example could be that one or more cells in a multicellular event can be classified as desired cells, such as cell X.

[0243] At 610, if the waveform is considered to belong to a multi-cell event (Y), the method moves to box 625. Otherwise, the waveform is considered to belong to a single-cell event (N), and the method moves to box 615.

[0244] At 615, the method determines one or more properties of the waveform corresponding to a single cell. For example, the integral or peak maximum of the waveform can be used to classify sperm cells as X cells or Y cells. Other properties, such as the slope of the rising and / or falling edges of the waveform, and / or the temporal symmetry of the waveform, may be used additionally or alternatively.

[0245] At 620, the method determines whether to select the cell based on the determined characteristics. If the cell is not selected for sorting (N), the method returns to box 605, where the waveform of the next cell event is analyzed. In one example, this allows desired cells to pass unimpeded through the cell processing apparatus for subsequent collection. Alternatively, desired cells may be subjected to sorting arrangements that could affect cell movement. For example, cell orientation may be affected by radiation pressure, the application of an electric field, or other biasing methods to ensure that cells are collected in the desired cell collection container. Otherwise, the cell is considered selected for sorting (Y), and the method moves to box 625. The method also returns to box 605 at 620Y, where the waveform of the next cell event is analyzed.

[0246] At 625, the method determines the offset delay and the selected time period. One or both of these variables can be based on the waveform width as previously described, or they can be based on predetermined values. These variables can also depend on whether the cellular event is a single-cell event or a multi-cell event.

[0247] In the example of single-cell events, the variable offset delay (OD) and the selection period (SP) can be determined based on one of the following:

[0248] 1) OD=POD and SP=PSP, where POD is the predetermined offset delay after the detected falling edge of the waveform, and PSP is the predetermined selected time period; or

[0249] 2) OD = PCTD - WW and SP = WW, where PCTD is the predetermined cell transition duration after the detected falling edge of the waveform, and WW is the waveform width; or

[0250] 3) OD = PCTD - aWW - b and PD = xWW + b, where PCTD is the predetermined cell transition delay after the detected falling edge of the waveform, WW is the waveform width, a is the distributable coefficient, and b is the distributable constant.

[0251] In the example of multicellular events, the variable offset delay (OD) and the selection period (SP) can be determined based on one of the following:

[0252] 3) OD = POD2 and SP = PSP, where POD2 is the predetermined offset delay after the detected falling edge of the waveform, and PSP is the predetermined selected time period; or

[0253] 4) OD = PCTD - WW and SP = WW, where PCTD is the predetermined cell transition duration after the detected falling edge of the waveform, and WW is the waveform width; or

[0254] 5) OD = PCTD - aWW - b and PD = xWW + b, where PCTD is the predetermined cell transition duration after the detected falling edge of the waveform, WW is the waveform width, a is the distributability coefficient, and b is the distributability constant.

[0255] At 630, the method selects one or more cells from a selected cellular event for sorting. This can be achieved by directly controlling the start and stop of a continuous or pulsed laser to coincide with the selected time period, for example, using an active Q-switch. In another specific implementation, a continuous or pulsed laser may already be in operation, and a switch such as an acousto-optic modulator can be used to apply the laser to the microfluidic flow only during the selected time period. In another example, sorting can be achieved by applying radiation pressure to the cells to change their orientation. In yet another example, sorting can be achieved by charging a droplet containing cells and attracting the charged droplet to a charged plate to adjust its trajectory.

[0256] Method 600 is configured to classify all cells in a multicellular event and selected cells in a single-cell event, wherein unselected cells in the single-cell event are unaffected. This means that some desired cells in the multicellular event can be selected and sorted, for example, by inactivation. However, overall, the concentration of selected cells will be reduced because all cells that cannot be identified as desired cells will be selected and sorted, for example, by inactivation. In an alternative example where at least some cells in the multicellular event can be distinguished and classified as desired or unwanted cells, the desired cells may also be spared from inactivation, and only the selected cells in the multicellular event may be inactivated.

[0257] Figure 8This is a graph of the waveform width relative to the selected time period. This can be used to determine the coefficients a and constant b, which can be set using the slope and y-intercept of the curve, respectively. This can be determined experimentally using different pulse widths and selected time periods. Example time periods from experiments are described; however, other values ​​can be alternatively obtained and / or used. Point 703 is the minimum waveform width (WW) of 9.96 μs and the minimum selected time period (SP) or laser on-time of Ops. This corresponds to a threshold below which no cellular events are identified. Point 706 corresponds to a single-cell event, where WW = 12.6 μs and SP = 3.64 μs. Point 709 corresponds to a two-cell event or a multi-cell event with two cells, where WW = 25.2 μs and SP = 21.3 μs. Point 712 corresponds to a three-cell event or a multi-cell event with three cells, where WW = 37.8 μs and SP = 38.9 μs. Based on the slope and intercept of the curve including these points 703-712, the coefficient (slope) a = 1.4, and the constant (y-intercept) b = -1.4. It should be noted that other two-cell and three-cell events may have different WW and SP values, for example, due to different cell sizes, overlaps, and / or spatial arrangements. These assignable values ​​a and b can be optionally determined to ensure that all single-cell events are exposed to only one laser pulse. These values ​​can be determined for different runs based on their own flow configuration and other processing parameters such as cell type and laser power.

[0258] Figure 9 An example of a controller 900 that can be used to implement a method for processing cells is illustrated. The controller 900 can be implemented as follows: Figure 1 The controller 135, however, the controller 900 can be used in different systems.

[0259] The controller 900 includes hardware 903 having a processor 906 and memory 909. The processor may be a microcontroller, an FPGA, or any other suitable hardware or a combination of hardware and software. The memory 909 includes first computer program instructions 912 that, when executed by the processor 906, cause the controller 900 to perform a plurality of steps 952-956. This can be implemented in conjunction with other hardware such as a laser (not shown). The memory 909 may alternatively include second computer program instructions 915 that, when executed by the processor 906, cause the controller 900 to perform a plurality of steps 962-966. This can also be implemented in conjunction with other hardware such as a laser (not shown).

[0260] At 952, the processor detects cellular events within the microfluidic flow by detecting the rising edge of the waveform in the received emitted signal associated with the microfluidic flow, followed by detecting the falling edge of the waveform. As previously discussed, the emitted signal can be generated by a detector 130 arranged around the microfluidic flow carrying cells, the presence of which can be detected in the interrogation region by the characteristics of scattered light or fluorescence emission after the microfluidic flow has been irradiated by a suitable radiation source such as infrared (IR) light or ultraviolet (UV) light. In one example, the infrared light is generated by a quantum cascade laser. The rising and falling edges of the waveform can be detected using the methods described above. Figure 2a and Figure 2b The methods discussed are for detection; however, other methods may be used alternatively.

[0261] At 954, the processor uses the corresponding waveform to classify the cell event into selected cell events. As mentioned herein, selected cell events can include any cell event identified as a multi-cell event or a single-cell event, where the associated single cell is an unwanted cell, such as a Y cell. Multi-cell events can be determined using various methods, such as having a waveform width exceeding a threshold, having multiple peaks, or not being identified as a single-cell event. Selected single-cell events can be determined using characteristics of the waveform, such as an integral within or above a selected cell threshold.

[0262] At 956, the processor initiates a sorting action on cells in the selected cellular event using an offset delay from the detected falling edge. This can be achieved by controlling a sorting laser or other inactivation component. During the selection period, the laser or inactivation component, or another sorting or inactivation action, is applied to the cells of the selected cellular event.

[0263] Using the detected falling edge provides an improved mechanism for controlling cell inactivation within a selected cell event, as this corresponds to the trailing edge or last cell in the selected cell event. This method of detecting and classifying single-cell or multi-cell events allows the selection period to be adapted based on many considerations, such as waveform width, which corresponds to the duration between the leading and trailing edges of one or more cells in the selected cell event. This allows for extending the selection period for a wider waveform width, for example, due to multiple closely grouped cells. Adjusting the offset delay allows for adjustment of the selection period, as the selection period should end at the trailing edge of one or more cells in the selected cell event to avoid affecting subsequent cells. This allows for higher enrichment of unselected (often desired) cells compared to selected (often unwanted) cells that will be sorted more effectively and efficiently. Using the detected falling edge to trigger the sorting or inactivation laser is also easier to implement than using the peak, thus reducing processing requirements and improving processing time, which in turn allows for improved control.

[0264] Other factors that can be used to adjust the offset delay and thus the selected time period may include inactivation type such as ablation compared to smaller damage, cell size and / or type, cell z-axis orientation, cell rate, diffusion (i.e., restriction) of core flow, and spacing (distance between each cell).

[0265] At 962, the processor uses the received emission signal associated with the microfluidic flow to detect cellular events within the microfluidic flow. As previously discussed, the detection of the rising and falling edges of the waveform can be used to detect the corresponding cellular events. The emission signal can be generated by a detector 130 arranged around the microfluidic flow carrying cells, the presence of which can be detected in the examination area by the characteristic of fluorescence emission after the microfluidic flow has been irradiated by a suitable radiation source such as infrared (IR) light or ultraviolet (UV) light. The rising and falling edges of the waveform can be detected using the characteristics described above regarding... Figure 2a or Figure 2b The methods discussed are for detection; however, other methods may be used alternatively.

[0266] At 964, the processor classifies cell events as selected or unselected cell events. Similar to step 954, selected cell events can include any cell event determined to be a multi-cell event or a single-cell event, where the associated single cell is an unwanted cell, such as a Y cell. Multi-cell events can be determined using various methods, such as having a waveform width exceeding a threshold, having multiple peaks, or not being identified as a single-cell event. Selected single-cell events can be determined using waveform characteristics, such as an integral within or above a selected cell threshold. Alternatively or additionally, unselected cell events can be single-cell events associated with a desired cell, where all other cell events are classified as selected cell events. Single-cell events can be determined by having a waveform width below a threshold, having only one peak, or not being a multi-cell event. Unselected single-cell events can be determined using waveform characteristics, such as an integral within a desired cell threshold.

[0267] At 966, the processor applies a sorting action to one or more cells in a selected cell event during a selected time period, the selection time period depending on the end of the selected cell event and the duration of the selected cell event. The end of the selected cell event can be determined based on the detected falling edge of the waveform associated with the selected cell event. The duration of the selected cell event can be determined based on the waveform width of the waveform associated with the selected cell event. These variables can be used to calculate the selection time period as previously described. During the selection time period, the selected cells are sorted using a sorting arrangement structure, such as by applying photon pressure using a laser, or by applying an inactivation action to the cells of the selected cell event, for example, via an inactivation component. In one example, the selection time period includes an inactivation period during which the inactivation component or the sorting arrangement structure inactivates one or more cells.

[0268] The ability to adapt the sorting process to different conditions by adjusting the selection time period improves the cell handling in the example. This allows for a higher enrichment of unselected cells compared to selected cells that will be sorted more efficiently and effectively and optionally inactivated.

[0269] This document describes a detector for detecting light emission from irradiated cells. The cells may be stained or unstained and contain one or more dyes that emit at a preferred wavelength. In one example, the detector includes detectors known in the art for collecting emission and converting it into a digital signal, such as photomultiplier tubes (PMTs), avalanche photodiodes, or other suitable devices for collecting light and converting it into analog or digital signals for signal processing. The detector may be connected to a suitable signal amplifier and / or analog-to-digital converter before sending the signal to a controller.

[0270] Multicellular events can result in waveforms with larger integral peak values ​​or waveform widths than those typically observed for single cells. This is due to additive effects or spacing of the emitted signals when detected by a detector. In one example, the invention includes a method for evaluating an increased peak value against a standard peak associated with a single-cell event and classifying that peak as either a multicellular or single-cell event. If the waveform is classified as a multicellular event, a determining step can be performed to determine a cell count, which indicates how many cells the multicellular event contains. For example, a multicellular event may contain two, three, or four overlapping cells.

[0271] The determination step may include one or a combination of the following: associating the waveform width, waveform integral, or number of detected peaks in the waveform with the number of cells. For example, in the case where the integral of a single Y cell is 100 and the integral of a single X cell is 104, a multi-cell event with an integral of 200 (+ / -2) can be determined as two Y cells, while a multi-cell event with an integral of 208 (+ / -2) can be determined as two X cells. Single-cell events can be used to continuously calibrate the integrals to be used. The waveforms of these single cells can be used to deconvolve the waveforms of the individual cells in the multi-cell event.

[0272] If the classification step determines that a multicellular event has occurred, the method further includes a differentiation step that distinguishes cells identified as present. This differentiation step distinguishes cells to identify the state of overlapping cells. For example, a two-cell multicellular event may include one of three cases:

[0273] i. Required + Required cells

[0274] ii. Required + Unnecessary cells, or

[0275] iii. Unnecessary + Unnecessary cells.

[0276] The selection of which group in the sorting arrangement can then be determined based on the expected enrichment of desired and unwanted cells. In one example, sperm cells stained with a DNA fluorescent dye can be used, where X is designated as desired cells and Y is designated as unwanted cells, and possible combinations include XX, XY, or YY. Given that sperm cell X has the highest DNA quality, the signal amplitude of the XX multicellular event is expected to exceed that of the XY event, and XY is expected to exceed that of the YY event. Based on the determination of the XX multicellular event, where the peak maximum or integral is greater than a sorting threshold, it can be decided to retain the XX multicellular event. For example, retention could mean that the cells are not selected cell events sorted via inactivation, or it could mean that they are collected using a sorting arrangement as described herein. The XY group can also be selected based on one or more selection criteria and the expected enrichment of the collected samples. The YY group (and optionally the XY group) can be designated as the selected cell events and processed according to the sorting method described herein, for example, the cells can be inactivated, not collected, or collected in unwanted cell collection containers. In another example, the Y cell can be the desired cell, so the selection criteria are reversed.

[0277] This technique is particularly useful in cases where cell differentiation is based on non-binary metrics (such as the fluorescence intensity of a first labeled cell relative to a second labeled cell with a different but non-zero standard fluorescence signal). In binary sorting cases, where X is labeled to emit an emission signal, but Y does not emit an emission signal, it is expected that a multi-cell event XY will generate the same signal as a single-cell event X. In this case, the multi-cell event classification step will fail to identify multiple cells. Therefore, this example of the invention is particularly useful when the emission signal intensity exhibited by the respective labeled cells differs from the total detectable intensity of any single cell by less than 50%.

[0278] In one example, if the sorting step determines that a multicellular event has occurred, the applied sorting action can be adjusted. For example, the power of the inactivating laser emitted in response to overlapping sperm cells is reduced. This has the effect of reducing the chance of collateral damage to the cells that are desired to be retained. Another example of adjusting the sorting action is to emit a lower-energy pulse when multiple cells are detected within a specific time / distance. This allows any energy dispersion or localized media vaporization or shock wave generated by the pulse to dissipate or move in the flow direction before the next pulse is applied. This technique can be implemented by having pulse groups consisting of multiple small pulses or multiple large pulses. The pulses within the pulse group are separated by one pulse frequency. This technique has the effect of intelligently triggering the sorting action to minimize collateral damage to cells and system components. In one example, the above-mentioned adjustment of the sorting action can be achieved by adjusting the power transmission properties through the beam splitter. For example, as with polarization or other beam splitters (e.g., regarding...). Figure 21 and Figure 23 As described in the description, a beam splitter can be associated with a beam that is directed to a microfluidic flow, or with a beam designed to be separated between two or more microfluidic flows.

[0279] When cells are flowed through a microfluidic flow, a highly constrained flow is generally desired—that is, minimal variation in the X and Y localization of the cells at a given Z position. This improves interrogation accuracy and generally enhances sorting operations, especially when cells are involved by focusing a sorting beam at specific X, Y, and Z positions. However, cell constraints remain complex. In one example, a waveform generated by the cell determines or indicates the lateral localization of the cell within the microfluidic flow. The X and Y coordinates of the cell relative to the average X and Y coordinates can be determined, and an offset value is calculated. This value provides information about the cell's localization relative to the normalized flow axis. The present invention provides a method for customizing cell targeting to minimize constraints. Thus, in one example, the present invention provides a method for selecting cells, the method comprising:

[0280] a. Measure the waveform associated with the cell to determine the X-axis and / or Y-axis deviation of the cell from the nominal axis of cell flow;

[0281] b. Adjust the selected action to account for X-axis and / or Y-axis deviations; and

[0282] c. Select the cell.

