Cell selection apparatus and methods

Abstract

In some examples there is provided a method of sorting cells within a microfluidic stream by adjusting an offset delay and / or a selection period for sorting cells within a microfluidic stream. In one aspect the method comprises detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; sorting one or more cells in the selected cell event over the selection period which follows the selected cell event by the offset delay; wherein the selection period and / or the offset delay is adjusted dependent on a waveform width of the waveform associated with the selected cell event.

Description

[0001] CELL SELECTION APPARATUS AND METHODS

[0002] 1. TECHNICAL FIELD

[0003] The present disclosure relates to cell selection and sorting within microfluidic systems. The methods provide enhanced sorting of biological cells such as sperm cells.

[0004] 2. BACKGROUND

[0005] The classification of biological cells having different characteristics is useful for many subsequent processes. For example, the classification of sperm cells into X and Y populations allows for downstream separation or sorting of these two populations. One category of sperm cells may be more desirable for certain types of animal farming. For example, bovine X sperm cells are preferred for the insemination of cows to produce predominantly female offspring for milking populations.

[0006] Challenges exist in the classification and sorting of bovine sperm cells including low sort efficiency, low enrichment and slow sorting speed. Low sort efficiency results in a low percentage of wanted cells (for example X sperm cells) being collected, compared with the total number of wanted cells introduced into the classification and sorting system. Low sort efficiency may be caused by a number of factors including poor orientation of cells for classification, inaccurate classification techniques, low efficiency sorting techniques as well as associated processes negatively impacting cell motility. Low enrichment means that the enrichment of a desired characteristic of the cells is less than expected or desirable. Slow sorting speed extends the time biological cells are outside of optimum storage conditions and can therefore also impact cell motility and viability.

[0007] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the inventions disclosed herein. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0008] It is an object of the invention to provide an improved method of processing cells within a microfluidic stream, or at least to provide the public with a useful choice of cell classifying and / or sorting methods. 3. SUMMARY OF THE INVENTION

[0009] In some examples, there is provided a method of timing the sorting of cells within a microfluidic stream. The method comprises detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; and sorting one or more cells in the selected cell event over a selection period which is dependent on a waveform width of the waveform associated with the selected cell event.

[0010] By sorting cells dependent on a waveform width of waveforms associated with cell events, the effectiveness and efficiency of sorting can be improved. The cell events may be associated with single cells or multiple closely grouped cells. Examples enable improved timing associated with the sorting of cells or other particles.

[0011] In some examples, the selection period and / or an offset period between the end of a selected cell event and the start of a corresponding selection period may be adjusted based on the waveform width of the cell event.

[0012] In some examples, the waveform width of a waveform may be determined by detecting a rising edge followed by a falling edge of the waveform in a cell emission signal received from the microfluidic stream and associated with the cell event. In other examples, the waveform width may be determined by detecting a peak in the waveform and using a predetermined duration before and after the peak. In another example, detecting a preceding rising edge or a following falling edge of the waveform may be performed, and the waveform width determined using the duration between the peak and the preceding rising edge or the following falling edge.

[0013] In some examples, the sorting one or more cells in the selected cell event over a selection period uses a pulsed sorting arrangement which generates regular pulses, and sorting a cell associated with the selected cell event is achieved by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream. The gate open period is controlled dependent on the detecting of the waveform associated with the cell event and the timing of one or more of the regular pulses.

[0014] In some examples selected cells may be sorted from unselected or unwanted cells by means of pressure asserted by the application of electromagnetic radiation to the selected and / or the unselected cells. In other examples, the selected or unselected cells may be deactivated using electromagnetic radiation.

[0015] In some examples, the cells may be bovine sperm cells and the sorting being used to separate X and Y cells. In some examples, the method may be implemented using a pulse sorting arrangement, such as a pulsed laser, and sorting the one or more cells. In some examples, sorting comprises controlling one or more gate open periods during the selection period, wherein during a said gate open period one or more regular pulses is directed to the microfluidic stream and the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0016] In some examples, the gate open period includes a switching delay, wherein the gate open period is controlled to avoid a regular pulse occurring during the switching delay. The switching delay corresponds to the time required for a switching device to switch from being fully open to being fully closed, or vice versa. For example, this may correspond to switching time required from the one or more regular pulses being directed to the microfluidic stream to the one or more regular pulses being directed away from the microfluidic stream, or vice versa.

[0017] In some examples the gate open period is controlled responsive to detection of a regular pulse following the detecting of the cell event, or the gate open period is controlled dependent on detection of a regular pulse before the detecting the cell event.

[0018] In some examples, the gate open period is started a start delay period after detecting the cell event or classifying the cell event as a selected cell event, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular pulses. The predetermined delay may be dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event. The variable delay may be dependent on detection of a regular pulse following the detecting the cell event. In some examples the variable delay may be calculated using a time difference between the classifying the cell event as a selected cell event and detection of a next regular pulse.

[0019] In some examples, there is provided a method of determining a z-axis orientation of a cell within a microfluidic stream. The method comprises detecting a cell within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell; determining a z-axis orientation of a cell by identifying a maxima in the waveform which corresponds to a first part of the cell and identifying a maxima in the waveform which corresponds to a second part of the cell; wherein the z-axis orientation of the cell is determined based on the order of the two maxima in the waveform. In some examples, there is provided a method of adjusting an offset delay and / or a selection period for sorting cells within a microfluidic stream. The method comprises detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; and sorting one or more cells in the selected cell event over the selection period which follows the selected cell event by the offset delay. The selection period and / or the offset delay is adjusted dependent on a waveform width of the waveform associated with the selected cell event.

[0020] In some examples, there is provided a method of processing cells within a microfluidic stream. The method comprises detecting a cell event within the microfluidic stream by detecting a rising edge followed by a falling edge of a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; and sorting one or more cells in the selected cell event using an offset delay from the detected falling edge.

[0021] In some examples, there is provided a method of processing cells within a microfluidic stream. The method comprises detecting cell events within the microfluidic stream using a received emission signal associated with the microfluidic stream; classifying the cell events as unselected or selected cell events; and sorting one or more cells in a selected cell event over a selection period which is dependent on an end of the selected cell event and a duration of the selected cell event.

[0022] There is also provided a computer program comprising processor instructions which when executed by a processor cause the processor to carry out any of the above methods. In some examples, there is provided a non-transitory medium on which the computer program is stored.

[0023] In some examples, there is provided an apparatus for timing the sorting of cells within a microfluidic stream. The apparatus comprises a processor and memory configured to detect a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classify the cell event as a selected cell event using the waveform; and sort one or more cells in the selected cell event over a selection period which is dependent on a waveform width of the waveform associated with the selected cell event.

[0024] In some examples, there is provided an apparatus for processing cells within a microfluidic stream. The apparatus comprises a processor and memory configured to detect cell events within the microfluidic stream using a received emission signal associated with the microfluidic stream; classify the cell events as unselected or selected cell events; and sort one or more cells in a selected cell event over a selection period which is dependent on an end of the selected cell event and a duration of the selected cell event.

[0025] In some examples, there is provided an apparatus for processing cells within a microfluidic stream. The apparatus comprises means for detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; means for directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; means for directing sorting electromagnetic radiation at selected cells within the microfluidic stream; and an optical component used for directing the interrogating electromagnetic radiation and / or the sorting electromagnetic radiation . The optical component is adjustable dependent on a characteristic of the waveform.

[0026] In some examples there is provided an apparatus for processing cells within a microfluidic stream. The apparatus comprises means for delivering the microfluidic stream; means for detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; means for directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; means for directing sorting electromagnetic radiation at selected cells within the microfluidic stream. The means for delivering the microfluidic stream is adjustable to vary a path of the microfluidic stream dependent on a characteristic of the waveform.

[0027] In some examples, there is provided a method of sorting cells within a microfluidic stream using a pulsed sorting arrangement which generates regular pulses. The method comprises detecting a cell event within the microfluidic stream; classifying the cell event as a selected cell event; and sorting a cell associated with the selected cell event by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream. The gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0028] In some examples, there is provided a method of timing the sorting of cells within a microfluidic stream wherein the sorting of cells comprises using a pulsed sorting arrangement which generates regular pulses. The method comprises detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; and sorting one or more cells in the selected cell event over a selection period which is dependent on a waveform width of the waveform associated with the selected cell event, wherein sorting one or more cells in the selected cell event is achieved by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream. The gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0029] In some examples, the sorting one or more cells in a selected cell event over a selection period is dependent on an end of the selected cell event and a duration of the selected cell event. In some examples, the selection period may include one or more gate open periods.

[0030] By controlling the application of some regular pulses to a microfluidic stream dependent on detection of a cell event and detection of one or more of the regular pulses, cells associated with the cell event are more accurately targeted. This improves the likelihood of sorting cells such as unwanted Y-sperm cells associated with the cell event and not sorting cells such as wanted X-sperm cells not associated with the cell event. This in turn improves sorting efficiency and reduces collateral damage to other cells not associated with the cell event.

[0031] In some examples the gate open period may include a switching delay that corresponds to a time required for a switching device to switch from the one or more regular pulses being directed to the microfluidic stream to the one or more regular pulses being directed away from the microfluidic stream, or vice versa; and the gate open period may be controlled to avoid a regular pulse occurring during the switching delay.

[0032] In some examples, the gate open period may be controlled responsive to detection of a regular pulse following the detecting of the cell event. Alternatively, the gate open period may be controlled dependent on detection of a regular pulse before the detecting the cell event. Detection of a regular pulse may be implemented in laser pulse-based systems using a photodetector.

[0033] In some examples, the gate open period may be started a start delay period after detecting the cell event or classifying the cell event as a selected cell event, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular pulses. The predetermined delay may be dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event. The variable delay may be dependent on detection of a regular pulse following the detecting the cell event. The variable delay may be calculated using a time difference between the classifying the cell event as a selected cell event and detection of a next regular pulse. In some examples, the gate open period may be one or more of the following: equal to or less than the inter-pulse period between the regular pulses; 30-70% of the interpulse period; 46-60%, or approximately 50% of the inter-pulse period.

[0034] In some examples, the gate open period is controlled to overlap a single pulse. The single pulse may be timed within a central portion of the gate open 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.

[0035] In some examples, the gate open period may be controlled to overlap two or more pulses in response to detecting a cell event associated with a single cell. In some examples, the gate open period may be controlled to overlap two or more pulses in response to detecting a cell event associated with a plurality of cells. The gate open period may be ended an end delay period after detecting the last cell in the cell event or classifying the cell event as a selected cell event, wherein the end delay period is dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event. The end delay period may comprise the transit time less a variable end delay dependent on a switching delay from the one or more regular pulses being directed to the microfluidic stream to the one or more regular pulses being directed away from the microfluidic stream, or vice versa. The variable end delay may be calculated in response to determining that a next pulse will coincide with the switching delay associated with ending the gate period.

[0036] In some examples, the pulsed sorting arrangement may comprise a pulsed laser generating regular laser pulses. The sorting may comprise nudging, deactivating or ablating a cell associated with the selected cell event using a said laser pulse. The gate open period may be associated with an optical switch controlled to switch laser pulses into and away from the microfluidic stream. The optical switch may comprise one or more of the following: an acousto-optic modulator; a spatial light modulator; an electrooptic deflector or an electro-optic modulator.

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

[0038] In some examples, there is provided a method of sorting cells within a plurality of microfluidic streams using a pulsed laser which generates regular laser pulses. The method comprises splitting the laser pulses into a plurality of beams each associated with a respective microfluidic stream; detecting a cell event within each microfluidic stream; classifying the cell event in each microfluidic stream as a selected cell event; and sorting a cell associated with the selected cell event in each microfluidic stream using the regular laser pulses of the respective beam.

[0039] By sharing a common source of laser pulses for sorting cells in multiple microfluidic streams, costs and complexity may be reduced, whilst also making the method / system more robust. This approach allows for miniaturisation and parallel processing which both improves throughput and speed, but also improves portability and / or handling.

[0040] In some examples, the regular laser pulses may be split into the plurality of beams using one or more beam splitters. The beam splitter(s) may be a polarising beam splitter and the split ratio of the beams is adjusted by adjusting a ratio of light polarised in a first plane to light polarised in a second plane. The amount of light in the first versus the second plane may be adjusted by a polarisation modifier.

[0041] In some examples, the method sorts cells within N microfluidic streams using N or N-l beam splitters or polarisation beam splitters. The method may comprise adjusting the power transmission properties of at least some of the beam splitters responsive to at least one of cell event properties such as detection, classification and / or sorting properties, and beam status such as inactivation of detecting, classifying and / or sorting for one or more of the microfluidic streams. In some examples, the method sorts cells within N microfluidic streams using N or N-l polarisation beam splitters. The method may comprise adjusting the polarisation of at least some of the N or N-l polarisation beam splitters responsive to at least one of cell event properties such as detection, classification and / or sorting properties, or beam status such as inactivation of detecting, classifying and / or sorting for one or more of the microfluidic streams.

[0042] In some examples, the sorting a cell associated with the selected cell event may comprise controlling a respective gate open period during which one or more of the regular laser pulses in a said beam is directed to the respective microfluidic stream; wherein the respective gate open period is controlled dependent on the detecting of the cell event for the respective microfluidic stream and the timing of one or more of the regular laser pulses. The respective gate open period may include a switching delay from the one or more regular laser pulses of the respective beam being directed to the respective microfluidic stream to the one or more regular laser pulses of the respective beam being directed away from the respective microfluidic stream, or vice versa. The respective gate open period may be controlled to avoid a regular laser pulse of the respective beam occurring during the switching delay.

[0043] In some examples, the respective gate open period may be controlled responsive to detection of a regular laser pulse following the detecting of the cell event in the respective microfluidic stream. Alternatively, the respective gate open period may be controlled dependent on detection of a regular laser pulse before the detecting the cell event.

[0044] In some examples, the respective gate open period may be started a start delay period after detecting the cell event in the respective microfluidic stream or classifying the cell event as a selected cell event for the respective microfluidic stream, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular laser pulses. The predetermined delay may be dependent on a transit time for a cell in the respective microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event in the respective microfluidic stream and the sorting location corresponding to the sorting a cell associated with the cell event of the respective microfluidic stream. The variable delay may be dependent on detection of a regular laser pulse following the detecting the cell event. The variable delay may be calculated using a time difference between the classifying the cell event as a selected cell event for the respective microfluidic stream and detection of a next regular laser pulse.

[0045] In some examples, the respective gate open period may be 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; 40-60%, or approximately 50% of the inter-pulse period.

[0046] In some examples, the respective gate open period may be controlled to overlap a single laser pulse. The single laser pulse may be timed within a central portion of the respective gate open period, the central portion comprising one of the following: the middle 80% of the respective gate period: the middle 50% of the respective gate period; the middle of the respective gate period.

[0047] In some examples, the respective gate open period may be controlled to overlap two or more laser pulses in response to detecting a cell event associated with a plurality of cells in the respective microfluidic stream. The respective gate open period may be ended an end delay period after detecting the last cell in the cell event or classifying the cell event as a selected cell event in the respective microfluidic stream, wherein the end delay period is dependent on a transit time for a cell in the respective microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event in the respective microfluidic stream and the sorting location corresponding to the sorting a cell associated with the cell event in the respective microfluidic stream. The end delay period may comprise the transit time less a variable end delay dependent on a switching delay from the one or more regular laser pulses of the respective beam being directed to the respective microfluidic stream to the one or more regular laser pulses being directed away from the respective microfluidic stream, or vice versa. The variable end delay may be calculated in response to determining that a next laser pulse will coincide with the switching delay associated with ending the respective gate period.

[0048] In some examples, the sorting may comprise nudging, deactivating or ablating a cell associated with the selected cell event using a said laser pulse.

[0049] In some examples, the respective gate open period may be associated with an optical switch controlled to switch laser pulses into and away from the respective microfluidic stream. The optical switch may comprise 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, there is provided a sorting apparatus for sorting cells within a microfluidic stream using a pulsed sorting arrangement which generates regular pulses. The apparatus comprises a pulsed sorting arrangement which generates regular pulses; a detection means for detecting a cell event within the microfluidic stream; a classifying means for classifying the cell event as a selected cell event; and a sorting means for sorting a cell associated with the selected cell event by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream. The gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0051] In some examples, there is provided a sorting apparatus for sorting cells within a plurality of microfluidic streams using a pulsed laser which generates regular laser pulses. The apparatus comprises a pulsed laser which generates regular laser pulses; a beam splitter for the laser pulses into a plurality of beams each associated with a respective microfluidic stream; one or more detection means for detecting a cell event within respective microfluidic streams; one or more classifying means for classifying the cell event in respective microfluidic streams as a selected cell event; and respective sorting means for sorting the selected cell event in respective microfluidic streams using the regular laser pulses.

[0052] In some examples, there is provided a method of timing the sorting of cells within a microfluidic stream. The method comprises detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; and sorting one or more cells in the selected cell event over a selection period which is dependent on a waveform width of the waveform associated with the selected cell event. Aspects of the inventions may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein that have known equivalents in the art to which a said invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0053] 4. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0055] FIG 1 is a schematic diagram of a system for processing cells according to some examples;

[0056] FIG 2a illustrates assessment of a single cell event waveform of a received emission signal according to some examples;

[0057] FIG 2b illustrates assessment of a multicell event waveform of a received emission signal according to some examples;

[0058] FIG 3a - 3d illustrate single and multi cell event waveforms of a received emission signal, peak detection and cell deactivation;

[0059] FIG 4 illustrates cell event waveforms of a received emission signal, falling edge detection and cell deactivation according to some examples;

[0060] FIG 5a illustrates a single cell event waveform of a received emission signal and laser pulses for sorting the cell of the single cell event according to some examples;

[0061] FIG 5b illustrates a multicell event waveform of a received emission signal and laser pulses for sorting the cells of the multicell event according to some examples;

[0062] FIG 6a illustrates a single cell event waveform of a received emission signal and laser pulses for sorting the cell of the single cell event according to some examples;

[0063] FIG 6b illustrates a multicell event waveform of a received emission signal and laser pulses for sorting the cells of the multicell event according to some examples;

[0064] FIG 7 illustrates a method of sorting selected cells according to some examples;

[0065] FIG 8 is a plot illustrating selection period against waveform width of a cell event according to some examples;

[0066] FIG 9 illustrates a controller for processing cells according to some examples; FilG 10 illustrates a sperm cell;

[0067] FIG 11 illustrates z-axis orientation dependent waveforms for a sperm cell according to some examples;

[0068] FIG 12a and 12b illustrate a waveform for a sperm cell in a head-first z-axis orientation and a tail-first z-axis orientation respectively, according to some examples;

[0069] FIG 13a - 13d illustrate z-axis orientation dependent waveforms for a sperm cell based on fluorescent emissions determined from different angles, according to some examples;

[0070] FIG 14 is a schematic diagram of a system for processing cells according to some examples;

[0071] FIG 15a - 15c illustrate single and multi-cell event waveforms of a received emission signal, peak detection, laser pulses and cell deactivation;

[0072] FIG 16 illustrates a control approach for a single cell event according to an example, including the timing of laser pulses, ablation amplitude, and control signalling including use of different control periods;

[0073] FIG 17 illustrates a control approach for a single cell event according to another example, including the timing of laser pulses, ablation amplitude, and control signalling including use of different control periods;

[0074] FIG 18 illustrates a control approach for a multicell cell event according to an example, including the timing of laser pulses, ablation amplitude, and control signalling including use of different control periods;

[0075] FIG 19 illustrates a control approach for a multicell event according to another example, including the timing of laser pulses, ablation amplitude, and control signalling including use of different control periods;

[0076] FIG 20 illustrates a method of sorting cells within a microfluidic stream according to some examples;

[0077] FIG 21-23 illustrate systems for processing cells according to some examples;

[0078] FIG 24 illustrates a controller for processing cells according to some examples;

[0079] FIG 25 illustrates a pulsed sorting arrangement according to some examples.

[0080] 5. DETAILED DESCRIPTION OF THE INVENTION

[0081] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0082] The term "about" as used herein means a reasonable amount of deviation of the modified term such that the end result is not significantly changed. For example, when applied to a value, the term should be construed as including a deviation of + / - 5% of the value.

[0083] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0084] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0085] The terms "can" and "may" are used interchangeably in the present disclosure, and indicate that the referred to element, component, structure, function, functionality, objective, advantage, operation, step, process, apparatus, system, device, result, or clarification, has the ability to be used, included, or produced, or otherwise stand for the proposition indicated in the statement for which the term is used (or referred to) for a particular example(s).

[0086] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.

[0087] Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one example, to A only (optionally including elements other than B); in another example, to B only (optionally including elements other than A); in yet another example, to both A and B (optionally including other elements); etc.

[0088] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, 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") can refer, in one example, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another example, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another example, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0089] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0090] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.

[0091] The following sets forth specific details, such as particular examples or examples for purposes of explanation and not limitation. It will be appreciated by one skilled in the art that other examples may be employed apart from these specific details. In some instances, detailed descriptions of well-known methods, nodes, interfaces, circuits, and devices are omitted so as not obscure the description with unnecessary detail. Those skilled in the art will appreciate that the functions described may be implemented in one or more nodes using hardware circuitry (e.g., analog and / or discrete logic gates interconnected to perform a specialized function, ASICs, PLAs, etc.) and / or using software programs and data in conjunction with one or more digital microprocessors or general purpose computers. Nodes that communicate using the air interface also have suitable radio communications circuitry. Moreover, where appropriate the technology can additionally be considered to be embodied entirely within any form of computer- readable memory, such as solid-state memory, magnetic disk, or optical disk containing an appropriate set of computer instructions that would cause a processor to carry out the techniques described herein. Hardware implementation may include or encompass, without limitation, digital signal processor (DSP) hardware, a reduced instruction set processor, hardware (e.g., digital or analogue) circuitry including but not limited to application specific integrated circuit(s) (ASIC) and / or field programmable gate array(s) (FPGA(s)), and (where appropriate) state machines capable of performing such functions. Memory may be employed to storing temporary variables, holding and transfer of data between processes, nonvolatile configuration settings, standard messaging formats and the like. Any suitable form of volatile memory and non-volatile storage may be employed including Random Access Memory (RAM) implemented as Metal Oxide Semiconductors (MOS) or Integrated Circuits (IC), and storage implemented as hard disk drives and flash memory.

[0092] Some or all of the described apparatus or functionality may be instantiated in cloud environments such as Docker, Kubenetes or Spark. This cloud functionality may be instantiated in the network edge, apparatus edge, in the local premises or on a remote server coupled via a network such as 4G or 5G. Alternatively, this functionality may be implemented in dedicated hardware.

[0093] The term "confinement" as referred to herein refers to the restriction of the cross- sectional shape and size of a flow of cells in a fluid stream. For example, the diameter of a circular section of the flow may be restricted or the dimensions of the major and minor axes of an elliptical section flow may be restricted which may result in a single narrow trajectory with minimal deviation in any polar axis of cells from a defined central longitudinal axis of the flow.

[0094] The term "rotational orientation" of asymmetric cells (including sperm cells) means the predominant angle of a face of a representative sample of said cells with respect to an axis substantially perpendicular to the axis of flow of the cells. Without any features imparting an orienting torque on the cells, it is expected that the rotational orientation of said face will be randomly distributed and facing any angle around 360°. A sample of cells that have had an orienting torque applied via an orienting force or feature will have a non-random angular rotational orientation that preferentially directs the face of the cell in a particular angle so that a predominant angle can be determined or observed.

[0095] The term "z-axis orientation" refers to the direction of travel of an elongate cell within a microfluidic stream. The z-axis aligns with the direction of flow. In these examples, elongate cells are defined by a longitudinal axis, which may facilitate a function or movement within a specific environment. This longitudinal axis runs lengthwise from one end of the cell to the other, defining the primary direction of the cell's extension and often influencing its movement and interaction with its surroundings. Where such elongated cells have different structures at each end of the longitudinal axis, they may be said to exhibit a z-axis orientation because one structure or form leads the direction of travel. For elongate cells which comprise a head and tail, such as sperm cells, the z- axis orientation designates whether the cells within the flow travel head-first, or tail-first.

[0096] "Cells" and "X-cells" are referred to herein as examples of particular types of particles that may be desirable to retain within a microfluidic sorting arrangement. Where the term cell is used herein, there is no requirement for the cell to be a living cell. Those of skill in the art will readily appreciate that the mention of X-cells is intended to be indicative of any other particles or cells that have characteristics suitable for interrogation and sorting according to the present invention. In particular, X-cells may be substituted herein for any type of cell, including substantially symmetric and asymmetric cells, neurons, red blood cells, tagged cells, viruses, or microbiota as will be known to those of skill in the art.

[0097] Whilst some examples are described with respect to cells, this disclosure is not limited to processing or sorting cells but may equally be applicable to other types of particles. Accordingly, the term "cell" or "cells" as referred to herein includes other types of particles suitable to be detected and sorted according to the methods and apparatus described herein. A particle, as used in this context, refers to any discrete unit that can be manipulated and sorted within a microfluidic system, predominantly fluorescently labelled particles. This includes, but is not limited to, chemical entities such as labelled compounds, biological entities such as cells, in particular sperm cells, DNA or RNA vectors or molecules, oligomers, proteins such as enzymes or antibodies, viruses, liposomes, plasmids, hormones, microvesicles, and exosomes; beads, for example those tagged with specific molecules for identification or separation purposes; nanoparticles including those used for drug delivery or imaging such as metal nanoparticles, quantum dots, carbon-based nanoparticles, polymeric nanoparticles, silica nanoparticles, and magnetic nanoparticles.

