A system and method for detection and collection of cells, organisms, droplets, bioparticles or other particles

Abstract

A system and method that flows a suspension containing cells, organisms, droplets bioparticles or other particles through a channel 5, interrogates them, e.g. images them with camera 4, and processes the data, e.g. image(s). When a target cell, organism, droplet, bioparticle or other particle of interest 16 is detected, the target is selectively collected from a collection zone 6 along the channel in a collection device 10, and made available for further dispensing, analysis, processing, or other use external to the channel.

Description

[0001] A System and Method for Detection and Collection of Cells, Organisms, Droplets, bioparticles or Other Particles

[0002] Field of the Invention.

[0003] The present invention relates generally to a system and method for detection and collection of cells, organisms, droplets, bioparticles and other particles from a heterogenous mixture to allow for further analysis, processing, or other use.

[0004] Background of the Invention.

[0005] There is often a need to analyze a given heterogenous mixture of cells, droplets or particles and to retrieve cells of a specific type from the mixture. Retrieving a specific cell, organism, droplet, bioparticle or other particle from a heterogeneous mixture can be a daunting, but necessary and important task. One example is finding and collecting sperm cells from the ejaculate or from testicular biopsies / surgeries. There are many millions of background cells aside from the desired sperm cells. The collected cells can then be used in fertility treatments. Another example is circulating tumor cells (CTC). Only a few cells may be present in a relatively large blood sample, and they may be similar in size and shape to the regular cells. Collecting CTCs individually allows for analysis of the mutations present in a single CTC. This is important to identify if there are multiple types of cancer cells present and their specific mutations.

[0006] Cells, particles or droplets can be sorted based on size, morphology, labelling / tagging (e.g. staining to a specific fluorescent marker, or binding to a specific target), or other physical characteristics, such as their response to an electric field, their density or deformability. Sorting cells, particles or droplets based on size or physical characteristics alone, e.g. filtration, density gradient centrifugation, can be inefficient when dealing with a very varied sample that may contain other cells, particles or droplets with similar characteristics.

[0007] Generally, the detection and retrieval of individual cells, particles or droplets of interest takes place in multiple stages. First, the cells, particles or droplets will be sorted. When actively sorting cells, particles or droplets, they will generally pass by a detector of some sort, whether bright-field, darkfield, fluorescent, holographic, electrodes etc. The cells, particles or droplets are generally then directed to one of a number of integrated subchannels based on an analysis of some sort. Once sorted, the cells, particles or droplets in each subchannel can be directed to some sort of outlet at the end of the channel, e.g. reservoir, open well, bin, traps etc., from which they subsequently need to be individually retrieved, or in the case of a fdter, the cells need to be removed from the fdter. Recovery rates of cells, particles or droplets are often low as cells, particles or droplets can stick / adsorb to the walls of the well or filter, or one or a number of cells, particles or droplets of interest can often end up being suspended, possibly all together, in relatively large amounts of fluid. Additionally, when removing the sorted cells, particles or droplets, e.g. with a pipette, they can stick to the surface or to the pipette, or get left behind in a minute amount of fluid that wasn’t successfully collected etc. Separating between the individual collected cells, particles or droplets in the total sorted volume is also a challenge. Even if the sorted cells, particles or droplets are in individual small volumes, for example encapsulated in droplets or traps, it can be difficult to effectively recover and manipulate the small droplets and sometimes some of the sorted cells, particles or droplets can be lost in the process.

[0008] In short, it is difficult to effectively detect and retrieve individual cells, particles or droplets of interest from a heterogeneous sample for uses, such as fertility treatment or single-cell analysis. It is an object of the present invention to provide an improved method for detection and collection of cells, organisms, droplets, bioparticles or other particles that aims to overcome, or at least alleviate, the above-mentioned problems.

[0009] It is a further object of the present invention to provide an improved system for detection and collection of cells, organisms, droplets, bioparticles or other particles that aims to overcome, or at least alleviate, the above-mentioned problems.

[0010] Summary of the Invention.

[0011] Herein is a summary of the invention. It should not be construed as limiting other implementations and embodiments.