[0283] Adjustments to the selection action may include adjusting the direction or power of the sorting beam to guide or focus it onto the identified cell location, taking into account calculated biases. This allows the sorting beam ablation laser to be directed to the cell location in the flow. Adjustments to the direction or power will typically involve a beam modulation / targeting system used to deflect the beam. Power adjustments can be made via a beam splitter (such as...). Figure 21 and Figure 23 The described beam splitter and optionally associated arrangement are used. The system can be an acousto-optic modulator, an electro-optic deflector, an electro-optic modulator, a galvanometer scanner, a polygon scanner, or different gates for different locations in the flow.

[0284] In another example, the waveform can indicate a lack of lateral alignment of cells with the nominal and desired flow axes. Therefore, in one example, the present invention provides a method for selecting cells, the method comprising:

[0285] a. Measure the waveform associated with the cell to determine alignment with the nominal axis of the cell flow;

[0286] b. Adjust the selection action based on the determined alignment; and

[0287] c. Select the cell.

[0288] When the selected action involves targeting cells with a sorting beam, adjustments may include one or more of the following:

[0289] a. Adjust the beam direction to XY or Z;

[0290] b. Adjust the beam focus on the XY or Z axis;

[0291] c. Adjust the shape of the beam;

[0292] d. Adjust the elongation ratio of the beam profile, such as the horizontal line across X;

[0293] e. Tilt to elongate the beam profile.

[0294] Those skilled in the art will understand that tilting the elongated beam from its initial X-axis orientation may result in a deflection in the Y or Z axes.

[0295] The pulse width of a single-cell event can be used to measure cell velocity in real time, enabling enhanced cell targeting. The latest cell velocity determined from the most recent single-cell event can then be applied to subsequent cell events (including multi-cell events) until the cell velocity is updated again. The offset delay can be automatically modulated based on cell velocity measurements derived solely from waveform width or other waveform characteristics.

[0296] Therefore, in one example, the present invention provides a method for selecting cells, the method comprising:

[0297] a. Measure waveforms associated with cellular events to determine cell velocity;

[0298] b. Calculate the offset delay based on the determined cell velocity;

[0299] c. Use offset delay to select one or more cells in a cell event.

[0300] Cell selection may also include sorting according to any sorting action provided herein. In another example, the offset delay is automatically updated based on the determined velocity of the second cell.

[0301] In another example, the present invention provides a method for selecting cells, the method comprising the following two items:

[0302] a. Adjust the selected action to account for X-axis and / or Y-axis deviations; and

[0303] b. Calculate the offset delay based on the determined cell velocity.

[0304] In this example, two previously described methods for improved targeting are combined to simultaneously optimize offset delay and laser focus localization. These methods work together to provide an enhanced cell selection approach.

[0305] Detector parameters (e.g., photomultiplier tubes) can be adjusted based on peak characteristics (e.g., peak maximum or integral). This ensures optimal detection by the detector and within the set range. This reduces the requirements for user input and detector calibration, especially during setup. For example, the detector voltage can be automatically adjusted to account for intensity variations in cellular events.

[0306] Therefore, in one example, the present invention provides a method for selecting cells, the method comprising:

[0307] a. Use a detector to detect emission signals from cells in a microfluidic flow;

[0308] b. Generate a waveform associated with the cell based on the emitted signal;

[0309] c. Adjust detector parameters based on waveform;

[0310] d. Use the adjusted parameters to detect cells;

[0311] e. Select the cell for sorting.

[0312] In this example, the detector parameter can be voltage. The waveform can provide intensity measurements. In this example, the detector voltage is automatically updated based on the intensity to enhance the detection of cellular properties such as fluorescence intensity.

[0313] Waveform characteristics can be used to adjust optics or flow positions to achieve enhanced interrogation and sorting, and to calibrate the cell handling system. For example, input can be provided to the motorized optics level to adjust based on peak characteristics (e.g., peak width, maximum value, or integral). This ensures the system optimally interrogates and sorts cells within set ranges. Automatic adjustment reduces the need for user input and potential errors.

[0314] Other waveform characteristics may include waveform shape, the number of peaks and their relative height or timing, waveform width, area under the waveform, maximum signal value, or waveform slope. These waveform characteristics can be used to determine the properties of the corresponding cell, such as whether it is an X-type or Y-type sperm cell, or the cell's longitudinal z-axis orientation (i.e., flow direction). Longitudinal z-axis orientation (e.g., tail-first or head-first) is related to... Figure 10 and Figure 11 To describe in more detail.

[0315] By determining one or more properties of a cell based on one or more characteristics of the cell's corresponding waveform, one or more parts of the interrogation and / or sorting system can be adjusted, for example, to optimize these systems.

[0316] In some examples, a subset of waveform characteristics can be used to classify detected cells into, for example, X or Y sperm cells, and different subsets of waveform characteristics can be used to adjust the sorting of selected cells or cell events. For example, the maximum signal value and integral of the waveform can be used to select cell events for sorting, and the waveform width and shape can be used to control the selected time period during which sorting occurs.

[0317] In one example, the present invention may provide a method for querying, selecting, and / or sorting cells within a microfluidic flow, the method comprising:

[0318] The cellular event is detected using waveforms from cellular emission signals received from and associated with cellular events within the microfluidic flow;

[0319] The electromagnetic radiation is directed to the cells within the microfluidic flow to promote responsive emission signals from these cells;

[0320] Sorting selected cells within a microfluidic flow (e.g., using sorting electromagnetic radiation);

[0321] The sorting process is adjusted based on the characteristics of the waveform.

[0322] In one example, the present invention provides a method for selecting cells, the method comprising:

[0323] a. Detecting emission signals from cells in a microfluidic flow;

[0324] b. Generate a waveform associated with the cell based on the emitted signal;

[0325] c. Based on waveform adjustment system components;

[0326] d. Use the adjusted system components to detect cells;

[0327] e. Select the cell for sorting.

[0328] In one example, the present invention provides a method for sorting cells, the method comprising:

[0329] a. Detecting emission signals from cells in a microfluidic flow;

[0330] b. Generate a waveform associated with the cell based on the emitted signal;

[0331] c. Based on waveform adjustment system components;

[0332] d. Use the modified system components to sort cells.

[0333] In this example, the system component can be an optical component used to generate, focus, position, or guide an interrogating or sorting beam. In another example, the system component can be a microfluidic chip, allowing adjustment to change the positioning of the microfluidic flow, for example, through an adjustable aperture as described in WO2024 / 102007. In yet another example, the system component can be an electrostatic or acoustic field generator arranged to disrupt cell pathways within droplets formed by the microfluidic flow. Waveforms can provide intensity measurements. In this example, the intensity measurement can indicate a focal position determined by a system component such as an optical component or the microfluidic flow, wherein the focal position is automatically updated based on intensity to enhance the detection of cellular properties such as fluorescence intensity.

[0334] In one example, the optical components may be continuous or pulsed lasers, laser pulse detectors, optical switches or gates, lenses, or movable mirrors. Various optical components may be arranged in free space or in a fiber optic arrangement where laser pulses or continuous beams are guided between one or more optical components using appropriate optical fibers.

[0335] In some examples, a pulse sorting arrangement that generates regular pulses is used to sort selected cellular events, such as detected sperm cells (e.g., Y sperm cells) classified as unwanted. This can be achieved by coordinating the regular pulses with a selection period or a gated opening period, during which one or more regular pulses are directed to a microfluidic flow carrying the cells to be sorted. In some examples, the selection period for sorting cellular events may include more than one gated opening period. For example, multiple cellular events can utilize multiple gated opening periods to target different cells within the cellular event, while reducing collateral damage to desired cells by using shorter-duration pulses that affect the microfluidic flow rather than performing more continuous sorting within the selection period. The concept of using multiple gated opening periods within a selection period advantageously enables more precise targeting of cells using laser pulses that are controllable at the level of a single pulse. This provides increased sorting fidelity by individually selecting (or not selecting) cells within the cellular event, then targeting the selected cells for sorting, and leaving unselected (i.e., desired) cells to be collected for downstream applications. Even in cases of partial cell overlap, the ability to detect and sort cells individually offers considerable benefit to the art by enhancing the enrichment of desired cells in the collected sample. Alternatively, the method described herein can increase throughput (i.e., increase cell selection and flow rate) when the normalized enrichment threshold is sufficient. Finally, the ability to selectively target cells using single-cell pulses reduces the likelihood of damage to non-target cells, thereby improving their viability.

[0336] In one example, pulsed lasers are used to sort selected cells or selected cellular events by at least one of the following methods: targeting a cell or a portion of a fluid substantially adjacent to the cell with a pulsed laser to alter the cell's trajectory or position; optical manipulation; optical trapping; holographic steering; application of photon pressure; laser inactivation; or laser ablation. This can be achieved by controlling an optical switch or gating to switch between directing regular laser pulses toward and away from the microfluidic flow according to the gating opening period.

[0337] In other examples, laser pulses can be directed into or away from the microfluidic flow or bubble-generating regions associated with it. The laser pulses generate bubbles that alter the flow path of selected cells or cell subpopulations (such as Y sperm cells). In other examples, laser pulses directed into the microfluidic flow can be used to "nudge" a cell subpopulation into a different flow path. In other examples, pulse sorting arrangements can use mechanisms other than lasers, such as generating regular electrostatic, electric, or magnetic field pulses. These pulses can be selectively directed into the microfluidic flow based on the detection and classification of cellular events. For example, a pulsed high-voltage signal can be switched to or isolated from a pair of electrodes between which the microfluidic flow flows. This switching allows one or more electrostatic pulses to be directed into the microfluidic flow, based on a gating opening period.

[0338] Figure 14 Examples of using Figure 1 The cell processing system 1400 uses some of the same components as the system but with a different sorting arrangement. Common components have the same reference numerals, and their detailed description and operation are as previously described. Specifically, the cell processing system 1400 includes a preparation station 105 that delivers prepared cells to an input arrangement structure 110 that delivers the cells to a microfluidic flow 115 for downstream processing. The microfluidic flow 115 can be a laminar flow having a predetermined cross-sectional size and transported within a flow environment. In an example, the flow environment may include a volume of gas (such as air) or microchannels that completely or partially surround the microfluidic flow. One or more irradiators 120 generate an interrogation or detection beam, such as an infrared (IR) or ultraviolet (UV) irradiator or other radiation device. The interrogation beam irradiates the cells within the microfluidic flow at the interrogation or detection region 125. The radiation to the cells causes emission signals, such as scattered light or fluorescence detected by one or more detectors 130. The measured characteristics of the detected emission signal generate one or more signals, which are forwarded to a controller 1435 that controls a pulse sorting arrangement to sort cells into two subpopulations (e.g., X and Y sperm cells). The sorted cells are collected in at least one collection container.

[0339] Controller 1435 may include a processor and memory, and is configured to interpret received emission signals in order to control pulse sorting arrangement 1440. In one example, the emission signal includes a fluorescence signal. Controller 1435 is configured to detect single-cell events, each single-cell event comprising a single cell within a microfluidic flow passing through interrogation region 125. A single-cell event results in the reception of light at detector 130, which generates a signal that will be used by the controller to detect the single-cell event, as described in more detail below. Controller 1435 may also be configured to detect multi-cell events, each multi-cell event comprising multiple tightly grouped cells within a microfluidic flow passing through interrogation region 125. The tightly grouped cells in a multi-cell event may not be individually resolved for some downstream processes.

[0340] In some examples, detecting cellular events or multicellular events can be as previously discussed. Figure 2a and Figure 2b This is achieved as described. In other examples, the detection of cellular events can be achieved using peak detection of fluorescence signals or any other known cell detection mechanism.

[0341] In some aspects, sorting selected cells or cellular events can include: laser targeting of cells, using fluid pressure to alter the trajectory or orientation of cells, mechanical sorting, piezoelectric actuation, dielectric electrophoresis of droplets, electrolysis or electroporation, optical manipulation, optical trapping, holographic steering, acoustic-assisted hydrodynamic focusing, application of photon pressure, acoustic deflection, laser inactivation, or laser ablation. Sorting can affect at least one of forces and torques on cells in a population to induce at least one of displacement and orientation of these cells relative to an axis defined by the flow direction of the microfluidic flow. When sorting involves the inactivation of cells associated with selected cellular events, this includes imparting energy to the cells associated with the selected cellular events, such energy being sufficient to damage the cells, thereby causing them to become non-viable.

[0342] In specific examples, the pulse sorting arrangement may include: microbubble-based sorting, such as using lasers, sparks, or hot steam; cell sorting based on pneumatic and solenoid valves, such as using polydimethylsiloxane (PDMS); or piezoelectric actuation, such as using PDMS valves. In another example, the pulse sorting arrangement may include a pulsed radiation source configured to radiate a microfluidic flow at the target region or sorting zone 1470 of the sorting beam. The pulsed radiation source is coordinated, and sometimes directed, to or near cells associated with selected cellular events to cause changes in the orientation, position, or direction of travel of said cells, or to achieve inactivation, including ablation or damage to selected cells within them. In this example, the radiation emitted from the radiation source that affects the cells is referred to as the “sorting beam.” In any of the examples provided herein, the sorting beam may include an elongated beam profile. For example, the elongated beam profile may include a line, an ellipse, a rectangle, or a rounded rectangle. In some examples, the pulsed radiation source of the pulse sorting arrangement comprises regular laser pulses, which can have a single pulse duration of 100 nanoseconds to 10 femtoseconds. Regular laser pulses can include inter-pulse intervals in the nanosecond, picosecond, or femtosecond range, for example, <10 pm to about 10 ns. The specific characteristics of the sorting beam can vary depending on the desired frequency, power, and wavelength. Furthermore, different cells may require different sorting beams for sorting. Those skilled in the art will be able to determine the desired frequency, power, and pulse duration to adapt the sorting beam to cell type and flow rate. However, in some examples, the sorting beam includes frequencies between about 100 kHz and 3000 kHz.

[0343] This arrangement can be used in combination with the previously described static timing selection trigger, adaptive timing selection trigger, or adaptive timing adaptive selection trigger.

[0344] In some examples, this arrangement is particularly useful for removing unwanted cells from a population containing both desired and unwanted cells. For example, during the generation of a cell population for CAR T-cell therapy, certain types of cells may be present that do not exhibit the desired phenotype. Cells in this first population (P1) are disrupted, degenerated, or rendered immobile by the sorting arrangement. An alternative population (P2) of cells not exhibiting the desired characteristics (e.g., desired cells not yet selected by the controller) remains undisturbed. The sorted or processed cells in the microfluidic flow 115 can then be collected in one or more collection containers 160 for further use. The sorting arrangement 140 thus provides a cell population (P2) rich in the desired characteristics. When using sperm cells, this desired population may include motile X cells.

[0345] In one example, the pulse sorting arrangement 1440 includes a suitably configured controller 1435, a pulsed laser 1445, a laser pulse detector 1450, and an optical switch or gating 1455. The pulsed laser 1445 generates regular laser pulses, which are detected by the laser pulse detector 1450, which issues an indication each time a laser pulse is detected. The laser pulse detector 1450 may include a photodiode or other type of photodetector. The various optical components 1445, 1450, and 1455 can be arranged in free space or in an optical fiber arrangement, wherein appropriate optical fibers are used to guide the laser pulses between one or more optical components.

[0346] Optical switch 1455 may include an acousto-optic modulator (AOM), an electro-optic modulator (EOM), a spatial light modulator (SLM) (such as a digital micromirror device (DMD)), or any other optical switch configured to direct incident laser pulses toward or away from the microfluidic flow 115 in sorting region 1470. Optical switch 1455 is controlled to allow incident laser pulses from pulsed laser 1445 toward the microfluidic flow according to a gate opening period controlled by controller 1435, so as to allow cell interaction with selected cellular events. At other times, incident laser pulses from pulsed laser 1445 are prevented from sorting cells in the microfluidic flow. This can be achieved by controlling the optical switch to direct the laser pulses away from the microfluidic flow, wherein directing away may include blocking by a filter, becoming opaque, or redirecting the incident laser pulses to another location, such as a beam collector 1465 that absorbs laser pulses unrelated to the gate opening period.

[0347] The optical switch 1455 can be controlled according to various control strategies described in detail below. The controller can control a driver (not shown), which in turn drives the optical switch; for example, an RF driver can be used to redirect the incident laser pulse into the microfluidic flow via the AOM.