[0098] The term "microfluidic stream" as referred to herein refers to a flow of liquid having at least one dimension at which surface forces dominate volumetric forces. In an example this may include a liquid stream having a sub-millimetre diameter or other cross- sectional dimension. In an example the microfluidic stream may be a continuous phase flow of liquid such as an unbroken stream 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. The microfluidic stream may alternatively or additionally comprise a dispersed flow of liquid drops.

[0099] The term "flow environment" as referred to herein refers to an environment which the microfluidic stream may flow through. An example includes a microchannel which may comprise a material such as glass forming an elongate lumen or pathway through which the microfluidic stream flows. The pathway may be fully encompassed by the material between each end of the pathway. The pathway may have at least one boundary exposing the microfluidic stream to a fluid environment, wherein the material interfaces with another boundary(s) of the microfluidic stream. In another example, the flow environment may be a fluid environment or volume which may be substantially static or which itself may be flowing. In this example, the microfluidic stream may not interface with a material substrate but be fully encompassed by the 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 which separates or enables the separation of a population of cells with a first characteristic from a population of cells with a different characteristic. It will be appreciated by those of skill in the art that various methods may be employed within the sorting arrangements described herein to achieve a collected cell fraction enriched in a desired characteristic. For example, use of electrostatic sorting methods, or microbubble-induced particle sorting will be known to those of skill in the art and are designed to accomplish the same task. Here a microbubble or droplet comprising a nudged cell may move laterally with respect to an axis corresponding to the direction of flow and do not, necessarily, remain entrained within separated fluid streams. While increasing a power of the radiation source or similar alternative modification of the sorting arrangement may increase the initial separation distance between the streams of microparticles, the ability to do so without impacting viability of cells may be desirable. Therefore, when using this method of sorting, careful control of the radiation source is desirable to ensure that selected cells are displaced into a different flow path compared with unselected cells and the selected cells are not rendered immotile or unviable as a result of the radiation. This displacement effect is preferably achieved by applying electromagnetic radiation, for example by way of a laser, to change the direction of cells from a first flow path to a different flow path. The particle flow path containing the selected or unselected cells may then be directed to a collection vessel and the particle flow containing the other population of unwanted cells discarded or collected in a second, different collection vessel.

[0101] The term "cell emission signal" is intended to refer to any signal that may be detected from a microfluidic stream and which is indicative of the presence of one or more cells within the microfluidic stream at a position corresponding to a detection or interrogation area. In one example, an ultraviolet beam is directed at the microfluidic stream and the presence of a cell or cells results in a fluorescent emission which is detected as a cell emission signal. The term "selected cell event" is intended to refer to the identification of a cell or a group of cells within a microfluidic stream which is or are intended for processing or handling which is different from the processing or handling that may be provided in respect of other cells not corresponding to a selected cell event. In one example, a selected cell event may correspond with an unwanted cell such as a Y sperm cell which may be intended for deactivation; this compared with other cells not corresponding to a selected cell event and where deactivation is not intended. Selected cell events may be detected using cell emission signals received from a microfluidic stream.

[0102] The term "selection period" is intended to refer to the duration over which a cell or cells corresponding to a selected cell event are processed. In one example this may be the period of application of a deactivating laser to the cell or cells. In one example the selection period may refer to the period over which a pulsed sorting arrangement which generates regular pulses is directed to the cell, cells or microfluidic stream containing said cell or cells. In some examples, this may be controlled by a gate open period which is dependent on detecting a cell event and the timing of the regular pulses, for example laser pulses. The selection period may include one or more gate open periods. In another example, the selection period may refer to the duration over which a non-deactivating laser, that may be pulsed or continuous, is applied to a cell or cells in order to push them from one laminar flow pathway into another laminar flow pathway within a microfluidic stream. This may in turn allow cells in the different laminar flow pathways to be separated or sorted from each other.

[0103] The term "gate open period" is intended to refer to a controllable period of a device or apparatus which is switchable or controllable to direct regular pulses to or away from a microfluidic stream. The gate open period indicates when the regular pulses are directed to the microfluidic stream. The gate open period may correspond to the "selection period" referred to herein, or may correspond to a part of the selection period in some examples where the selection period includes two or more gate open periods. For example, an optical switch or gate may be controlled to allow incoming laser pulses to be directed to the microfluidic stream during the gate open period and to block or direct laser pulses away from the microfluidic stream outside of the gate open period. Some controllable devices may be associated with a switching delay during which time part of the power of a regular pulse may be directed to the microfluidic stream. Depending on implementation, this switching delay may or may not be accounted for when controlling the gate open period.

[0104] The term "regular pulses" is intended to refer to pulses of an applied physical phenomenon used to interact with some cells in order to enable sorting of one population of cells from another population of cells flowing in a microfluidic stream. The pulses are regular in the sense that they have a common inter-pulse distance or period between adjacent pulses. An example applied physical phenomenon is a laser in which the regular pulses refer to a brief and controlled emission of laser energy that occurs at a regular interval followed by a period of no emission before the next pulse. These emissions can last anywhere from microseconds to femtoseconds, depending on the laser's design.

[0105] Each pulse delivers concentrated energy over a short time, which can result in high peak power during those intervals, making pulsed lasers useful for applications requiring precision and high-intensity light in short, controlled phases. These pulses may be directed in beams which may be harnessed for sorting cells in a microfluidic stream. These regular pulses may be generated as regular pulses using a controllable device or may be generated by switching a continuous beam of energy. Example regular pulses include laser pulses and electrostatic or electromagnetic pulses. The pulses may be separated from adjacent pulses by an inter-pulse period.

[0106] The term "transit time" is intended to refer to the time taken for a cell in a microfluidic stream to flow from an interrogation or cell detection zone where it is exposed to detection radiation to a sorting zone or region. The sorting zone or region may comprise a sorting arrangement configured to expose the cell(s) to at least one regular pulse for the purpose of sorting the cell based on the detection.

[0107] The term "nudging" is intended to refer to the application of at least one of a force and torque on a cell in one population so as to induce at least one of displacing and orienting these cells relative to an axis defined by the direction of the microfluidic stream flow. This may change the trajectory of one population of cells compared with a different population such that they may be separately collected or otherwise further processed. In some examples, this displacing or orienting may be implemented using pressure applied to a selected or non-selected cell by an impinging laser or a bubble directed at the cell.

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

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

[0110] FIG 1 illustrates a cell processing system 100 comprising a preparation station 105 which delivers prepared cells to an input arrangement 110 which delivers the cells into a microfluidic stream 115 for downstream processing. The microfluidic stream 115 may be a laminar flow having a predetermined range of cross-sectional dimensions and carried within a flow environment. In an example the flow environment may comprise a volume of gas such as air, or a microchannel fully or partially enclosing the microfluidic stream. One or more illuminators 120 generate an interrogation beam, for example an infra-red (IR) or ultraviolet (UV) illuminator or other irradiation devices. The interrogation beam irradiates the cells within the microfluidic stream at an interrogation area 125.

[0111] Irradiation of the cells causes emission signals such as scattered or fluorescent light which is detected by one or more detectors 130. Measured characteristics of the detected emission signals generate one or more signals which are forwarded to a controller 135.

[0112] In one example, the interrogation beam comprises at least one laser source configured to deliver light to a cell in order to induce bond vibrations in the DNA of said cell. In this example, the emission signal is detected and provides a signature of the bond vibrations which is used to calculate a DNA content carried by the sperm cell. This technique can be used to identify the sperm cell as carrying an X-chromosome or Y-chromosome. In one example the emission signal comprises at least one of resonant mid-infrared absorption, non-resonant mid-infrared absorption, and scattering by the cell. Any of these emission signals may be used to determine a property of the cell. Another example comprises the use of a quantum cascade laser (QCL) to deliver light to a cell within the microfluidic stream to induce resonant mid-IR absorption by DNA, one or more analytes of the cell, or another cellular component. Following interrogation, a characteristic signature of transmitted mid-infrared wavelength light can be detected using a suitable detector such as a mid-infrared detector. The signature provides an indication of the quantity or identity of DNA, analytes, or other cellular components (collectively "cellular elements") within the cell which can be used to identify one or more cell characteristics.

[0113] The controller 135 may comprise a processor and memory and is configured to interpret received emission signals in order to control a sorting arrangement 140. In one example, the emission signal comprises a fluorescence signal. The controller 135 is configured to detect single cell events each comprising the passing of a single cell within the microfluidic stream past the interrogation area 125. This results in light received at the detector 130 which generates a signal to be used by the controller to detect a single cell event, as described in more detail below. The controller 135 may also be configured to detect multicell events each comprising the passing of a closely grouped plurality of cells within the microfluidic stream past the interrogation area 125. The closely grouped cells of a multicell event may not be able to be individually resolved into single cell events. The controller may also be configured to classify the cell events into selected and unselected cell events. This may correspond to classifying cells within at least some of these cell events into different populations Pl and P2 depending on analysis of emission signals associated with those 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 the selected cells or cells events may include: targeting the cell with a laser, use of fluid pressure to change the trajectory or position of the cell, mechanical sorting, piezoelectric actuation, dielectrophoresis of droplets, electrolysis or electroporation, optical manipulation, optical trapping, holographic steering, acoustic- assisted hydrodynamic focusing, application of photonic pressure, acoustic deflection, laser deactivation or laser ablation. Sorting may effect at least one of a force and torque on a cell in one population so as to induce at least one of displacing and orienting these cells relative to an axis defined by the direction of the microfluidic stream flow. Where sorting comprises the deactivation of cells associated with selected cell events, this comprises imparting energy into the cells associated with the selected cell events, the imparted energy sufficient to damage the cells leading to their unviability.

[0115] In particular examples, the sorting arrangements may include: microbubble-based sorting, for example using lasers, spark or thermal vapour; pneumatic and solenoid valve based cell sorting for example using polydimethylsiloxane PDMS; or piezoelectric actuation for example using PDMS valves. In further examples, the sorting arrangement may include a radiation source configured to irradiate the microfluidic stream at a sorting beam target area 145. The radiation source is directed at or near cells associated with selected cell events in order to cause a change in the orientation, position or direction of travel of said cells, or to achieve deactivation comprising ablation or damage to selected cells within it. In this example the emission of radiation from the radiation source which causes an effect on the cells is referred to as a "sorting beam". In any of the examples provided herein, the sorting beam may comprise an elongated beam profile. For example, the elongated beam profile may comprise a line, an ellipse, a rectangle, or a rounded rectangle. In some examples, the radiation source of the sorting arrangement comprises a laser and can be configured for pulsing operation. The laser may comprise a nanosecond, picosecond or femtosecond laser. The specific characteristics of the sorting beam may be varied according to the required frequency, power and wavelength. In turn, various cells may require different sorting beams to achieve sorting. Those of skill in the art will be able to determine the required frequency, power and pulse duration to adapt a sorting beam to a cell type and flow speed. However, in some examples, the sorting beam comprises a frequency between 100 and 3000kHz. A switch is used to control the emission of the sorting beam towards the microfluidic stream. In one example, the switch comprises an acousto-optic modulator or a pockels cell or an electro-optic deflector or an electro-optic modulator. The switch is used to quickly control the emission of the laser towards the cells at an appropriate time, and for a desired period - referred to herein as the selection period. In some examples the laser may be pulsed with the selection period including one or more gate open periods during which one or more laser pulses are directed at the microfluidic stream. As described in more detail below, the gate open period or periods are controlled dependent on detecting a cell event for sorting and the timing of the laser pulses. In some examples, the system also includes at least one of free-space optics, fibre-optics, and other waveguides, configured to direct and focus the radiation from the radiation source onto the microfluidic fluid flow.

[0116] This arrangement is particularly useful for removing unwanted cells within a population comprising both wanted and unwanted cells. For example, during the production of cell populations for CAR T-cell therapy there may be certain types of cells that do not exhibit the desired phenotype. Cells in this first population (Pl) are destroyed, denatured or rendered immotile by the sorting arrangement. The alternative population that does not exhibit the desired characteristic in the microfluidic stream 115, such as desirable cells that have not been selected by the controller, are left undisturbed (P2). The sorted or processed cells in the microfluidic stream 115 may then be collected in one or more collection vessels 160 for further use. The sorting arrangement 140 thereby provides a population of cells (P2) enriched with a desired characteristic. Where sperm cells are used, this desirable population may comprise motile X cells.

[0117] In one example, the sorting beam comprises a laser configured to deactivate a cell. Deactivation is the process of transferring energy to the cell sufficient to permanently render the cell lacking in viability for its normal function or purpose. For example, sperm cells may be deactivated to rapidly induce permanent immotility, or they may be deactivated to "prime" them to be incapable of surviving downstream processes such as freezing and thawing. In the former, the deactivation may involve complete ablation meaning the cell surface membrane is ruptured which destroys cell integrity. During "priming", the cell surface membrane remains substantially intact, even though motility may be reduced or cease. Deactivation may also include damaging the DNA in the cells, for example through UV irradiation, or compromising the integrity of external structures of the cell essential for survival, for example disrupting or removing the flagellum (tail) of sperm cells resulting in lack of viability.

[0118] Downstream of the sorting arrangement 140, the cells may be collected in one or more collection vessels 160pl, 160p2. Where selected or unselected cells are separated into different flow paths using force or torque applied to at least one of these two populations, wanted and unwanted cells may be separated using microfluidic channels or other mechanisms such that these two populations of cells are directed to respective collection vessels 160pl, 160p2. In other examples, where selected cells are deactivated but remain within the same flow path as unselected cells, all cells (or cell remnants) may be directed to a single collection vessel 160pl having a higher concentration of viable unselected cells (e.g. wanted, intact cells) to selected cells (e.g. unwanted, deactivated cells) compared to the concentration of viable wanted cells to unwanted cells upstream of the sorting arrangement 140.

[0119] Referring to FIG 2a, a waveform or pulse corresponding to a single event is shown. The waveform extends along the time axis t and the height of the waveform may correspond to an amplitude of a received cell emission signal associated with a microfluidic stream carrying cells. The waveform 800a includes a single peak 842a and initially rises from a baseline 822 before falling again to the baseline. The peak in the waveform corresponds to the detection of the head and / or body of a cell in a single cell event. In the example of a sperm cell, the cell comprises a relatively large head, a smaller mid-section and a long thin tail - in the case of a sperm cell the peak primarily corresponds to the head part of the cell.

[0120] A rising edge threshold 824 is a waveform height or amplitude which is used for detecting a rising edge of a waveform. When the received emission signal rises from a baseline 822 above the rising edge threshold, a rising edge 860 is detected at time tr. The baseline 822 may be zero, a signal noise floor, or any other suitable datum. The rising edge threshold 824 may be determined using experimentation or trial and error. In one example, the rising edge threshold or the falling edge threshold (described below) is set by: a. determining a maximum amplitude of a background noise level; b. determining a minimum amplitude of a cell-generated waveform; c. setting a threshold level to be greater than a., and less than b.

[0121] A falling edge 865 is detected at time tf, where the intensity drops from a peak maxima 842a and falls below the falling edge threshold 864 at the falling edge (tf) 865. The falling edge threshold may be dependent on the waveform 800a, for example being set at the average height 852 of the waveform or a percentage of the maximum height of the waveform. In one example, the falling edge threshold 865 is set lower than the rising edge threshold 860. This is to reduce the possibility of an instantaneous false trigger due to high frequency noise which might rapidly fluctuate above and then below the threshold. 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 will be appreciated that the waveform width for single cell events will be similar but that the waveform width for multicell events will vary depending on the number of closely grouped cells and their spatial distribution within the microfluidic stream. A multicell event may be detected by assessing whether a waveform width exceeds a pre-determined time, where that time is specific to a single cell event.

[0122] Referring to FIG 2b, a waveform or pulse corresponding to a multicell event is shown. The waveform extends along the time axis t and the height of the waveform may correspond to an amplitude of a received emission signal associated with a microfluidic stream carrying cells. Peaks in the waveform correspond to cell events, which in this multicell event correspond to multiple closely grouped cells. Depending on how the cells within a multicell event are positioned in the X, Y and Z axes of the microfluidic stream as well as how they are oriented within the microfluidic stream, not all cells may give rise to a peak. For example, where a cell is substantially transparent the beam may diffract around the cell and result in both signals merging into a single large peak when they are close together or overlapping. The waveform 800b includes three peaks 842b, 844b and 846b and initially rises from a baseline 822 before falling again to the baseline.

[0123] As noted above, the rising edge threshold 824 is a waveform height or amplitude which is used for detecting a rising edge of a waveform. When the received emission signal rises from a baseline 822 above the rising edge threshold, a rising edge 860 is detected at time tr.

[0124] A falling edge 865 is detected at time tf, and uses a falling edge threshold 866 to determine whether one of the intensity downslopes after the peaks 842, 844, 846 exceeds this threshold. The falling edge threshold may be set in a number of ways to trigger a falling edge detection and, therefore indicate a cell event: a. In one example, the falling edge threshold 866 is defined by an emission intensity metric at a pre-determined height above a calibrated baseline 822. For example this may be a predetermined percentage of the maximum or average waveform height over representative number of waveforms measured. In this example, the when the intensity drops below this absolute value, a falling edge 865 is detected. b. In another example, the falling edge threshold 866 is defined by a relative intensity drop 858b from a peak maxima 846b. c. In another example, the falling edge threshold is dependent on an average height of waveforms corresponding to similar cells, for example being set at a predetermined percentage (for example 50%) of the mean peak height maxima 852 of a representative number of previously measured waveforms.

[0125] Either method of defining the falling edge threshold (and therefore the falling edge event trigger) may be used within the methods of the invention described herein.

[0126] The falling edge threshold 866 used for a multicell event may be different to that used for a single cell event, for example to accommodate peaks of similar or different heights being adjacent within the waveform. The downslope after the second peak 844b extends down a distance or height 856 which in this example is insufficient to exceed the falling edge threshold 866 and hence does not trigger detection of a falling edge. The downslope after the third peak 846b extends past the falling edge threshold and hence triggers detection of a falling edge 865. In one example, the falling edge threshold 866 is set lower than the rising edge threshold 824. This is to reduce the possibility of an instantaneous false trigger due to high frequency noise which might rapidly fluctuate above and then below the threshold.

[0127] 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 will be appreciated that the waveform width for single cell events will be similar but that the waveform width for multicell events will vary depending on the number of closely grouped cells and their spatial distribution within the microfluidic stream.

[0128] FIG 3a-d illustrate cell event waveforms of a received emission signal, peak detection and cell deactivation. The emission signals described herein may comprise a fluorescent emission signal. A single cell event waveform 210s is illustrated in FIG 3a and multicell event waveforms 210m are illustrated FIG 3b-d. The single cell event is associated with a single cell 205s passing through a cell processing system and the multicell event is associated with a closely grouped plurality of cells 205m passing through the system. The closely grouped cells 205m may overlap so that it is not easy or possible to individually resolve them, for example using respective single cell event waveforms.

[0129] For the single cell event, a peak detection signal 215s is detected in the waveform. A peak detection signal may be triggered by a number of means. For example this may be detected by: a. determining the maximum height or amplitude of the received emission signal, for example by determining when an emission signal rises above a rising edge threshold then the gradient reaches 0; b. determining a timepoint when an emission signal falls below a falling edge threshold after having first exceeded a rising edge threshold; or c. by generating a running average of the amplitude or intensity of the received emission signal over a short window, and generating the peak detection signal 215s when whenever the current amplitude falls below the running average.

[0130] In one example shown in Figure 3a, a falling edge threshold 212 is shown. In one example, this may be a predetermined percentage of the maximum or average waveform height or intensity over a representative number of waveforms measured over a local window.

[0131] For this method of peak detection, it can be seen that the actual peak maxima precedes detection of the falling edge and this difference defines a "detection delay time". If needed, this detection delay time may be accommodated using an estimate of the detection delay time between peak and detection of the peak. This detection delay time may be estimated or determined experimentally, or in any other suitable manner. A cell transit duration is defined by the time between detection of the cell and the cell passing the influence of the sorting arrangement. This duration may be determined experimentally, for example by selecting all cells for sorting, and maximising the total number of cells sorted. For example all cells are targeted for sorting using a deactivation laser, and the number of cells actually deactivated can be measured while varying the duration to achieve the maximal deactivated cells. This predetermined cell transit duration (PCTD), for a given cell velocity and distance between the interrogation beam in the interrogation area 125 and sorting arrangement 140, may then be used to set the end of a selection period or ON time for a particular cell event (when the cell can no longer be influenced by the sorting arrangement). Various other peak detection and sorting methods may alternatively be used.

[0132] A trigger signal 220s is activated for a selection period, pulse or ON period which begins after a determined offset delay or time after the detected peak at 215s in order to coincide with the cell being selected for sorting at 205s. In this example, the selection period and the offset delay are both predetermined, for example a selection period of Ips and an offset delay has a predetermined duration, for example 61ps. The end of the selection period may be set at the predetermined cell transit duration following detection of the cell event. This type of triggering is referred to herein as single cell statically timed selection triggering, however adaptively timed selection triggering modes in which the offset delay and / or the selection period may be adjusted may alternatively be used as described in more detail further below.

[0133] In the present statically timed selection triggering example, the predetermined offset delay may correspond to the duration between the cell passing the interrogation area 125 and the sorting arrangement 140 of FIG 1 - the predetermined cell transit duration (PCTD), less a predetermined selection period or ON time. In one example comprising an electrostatic cell sorting arrangement, the predetermined offset delay may correspond to a drop delay value meaning the duration between the cell passing the interrogation area 125 and reaching a droplet break-off point where a charge is applied to the droplet that contains this target cell for downstream sorting. In another example where the selection leads to activation of a deactivation laser, the predetermined offset delay may correspond to the time taken for the cell to move from the interrogation beam to the start of a sorting area or deactivation beam.

[0134] Referring to FIG 3b, in one example of a multicell event comprising cells A, B and C, a 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 falls past a predetermined falling edge threshold shown as 212m. In this case only one peak is resolved due to the falling edge threshold only being crossed once - the peak corresponding to cell A. This results in cells B and C not being assessed for sorting.

[0135] In an alternative detection method shown in FIG 3c, the falling edge thresholds 212-1 and 212-2 are variable and may be set by being a specified intensity drop from a peak maxima. The peak maxima may be detected by any means, for example when the gradient is 0. In this case, two peaks are detected corresponding to cells A and B. In this case it can be seen that there are two well resolved peaks which are detected, although a third peak which is not detected can also be seen.

[0136] A laser trigger signal 220m for the detected falling edges or local maxima of the multicell event includes a pulse or pulses 221 and 222 a predetermined offset delay or time after the detected peak(s) in 215m in order to coincide with the cells 205m being selected for sorting, for example via deactivation. The predetermined offset delay may correspond to the duration between the detected cell(s) passing the interrogation area 125 and the sorting arrangement 140 of FIG 1. Each selection period or laser ON period may be a predetermined duration. The sorting action applied to the cell is indicated in the figures by a star above the cell. In FIG. 3c, it can be seen that the selection period or laser ON period for the laser trigger signal on the right 221 coincides with a predetermined offset delay following cell A. The detected cell and the selection period or laser ON period on the left 222 coincides with a predetermined offset delay following detected cell B. As the undetected cell C is outside of the selection period, it is not selected for sorting or acted on by the laser during the laser ON period. The waveform corresponding to peak C is not assessed or acted on. This can lead to inaccurate measures of cell number, and can cause contamination of a collected sample if cell C is a cell that should have been selected for sorting. This illustrates a problem that can occur in multicell events where the closely grouped cells cannot be well resolved, and not all cells may be acted upon.

[0137] In particular, where there is only one falling edge to trigger on during multi-cell event as shown in figure 3b, cells in a multicell event may not be selected and therefore acted upon to enhance the purity of a collected cell sample.

[0138] Referring to FIG 3d, in one example of a multicell event, a single selection period may be triggered following detection of the end of the multicell event. The duration of the single selection period may be pre-determined to align with an expected period for multiple, overlapping waveforms to start and finish. For example, by determining an average time from a rising edge to a falling edge. However, as shown in FIG. 3d, the falling edge threshold 212m causes detection 215m of two cells despite three cells actually being present. The triggering of a single selection period or ON pulse with a predetermined offset delay after detection of the end of a multicell event is referred to herein as multicell statically timed selection triggering.

[0139] The proximity of time between the two cells may trigger the multicell mode. Despite two cells being detected, and the selection period or laser ON period 223 being extended to select cells A and B for sorting, cell C is not selected.

[0140] In one example, a predetermined offset delay such as 61ps and a predetermined selection period or ON period such as Ips may be employed.

[0141] FIG 4 illustrates cell event waveforms of a received emission signal, peak detection and cell deactivation according to some examples. A single cell event waveform 310s is illustrated on the left and a multicell event waveform 310m is illustrated on the right. The single cell event is associated with a single cell 305s passing through a cell processing system and the multicell event is associated with a closely grouped plurality of cells 305m passing through the system.

[0142] It is noted that sorting, for example via deactivation, is only associated with selected cell events and that cell events not classified as selected may not be subject to sorting actions such as deactivation. Whether or not a cell event is classified as selected or unselected may be configured using various factors such as whether the cell event is a single cell event or a multicell event and / or whether a cell or cells associated with the cell event are wanted cells or unwanted cells. In one example, X-cells may be unselected (i.e. wanted) and Y-cells may be selected (i.e. unwanted).

[0143] For the selected single cell event, a detected falling edge 315s of the received emission signal is determined at the end of the waveform 310s as described below. A laser trigger signal 320s includes a pulse, ON period or selection period 325s which starts after an offset delay 330s after the detected falling edge 315s. For the selected multicell event, a detected falling edge 315m of the received emission signal is determined at the end of the waveform 310m. A laser trigger signal 320m includes a pulse, ON period or selection period 325m which starts after an offset delay 330m after the detected falling edge 315m. Example selection triggering modes for single cell and multicell events are described in more detail below.