[0012] According to a first aspect of the present invention there is provided a system for the detection and separation of one or more cells, particles or droplets of interest from a suspension containing a mixture of cells, particles or droplets, the system comprising: at least one inlet in fluid communication with at least one channel for flow of the suspension containing the one or more cells, particles or droplets; at least one interrogation module comprising an interrogation device for interrogation of the cells, particles or droplets to provide data relating to the cells, particles or droplets; at least one computing module comprising a processor that analyzes the data obtained by the at least one interrogation module and is configured to detect at least one cell, particle or droplet of interest and provide an output concerning at least one characteristic of a particle, droplet or cell of interest to enable selective collection of the particle, droplet or cell of interest from the at least one channel, and at least one external collection device connectable to at least one of the at least one channel, wherein the collection device when connected to the at least one channel selectively collects, based on said interrogation data, the detected at least one cell, particle or droplet of interest during its passage through the at least one channel from at least one collection zone provided along at least one of the at least one channel, meaning between an initial point and a terminal point of the channel, whereby when cells, particles or droplets are not being collected and no valves or other means are preventing flow, the suspension of cells, particles or droplets can flow past the at least one collection zone.

[0013] The system of the present invention collects the at least one cell, particle or droplet of interest based on said interrogation data without first: trapping the detected cell, particle or droplet at the collection zone, or sending it to a well or to the end of the channel. This means that, when no valves or other means are preventing flow, the suspension of cells, particles or droplets can flow past the at least one collection zone when cells or particles are not being collected.

[0014] In the context of this disclosure, the term “cell” can refer to any type of cell, particle or organism, and “selectively collect at least one detected cell, particle or droplet of interest” can also include collecting other background cells, particles or droplets, or other cells, particles or droplets of interest in the vicinity of the detected cell, particle or droplet of interest along with the detected cell, particle or droplet of interest. Furthermore, when multiple features embodiments or claims reference to “at least one channel” or “the at least one channel” it may include multiple channels, with some channels having one or more of the features but not necessarily all of the features (this also applies to other uses of “at least one” for other features), wherein the selective collection of the particle, droplet or cell of interest from the at least one channel, and the at least one collection device connectable to the at least one channel includes each channel having a collection device, some of the channels having a collection device or only a single channel having a collection device.

[0015] In embodiments the cells particles or droplets of interest are directly collected from at least one collection zone located in at least one enclosed channel or in at least one enclosed portion of the at least one channel. In embodiments, the at least one channel comprises a main channel for flow of the suspension with interrogation taking place in the main channel and at least one collection zone is located in the main channel. Optionally the at least one collection device is directly connectable to the main channel and optionally the at least one cell, particle or droplet of interest is directly collected from the at least one collection zone of the main channel. However, in other embodiments, at least one other channel may be provided in selective fluid communication with the main channel for receiving at least a portion of the suspension for at least one or more of the following: interrogation, analysis or collection.

[0016] The collection device is preferably selectively activated to enable selective collection, based on said interrogation data, the detected at least one cell, particle or droplet of interest during its passage through the at least one channel. Following selective collection in the collection device, the contents are retained in the collection device, optionally for storage and, optionally, for later dispensing.

[0017] In preferred embodiments, the cells, particles or droplets are focused in the channel, either before interrogation to optimize the interrogation and / or after the interrogation to facilitate the retrieval of the at least one detected cell, particle or droplet of interest with less background cells or particles, or junk. For example, the cells, particles or droplets may be focused towards a horizontal or vertical center of the channel, or focused to a wide thin layer or to a spaced single file line. The system may also be configured to induce spacing between the cells, particles or droplets in the suspension. In embodiments, this may be achieved by having additional inlets to introduce fluid into the channel, for example above and / or below the suspension, by having a change of dimensions of the channel or through external means such as using electric fields or acoustic focusing.

[0018] The cell, particle or droplet suspension may be focused into a wide thin layer in the at least one channel with sheath flow above and / or below, preferably wherein the focusing is achieved with at least one sheath flow inlet upstream and / or downstream from the suspension inlet, preferably wherein the suspension is also focused away from the sidewalls of the at least one channel, optionally wherein this is achieved by the suspension inlet being narrower than the at least one channel.