[0348] Figure 15a A control method for sorting single-cell events is illustrated. A fluorescence signal 1510s from detector 130 is received by controller 1435 and used to detect single-cell events. In this example, peak detection is used to detect cell events, where a rapid rise and subsequent fall in signal amplitude is used to detect single-cell events, as illustrated in peak detection signal 1515s. In other examples, different single-cell event detection methods may be used, such as those related to... Figure 2a As described.

[0349] The controller 1435 uses this characteristic and optionally other characteristics of the signal waveform to determine whether the detected cell event corresponds to a desired cell (e.g., an X sperm cell) or an unwanted cell (e.g., a Y sperm cell). Detected cell events corresponding to unwanted cells are classified as selected cell events to be sorted by the pulse sorting arrangement 1440. Cell events not classified as selected cell events may include cells that are allowed to pass through the sorting area 1470 without any sorting interaction.

[0350] Pulsed laser 1445 generates regular laser pulses, as illustrated in 1525. Controller 1435 controls optical switch 1465 to allow pulses to be directed into the microfluidic flow according to a gating opening period 1520s. As illustrated in 1530s, this allows a controlled or specified number of pulses (e.g., a single laser pulse only) to be directed into the microfluidic flow to sort cells 205s associated with selected cellular events. In other words, the size of the gating opening period is set and its timing is coordinated or synchronized with the laser pulses so that only a controlled or specified number of pulses, in this case a single laser pulse 1530, pass through during the gating opening period 1520. All other laser pulses 1525 are directed away from (including blocked from) the microfluidic flow, resulting in them not interacting with or sorting cells in the microfluidic flow. This arrangement uses a controlled number of specified laser pulses (e.g., a single specified laser pulse only) to sort cells; this reduces collateral damage to other nearby cells. In other words, adjacent cells of a potentially desired cell will not be sorted or damaged by one or more specified pulses.

[0351] The laser pulses are configured to be sufficiently regular to allow one or more designated pulses to target unwanted cells 205 regardless of the flow velocity. The applied pulses can also be sufficiently configured, for example, to achieve sorting (such as by nudge, inactivation, or cell ablation) with sufficient power, thereby inactivating the unwanted cells. Depending on the specific implementation of the optical switch, there may be a switching delay associated with the optical switch alternating between directing the laser pulses into and away from the microfluidic flow (or vice versa). This can affect the gating request signal applied to the optical switch by the controller 1435, as described below.

[0352] Figure 15bAn example of a control method for sorting multicellular events is illustrated. A fluorescence signal 1510m from detector 130 is received by controller 1435 and used to detect multicellular events. In this example, peak detection is used to detect multiple closely spaced cells that together correspond to a multicellular event. Multicellular events can be defined in different ways, such as comprising fluorescence signal peaks within a predetermined time interval. Each peak itself corresponds to a rapid rise and subsequent fall in signal amplitude, as illustrated by the peak detection signal 1515m. In other examples, different multicellular event detection methods may be used, such as those related to… Figure 2b As described.

[0353] The controller 1435 uses the fluorescence signal and optionally other characteristics of the signal waveform to determine whether a detected cellular event corresponds to a desired cell (e.g., an X sperm cell) or an unwanted cell (e.g., a Y sperm cell). Detected multi-cell events corresponding to unwanted cells can be classified as selected cellular events to be sorted by the pulse sorting arrangement 1440. Selected cellular events can correspond to multi-cell events where only one or some cells are unwanted, but also include other cells that may be considered desirable. Multi-cell events not classified as selected cellular events may include cells that are allowed to pass through the sorting area 1470 without any sorting interaction.

[0354] A pulsed laser 1445 generates regular laser pulses, as illustrated in 1525. A controller 1435 controls an optical switch 1455 to allow a designated pulse to be directed into the microfluidic flow according to multiple gating periods 1520m. As illustrated in 1530m, during each gating period 1530m, only a single designated laser pulse is directed into the microfluidic flow to sort the corresponding individual cell of a selected multicellular event 205m. In other words, the size of the gating period is set and its timing is coordinated or synchronized with the laser pulses so that only the designated single laser pulse passes through during that gating period. All other laser pulses 1525 are blocked and cannot interact with or sort the cells in the microfluidic flow. This arrangement using a designated pulse (e.g., only a single laser pulse) to sort cells reduces collateral damage to other nearby cells. In other words, adjacent cells of a potentially desired cell will not be sorted or affected by the designated pulse.

[0355] Figure 15cAnother control method for sorting multi-cell events is illustrated. Fluorescence signal 1510x from detector 130 is received by controller 1435 and used to detect multi-cell events 1515mx and single-cell events 1515sx. In this example, peak detection is used to detect multiple closely spaced cells, which together correspond to multi-cell events. Peaks with larger spacing on the right are detected as individual single-cell events. Multi-cell events can be defined in different ways, such as including fluorescence signal peaks within a predetermined time interval. Each peak itself corresponds to a rapid rise and subsequent fall in signal amplitude. In other examples, different multi-cell event detection methods, such as those related to… Figure 2b As described.

[0356] The controller 1435 uses this characteristic and optionally other characteristics of the signal waveform to determine whether a detected cellular event corresponds to a desired cell (e.g., an X sperm cell) or an unwanted cell (e.g., a Y sperm cell). Detected multi-cell events or single-cell events corresponding to unwanted cells are classified as selected cellular events to be sorted by the pulse sorting arrangement 1440. Selected cellular events may correspond to multi-cell events, where only one or some cells are unwanted, but also include other cells that may be considered desirable. Multi-cell events not classified as selected cellular events may include cells that are allowed to pass through the sorting area 1470 without any sorting interaction.

[0357] Pulsed laser 1445 generates regular laser pulses, as illustrated in 1515. Controller 1435 controls optical switch 1455 to allow multiple pulses to pass through the microfluidic flow according to a gating opening period 1520mx corresponding to the detected multicellular event 1515mx. The figure also illustrates the sorting of single-cell events 1515sx, which is discussed above regarding... Figure 15a A more comprehensive description.

[0358] and Figure 15b The control method differs; in this example, a single, extended gating opening period is used to sort cells associated with a selected multicellular event, during which more than one regular laser pulse is directed into the microfluidic flow. The time within the sorting zone 1470 will depend on the flow rate of the microfluidic flow. The inter-pulse interval between this flow rate and the laser pulse can be configured such that a portion of each cell in the multicellular event will coincide with the laser pulse during the gating period, as described in more detail below. In some examples where the cells are sperm cells, the flow rate and inter-pulse interval can be configured such that at least one laser pulse coincides with the passage of the sperm cell head within the sorting zone.

[0359] In some examples, the size of the gating opening period is set and its timing is coordinated or synchronized with the laser pulses so that only a designated pulse, such as a single laser pulse, passes through each cell during that gating period. All other laser pulses 1525 are directed away from or blocked, unable to interact with or sort the cells in the microfluidic flow. This arrangement, which uses only a designated laser pulse to sort cells, reduces collateral damage to other cells near multicellular events. In other words, neighboring cells of a potentially desired cell are not sorted or damaged by the laser pulse.

[0360] Figure 16 A control signaling timing diagram according to the example is illustrated. The control signaling diagram illustrates relative timing or various signals and can be implemented by a suitably programmed processor or a suitably configured FPGA (such as processor 1435). The first line 1611 illustrates a trigger signal 1621 corresponding to detecting a cell event and classifying it as a selected cell event. The trigger signal can be any suitable signal provided by the cell event detection and / or classification function, which can be implemented by, for example, a suitably programmed processor or a suitably configured FPGA; this can be the same or different processor used to implement the other signaling illustrated. The second line corresponds to a gating timing component 1613, which controls different delay periods and the laser pulse detection signal 1623 (also referred to as a synchronous output clock) to determine a gating open request 1625. The gating timing represents a functionality within a processor or FPGA configured to generate the illustrated signals in response to input signals such as the trigger signal 1621 and the laser pulse detection signal 1623. The laser pulse detection signal 1623 corresponds to a specific laser pulse having the intensity or amplitude illustrated on line 1617. In this example, these regular laser pulses have an inter-pulse period of 1 µs to aid in operational illustration, but the example is not limited to such a time frame.

[0361] Gating request period 1637 is initiated by gating open request 1625, which is a signal arranged to control the optical switch to open the optical gating for the duration of the gating request period. This corresponds to gating open period 1646, during which laser pulses incident on the optical switch are guided into the microfluidic flow. Line 1615 provides the gating state, and dashed line 1645 indicates the transition from a fully closed state 1640 to a fully open state 1642. In some examples, the optical switch may cause a switching delay between fully open and fully closed (or vice versa), as can be seen in the slanted lines of period waveform 1645. During this period, the laser may experience power reduction, misdirection, or defocus. The power of a single laser pulse incident on the target that occurs during the switching delay (i.e., from fully closed to fully open) may appear to be attenuated, and therefore the laser pulse guided into the microfluidic flow by the optical switch will be delivered to the microfluidic flow with reduced power. This can be problematic because some pulses may not be sufficient to sort cells.

[0362] Trigger signal 1621 induces gating request 1622 at gating control unit, which controls an initiation delay period 1631 between gating request 1622 and the initiation of gating opening period 1640. This initiation delay period 1631 is determined based on a predetermined delay period 1633 and a variable delay period 1635. The predetermined delay period 1633 corresponds to the transit time of the cell from detection zone 125 to sorting zone 1470. This will depend on the flow rate of the microfluidic stream and the distance between the two zones. A small margin can be allowed for the signal processing time required for detection and sorting. An example predetermined delay period of 2.75 μs is used in this example to aid in operational illustration, but other examples are not limited to such a time frame. The variable delay period 1635 depends on the timing of the next pulse detection signal 1623 following gating request 1622. By timing the gating open request 1625 to a predetermined period 1631 following the next laser pulse, the gating open period 1646 should coincide with the subsequent laser pulse and the cell corresponding to the selected cell event of the trigger signal 1621. The gating open request 162 and the gating request period 1637 can also be generated with consideration of switching delays, as described in more detail below.

[0363] In this example, for illustrative purposes, the trigger signal 1621 substantially coincides with the gating signal 1622. The variable delay period is the time between the gating request 1622 and the next pulse detection signal 1623. The pulse detection signal 1623 can be generated by, for example, a photodiode, and in one example, can be 0.5 μs. In this case, the activation delay period 1631 for requesting the optical switch to open is this variable delay of 0.5 μs plus a predetermined delay of 2.75 μs, such that the total request or activation delay period from the gating request 1622 to the gating open request at 1625 is 3.25 μs. In this example, the duration of the gating open period 1637 corresponds to the activation of the gating open period to allow a specified laser pulse to be directed into the microfluidic flow until the activation of the gating close switching delay 1645. In one example, this configuration can be used to implement... Figures 3a to 3d , Figure 5a , Figure 5b , Figure 6a , Figure 6b or Figure 15b The control and timing strategies are described.

[0364] In the illustrative example, the head of sperm cell 1605 has a length of approximately 10 μm, as shown in 1641, and will be continuously ablated within the target area for 1 μs at a flow rate of 10 m / s. By providing a gated opening period of less than or equal to 1 μs for this target period, during which at most one laser pulse with a pulse frequency of 1 μs will strike the head of the sperm cell. The gated opening period can be aligned such that its midpoint corresponds to the laser pulse. This avoids laser pulses occurring during one of the switching delays. In one example, the gated opening period can be 50% of the inter-pulse period, i.e., 0.5 μs. In another example, the gated period is within 30%–70% of the inter-pulse period, or within 46%–60% of the inter-pulse period.

[0365] Gating state line 1615 illustrates beam attenuation performed by an optical switch. When the optical switch or gating is fully open, all or nearly all the power of the laser pulse is directed to the microfluidic flow to sort cells. When the optical switch or gating is closed, no laser pulse power is directed to the microfluidic flow. It can be seen that there is a finite time for the optical switch to open and close, which corresponds to a switching delay and can cause beam attenuation. If a laser pulse occurs during this switching delay, only a portion of the laser pulse's power or intensity will be directed to the microfluidic flow. This can be problematic because a portion of the laser pulse may be insufficient to sort (e.g., by nudge or deactivation) unwanted cells and / or may affect adjacent desired cells.

[0366] This problem can be mitigated by timing the gating opening period so that the laser pulse falls within its central portion. In one example, the gating period can be adjusted so that the laser pulse coincides with the center of the gating period, or falls within the middle 80% or the middle 50% of the gating period.

[0367] Figure 17 A control signaling timing diagram based on another example is shown. The diagram is similar to... Figure 16 The figure shows features such as trigger line 1711, which exemplifies trigger signal 1721; second gating timing line 1713, which exemplifies delayed relative timing; laser pulse detection signal 1723; and gating open request 1725.

[0368] In this example, the next laser pulse detection signal 1723 is closer to the gating request 1722, and a shorter predetermined time period 1733, together with a variable delay 1735 calculated in a different manner, is used to ensure that the gating request time period 1737 coincides with the laser pulse and cell from the selected cell event. The shorter predetermined delay 1733 may be due to the shorter beam distance between the detection area 125 and the sorting area 1470 and / or the higher flow rate of the microfluidic flow. In this example, the variable delay 1735 between the gating request 1722 and the next laser pulse detection signal 1723 can be 0.25 μs, and with a predetermined delay of 0.75 μs, the initiation delay time period 1731 is 1 μs after the gating request 1721. Furthermore, gating opening is achieved by an optical switch and coincides with the gating opening request 1725 to provide a substantially pulse-centric gating opening time period 1746. In one example, this configuration can be used to implement regarding Figures 3a to 3d , Figure 5a , Figure 5b , Figure 6a , Figure 6b or Figure 15b The control and timing strategies are described.

[0369] Figure 18 An example of a control signaling timing diagram for multi-cell events is illustrated. In this example, the gating request period 1837 is extended over more than a single laser pulse so that it coincides with more than one cell 1805. This is similar to the approach regarding... Figure 15c The situation described. Figure 18 The diagram is similar to Figure 16 or Figure 17 The figure shows that features similarly marked include a first trigger line 1811 exemplifying trigger signal 1821, a second gating timing line 1813 exemplifying a delayed relative timing, a laser pulse detection signal 1823, and a gating request period 1837.

[0370] In this example, the extended gating request period 1837 corresponds to sorting two unwanted cells (or unwanted cells and desired or unsorted cells) in a multicellular event. As can be seen, the gating request period 1837 is extended over two complete laser pulses, illustrated by the full solid line on 1817. However, due to the switching delay of the optical switch after the end of the gating request period, the optical switch partially allows a third laser pulse to pass through to the microfluidic flow, as shown by the partial solid and partial dashed lines on 1817. This can be addressed by shortening the gating open request signal, where it is determined that the switching delay of the optical switch will coincide with the regular laser pulse. This can be achieved by estimating the next laser pulse based on past laser pulses and determining whether it will fall within a window that begins at the end of the gating request period and has a duration corresponding to the switching delay. If this occurs, the gating request period can be shortened by an amount corresponding to the switching delay, or by an even longer amount, which can depend on other factors such as the duration of the inter-pulse period and the flow rate. Alternatively, the gating open request 1825 can be initiated earlier by shortening the variable or predetermined delay period. In one example, this configuration can be used to implement... Figures 3a to 3d , Figure 5a , Figure 5b , Figure 6a , Figure 6b or Figure 15b The control and timing strategies are described.

[0371] Figure 19 Another control signaling timing diagram for multicellular events is illustrated, based on another example. Figure 18 Similar to the example, the gating request period 1937 is extended over more than a single laser pulse to coincide with more than one cell in a multicellular event. However, in this example, the gating request period 1937, which is typically used to signal when the optical switch is turned on and off, is overridden. In this example, the optical switch is controlled to turn off after the first laser pulse and turn on again before the next laser pulse, allowing two laser pulses to enter the microfluidic flow and sort two corresponding cells. This can be achieved by using a laser pulse detection signal 1923 to allow or prevent the operation of the gating open request signal on the optical switch. In one example, this configuration can be used to implement regarding Figures 3a to 3d , Figure 5a , Figure 5b , Figure 6a , Figure 6b or Figure 15bThe control and timing strategy is described. The optical switch can be configured to predict whether a gating close request will occur during a switching delay period preceding a subsequent pulse, and if so, to prevent gating open to direct the subsequent pulse into the microfluidic flow. This avoids unwanted portions of the pulse being directed during the switching delay.