[0144] It can be seen that the selection period 325m for the multicell event is of longer duration than the selection period 325s for the single cell event. This allows for multiple cells within the multicell event to be sorted, for example via deactivation. The end of the selection period may be a predetermined time or duration following the end of the waveform 310s, 310m detected using the falling edge of the generated waveform. In an example, this may correspond to a time difference between the last (or only) cell in the cell event passing through the inspection area 125 of the system of FIG 1, and that cell flowing along the microfluidic stream past the sorting beam 140. The offset period 330m for the multicell event is correspondingly shorter than the offset period 330s for the single cell event as the selection period or ON pulse is longer in the multicell event.

[0145] The waveform associated with a cell event may be used to classify the waveform and / or the cell or cells associated with the waveform. A characteristic of a waveform may be used to classify the associated cell event as unselected or selected.

[0146] The waveform has a waveform width (or a pulse width PW) corresponding to the time difference between the detected rising and falling edges.

[0147] In one example, an integral of the waveform may be determined and used to classify cells, for example as unselected X-cells and selected Y-cells. The integral represents the total light collected during the time the cell traverses the interrogation beam. This measurement can be more stable and less susceptible to noise compared to peak maxima. The integral of the waveform is calculated between two time points which may be labelled a and b. In one example, a and b are equidistant before and after the peak maxima. In another example, a and b are selected to intersect with a threshold selected to be above a background noise level detected by the detectors.

[0148] In one example, the peak maxima or height is used to classify cells. This corresponds to the maximum amplitude of the signal generated when a cell passes through the interrogation beam. It is proportional to the intensity of the light scatter or fluorescence emanating from the cell.

[0149] Cell events may be classified as selected or unselected depending on whether they comprise unwanted or wanted cells. Where the classification of cells within a cell event cannot be determined, the cell event may be classified as selected. This may occur where the cells of a multicell event are too closely grouped together to be individually resolvable and classified. Sorting of the unwanted or wanted cell or cells of a cell event may then be performed by the sorting arrangement dependent on the classification of the cells or cell events.

[0150] The cells 305m all fall within the selection period so are all selected for sorting. In some examples, this may be desirable. For example when selecting cells with an unwanted characteristic for deactivation, it may be preferable to deactivate any cells that cannot be resolved, rather than have those cells potentially contaminate the wanted cells that are not deactivated, and reduce the enrichment of the wanted characteristic. However, this strategy of deactivating all unresolvable cells has the effect of potentially wasting cells that, if resolved, would be deemed to be wanted cells.

[0151] The methods described herein may provide one or more advantages including:

[0152] 1) enhanced enrichment of a desired cell characteristic by enhancing the sorting of cells. In one example, the sorting action prevents undesired cells from progressing (especially during multi-cell events).

[0153] 2) Enhanced cell throughput for a constant cell selection metric. I.e. less cells of the undesired type are allowed through which means a higher concentration of cells can be collected using the sorting arrangement. This has the effect of increasing speed of the sample through the system. This advantage can be particularly important where cells deteriorate based on time spent flowing through the cell processing system. Therefore enhanced cell throughput can ultimately lead to higher viability of the collected cells due to the decreased time of the sample flowing through the system.

[0154] 3) Real-time detection of the quality / characteristics of the cells and media. For example a) Sample quality, b) Cell quality c) Sheath or buffer fluid quality.

[0155] In some examples, the offset delay from detecting a single or multi cell event until triggering a selection period for the cell event may be predetermined, i.e. static, for example 61ps and 5ps. As referred to herein, this form of triggering is referred to as statically timed selection triggering with a single cell event example shown in FIG 3a and a multicell event example shown in FIGs 3b-d. The actual times will depend on the set up of the cell processing system used, for example the velocity of the microfluidic stream in FIG 1 and the distance between the inspection area 125 and the sorting arrangement 140.

[0156] In the following examples, the offset delay or timing for triggering the selection period is adaptive. FIG 5a illustrates the application of a sorting action for a single cell event and shows plots of, from upper to lower, a received emission signal (signal), a sorting trigger signal (trigger), a laser pulses (laser), and a cell - grey when interrogated to generate a waveform in the received emission signal, for example at inspection area 125, and black when sorted via deactivation, for example at sorting arrangement 140.

[0157] The received emission signal includes a waveform corresponding to detection of a single cell event - the grey cell. The waveform includes a detected rising edge and a detected falling edge. The waveform has a waveform width (or a 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 starts after an offset delay after the detected falling edge. Example times for the waveform width (15ps), the offset delay (51ps) and the selection period or ON time (15ps) are shown, however it will be appreciated that other durations for these parameters may be employed.

[0158] In this example, an adaptively timed selection triggering mode is used. In this triggering mode, the selection period or ON time (e.g. 15ps) is set to be the same or similar to the waveform width (15ps). In the example of FIG 5a, the selection period corresponds with three laser pulses as indicated, resulting in three interactions with the cell - illustrated as stars. The use of an extended selection period in FIG 5a compared with the shorter selection period in the example of FIG 3a may be used in different ways. For example, the additional laser pulses may be used to impart additional energy into the selected cell in order to improve the likelihood of its deactivation. Similarly, the use of a longer selection period may increase the likelihood that a laser pulse impacts a particular part of the cell such as the cell body which may improve deactivation for example. On the other hand, a laser with lower power may be utilised where the energy imparted to the cell is spread across a longer selection period which still results in deactivation of the targeted cell but may result in less collateral damage to other cells - for example an unselected or wanted cell (not shown) may be oriented such that its tail overlaps part of the cell being deactivated. In this scenario, the reduction of power of the laser may reduce its effect on the tail of the wanted cell whilst still being sufficient to deactivate the cell selected for deactivation.

[0159] In other examples the offset delay and / or the ON time or selection period may be adjusted according to factors such as the number or type of cells, size of cells, rotational orientation, z-axis orientation or the type of sorting used such as deactivation via ablation or lesser damage but still sufficient to deactivate the cell. For example, a longer ON time or selection period may be used such as 35ps. This may allow for greater energy to be input into the cells which may result in complete ablation (i.e. lysis of the cell membrane) compared with less energy input which may only result in cell damage resulting in eventual unviability i.e. priming of the cell. In this case a shorter offset delay is used, the previously predetermined offset less the increase in ON time. Alternatively a different predetermined offset delay may be used corresponding to the end of the selection period (when the cell is past the sorting process), less the selection period - for example 66 - 15 = 51ps. In one example, the ON time may be modified to minimise the chance of collateral damage to bystander cells. For example, the selection period or ON time may be reduced to avoid impacting on cells outside the cell event. For example, a wanted sperm cell may have a tail oriented in such a way that this extends into the (unwanted) cell event, and which would otherwise incur unwanted damage from the laser pulses if the selection period was not reduced. Similarly, heating of fluid surrounding targeted cells may impact nearby wanted cells, and therefore reducing the selection period can mitigate this effect.

[0160] In another example, the ON time may be modified to compensate for cell positioning variation, for example if a cell is detected to be positioned out of a focal point of a sorting beam, the ON time may be increased to achieve greater energy input to the cell to account for the lower intensity. In another example, the offset delay is varied to account for a cell's z-axis orientation (head-to-tail, tail-to-head, off-axis etc).

[0161] In some examples, the selection period may correspond to a gate open period and may be timed to extend over multiple laser pulses from a regular pulsed laser sorting arrangement. In one example as described further in relation to FIG 16 and 17, the gate open period within the selection period is timed to ensure a full laser pulse is directed to the cell or microfluidic stream. This concept of timing and selecting pulses during the selection period may further be extended to multiple cell events and multiple pulses as described in relation to Figure 18 and 19. In these examples, an optical switch may be used and controlled to only be open for a predetermined gate open period about each laser pulse during the selection period.

[0162] In some examples, the sorting beam comprises a deactivation laser comprising a pulsing laser such as a nanosecond, picosecond or femtosecond laser. In one example, the pulse duration is between about 10 femtoseconds to 100 nanoseconds. Those of skill in the art will appreciate that a different pulse duration will affect laser characteristics such as the energy and power output, and will affect the physical behaviour of the interaction between cells and the laser beam. In some examples, pulse duration may also inform other aspects such as repetition rate and beam quality.

[0163] In one example, the sorting beam comprises a deactivation laser comprising a wavelength in the IR, UV, or visible wavelength range. In another example the sorting beam comprises an x-ray or gamma ray wavelength. In one example, the sorting beam comprises a wavelength of between about 100-400 nm, 380 to 780 nm, or 760 nm to 10pm. In some examples, a sorting beam with a UV wavelength is employed. This has the advantage of exciting certain DNA stains (e.g. Hoechst-33342) commonly used for interrogation of cells. As such, stained cells will absorb better which may provide advantages in being able to use a lower power beam versus other, non-UV wavelengths.

[0164] The sorting beam may be controlled to be directed to the microfluidic stream during the selection period or ON time, or gate open period. The number of laser pulses applied to the microfluidic stream (and any cells coincident with the laser) will depend on the selection period, timing and inter-pulse period. In the example shown in FIG 6a, one laser pulse is applied to the (black) cell however in other configurations multiple laser pulses may be applied to the cell where the selection period is longer and / or where this starts at the same or similar time to a laser pulse so that two or more laser pulses may be included within the selection period - again depending on configuration parameters such as the laser pulse frequency and the duration of the selection period.

[0165] In one example, a z-axis length of a cell is measured and a laser with sufficient rep rate is switched on prior to or concurrently with the cell entering the sorting beam target area 145 and turned off after the cell (or a part of the cell) leaves the sorting beam target area. The duration between switching the laser ON and OFF (also referred to herein as the selection period or gate open period) or length of the target area may be a function of the cell length to ensure that the cell receives the pre-determined number of pulses. Optionally, the sorting beam target area may be one cell length greater than the cell length to account for variations in the cell speed. It will be appreciated by those of skill in the art that the reference to switching the laser ON or OFF herein is intended to indicate the cell being allowed to interact with the cell or microfluidic stream, and is not indicative of the power status of the laser itself. The switching from ON to OFF and vice versa may be achieved by a switching arrangement such as a pulse picker in which the laser may be directed away, or blocked, during the OFF period, and directed towards the cell or microfluidic stream during the ON period or gate open period.

[0166] The selection period may be implemented using an acousto-optic modulator or other switch to control the emission of the laser pulses towards the microfluidic stream during the selection period - indicated by the black laser pulses inside the "ON time" delineators for example in figures 5 and 6. At other times the laser pulse may be directed elsewhere or absorbed / blocked - indicated by the grey laser pulses outside the "ON time" delineators for example in figures 5 and 6.

[0167] In some examples the laser may be adapted for low absorption in water (or other carrier medium) and high absorption by cells where deactivation-based sorting is employed.

[0168] In some examples, a high repetition rate for pulsed lasers may be employed, which may allow for reduced selection period or ON duration. In this instance, high repetition rate means greater than one pulse per cell head z-axis length. For example, for a selection period of >10ps a laser of 200-500 kHz could be used to achieve multi-shot cell targeting. In another example, for a selection period of 0.5-5ps a laser of 500-5000 kHz could be used to achieve multi-shot cell targeting.

[0169] Other examples may employ one or more of the following laser characteristics: a smaller focal spot in the z-axis (direction of travel); high beam quality, such as low M2; homogeneous illumination across the stream (line profile); sufficient fluence to ablate cells.

[0170] FIG 5b illustrates sorting for a multicell event using an adaptively timed selection triggering process. It shows plots of, from upper to lower, a received emission signal (signal), a trigger signal (trigger), a pulsing laser (laser), and cells - grey when interrogated to generate a waveform in the received emission signal, for example at inspection area 125 and black when sorted, for example at sorting arrangement 140.

[0171] As noted previously, the predetermined cell transit duration is the time taken for a cell to traverse from detection corresponding to its presence in an interrogation area or detecting location to being influenceable by a sorting process such as application of a laser or charged electrostatic sorting plates when the cell is in a sorting area or location. This will depend on the configuration of the cell processing system used, for example the velocity of the microfluidic stream in FIG 1 and the distance between the inspection area 125 and the sorting arrangement 140.

[0172] The received emission signal includes a waveform corresponding to detection of a multicell event - the grey cells. The waveform includes a detected rising edge and a detected falling edge which exceeds a falling edge threshold as previously described. The waveform has a waveform width (or a 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 starts after an offset delay after the detected falling edge. The end of the selection period may correspond with the cell detection (e.g. using the previously described falling edge threshold technique) plus the predetermined cell transit duration. The offset delay may be calculated by subtracting the waveform width from the predetermined cell transit duration. Example times for the waveform width (34ps), the offset delay (32ps) and the selection period or ON time (34.5ps) are shown, however it will be appreciated that other durations for these parameters may be employed. For example the waveform width may be less than 50ps, for example in a range from 0.1 to 50ps. In some examples the offset delay may range from 0.5 to 50ps, and the selection period or ON time may range from 0.5-100ps. These values depend on a range of factors. For example the distance between the inter-beam distance between the interrogation beam and the sorting beam, the speed of the cells through the system, processor speed to resolve and determine whether to select a cell, actuation time of any sorting arrangement, pump capacity, microfluidic pressure limitations or other factors. Those of skill in the art will appreciate the variations to achieve effective targeting of the cells based on these parameters and reasonable experimentation. Assessment of the effect of varying the parameters to determine the result of cell targeting can be achieved via methods including visual or cell fluorescence detection during sorting, or downstream measures of the percent purity of the selected or unselected cells according to known methods.

[0173] In this example, as with the single cell event of FIG 5a, an adaptively timed selection triggering mode is used. In this triggering mode, the selection period or ON time (e.g. 15ps) is set to be the same or similar to the waveform width (15ps). The offset period is calculated using the predetermined duration corresponding to the time for a cell to traverse from an inspection area, where it is detected, to the end of a sorting or laser activation area. As noted previously, the actual times will depend on the set up of the cell processing system used, for example the velocity of the microfluidic stream in FIG 1 and the distance between the inspection area 125 and the sorting arrangement 140. In this example, the predetermined duration is 66ps, after which a cell is no longer influenced by a sorting beam. The offset delay is calculated from this predetermined duration less the waveform width 34ps - in this example being 66ps - 34ps = 32ps. This enables the sorting beam to act on all cells within the multicell event.

[0174] In the example of FIG 5b, the selection period corresponds with seven laser pulses as indicated, resulting in seven interactions with the cells - illustrated as stars. The use of an extended selection period in FIG 5b compared with the shorter selection period in the example of any of FIGs 3b-3d enables all cells within the multicell event to be targeted by the laser pulses.

[0175] In the example of FIG 5b, the offset delay and / or the ON time or selection period are adjusted depending on the waveform width. As noted above, in this example the selection period or ON time is the waveform width 34ps and the offset delay is the predetermined cell transit duration (PCTD) less the selection period (or the waveform width when this is the same) for example 66 (predetermined) - 34 (width) = 32ps. This may be described by the equation :

[0176] Equation 1: OD = PCTD - WW where OD is the offset delay, PCTD is the predetermined cell transit duration and WW is the waveform width or duration. This approach enables the duration of the sorting process to correspond with the period over which the cells are influenceable by the sorting process - in other words the duration of the waveform width corresponds to a time when cells of a multicell event are detected and the sorting process is applied for this same time (the selection period) but delayed to allow the detected cells to arrive at the sorting process. Such an arrangement may ensure that all cell heads of detected sperm cells pass through the focal spot of a deactivation laser.

[0177] The selection period may be adjusted based on factors such as the type of cells, their size, z-axis orientation or the type of sorting used such as ablation, priming or other methods. For example, an extended ON time may allow for greater energy to be input into the cells which may result in complete ablation, whereas a reduced ON time may reduce the likelihood of the collateral damage of nearby wanted cells. The selection period may correspond to a gate open period as described in relation to figures 16-19 using a regular pulsed laser in which an optical switch is used to direct selected pulses to the cells or microfluidic stream at specific intervals. This achieves enhanced sorting and reduces collateral damage to wanted cells.

[0178] In one example, the waveform width is greater than the predetermined delay offset, for example where a clump of cells provides a multitude of overlapping emission events. In this instance, the calculation above for the delay offset period may result in a negative time, or the calculation may fail to complete prior to the cell passing a sorting beam location. To avoid this happening, the system may be configured to detect a long multicell event and initiate the selection period as soon the waveform width exceeds a threshold waveform width, e.g. the predetermined delay.

[0179] In another example, the offset delay may be based on a proportion of the waveform width. As referred to herein, this process is referred to as adaptively timed adaptive selection triggering. It may be described by:

[0180] Equation 2: OD = PCTD - aWW - b where PCTD is the predetermined cell transit duration, a is a coefficient multiplied with the waveform width WW, and b is a constant.

[0181] FIG 6a illustrates the application of a sorting action for a single cell event and shows plots of, from upper to lower, a received emission signal (signal), a sorting trigger signal (trigger), a laser pulses (laser), and a cell - grey when interrogated to generate a waveform in the received emission signal, for example at a detecting location or inspection area 125, and black when sorted via deactivation, for example at sorting location or arrangement 140.

[0182] As previously described, the received emission signal includes a waveform corresponding to detection of a single cell event - the grey cell. The waveform includes a detected rising edge and a detected falling edge. The waveform has a waveform width (or a 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 starts after an offset delay after the detected falling edge. In this example, an adaptively timed adaptive selection triggering mode is used. In this triggering mode, the selection period or ON time (e.g. 4ps) is calculated based on a proportion of the waveform width (e.g. 15us). The selection period may be calculated by multiplying the waveform width by a coefficient a and adding a constant b. This is described by the equation :

[0183] Equation 3: SP (selection period) = aWW (coefficient x waveform width) + c (constant)

[0184] Similarly, the offset delay may be calculated by multiplying the waveform width by the coefficient and subtracting this and the constant from the predetermined cell transit delay. This is described by the equation shown above for OD. Determining the coefficient a and the constant b are described in more detail below with reference to FIG 8.

[0185] In the example of FIG 6a, the selection period corresponds with one laser pulse as indicated, resulting in a single interaction with the cell - illustrated by the star. The use of a shorter selection period in FIG 6a compared with the longer selection period in the example of FIG 5a may be used in different ways. For example, the reduced number of laser pulses may avoid collateral damage to nearby wanted cells, including wanted cells which may partially overlap with the targeted cell. A reduced number of laser pulses may be used to avoid completely ablating the cell whilst still causing its eventual deactivation (priming as previously described).

[0186] FIG 6b illustrates the application of a sorting action for a multi cell event and shows plots of, from upper to lower, a received emission signal (signal), a sorting trigger signal (trigger), a laser pulses (laser), and a cell - grey when interrogated to generate a waveform in the received emission signal, for example at inspection area 125, and black when sorted via deactivation, for example at sorting arrangement 140.

[0187] As previously described, the received emission signal includes a waveform corresponding to detection of a multicell event - the grey cells. The waveform includes a detected rising edge and a detected falling edge. The waveform has a waveform width (or a 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 starts after an offset delay after the detected falling edge. In this example, an adaptively timed adaptive selection triggering mode is used. In this triggering mode, the selection period or ON time (e.g. 34ps) is calculated based on a proportion of the waveform width. The selection period may be calculated by multiplying the waveform width by a coefficient a and adding a constant b. This is described by equation 3 above. Similarly, the offset delay may be calculated by multiplying the waveform width by the coefficient and subtracting this and the constant from the predetermined cell transit delay. This is described by equation 2 above.

[0188] In the example of FIG 6b, the selection period corresponds with six laser pulses as indicated, resulting in six interactions with the cell - illustrated by the stars. The use of a shorter selection period in FIG 6b compared with the longer selection period in the example of FIG 5b may be used in different ways. For example, the reduced number of laser pulses may avoid collateral damage to nearby wanted cells, including wanted cells which may partially overlap with the targeted cells of the multicell event. A reduced number of laser pulses may be used to avoid completely ablating the cells whilst still causing their eventual deactivation (priming as previously described).

[0189] As described in relation to FIG 5a, 5b, 6a, 6b, the sorting beam may be controlled to be directed to the microfluidic stream during the selection period or ON time. The number of laser pulses applied to the microfluidic stream (and any cells coincident with the laser) will depend on the selection period and timing. In the examples shown in FIG 5a and 5b (adaptively timed selection triggering), more laser pulses are applied to the (black) cell(s) than in the examples of FIG 6a and 6b (adaptively timed adaptive selection triggering). In other configurations different numbers of laser pulses may be applied for the same (or different) waveform widths. These laser pulses may be sufficient to sort or deactivate all cells in the multicell event.

[0190] In some examples, the laser pulses are selected according to the methods described in relation to FIGs 16-19.

[0191] The selection period may be implemented using an acousto-optic modulator or other switch to control the emission of the laser pulses towards the microfluidic stream during the selection period. At other times the laser pulse may be directed elsewhere or absorbed / blocked - indicated by the grey laser pulses.

[0192] Whilst some examples determine waveform widths for a cell event by using a rising and falling edge of the waveform of the cell event, other examples may use other approaches. In one example, a peak or maxima may be determined for a waveform and a predetermined duration or "width" added before and after the peak in order to provide an estimated waveform width that may be used in the previously described adaptations of the selection period and / or the delay offset. These additional predetermined durations may be determined based on experimentation and / or analysis of the waveforms of multiple cell events. In some examples the predetermined durations applied before and after the peak may be different. In some examples, where the cell event comprises multiple peaks, the predetermined period may be added after the last peak and before the first peak in order to determine the total waveform width of the multicell event.

[0193] In other examples, a duration or period after a peak may be determined using a falling edge of the waveform as previously described. This duration or period may then additionally be added before the detected peak (as well as after the peak) in order to determine an estimated waveform width. In the case of multicell events with multiple peaks, the duration between the last peak and the falling edge may then also be added before the first peak and any intermediate peaks in order to determine the waveform width.

[0194] As shown in Figure 10, sperm cells comprise a head 1007, mid-piece 1008 and tail 1011. The tail can be divided into a principal piece 1009 and an end piece 1010. The head comprises a plasma membrane 1001, acrosome 1002 and nucleus 1003. The nucleus holds genomic DNA. The head is connected to the midpiece by a connecting piece 1004. The midpiece contains mitochondria 1005 in a mitochondrial sheath which helically wraps the midpiece of the tail and supplies the energy the tail needs to move. The tail moves with whip-like movements back and forth to propel the sperm towards the egg. The mitochondria contain their own genome (mitochondrial DNA"mtDNA") on which a limited number of genes are encoded. There are 5-10 copies of mtDNAs in one mitochondrion, and 1000-5000 copies in one cell in the case of the somatic cell. The spermatozoon has approximately 50-75 mitochondria, and each mitochondrion contains, on average, one copy of mtDNA.

[0195] The inventors have surprisingly found that mtDNA can be used to enhance sorting and targeting of sperm cells stained with DNA-specific stains. This technique involves detection of a waveform with sufficient fidelity to identify genomic DNA (gDNA) from the nucleus which yields a gDNA peak, and mitochondrial DNA (mtDNA) from the midpiece. The relationship between the mtDNA peak and the gDNA enables a user to determine at least one property of the cell selected from the group consisting of: z-axis orientation (i.e. head-first or tail first); alignment of the cell along its longitudinal axis (head to tail) with respect to a z-axis direction of flow.

[0196] The emissions characteristic of mtDNA are significantly less than the emissions characteristic of gDNA due to the corresponding lower amount of DNA present in the mid-piece versus the head of a sperm cell. Accordingly, the cell emission detected will result in a significantly smaller peak compared to a de novo gDNA peak such as gDNA from a semi-overlapping, overlapping or adjacent cell head. The cell emission magnitude may be determined by any appropriate method such as peak integral, or maxima. Differentiating between the gDNA and mtDNA peaks may be carried out by any suitable method. In some examples, the peaks are differentiated as follows:

[0197] 1. Calculate centroid of waveform and compare to the maxima. If the maxima comes before the centroid, the cell is travelling head-to-tail, while if the maxima comes after the centroid, the cell is travelling tail-to-head.

[0198] 2. Similar to the methods described in relation to Figure 3d, a high falling edge threshold is set 212m that will occur after a first peak (either mtDNA or gDNA) and before the second peak (either mtDNA or gDNA). A further falling edge threshold is set much lower which is only triggered when the downslope approaches the baseline, for example as shown in Fig. 3b 212m. For a single sperm cell, the period between the first and second falling edges will be within a well-defined range where the peaks correspond to gDNA and mtDNA and may be referred to as the head to mid-piece transit period "HMTP". This HTMP should be predictable and within a constant range for a given flow speed, and with sperm cells travelling head-to-tail or tail-to-head and being substantially aligned with the Z-axis of flow. In order to differentiate a single cell with two peaks (relating to mtDNA and gDNA) from a multi-cell event, the time between the falling edges (or peak maxima) can be measured and assessed against the pre-determined HMTP. If the time is outside the HMTP range, this indicates it is a multi-cell event. If is it within the range, this indicates with a high probability that the peaks correspond to mtDNA and gDNA. As such, this is a mechanism to avoid false positive detection of multi-cell events when attempting to determine cell z-axis orientation.

[0199] In order to detect the emissions of the midpiece and differentiate it from the emissions of the gDNA in the nucleus, the beam and emissions must provide sufficient fidelity. This fidelity can be achieved in a number of ways.

[0200] In one example the interrogation beam width is less than or equal to half the length of the head when measured along a longitudinal head to tail axis. A bovine sperm cell head is approximately 8-llpm long. Therefore in one example, the interrogation beam width comprises less than about 5pm. Increased fidelity can generally be achieved by a narrower beam, for example less than a third of the head length, for example 4pm or less than 3pm.

[0201] In another example, the emission signal from the mtDNA is separated from the gDNA emission signal.