[0019] The at least one interrogation module preferably comprises at least one of a sensor or an imaging device, such as a camera to provide data relating to the cells, particles or droplets as they pass through the channel. Preferably, the interrogation module is configured to take multiple images and / or multiple imaging devices are provided. The suspension may be interrogated one or multiple times during its passage through the channel and / or at one or more angles and / or at one or more positions in the at least one channel.

[0020] The output concerning a characteristic of the at least one cell, particle or droplet of interest may comprise, for example at least one morphological characteristic or property of the cell, particle or droplet, its position and / or its velocity.

[0021] It is to be appreciated that other types of interrogation may be carried out on the suspension in addition to, or instead of, imaging of the cells, particles or droplets in the suspension. In embodiments, the cells, particles or droplets may be interrogated in one or a combination of many different ways, for example, imaged in brightfield, darkfield or fluorescence, forward scatter, side scatter, reflected, or transmitted light or signal measured, holographic imaging, acoustic characterization, dielectrophoretic response, electrical properties, such as resistance or capacitance, measured etc. The data may also be the determination of the cell’s, particle’s or droplet’s response to a stimulus such as an external force or some sort of physical reaction. For example, specific biochemical reactions or binding to a surface, antibody or particle, movement to a specific location in the channel due to an acoustic, dielectrophoretic force or some other selective focusing technique etc. Interrogation of any type of data, alone or in combination, that can be utilized to determine whether a specific cell, particle or droplet of interest is present in a sample may be used in the present invention.

[0022] Preferably the computing module includes at least one processor capable of analyzing the data provided by the interrogation module and provides an output concerning the detection of a particle, droplet or cell of interest to enable automatic selective collection of the particle, droplet or cell of interest from the collection area of the at least one channel. Preferably the cells, droplets or particles of interest are detected in real time. More preferably, the analysis of the data is carried out implementing artificial intelligence techniques, such as machine learning, deep learning, neural networks, or cognitive computing. The methods carried out by a processor of the computing module preferably include pre-trained models for recognizing cells of interest and their parameters as well as adaptable models capable of self-learning or categorizing diverse cell, droplet or particle types, with or without prior training.

[0023] In preferred embodiments of the invention, the interrogation module and computing module are configured such that the data obtained from interrogation, e.g. images are analyzed to detect the velocity and position of the at least one cell, droplet or particle. The information on the velocity and position of the cell, droplet or particle may then be used to determine a future position and / or velocity of the cell, droplet or particle, thereby enabling subsequent interrogation, sorting and / or collection. In embodiments, the interrogation module, e.g. sensor or camera or other interrogation device, or the channel itself, may be moved, or one or more additional interrogation modules, e.g. sensors or cameras, may be used for subsequent additional interrogation(s), e.g. imaging, of the cell, droplet or particle for multi-stage sorting or to facilitate the retrieval of the at least one detected cell, droplet or particle of interest with less background cells, droplets or particles, or junk.

[0024] The system is preferably configured to enable the flow of the suspension containing the at least one detected cell, droplet or particle of interest to be controlled at one or multiple points along the at least one channel, for example to be reduced, reversed stopped for further interrogation, e.g. imaging, sorting or for collection. In embodiments, this may be achieved by changing the channel geometry, stopping or slowing the pumps or other actuation mechanisms acting directly (for example the mechanisms pumping the suspension) or indirectly (for example pressurizing a side channel) on the suspension in the at least one channel, or using valves. A preferred embodiment entails stopping the cell, droplet or particle of interest at the collection zone for collection. The system may be configured to carry out the procedure automatically, actively based on the original interrogation, or actively using subsequent interrogation(s). The system may also be dynamic, continuously modifying using ongoing interrogation for more precise positioning.

[0025] In some embodiments, at least one valve is provided in one or more of the at least one channel, wherein the at least one valve may be closed to isolate the at least one detected cell, droplet or particle of interest on its own or along with some surrounding cells, droplets or particles. In some embodiments, this enables continued interrogation of the rest of the suspension of cells, droplets or particles while the detected cell, droplet or particle of interest is collected by the collection device, optionally wherein the at least one cell, droplet or particle of interest is first separated from the suspension, e.g. to a subsidiary channel, or a different portion of the channel, alone or along with some surrounding cells, droplets or particles prior to collection, optionally wherein at least one collection zone is located in one of the at least one subsidiary channel.