[0372] Figure 19 The diagram is similar to Figure 16 , Figure 17 or Figure 18 The figure shows features such as a first trigger line 1911, which exemplifies a trigger or request signal 1921; a second gating timing line 1913, which exemplifies a delayed relative timing; a laser pulse detection signal 1923; and a gating request period 1937.

[0373] In this example, the gating open request signal 1937 corresponds to sorting two unwanted cells (or an unwanted cell and a desired or unsorted cell) in a multi-cell event. As can be seen, it is similar to... Figure 18 The gating request period 1937 is extended over two complete laser pulses, illustrated by the solid line at 1917. However, during the gating open request signal 1937, the optical switch is additionally controlled to open only during a predetermined gating open period 1941 near each laser pulse. This can be achieved by opening the optical gate halfway through the predetermined gating open period 1941 before the estimated time of the next laser pulse and closing the optical gate after the predetermined gating open period 1941. For example, for a 1µs inter-pulse period, the predetermined gating open period 1941 could be 0.5µs, thus the optical switch is controlled to open 0.25µs before the next laser pulse. This method avoids the aforementioned... Figure 18 The laser pulse described is because the optical switch is fully turned on when the laser pulse occurs.

[0374] In the previous example, the gating opening period was controlled in response to the detection of a regular pulse after a cell event was detected—for example, a predetermined delay could be implemented after both the cell event detection and the next regular pulse detection. In some other examples, the gating opening period could be controlled based on the detection of a regular pulse before a cell event was detected. This could be achieved by using the detection of a regular pulse to trigger a countdown timer, causing the inter-pulse period to expire with a predetermined delay. If a cell event is detected within the inter-pulse period after the countdown timer is triggered, the expiration of that timer is used to trigger a gating opening request 1625 to control the optical switch. The countdown timer can be started for each pulse detection. If no cell event is detected within the inter-pulse period after a pulse detection, cell event detection is monitored for the next countdown timer (i.e., the inter-pulse period after the next pulse detection), and so on, until a cell event detection falls within the inter-pulse period of one of the countdown timers.

[0375] Figure 20 The illustration shows a method according to an example, which uses a pulsed laser sorting arrangement structure to sort cells in a microfluidic flow using regular laser pulses. Method 2000 can be applied to, for example... Figure 14 This method is implemented in the cell sorting device of the cell processing system 1400, however, the method can be alternatively used in other cell sorting devices.

[0376] In 2005, Method 2000 detected cellular events. This can be used... Figure 14 The detection devices 120 and 130, along with appropriate signal processing, are used to achieve, for example, identifying pulses or peaks in the signal from the detection devices or predetermined signal waveforms as previously described. However, other methods for detecting cellular events may be alternatively employed.

[0377] In 2010, in some examples, the method determined whether the detected cellular event was a multi-cell event. However, in other examples, this process could be omitted, allowing the method to detect only single-cell events. Multi-cell events can be determined based on signal waveforms as previously described or by determining that individual signal pulses are within a predetermined distance from each other, indicating that a group of closely spaced cells has been detected.

[0378] In 2015, this method determined one or more characteristics of one or more cells associated with cellular events. In one example, this could involve determining whether one or more cells contain unwanted cells, such as Y sperm cells as previously described.

[0379] At 2020, if the cell event includes unwanted cells based on the determined characteristics, the method classifies the cell event as a selected cell event (2020Y). If the detected cell event is not classified as a selected cell event (2020N), the method returns to 2005 to detect another cell event. If the detected cell event is classified as a selected cell event (2020Y), the method sends a gating request signal to 2030. The method also returns to 2005 to detect another cell event.

[0380] At point 2025, the method receives a pulse detection signal corresponding to the detected laser pulse. These pulse detection signals, along with the sorting signal, are provided to the optical switching control process 2030.

[0381] At point 2030, the method generates a gating opening request for the optical switch. The above already provides examples regarding... Figures 16 to 19 Examples of different optical switch controls are discussed.

[0382] At 2025, the method opens (and closes) the optical switch based on a gating open request from 2030. In one example, this can be achieved by starting (and stopping) the RF signal driving the AOM, which causes the AOM to redirect incoming laser pulses into the microfluidic flow to sort cells associated with selected cellular events. However, alternative mechanisms for sorting cells associated with selected cellular events can be implemented alternatively.

[0383] Some examples offer one or more advantages by using the described gated opening control strategy with a pulse sorting arrangement. This approach reduces collateral damage to the desired cells while also allowing the sorting of multiple cells in multi-cell events. This, in turn, provides:

[0384] 1) Enhance the enrichment of desired cellular properties by improving cell sorting. In one example, the sorting action prevents unwanted cell progression (especially during multicellular events).

[0385] 2) Increase cell throughput while keeping cell selection metrics constant. That is, allow fewer unwanted cell types to pass through, meaning that a sorting arrangement can be used to collect higher cell concentrations. This has the effect of increasing the speed at which the sample passes through the system. This advantage can be particularly important when cells degrade based on the time spent flowing through the cell processing system. Therefore, increased cell throughput can ultimately lead to more viable collected cells due to the reduced sample flow time through the system.

[0386] 3) Real-time monitoring of cell and culture medium quality / characteristics. For example...

[0387] a) Sample quality,

[0388] b) Cell quality

[0389] c) Mass of the sheath fluid or buffer fluid.

[0390] Figure 21 An apparatus for splitting a sorting beam into multiple channels to sort cells in a corresponding microfluidic flow is shown. In this example, eight channels or microfluidic flows are shown flowing through eight sorting units or sorting arrangements 2155A-H; however, different numbers of channels can be implemented. Laser pulses from a pulsed sorting laser 2140 are split into eight beams using multiple beam splitters 2190A-H. The beam splitters can be polarization beam splitters. Each beam splitter transmits a portion of the light (e.g., S-polarized or P-polarized). Each sorting unit 2155A-H can be associated with a beam splitter 2190A-H, which may include elements for controlling whether incoming sorting laser pulses are directed to their respective microfluidic flows. The pulsed sorting arrangement as described previously can be employed; however, other sorting arrangements can be used alternatively.

[0391] A beam splitter is a component used to modify the power characteristics of a laser beam by dividing it into two or more separate parts. The division of the laser beam allows for control over the power distribution between different optical paths. For example, a beam splitter with a 50:50 split ratio will distribute 50% of the laser power along one path and 50% along another. This ratio can be adjusted, such as to 70:30 or 90:10, to control the amount of power directed into each path.

[0392] The beam splitter can also be adjusted to cause a reduction in the power of the original laser beam. As the beam is split, the power of each output beam may decrease or attenuate. This attenuation can be used to reduce or stop the power delivered to the sorting unit. In some examples, the beam splitter can be polarization-sensitive, allowing it to split the laser power based on the polarization state of the beam. This enables selective power control, where different polarization components of the laser beam are guided along separate optical paths, each with a different power level.

[0393] Furthermore, some beam splitters can be designed to divide the laser beam based on its spectral characteristics, such as wavelength. This allows different power levels to be directed into different paths based on the wavelength component of the laser beam, thus providing greater flexibility in managing power distribution in more complex optical systems. Therefore, in one example, the power transfer properties of the beam splitter are adjusted to segment the power based on detected cellular properties, such as fluorescence, waveform width, z-axis orientation, or cell location.

[0394] For a polarization beamsplitter that transmits P-polarized light, S-polarized light is reflected and guided to one of the eight sorting arrangements. The degree of S-polarization or P-polarization is determined by a polarization modulator (such as a half-wave plate), which can be placed before each polarization beamsplitter, such as... Figure 23 As shown. Each polarization modulator can be arranged to modulate the beam entering each beamsplitter 2190A-H. Each polarization modulator is tuned to control the power of the corresponding sorted beam guided to each sorting unit or arrangement structure. In one example, this can be achieved by rotating the polarization modulator while simultaneously measuring the power at a photodetector located before or after the beamsplitter. Some portions of the sorted beam are split by the beamsplitter and guided to the corresponding sorting arrangement structure, where the transmitted beam is continued to the next beamsplitter. The final beamsplitter 2190A can simply be a mirror (or a suitably routed fiber), where it is desirable to guide the entire remaining power of the beam to the pulse pickup. In one example, the polarization modulator is tuned such that the power of the laser pulse is uniformly split between the microfluidic channels.

[0395] Although polarization modulators have been mentioned herein, those skilled in the art will understand that other power regulation components can be used to influence the regulation of power transmission through the beam splitter. For example, a half-wave plate or a Pockels cell can be used. The latter has the utility of both picking up pulses to be directed into the microfluidic flow and modulating the power transmitted to the split beam.

[0396] If one of the channels is not needed, for example because one of the microfluidic flows stops or the sorting arrangement requires maintenance, the power transmission properties of the laser beam can be adjusted, for example, via a polarization modulator and / or beam splitter. In some examples, the power split between channels can be adjusted based on how many channels are active. The polarization control of the beam splitter can be adjusted automatically in response to detecting the state of the channels (i.e., whether they are in use or faulty).

[0397] In some examples, the method uses N or N-1 beamsplitters or polarization beamsplitters to sort cells within N microfluidic flows. The method may include adjusting the power transfer properties of at least one of the beamsplitters in response to cell event properties (such as detection, classification, and / or sorting properties) or for one or more beam states (such as deactivation of detection, classification, and / or sorting for one or more).

[0398] Adjusting power transfer properties means adjusting properties related to the beam power directed to one or more microfluidic flows. Power can be increased or decreased. The power of a first beam transmitted through a beamsplitter and directed to a first microfluidic flow can be adjusted independently of the power of a second beam directed to one or more other microfluidic flows. In one example, the inventors have found that beam power adjustment for a specific cellular event can be achieved based on detected cellular event properties and / or orientation. In another example, the inventors have found that beam power adjustment can be advantageously achieved based on beam state or waveform width. For example, detection, classification, and / or sorting feedback indicating that cellular event properties (such as multi-cell detection, sorting, orientation, localization) or cell selection metrics (such as X or Y cell enrichment) at one or more microfluidic flows in the microfluidic flow are below a predetermined threshold can be identified. Based on this feedback, the beamsplitter can be adjusted to adjust the power transfer properties of one or more microfluidic flows in the microfluidic flow. For example, feedback indicating inactive or invalid detection, classification, or sorting in a microfluidic flow, based on user input or an automated response, can be used to increase or decrease the power of that flow. In one example, waveform width or other waveform characteristics are used as a signal to control the adjustment of beam power attributes. The power transfer attributes of one or more beams may respond to cellular event attributes or beam states. In one example, cellular event attributes may relate to at least one of detection, classification, or sorting attributes, and beam states may relate to at least one of deactivation of detection, classification, and / or sorting for one or more of a plurality of microfluidic flows.

[0399] Power transfer can be adjusted based on detected cell properties and z-axis orientation, which can be detected by waveform or waveform width. Power transfer can be adjusted to a single microfluidic flow, multiple microfluidic flows, or a single beam that is further segmented to be directed into multiple microfluidic flows. In one example, a method for sorting cells within multiple microfluidic flows using a laser is provided, the method comprising:

[0400] A beam splitter is used to split the laser into multiple beams, each of which is associated with a corresponding microfluidic flow.

[0401] Detect cellular events within each microfluidic flow;

[0402] Classify the cellular events in each microfluidic flow into selected cellular events;

[0403] Analyze the cell event to determine the cell event attributes or beam state;

[0404] Adjust at least one power transmission attribute of the beam splitter based on cell event attributes or beam state;

[0405] One of the multiple beams is used to sort cells in each microfluidic flow that are associated with selected cellular events.

[0406] Figure 22 An exemplary cell processing system according to an example is shown. Cell processing system 2200 includes multiple sorting channels, each for sorting a corresponding microfluidic flow. System 220 includes a multiple pulsed sorting arrangement structure sharing a common sorting laser beam. In this way, cell processing system 2200 can reduce costs and improve robustness and simplicity by reducing the number of lasers required to process multiple microfluidic flows in parallel.

[0407] The cell processing system 2200 includes a single sorting laser 2240 and a single interrogation or detection laser 2230. The sorting laser 2240 may be a pulsed laser that generates laser pulses, and the detection laser 2230 may be a continuous-wave or quasi-continuous-wave laser. The sorting laser pulse is split between two channels by a beam splitter 2290B, such as a half-silvered mirror, a polarization beam splitter (e.g., in a free-space arrangement), or a fused biconical (FBT) beam splitter (in a fiber optic arrangement). Each sorted beam is directed to corresponding sorting devices 2285A and 2285B, each sorting device sorting cells in a corresponding microfluidic flow. This may involve using mirror 2290A to change the direction of the corresponding split beam in a free-space arrangement, or a suitable fiber optic route in a fiber optic arrangement.

[0408] The detection laser can be split between two or more channels using a beam splitter as shown in 2295B (such as a half-silvered mirror, a polarizing beam splitter, or a fused biconical (FBT) beam splitter). The power split ratio between channels can be approximately equal, for example, 50% for each in the example shown, and 25% for a system with four channels or microfluidic flow.

[0409] Each sorting device 2285A, 2285B can be associated with a controller 2255A, 2255B, which controls whether incoming sorting laser pulses are directed to their respective microfluidic flows. A pulse sorting arrangement as previously described can be used; however, other sorting arrangements may be alternatively employed. Figure 23An apparatus for splitting a sorting beam into multiple channels to sort cells in a corresponding microfluidic flow is shown. In this example, four channels or microfluidic flows are shown; however, different numbers of channels can be implemented. A laser pulse from a pulsed sorting laser 2340 is split into four beams using beam splitters such as polarization beam splitters 2390A, 2390B, 2390C, and 2390D. Each beam splitter transmits a portion of the light (e.g., S-polarized or P-polarized). In the case of transmitting P-polarized light, S-polarized light is reflected and guided to one of the four sorting arrangements. The degree of S- or P-polarization is determined by a polarization modulator such as a half-wave plate.

[0410] Figure 23 Four polarization modulators 2380A-D are shown, arranged to modulate the beam entering each beam splitter 2390A-D. Each polarization modulator is tuned to control the power of the corresponding sorted beam guided to each sorting arrangement. This can be achieved by rotating the polarization modulator while simultaneously measuring the power at a photodetector located before or after the pulse pickup 2355A-D. A portion of the sorted beam is split by the beam splitter and guided to the corresponding sorting arrangement, where the transmitted beam continues to the next beam splitter. The final beam splitter 2390A can simply be a mirror (or a suitably routed fiber), where it is desirable to guide the entire remaining power of the beam to the pulse pickup. In one example, the polarization modulators are tuned such that the power of the laser pulse is evenly divided among the four channels.

[0411] If one of the channels is not needed, for example because one of the microfluidic flows stops or the sorting arrangement requires maintenance, the power of the laser beam or other power transmission properties can be adjusted, for example, via a polarization modulator. In some examples, the power splitting between channels can be adjusted based on how many channels are active. Control of the beam splitter can be adjusted automatically in response to detecting the state of the channels (i.e., whether they are in use or faulty). Regarding... Figure 21 Further examples of adjusting power transmission properties are provided, and those skilled in the art will understand that such examples are also applicable to this sorting system and other sorting systems.

[0412] Figure 24 A controller 2400, according to some examples, is shown that can be used to implement methods for processing cells. The controller 2400 can be implemented as... Figure 14 The controller 1435, however, the controller 1400 can be used in different systems.

[0413] The controller 2400 includes hardware 2403 having a processor 2406 and memory 2409. The processor may be a microcontroller, an FPGA, or any other suitable hardware or a combination of hardware and software. The memory 2409 includes first computer program instructions 2412 that, when executed by the processor 2406, cause the controller 2400 to perform a plurality of steps 2452-2456. This can be implemented in conjunction with other hardware such as a laser (not shown). The memory 2409 may additionally or alternatively include second computer program instructions 2415 that, when executed by the processor 2406, cause the controller 2400 to perform a plurality of steps 2462-2466. This can be implemented in conjunction with other hardware such as a laser (not shown). The memory 2409 may additionally or alternatively include third computer program instructions 2417 that, when executed by the processor 2406, cause the controller 2400 to perform a plurality of steps 2472-2476. This can be implemented in conjunction with other hardware such as a laser (not shown).

[0414] At 2452, the processor detects cellular events within the microfluidic flow by detecting the rising edge of the waveform in the received emitted signal associated with the microfluidic flow, followed by detecting the falling edge of the waveform, or more generally by detecting signal peaks with certain characteristics, such as minimum amplitude. As previously discussed, the emitted signal can be generated by detectors 130 arranged around the microfluidic flow carrying cells, the presence of which can be detected in the interrogation region by the characteristics of scattered light or fluorescence emission after the microfluidic flow has been irradiated by a suitable radiation source, such as infrared (IR) light or ultraviolet (UV) light. In one example, the infrared light is generated by a quantum cascade laser.