[0202] In a further example, properties of the mtDNA emission signal are differentiated from properties of the gDNA emission signal. For example an mtDNA specific stain is used that provides a different emission signal when compared to the gDNA emission signal. In one example a mitochondria-specific stain is used to induce an emission signal. This stain may or may not stain the mtDNA; it may also stain other cellular components specific to mitochondria. In a further example a mitochondrial (non-mtDNA) stain may be used in combination with a mtDNA emitting stain to induce an increased emission intensity to effectively determine the at least one property of the cell such as those described above.

[0203] It will be understood by those of skill in the art that the concept of detecting DNA or other cellular components from multiple longitudinal cell locations to understand cell properties such as Z-axis orientation may also apply to other cells with components such as DNA found at multiple longitudinal locations using similar techniques.

[0204] In some situations, a cell head may overlap with a sperm tail and the sperm head is desired to be hit. This may cause the overlapping sperm's tail to also be hit as shown in 510, figure 5b. In one example, the invention provides a method of selecting a cell comprising: a. determining the z-axis orientation (i.e. flowing head first or tail first) of a cell based on the shape of the waveform; b. determining a selection period wherein the selection period is timed to start based on the determination of z-axis orientation; and c. selecting the cell.

[0205] The selection of the cell may be according to any selection or deactivation method described herein. The start of the selection period is adjusted to select a preferred portion of the cell. For example the selection period may be timed to select only the head and not the tail, or only the tail and not the head. Selecting the head of a cell e.g. by deactivation laser is more likely to cause rupture of the cell membrane. This may cause cell components such as DNA to be released into the media. Free DNA or other cell components may be undesirable, for example where those components may be detrimental to retained cell health, or cause interference with analysis of the remaining cells or cell media. Accordingly, this technique has the potential to improve cell sorting and analytical techniques. Based on the determination of z-axis orientation, an action associated with the selection period may be adjusted. For example a laser intended to cause a change in the orientation, position or direction of travel of said cells, or a deactivation laser may be adjusted to achieve one or more of a decrease in laser power, an increase in laser power, or a variation in laser direction.

[0206] In another example the method of selecting a cell by determination of z-axis orientation may be combined with techniques described herein to determine a multi-cell event. In this example, a first cell of a multi-cell event may be determined to be in a first z- orientation, and a second or further cell of a multi-cell event may be determined to be in a second z-axis orientation. Based on the respective z-axis orientations, an action associated with the selection period may be adjusted. For example a tail of a first cell travelling in a head first z-axis orientation may overlap with a head of a second cell travelling in a tail first z-axis orientation as shown in 510. Selecting the first cell, for example via a deactivation laser may harm the second cell due to the likelihood of damage to the tail of the second cell. Therefore in this instance it may be preferable to lower the deactivation power, or to omit to fire the deactivation to allow both cells to pass. Alternatively, a decision may be taken to extend a selection period to cover (i.e. deactivate) both cells.

[0207] In an alternative example, a first cell travelling in a tail first z-axis orientation may be selected according to selection criteria, and this cell may be close, or overlap with a second cell travelling in a head first z-axis orientation that is not selected. In this example, the selection period may be shortened to minimise the chance of selecting, for example damaging, the second cell. In this example, a first part of the head of the first cell is targeted while leaving a latter, overlapping part of that head untouched to decrease the likelihood of damage to the overlapping second cell.

[0208] Referring to FIG 11, two waveforms are shown with representative cells also illustrated. Each cell 1105f and 1105g comprises a cell head 1106f and 1106g, a mid-piece 1107f and 1107g, and a tail 1108f and 1108g. These cell parts are described in more detail below. Each cell 1105f, 1105g is associated with a high-resolution waveform lllOf, 1110g respectively. In these high-resolution waveforms, it can be seen that each comprises two peaks or maxima, a higher maxima or peak 1112f, 1112g corresponding to the cell head 1106f, 1106g respectively, and a lower maxima peak 1114f, 1114g corresponding to the mid-piece 1107f, 1107g respectively. The waveform can be considered as a series of cell emission signal values which are all above (or deviated from) a signal value baseline (e.g. zero) and where a first maxima and a second maxima correspond to respective local maximum signal values or amplitudes within the waveform corresponding to DNA in the cell head or DNA in the cell mid-piece - the maxima associated with the cell head having a higher signal value or amplitude than the maxima associated with the cell mid-piece. The word "above" in this context is intended to convey a deviation from the baseline and so could refer to negative signal values depending on the particular implementation of the signal value determination circuitry. In some examples, a waveform may be further defined by a series of signal values which, following the first maxima, are above 10% or 50% of the signal value of that first maxima. In other examples, the waveform may be defined by a maximum duration after the first maxima and / or when the signal values of the series of signal values return to within a predetermined offset from the baseline. By identifying the different height maxima in the same waveform, the order of different parts of the cell can be determined. For example if the first maxima is higher (cell head) than the second maxima (cell mid-piece), then the cell is oriented head first in the direction of travel. On the other hand, if the first maxima is lower (cell mid-piece) than the second maxima (cell head), then the cell is oriented tail first.

[0209] From FIG 11, it can also be seen that the cell z-axis orientation in the z-direction or direction of cell travel affects the shape of the corresponding waveform. The cell 1105f on the left is oriented with the tail first, resulting in a waveform lllOf with the lower peak 1112f occurring before the higher peak 1114f. Conversely, the cell 1105g on the right is oriented head first, resulting in a waveform 1110g with the lower peak 1114g occurring after the higher peak 1112g. Therefore, by examining the waveform of single cell events, the z-axis orientation of the corresponding cell may be determined.

[0210] It can be observed in Figure 11 that the cell direction of travel is from right to left in the depicted figure. As the cells move past a stationary detector, for example left of the figure, the head 1106f of cell 1105f will produce emissions that register as a peak readout lllOf prior to the mid-piece 1107f which registers as the smaller peak 1114f after the larger peak.

[0211] Figure 12a and 12b show actual waveforms detected from cells. The mid-piece emission signal is observable as minor peaks 1201a and 1201b adjacent a major peak correlated to the head 1202a and 1202b. The background noise threshold 1203 is also observable. The waveforms shown in figures 12a and 12b were obtained by using an interrogation beam of approximately 3pm. To achieve accurate resolution of the major and minor peaks, it is useful to use a beam width of less than the width of the sperm head.

[0212] Accordingly, in some examples the beam width is less than the length of the sperm head being analysed. In some examples, the beam width is less than about 10pm. The position of the minor peak 1201a in fig 12a to the left of the major peak 1202a is indicative of the z-axis orientation of the cell which is correlated to the overlaid depiction of the cell 1204 and the corresponding mid-piece 1205.

[0213] Figures 13a-d show further examples of waveforms detecting the head and mid-piece of sperm cells in a sequential manner. Sperm cells have been overlaid on the waveforms to indicate the observed z-axis orientation of the sperm. The two waveforms on each figure are overlaid waveforms detected from orthogonal detectors. In one example, the information from the two detectors is combined to determine with higher accuracy the z- axis orientation versus data from a single detector. The upper (higher maxima) waveform 1305 on each figure corresponds to a first detector and the lower waveform 1310 corresponds to fluorescence detected from a second detector.

[0214] Figure 13a illustrates a difference between the first detector waveform shape 1305 and the second detector waveform shape 1310. In contrast, Fig. 13b shows a first detector waveform shape 1305 substantially the same as the second detector waveform shape 1310. The similarity in waveform shape is believed to be due to the x- or y-axis orientation of the cell. X-axis orientation means the predominant direction of the cell head-to-tail in the x-axis (across the microfluidic stream) and y-axis orientation means the predominant direction of the cell head in the y-axis (up and down in the stream). Accordingly, a method of determining x- or y-axis orientation is provided by comparing the similarity of the first waveform 1305 and the second waveform 1310 to determine an x- or y-orientation with respect to the first and / or second detector. Based on the x- or y-axis orientation, at least one cell sorting parameter may be adjusted to improve cell selection, targeting or sorting. In one example, a correlation parameter between the two waveforms may be determined and if this exceeds a threshold, this may be indictive of one orientation whereas if the correlation parameter is below that threshold or another lower threshold, this may be indicative of the other orientation. The correlation parameter may be a waveform cross-correlation coefficient, although other measures may alternatively be used.

[0215] This additional information on x, y, or z-axis cell orientation may be used to adjust the selection processing of cell events. For example, if a wanted cell which is oriented tail first is determined to be within a predetermined distance of a cell event (single or multiple), then this indicates an overlap between the wanted cell and an unwanted or targeted cell or cells. This determination of an overlap may then be used to adjust the selection processing of the preceding unwanted cell event, for example in order to avoid affecting the tail of the following wanted cell.

[0216] In one example, this adjusted processing may be to prevent selection processing of the preceding unwanted cell event. Whilst this will allow the cells of the unwanted cell event through without selection processing, it will also avoid potentially damaging the tail and hence the viability of the following wanted cell.

[0217] In another example, where an overlapping wanted cell is determined, the adaptive triggering may be switched from adaptively timed selection triggering for selection processing of the preceding cell event (FIG 5a or 5b) to adaptively timed adaptive selection triggering (FIG 6a or 6b). This effectively shortens the selection period as previously described which may avoid the tail of the following wanted cell being damaged. FIG 7 illustrates a method of processing cells according to some examples. The method 600 may be implemented in any suitable apparatus such as the controller 135 of FIG 1. The method analyses cell events and determines whether to sort cells within these cell events.

[0218] At 605, the method detects a rising edge and a following falling edge of a waveform, as well as determining the waveform width or duration of the waveform. This may be implemented using the approach described with respect to FIG 2a and 2b, however other approaches may alternatively be employed.

[0219] At 610, the method determines whether the waveform corresponds to a multicell event comprising multiple closely grouped cells. In such an event, it may not be possible to fully resolve each cell. By comparison, a single cell event may comprise a single cell resulting in a well-defined waveform of an expected pattern and range of waveform widths and / or heights. Cell events which do not meet these expectations may be classified as multicell events. Alternatively, multicell events may be determined from one or more characteristics of the waveform such as a waveform width greater than a multicell event threshold, more than one peak detected or other characteristics.

[0220] In this method, a multicell event is considered a selected cell event, however in other examples further analysis of the waveform may be performed in order to classify the multicell event as unselected or selected. For example, in some multicell events it may be possible to determine sufficient information about the cells within the multicell event in order to classify the event as wanted and therefore not select those cells for deactivation, or sort them into a different vessel for wanted cells. An example may be that one or more of the cells in the multicell event can be classified as wanted cells, such as X-cells for example.

[0221] At 610, if the waveform is considered as belonging to a multicell event (Y), the method moves to block 625. Otherwise, the waveform is considered as belonging to a single cell event (N) and the method moves to block 615.

[0222] At 615, the method determines one or more characteristics of the waveform, which corresponds with a single cell. For example, an integral or peak maxima of the waveform may be used to classify a sperm cell as an X-cell or a Y-cell. Other characteristics may additionally or alternatively be used, for example the slope of the rising and / or falling edge of the waveform, and / or the temporal symmetry of the waveform.

[0223] At 620, the method determines whether the cell is selected based on the determined characteristic(s). If the cell is not selected for sorting (N), the method returns to block 605 where a waveform of a next cell event is analysed. In one example, this allows a wanted cell to pass through a cell processing apparatus unaffected, for subsequent collection. Alternatively, the wanted cell may be subjected to a sorting arrangement that may influence the movement of the cell. For example the cell direction may be influenced by radiation pressure, application of an electric field, or other biasing methods to cause the cell to be collected in a wanted cell collection vessel. Otherwise, the cell is considered selected for sorting (Y) and the method moves to block 625. The method at 620Y also returns to block 605 where a waveform of a next cell event is analysed.

[0224] At 625, the method determines an offset delay and a selection period. One or both of these variables may be based on the waveform width as previously described, or may be based on predetermined values. These variables may also be dependent on whether the cell event is a single cell event or a multicell event.

[0225] In an example for a single cell event, the offset delay (OD) and selection period (SP) variables may be determined according to one of the following:

[0226] 1) OD = POD and SP = PSP where POD is a predetermined offset delay following a detected falling edge of the waveform and PSP is a predetermined selection period; or

[0227] 2) OD = PCTD - WW and SP = WW, where PCTD is a predetermined cell transit duration following a detected falling edge of the waveform and WW is the waveform width; or

[0228] 3) OD = PCTD - aWW - b and PD = xWW + b, where PCTD is the predetermined cell transit delay following a detected falling edge of the waveform, WW is the waveform width, a is an assignable coefficient and b is an assignable constant.

[0229] In an example for a multicell event, the offset delay (OD) and selection period (SP) variables may be determined according to one of the following:

[0230] 3) OD = POD2 and SP = PSP where POD2 is a predetermined offset delay following a detected falling edge of the waveform and PSP is a predetermined selection period; or

[0231] 4) OD = PCTD - WW and SP = WW, where PCTD is the predetermined cell transit duration following a detected falling edge of the waveform and WW is the waveform width; or

[0232] 5) OD = PCTD - aWW - b and PD = xWW + b, where PCTD is the predetermined cell transit duration following a detected falling edge of the waveform, WW is the waveform width, a is an assignable coefficient and b is an assignable constant. At 630, the method selects the cell or cells of the selected cell event for sorting. This may be implemented by directly controlling a continuous or pulsed laser to start and stop lasing to coincide with the selection period, for example using an active-q switch. In another implementation, a continuous or pulsed laser may already be in operation and a switch such as an acousto-optic modulator may be used to apply the laser to the microfluidic stream only during the selection period. In another example, sorting may be achieved by applying radiation pressure to the cell to cause it to change direction. In another example, sorting may be achieved by charging a droplet containing the cell and causing the charged droplet to be attracted to charged plates to adjust its trajectory.

[0233] The method 600 is configured to sort all cells in multicell events as well as selected cells in single cell events, with unselected cells in single cell events being unaffected. This means that some wanted cells in multicell events may be selected and sorted, for example via deactivation. However overall the concentration of selected cells will be decreased as all cells which cannot be determined as wanted cells will be selected and sorted for example via deactivation. In alternative examples where at least some cells in multicell events can be distinguished and classified as wanted or unwanted, these wanted cells may also be spared deactivation whilst only the selected cells of the multicell event are deactivated.

[0234] FIG 8 is a plot of waveform width against selection period. This may be used to determine the coefficient a and the constant b which can be set using the slope and y- intercept respectively of the curve. This may be determined experimentally using different pulse widths and selection periods. Example time periods for an experiment are described, however other values could alternatively be obtained and / or used. Point 703 is a minimum waveform width (WW) of 9.96ps and a minimum selection period (SP) or laser ON time of Ops. This corresponds to a threshold below which there is no recognised cell event. Point 706 corresponds to a single cell event where WW = 12.6ps and SP = 3.64ps. Point 709 corresponds to a double cell event or a multicell event having two cells where WW = 25.2ps and SP = 21.3ps. Point 712 corresponds to a triple cell event or a multicell event having three cells where WW = 37.8ps and SP = 38.9ps. From the slope and intercept of the curve comprising these points 703 - 712, coefficient (slope) a = 1.4 and constant (y-intercept) b = -1.4. It is noted that other double and triple cell events may have different WW and SP values for example due to different cell size, overlapping and / or spatial arrangements. These assignable values a and b may alternatively be determined to ensure that all single cell events are exposed to only one laser pulse. These values may be determined for different runs, based on their own flow configuration and other processing parameters such as type of cells and power of laser. FIG 9 illustrates a controller 900 which may be used to implement a method of processing cells according to some examples. This controller 900 may be implemented as the controller 135 of FIG 1, however this controller 900 may be used in different systems.

[0235] The controller 900 comprises hardware 903 having a processor 906 and memory 909. The processor may be a microcontroller, FPGA or any other suitable hardware or combination of hardware and software. The memory 909 comprises first computer program instructions 912 which when executed by the processor 906, cause the controller 900 to carry out a number of steps 952 - 956. This may be implemented in conjunction with other hardware such as a laser (not shown). The memory 909 may additionally or alternatively comprise second computer program instructions 915 which when executed by the processor 906, cause the controller 900 to carry out a number of steps 962 - 966. This may be implemented in conjunction with other hardware such as a laser (not shown).

[0236] At 952, the processor detects a cell event within a microfluidic stream by detecting a rising edge of a waveform followed by a falling edge of the waveform in a received emission signal associated with the microfluidic stream. As previously discussed, the emission signal may be generated by detectors 130 arranged about a microfluidic stream carrying cells, the presence of which can be detected in an interrogation area by the characteristic release of scattered light or fluorescent emissions following irradiation of the microfluidic stream by a suitable source of radiation such as infrared (IR) 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 may be detected using the approach discussed above with respect to FIG 2a and 2b, however other methods may alternatively be used.

[0237] At 954, the processor classifies the cell event as a selected cell event using the respective waveform. As referred to herein, a selected cell event may include any cell event determined to be a multicell event or a single cell event where the associated single cell is an unwanted cell, such as a Y-cell. Multicell events may be determined using various methods such as having a waveform width over a threshold, having multiple peaks or not being identified as a single cell event. Selected single cell events may be determined using a characteristic of the waveform, such as an integral which is within or above a selected cell threshold.

[0238] At 956, the processor initiates a sorting action on the cells in the selected cell event using an offset delay from the detected falling edge. This may be implemented by controlling a sorting laser or other deactivating means. During a selection period, application of a laser or a deactivating means, or application of another sorting or deactivating action is performed on cells of the selected cell event.

[0239] The use of a detected falling edge provides an improved mechanism for controlling deactivation of cells within a selected cell event as this corresponds to the trailing edge of the cell or the last cell in the selected cell event. This method of detecting and classifying single or multi-cell events enables the selection period to be adapted based on a number of considerations such as the waveform width which corresponds to the duration between the leading and trailing edges of the cell or cells in the selected cell event. This enables the selection period to be extended for wider waveform widths, for example due to multiple closely grouped cells. Adjusting the offset delay allows the selection period to be adjusted as the selection period should finish at the trailing edge of the cell or cells in the selected cell event, in order to avoid affecting subsequent cells. This enables higher enrichment of unselected (typically wanted) cells, compared with selected (typically unwanted) cells which will be more effectively and efficiently sorted. The use of a detected falling edge to trigger a sorting or deactivation laser is also easier to implement compared with using peaks, thereby lowering the processing requirement and improving the processing time, in turn allowing for improved control.

[0240] Other factors which may be used for adjusting the offset delay, and hence the selection period, may include the type of deactivation such as ablation compared with lesser damage, the size and / or type of cells, the z-axis orientation of the cells, cell speed, spread of a core stream (i.e. confinement), and pitch (distance between each cell).

[0241] At 962, the processor detects cell events within a microfluidic stream using a received emission signal associated with the microfluidic stream. As previously discussed, detection of rising and falling edges of a waveform may be used to detect respective cell events. The emission signal may be generated by detectors 130 arranged about a microfluidic stream carrying cells, the presence of which can be detected in an inspection area by the characteristic release of fluorescent emissions following irradiation of the microfluidic stream by a suitable source of radiation such as infrared (IR) or ultraviolet (UV) light. The rising and falling edges of the waveform may be detected using the approach discussed above with respect to FIG 2a or 2b, however other methods may alternatively be used.

[0242] At 964, the processor classifies the cell events as selected or unselected cell events. As with step 954, a selected cell event may include any cell event determined to be a multicell event or a single cell event where the associated single cell is an unwanted cell, such as a Y-cell. Multicell events may be determined using various methods such as having a waveform width over a threshold, having multiple peaks or not being identified as a single cell event. Selected single cell events may be determined using a characteristic of the waveform, such as an integral which is within or above a selected cell threshold. Alternatively or additionally, an unselected cell event may be a single cell event associated with a wanted cell, with all other cell events being classified as selected cell events. A single cell event may be determined as having a waveform width below a threshold, having only one peak or not being a multicell event. Unselected single cell events may be determined using a characteristic of the waveform, such as an integral which is within a wanted cell threshold.

[0243] At 966, the processor applies a sorting action to one or more cells in a selected cell event over a selection period which is dependent on an end of the selected cell event and a duration of the selected cell event. The end of a selected cell event may be determined from a detected falling edge of a waveform associated with the selected cell event. The duration of the selected cell event may be determined from a waveform width of a waveform associated with the selected cell event. These variables may be used as previously described to calculate the selection period. During the selection period, a sorting arrangement is used to sort the selected cell, for example a laser to apply photonic pressure, or a deactivating action is applied to cells of the selected cell event for example via a deactivating means. In one example, the selection period comprises a deactivation period during which a deactivating means or sorting arrangement deactivates the cell or cells.

[0244] The ability to adapt a sorting action by adapting the selection period to different conditions improves the processing of cells in examples. This enables higher enrichment of unselected cells, compared with selected cells which will be more effectively and efficiently sorted and optionally deactivated.

[0245] Detectors are described herein for the detection of light emissions from irradiated cells. The cells may be stained or unstained and comprise one or more dyes which emit at preferred wavelengths. In one example, the detector comprises detectors known in the art for achieving the collection and transformation of emissions into a digital signal, for example a photomultiplier tube (PMT), an avalanche photodiode, or another suitable apparatus which collects light and translates it into an analogue or digital signal for signal processing. The detector may be connected to a suitable signal amplifier and / or analogue-to-digital converter prior to sending the signal to the controller.

[0246] A multi-cell event may result in the generation of a waveform with an integral, peak maxima or waveform width greater than those typically observed for single cells. This arises due to the additive effect or spacing apart of the emission signal when detected by the detector. In one example, the invention comprises a method of assessing the increased peak maxima against a standard peak associated with a single cell event, and classifying the peak as a multi-cell event or a single cell event. Where the waveform is classified as a multi-cell event, a determination step may be carried out to determine the cell count which means how many cells the multi-cell event comprises. For example the multi-cell event may comprise two, three or four overlapping cells.

[0247] The determination step may include one or a combination of the following : correlating the waveform width, the waveform integral or the number of detected peaks in the waveform with the number of cells. For example, where the integral of a single Y cell is 100 and the integral of a single X cell is 104, a multicell event having an integral of 200 (+ / - 2) may be determined to be two Y cells whereas a multicell event having an integral of 208 (+ / - 2) may be determined to be two X cells. Single cell events may be used to continuously calibrate the integral to be used. The waveforms of these single cells may be used to deconvolve the waveforms of individual cells in a multicell event.

[0248] Where the classification step determines that a multi-cell event has occurred, the method further comprises a discrimination step to discriminate between the cells determined to be present. This discrimination step discriminates between the cells to identify the status of the overlapping cells. For example a two-cell multi-cell event may comprise one of three situations: i. wanted+wanted cells, ii. wanted-i- unwanted cells, or iii. unwanted+unwanted cells.

[0249] A decision may then be made as to which of the groups is selected at the sorting arrangement based on the desired enrichment of wanted vs unwanted cells. In an example, sperm cells stained with a DNA fluorescing dye may be used where X is designated as a wanted cell, and Y is designated as an unwanted cell, and where the possible combinations comprise either X-X, X-Y, or Y-Y. Given that X sperm cells have greatest DNA mass, the signal amplitude for X-X multi-cell events is expected to exceed X-Y events, and X-Y is expected to exceed Y-Y events. Based on the determination of an X-X multi-cell event, where the peak maxima or integral is greater than a sorting threshold, a decision may be made to retain the X-X multicell event. For example retention may mean that the cells are not a selected cell event which undergoes sorting via deactivation, or it may mean that they are collected using a sorting arrangement as described herein. The X-Y group may also be selected based on one or more a selection criteria and the desired enrichment of the collected sample. The Y-Y group (and optionally the X-Y group) may be designated as a selected cell event and be processed according to the sorting methods described herein, for example the cells may be deactivated, not collected, or collected in an unwanted cell collection vessel. In another example, the Y cells may be wanted cells and therefore the selection criteria are reversed.

[0250] This technique is particularly useful where cell discrimination is based on non-binary metrics, such as the degree of fluorescence of a first tagged cell versus a second tagged cell with a different but non-zero standard fluorescence signal. In a binary sorting situation, where X is tagged to emit an emission signal, but Y does not emit an emission signal, a multi-cell event X-Y would be expected to generate the same signal as a single cell event X. In this case, the multi-cell event classification step would fail to identify multiple cells. Accordingly, this example of the invention has particular utility where the respective labelled cells exhibit an emission signal intensity that differs by less than 50% of the total detectable intensity of either cell.

[0251] In one example, where a classification step determines that a multi-cell event has occurred, the sorting action applied may be adjusted. For example a power of a deactivation laser emitted in response to an overlapping sperm cell is reduced. This has the effect of reducing the chance of collateral damage to the cell that is desired to be kept. Another example of an adjustment to the sorting action is to emit lower energy pulses when multiple cells are detected within a certain time / distance. This enables any energy dispersion or localised media gasification or shockwave produced by the pulse to dissipate or be moved in the direction of flow prior to the next pulse being applied. This technique may be achieved by having a pulse set composed of multiple small pulses or multiple large pulses. The pulses within a pulse set are separated by a pulse frequency. This technique has the effect of intelligently triggering a sorting action to minimise collateral damage to cell and system components. In one example, the adjustment of the sorting action described above may be achieved by adjustment of the power transfer properties through a beam splitter. For example as described in relation to a polarising or other beam splitter for example in relation to FIG 21 and 23. The beam splitter may be associated with a beam directed to a microfluidic stream, or a beam intended to be split between two or more microfluidic streams.