[0026] In some embodiments, a valve can refer to a mechanical valve, an electrowetting based valve, a pressure based valve, a deformable membrane based valve or any other mechanism known in the art for locally preventing, modifying or controlling a flow of fluid.

[0027] Alternatively, or additionally, one or more inlets or side inlet channels may be provided in selective fluid communication with the main channel that enables a portion of the channel upstream and / or downstream of the at least one detected cell, droplet or particle of interest to be flushed and cleaned from background cells, droplets particles or junk, prior to collecting the detected cell, droplet or particle of interest. This can facilitate the retrieval of the detected cell, droplet or particle of interest with less background cells, droplets or particles, or junk. It can also enable collection of the cell, droplet or particle of interest along with a specific buffer or fluid.

[0028] In embodiments, a portion of the at least one channel downstream and / or upstream of the collection zone may be closed with a valve during collection alternatively or in addition at least one inlet or side inlet channel near the collection zone opened so that when the detected cell, droplet or particle of interest is collected, fluid is also drawn from the inlet or side inlet channel. The fluid may be provided passively, e.g. the collection action draws the fluid during collection, or alternatively, the inlet or side inlet can actively introduce the fluid. The fluid may be introduced before, during or after collection.

[0029] In some embodiments, an immiscible fluid, e.g. oil, can be introduced, actively or passively into the channel through one or more of the above-mentioned inlet or side inlet, before, during or after collection, so that when the at least one detected cell, particle or droplet of interest is collected, it is automatically encapsulated in the collection device by drawing in the suspension fluid followed by the immiscible fluid.

[0030] In preferred embodiments, the at least one external collection device comprises at least one collection tube connectable to the at least one channel, preferably being connectable at or near the collection zone. The collection zone may comprise a given volume or length of the channel where the channel is adapted to receive the collection device. It may also refer to an area where the cell, particle or droplet of interest is located or directed to, and from where it is then collected. The collection zone may comprise part of the main channel or may be provided in a subsidiary channel in selective or continuous fluid communication with the main channel. For example, a valve may be provided between the main channel and a subsidiary channel for selective fluid communication.

[0031] It is to be appreciated that the or each collection device is a separate, independent, external collection device connectable to the at least one channel between its initial and terminal points. In preferred embodiments, the collection device is removably connectable to the at least one channel between its initial and terminal points. For example, the collection device may be a plug -on device connectable to the channel at or near the at least one collection zone. The channel generally extends between an initial point, generally being where the suspension, buffer or other fluid enters the channel, to a terminal point corresponding to the end of the channel generally furthest from the initial point (generally corresponding to a final outlet of the channel). However, it is to be appreciated that these points may be at other locations along the channel.

[0032] The at least one collection device may be connected to the at least one channel by a connector or a side port, e.g. a lateral protrusion of the channel or side channel near the collection zone.

[0033] Preferably, the at least one collection device is flexible to enable external manipulation of the fluid contained within, for example a peristaltic pump, as well as to manipulate its ends using for example a robotic arm to the destination place of dispensing. In embodiments, the collection device is a flexible tube, optionally wherein the collection device is transparent so as to enable visualization of its contents and in particular the cells, particles or droplets of interests contained within. Optionally the collection device is made of a biocompatible material, preferably having specific wetting properties.

[0034] In embodiments, the system further comprises a reservoir for holding the suspension of cells, droplets or particles prior to their introduction into the channel, said reservoir comprising, for example, at least one of a tube, container, well or syringe, preferably further comprising means for moving or rotating either the suspension and / or the reservoir continuously or periodically to prevent the cells, droplets or particles from settling while in the reservoir while waiting to enter the at least one channel. The inlet or inlet tube may be connected to the reservoir, e.g. syringe or container, holding the suspension of cells, droplets or particles by a rotating joint that allows the reservoir to rotate without rotating the inlet or inlet tube. Alternatively, or additionally, the inlet tube may be attached to the inlet and / or reservoir by a rotating joint that allows the inlet tube to rotate without moving the inlet and / or reservoir.