[0415] At 2454, the processor classifies the cell event into selected cell events, for example, by using a corresponding waveform. As mentioned herein, selected cell events can include any cell event identified as a multi-cell event or a single-cell event, wherein the associated single cell is an unwanted cell, such as a Y cell. Various methods can be used to determine multi-cell events, such as having a waveform width exceeding a threshold, having multiple peaks, or not being identified as a single-cell event. Selected single-cell events can be determined using characteristics of the waveform, such as an integral within or above a selected cell threshold.

[0416] At 2456, the processor initiates a sorting action on cells in a selected cellular event by controlling a gating opening period during which one or more regular pulses are directed into the microfluidic flow. The gating opening period depends on the detection of the cellular event and the timing of the one or more regular pulses. In some examples, the regular pulses used for sorting can be implemented using a pulsed laser; however, as previously described, other implementations are also possible.

[0417] At 2472, the processor detects cellular events within multiple microfluidic flows by detecting the rising edge of a waveform in a received emitted signal associated with the microfluidic flow, followed by detecting the falling edge of the waveform, or more generally by detecting a signal peak with certain characteristics, such as a minimum amplitude. Cellular events in different microfluidic flows can be detected at the same or different times. As previously discussed, the emitted signal can be generated by detectors 130 arranged around each microfluidic flow carrying cells, the presence of which can be detected in the interrogation region by the characteristics of scattered light or fluorescence emission after the microfluidic flow has been irradiated by a suitable radiation source such as infrared (IR) light or ultraviolet (UV) light. In one example, the infrared light is generated by a quantum cascade laser. In another example, regular laser pulses can be used, and in some examples, these regular laser pulses can also be used to sort selected cellular events.

[0418] At 2474, the processor, for example, classifies the detected cellular events into selected cellular events using a corresponding waveform. As mentioned herein, selected cellular events can include any cellular event identified as a multi-cell event or a single-cell event, wherein the associated single cell is an unwanted cell, such as a Y cell. Multi-cell events can be determined using various methods, such as having a waveform width exceeding a threshold, having multiple peaks, or not being identified as a single-cell event. Selected single-cell events can be determined using characteristics of the waveform, such as an integral within or above a selected cell threshold.

[0419] At position 2476, the processor initiates a sorting action for cells in selected cellular events across multiple microfluidic flows. This can be achieved using a regular laser pulse from a single source, split into multiple beams, each associated with a corresponding sorting device for a different microfluidic flow. This allows for parallel sorting of cells across multiple microfluidic flows. In one example, this can be achieved by controlling the gating periods of multiple microfluidic flows, during which one or more regular pulses are directed to the corresponding microfluidic flow. The gating period for each microfluidic flow depends on the detection of cellular events in the corresponding microfluidic flow and the timing of one or more regular pulses.

[0420] Figure 25A sorting apparatus according to an example is shown, which uses a pulse sorting arrangement that generates regular pulses to sort cells within a microfluidic flow. In this example, the sorting apparatus 2500 includes a pulsed laser 2505 that generates regular laser pulses. The laser pulses are directed to an optical switch or gating 2510, which controls or switches the incoming laser pulses to direct them into or away from the microfluidic flow (or at least the cells within the microfluidic flow)—the switch or gating is open when a laser pulse is directed into the microfluidic flow, and closed when a laser pulse is not directed into the microfluidic flow.

[0421] Optical switch 2510 is also controlled to direct incoming laser pulses away from the microfluidic flow, or at least away from cells flowing within the microfluidic flow—a switch or gating closure. Control is achieved using a gating open request during a selected time period, during which the optical switch is requested or controlled to open. In this example, the optical switch is an AOM that toggles between directing a laser pulse or beam in one direction toward the microfluidic flow (with an open position for a 1st-order beam shown at 2515) and in another direction away from the microfluidic flow (with a closed position for a 0th-order beam shown at 2515).

[0422] The inventors have discovered that a beam focusing device (not shown) can be used to narrow the laser pulse entering an optical switch, thereby enabling better control via the optical switch. A beam expander (not shown) between the optical switch and the microfluidic flow can be used to extend the laser pulse to improve the sorting interaction with cells in the microfluidic flow.

[0423] The sorting device 2500 also includes a controller 2520, a synchronization circuit 2525, and an optical switch driver 2530. The optical switch driver 2530 may include a radio frequency (RF) driver that, during operation, deflects incoming laser pulses into the AOM (Alternating Optical Oscillator). The synchronization circuit 2525 can be implemented using a photodiode or other photodetector positioned to intercept a portion of the laser pulse, for example, by using a beam splitter to direct a low-power beam of the laser pulse to the photodetector. The synchronization circuit 2525 can be implemented using an AND gate that receives inputs from the controller 2520 and the photodetector. When a gate opening request from the controller is ON and a laser pulse detection signal from the photodetector is ON, the AND gate outputs an ON gate opening request, which is sent to the RF driver 2530 to generate an RF driver signal to open the optical switch. This implementation corresponds to... Figure 19The control method involves a synchronization circuit that overrides the gate opening request. This control method can be implemented using alternative circuit architectures. In other examples, the output from the synchronization circuit 2525 may simply be the output signal from the photodetector, which is fed as input to the controller 2520 to control the generation of a gate opening request directly output to the RF driver.

[0424] Controller 2520 can be configured to implement already related Figures 16 to 19 One of the control strategies discussed; and receiving a sorting signal as a trigger to sort selected cell events based on cell event detection and classification.

[0425] Example

[0426] The following examples illustrate implementation schemes of the present invention.

[0427] Purpose

[0428] The effects of different cell-targeting technologies on the overall enrichment of selected cells were compared, while also considering the viability and motility of the collected cells.

[0429] method

[0430] Sperm cells were collected from bulls and stained using Hoechst-33342 according to the sample preparation and staining conditions described in the following literature: Garner et al. (2013-Sex-Sorting Sperm Using Flow Cytometry / Cell Sorting, Methods in Molecular Biology (Clifton, NJ), Vol. 927, pp. 279-295). Cells were passed through a microfluidic focusing device and then through a UV interrogating laser (~355 nm). A photomultiplier tube detector collected cellular fluorescence emission from each cell and converted them into digital signals, which were then transmitted to a signal processor. The signal processor determined waveforms exceeding an event threshold and calculated the integral of each waveform. Cells exceeding the cell selection threshold were selected and inactivated using a sorting beam. The following sorting strategies were compared:

[0431] 1. Static Timing Select Trigger (STST) - After positive selection of a single-cell or multi-cell event, a single laser pulse is emitted at the cell using a constant offset delay, which is timed to coincide with the time it takes for one or more cells to pass through the focus of the sorting beam. This mode cannot distinguish between single-cell and multi-cell events.

[0432] 2. Adaptive Timing Selective Trigger (ATST) - After positive selection of cellular or multicellular events, a constant offset delay is used to trigger the laser for a duration corresponding to the waveform width for a time t. This constant offset delay is timed to coincide with the time it takes for a cell to pass through the focus of the sorting beam.

[0433] 3. Adaptive Timing Adaptive Selection Trigger (ATAST) - After positive selection of cellular or multicellular events, the laser is triggered after an adjusted offset delay (OD) (Equation 2 above) for a continuous selection period t (Equation 3 above).

[0434] Cells were collected and subjected to an "upstream" procedure to separate motile sperm from inactive sperm. This ensured that only cells not selected for sorting and inactivation were analyzed during enrichment analysis. DNA was lysed, extracted, and precipitated according to standard methods. Enrichment was assessed by comparing X-chromosome and Y-chromosome-specific genes with reference genes.

[0435] Assess the viability of the collected cells.

[0436] Two experiments were conducted. Experiment 1 evaluated the enrichment of cells not selected for sorting when using the three main cell-targeting technologies. Experiment 2 evaluated the enrichment of cells not selected for sorting when using static timed selection triggering (STST) and adaptive timed selection triggering (ATST).

[0437] Results of Experiment 1

[0438] Using STST as a reference, the enrichment results when using ATST and ATAST are as follows:

[0439] ATST showed that the purity of X cells increased by 8.60% (+ / - 0.98%).

[0440] ATAST showed that the purity of X cells increased by 7.07% (+ / - 1.41%).

[0441] Cell viability and motility did not show significant differences when treated with different methods.

[0442] Results of Experiment 2

[0443] Using STST as a reference, the enrichment when using ATST is as follows:

[0444] ATST showed that X cell purity increased by 6.51% (+ / - 1.53%).

[0445] Cell viability and motility did not show significant differences when treated with different methods.

[0446] in conclusion

[0447] Compared to STST, both Adaptive Timed Selection Trigger (ATST) and Adaptive Timed Adaptive Selection Trigger (ATAST) resulted in significantly higher X cell purity. This indicates that significantly more cells of the desired type (i.e., those not selected for sorting) were collected.

[0448] Cell viability and motility were not significantly different when treated with different methods, indicating that these techniques have no adverse effects on cell health.

[0449] Any and all references to publications or other documents, including but not limited to patents, patent applications, articles, web pages, books, etc., presented anywhere in this application, are incorporated herein by reference in their entirety.

[0450] As noted elsewhere, the disclosed embodiments of the invention are described for illustrative purposes only and are not restrictive. Other examples are possible and covered by this disclosure, as will be apparent from the teachings contained herein. Therefore, the breadth and scope of this disclosure should not be limited by any of the foregoing examples, but should be defined solely by the claims supported by this disclosure and their equivalents. Furthermore, the examples disclosed in this subject matter may include methods, systems, and apparatuses / devices, and may also include any and all elements from any other disclosed methods, systems, and apparatuses, including any and all elements corresponding to combination event-determining systems, apparatuses, and methods. In other words, elements from one or more disclosed examples may be interchangeable with elements from other disclosed examples. Moreover, one or more features / elements of a disclosed example may be removed and still result in patentable subject matter (and thus, more examples disclosed in this subject matter). Moreover, some examples correspond to systems, apparatuses, and methods that are specifically missing one and / or another element, structure, and / or step (if applicable) compared to the teachings of the prior art, and thus represent patentable subject matter and can be distinguished from it (i.e., claims for such examples may include one or more negative limitations to indicate that the prior art teachings lack one or more features).

[0451] The various inventive concepts disclosed herein can be embodied in one or more methods (as described above). Actions performed as part of a method can be ordered in any suitable manner. Thus, examples can be constructed in which actions are performed in an order different from the illustrative order, which may include performing several actions simultaneously, even if they are shown as sequential actions in the illustrative embodiments.

[0452] The following numbered clauses define specific aspects and implementation schemes envisioned herein: Clauses :

[0453] 1. A method for sorting cells in a microfluidic flow using a pulse sorting arrangement structure that generates regular pulses, the method comprising:

[0454] Detecting cellular events within the microfluidic flow;

[0455] The cellular events are classified into selected cellular events;

[0456] Cells associated with the selected cellular events are sorted by controlling the gating opening period, during which one or more of the regular pulses are directed to the microfluidic flow;

[0457] The gating opening period is controlled based on the detection of the cellular events and the timing of one or more regular pulses in the regular pulses.

[0458] 2. The method described in Clause 1:

[0459] The gating opening period includes a switching delay between the introduction of one or more regular pulses into the microfluidic flow and the introduction of one or more regular pulses out of the microfluidic flow; and

[0460] The gating opening period is controlled to prevent regular pulses from occurring during the switching delay.

[0461] 3. The method according to Clause 1 or 2, wherein the gating opening period is controlled in response to the detection of a regular pulse following the detection of the cellular event.

[0462] 4. The method according to Clause 1 or 2, wherein the gating opening period is controlled based on the detection of a regular pulse prior to the detection of the cellular event.

[0463] 5. The method according to any of the preceding clauses, the method comprising initiating the gating opening period after detecting the cell event or classifying the cell event as the selected cell event, wherein the initiation delay period includes a predetermined delay and a variable delay depending on the timing of the one or more rule pulses in the rule pulses.

[0464] 6. The method described in Clause 5:

[0465] The predetermined delay depends on the transit time of a cell in the microfluidic flow between a detection position and a sorting position, the detection position corresponding to the detection of a cell event and the sorting position corresponding to the sorting of cells associated with the cell event;

[0466] And the variable delay therein depends on the detection of the regular pulse after the detection of the cellular event.

[0467] 7. The method according to Clause 6, wherein the variable delay is calculated using the time difference between classifying the cell event as a selected cell event and the detection of the next rule pulse.

[0468] 8. The method according to any of the preceding clauses, wherein the gating opening period is one or more of the following: equal to or less than the inter-pulse period between the regular pulses; 30%-70% of the inter-pulse period; 40%-60% of the inter-pulse period, or about 50%.

[0469] 9. The method according to any of the preceding clauses, wherein the gating opening period is controlled to overlap with a single pulse.

[0470] 10. The method according to Clause 9, wherein the single pulse is timed within a central portion of the gating opening period, the central portion comprising one of the following: the middle 80% of the gating period; the middle 50% of the gating period; or the middle of the gating period.

[0471] 11. The method according to any one of clauses 1 to 8, wherein the gating opening period is controlled to overlap with two or more pulses in response to the detection of a cellular event associated with a plurality of cells.

[0472] 12. The method according to Clause 11, the method comprising ending the gating opening period after detecting the last cell in the cell event or classifying the cell event as a selected cell event, wherein the ending delay period depends on the transit time of a cell in the microfluidic flow traveling between a detection position and a sorting position, the detection position corresponding to detecting a cell event and the sorting position corresponding to sorting cells associated with the cell event.

[0473] 13. The method according to Clause 12, wherein the end delay period comprises the transit time minus a variable end delay, the variable end delay depending on the switching delay between the one or more regular pulses being directed into the microfluidic flow and the one or more regular pulses being directed out of the microfluidic flow.

[0474] 14. The method according to Clause 13, wherein the variable end delay is calculated in response to determining that the next pulse will coincide with the switching delay associated with the end of the gating period.

[0475] 15. The method according to any of the preceding clauses, wherein the pulse sorting arrangement structure includes a pulsed laser that generates regular laser pulses.

[0476] 16. The method according to Clause 15, wherein the sorting includes using the laser pulse to nudge, inactivate, or ablate cells associated with the selected cellular events.

[0477] 17. The method according to Clause 16, wherein the gating opening period is associated with an optical switch, the optical switch being controlled to switch laser pulses into and out of the microfluidic flow.

[0478] 18. The method according to Clause 17, wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector; or an electro-optic modulator.

[0479] 19. The method according to any one of clauses 15 to 18, wherein the regular laser pulses are further used to sort cells in the second microfluidic flow that are associated with selected cellular events.

[0480] 20. The method according to any of the preceding clauses, wherein the cell is a sperm cell.

[0481] 21. A method for sorting cells within a plurality of microfluidic flows using a pulsed laser that generates regular laser pulses, the method comprising:

[0482] The laser pulse is split into multiple beams, each beam being associated with a corresponding microfluidic flow;

[0483] Detect corresponding cellular events within at least two of the plurality of microfluidic flows;

[0484] Each detected cellular event in each corresponding microfluidic flow is individually classified as a selected cellular event;

[0485] By independently controlling regular laser pulses of the corresponding beams entering the respective microfluidic flows, one or more cells in at least two microfluidic flows associated with the selected cellular events are sorted.

[0486] 22. The method according to Clause 21, wherein one or more beam splitters are used to split the regular laser pulse into the plurality of beams.

[0487] 23. The method according to Clause 22, wherein the beam splitter is a polarization beam splitter, and the beam splitting ratio of the beam is adjusted by adjusting the ratio of light polarized in a first plane to light polarized in a second plane.

[0488] 24. The method according to Clause 23, wherein the amount of light in the first plane relative to the second plane is adjusted by a polarization modulator.

[0489] 25. The method according to Clause 23 or 24, the method being used to sort cells within a plurality of microfluidic streams using at least one beam splitter, the method comprising adjusting the power transmission properties of at least one beam splitter in response to cell event attributes or beam states for one or more microfluidic streams in the plurality of microfluidic streams, optionally wherein the cell event attributes include at least one of detection, classification, or sorting attributes, optionally wherein the beam states include at least one of deactivation of detection, classification, and / or sorting for one or more microfluidic streams in the plurality of microfluidic streams.