[0252] When flowing cells through a microfluidic stream, it is generally desirable to have a highly confined stream - i.e. there is minimal variation in the X and Y positioning of a cell at a given Z position. This enhances the interrogation accuracy, and generally enhances sorting actions, especially if they involve the cell passing through a sorting beam which will be focused on a specific X, Y and Z position. However, confinement of cells remains complex. In one example, the waveform generated by a cell is determinative or indicative of the lateral positioning of a cell within a microfluidic stream. The X and Y coordinates of a cell with respect to an average X and Y co-ordinate may be determined and an offset value calculated. This value provides positioning information of the cell with respect to a normalised axis of flow. The present invention provides a method of customising the targeting of cells to minimise variations in confinement. Accordingly, in one example, the invention provides a method of selecting a cell comprising: a. measuring a waveform associated with a cell to determine an X- and / or Y-axis deviation of the cell from a nominal axis of cell flow; b. adjusting a selection action to account for the X-and / or Y-axis deviation; and c. selecting the cell.

[0253] The adjustment of selection action may comprise adjusting the direction or power of a sorting beam to direct or focus the beam on the determined position of the cell which takes into account the calculated deviation. This enables the sorting beam ablation laser to be directed to the cell position in the stream. The adjustment of the direction or power will typically involve a beam modulation / targeting system to deflect the beam. The power adjustment may be by way of a beam splitter such as a beam splitter and optionally associated arrangement as described in relation to FIG 21 and 23. This system may be an acousto-optic modulator, an electrooptic deflector, electro-optic modulator, a Galvanometer scanner, a polygon scanner, or different gates aimed at different positions in the stream.

[0254] In another example, the waveform may be indicative of a lack of lateral alignment of a cell with a nominal and desirable axis of flow. Accordingly, in one example, the invention provides a method of selecting a cell comprising: a. measuring a waveform associated with a cell to determine an alignment with a nominal axis of cell flow; b. adjusting a selection action based on the determined alignment; and c. selecting the cell.

[0255] When the selection action comprises targeting the cell with a sorting beam, the adjusting may comprises one or more of: a. adjusting the beam direction X-Y or Z. b. adjusting the beam focal point in X-Y or Z. c. adjusting the shape of the beam. d. adjusting the elongation scale of an elongated beam profile, for example a transverse line spanning X. e. tilting an elongated beam profile.

[0256] It will be appreciated by those of skill in the art that tilting an elongated beam from an initial X-axis orientation may cause a deflection in Y or Z. The pulse width of a single cell event can be used to measure the speed of cells in realtime which can lead to enhanced cell targeting. The latest cell speed determined from a latest single cell event can then be applied to subsequent cell events, including multi cell events, until the cell speed is again updated. The offset delay can be automatically modulated based on measured cell velocity solely from the waveform width or other waveform characteristics.

[0257] Accordingly, in one example, the invention provides a method of selecting a cell comprising: a. measuring a waveform associated with a cell event to determine the velocity of a cell; b. calculating an offset delay based on the determined velocity of the cell; c. selecting one or more cells in the cell event using an offset delay.

[0258] Selection of the cells may further comprise sorting according to any of the sorting actions provided herein. In a further example, the offset delay is automatically updated based on a determined velocity of a second cell.

[0259] In a further example, the invention provides a method of selecting a cell comprising both: a. adjusting a selection action to account for the X-and / or Y-axis deviation; and b. calculating an offset delay based on a determined velocity of the cell.

[0260] In this example, the two previously described methods of improving targeting are combined to both optimise the offset delay, and to optimise the laser focal point positioning. These methods work in tandem to provide an enhanced cell selection method.

[0261] The detector (e.g. photomultiplier tube) parameters can be adjusted based on the peak characteristics (e.g. peak maxima or integral). This ensures that the detector is detecting optimally and within a set range. This reduces the requirement for user input and calibration of the detector, especially during set-up. For example the detector voltage could be automatically adjusted to account for the variation in intensity of a cell event.

[0262] Accordingly, in one example, the invention provides a method of selecting a cell comprising: a. detecting an emission signal from a cell in a microfluidic stream using a detector; b. generating a waveform associated with the cell based on the emission signal; c. adjusting a detector parameter based on the waveform; d. using the adjusted parameter to detect a cell; e. selecting the cell for sorting. In this example, the detector parameter may be voltage. The waveform may provide an intensity measurement. In this example, the detector voltage is automatically updated based on the intensity to enhance the detection of a cell property such as fluorescence intensity.

[0263] Waveform characteristics can be used to adjust optics or flow position to enable enhanced interrogation and sorting, and to calibrate the cell processing system. For example an input may be provided to a motorised optics stage to be adjusted based on the peak characteristics (e.g. peak width, maxima or integral). This would ensure that the system is interrogating and sorting cells optimally and within a set range. Automatic adjustment reduces the requirement for user input and potential error.

[0264] Other waveform characteristics may include the waveform shape, the number of peaks and their relative heights or timing, waveform width, the area under the waveform, the maximum signal value, or waveform slope. These waveform characteristics can be used to determine properties of the corresponding cell - for example whether it is an X or Y type sperm cell, or the cells longitudinal z-axis orientation (i.e. the direction of flow). Longitudinal z-axis orientation, for example tail-first or head-first, is described in more detail with respect to FIG 10 and 11.

[0265] By determining one or more properties of a cell based on one or more characteristics of its corresponding waveform, one or more parts of the interrogation and / or sorting system may be adjusted, for example to optimise these systems.

[0266] In some examples, a subset of waveform characteristics may be used to classify a detected cell, for example as an X or Y sperm cell, and a different subset of waveform characteristics may be used for adjusting sorting of selected cells or cell events. For example, maximum signal value and integral of the waveform may be used to select a cell event for sorting, and waveform width and shape may be used to control a selection period over which the sorting occurs.

[0267] In one example, the invention may provide a method of interrogating, selecting and / or sorting a cell within a microfluidic stream, comprising: detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; sorting the selected cells within the microfluidic stream (for example using sorting electromagnetic radiation); wherein the sorting is adjusted dependent on a characteristic of the waveform.

[0268] In one example, the invention provides a method of selecting a cell comprising: a. detecting an emission signal from a cell in a microfluidic stream; b. generating a waveform associated with the cell based on the emission signal; c. adjusting a system component based on the waveform; d. using the adjusted system component to detect a cell; e. selecting the cell for sorting.

[0269] In one example, the invention provides a method of sorting a cell comprising: a. detecting an emission signal from a cell in a microfluidic stream; b. generating a waveform associated with the cell based on the emission signal; c. adjusting a system component based on the waveform; d. using the adjusted system component to sort the cell.

[0270] In this example, the system component may be an optical component to generate, focus, position or direct an interrogation or sorting beam. In another example, the system component may be a microfluidic chip such that adjustment varies the positioning of the microfluidic stream, for example by way of an adjustable aperture as described in W02024 / 102007. In another example, the system component may be an electrostatic or acoustic field generator arranged to defect the path of cells within droplets formed from the microfluidic stream. The waveform may provide an intensity measurement. In this example, the intensity measurement may be indicative of the focal point position determined by a system component such as an optical component, or a microfluidic stream, wherein said focal point position is automatically updated based on the intensity to enhance the detection of a cell property such as fluorescence intensity.

[0271] In an example, the optical component may be a continuous or pulsed laser, a laser pulse detector, an optical switch or gate, a lens or a moveable mirror. The various optical components may be arranged in free-space, or in a fibre arrangement where laser pulses or continuous beams are guided between one or more of the optical components using respective optical fibres.

[0272] In some examples, a pulsed sorting arrangement which generates regular pulses is used for sorting selected cell events, such as detected sperm cells which are classified as unwanted (e.g. Y-sperm cells). This may be implemented by coordinating the regular pulses with a selection period or gate open period during which one or more of the regular pulses is directed to a microfluidic stream carrying cells to be sorted. In some examples, a selection period over which a cell event is sorted may comprise more than one gate open period. For example, a multicell event may utilise multiple gate open periods to target different cells within the multicell event, whilst at the same time reducing collateral damage to wanted cells due to the use of shorter duration pulses impacting the microfluidic stream instead of more continuous sorting over the selection period. The concept of using multiple gate open periods within a selection period advantageously enables more accurate targeting of cells using laser pulses controllable at the level of a single pulse. This provides increased sorting fidelity by individually selecting (or not selecting) cells within a multi-cell event, then targeting selected cells for sorting, and leaving unselected (i.e. wanted) cells to be collected for downstream applications. The ability to detect and sort cells one-by-one, even where cells partially overlap provides considerable benefits on the art by enhancing wanted cell enrichment in the collected sample. Alternatively, where a standardised enrichment threshold is sufficient, the methods described herein enable enhanced throughput (i.e. increased cells selection and flow rate). Finally, the ability to selectively target cells using single cell pulses reduces the likelihood of damage to non-target cells which increases their viability.

[0273] In one example, a pulsed laser is used to sort the selected cells or selected cell events by way of at least one of the following methods: targeting the cell or a portion of fluid substantially adjacent the cell with the pulsed laser to change the trajectory or position of the cell; optical manipulation; optical trapping; holographic steering; application of photonic pressure; laser deactivation or laser ablation. This may be implemented by controlling an optical switch or gate to switch between directing the regular laser pulses towards and away from the microfluidic stream according to the gate open period.

[0274] In other examples, laser pulses may be directed to or away from bubble creating regions in or associated with the microfluidic stream. The laser pulses create bubbles which are employed to change the flow path of a selected cell or a sub-population of cells, such as Y-sperm cells. In other examples, laser pulses directed to the microfluidic stream may be used to "nudge" one sub-population of cells into a different flow path. In other examples, the pulsed sorting arrangement may use mechanisms other than lasers, for example generating regular electrostatic, electric field or magnetic field pulses. These pulses may be selectively directed to a microfluidic stream dependent on the detection and classification of cell events. For example, a pulsing high voltage signal may be switched to or isolated from a pair of electrodes between which a microfluidic stream flows. This switching allows one or more electrostatic pulses to be directed at the microfluidic stream and is based on a gate open period.

[0275] FIG 14 illustrates a cell processing system 1400 which uses some of the same components of the system of FIG 1, but uses a different sorting arrangement. The common components have the same reference numerals and their detailed description and operation is as previously described. In particular, the cell processing system 1400 comprises a preparation station 105 which delivers prepared cells to an input arrangement 110 which delivers the cells into a microfluidic stream 115 for downstream processing. The microfluidic stream 115 may be a laminar flow having a predetermined range of cross-sectional dimensions and carried within a flow environment. In an example the flow environment may comprise a volume of gas such as air, or a microchannel fully or partially enclosing the microfluidic stream. One or more illuminators 120 generate an interrogation or detection beam, for example an infra-red (IR) or ultraviolet (UV) illuminator or other irradiation devices. The interrogation beam irradiates the cells within the microfluidic stream at an interrogation or detection zone 125. Irradiation of the cells causes emission signals such as scattered or fluorescent light which is detected by one or more detectors 130. Measured characteristics of the detected emission signals generate one or more signals which are forwarded to a controller 1435 which controls a pulsed sorting arrangement for sorting cells into two sub-populations (e.g. X and Y sperm cells). Sorted cells are collected in at least one collection vessels.

[0276] The controller 1435 may comprise a processor and memory and is configured to interpret received emission signals in order to control the pulsed sorting arrangement 1440. In one example, the emission signal comprises a fluorescence signal. The controller 1435 is configured to detect single cell events each comprising the passing of a single cell within the microfluidic stream past the interrogation area 125. A single cell event results in light received at the detector 130 which generates a signal to be used by the controller to detect the single cell event, as described in more detail below. The controller 1435 may also be configured to detect multicell events each comprising the passing of a closely grouped plurality of cells within the microfluidic stream past the interrogation area 125. The closely grouped cells of a multicell event may not be able to be individually resolved for some downstream processes.

[0277] In some examples, the detecting a cell event or a multicell event may be implemented as previously described with respect to FIG 2a and 2b. In other examples, detecting a cell event may be implemented using peak detection of the fluorescent signal or any other known cell detection mechanism.

[0278] In some aspects, sorting the selected cells or cells events may include: targeting the cell with a laser, use of fluid pressure to change the trajectory or position of the cell, mechanical sorting, piezoelectric actuation, dielectrophoresis of droplets, electrolysis or electroporation, optical manipulation, optical trapping, holographic steering, acoustic- assisted hydrodynamic focusing, application of photonic pressure, acoustic deflection, laser deactivation or laser ablation. Sorting may effect at least one of a force and torque on a cell in one population so as to induce at least one of displacing and orienting these cells relative to an axis defined by the direction of the microfluidic stream flow. Where sorting comprises the deactivation of cells associated with selected cell events, this comprises imparting energy into the cells associated with the selected cell events, the imparted energy sufficient to damage the cells leading to their unviability.

[0279] In particular examples, the pulsed sorting arrangements may include: microbubblebased sorting, for example using lasers, spark or thermal vapour; pneumatic and solenoid valve based cell sorting for example using polydimethylsiloxane PDMS; or piezoelectric actuation for example using PDMS valves. In further examples, the pulsed sorting arrangement may include a pulsed radiation source configured to irradiate the microfluidic stream at a sorting beam target area or sorting zone 1470. The pulsed radiation source is coordinated to sometimes be directed at or near cells associated with selected cell events in order to cause a change in the orientation, position or direction of travel of said cells, or to achieve deactivation comprising ablation or damage to selected cells within it. In this example the emission of radiation from the radiation source which causes an effect on the cells is referred to as a "sorting beam". In any of the examples provided herein, the sorting beam may comprise an elongated beam profile. For example, the elongated beam profile may comprise a line, an ellipse, a rectangle, or a rounded rectangle. In some examples, the pulsed radiation source of the pulsed sorting arrangement comprises regular laser pulses that may have a single pulse duration of 100 nanosecond to 10 femtoseconds. The regular laser pulses may comprise inter-pulse periods in the nanosecond, picosecond or femtosecond range, for example <10pm to about 10ns. The specific characteristics of the sorting beam may be varied according to the required frequency, power and wavelength. In turn, various cells may require different sorting beams to achieve sorting. Those of skill in the art will be able to determine the required frequency, power and pulse duration to adapt a sorting beam to a cell type and flow speed. However, in some examples, the sorting beam comprises a frequency between about 100 and 3000kHz.

[0280] This arrangement may be used in combination with the previously described statically timed selection triggering, adaptively timed selection triggering, or adaptively timed adaptive selection triggering.

[0281] In some examples, this arrangement is particularly useful for removing unwanted cells within a population comprising both wanted and unwanted cells. For example, during the production of cell populations for CAR T-cell therapy there may be certain types of cell that do not exhibit the desired phenotype. Cells in this first population (Pl) are destroyed, denatured or rendered immotile by the sorting arrangement. The alternative population that does not exhibit the desired characteristic in the microfluidic stream 115, such as desirable cells that have not been selected by the controller, are left undisturbed (P2). The sorted or processed cells in the microfluidic stream 115 may then be collected in one or more collection vessels 160 for further use. The sorting arrangement 140 thereby provides a population of cells (P2) enriched with a desired characteristic. Where sperm cells are used, this desirable population may comprise motile X cells.

[0282] In one example, the pulsed sorting arrangement 1440 comprises a suitably configured controller 1435, a pulsed laser 1445, a laser pulse detector 1450, and an optical switch or gate 1455. The pulsed laser 1445 generates regular laser pulses which are detected by the laser pulse detector 1450 which indicates each time a laser pulse is detected. The laser pulse detector 1450 may comprise a photodiode or other type of photodetector. The various optical components 1445, 1450, 1455 may be arranged in free-space, or in a fibre arrangement where laser pulses are guided between one or more of the optical components using respective optical fibres.

[0283] The optical switch 1455 may comprise an acousto-optic modulator (AOM), an electrooptic modulator (EOM), a spatial light modulator (SLM) such as a digital micromirror device (DMD) or any other optical switch configured to direct an incoming laser pulse to or away from the microfluidic stream 115 in the sorting zone 1470. The optical switch 1455 is controlled to allow incoming laser pulses from the pulsed laser 1445 towards the microfluidic stream according to a gate open period controlled by the controller 1435, in order to interact with cells associated with selected cell events. At other times, incoming laser pulses from the pulsed laser 1445 are prevented from sorting the cells in the microfluidic stream. This may be achieved by controlling the optical switch to direct a laser pulse away from the microfluidic stream, where directing away may comprise blocking by a filter, becoming opaque or by re-directing the incoming laser pulses to another location, such as a beam dump 1465 which absorbs the laser pulses not associated with a gate open period.

[0284] The optical switch 1455 may be controlled according to various control strategies as described in detail below. The controller may control a driver (not shown) which in turn drives the optical switch - for example an RF driver may be used to cause an AOM to redirect incoming laser pulses to the microfluidic stream.

[0285] FIG 15a illustrates a control approach for sorting a single cell event. A fluorescent signal 1510s from detectors 130 is received by the controller 1435 and used to detect a single cell event. In this example, peak detection is used to detect a cell event in which a rapid rising and subsequent falling of the signal amplitude is used to detect a single cell event - illustrated in the peak detection signal 1515s. In other examples, different methods of single cell event detection may be used, such as that described with respect to FIG 2a.

[0286] The controller 1435 uses this and optionally other characteristics of the signal waveform to determine whether the detected cell event corresponds to a wanted cell (e.g. X sperm cell) or an unwanted cell (e.g. Y sperm cell). Detected cell events corresponding to unwanted cells are classified as selected cell events which are to be sorted by the pulsed sorting arrangement 1440. Cell events not classified as selected cells events may comprise cells that are allowed to pass the sorting zone 1470 without any sorting interaction.

[0287] The pulse laser 1445 generates regular laser pulses as illustrated at 1525. The controller 1435 controls the optical switch 1465 to allow the pulses to be directed to the microfluidic stream according to a gate open period 1520s. As illustrated at 1530s, a controlled or designated number of pulses, e.g. only a single laser pulse, are allowed to be directed to the microfluidic stream to sort the cell 205s associated with the selected cell event. In other words, the gate open period is sized and its timing is coordinated or synchronised with the laser pulses in order to pass only a controlled or designated number of pulses, in this case a single laser pulse 1530, during that gate open period 1520. All other laser pulses 1525 are directed away (including being blocked) from the microfluidic stream which results in them not interacting with or sorting cells in the microfluidic stream. This arrangement uses a controlled number of designated laser pulses, for example only a single designated laser pulse, to sort cells; which reduces collateral damage to other nearby cells. In other words, adjacent cells which may be wanted cells are not sorted or damaged by the designated pulse or pulses.

[0288] The laser pulses are configured to be sufficiently regular such that a designated pulse or pulses is allowed to target the unwanted cell 205, regardless of the flow speed. The pulses applied may also be sufficiently configured to deactivate that unwanted cell for example by having sufficient power to effect sorting such as by nudging, deactivation or cell ablation. Depending on the optical switch implementation, there may be a switching delay associated with the optical switch changing from directing laser pulses to and away from the microfluidic stream, or vice versa. This may affect a gate request signal applied by the controller 1435 to the optical switch, as described below.

[0289] FIG 15b illustrates a control approach for sorting a multi-cell event. A fluorescent signal 1510m from detectors 130 is received by the controller 1435 and used to detect a multicell event. In this example, peak detection is used to detect multiple closely spaced cells which together correspond to a multi-cell event. The multi-cell event may be defined in different ways, for example as comprising fluorescent signal peaks within a predetermined time of each other. The individual peaks themselves correspond to rapid rising and subsequent falling of the signal amplitude - illustrated in the peak detection signals shown 1515m. In other examples, different methods of multi-cell event detection may be used, such as that described with respect to FIG 2b. The controller 1435 uses the fluorescent signal and optionally other characteristics of the signal waveform to determine whether the detected cell event corresponds to a wanted cell (e.g. X sperm cell) or an unwanted cell (e.g. Y sperm cell). Detected multi-cell events corresponding to unwanted cells may be classified as selected cell events which are to be sorted by the pulsed sorting arrangement 1440. A selected cell event may correspond to a multi-cell event in which only one or only some of the cells are unwanted but also contains other cells which may be considered wanted. Multi-cell events not classified as selected cells events may comprise cells that are allowed to pass the sorting zone 1470 without any sorting interaction.

[0290] The pulsed laser 1445 generates regular laser pulses as illustrated at 1525. The controller 1435 controls the optical switch 1455 to allow designated pulses to be directed to the microfluidic stream according to multiple gate periods 1520m. As illustrated at 1530m, only a single designated laser pulse is directed to the microfluidic stream during each gate open period 1530m in order to sort respective single cells of the selected multi-cell event 205m. In other words, the gate open period is sized and its timing is coordinated or synchronised with the laser pulses in order to pass only designated single laser pulses during that gate period. All other laser pulses 1525 are blocked from interacting with or sorting cells in the microfluidic stream. This arrangement in which designated pulses, e.g. only a single laser pulse, are used to sort a cell reduces collateral damage to other nearby cells. In other words, adjacent cells which may be wanted cells are not sorted or affected by the designated pulses.

[0291] FIG 15c illustrates another control approach for sorting a multi-cell event. A fluorescent signal 1510x from detectors 130 is received by the controller 1435 and used to detect a multi-cell event 1515mx and a single cell event 1515sx. In this example, peak detection is used to detect multiple closely spaced cells which together correspond to a multi-cell event. The more separated peak to the right is detected as a separate single cell event. The multi-cell event may be defined in different ways, for example as comprising fluorescent signal peaks within a predetermined time of each other. The individual peaks themselves correspond to rapid rising and subsequent falling of the signal amplitude. In other examples, different methods of multi-cell event detection may be used, such as that described with respect to FIG 2b.

[0292] The controller 1435 uses this and optionally other characteristics of the signal waveform to determine whether the detected cell event corresponds to a wanted cell (e.g. X sperm cell) or an unwanted cell (e.g. Y sperm cell). Detected multi-cell events or single cell events corresponding to unwanted cells are classified as selected cell events which are to be sorted by the pulsed sorting arrangement 1440. A selected cell event may correspond to a multi-cell event in which only one or only some of the cells are unwanted but also contains other cells which may be considered wanted. Multi-cell events not classified as selected cell events may comprise cells that are allowed to pass the sorting zone 1470 without any sorting interaction.

[0293] The pulse laser 1445 generates regular laser pulses as illustrated at 1515. The controller 1435 controls the optical switch 1455 to allow multiple pulses through to the microfluidic stream according to gate open period 1520mx which corresponds to the detected multicell event 1515mx. The figure also illustrates the sorting of a single cell event 1515sx which is more fully described above with respect to FIG 15a.

[0294] Unlike the control approach of FIG 15b, in this example cells associated with selected multi-cell events are sorted using a single extended gate open period in which more than one of the regular laser pulses are directed at the microfluidic stream. The time in the sorting zone 1470 will depend on the flow rate of the microfluidic stream. This flow rate and the inter-pulse period between laser pulses can be configured such that some part of each cell of the multicell event will coincide with a laser pulse during the gate period, as described in more detail below. In some examples where the cells are sperm cells, the flow-rate and inter-pulse period may be configured such that at least one laser pulse coincides with the passing of a sperm cell head within the sorting zone.

[0295] In some examples, the gate open period is sized and its timing is coordinated or synchronised with the laser pulses in order to pass only designated pulses, e.g. a single laser pulse, for each cell during that gate period. All other laser pulses 1525 are directed away or blocked from interacting with or sorting cells in the microfluidic stream. This arrangement in which only designated laser pulses are used to sort cells reduces collateral damage to other cells nearby to the multi-cell event. In other words, adjacent cells which may be wanted cells are not sorted or damaged by the laser pulse.

[0296] FIG 16 illustrates a control signalling timing diagram according to an example. The control signalling diagram illustrates the relative timing or various signals and may be implemented by a suitably programmed processor or a suitably configured FPGA for example, such as processor 1435. The first line 1611 illustrates a trigger signal 1621 corresponding to the detection and classifying of a cell event as a selected cell event. The trigger signal may be any suitable signal provided by a cell event detection and / or classification function, which may be implemented by a suitably programmed processor or a suitably configured FPGA for example; this may be the same or a different processor to that used for implementing the other signalling illustrated. The second line corresponds to a gate timing means 1613 which controls different delay periods and laser pulse detection signals 1623, also known as a sync-out clock, to determine a gate open request 1625. The gate timing represents functionality within a processor or FPGA which is configured to generate the various signals illustrated in response to incoming signals such as the trigger signal 1621 and a laser pulse detection signal 1623. The laser pulse detection signals 1623 correspond to respective laser pulses having their intensity or amplitude illustrated on line 1617. These regular laser pulses have an inter-pulse period of lus in this example to aid with operational explanation, but examples are not restricted to such a time frame.

[0297] The gate request period 1637 is initiated by a gate open request 1625 which is a signal arranged to control an optical switch to open an optical gate for the duration of the gate request period. This corresponds with a gate open period 1646 during which the laser pulses incident on the optical switch are directed to the microfluidic stream. Line 1615 provides the gate status - the dotted line 1645 indicates the transition from a fully closed state 1640 to fully open 1642. In some examples, the optical switch may incur a switching delay between being fully open and fully closed (or vice versa) as can be seen in the sloped lines of the period waveform 1645. During this period, the laser may experience a reduction in power, misdirection or defocusing. The power of a single laser pulse incident on a target occurring during a switching delay (i.e. from fully closed to fully open), may appear to be attenuated and therefore laser pulses directed to the microfluidic stream by the optical switch will be passed to the microfluidic stream with reduced power. This can be problematic as a partial pulse may be insufficient to sort a cell.