[0035] In embodiments, at least one other fluids can be introduced into the at least one collection device or upstream or downstream from the collection device, before and / or after the at least one cell, droplet or particle of interest has been collected into the collection device to isolate the fluid containing the at least one cell, droplet or particle of interest as a discrete droplet or plug of fluid in the collection device, optionally wherein the at least one other fluid is injected through a port in the collection device, optionally wherein at least one of the at least one other fluids is immiscible with the fluid in the channel. In the case of a suspension of droplets, the other fluid can be the same as the carrier fluid and different from the droplet fluid, it can be the same as the droplet fluid but different from the carrier fluid, or it can be an entirely different fluid. In embodiments, the at least one collection device such as a collection tube may be prefilled with a fluid, for example oil. This fluid may lubricate the device or tube, prevent adhesion of cells, droplets or particles to the tube and can assist in transporting droplets or plugs of fluid containing collected cell(s), droplet(s) or particle(s) of interest in a controlled way through the device or tube. The fluid may be immiscible with the fluid in the at least one channel.

[0036] In preferred embodiments, the at least one collection device, for example at least one collection tube, may be disconnected from the channel and the at least one collected cell, droplet or particle of interest may be incubated or cryopreserved in the at least one collection device.

[0037] In some embodiments, the collection device can include various means to further manipulate or interrogate the at least one collected cell, particle or droplet of interest or a plug of fluid containing it, e.g. impedance detection, degassing, volume reduction, for example by capillary wicking, of the collected droplet or plug via absorbent or porous areas etc. For example, in some embodiments, volume reduction can help by removing some of the fluid in the plug or droplet thus lowering the fluid volume to particle ratio without affecting the osmolality or other properties of the fluid. This can be useful to make it easier to identify the cell or particle in the plug or droplet making it easier to retrieve once the plug or droplet is dispensed.

[0038] In embodiments, the collected at least one cell, droplet or particle of interest may be dispensed individually on a substrate, such as a petri dish or a glass slide. In embodiments, the detected cell, droplet or particle of interest, or plug or droplet of fluid containing the cell, droplet or particle of interest may be collected at one end of the collection device and deposited on one or more substrates, e.g. a petri dish or glass slide, from another port of the collection device, with or without disconnecting it from the channel. Alternatively, it may be dispensed from the same port after it is disconnected from the channel.

[0039] In embodiments, the at least one collection device may be manipulated so that at least one specific cell, droplet or particle of interest, or droplet or plug of fluid containing the at least one cell, droplet or particle of interest may be dispensed from the at least one collection device onto a specific location on one or more substrates, e.g. a petri dish or glass slide. The dispensing location may be chosen based on previously collected data, e.g. images of the at least one cell, droplet or particle of interest, optionally wherein the positioning and / or dispensing is done automatically. In preferred embodiments of the present invention, the channel, the interrogation module and the collection zone are incorporated into a single channel and the at least one cell, droplet or particle of interest is directly collected from the channel by the collection device which is connected to the channel, or to a side port off of the channel. In some embodiments, the collection is done without the at least one cell, droplet or particle of interest being first sorted nor separated from the rest of the suspension of cells, droplets or particles to a different channel, portion of the channel or a subchannel. However, this does not preclude focusing the cells, droplets or particles, or inducing spacing between the cells, droplets or particles in the suspension of cells, droplets or particles.

[0040] In some embodiments, the at least one cell, droplet or particle of interest is collected without first: marking, labeling, trapping nor encapsulating the at least one cell, droplet or particle of interest.

[0041] In embodiments, the channel may be provided in or on a fluidic chip, such as a glass slide and a polymer with a channel embossed in it or an injection molded chip.