[0490] 26. The method according to any one of clauses 21 to 25, wherein sorting cells associated with the selected cellular event comprises:

[0491] Controlling a corresponding gating opening period, during which one or more regular laser pulses in the corresponding beam are guided to the corresponding microfluidic flow;

[0492] The corresponding gating opening period is controlled based on the detection of the cellular events of the corresponding microfluidic flow and the timing of one or more regular laser pulses in the regular laser pulses.

[0493] 27. The method according to Clause 26, wherein the corresponding gating opening period includes a switching delay between when the one or more regular laser pulses of the corresponding beam are guided into the corresponding microfluidic flow and when the one or more regular laser pulses of the corresponding beam are guided away from the corresponding microfluidic flow; and

[0494] The corresponding gating opening period is controlled to prevent regular laser pulses of the corresponding beam from occurring during the switching delay.

[0495] 28. The method according to clause 26 or 27, wherein the corresponding gating opening period is controlled in response to the detection of a regular laser pulse following the detection of the cellular event in the corresponding microfluidic flow.

[0496] 29. The method according to clause 26 or 27, wherein the corresponding gating opening period is controlled based on the detection of regular laser pulses prior to the detection of the cellular event.

[0497] 30. The method according to any one of clauses 26 to 29, said method comprising:

[0498] The corresponding gating opening period is initiated after a certain start delay period following the detection of the cellular event in the corresponding microfluidic flow or the classification of the cellular event into a selected cellular event for the corresponding microfluidic flow.

[0499] The aforementioned start-up delay period includes a predetermined delay and a variable delay that depends on the timing of one or more of the regular laser pulses.

[0500] 31. The method described in accordance with Clause 30:

[0501] The predetermined delay depends on the transit time of a cell in the corresponding microfluidic flow between a detection position and a sorting position, the detection position corresponding to detecting a cell event in the corresponding microfluidic flow, and the sorting position corresponding to sorting cells associated with the cell event in the corresponding microfluidic flow.

[0502] And the variable delay depends on the detection of the regular laser pulse after the detection of the cellular event.

[0503] 32. The method according to Clause 31, wherein the variable delay is calculated using the time difference between classifying the cellular event as a selected cellular event for the corresponding microfluidic flow and the detection of the next regular laser pulse.

[0504] 33. The method according to any one of clauses 26 to 32, wherein the corresponding gating opening period is one or more of the following: equal to or less than the inter-pulse period between the regular laser pulses; 30%-70% of the inter-pulse period; 46%-60% or about 50% of the inter-pulse period.

[0505] 34. The method according to any one of clauses 26 to 33, wherein the corresponding gating opening period is controlled to overlap with a single laser pulse.

[0506] 35. The method according to Clause 34, wherein the single laser pulse is timed within the central portion of the corresponding gating opening period, the central portion comprising one of the following: the middle 80% of the corresponding gating period; the middle 50% of the corresponding gating period; or the middle of the corresponding gating period.

[0507] 36. The method according to any one of clauses 26 to 33, wherein the respective gating opening period is controlled to overlap with two or more laser pulses in response to the detection of a cellular event associated with a plurality of cells in the respective microfluidic flow.

[0508] 37. The method according to Clause 36, the method comprising ending the corresponding gating opening period after detecting the last cell in the cellular event or classifying the cellular event as a selected cellular event in the corresponding microfluidic flow by a certain end delay period, wherein the end delay period depends on the transit time of the cells in the corresponding microfluidic flow traveling between a detection position and a sorting position, the detection position corresponding to detecting the cellular event in the corresponding microfluidic flow, and the sorting position corresponding to sorting the cells in the corresponding microfluidic flow associated with the cellular event.

[0509] 38. The method according to Clause 39, wherein the end delay period comprises the transit time minus a variable end delay, the variable end delay depending on the switching delay between the one or more regular laser pulses of the corresponding beam being guided into the corresponding microfluidic flow and the one or more regular laser pulses being guided away from the corresponding microfluidic flow.

[0510] 39. The method according to Clause 38, wherein the variable end delay is calculated in response to determining that the next laser pulse will coincide with a switching delay associated with the end of the corresponding gating period.

[0511] 40. The method according to any one of clauses 26 to 39, wherein the sorting comprises using the laser pulse to nudge, inactivate or ablate cells associated with the selected cellular event.

[0512] 41. The method according to any one of clauses 26 to 40, wherein the respective gating opening period is associated with an optical switch, the optical switch being controlled to switch laser pulses into and out of the respective microfluidic flow.

[0513] 42. The method according to Clause 41, wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector; or an electro-optic modulator.

[0514] 43. The method according to any one of clauses 21 to 42, wherein the cell is a sperm cell.

[0515] 44. A sorting apparatus for sorting cells in a microfluidic flow using a pulse sorting arrangement that generates regular pulses, the apparatus comprising:

[0516] A pulse sorting arrangement structure for generating regular pulses;

[0517] Detection components for detecting cellular events within the microfluidic flow;

[0518] A classification component for classifying the cellular events into selected cellular events;

[0519] A sorting component for sorting cells associated with the selected cellular events by controlling a gating opening period, during which one or more of the regular pulses are directed to the microfluidic flow;

[0520] The gating opening period is controlled based on the detection of the cellular events and the timing of one or more regular pulses in the regular pulses.

[0521] 45. The sorting device as described in Clause 44:

[0522] The gating opening period includes a switching delay between the introduction of one or more regular pulses into the microfluidic flow and the introduction of one or more regular pulses out of the microfluidic flow; and

[0523] The gating opening period is controlled to prevent regular pulses from occurring during the switching delay.

[0524] 46. ​​The sorting device according to clause 44 or 45, wherein the gating opening period is controlled in response to the detection of a regular pulse following the detection of the cellular event.

[0525] 47. The sorting device according to clause 44 or 45, wherein the gating opening period is controlled based on the detection of a regular pulse prior to the detection of the cellular event.

[0526] 48. The sorting device according to any one of clauses 44 to 47, wherein the sorting device is configured to initiate the gating opening period after detecting the cell event or classifying the cell event as a selected cell event for a certain initiation delay period, wherein the initiation delay period includes a predetermined delay and a variable delay depending on the timing of the one or more rule pulses in the rule pulses.

[0527] 49. The sorting device as described in Clause 48:

[0528] The predetermined delay depends on the transit time of a cell in the microfluidic flow between a detection position and a sorting position, the detection position corresponding to the detection of a cell event and the sorting position corresponding to the sorting of cells associated with the cell event;

[0529] And the variable delay therein depends on the detection of the regular pulse after the detection of the cellular event.

[0530] 50. The sorting device according to Clause 49, wherein the variable delay is calculated using the time difference between classifying the cell event as the selected cell event and the detection of the next rule pulse.

[0531] 51. The sorting device according to any one of clauses 44 to 50, wherein the gate opening period is one or more of the following: equal to or less than the inter-pulse period between the regular pulses; 30%-70% of the inter-pulse period; 46%-60% of the inter-pulse period, or about 50%.

[0532] 52. The sorting device according to any one of clauses 44 to 51, wherein the gate opening period is controlled to overlap with a single pulse.

[0533] 53. The sorting device according to Clause 52, wherein the single pulse is timed within the central portion of the gate opening period, the central portion comprising one of the following: the middle 80% of the gate period; the middle 50% of the gate period; the middle of the gate period.

[0534] 54. The sorting device according to any one of clauses 44 to 51, wherein the gating opening period is controlled to overlap with two or more pulses in response to the detection of a cellular event associated with a plurality of cells.

[0535] 55. The sorting device according to Clause 54, wherein the sorting device is configured to end the gating opening period after detecting the last cell in the cell event or classifying the cell event as the selected cell event, wherein the end delay period depends on the transit time of the cell in the microfluidic flow between a detection position and a sorting position, the detection position corresponding to the detection of the cell event and the sorting position corresponding to the sorting of the cell associated with the cell event.

[0536] 56. The sorting device according to Clause 55, wherein the end delay period comprises the transit time minus a variable end delay, the variable end delay depending on the switching delay between the one or more regular pulses being directed into the microfluidic flow and the one or more regular pulses being directed out of the microfluidic flow.

[0537] 57. The sorting device according to Clause 56, wherein the variable end delay is calculated in response to determining that the next pulse will coincide with the switching delay associated with the end of the gating period.

[0538] 58. The sorting apparatus according to any one of clauses 44 to 57, wherein the pulse sorting arrangement includes a pulsed laser that generates regular laser pulses.

[0539] 59. The sorting apparatus according to Clause 58, wherein the sorting includes using the laser pulse to nudge, inactivate, or ablate cells associated with the selected cellular event.

[0540] 60. The sorting device according to clause 58 or 59, the sorting device comprising an optical switch controlled to switch a laser pulse into and out of the microfluidic flow according to the gating opening period.

[0541] 61. The sorting device according to Clause 60, wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector; or an electro-optic modulator.

[0542] 62. The sorting apparatus according to Clause 61, wherein the acousto-optic modulator comprises a tellurium crystal or a silicon dioxide crystal.

[0543] 63. The sorting device according to any one of clauses 60 to 62, wherein the sorting device comprises one or more of the following:

[0544] A beam shortener, the beam shortener being arranged to narrow the regular laser pulse incident on the optical switch; and

[0545] A beam expander is arranged to amplify the laser pulses directed to the microfluidic flow.

[0546] 64. The sorting apparatus according to any one of clauses 58 to 63, wherein the pulsed laser is configured to generate regular laser pulses at a predetermined rate between 1 ps and 1 ms.

[0547] 65. The sorting apparatus according to any one of clauses 58 to 64, the sorting apparatus comprising a photodetector for detecting regular laser pulses.

[0548] 65a. The sorting apparatus according to Clause 65, wherein the photodetector comprises a photodiode, preferably an avalanche photodiode.

[0549] 66. The sorting apparatus according to any one of clauses 58 to 65a, wherein the regular laser pulses are further used to sort cells in a second microfluidic flow that are associated with selected cellular events.

[0550] 67. The sorting device according to any one of clauses 44 to 66, wherein the sorting device is configured for sorting sperm cells.

[0551] 68. A sorting apparatus for sorting cells within a plurality of microfluidic flows using a pulsed laser that generates regular laser pulses, the apparatus comprising:

[0552] A pulsed laser that generates regular laser pulses;

[0553] A beam splitter for splitting the laser pulse into multiple beams, each beam being associated with a corresponding microfluidic flow;

[0554] One or more detection components for detecting cellular events within a corresponding microfluidic flow;

[0555] One or more classification components for classifying the cellular events in a corresponding microfluidic flow into selected cellular events;

[0556] A corresponding sorting component for sorting the selected cellular events in the corresponding microfluidic flow using the regular laser pulses.

[0557] 69. The sorting apparatus according to Clause 68, wherein the beam splitter is a polarization beam splitter, and the beam splitting ratio is adjusted by adjusting the ratio of light polarized in a first plane to light polarized in a second plane.

[0558] 70. The sorting apparatus according to clause 68 or 69, the sorting apparatus further comprising at least one of a polarization modulator, a power adjustment component, a half-wave plate, or a Pockels cell arranged to adjust the amount of light in the first plane relative to the second plane.

[0559] 71. A sorting device according to any one of claims 68 to 70, the sorting device comprising at least one beam splitter for sorting cells within the plurality of microfluidic streams, the sorting device being configured to adjust the power transmission of at least some of the beam splitters in response to a cell event attribute or beam state for one or more microfluidic streams in the microfluidic streams, optionally wherein the cell event attribute includes at least one of detection, classification, or sorting attributes, optionally wherein the beam state includes at least one of deactivation of detection, classification, and / or sorting for one or more microfluidic streams in the microfluidic streams.

[0560] 72. The sorting apparatus according to any one of clauses 68 to 71, wherein the respective sorting component is configured to:

[0561] Controlling a corresponding gating opening period, during which one or more regular laser pulses in the corresponding beam are guided to the corresponding microfluidic flow;

[0562] The corresponding gating opening period is controlled based on the detection of the cellular events of the corresponding microfluidic flow and the timing of one or more regular laser pulses in the regular laser pulses.

[0563] 73. The sorting device according to clause 72, wherein the corresponding gating opening period includes a switching delay between when the one or more regular laser pulses of the corresponding beam are guided into the corresponding microfluidic flow and when the one or more regular laser pulses of the corresponding beam are guided away from the corresponding microfluidic flow; and

[0564] The corresponding gating opening period is controlled to prevent regular laser pulses of the corresponding beam from occurring during the switching delay.

[0565] 74. The sorting device according to clause 72 or 73, wherein the corresponding gating opening period is controlled in response to the detection of a regular laser pulse following the detection of the cellular event in the corresponding microfluidic flow.

[0566] 75. The sorting device according to clause 72 or 73, wherein the corresponding gating opening period is controlled based on the detection of regular laser pulses prior to the detection of the cellular event.

[0567] 76. The sorting device according to any one of clauses 72 to 75, the sorting device comprising initiating the corresponding gating opening period after detecting the cellular event in the corresponding microfluidic flow or classifying the cellular event as a selected cellular event for the corresponding microfluidic flow by a certain initiation delay period, wherein the initiation delay period comprises a predetermined delay and a variable delay depending on the timing of the one or more regular laser pulses in the regular laser pulses.

[0568] 77. The sorting device as described in Clause 76:

[0569] The predetermined delay depends on the transit time of a cell in the corresponding microfluidic flow between a detection position and a sorting position, the detection position corresponding to detecting a cell event in the corresponding microfluidic flow, and the sorting position corresponding to sorting cells associated with the cell event in the corresponding microfluidic flow.

[0570] And the variable delay depends on the detection of the regular laser pulse after the detection of the cellular event.

[0571] 78. The sorting device according to Clause 77, wherein the variable delay is calculated using the time difference between classifying the cellular event as a selected cellular event for the corresponding microfluidic flow and the detection of the next regular laser pulse.

[0572] 79. The sorting device according to any one of clauses 72 to 78, wherein the corresponding gate opening period is one or more of the following: equal to or less than the inter-pulse period between the regular laser pulses; 30%-70% of the inter-pulse period; 46%-60% of the inter-pulse period, or about 50%.

[0573] 80. The sorting device according to any one of clauses 72 to 79, wherein the corresponding gate opening period is controlled to overlap with a single laser pulse.

[0574] 81. The sorting device according to Clause 80, wherein the single laser pulse is timed within the central portion of the corresponding gate opening period, the central portion comprising one of the following: the middle 80% of the corresponding gate period; the middle 50% of the corresponding gate period; or the middle of the corresponding gate period.

[0575] 82. The sorting device according to any one of clauses 72 to 81, wherein the corresponding gating opening period is controlled to overlap with two or more laser pulses in response to the detection of a cellular event associated with a plurality of cells in the corresponding microfluidic flow.

[0576] 83. The sorting device according to Clause 82, wherein the sorting device is configured to end the corresponding gating opening period after detecting the last cell in the cellular event or classifying the cellular event as a selected cellular event in the corresponding microfluidic flow by a certain end delay period, wherein the end delay period depends on the transit time of the cells in the corresponding microfluidic flow traveling between a detection position and a sorting position, the detection position corresponding to detecting a cellular event in the corresponding microfluidic flow, and the sorting position corresponding to sorting cells in the corresponding microfluidic flow associated with the cellular event.

[0577] 84. The sorting apparatus according to Clause 83, wherein the end delay period comprises the transit time minus a variable end delay, the variable end delay depending on the switching delay between the one or more regular laser pulses of the corresponding beam being guided into the corresponding microfluidic flow and the one or more regular laser pulses being guided away from the corresponding microfluidic flow.

[0578] 85. The sorting device according to Clause 84, wherein the variable end delay is calculated in response to determining that the next laser pulse will coincide with a switching delay associated with the end of the corresponding gated period.

[0579] 86. The sorting apparatus according to any one of clauses 72 to 85, wherein the sorting includes using the laser pulse to nudge, inactivate or ablate cells associated with the selected cellular event.

[0580] 87. The sorting device according to any one of clauses 72 to 86, the sorting device comprising an optical switch controlled to switch a laser pulse into and out of the microfluidic flow according to the corresponding gating opening period.

[0581] 88. The sorting device according to Clause 87, wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector; or an electro-optic modulator.

[0582] 89. The sorting apparatus according to Clause 88, wherein the acousto-optic modulator comprises a tellurium crystal or a silicon dioxide crystal.