[0298] The trigger signal 1621 causes a gate request 1622 at the gate control means which controls a start delay period 1631 between the gate request 1622 and the start of the gate open period 1640. This start 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 for cells to travel from the detection zone 125 to the sorting zone 1470. This will depend on the flow rate of the microfluidic stream and the distance between these two regions. A small allowance may be made for the signal processing time required for detection and classification. An example predetermined delay period is used in this example 2.75ps to aid with operational explanation, but other examples are not restricted to such a time frame. The variable delay period 1635 is dependent on the timing of the next pulse detection signal 1623 after the gate request 1622. By timing the gate open request 1625 to be the predetermined period 1631 after the next laser pulse, the gate open period 1646 should coincide with a subsequent laser pulse and a cell of the selected cell event corresponding to the trigger signal 1621. Consideration of the switching delays may also be employed to generate the gate open request 162 and gate request period 1637, as described in more detail below. In this example, provided for explanatory purposes, the trigger signal 1621 is substantially coincident with the gate signal 1622. The variable delay period is the time between the gate request 1622 and the next pulse detection signal 1623. The pulse detection signal 1623 may be generated by a photodiode for example, and in one example may be 0.5ps. In this situation, the start delay period 1631 for requesting the optical switch to open is this variable delay 0.5ps plus a predetermined delay of 2.75ps, making a total request or start delay period of 3.25ps from the gate request 1622 until the gate open request at 1625. The duration of the gate open period 1637 in this example corresponds with the start of the gate open period to allow designated laser pulses to be directed to the microfluidic stream until the start of the gate closing switching delay 1645. In one example this configuration can be used to implement the control and timing strategies described with respect to FIGs 3a to 3d, FIG 5a, 5b, 6a, 6b or 15b.

[0299] In an explanatory example, the head of a sperm cell 1605 has a length of approximately 10pm as shown at 1641 and will be within an ablation laser target zone for lus at a flow rate of lOm / s. By providing a gate open period which is less than or equal to this target period of lus, a maximum of one laser pulses at a Ips pulse frequency will hit the head of the sperm cell during this period. The gate open period may be aligned such that its mid-point corresponds with a laser pulse. This avoids a laser pulse occurring during one of the switching delays. In one example the gate open period may be 50% of the interpulse period, that is 0.5us. In one example, the gate period is within 30-70% of the inter-pulse period, or within 46-60% of the inter-pulse period.

[0300] Gate status line 1615 illustrates the beam attenuation performed by the optical switch. When the optical switch or gate is fully open, the full or near full power of the laser pulse is directed to the microfluidic stream to sort the cell. When the optical switch or gate is closed, no laser pulse power is directed to the microfluidic stream. It can be seen that there is a finite time over which the optical switch opens and closes which corresponds to the switching delay and which can cause beam attenuation. If a laser pulse occurs during this switching delay, only part of the power or intensity of the laser pulse will be directed to the microfluidic stream. This may be problematic as a partial laser pulse may be insufficient to sort (e.g. by nudging or deactivation) an unwanted cell and / or may affect an adjacent wanted cell.

[0301] By timing the gate open period such that a laser pulse falls within its central portion, this issue can be mitigated. In one example, the gate period may be adjusted so that the laser pulse coincides with the centre of the gate period, or in the central 80% of the gate period, or within the central 50% of the gate period. FIG 17 illustrates a control signalling timing diagram according to another example. The diagram is similar to that of FIG 16 with similarly labelled features including a trigger line 1711 illustrating a trigger signal 1721, a second, gate timing line 1713 which illustrates the relative timings of delays, laser pulse detection signals 1723 and a gate open request 1725.

[0302] In this example, the next laser pulse detection signal 1723 is closer to the gate request

[0303] 1722 and a shorter predetermined period 1733 is used together with a differently calculated variable delay 1735 to ensure that the gate request period 1737 coincides with a laser pulse and cell from a selected cell event. The shorter predetermined delay 1733 may be due to a shorter inter-beam distance between detection and sorting zones 125, 1470 and / or a higher flow rate of the microfluidic stream. In this example, the variable delay 1735 between gate request 1722 and the next laser pulse detection signal

[0304] 1723 may be 0.25ps and where a predetermined delay of 0.75ps used, the start delay period 1731 is Ips after the gate request 1721. In turn, the gate opening is effected by the optical switch and coincides with the gate open request 1725 to provide a gate open period 1746 substantially centred on the pulse. In one example this configuration can be used to implement the control and timing strategies described with respect to FIGs 3a to 3d, FIG 5a, 5b, 6a, 6b or 15b.

[0305] FIG 18 illustrates a control signalling timing diagram according to another example for use with a multi-cell event. In this example the gate request period 1837 extends over more one than a single laser pulse in order for this to coincide with more than one cell 1805. This is similar to the situation described with respect to FIG 15c. The diagram of FIG 18 is similar to that of FIG 16 or 17 with similarly labelled features including a first, trigger line 1811 illustrating a trigger signal 1821, a second, gate timing line 1813 which illustrates the relative timings of delays, laser pulse detection signals 1823 and the gate request period 1837.

[0306] In this example, the extended gate request period 1837 corresponds to sorting two unwanted cells (or an unwanted cell and a wanted or unclassified cell) in a multicell event. As can be seen, the gate request period 1837 extends over two full laser pulses illustrated by the full solid lines on 1817. However, due to the switching delay of the optical switch after the end of the gate request period terminates, the optical switch partially allows a third laser pulse through to the microfluidic stream - shown by the part solid and part dashed line on 1817. This may be addressed by shortening the gate open request signal where it is determined that the switching delay of the optical switch will coincide with a regular laser pulse. This may be implemented by estimating the next laser pulse based on past laser pulses and determining whether this will fall within a window starting at the end of the gate request period and having a duration corresponding to the switching delay. If this occurs, the gate request period may be shortened by an amount corresponding to the switching delay, or a longer amount which may be dependent on other factors such as the duration of the inter-pulse period and the flow rate. Alternatively, the gate open request 1825 may be initiated earlier, optionally by shortening the variable or predetermined delay period. In one example this configuration can be used to implement the control and timing strategies described with respect to FIGs 3a to 3d, FIG 5a, 5b, 6a, 6b or 15b.

[0307] FIG 19 illustrates another control signalling timing diagram according to another example for use with a multi-cell event. As with the example of FIG 18, the gate request period 1937 extends over more than a single laser pulse in order for this to coincide with more than one cell of the multicell event. However, in this example the gate request period 1937, which is normally used to signal when to open and close the optical switch, is overridden. In this example the optical switch is controlled to close after the first laser pulse and open again before the next laser pulse so that two laser pulses are allowed to pass into the microfluidic stream and sort two respective cells. This may be implemented by using the laser pulse detection signals 1923 to allow or prevent operation of the gate open request signal on the optical switch. In one example this configuration can be used to implement the control and timing strategies described with respect to FIGs 3a to 3d, FIG 5a, 5b, 6a, 6b or 15b. The optical switch may be configured to predict whether the gate close request will occur within a switching delay period prior to a subsequent pulse, and if so, to prevent the gate from opening for a subsequent pulse to be directed to the microfluidic stream. This avoids an unwanted partial pulse being directed during the switching delay.

[0308] The diagram of FIG 19 is similar to that of FIG 16, 17 or 18 with similarly labelled features including a first trigger line 1911 illustrating a trigger or request signal 1921, a second gate timing line 1913 which illustrates the relative timings of delays, laser pulse detection signals 1923 and the gate request period 1937.

[0309] In this example, the gate open request signal 1937 corresponds to sorting two unwanted cells (or an unwanted cell and a wanted or unclassified cell) in a multicell event. As can be seen, similar to FIG 18 the gate request period 1937 extends over two full laser pulses illustrated by the full solid lines on 1917. However, the optical switch is additionally controlled to only be open for a predetermined gate open period 1941 about each laser pulse during the gate open request signal 1937. This may be implemented by opening the optical gate half of this predetermined gate open period 1941 before an estimated time of the next laser pulse and closing the optical gate at the predetermined gate open period 1941 later. For example, for an inter-pulse period of lus, the predetermined gate open period 1941 may be 0.5us so the optical switch is controlled to open 0.25us before the next laser pulse. This approach avoids the partial laser pulses noted above with respect to FIG 18 as the optical switch is fully open when the laser pulse occurs.

[0310] In the previous examples, the gate open period is controlled responsive to detection of a regular pulse following detection of a cell event - for example the predetermined delay may be implemented following both the cell event detection and the next regular pulse detection. In some other examples, the gate open period may be controlled dependent on detection of a regular pulse before detecting the cell event. This could be implemented by using detection of a regular pulse to trigger a countdown timer expiring the inter-pulse period plus the predetermined delay later. If detection of a cell event occurs within the inter-pulse period following the triggering of the countdown timer, the expiration of this timer is then used to trigger the gate open request 1625 which controls the optical switch. Countdown timers may be started for each pulse detection. If cellevent detection does not occur within the inter-pulse period following one pulse detection, then cell-event detection is monitored for the next countdown timer (i.e. the inter-pulse period following the next pulse detection) and so on until a cell event detection falls within the inter-pulse period of one of the countdown timers.

[0311] FIG 20 illustrates a method according to an example of sorting cells in a microfluidic stream using a pulsed laser sorting arrangement which generates regular laser pulses. The method 2000 may be implemented in a cell sorting apparatus such as the cell processing system 1400 of FIG 14, however the method may alternatively be used in other cell sorting apparatus.

[0312] At 2005, the method 2000 detects a cell event. This may be implemented using the detection apparatus 120 and 130 of FIG 14 together with appropriate signal processing such as identifying a pulse or peak in a signal from the detection apparatus or a predetermined signal waveform as previously described. However other methods of detecting cell events may alternatively be employed.

[0313] At 2010, in some examples the method determines whether the detected cell event is a multicell event. However in other examples this process may be omitted so that the method only detects single cell events. Multicell events may be determined based on signal waveform as previously described or by determining that individual signal pulses are within a predetermined distance of each other, indicating that a group of closely spaced cells have been detected.

[0314] At 2015, the method determines one or more characteristics of the cell or cells associated with the cell event. In an example this may involve determining whether the cell or cells comprise unwanted cells, such as Y-sperm cells as previously described. At 2020, the method classifies the cell event as a selected cell event (2020Y) if this comprises unwanted cells based on the determined characteristics. 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 gate request signal to 2030. The method also returns to 2005 to detect another cell event.

[0315] At 2025, the method receives pulse detection signals corresponding to detected laser pulses. These pulse detection signals are provided together with a sort signal to an optical switch control process 2030.

[0316] At 2030, the method generates a gate open request for the optical switch. Examples of different optical switch control have been discussed above for example with respect to FIG 16-19.

[0317] At 2025, the method opens (and closes) the optical switch based on the gate open request from 2030. In an example, this may be implemented by started (and stopping) an RF signal which drives an AOM, which causes the AOM to redirect incoming laser pulses into the microfluidic stream to sort cells associated with the selected cell event. However, alternative mechanisms for sorting cells associated with selected cell events may alternatively be implemented.

[0318] Some examples provide one or more advantages by using the described gate open control strategy with pulsed sorting arrangements. This approach reduces collateral damage to wanted cells whilst also allowing for sorting of multiple cells in a multi-cell event. This in turn provides for:

[0319] 1) enhanced enrichment of a desired cell characteristic by enhancing the sorting of cells. In one example, the sorting action prevents undesired cells from progressing (especially during multi-cell events).

[0320] 2) Enhanced cell throughput for a constant cell selection metric. I.e. less cells of the undesired type are allowed through which means a higher concentration of cells can be collected using the sorting arrangement. This has the effect of increasing speed of the sample through the system. This advantage can be particularly important where cells deteriorate based on time spent flowing through the cell processing system. Therefore enhanced cell throughput can ultimately lead to higher viability of the collected cells due to the decreased time of the sample flowing through the system.

[0321] 3) Real-time detection of the quality / characteristics of the cells and media. For example a) Sample quality, b) Cell quality c) Sheath or buffer fluid quality.

[0322] FIG 21 illustrates apparatus for splitting sorting beams into multiple channels for sorting cells in respective microfluidic streams. In this example eight channels or microfluidic streams flowing through eight sorting units or sorting arrangements 2155A-H are illustrated, however different numbers of channels may be implemented. Laser pulses from a pulsed sorting laser 2140 are split into eight beams using multiple beam splitters 2190A-H. The beam splitter may be a polarising beam splitter. Each beam splitter transmits a portion of light (e.g. either S-polarised or P-polarised). Each sorting unit 2155A-H may be associated with a beam splitter 2190A-H which may comprise elements of a pulse picker or controller used to control whether or not the incoming sorting laser pulses are directed to their respective microfluidic streams. A pulsed sorting arrangement as previously described may be employed, however other sorting arrangements may alternatively be used.

[0323] A beam splitter is a component that can be employed to modify the power characteristics of a laser beam by dividing the beam into two or more separate parts. The division of the laser beam enables control over the distribution of power 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 70:30 or 90: 10, to control the amount of power directed into each path.

[0324] The beam splitter may also be adjusted to cause attenuation of the original laser beam's power. As the beam is divided, the power of each output beam may be reduced or attenuate. This attenuation can be used to reduce of cease the power delivered to sorting units. In some examples, the beam splitter may be polarisation-sensitive, allowing them to split the laser power based on the polarisation state of the beam. This enables selective power control, where different polarisation components of the laser beam are directed along separate optical paths, each with a varying level of power.

[0325] Furthermore, certain beam splitters can be designed to divide the laser beam according to its spectral characteristics, such as wavelength. This allows different power levels to be directed into distinct paths based on the wavelength components of the laser beam, offering greater flexibility in managing the power distribution in more complex optical systems. Accordingly, in one example power transmission properties of the beam splitter are adjusted to split the power dependent on the detected cell properties such as fluorescence, waveform width, z-axis orientation or position of the cell. For a polarising beam splitter, where the P-polarised light is transmitted, the S-polarised light is reflected and directed to one of the eight sorting arrangements. The degree of S- or P-polarised light is determined by a polarisation-modifier such as a half-wave plate which may be placed prior to each polarising beam splitter as shown in FIG 23. Each polarisation modifier may be arranged to modify the beam entering each beam splitters 2190A-H. Each polarisation modifier is tuned to control the power of the respective sorting beams directed to each sorting unit or arrangement. In one example, this can be achieved by rotating the polarisation modifiers while measuring the power at a photodetector positioned prior to or after the beam splitter. Portions of the sorting beam are split by the beam splitters and directed to a respective sorting arrangement with the transmitted beam forwarded onto the next beam splitter. The final beam splitter 2190A may simply be a mirror (or a suitably routed fibre) where the entire remaining power of the beam is desired to be directed to the pulse picker. In one example, the polarisation modifiers are tuned such that the power of the laser pulses is split evenly between the microfluidic channels.

[0326] Although polarisation modifiers are referred to herein, those of skill in the art will appreciate that other power adjustment means may be used to affect the adjustment of power transfer through the beam splitter. For example a half wave plate or a pockels cell could be used. The latter has utility in being able to both pick pulses to be directed to the microfluidic stream, as well as modulate the power transferred to the split beams.

[0327] If one of the channels is not required, for example because one of the microfluidic streams stops flowing or the sorting arrangement requires maintenance, the power transfer properties of the laser beam may be adjusted, for example via the polarisation modifiers and / or the beam splitter. In some examples the power split between the channels may be adjusted depending on how many channels are active. Control of the polarisation of the beamsplitters may be adjusted by automatically adjusting the polarisation modifiers in response to detecting the status of the channels, i.e. whether or not they are being used or faulty.

[0328] In some examples, the method sorts cells within N microfluidic streams using N or N-l beam splitters or polarisation beam splitters. The method may comprise adjusting the power transfer properties of at least one of the beam splitters responsive to cell event properties such as detection, classification and / or sorting properties, or for one or more beam status such as inactivation of detecting, classifying and / or sorting for one or more

[0329] Adjusting the power transfer properties means adjusting a property relating to beam power directed to one or more microfluidic streams. The power may be increased or decreased. A first beam power, transferred through a beam splitter and directed to a first microfluidic stream may be adjusted independently of a second beam power directed to one or more further microfluidic streams. In one example, the inventors have found that cell-event-specific beam power adjustment can be achieved dependent on a detected cell event property and / or orientation. In another example, the inventors have found that beam power adjustment is beneficially achieved based on beam status or waveform width. For example detection, classification and / or sorting feedback may be identified which indicates that cell event properties such as multiple cell detection, sorting, orientation, positioning or a cell selection metric such as X- or Y cell enrichment at one or more of the microfluidic streams is below a pre-determined threshold. Based on this feedback, the beam splitter can be adjusted to adjust the power transfer properties for one or more of the microfluidic streams. For example feedback indicating inactive or ineffective detection, classification or sorting in one microfluidic stream can be used to increase or reduce power to that stream, based on user input or an automated response. In one example the waveform width or other waveform characteristics are used as a signal to control adjustment of the beam power properties. The power transfer properties of one or more of the beams may be responsive to a cell event property or a beam status. In an example, the cell event property may relate to at least one of detection, classification or sorting properties, and the beam status relate to at least one of inactivation of detecting, classifying and / or sorting for one or more of the plurality of microfluidic streams.

[0330] The power transfer adjustment can be achieved dependent on the detected cell properties and z-axis orientation which may be detected by waveform or waveform width. The power transfer may be adjusted to a single microfluidic stream, or to multiple microfluidic streams, or to a single beam which is further split to be directed to multiple microfluidic streams. In one example, there is provided a method of sorting cells within a plurality of microfluidic streams using a laser, the method comprising: splitting the laser into a plurality of beams using a beam splitter, each of the plurality of beams being associated with a respective microfluidic stream; detecting a cell event within each microfluidic stream; classifying the cell event in each microfluidic stream as a selected cell event; analysing the cell event to determine a cell event property or beam status; adjusting at least one power transfer property of the beam splitter based on the cell event property or beam status; sorting a cell associated with the selected cell event in each microfluidic stream using one of the plurality of beams. FIG 22 illustrates an example cell processing system according to an example. The cell processing system 2200 comprises multiple sorting channels each for sorting a respective microfluidic stream. The system 220 comprises multiple pulsed sorting arrangements that share a common sorting laser beam. In this way the cell processing system 2200 is able to reduce costs and increase robustness and simplicity by reducing the number of lasers required to process multiple microfluidic streams in parallel.

[0331] The cell processing system 2200 comprises a single sorting laser 2240 and a single interrogation or detection laser 2230. The sorting laser 2240 may be a pulsed laser which generates laser pulses and the detection laser 2230 may be a continuous wave or quasi-continuous wave laser. The sorting laser pulses are split between two channels by a beam splitter 2290B such as a half-silvered mirror, a polarising beam splitter for example in a free space arrangement or a fused biconical taper (FBT) splitter in a fibre arrangement. Each sorting beam is directed to a respective sorting apparatus 2285A and 2285B each of which sorts cells in respective microfluidic streams. This may involve the use of mirrors 2290A to change direction of respective split beams in a free space arrangement, or a suitable optical fibre routing in a fibre arrangement.

[0332] The detection laser may be split between two or more channels by a beam splitter as shown in 2295B such as a half-silvered mirror, a polarising beam splitter or a fused biconical taper (FBT) splitter. The power split ratio may be approximately equal between channels, for example 50% each in the illustrated example, and 25% each for a system with four channels or microfluidic streams.

[0333] Each sorting apparatus 2285A, 2285B may be associated with a controller 2255A, 2255B used to control whether or not the incoming sorting laser pulses are directed to their respective microfluidic streams. A pulsed sorting arrangement as previously described may be employed, however other sorting arrangements may alternatively be used. FIG 23 illustrates apparatus for splitting sorting beams into multiple channels for sorting cells in respective microfluidic streams. In this example four channels or microfluidic streams are illustrated, however different numbers of channels may be implemented. Laser pulses from a pulsed sorting laser 2340 are split into four beams using a beam splitter such as a polarising beam splitter 2390A, 2390B, 2390C, 2390D. Each beam splitter transmits a portion of light (e.g. either S-polarised or P-polarised). Where the P- polarised light is transmitted, the S-polarised light is reflected and directed to one of the four sorting arrangements. The degree of S- or P-polarised light is determined by a polarisation-modifier such as a half-wave plate.

[0334] Figure 23 shows four polarisation modifiers 2380A-D which are arranged to modify the beam entering each beam splitters 2390A-D. Each polarisation modifier is tuned to control the power of the respective sorting beams directed to each sorting arrangement. This can be achieved by rotating the polarisation modifiers while measuring the power at a photodetector positioned prior to or after the pulse picker 2355A-D. Portions of the sorting beam are split by the beam splitters and directed to a respective sorting arrangement with the transmitted beam forwarded onto the next beam splitter. The final beam splitter 2390A may simply be a mirror (or a suitably routed fibre) where the entire remaining power of the beam is desired to be directed to the pulse picker. In one example, the polarisation modifiers are tuned such that the power of the laser pulses is split evenly between the four channels.

[0335] If one of the channels is not required, for example because one of the microfluidic streams stops flowing or the sorting arrangement requires maintenance, the power or other power transfer properties of the laser beam may be adjusted, for example via the polarisation modifiers. In some examples the power split between the channels may be adjusted depending on how many channels are active. Control of the beamsplitters may be adjusted by automatically adjusting the beam splitter and / or the polarisation modifiers in response to detecting the status of the channels, i.e. whether or not they are being used or faulty. Further examples of adjusting the power transfer properties are provided in relation to FIG 21 and those of skill in the art will appreciate that such examples are also applicable to this and other sorting systems.

[0336] FIG 24 illustrates a controller 2400 which may be used to implement a method of processing cells according to some examples. This controller 2400 may be implemented as the controller 1435 of FIG 14, however this controller 1400 may be used in different systems.

[0337] The controller 2400 comprises hardware 2403 having a processor 2406 and memory 2409. The processor may be a microcontroller, FPGA or any other suitable hardware or combination of hardware and software. The memory 2409 comprises first computer program instructions 2412 which when executed by the processor 2406, cause the controller 2400 to carry out a number of steps 2452 - 2456. This may be implemented in conjunction with other hardware such as a laser (not shown). The memory 2409 may additionally or alternatively comprise second computer program instructions 2415 which when executed by the processor 2406, cause the controller 2400 to carry out a number of steps 2462 - 2466. This may be implemented in conjunction with other hardware such as a laser (not shown). The memory 2409 may additionally or alternatively comprise third computer program instructions 2417 which when executed by the processor 2406, cause the controller 2400 to carry out a number of steps 2472 - 2476. This may be implemented in conjunction with other hardware such as a laser (not shown). At 2452, the processor detects a cell event within a microfluidic stream by detecting a rising edge of a waveform followed by a falling edge of the waveform in a received emission signal associated with the microfluidic stream, or more generally by detecting a signal peak which has certain characteristics such as a minimum amplitude. As previously discussed, the emission signal may be generated by detectors 130 arranged about a microfluidic stream carrying cells, the presence of which can be detected in an interrogation area by the characteristic release of scattered light or fluorescent emissions following irradiation of the microfluidic stream by a suitable source of radiation such as infrared (IR) or ultraviolet (UV) light. In one example, the infrared light is generated by a quantum cascade laser.

[0338] At 2454, the processor classifies the cell event as a selected cell event, for example by using the respective waveform. As referred to herein, a selected cell event may include any cell event determined to be a multicell event or a single cell event where the associated single cell is an unwanted cell, such as a Y-cell. Multicell events may be determined using various methods such as having a waveform width over a threshold, having multiple peaks or not being identified as a single cell event. Selected single cell events may be determined using a characteristic of the waveform, such as an integral which is within or above a selected cell threshold.

[0339] At 2456, the processor initiates a sorting action on the cells in the selected cell event by controlling a gate open period during which one or more regular pulses is directed to the microfluidic stream. The gate open period is dependent on the detection of the cell event and the timing of the one or more regular pulses. In some examples, the regular pulses used for sorting may be implemented using a pulse laser, however other implementations are possible as previously described.

[0340] At 2472 the processor detects a cell event within a plurality of microfluidic streams by detecting a rising edge of a waveform followed by a falling edge of the waveform in a received emission signal associated with the microfluidic stream, or more generally by detecting a signal peak which has certain characteristics such as a minimum amplitude. The cell events in different microfluidic streams may be detected at the same or different times. As previously discussed, the emission signals may be generated by detectors 130 arranged about each microfluidic stream carrying cells, the presence of which can be detected in an interrogation area by the characteristic release of scattered light or fluorescent emissions following irradiation of the microfluidic stream by a suitable source of radiation such as infrared (IR) or ultraviolet (UV) light. In one example, the infrared light is generated by a quantum cascade laser. In another example, regular laser pulses may be employed and in some examples these regular laser pulses may also be used for sorted selected cell events. At 2474, the processor classifies the detected cell events as a selected cell event for example by using the respective waveform. As referred to herein, a selected cell event may include any cell event determined to be a multicell event or a single cell event where the associated single cell is an unwanted cell, such as a Y-cell. Multicell events may be determined using various methods such as having a waveform width over a threshold, having multiple peaks or not being identified as a single cell event. Selected single cell events may be determined using a characteristic of the waveform, such as an integral which is within or above a selected cell threshold.

[0341] At 2476, the processor initiates sorting actions on the cells in the selected cell events in the plurality of microfluidic streams. This may be implemented using regular laser pulses from a single source and which are split into a plurality of beams each associated with a respective sorting apparatus for the different microfluidic streams. This allows sorting in parallel of cells in multiple microfluidic streams. In one example, this may be implemented by controlling a gate open period for multiple microfluidic streams and during which one or more regular pulses is directed to the respective microfluidic stream. The gate open period for each microfluidic stream is dependent on the detection of the cell event for the respective microfluidic stream and the timing of the one or more regular pulses.

[0342] FIG 25 illustrates a sorting apparatus according to an example for sorting cells within a microfluidic stream using a pulsed sorting arrangement which generates regular pulses. In this example, the sorting apparatus 2500 comprises a pulsed laser 2505 which generates regular laser pulses. The laser pulses are directed to an optical switch or gate 2510 which is controlled or switched to direct incoming laser pulses into or away from the microfluidic stream (or at least cells within the microfluidic stream) - the switch or gate is open when directing laser pulses into the microfluidic stream and closed when not directing laser pulses into the microfluidic stream.