[0042] According to a second aspect of the present invention there is provided a method for the detection and separation of one or more cells, particles or droplets of interest from a suspension containing a mixture of cells, particles or droplets, the method comprising the steps of: passing a suspension containing a mixture of cells, particles or droplets including at least one cell or particle of interest through at least one channel; interrogating the cells, particles or droplets at least once as the suspension passes through the at least one channel to provide data relating to the cells, particles or droplets; analyzing the data obtained to detect at least one cell, particle or droplet of interest in the suspension and providing an output concerning at least one characteristic of a particle, droplet or cell of interest to enable selective collection of the particle, droplet or cell of interest from the at least one channel; and selectively collecting, based on said interrogation data, the at least one cell, particle or droplet of interest from the suspension during its passage along the at least one channel from a collection zone provided along the at least one channel, the at least one cell or particle of interest being collected in at least one collection device connectable to the at least one channel between an initial and a terminal point of the channel, whereby when cells, particles or droplets are not being collected and no valves or other means are preventing flow, the suspension of cells, particles or droplets can flow past the at least one collection zone. Preferably, all four steps of the method according to the second aspect of the present invention take place in a single channel and the at least one detected cell or particle of interest is collected by a collection device, such as a collection tube, without the detected cell, particle or droplet of interest being first separated from the rest of the suspension of cells, particles or droplets to a different portion of the channel nor to a different channel or specific sub-channel before being collected in the collection device. More preferably, the method includes selectively attaching the collection device to the channel between the initial and terminal points for collection of the at least one cell, particle or droplet of interest and then, in some cases removing or disconnecting the collection device from the channel. Preferably, selective activation of the collection device is required to selectively collect, based on said interrogation data, the at least one cell, particle or droplet of interest from the suspension during its passage along the at least one channel.

[0043] Preferably, the method includes one or both the steps of (i) focusing the cells, particles or droplets in the suspension for interrogation, detection and / or collection and (ii) inducing spacing between the cells, particles or droplets in the suspension of cells, particles or droplets for interrogation, detection and / or collection. The flow of the suspension through the channel may also be controlled before, after and / or during interrogation, detection and / or collection.

[0044] The suspension of cells, particles or droplets may be held in a reservoir prior to their introduction into the channel, for example a syringe or container. Preferably, the suspension may be moved continuously or periodically to prevent the cells, particles or droplets from settling while waiting to enter the at least one channel.

[0045] In preferred embodiments of the method according to the second aspect of the invention, the interrogation step comprises at least imaging of the suspension to provide data in the form of images for analysis. More preferably, the images are analyzed to detect the velocity and position of the at least one cell, particle or droplet of interest in the suspension. More preferably still, this data on the velocity and position of the at least one cell, particle or droplet of interest is used to determine a future position and / or velocity of the cell, particle or droplet and / or the required manipulations to the flow to enable subsequent interrogation, sorting and / or collection.

[0046] In embodiments wherein the method includes isolating the at least one detected cell, particle or droplet of interest on its own or along with some surrounding cells, particles or droplets, possibly by opening or closing a valve, preferably wherein the system is configured to enable continued interrogation of the rest of the suspension of cells, particles or droplets while the at least one detected cell, particle or droplet of interest is collected by the collection device, optionally wherein at least one subsidiary channel is provided for separation of the at least one cell, particle or droplet of interest from the suspension, alone or along with some surrounding cells, particles or droplets, prior to collection, optionally wherein at least one collection zone is located in one of the at least one subsidiary channel .

[0047] In embodiments wherein the method includes the step of focusing the cells, particles or droplets, the cells, particles or droplets may be focused towards a horizontal and / or vertical center of the channel, focused to a wide layer or spaced single fde line before the interrogation step, e.g. imaging, to optimize the interrogation. Alternatively, or additionally, the cells, particles or droplets may be focused after the interrogation step to facilitate the removal of the at least one detected cell, particle or droplet of interest with less background cells or particles, or junk.

[0048] The interrogation step may include multiple interrogation steps. In embodiments, the interrogation step may include moving either the interrogation module, e.g. sensor or camera, or the channel, or by using one or more additional interrogation modules, e.g. sensors or cameras to provide additional data from the interrogation.

[0049] Preferably, the analysis step is carried out using artificial intelligence techniques, such as machine learning, deep learning, neural networks, or cognitive computing. The methods used may encompass pre-trained models for recognizing cells / particles / droplets of interest and their parameters as well as adaptable models capable of self-learning or categorizing diverse cell, particle or droplet types, with or without prior training.