[0583] 90. A sorting device according to any one of clauses 87 to 89, wherein the sorting device comprises one or more of the following:

[0584] A beam shortener, the beam shortener being arranged to narrow the regular laser pulse incident on the optical switch; and

[0585] A beam reducer, which is arranged to amplify the laser pulses directed to the microfluidic flow.

[0586] 91. The sorting apparatus according to any one of clauses 72 to 90, said sorting apparatus comprising a photodetector for detecting regular laser pulses.

[0587] 92. The sorting apparatus according to any one of clauses 68 to 91, wherein the pulsed laser is configured to generate regular laser pulses at a predetermined rate between 1 ms and 1 ps.

[0588] 93. The sorting device according to any one of clauses 68 to 92, wherein the sorting device is configured for sorting sperm cells.

[0589] 94. A computer program comprising processor instructions that, when executed by a processor, cause the processor to perform the method according to any one of claims 1 to 43.

[0590] 95. A method for timing the sorting of cells within a microfluidic flow, the method comprising:

[0591] The cellular events are detected using waveforms from cellular emission signals received from and associated with cellular events within the microfluidic flow;

[0592] The waveform is used to classify the cellular events into selected cellular events;

[0593] One or more cells from the selected cell events are sorted within a selected time period, the selected time period depending on the waveform width of the waveform associated with the selected cell events.

[0594] 96. The method according to Clause 95, wherein the selected cellular events include unwanted cells.

[0595] 97. The method according to clause 95 or 96, wherein the cell emission signal is a fluorescence signal.

[0596] 98. The method according to any one of clauses 95 to 97, wherein the sorting includes inactivating the one or more cells in the selected cellular event.

[0597] 99. The method according to any one of clauses 95 to 98, wherein the duration of the selected time period depends on the waveform width.

[0598] 100. The method according to any one of clauses 95 to 99, wherein the selected period begins after a certain offset delay following the end of the waveform.

[0599] 101. The method according to Clause 100, wherein the offset delay depends on the waveform width.

[0600] 102. The method according to clause 101, wherein the offset delay is determined based on one or more of the following:

[0601] OD = POD1 - WW;

[0602] OD = POD2 - xWW - b;

[0603] Where OD is the determined offset delay, POD1 and POD2 are allocatable predetermined offset delays, WW is the waveform width, x is the allocatable coefficient, and b is the allocatable constant.

[0604] 103. The method according to clause 102, wherein the selection of the time period is determined based on the following:

[0605] SP = xWW + b;

[0606] SP represents the selected time period.

[0607] 104. The method according to any one of clauses 95 to 100, wherein the sorting comprises applying a laser beam to the microfluidic flow during the selected time period to inactivate cells within the selected cellular event.

[0608] 105. The method according to Clause 104, wherein the laser is applied with one or more discrete pulses having a duration shorter than the selected time period.

[0609] 106. The method according to clause 105, wherein the laser is configured to periodically generate pulses, and the selected time period depends on the frequency of the pulses applied to the microfluidic flow.

[0610] 107. The method according to any one of clauses 104 to 106, the method comprising applying the laser to the microfluidic flow using an acousto-optic modulator.

[0611] 108. The method according to any one of clauses 104 to 108, wherein the laser is a picosecond laser.

[0612] 109. The method according to any one of claims 95 to 108, wherein the sorting includes controlling one or more gating opening periods during the selection period, wherein during the gating opening period, one or more regular pulses are directed to the microfluidic flow, and wherein the gating opening period is controlled based on the detection of the cellular event and the timing of one or more of the regular pulses.

[0613] 110. The method according to any one of clauses 95 to 109, wherein classifying cellular events into selected cellular events using corresponding waveforms comprises determining at least one of the following:

[0614] a. The corresponding waveform has a waveform width exceeding the multi-cell event threshold; and

[0615] b. The corresponding waveform has characteristics corresponding to the selected cell.

[0616] 111. The method according to any one of clauses 95 to 110, wherein the waveform width is determined by detecting the rising edge, followed by the falling edge, of the waveform in a cell emission signal received from the microfluidic flow and associated with the cellular event.

[0617] 112. The method according to Clause 111, wherein the detected rising edge corresponds to a received transmitted signal whose rise above the baseline exceeds a rising edge threshold.

[0618] 113. The method according to clause 111 or 112, wherein the detected falling edge corresponds to a received transmitted signal that falls beyond a falling edge threshold.

[0619] 114. The method according to Clause 113, wherein the falling edge threshold is a predetermined percentage of the average height of the corresponding waveform or a series of waveforms.

[0620] 115. The method according to any one of clauses 95 to 114, wherein the waveform width is determined by detecting a peak in the waveform and detecting a previous rising edge or a subsequent falling edge of the waveform, and using the duration between the peak and the previous rising edge or the subsequent falling edge to determine the waveform width.

[0621] 116. The method according to any one of clauses 95 to 115, wherein the waveform width is determined by detecting a peak in the waveform and a predetermined duration before and / or after the peak.

[0622] 117. The method according to any one of clauses 95 to 117, wherein the cell is a sperm cell, and the selected cellular events include at least one of the following:

[0623] Sperm cells classified as Y sperm cells;

[0624] Multicellular events involving multiple sperm cells.

[0625] 118. The method according to any one of clauses 95 to 117, the method comprising using the waveform of the cell event to determine the z-axis of the cell in the cell event.

[0626] 119. The method according to Clause 118, wherein classifying the cell event as a selected cell event depends on the z-axis orientation of the cell in the cell event.

[0627] 120. The method according to clause 118 or 119, wherein determining the z-axis orientation of the cell includes identifying a first maximum value in the waveform corresponding to a first portion of the cell and identifying a second maximum value in the waveform corresponding to a second portion of the cell, and wherein the z-axis orientation of the cell is determined based on the order of the first maximum value and the second maximum value within the waveform.

[0628] 121. The method according to Clause 120, wherein the first maximum value and the second maximum value have different amplitudes, and wherein the z-axis orientation of the cell is determined based on whether the amplitude of the first maximum value is greater than the amplitude of the second maximum value.

[0629] 122. The method according to Clause 121, wherein the maximum value with a larger amplitude corresponds to the head of the sperm cell, and the maximum value with a smaller amplitude corresponds to the middle segment of the sperm cell.

[0630] 123. The method according to any one of clauses 120 to 122, wherein the waveform in the cell emission signal comprises a series of signal values ​​above a signal value baseline, and wherein the two maximum values ​​correspond to corresponding local maximum signal values ​​in the series of signal values.

[0631] 124. The method according to any one of clauses 120 to 123, wherein the signal value between the two maximum values ​​in the series of signal values ​​is 50% higher than the signal value of the first maximum value.

[0632] 125. The method according to any one of clauses 120 to 124, wherein the cell is a sperm cell, and the first portion of the cell is the head of the sperm cell, and the second portion of the cell is the midsection of the sperm cell.

[0633] 126. A method for determining the z-axis orientation of cells within a microfluidic flow, the method comprising:

[0634] The cells are detected using waveforms from cell emission signals received from and associated with cells within the microfluidic flow;

[0635] The z-axis orientation of the cell is determined by identifying the first maximum value corresponding to the first part of the waveform and the second maximum value corresponding to the second part of the waveform.

[0636] The z-axis orientation of the cell is determined based on the order of the first maximum value and the second maximum value within the waveform.

[0637] 127. The method according to Clause 126, wherein the first maximum value and the second maximum value have different amplitudes, and wherein the z-axis orientation of the cell is determined based on whether the amplitude of the first maximum value is greater than the amplitude of the second maximum value.

[0638] 128. The method according to Clause 127, wherein the maximum value with a larger amplitude corresponds to the head of the sperm cell, and the maximum value with a smaller amplitude corresponds to the middle segment of the sperm cell.

[0639] 129. The method according to any one of clauses 126 to 128, wherein the waveform in the cell emission signal comprises a series of signal values ​​above a signal value baseline, and wherein the two maximum values ​​correspond to corresponding local maximum signal values ​​in the series of signal values.

[0640] 130. The method according to any one of clauses 126 to 129, wherein the signal value between the two maximum values ​​in the series of signal values ​​is 50% higher than the signal value of the first maximum value.

[0641] 131. A method for adjusting the offset delay and / or selection period for sorting cells within a microfluidic flow, the method comprising:

[0642] The cellular events are detected using waveforms from cellular emission signals received from and associated with cellular events within the microfluidic flow;

[0643] The waveform is used to classify the cellular events into selected cellular events;

[0644] One or more cells from the selected cell event are sorted within the selected period after the offset delay following the selected cell event;

[0645] The selected time period and / or the offset delay are adjusted based on the waveform width of the waveform associated with the selected cell event.

[0646] 132. An apparatus for adjusting an offset delay and / or selection period for sorting cells within a microfluidic flow, the apparatus comprising a processor and a memory configured to:

[0647] The cellular events are detected using waveforms from cellular emission signals received from and associated with cellular events within the microfluidic flow;

[0648] The waveform is used to classify the cellular events into selected cellular events;

[0649] The sorting unit is controlled to sort one or more cells in the selected cell event within the selection period after the offset delay following the selected cell event;

[0650] The selected time period and / or the offset delay are adjusted based on the waveform width of the waveform associated with the selected cell event.

[0651] 133. The method of claim 132, wherein the sorting includes controlling one or more gating opening periods during the selection period, wherein during the gating opening period, one or more regular pulses are directed to the microfluidic flow, and wherein the gating opening period is controlled based on the detection of the cellular event and the timing of one or more of the regular pulses.

[0652] 134. A computer program comprising processor instructions that, when executed by a processor, cause the processor to perform the method according to any one of claims 95 to 133.

[0653] 135. An apparatus for timing the sorting of cells within a microfluidic flow, the apparatus comprising a processor and a memory configured to:

[0654] The cellular events are detected using waveforms from cellular emission signals received from and associated with cellular events within the microfluidic flow;

[0655] The waveform is used to classify the cellular events into selected cellular events;

[0656] The sorting component is controlled to sort one or more cells in the selected cell events within a selected time period, the selected time period depending on the waveform width of the waveform associated with the selected cell events.

[0657] 136. The apparatus according to clause 135, wherein the duration of the selected time period depends on the waveform width.

[0658] 137. The apparatus according to clause 135 or 136, wherein the selection period begins after a certain offset delay following the end of the waveform.

[0659] 138. The apparatus according to clause 137, wherein the offset delay depends on the waveform width.

[0660] 139. The apparatus according to any one of clauses 135 to 138, wherein the apparatus is configured to determine the waveform width by detecting a rising edge, followed by a falling edge, of the waveform in a cell emission signal received from the microfluidic flow and associated with the cellular event.

[0661] 140. An apparatus for processing cells within a microfluidic flow, the apparatus comprising a processor and a memory, the processor and memory being configured to:

[0662] Received emission signals associated with the microfluidic flow are used to detect cellular events within the microfluidic flow;

[0663] The cellular events are classified as unselected or selected cellular events;

[0664] The sorting component is controlled to sort one or more cells in a selected cellular event within a selected time period, the selected time period depending on the end of the selected cellular event and the duration of the selected cellular event.

[0665] 141. The apparatus according to any one of clauses 135 to 140, said apparatus comprising:

[0666] Components for directing interrogating electromagnetic radiation into cells within the microfluidic flow to promote responsive emission signals from the cells;

[0667] Components used to direct sorting electromagnetic radiation to selected cells within the microfluidic flow.

[0668] 142. The apparatus according to clause 141, the apparatus comprising optical components for guiding the interrogating electromagnetic radiation and / or the sorting electromagnetic radiation;

[0669] The optical components described therein can be adjusted according to the characteristics of the waveform.

[0670] 143. The apparatus according to clause 142, wherein the positioning of the optical component relative to the microfluidic flow is adjustable.

[0671] 144. The apparatus according to clause 142 or 143, wherein the characteristics of the waveform include one or more of the following characteristics of a plurality of waveforms associated with corresponding plurality of cellular events: maximum waveform intensity; maximum waveform width; integral of the plurality of waveforms.

[0672] 145. The apparatus according to any one of clauses 135 to 144, said apparatus comprising:

[0673] Components used to deliver the microfluidic flow;

[0674] The component used to deliver the microfluidic flow is adjustable to change the path of the microfluidic flow according to the characteristics of the waveform.

[0675] 146. The apparatus according to clause 145, wherein the component for delivering the microfluidic flow is a microfluidic chip.

[0676] 147. The apparatus according to clause 145 or 146, wherein the characteristics of the waveform include one or more of the following characteristics of a plurality of waveforms associated with corresponding plurality of cellular events: maximum waveform intensity; maximum waveform width; integral of the plurality of waveforms.

[0677] 148. An apparatus for processing cells within a microfluidic flow, the apparatus comprising:

[0678] Components for detecting cellular events using waveforms in cellular emission signals received from and associated with cellular events within the microfluidic flow;

[0679] Components for directing interrogating electromagnetic radiation into cells within the microfluidic flow to promote responsive emission signals from the cells;

[0680] Components for directing sorting electromagnetic radiation to selected cells within the microfluidic flow;

[0681] Optical components used to guide the interrogating electromagnetic radiation and / or the sorting electromagnetic radiation;

[0682] The optical components described therein can be adjusted according to the characteristics of the waveform.

[0683] 149. The apparatus according to clause 148, wherein the optical component is a laser for generating the sorted electromagnetic radiation, and wherein the power of the laser is adjustable according to the characteristics of the waveform.

[0684] 150. The apparatus according to Clause 149, wherein the positioning of the optical component relative to the microfluidic flow is adjustable.

[0685] 151. The apparatus according to clause 149 or 150, wherein the characteristic of the waveform includes one or more of the following: maximum waveform intensity; maximum waveform width; integral of the waveform; shape of the waveform; slope of the waveform; number of peaks of the waveform.

[0686] 152. An apparatus for processing cells within a microfluidic flow, the apparatus comprising:

[0687] Components used to deliver the microfluidic flow;

[0688] Components for detecting cellular events using waveforms in cellular emission signals received from and associated with cellular events within the microfluidic flow;

[0689] Components for directing interrogating electromagnetic radiation into cells within the microfluidic flow to promote responsive emission signals from the cells;

[0690] Components for directing sorting electromagnetic radiation to selected cells within the microfluidic flow;

[0691] The component used to deliver the microfluidic flow is adjustable to change the path of the microfluidic flow according to the characteristics of the waveform.

[0692] 153. The apparatus according to clause 152, wherein the component for delivering the microfluidic flow is a microfluidic chip.

[0693] 154. The apparatus according to clause 152 or 153, wherein the characteristics of the waveform include one or more of the following characteristics of a plurality of waveforms associated with corresponding plurality of cellular events: maximum waveform intensity; maximum waveform width; integral of the plurality of waveforms.

Claims

1. A method for adjusting the offset delay and / or selection period for sorting cells within a microfluidic flow, the method comprising: The cellular events are detected using waveforms from cellular emission signals received from and associated with cellular events within the microfluidic flow; The waveform is used to classify the cellular events into selected cellular events; One or more cells from the selected cell event are sorted within the selected period after the offset delay following the selected cell event; The selected time period and / or the offset delay are adjusted based on the waveform width of the waveform associated with the selected cell event.

2. The method according to any of the preceding claims, wherein the sorting comprises inactivating the one or more cells in the selected cellular events.

3. The method of claim 1, wherein the selected time period begins after the offset delay following the end of the waveform.

4. The method of claim 3, wherein the offset delay is determined according to one or more of the following: OD = POD1 - WW; OD = POD2 - xWW - b; Where OD is the determined offset delay, POD1 and POD2 are allocatable predetermined offset delays, WW is the waveform width, x is the allocatable coefficient, and b is the allocatable constant, and / or The selected time period is determined based on the following: SP = xWW + b; SP represents the selected time period.

5. The method according to any one of claims 1 to 4, wherein: The sorting includes applying a laser beam to the microfluidic flow during the selected time period to inactivate cells within the selected cellular event, and wherein the laser is applied in one or more discrete pulses having a duration shorter than the selected time period, and / or The sorting includes controlling one or more gating opening periods during the selected period, wherein during the gating opening period, one or more regular pulses are directed to the microfluidic flow, and wherein the gating opening period is controlled based on the detection of the cellular event and the timing of one or more of the regular pulses.