[0343] The optical switch 2510 is also controlled to direct the incoming laser pulses away from the microfluidic stream, or at least away from cells flowing in the microfluidic stream - the switch or gate is closed. Control is achieved using a gate open request during a selection period during which the optical switch is requested or controlled to open. In this example, the optical switch is an AOM which switches between directing the laser pulses or beam in one direction towards the microfluidic stream (open position with 1storder beam shown at 2515) and in another direction away from the microfluidic stream (closed position with 0thorder beam as shown at 2515).

[0344] The inventors have found that a beam focusing device (not shown) may be used to narrow the laser pulses into the optical switch for better control by the optical switch. A beam expander (not shown) between the optical switch and microfluidic stream may be used to expand the laser pulses for better sorting interaction with cells in the microfluidic stream.

[0345] The sorting apparatus 2500 also comprises a controller 2520, a sync circuit 2525 and an optical switch driver 2530. The optical switch driver 2530 may comprise a Radio Frequency (RF) driver which, when operating, causes the AOM to deflect incoming laser pulses. The sync circuit 2525 may be implemented using a photodiode or other photodetector positioned to intercept part of the laser pulses, for example using a splitter which directs a low power beam of the laser pulse to the photodetector. The sync circuit 2525 may be implemented with an AND gate which receives inputs from the controller 2520 and the photodetector. When both a gate open request from the controller is ON and a laser pulse detected signal from the photodetector is ON, the AND gate will output an ON gate open request which is sent to the RF driver 2530 to generate RF driver signals to OPEN the optical switch. This implementation corresponds to the control approach of FIG 19 where the sync circuit overrides the gate open request. Alternative circuit architectures may be used to implement this control approach. In other examples, the output from the sync circuit 2525 may simply be the output signal from the photodetector which is fed to the controller 2520 as an input to control generation of a gate open request which is output directly to the RF driver.

[0346] The controller 2520 may be configured to implement one of the control strategies already discussed with respect to FIG 16-19; and receives a sort signal as a trigger to sort a selected cell event, based on a cell event detection and classification.

[0347] Example

[0348] The following example illustrates embodiments of the invention.

[0349] Aim

[0350] To compare the effect of different cell targeting techniques on the overall enrichment of preferred cells, while taking into account the viability and motility of collected cells.

[0351] Methodology

[0352] Sperm cells were collected from a bull and stained using Hoechst-33342 according to sample preparation and staining conditions described in Garner et al. (2013 - Sex- Sorting Sperm Using Flow Cytometry / Cell Sorting, Methods in molecular biology (Clifton, N .), Vo. 927, pages 279-295). The cells passed through a microfluidic focusing apparatus then passed in front of a UV interrogation laser (~355nm). A photomultiplier tube detector collected the cellular fluorescence emissions from each cell and these were converted to digital signals and passed to a signal processor. The signal processor determined waveforms which exceeded an event threshold and the integral of each waveform was calculated. Cells which exceeded a cell selection threshold were selected for deactivation using a sorting beam. The following sorting strategies were compared:

[0353] 1. Statically timed selection triggering (STST) - following positive selection of a single cell event or multi-cell event, a single laser pulse was fired at the cell using a constant offset delay timed to coincide with the cell or cells passing the focal point of the sorting beam. This mode cannot discriminate between single or multi-cell events.

[0354] 2. Adaptively timed selection triggering (ATST) - following positive selection of a cell-event or multi-cell event, the laser was triggered for a time t corresponding to the width of the waveform using a constant offset delay timed to coincide with the cell passing the focal point of the sorting beam.

[0355] 3. Adaptively timed adaptive selection triggering (ATAST) - following positive selection of a cell-event or multi-cell event, the laser was triggered after an adjusted offset delay (OD) (Equation 2 above) for a selection period t (Equation 3 above).

[0356] Cells were collected and subjected to a "swim-up" procedure to separate motile from non-motile sperm. This ensures that only cells that were not selected for sorting and deactivated are analysed during enrichment analysis. DNA was lysed, extracted and precipitated according to standard methods. Enrichment was assessed by detection of X-chromosome and Y-chromosome specific genes versus a reference gene.

[0357] Viability of the collected cells was assessed.

[0358] Two trials were conducted. Trial 1 assessed the enrichment of cells not selected for sorting using the three main cell targeting techniques. Trial 2 assessed the enrichment of cells not selected for sorting using the Statically timed selection triggering (STST) and Adaptively timed selection triggering (ATST).

[0359] Trial 1 Results

[0360] Using STST as a reference, enrichment using ATST and ATAST was as follows:

[0361] ATST showed an 8.60% (+ / - 0.98%) increase in X-cell purity.

[0362] ATAST showed a 7.07% (+ / - 1.41%) increase in X-cell purity.

[0363] Viability and motility of the cells processed using the different methods did not exhibit significant differences.

[0364] Trial 2 Results Using STST as a reference, enrichment using ATST was as follows:

[0365] ATST showed a 6.51% (+ / - 1.53%) increase in X-cell purity.

[0366] Viability and motility of the cells processed using the different methods did not exhibit significant differences.

[0367] Conclusions

[0368] Both Adaptively timed selection triggering (ATST) and Adaptively timed adaptive selection triggering (ATAST) resulted in significantly higher X-cell purity compared to STST. This indicates that substantially more cells of the desired type (i.e. those not selected for sorting) were collected.

[0369] Viability and motility of the cells processed using the different methods did not exhibit significant differences indicating that the techniques did not have a detrimental effect on cell health.

[0370] Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety.

[0371] As noted elsewhere, the disclosed inventive examples have been described for illustrative purposes only and are not limiting. Other examples are possible and are covered by the disclosure, which will be apparent from the teachings contained herein. Thus, the breadth and scope of the disclosure should not be limited by any of the abovedescribed examples but should be defined only in accordance with claims supported by the present disclosure and their equivalents. Moreover, examples of the subject disclosure may include methods, systems and apparatuses / devices which may further include any and all elements from any other disclosed methods, systems, and devices, including any and all elements corresponding to binding event determinative systems, devices and methods. In other words, elements from one or another disclosed examples may be interchangeable with elements from other disclosed examples. In addition, one or more features / elements of disclosed examples may be removed and still result in patentable subject matter (and thus, resulting in yet more examples of the subject disclosure). Also, some examples correspond to systems, devices and methods which specifically lack one and / or another element, structure, and / or steps (as applicable), as compared to teachings of the prior art, and therefore, represent patentable subject matter and are distinguishable therefrom (i.e., claims directed to such examples may contain one or more negative limitations to note the lack of one or more features prior art teachings). Various inventive concepts disclosed herein may be embodied as one or more methods (as so noted). The acts performed as part of the method may be ordered in any suitable way. Accordingly, examples may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative examples.

[0372] The following numbered clauses define particular aspects and embodiments contemplated herein: Clauses:

[0373] 1. A method of sorting cells within a microfluidic stream using a pulsed sorting arrangement which generates regular pulses, the method comprising: detecting a cell event within the microfluidic stream; classifying the cell event as a selected cell event; sorting a cell associated with the selected cell event by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream; wherein the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0374] 2. The method of clause 1 : wherein the gate open period includes a switching delay between the one or more regular pulses being directed to the microfluidic stream and the one or more regular pulses being directed away from the microfluidic stream; and wherein the gate open period is controlled to avoid a regular pulse occurring during the switching delay.

[0375] 3. The method of clause 1 or 2, wherein the gate open period is controlled responsive to detection of a regular pulse following the detecting of the cell event.

[0376] 4. The method of clause 1 or 2, wherein the gate open period is controlled dependent on detection of a regular pulse before the detecting the cell event.

[0377] 5. The method of any one preceding clause, comprising starting the gate open period a start delay period after detecting the cell event or classifying the cell event as a selected cell event, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular pulses.

[0378] 6. The method of clause 5: wherein the predetermined delay is dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event; and wherein the variable delay is dependent on detection of a regular pulse following the detecting the cell event.

[0379] 7. The method of clause 6, wherein the variable delay is calculated using a time difference between the classifying the cell event as a selected cell event and detection of a next regular pulse.

[0380] 8. The method of any one preceding clause, wherein the gate open 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 approximately 50% of the interpulse period.

[0381] 9. The method of any one preceding clause, wherein the gate open period is controlled to overlap a single pulse.

[0382] 10. The method of clause 9, wherein the single pulse is timed within a central portion of the gate open 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.

[0383] 11. The method of any one of clauses 1 to 8, wherein the gate open period is controlled to overlap two or more pulses in response to detecting a cell event associated with a plurality of cells.

[0384] 12. The method of clause 11, comprising ending the gate open period an end delay period after detecting the last cell in the cell event or classifying the cell event as a selected cell event, wherein the end delay period is dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event.

[0385] 13. The method of clause 12, wherein the end delay period comprises the transit time less a variable end delay dependent on a switching delay between the one or more regular pulses being directed to the microfluidic stream and the one or more regular pulses being directed away from the microfluidic stream. 14. The method of clause 13, wherein the variable end delay is calculated in response to determining that a next pulse will coincide with the switching delay associated with ending the gate period.

[0386] 15. The method of any one preceding clause, wherein the pulsed sorting arrangement comprises a pulsed laser generating regular laser pulses.

[0387] 16. The method of clause 15, wherein the sorting comprises nudging, deactivating or ablating a cell associated with the selected cell event using a said laser pulse.

[0388] 17. The method of clause 16, wherein the gate open period is associated with an optical switch controlled to switch laser pulses into and away from the microfluidic stream.

[0389] 18. The method of 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.

[0390] 19. The method of any one of clauses 15 to 18, wherein the regular laser pulses are additionally used for sorting a cell associated with a selected cell event in a second microfluidic stream.

[0391] 20. The method of any one preceding clause, wherein the cells are sperm cells.

[0392] 21. A method of sorting cells within a plurality of microfluidic streams using a pulsed laser which generates regular laser pulses, the method comprising: splitting the laser pulses into a plurality of beams each associated with a respective microfluidic stream; detecting a respective cell event within at least two of the plurality of microfluidic streams; separately classifying each of the detected cell events in each respective microfluidic stream as a selected cell event; sorting one or more cells associated with the selected cell event in the at least two microfluidic streams by independently controlling the regular laser pulses of the respective beam into the respective microfluidic stream.

[0393] 22. The method of clause 21, wherein the regular laser pulses are split into the plurality of beams using one or more beam splitters. 23. The method of clause 22, wherein the beam splitter is a polarising beam splitter and the split ratio of the beams is adjusted by adjusting a ratio of light polarised in a first plane to light polarised in a second plane.

[0394] 24. The method of clause 23 wherein the amount of light in the first versus the second plane is adjusted by a polarisation modifier.

[0395] 25. The method of clause 23 or 24 for sorting cells within a plurality of microfluidic streams using at least one beam splitter, the method comprising adjusting the power transfer properties of at least one beam splitter responsive to a cell event property or a beam status for one or more of the microfluidic streams, optionally wherein the cell event property comprises at least one of detection, classification or sorting properties, optionally wherein the beam status comprises at least one of inactivation of detecting, classifying and / or sorting for one or more of the plurality of microfluidic streams.

[0396] 26. The method of any one of clauses 21 to 25, wherein the sorting a cell associated with the selected cell event comprises: controlling a respective gate open period during which one or more of the regular laser pulses in a respective beam is directed to the respective microfluidic stream; wherein the respective gate open period is controlled dependent on the detecting of the cell event for the respective microfluidic stream and the timing of one or more of the regular laser pulses.

[0397] 27 The method of clause 26, wherein the respective gate open period includes a switching delay between the one or more regular laser pulses of the respective beam being directed to the respective microfluidic stream and the one or more regular laser pulses of the respective beam being directed away from the respective microfluidic stream; and wherein the respective gate open period is controlled to avoid a regular laser pulse of the respective beam occurring during the switching delay.

[0398] 28. The method of clause 26 or 27, wherein the respective gate open period is controlled responsive to detection of a regular laser pulse following the detecting of the cell event in the respective microfluidic stream.

[0399] 29. The method of clause 26 or 27, wherein the respective gate open period is controlled dependent on detection of a regular laser pulse before the detecting the cell event.

[0400] 30. The method of any one of clauses 26 to 29, comprising: starting the respective gate open period a start delay period after detecting the cell event in the respective microfluidic stream or classifying the cell event as a selected cell event for the respective microfluidic stream, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular laser pulses.

[0401] 31. The method of clause 30: wherein the predetermined delay is dependent on a transit time for a cell in the respective microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event in the respective microfluidic stream and the sorting location corresponding to the sorting a cell associated with the cell event of the respective microfluidic stream; and wherein the variable delay is dependent on detection of a regular laser pulse following the detecting the cell event.

[0402] 32. The method of clause 31, wherein the variable delay is calculated using a time difference between the classifying the cell event as a selected cell event for the respective microfluidic stream and detection of a next regular laser pulse.

[0403] 33. The method of any one of clauses 26 to 32, wherein the respective gate open 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 approximately 50% of the inter-pulse period.

[0404] 34. The method of any one of clauses 26 to 33, wherein the respective gate open period is controlled to overlap a single laser pulse.

[0405] 35. The method of clause 34, wherein the single laser pulse is timed within a central portion of the respective gate open period, the central portion comprising one of the following: the middle 80% of the respective gate period: the middle 50% of the respective gate period; the middle of the respective gate period.

[0406] 36. The method of any one of clauses 26 to 33, wherein the respective gate open period is controlled to overlap two or more laser pulses in response to detecting a cell event associated with a plurality of cells in the respective microfluidic stream.

[0407] 37. The method of clause 36, comprising ending the respective gate open period an end delay period after detecting the last cell in the cell event or classifying the cell event as a selected cell event in the respective microfluidic stream, wherein the end delay period is dependent on a transit time for a cell in the respective microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event in the respective microfluidic stream and the sorting location corresponding to the sorting a cell associated with the cell event in the respective microfluidic stream.

[0408] 38. The method of clause 39, wherein the end delay period comprises the transit time less a variable end delay dependent on a switching delay between the one or more regular laser pulses of the respective beam being directed to the respective microfluidic stream and the one or more regular laser pulses being directed away from the respective microfluidic stream.

[0409] 39. The method of clause 38, wherein the variable end delay is calculated in response to determining that a next laser pulse will coincide with the switching delay associated with ending the respective gate period.

[0410] 40. The method of any one of clauses 26 to 39, wherein the sorting comprises nudging, deactivating or ablating a cell associated with the selected cell event using a said laser pulse.

[0411] 41. The method of any one of clauses 26 to 40, wherein the respective gate open period is associated with an optical switch controlled to switch laser pulses into and away from the respective microfluidic stream.

[0412] 42. The method of 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.

[0413] 43. The method of any one of clauses 21 to 42, wherein the cells are sperm cells.

[0414] 44. A sorting apparatus for sorting cells within a microfluidic stream using a pulsed sorting arrangement which generates regular pulses, the apparatus comprising: a pulsed sorting arrangement which generates regular pulses; a detection means for detecting a cell event within the microfluidic stream; a classifying means for classifying the cell event as a selected cell event; a sorting means for sorting a cell associated with the selected cell event by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream; wherein the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0415] 45. The sorting apparatus of clause 44: wherein the gate open period includes a switching delay between the one or more regular pulses being directed to the microfluidic stream and the one or more regular pulses being directed away from the microfluidic stream; and wherein the gate open period is controlled to avoid a regular pulse occurring during the switching delay.

[0416] 46. The sorting apparatus of clause 44 or 45, wherein the gate open period is controlled responsive to detection of a regular pulse following the detecting of the cell event.

[0417] 47. The sorting apparatus of clause 44 or 45, wherein the gate open period is controlled dependent on detection of a regular pulse before the detecting the cell event.

[0418] 48. The sorting apparatus of any one of clauses 44 to 47, configured to start the gate open period a start delay period after detecting the cell event or classifying the cell event as a selected cell event, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular pulses.

[0419] 49. The sorting apparatus of clause 48: wherein the predetermined delay is dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event; and wherein the variable delay is dependent on detection of a regular pulse following the detecting the cell event.

[0420] 50. The sorting apparatus of clause 49, wherein the variable delay is calculated using a time difference between the classifying the cell event as a selected cell event and detection of a next regular pulse.

[0421] 51. The sorting apparatus of any one of clauses 44 to 50, wherein the gate open 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%, or approximately 50% of the inter-pulse period.

[0422] 52. The sorting apparatus of any one of clauses 44 to 51, wherein the gate open period is controlled to overlap a single pulse.

[0423] 53. The sorting apparatus of clause 52, wherein the single pulse is timed within a central portion of the gate open 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.

[0424] 54. The sorting apparatus of any one of clauses 44 to 51, wherein the gate open period is controlled to overlap two or more pulses in response to detecting a cell event associated with a plurality of cells.

[0425] 55. The sorting apparatus of clause 54, configured to end the gate open period an end delay period after detecting the last cell in the cell event or classifying the cell event as a selected cell event, wherein the end delay period is dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event.

[0426] 56. The sorting apparatus of clause 55, wherein the end delay period comprises the transit time less a variable end delay dependent on a switching delay between the one or more regular pulses being directed to the microfluidic stream and the one or more regular pulses being directed away from the microfluidic stream.

[0427] 57. The sorting apparatus of clause 56, wherein the variable end delay is calculated in response to determining that a next pulse will coincide with the switching delay associated with ending the gate period.

[0428] 58. The sorting apparatus of any one of clauses 44 to 57, wherein the pulsed sorting arrangement comprises a pulsed laser generating regular laser pulses.

[0429] 59. The sorting apparatus of clause 58, wherein the sorting comprises nudging, deactivating or ablating a cell associated with the selected cell event using a said laser pulse.

[0430] 60. The sorting apparatus clause 58 or 59, comprising an optical switch controlled to switch laser pulses into and away from the microfluidic stream dependent on the gate open period.

[0431] 61. The sorting apparatus of clause 60, wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electrooptic deflector or an electro-optic modulator.

[0432] 62. The sorting apparatus of clause 61, wherein the acousto-optic modulator comprises a Tellurium Dioxide crystal or a Silicon Dioxide crystal.

[0433] 63. The sorting apparatus of any one of clauses 60 to 62, comprising one or more of: a beam narrower arranged to narrow the regular laser pulses incident on the optical switch; and a beam expander arranged to expand laser pulses directed to the microfluidic stream.

[0434] 64. The sorting apparatus of any one of clauses 58 to 63, wherein the pulsed laser is configured to generate regular laser pulses at a predetermined rate between Ips and 1ms.

[0435] 65. The sorting apparatus of any one of clauses 58 to 64, comprising a photodetector for detection of a regular laser pulse.

[0436] 65a. The sorting apparatus of clause 65, wherein the photodetector comprises a photodiode, preferably an avalanche photodiode.

[0437] 66. The sorting apparatus of any one of clauses 58 to 65a, wherein the regular laser pulses are additionally used for sorting a cell associated with a selected cell event in a second microfluidic stream.

[0438] 67. The sorting apparatus of any one of clauses 44 to 66, configured for sorting sperm cells.

[0439] 68. A sorting apparatus for sorting cells within a plurality of microfluidic streams using a pulsed laser which generates regular laser pulses, the apparatus comprising: a pulsed laser which generates regular laser pulses; a beam splitter for splitting the laser pulses into a plurality of beams each associated with a respective microfluidic stream; one or more detection means for detecting a cell event within respective microfluidic streams; one or more classifying means for classifying the cell event in respective microfluidic streams as a selected cell event; respective sorting means for sorting the selected cell event in respective microfluidic streams using the regular laser pulses.

[0440] 69. The sorting apparatus of clause 68, wherein the beam splitter is a polarising beam splitter and the split ratio of the beams is adjusted by adjusting a ratio of light polarised in a first plane to light polarised in a second plane. 70. The sorting apparatus of clause 68 or 69, further comprising at least one of a polarisation modifier arranged to adjust the amount of light in the first versus the second plane, a power adjustment means, a half wave plate or a pockels cell.

[0441] 71. The sorting apparatus of any one of clauses 68 to 70, comprising at least one beam splitters for sorting cells within the plurality of microfluidic streams, the sorting apparatus configured to adjust the power transfer of at least some of the beam splitters responsive to a cell event property or a beam status for one or more of the microfluidic streams, optionally wherein the cell event property comprises at least one of detection, classification or sorting properties, optionally wherein the beam status comprises at least one of inactivation of detecting, classifying and / or sorting for one or more of the microfluidic streams.

[0442] 72. The sorting apparatus of any one of clauses 68 to 71, wherein the respective sorting means are configured to: control a respective gate open period during which one or more of the regular laser pulses in a respective beam is directed to the respective microfluidic stream; wherein the respective gate open period is controlled dependent on the detecting of the cell event for the respective microfluidic stream and the timing of one or more of the regular laser pulses.

[0443] 73. The sorting apparatus of clause 72, wherein the respective gate open period includes a switching delay between the one or more regular laser pulses of the respective beam being directed to the respective microfluidic stream and the one or more regular laser pulses of the respective being directed away from the respective microfluidic stream; and wherein the respective gate open period is controlled to avoid a regular laser pulse of the respective beam occurring during the switching delay.

[0444] 74. The sorting apparatus of clause 72 or 73, wherein the respective gate open period is controlled responsive to detection of a regular laser pulse following the detecting of the cell event in the respective microfluidic stream.

[0445] 75. The sorting apparatus of clause 72 or 73, wherein the respective gate open period is controlled dependent on detection of a regular laser pulse before the detecting the cell event.

[0446] 76. The sorting apparatus of any one of clauses 72 to 75, comprising starting the respective gate open period a start delay period after detecting the cell event in the respective microfluidic stream or classifying the cell event as a selected cell event for the respective microfluidic stream, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular laser pulses.

[0447] 77. The sorting apparatus of clause 76: wherein the predetermined delay is dependent on a transit time for a cell in the respective microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event in the respective microfluidic stream and the sorting location corresponding to the sorting a cell associated with the cell event of the respective microfluidic stream; and wherein the variable delay is dependent on detection of a regular laser pulse following the detecting the cell event.

[0448] 78. The sorting apparatus of clause 77, wherein the variable delay is calculated using a time difference between the classifying the cell event as a selected cell event for the respective microfluidic stream and detection of a next regular laser pulse.

[0449] 79. The sorting apparatus of any one of clauses 72 to 78, wherein the respective gate open 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 approximately 50% of the inter-pulse period.

[0450] 80. The sorting apparatus of any one of clauses 72 to 79, wherein the respective gate open period is controlled to overlap a single laser pulse.

[0451] 81. The sorting apparatus of clause 80, wherein the single laser pulse is timed within a central portion of the respective gate open period, the central portion comprising one of the following: the middle 80% of the respective gate period: the middle 50% of the respective gate period; the middle of the respective gate period.

[0452] 82. The sorting apparatus of any one of clauses 72 to 81, wherein the respective gate open period is controlled to overlap two or more laser pulses in response to detecting a cell event associated with a plurality of cells in the respective microfluidic stream.

[0453] 83. The sorting apparatus of clause 82, configured to end the respective gate open period an end delay period after detecting the last cell in the cell event or classifying the cell event as a selected cell event in the respective microfluidic stream, wherein the end delay period is dependent on a transit time for a cell in the respective microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event in the respective microfluidic stream and the sorting location corresponding to the sorting a cell associated with the cell event in the respective microfluidic stream.

[0454] 84. The sorting apparatus of clause 83, wherein the end delay period comprises the transit time less a variable end delay dependent on a switching delay between the one or more regular laser pulses of the respective beam being directed to the respective microfluidic stream and the one or more regular laser pulses being directed away from the respective microfluidic stream.

[0455] 85. The sorting apparatus of clause 84, wherein the variable end delay is calculated in response to determining that a next laser pulse will coincide with the switching delay associated with ending the respective gate period.

[0456] 86. The sorting apparatus of any one of clauses 72 to 85, wherein the sorting comprises nudging, deactivating or ablating a cell associated with the selected cell event using a said laser pulse.

[0457] 87. The sorting apparatus of any one of clauses 72 to 86, comprising an optical switch controlled to switch laser pulses into and away from the microfluidic stream dependent on the respective gate open period.

[0458] 88. The sorting apparatus of clause 87, wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electrooptic deflector or an electro-optic modulator.

[0459] 89. The sorting apparatus of clause 88, wherein the acousto-optic modulator comprises a Tellurium Dioxide crystal or a Silicon Dioxide crystal.

[0460] 90. The sorting apparatus of any one of clauses 87 to 89, comprising one or more of: a beam narrower arranged to narrow the regular laser pulses incident on the optical switch; and a beam narrower arranged to expand laser pulses directed to the microfluidic stream.

[0461] 91. The sorting apparatus of any one of clauses 72 to 90, comprising a photodetector for detection of a regular laser pulse.

[0462] 92. The sorting apparatus of any one of clauses 68 to 91, wherein the pulsed laser is configured to generate regular laser pulses at a predetermined rate between 1ms and Ips.

[0463] 93. The sorting apparatus of any one of clauses 68 to 92, configured for sorting sperm cells. 94. A computer program comprising processor instructions which when executed by a processor cause the processor to carry out the method of any one of clauses 1 to 43.

[0464] 95. A method of timing the sorting of cells within a microfluidic stream, the method comprising: detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; sorting one or more cells in the selected cell event over a selection period which is dependent on a waveform width of the waveform associated with the selected cell event.

[0465] 96. The method of clause 95, wherein the selected cell event comprises unwanted cells.

[0466] 97. The method of clause 95 or 96, wherein the cell emission signal is a fluorescence signal.

[0467] 98. The method of any one clauses 95 to 97, wherein the sorting comprises deactivating the one or more cells in the selected cell event.