[0050] The subsequent interrogation steps may be used for multi-stage sorting or to facilitate the retrieval of the at least one detected cell, particle or droplet of interest with less background cells, particles or droplets, or junk.

[0051] In embodiments the method further comprises holding the suspension of cells, particles or droplets in a reservoir, said reservoir comprising, for example, at least one of a tube, container, well or syringe, prior to their introduction into the channel, preferably further comprising moving or rotating the suspension and / or the reservoir continuously or periodically to prevent the cells, particles or droplets from settling while in the reservoir.

[0052] In embodiments, the rate of flow of suspension containing the at least one detected cell, particle or droplet of interest is slowed or stopped at specific points or regions in the at least one channel for further interrogation, e.g. imaging, for sorting and / or for collection. For example, the rate of flow may be altered by changing the channel geometry, stopping or slowing pumps that affect flow and / or or using valves. This can be done automatically, actively based on the original interrogation, or actively using subsequent interrogation(s). The process can also be dynamic, continuously modifying using ongoing interrogation for more precise positioning.

[0053] In embodiments, the at least one detected cell, particle or droplet of interest, can be manipulated directly to facilitate more accurate interrogation, e.g. imaging, sorting and / or collection, e.g. using physical means such as acoustic, magnetic, electrical, hydrodynamic, optical or dielectrophoretic manipulation to accurately position it at the collection point. This can also be done automatically, actively based on the original interrogation, or actively using subsequent interrogation(s). The process may also be dynamic, continuously modifying using ongoing interrogation for more precise positioning.

[0054] In some embodiments, the method may comprise further steps for the manipulation or interrogation of the at least one collected cell, particle or droplet of interest or a plug of fluid containing it, e.g. these steps may include one or more of impedance detection, degassing, volume reduction, for example by capillary wicking, of the collected droplet or plug via absorbent or porous areas etc.

[0055] The method according to the second aspect of the present invention may include a flushing step to introduce a clean fluid (such as a buffer fluid) into a portion of the channel downstream from the at least one detected cell, particle or droplet of interest thereby cleaning it from background cells, particles, droplets or junk, prior to collecting the at least one detected cell, particle or droplet of interest. This can facilitate the retrieval of the at least one detected cell, particle or droplet of interest with less background cells / particles / droplets, or junk. It can also enable collection of the at least one cell, particle or droplet of interest along with a specific buffer or fluid.

[0056] In embodiments, a portion of the at least one channel upstream and / or downstream of the collection zone may be closed with a valve and at least one inlet or side inlet channel near the collection zone may be opened so that when the at least one cell, particle or droplet of interest is collected, clean fluid is also drawn from the at least one inlet or side inlet channel. The fluid can be provided passively, e.g. the collection action draws the fluid during collection, alternatively, the inlet or side inlet can actively introduce the fluid. The fluid can be introduced before, during or after collection. Preferably, the collection device is prefilled, possibly with a fluid, such as a carrier fluid or an immiscible fluid, for example oil. This fluid, can lubricate the tube, prevent adhesion of cells, particles or droplets to the collection device and can assist in transporting droplets or plugs of fluid containing collected cells, particles or droplets of interest in a controlled way through the collection device.

[0057] In embodiments, at least one other fluid, including air, may be introduced into the at least one collection device, before and / or after the at least one cell, particle or droplet of interest has been collected into the collection device, effectively isolating the fluid containing the at least one cell, particle or droplet of interest as a discrete droplet or plug of fluid in the collection device. Preferably wherein at least one of the at least one other fluids is immiscible with the fluid in the channel.

[0058] Once the at least one cell, particle or droplet of interest has been directed and collected into the collection device, such as a collection tube, the collector tube may be disconnected from the channel and the collected cell, particle or droplet of interest may be incubated or cryopreserved in the tube. The method may further comprise the step of individually dispensing each droplet or plug of fluid containing the at least one cell, particle or droplet of interest onto a substrate, such as a petri dish or glass slide. In embodiments, the at least one detected cell, particle or droplet of interest, or droplet or plug of fluid containing the cell, particle or droplet of interest may be collected at one end of the collection device and dispensed onto a substrate from another end or port of the collection device with or without disconnecting the device. Alternatively, the device can be disconnected and the dispensing can be from the same end through which they were collected. The dispensing step may also include manipulating the collection tube to deposit the at least one cell, particle or droplet of interest, or droplet or plug of fluid containing the at least one cell, particle or droplet of interest onto a specific location on the substrate. The location may be chosen, manually or automatically based on previously collected data, e.g. images, of the at least one cell, particle or droplet of interest.