6. The method according to any one of claims 1 to 5, wherein classifying cellular events into selected cellular events using corresponding waveforms comprises determining at least one of the following: a. The corresponding waveform has a waveform width exceeding the multi-cell event threshold; and b. The corresponding waveform has characteristics corresponding to the selected cell.

7. The method according to any one of claims 1 to 6, wherein the waveform width is determined by at least one of the following: a. Detecting the rising edge, followed by the falling edge, of the waveform in the cell emission signal received from the microfluidic flow and associated with the cellular event; and b. Detect a peak in the waveform and detect a previous rising edge or a subsequent falling edge of the waveform, and use the duration between the peak and the previous rising edge or the subsequent falling edge to determine the waveform width, or c) detect a peak in the waveform and a predetermined duration before and / or after the peak.

8. The method according to any one of claims 1 to 7, wherein the cell is a sperm cell, and the selected cellular events include at least one of the following: Sperm cells classified as Y sperm cells; Multicellular events involving multiple sperm cells.

9. The method of claim 8, the method comprising using the waveform of the cell event to determine the z-axis orientation of the cell in the cell event, and wherein classifying the cell event as a selected cell event depends on the z-axis orientation of the cell in the cell event.

10. The method of claim 10 or 9, wherein determining the z-axis orientation of the cell includes identifying a first maximum value in the waveform corresponding to a first portion of the cell and identifying a second maximum value in the waveform corresponding to a second portion of the cell, and wherein the z-axis orientation of the cell is determined based on the order of the first maximum value and the second maximum value within the waveform.

11. The method of claim 10, wherein: The first maximum value and the second maximum value have different amplitudes, and wherein the z-axis orientation of the cell is determined based on whether the amplitude of the first maximum value is greater than the amplitude of the second maximum value; and / or The maximum value with a larger amplitude corresponds to the head of the sperm cell, and the maximum value with a smaller amplitude corresponds to the middle segment of the sperm cell; And / or The waveform in the cell-emitted signal includes a series of signal values ​​above the signal value baseline, and two of the maximum values ​​correspond to the respective local maximum signal values ​​in the series of signal values.

12. A method for determining the z-axis orientation of cells within a microfluidic flow, the method comprising: The cells are detected using waveforms from cell emission signals received from and associated with cells within the microfluidic flow; The z-axis orientation of the cell is determined by identifying the first maximum value corresponding to the first part of the waveform and the second maximum value corresponding to the second part of the waveform. The z-axis orientation of the cell is determined based on the order of the first maximum value and the second maximum value within the waveform.

13. An apparatus for timing the sorting of cells within a microfluidic flow, the apparatus comprising a processor and a memory configured to: The cellular events are detected using waveforms from cellular emission signals received from and associated with cellular events within the microfluidic flow; The waveform is used to classify the cellular events into selected cellular events; The sorting component is controlled to sort one or more cells in the selected cell events within a selected time period, the selected time period depending on the waveform width of the waveform associated with the selected cell events.

14. The apparatus of claim 13, wherein the duration of the selected time period depends on the waveform width, and / or wherein the selected time period begins after a certain offset delay following the end of the waveform, wherein optionally the offset delay depends on the waveform width.

15. The apparatus of claim 13 or 14, wherein the apparatus is configured to determine the waveform width by detecting a rising edge, followed by a falling edge, of the waveform in a cell emission signal received from the microfluidic flow and associated with the cellular event.

16. The apparatus according to any one of claims 13 to 15, wherein the apparatus comprises: An interrogation component for directing interrogating electromagnetic radiation into cells within the microfluidic flow to promote responsive emission signals from the cells; A sorting component for directing sorting electromagnetic radiation into selected cells within the microfluidic flow; The sorting component and / or the interrogation component include optical components for guiding the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation; The optical components described therein can be adjusted according to the characteristics of the waveform.

17. The apparatus of claim 16, wherein the characteristics of the waveform include one or more of the following characteristics of a plurality of waveforms associated with corresponding plurality of cellular events: maximum waveform intensity; maximum waveform width; integral of the waveform; number of peaks of the waveform; shape of the waveform.

18. The apparatus according to any one of claims 13 to 17, wherein the apparatus comprises: Components used to deliver the microfluidic flow; The component used to deliver the microfluidic flow is adjustable to change the path of the microfluidic flow according to the characteristics of the waveform.

19. An apparatus for processing cells within a microfluidic flow, the apparatus comprising: Components for detecting cellular events using waveforms in cellular emission signals received from and associated with cellular events within the microfluidic flow; Components for directing interrogating electromagnetic radiation into cells within the microfluidic flow to promote responsive emission signals from the cells; Components for sorting the selected cells within the microfluidic flow; Optical components used to guide the inquiry of electromagnetic radiation; The optical components described therein can be adjusted according to the characteristics of the waveform.

20. The apparatus of claim 19, wherein the sorting component includes a component for directing sorting electromagnetic radiation to the selected cells, and wherein the optical component is used to direct the sorting electromagnetic radiation.

21. The apparatus of claim 20, wherein the optical component is a laser for generating the sorted electromagnetic radiation, and wherein the power of the laser is adjustable according to the characteristics of the waveform.

22. The apparatus of claim 21, wherein the characteristic of the waveform includes one or more of the following: maximum waveform intensity; maximum waveform width; integral of the waveform; shape of the waveform; slope of the waveform; number of peaks of the waveform.

23. A method for sorting cells in a microfluidic flow using a pulse sorting arrangement that generates regular pulses, the method comprising: Detecting cellular events within the microfluidic flow; The cellular events are classified into selected cellular events; Cells associated with the selected cellular events are sorted by controlling the gating opening period, during which one or more of the regular pulses are directed to the microfluidic flow; The gating opening period is controlled based on the detection of the cellular events and the timing of one or more regular pulses in the regular pulses.

24. The method according to claim 23: The gating opening period includes a switching delay. The gating opening period is controlled to prevent regular pulses from occurring during the switching delay; Optionally, the gate opening period occurs as part of a selected period according to any one of claims 1 to 8.

25. The method of claim 24, wherein the switching delay corresponds to the time required for the switching device to switch the one or more regular pulses from being directed into the microfluidic flow to being directed away from the microfluidic flow, or vice versa.

26. The method of any one of claims 23 to 25, wherein the gating opening period is controlled in response to the detection of a regular pulse after the detection of the cellular event, or wherein the gating opening period is controlled based on the detection of a regular pulse before the detection of the cellular event.

27. The method of any one of claims 23 to 26, the method comprising initiating the gating opening period after detecting the cell event or classifying the cell event as a selected cell event by a certain initiation delay period, wherein the initiation delay period includes a predetermined delay and a variable delay, wherein the variable delay depends on the timing of the one or more rule pulses in the rule pulses.

28. The method according to claim 27: The predetermined delay depends on the transit time of a cell in the microfluidic flow between a detection position and a sorting position, the detection position corresponding to the detection of a cell event and the sorting position corresponding to the sorting of cells associated with the cell event; And wherein the variable delay depends on the detection of a regular pulse after the detection of the cell event; wherein optionally the variable delay is calculated using the time difference between classifying the cell event as a selected cell event and the detection of the next regular pulse.

29. The method according to any one of claims 23 to 28, wherein: The gate opening period is one or more of the following: equal to or less than the inter-pulse period between the regular pulses; 30%-70% of the inter-pulse period; 40%-60% or about 50% of the inter-pulse period; and / or The gating opening period is controlled to overlap with a single pulse, wherein the single pulse is timed within the central portion of the gating opening period, the central portion comprising one of the following: the middle 80% of the gating period; the middle 50% of the gating period; or the middle of the gating period.

30. The method of any one of claims 23 to 29, wherein the gating opening period is controlled to overlap with two or more pulses in response to the detection of a cellular event associated with a plurality of cells; Optionally, the method includes ending the gating opening period after a certain end delay period following the detection of the last cell in the cell event or after classifying the cell event as a selected cell event. The termination delay period depends on the transit time of the cells in the microfluidic flow between the detection position and the sorting position, the detection position corresponding to the detection of a cell event and the sorting position corresponding to the sorting of cells associated with the cell event; Optionally, the end delay period includes the transit time minus a variable end delay, which depends on a switching delay corresponding to the time required for the switching device to switch the one or more regular pulses from being guided into the microfluidic flow to being guided away from the microfluidic flow, or vice versa. Optionally, the variable end delay is calculated in response to determining that the next pulse will coincide with the switching delay associated with the end of the gating period.

31. The method according to any one of claims 23 to 30, wherein the pulse sorting arrangement includes a pulsed laser that generates regular laser pulses; and wherein optionally the sorting includes using the laser pulses to nudge, inactivate, or ablate cells associated with the selected cellular events.

32. The method of claim 31, wherein the gating opening period is associated with an optical switch controlled to switch a laser pulse into and out of the microfluidic flow; and optionally wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector; or an electro-optic modulator; Optionally, the regular laser pulses are further used to sort cells in the second microfluidic flow that are associated with selected cellular events.

33. A method for sorting cells within a plurality of microfluidic flows using a pulsed laser that generates regular laser pulses, the method comprising: The laser pulse is split into multiple beams, each beam being associated with a corresponding microfluidic flow; Detect corresponding cellular events within at least two of the plurality of microfluidic flows; Each detected cellular event in each corresponding microfluidic flow is individually classified as a selected cellular event; By independently controlling regular laser pulses of the corresponding beams entering the respective microfluidic flows, one or more cells in at least two microfluidic flows associated with the selected cellular events are sorted.

34. The method of claim 33, wherein one or more beam splitters are used to split the regular laser pulse into the plurality of beams.

35. The method of claim 34, wherein the beam splitter is a polarization beam splitter, and the beam splitting ratio is adjusted by adjusting the ratio of light polarized in a first plane to light polarized in a second plane; optionally, wherein the amount of light in the first plane relative to the second plane is adjusted by a polarization adjuster.

36. The method of claim 34, wherein the method is used to sort cells within a plurality of microfluidic flows using at least one beam splitter, the method comprising: The power transmission properties of at least one beam splitter are adjusted in response to cellular event properties or beam states of one or more microfluidic flows in the microfluidic flow. Optionally, the cellular event attribute includes at least one of detection, classification, or sorting attributes, and optionally, the beam state includes at least one of deactivation of detection, classification, and / or sorting for one or more of the plurality of microfluidic flows.

37. The method of any one of claims 32 to 36, wherein sorting cells associated with the selected cellular events comprises: Controlling a corresponding gating opening period, during which one or more regular laser pulses in the corresponding beam are guided to the corresponding microfluidic flow; The corresponding gating opening period is controlled based on the detection of the cellular events of the corresponding microfluidic flow and the timing of one or more regular laser pulses in the regular laser pulses; Optionally, the corresponding gating opening period includes a switching delay corresponding to the time required for the switching device to switch from being guided by one or more regular laser pulses of the corresponding beam to being guided away from the corresponding microfluidic flow, or vice versa; and The corresponding gating opening period is controlled to prevent regular laser pulses of the corresponding beam from occurring during the switching delay.

38. The method of claim 37, wherein the corresponding gating opening period is controlled in response to the detection of a regular laser pulse after the detection of the cellular event in the corresponding microfluidic flow; or wherein the corresponding gating opening period is controlled based on the detection of a regular laser pulse before the detection of the cellular event.

39. The method according to any one of claims 32 to 38, the method comprising: The corresponding gating opening period is initiated after a certain start delay period following the detection of the cellular event in the corresponding microfluidic flow or the classification of the cellular event into a selected cellular event for the corresponding microfluidic flow. The aforementioned start-up delay period includes a predetermined delay and a variable delay that depends on the timing of one or more of the regular laser pulses.

40. The method according to claim 39: The predetermined delay depends on the transit time of a cell in the corresponding microfluidic flow between a detection position and a sorting position, the detection position corresponding to detecting a cell event in the corresponding microfluidic flow, and the sorting position corresponding to sorting cells associated with the cell event in the corresponding microfluidic flow. And the variable delay depends on the detection of the regular laser pulse after the detection of the cellular event; Optionally, the variable delay is calculated using the time difference between classifying the cellular event as a selected cellular event for the corresponding microfluidic flow and the detection of the next regular laser pulse.

41. The method according to any one of claims 23 to 40, wherein the cell is a sperm cell.

42. A sorting apparatus for sorting cells in a microfluidic flow using a pulse sorting arrangement that generates regular pulses, the apparatus comprising: A pulse sorting arrangement structure for generating regular pulses; Detection components for detecting cellular events within the microfluidic flow; A classification component for classifying the cellular events into selected cellular events; A sorting component for sorting cells associated with the selected cellular events by controlling a gating opening period, during which one or more of the regular pulses are directed to the microfluidic flow; The gating opening period is controlled based on the detection of the cellular events and the timing of one or more regular pulses in the regular pulses.

43. The sorting device according to claim 42: The gating opening period includes a switching delay, which corresponds to the time required for the switching device to switch from being guided by the one or more regular pulses to being guided away from the microfluidic flow, or vice versa; and The gating opening period is controlled to prevent regular pulses from occurring during the switching delay.

44. The sorting apparatus according to claim 41 or 43, the sorting apparatus comprising an optical switch controlled to switch a laser pulse into and out of the microfluidic flow according to the gating opening period; and optionally... The optical switch described herein includes one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector or an electro-optic modulator; and optionally the acousto-optic modulator includes a tellurium dioxide crystal or a silicon dioxide crystal.

45. The sorting apparatus according to any one of claims 41 to 44, wherein the sorting apparatus comprises one or more of the following: A beam shortener, the beam shortener being arranged to narrow the regular laser pulse incident on the optical switch; and A beam expander is arranged to amplify the laser pulses directed to the microfluidic flow.

46. ​​The sorting apparatus according to any one of claims 41 to 45, wherein the pulsed laser is configured to generate regular laser pulses at a predetermined rate between 1 ps and 1 ms.

47. The sorting apparatus according to any one of claims 41 to 46, the sorting apparatus comprising a photodetector for detecting regular laser pulses; and optionally wherein the photodetector comprises a photodiode, preferably an avalanche photodiode.

48. A sorting apparatus for sorting cells within a plurality of microfluidic flows using a pulsed laser that generates regular laser pulses, the apparatus comprising: A pulsed laser that generates regular laser pulses; A beam splitter for splitting the laser pulse into multiple beams, each beam being associated with a corresponding microfluidic flow; One or more detection components for detecting cellular events within a corresponding microfluidic flow; One or more classification components for classifying the cellular events in a corresponding microfluidic flow into selected cellular events; A corresponding sorting component for sorting the selected cellular events in the corresponding microfluidic flow using the regular laser pulses.

49. The sorting apparatus according to claim 48, wherein: a) The beam splitter is a polarization beam splitter, and the beam splitting ratio is adjusted by adjusting the ratio of light polarized in the first plane to light polarized in the second plane; and / or b) The sorting device further includes at least one of a polarization modulator, a power adjustment component, a half-wave plate, or a Polkers cell arranged to adjust the amount of light in the first plane relative to the second plane; and / or c) The sorting device includes at least one beam splitter for sorting cells within the plurality of microfluidic flows, the sorting device being configured to adjust the power transmission of at least some of the beam splitters in response to cell event attributes or beam states for one or more microfluidic flows in the microfluidic flows, optionally wherein the cell event attributes include at least one of detection, classification, or sorting attributes, optionally wherein the beam states include at least one of deactivation of detection, classification, and / or sorting for one or more microfluidic flows in the microfluidic flows.

50. The sorting apparatus according to claim 48, wherein the sorting apparatus includes an optical switch controlled to switch a laser pulse into and out of the microfluidic flow according to the corresponding gating opening period; and optionally wherein the optical switch includes one or more of the following: an acousto-optic modulator; a spatial light modulator; an electro-optic deflector or an electro-optic modulator; and optionally wherein the acousto-optic modulator includes a tellurium dioxide crystal or a silicon dioxide crystal.

51. The sorting apparatus according to any one of claims 48 to 50, wherein the sorting apparatus comprises one or more of the following: A beam shortener, the beam shortener being arranged to narrow the regular laser pulse incident on the optical switch; and A beam reducer, which is arranged to amplify the laser pulses directed to the microfluidic flow.

52. The sorting apparatus according to any one of claims 48 to 51, the sorting apparatus comprising a photodetector for detecting regular laser pulses; and optionally wherein the photodetector comprises a photodiode, preferably an avalanche photodiode.

53. A computer program comprising processor instructions that, when executed by a processor, cause the processor to instruct the method according to any one of claims 1 to 12 or 23 to 41.

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