[0468] 99. The method of any one of clauses 95 to 98, wherein a duration of the selection period is dependent on the waveform width.

[0469] 100. The method of any one clauses 95 to 99, where the selection period begins after an offset delay following the end of the waveform.

[0470] 101. The method of clause 100, wherein the offset delay is dependent on the waveform width.

[0471] 102. The method of clause 101, wherein the offset delay is determined according to one or more of the following:

[0472] OD = POD1 - WW;

[0473] OD = POD2 - xWW - b; where OD is the determined offset delay, POD1 and POD2 are assignable predetermined offset delays, WW is the waveform width, x is an assignable coefficient and b is an assignable constant.

[0474] 103. The method of clause 102, wherein the selection period is determined according to: SP = xWW + b; where SP is the selection period.

[0475] 104. The method of any one of clauses 95 to 100, wherein the sorting comprises applying a laser beam to the microfluidic stream during the selection period to deactivate cells within the selected cell event.

[0476] 105. The method of clause 104, wherein the laser is applied in one or more discrete pulses having a shorter duration than the selection period.

[0477] 106. The method of clause 105, wherein the laser is configured to generate pulses periodically and the selection period is dependent on the frequency of pulses applied to the microfluidic stream.

[0478] 107. The method of any one of clauses 104 to 106, comprising using an acousto-optic modulator to apply the laser to the microfluidic stream.

[0479] 108. The method of any one of clauses 104 to 108, wherein the laser is a picosecond laser.

[0480] 109. The method of any one preceding of clauses 95 to 108, wherein the sorting comprising controlling one or more gate open periods during the selection period, wherein during a said gate open period one or more regular pulses is directed to the microfluidic stream and wherein the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0481] 110. The method of any one of clauses 95 to 109, wherein classifying a cell event as a selected cell event using the respective waveform comprises determining at least one of the following: a. the respective waveform has a waveform width exceeding a multicell event threshold; and b. the respective waveform has a characteristic corresponding to a selected cell.

[0482] 111. The method of any one of clauses 95 to 110, wherein the waveform width is determined by detecting a rising edge followed by a falling edge of the waveform in a cell emission signal received from the microfluidic stream and associated with the cell event.

[0483] 112. The method of clause 111, wherein the detected rising edge corresponds to the received emission signal rising above a rising edge threshold above a baseline. 113. The method of clause 111 or 112, wherein the detected falling edge corresponds to the received emission signal falling more than a falling edge threshold.

[0484] 114. The method of clause 113, wherein the falling edge threshold is a predetermined percentage of the average height of the respective waveform or a series of waveforms.

[0485] 115. The method of any one of clauses 95 to 114, wherein the waveform width is determined by detecting a peak in the waveform and detecting a preceding rising edge or a following falling edge of the waveform, and using the duration between the peak and the preceding rising edge or the following falling edge to determine the waveform width.

[0486] 116. The method of 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.

[0487] 117. The method of any one of clauses 95 to 117, wherein the cells are sperm cells and the selected cell event comprises at least one of the following: a sperm cell classified as a Y sperm cell; a multicell event comprising a plurality of sperm cells.

[0488] 118. The method of any one of clauses 95 to 117, comprising determining a z-axis of a cell in a said cell event using the waveform of the cell event.

[0489] 119. The method of clause 118, wherein classifying the said cell event as a selected cell event is dependent on said z-axis orientation of the cell in the said cell event.

[0490] 120. The method of clause 118 or 119, wherein determining the z-axis orientation of the cell comprises identifying a first maxima in the waveform which corresponds to a first part of the cell and identifying a second maxima in said waveform which corresponds to a second part of the cell, and wherein the z-axis orientation of the cell is determined based on the order of the first and the second maxima within the waveform.

[0491] 121. The method of clause 120, wherein the first and second maxima have different amplitudes, and wherein the z-axis orientation of the cell is determined based whether the amplitude of first maxima is greater than the amplitude of the second maxima.

[0492] 122. The method of clause 121, wherein the maxima having a greater amplitude corresponds to a head of a sperm cell and the maxima having a smaller amplitude corresponds to a mid-piece of the sperm cell.

[0493] 123. The method of 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 maxima correspond to respective local maximum signal values in the series of signal values.

[0494] 124. The method of any one of clauses 120to 123, wherein the signal values of the series of signal values between the two maxima are above 50% of the signal value of the first maxima.

[0495] 125. The method of any one of clauses 120 to 124, wherein the cell is a sperm cell and the first part of the cell is a head of the sperm cell and the second part of the cell is a mid-piece of the sperm cell.

[0496] 126. A method of determining a z-axis orientation of a cell within a microfluidic stream, the method comprising: detecting a cell within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell; determining a z-axis orientation of a cell by identifying a first maxima in the waveform which corresponds to a first part of the cell and identifying a second maxima in said waveform which corresponds to a second part of the cell; wherein the z-axis orientation of the cell is determined based on the order of the first and the second maxima within the waveform.

[0497] 127. The method of clause 126, wherein the first and second maxima have different amplitudes, and wherein the z-axis orientation of the cell is determined based whether the amplitude of first maxima is greater than the amplitude of the second maxima.

[0498] 128. The method of clause 127, wherein the maxima having a greater amplitude corresponds to a head of a sperm cell and the maxima having a smaller amplitude corresponds to a mid-piece of the sperm cell.

[0499] 129. The method of 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 maxima correspond to respective local maximum signal values in the series of signal values.

[0500] 130. The method of any one of clauses 126 to 129, wherein the signal values of the series of signal values between the two maxima are above 50% of the signal value of the first maxima.

[0501] 131. A method of adjusting an offset delay and / or a selection period for sorting cells within a microfluidic stream, the method comprising: detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; sorting one or more cells in the selected cell event over the selection period which follows the selected cell event by the offset delay; wherein the selection period and / or the offset delay is adjusted dependent on a waveform width of the waveform associated with the selected cell event.

[0502] 132. An apparatus for adjusting an offset delay and / or a selection period for sorting cells within a microfluidic stream, the apparatus comprising a processor and memory configured to: detect a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classify the cell event as a selected cell event using the waveform; control a sorting means to sort one or more cells in the selected cell event over the selection period which follows the selected cell event by the offset delay; wherein the selection period and / or the offset delay is adjusted dependent on a waveform width of the waveform associated with the selected cell event.

[0503] 133. The method of claim 132, wherein the sorting comprising controlling one or more gate open periods during the selection period, wherein during a said gate open period one or more regular pulses is directed to the microfluidic stream and wherein the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

[0504] 134. A computer program comprising processor instructions which when executed by a processor cause the processor to carry out the method of any one of clauses 95 to 133.

[0505] 135. An apparatus for timing the sorting of cells within a microfluidic stream, the apparatus comprising a processor and memory configured to: detect a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classify the cell event as a selected cell event using the waveform; control a sorting means to sort one or more cells in the selected cell event over a selection period which is dependent on a waveform width of the waveform associated with the selected cell event. 136. The apparatus of clause 135, wherein a duration of the selection period is dependent on the waveform width.

[0506] 137. The apparatus of clause 135 or 136, where the selection period begins after an offset delay following the end of the waveform.

[0507] 138. The apparatus of clause 137, wherein the offset delay is dependent on the waveform width.

[0508] 139. The apparatus of any one of clauses 135 to 138, 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 stream and associated with the cell event.

[0509] 140. An apparatus for processing cells within a microfluidic stream, the apparatus comprising a processor and memory configured to: detect cell events within the microfluidic stream using a received emission signal associated with the microfluidic stream; classify the cell events as unselected or selected cell events; control a sorting means to sort one or more cells in a selected cell event over a selection period which is dependent on an end of the selected cell event and a duration of the selected cell event.

[0510] 141. The apparatus of any one of clauses 135 to 140, comprising: means for directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; means for directing sorting electromagnetic radiation at selected cells within the microfluidic stream.

[0511] 142. The apparatus clause 141, comprising an optical component used for directing the interrogating electromagnetic radiation and / or the sorting electromagnetic radiation; wherein the optical component is adjustable dependent on a characteristic of the waveform.

[0512] 143. The apparatus of clause 142, wherein the position of the optical component relative to the microfluidic stream is adjustable.

[0513] 144. The apparatus of clause 142 or 143, wherein the characteristic of the waveform comprises one or more of the following characteristics of a plurality of waveforms associated with a respective plurality of cell events: a maximum waveform intensity; a maximum waveform width; an integral of the plurality of waveforms.

[0514] 145. The apparatus of any one of clauses 135 to 144, comprising: means for delivering the microfluidic stream; wherein the means for delivering the microfluidic stream is adjustable to varying a path of the microfluidic stream dependent on a characteristic of the waveform.

[0515] 146. The apparatus of clause 145, wherein the means for delivering the microfluidic stream is a microfluidic chip.

[0516] 147. The apparatus of clause 145 or 146, wherein the characteristic of the waveform comprises one or more of the following characteristics of a plurality of waveforms associated with a respective plurality of cell events: a maximum waveform intensity; a maximum waveform width; an integral of the plurality of waveforms.

[0517] 148. An apparatus for processing cells within a microfluidic stream, the apparatus comprising: means for detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; means for directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; means for directing sorting electromagnetic radiation at selected cells within the microfluidic stream; an optical component used for directing the interrogating electromagnetic radiation and / or the sorting electromagnetic radiation; wherein the optical component is adjustable dependent on a characteristic of the waveform.

[0518] 149. The apparatus of clause 148, wherein the optical component is a laser for generating the sorting electromagnetic radiation and wherein the power of the laser is adjustable dependent on the characteristic of the waveform.

[0519] 150. The apparatus of clause 149, wherein the position of the optical component relative to the microfluidic stream is adjustable.

[0520] 151. The apparatus of clause 149 or 150, wherein the characteristic of the waveform comprises one or more of the following: a maximum waveform intensity; a maximum waveform width; an integral of the waveform; a shape of the waveform; a slope of the waveform; a number of peaks of the waveform.

[0521] 152. An apparatus for processing cells within a microfluidic stream, the apparatus comprising: means for delivering the microfluidic stream; means for detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; means for directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; means for directing sorting electromagnetic radiation at selected cells within the microfluidic stream; wherein the means for delivering the microfluidic stream is adjustable to varying a path of the microfluidic stream dependent on a characteristic of the waveform.

[0522] 153. The apparatus of clause 152, wherein the means for delivering the microfluidic stream is a microfluidic chip.

[0523] 154. The apparatus of clause 152 or 153, wherein the characteristic of the waveform comprises one or more of the following characteristics of a plurality of waveforms associated with a respective plurality of cell events: a maximum waveform intensity; a maximum waveform width; an integral of the plurality of waveforms.

Claims

EXEMPLARY CLAIMS OF THE DISCLOSURE:

1. A method of adjusting an offset delay and / or a selection period for sorting cells within a microfluidic stream, the method comprising: detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classifying the cell event as a selected cell event using the waveform; sorting one or more cells in the selected cell event over the selection period which follows the selected cell event by the offset delay; wherein the selection period and / or the offset delay is adjusted dependent on a waveform width of the waveform associated with the selected cell event.

2. The method of any one preceding claim, wherein the sorting comprises deactivating the one or more cells in the selected cell event.

3. The method of claim 1, where the selection 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 assignable predetermined offset delays, WW is the waveform width, x is an assignable coefficient and b is an assignable constant, and / or wherein the selection period is determined according to:SP = xWW + b; where SP is the selection period.

5. The method of any one of claims 1 to 4, wherein: the sorting comprises applying a laser beam to the microfluidic stream during the selection period to deactivate cells within the selected cell event, and wherein the laser is applied in one or more discrete pulses having a shorter duration than the selection period, and / orthe sorting comprises controlling one or more gate open periods during the selection period, wherein during a said gate open period one or more regular pulses is directed to the microfluidic stream and wherein the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

6. The method of any one of claims 1 to 5, wherein classifying a cell event as a selected cell event using the respective waveform comprises determining at least one of the following: a. the respective waveform has a waveform width exceeding a multicell event threshold; and b. the respective waveform has a characteristic corresponding to a selected cell.

7. The method of any one of claims 1 to 6, wherein the waveform width is determined by at least one of: a. detecting a rising edge followed by a falling edge of the waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; and b. detecting a peak in the waveform and detecting a preceding rising edge or a following falling edge of the waveform, and using the duration between the peak and the preceding rising edge or the following falling edge to determine the waveform width, orc) detecting a peak in the waveform and a predetermined duration before and / or after the peak.

8. The method of any one of claims 1 to 7, wherein the cells are sperm cells and the selected cell event comprises at least one of the following: a sperm cell classified as a Y sperm cell; a multicell event comprising a plurality of sperm cells.

9. The method of claim 8, comprising determining a z-axis orientation of a cell in a said cell event using the waveform of the cell event, and wherein classifying the said cell event as a selected cell event is dependent on said z-axis orientation of the cell in the said cell event.

10. The method of claim 10 or 9, wherein determining the z-axis orientation of the cell comprises identifying a first maxima in the waveform which corresponds to a first part of the cell and identifying a second maxima in said waveform which corresponds toa second part of the cell, and wherein the z-axis orientation of the cell is determined based on the order of the first and the second maxima within the waveform.

11. The method of claim 10, wherein: the first and second maxima have different amplitudes, and wherein the z-axis orientation of the cell is determined based whether the amplitude of first maxima is greater than the amplitude of the second maxima; and / or the maxima having a greater amplitude corresponds to a head of a sperm cell and the maxima having a smaller amplitude corresponds to a mid-piece of the sperm cell; and / or the waveform in the cell emission signal comprises a series of signal values above a signal value baseline, and wherein the two maxima correspond to respective local maximum signal values in the series of signal values.

12. A method of determining a z-axis orientation of a cell within a microfluidic stream, the method comprising: detecting a cell within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell; determining a z-axis orientation of a cell by identifying a first maxima in the waveform which corresponds to a first part of the cell and identifying a second maxima in said waveform which corresponds to a second part of the cell; wherein the z-axis orientation of the cell is determined based on the order of the first and the second maxima within the waveform.

13. An apparatus for timing the sorting of cells within a microfluidic stream, the apparatus comprising a processor and memory configured to: detect a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; classify the cell event as a selected cell event using the waveform; control a sorting means to sort one or more cells in the selected cell event over a selection period which is dependent on a waveform width of the waveform associated with the selected cell event.

14. The apparatus of claim 13, wherein a duration of the selection period is dependent on the waveform width, and / or wherein the selection period begins after anoffset delay following the end of the waveform, wherein optionally the offset delay is dependent on the waveform width.

15. The apparatus of claims 13 or 14, 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 stream and associated with the cell event.

16. The apparatus of any one of claims 13 to 15, comprising: interrogating means for directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; sorting means for directing sorting electromagnetic radiation at selected cells within the microfluidic stream; wherein the sorting means and / or the interrogating means comprising an optical component used for directing the interrogating electromagnetic radiation and / or the sorting electromagnetic radiation; wherein the optical component is adjustable dependent on a characteristic of the waveform.

17. The apparatus of claim 16, wherein the characteristic of the waveform comprises one or more of the following characteristics of a plurality of waveforms associated with a respective plurality of cell events: a maximum waveform intensity; a maximum waveform width; an integral of the waveform; the number of peaks of the waveform; the shape of the waveforms.

18. The apparatus of any one of claims 13 to 17, comprising: means for delivering the microfluidic stream; wherein the means for delivering the microfluidic stream is adjustable to varying a path of the microfluidic stream dependent on a characteristic of the waveform.

19. An apparatus for processing cells within a microfluidic stream, the apparatus comprising: means for detecting a cell event within the microfluidic stream using a waveform in a cell emission signal received from the microfluidic stream and associated with the cell event; means for directing interrogating electromagnetic radiation at cells within the microfluidic stream to promote responsive emission signals from the cells; means for sorting the selected cells within the microfluidic stream;an optical component used for directing the interrogating electromagnetic radiation; wherein the optical component is adjustable dependent on a characteristic of the waveform.

20. The apparatus of claim 19, wherein the means for sorting comprises means for directing sorting electromagnetic radiation at the selected cells, and wherein the optical component is used for directing the sorting electromagnetic radiation.

21. The apparatus of claim 20, wherein the optical component is a laser for generating the sorting electromagnetic radiation and wherein the power of the laser is adjustable dependent on the characteristic of the waveform.

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

23. A method of sorting cells within a microfluidic stream using a pulsed sorting arrangement which generates regular pulses, the method comprising: detecting a cell event within the microfluidic stream; classifying the cell event as a selected cell event; sorting a cell associated with the selected cell event by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream; wherein the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

24. The method of claim 23: wherein the gate open period includes a switching delay wherein the gate open period is controlled to avoid a regular pulse occurring during the switching delay; optionally wherein the gate open period occurs as part of the selection period of any one of claims 1 to 8.

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

26. The method of any one of claims 23 to 25, wherein the gate open period is controlled responsive to detection of a regular pulse following the detecting of the cell event, or wherein the gate open period is controlled dependent on detection of a regular pulse before the detecting the cell event.

27. The method of any one of claims 23 to 26, comprising starting the gate open period a start delay period after detecting the cell event or classifying the cell event as a selected cell event, wherein the start delay period comprises a predetermined delay and a variable delay, wherein the variable delay is dependent on the timing of the one or more of the regular pulses.

28. The method of claim 27: wherein the predetermined delay is dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event; and wherein the variable delay is dependent on detection of a regular pulse following the detecting the cell event; wherein optionally the variable delay is calculated using a time difference between the classifying the cell event as a selected cell event and detection of a next regular pulse.

29. The method of any one of claims 23 to 28, wherein: the gate open 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 approximately 50% of the inter-pulse period; and / or the gate open period is controlled to overlap a single pulse, and wherein the single pulse is timed within a central portion of the gate open 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.

30. The method of any one of claims 23 to 29, wherein the gate open period is controlled to overlap two or more pulses in response to detecting a cell event associated with a plurality of cells; and optionally comprising ending the gate open period an end delay period after detecting the last cell in the cell event or classifying the cell event as a selected cell event, wherein the end delay period is dependent on a transit time for a cell in the microfluidic stream to travel between a detecting location and a sorting location, thedetecting location corresponding to the detecting a cell event and the sorting location corresponding to the sorting a cell associated with the cell event; and optionally wherein the end delay period comprises the transit time less a variable end delay dependent on a switching delay corresponding to a time required for a switching device to switch the one or more regular pulses from being directed to the microfluidic stream to the one or more regular pulses being directed away from the microfluidic stream, or vice versa; and optionally wherein the variable end delay is calculated in response to determining that a next pulse will coincide with the switching delay associated with ending the gate period.

31. The method of any one of claims 23 to 30, wherein the pulsed sorting arrangement comprises a pulsed laser generating regular laser pulses; and wherein optionally the sorting comprises nudging, deactivating or ablating a cell associated with the selected cell event using a said laser pulse.

32. The method of claim 31, wherein the gate open period is associated with an optical switch controlled to switch laser pulses into and away from the microfluidic stream; 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; and optionally wherein the regular laser pulses are additionally used for sorting a cell associated with a selected cell event in a second microfluidic stream.

33. A method of sorting cells within a plurality of microfluidic streams using a pulsed laser which generates regular laser pulses, the method comprising: splitting the laser pulses into a plurality of beams each associated with a respective microfluidic stream; detecting a respective cell event within at least two of the plurality of microfluidic streams; separately classifying each of the detected cell events in each respective microfluidic stream as a selected cell event; sorting one or more cells associated with the selected cell event in the at least two microfluidic streams by independently controlling the regular laser pulses of the respective beam into the respective microfluidic stream.

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

35. The method of claim 34, wherein the beam splitter is a polarising beam splitter and the split ratio of the beams is adjusted by adjusting a ratio of light polarised in a first plane to light polarised in a second plane; optionally wherein the amount of light in the first versus the second plane is adjusted by a polarisation modifier.

36. The method of claim 34 for sorting cells within a plurality of microfluidic streams using at least one beam splitter, the method comprising: adjusting the power transfer properties of at least one beam splitter responsive to a cell event property or a beam status for one or more of the microfluidic streams, optionally wherein the cell event property comprises at least one of detection, classification or sorting properties, optionally wherein the beam status comprises at least one of inactivation of detecting, classifying and / or sorting for one or more of the plurality of microfluidic streams.

37. The method of any one of claims 32 to 36, wherein the sorting a cell associated with the selected cell event comprises: controlling a respective gate open period during which one or more of the regular laser pulses in a respective beam is directed to the respective microfluidic stream; wherein the respective gate open period is controlled dependent on the detecting of the cell event for the respective microfluidic stream and the timing of one or more of the regular laser pulses; and optionally wherein the respective gate open period includes a switching delay corresponding to a time required for a switching device to switch from the one or more regular laser pulses of the respective beam being directed to the respective microfluidic stream to the one or more regular laser pulses of the respective beam being directed away from the respective microfluidic stream, or vice versa; and wherein the respective gate open period is controlled to avoid a regular laser pulse of the respective beam occurring during the switching delay.

38. The method of claim 37, wherein the respective gate open period is controlled responsive to detection of a regular laser pulse following the detecting of the cell event in the respective microfluidic stream; or wherein the respective gate open period is controlled dependent on detection of a regular laser pulse before the detecting the cell event.

39. The method of any one of claims 32 to 38, comprising :starting the respective gate open period a start delay period after detecting the cell event in the respective microfluidic stream or classifying the cell event as a selected cell event for the respective microfluidic stream, wherein the start delay period comprises a predetermined delay and a variable delay dependent on the timing of the one or more of the regular laser pulses.

40. The method of claim 39: wherein the predetermined delay is dependent on a transit time for a cell in the respective microfluidic stream to travel between a detecting location and a sorting location, the detecting location corresponding to the detecting a cell event in the respective microfluidic stream and the sorting location corresponding to the sorting a cell associated with the cell event of the respective microfluidic stream; and wherein the variable delay is dependent on detection of a regular laser pulse following the detecting the cell event; and optionally wherein the variable delay is calculated using a time difference between the classifying the cell event as a selected cell event for the respective microfluidic stream and detection of a next regular laser pulse.

41. The method of any one of claims 23 to 40, wherein the cells are sperm cells.

42. A sorting apparatus for sorting cells within a microfluidic stream using a pulsed sorting arrangement which generates regular pulses, the apparatus comprising: a pulsed sorting arrangement which generates regular pulses; a detection means for detecting a cell event within the microfluidic stream; a classifying means for classifying the cell event as a selected cell event; a sorting means for sorting a cell associated with the selected cell event by controlling a gate open period during which one or more of the regular pulses is directed to the microfluidic stream; wherein the gate open period is controlled dependent on the detecting of the cell event and the timing of one or more of the regular pulses.

43. The sorting apparatus of claim 42: wherein the gate open period includes a switching delay corresponding to a time required for a switching device to switch from the one or more regular pulses being directed to the microfluidic stream to the one or more regular pulses being directed away from the microfluidic stream, or vice versa; and wherein the gate open period is controlled to avoid a regular pulse occurring during the switching delay.

44. The sorting apparatus claim 41 or 43, comprising an optical switch controlled to switch laser pulses into and away from the microfluidic stream dependent on the gate open period; and optionally wherein the optical switch comprises one or more of the following: an acoustooptic modulator; a spatial light modulator; an electro-optic deflector or an electro-optic modulator; and optionally wherein the acousto-optic modulator comprises a Tellurium Dioxide crystal or a Silicon Dioxide crystal.

45. The sorting apparatus of any one of claims 41 to 44, comprising one or more of: a beam narrower arranged to narrow the regular laser pulses incident on the optical switch; and a beam expander arranged to expand laser pulses directed to the microfluidic stream.

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

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

48. A sorting apparatus for sorting cells within a plurality of microfluidic streams using a pulsed laser which generates regular laser pulses, the apparatus comprising: a pulsed laser which generates regular laser pulses; a beam splitter for splitting the laser pulses into a plurality of beams each associated with a respective microfluidic stream; one or more detection means for detecting a cell event within respective microfluidic streams; one or more classifying means for classifying the cell event in respective microfluidic streams as a selected cell event; respective sorting means for sorting the selected cell event in respective microfluidic streams using the regular laser pulses.

49. The sorting apparatus of claim 48, wherein: a) the beam splitter is a polarising beam splitter and the split ratio of the beams is adjusted by adjusting a ratio of light polarised in a first plane to light polarised in a second plane; and / orb) the sorting apparatus further comprises at least one of a polarisation modifier arranged to adjust the amount of light in the first versus the second plane, a power adjustment means, a half wave plate or a pockels cell; and / or c) the sorting apparatus comprises at least one beam splitter for sorting cells within the plurality of microfluidic streams, the sorting apparatus configured to adjust the power transfer of at least some of the beam splitters responsive to a cell event property or a beam status for one or more of the microfluidic streams, optionally wherein the cell event property comprises at least one of detection, classification or sorting properties, optionally wherein the beam status comprises at least one of inactivation of detecting, classifying and / or sorting for one or more of the microfluidic streams.

50. The sorting apparatus of claim 48 or 48, comprising an optical switch controlled to switch laser pulses into and away from the microfluidic stream dependent on the respective gate open period; and optionally wherein the optical switch comprises one or more of the following: an acousto-optic modulator; a spatial light modulator; an electrooptic deflector or an electro-optic modulator; and optionally wherein the acousto-optic modulator comprises a Tellurium Dioxide crystal or a Silicon Dioxide crystal.

51. The sorting apparatus of any one of claims 48 to 50, comprising one or more of: a beam narrower arranged to narrow the regular laser pulses incident on the optical switch; and a beam narrower arranged to expand laser pulses directed to the microfluidic stream.

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

53. A computer program comprising processor instructions which when executed by a processor cause the processor to instruct the method of any one of claims 1 to 12, or 23 to 41. no

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