[0059] It is to be appreciated that in embodiments of the method or system of the present invention where there are multiple channels there need not be a collection device for each channel. There could be as little as a single collection device attached to one of the channels. There is also no upper limit to the number of collection devices and there may be multiple collection devices attached to a single channel.

[0060] The system and method according to the first and second aspects of the present invention may used to separate any type of cell, particle or droplet of interest from a suspension of cells, particles or droplets. However, the invention is particularly applicable for the detection and collection of at least one of the following cells of interest: circulating tumour cells, sperm cells, immature sperm cells (such as round spermatids) or bacteria.

[0061] It is to be appreciated that the system and method may be automated to provide a convenient and quick throughput for the detection and collection of cells, particles or droplets of interest from a suspension containing a mixture of cells and / or particles and / or droplets including those of interest.

[0062] Brief Description of the Invention.

[0063] For a better understanding of the present invention and to show more clearly how it may be carried into effect, reference will now be made by way of example only, to the accompanying drawings in which:

[0064] FIG. l is a schematic overview of an apparatus for the collection of cells of interest according to one embodiment of the present invention;

[0065] FIG. 2 is a schematic overview of a plurality of plugs containing cells of interest collected using the apparatus shown in Figure 1 and dispensed as individual droplets onto a substrate, in this case onto a petri dish below a layer of oil;

[0066] FIG. 3 A is an isometric overview of the apparatus without the collection device shown in Figure 1;

[0067] FIG. 3B is a top view of the apparatus shown in Figure 3A;

[0068] FIG. 4 is a cross-sectional view showing some features of the apparatus shown in Figures 1, 3A and 3B, specifically the focusing of the suspension of cells in a thin layer at the vertical center of the channel;

[0069] FIG. 5A is a photograph of an experimental setup of an embodiment of the apparatus of the present invention showing the channel and the collection device sitting on a microscope;

[0070] FIG. 5B is a photograph of a petri dish containing retrieved cells of interest in individual droplets under oil;

[0071] FIG. 5C is a sketch of the photograph of the experimental setup shown in FIG.5A;

[0072] FIG. 5D is a sketch of the photograph of the petri dish shown in FIG. 5B;

[0073] FIG. 6A, Fig.6B and Fig.6C are a sequence of images that show the identification, retrieval and dispensing of a cell of interest (in this case a sperm cell) using the experimental setup of an embodiment shown in FIG. 5A and 5C, wherein Fig.6A shows a sperm cell of interest imaged while it is flowing through the channel; Fig. 6B shows the sperm cell imaged after it is automatically stopped near the collection zone; and Fig. 6C shows the sperm cell after being collected and subsequently dispensed in a droplet in a petri dish;

[0074] FIG. 7 is a top view of an apparatus for the collection of cells of interest according to another embodiment of the present invention; and

[0075] FIG. 8 is a top plan view of an apparatus for the collection of cells according to yet a further embodiment of the present invention.

[0076] Note that throughout the drawings, the same reference numbers are used to indicate similar, but not necessarily identical, elements. The figures may not be to scale. Furthermore, the drawings present examples and / or implementations that align with the description, but the description is not confined to the examples and / or implementations shown in the drawings

[0077] Detailed Description of the Invention.

[0078] There is a need to sort out rare cells, droplets or particles from a mixture of cells, particles or droplets in a number of areas, such as fertility and oncology. However, sorting them alone is not enough. In many cases the individual sorted cell, particle or droplet is needed for subsequent use processing or analysis, for example single cell analysis, fertility treatments etc. Cell sorters for example, generally sort cells of interest by directing them to an alternate outlet at the end of the channel, e.g. reservoir, open well, bin, traps etc., from which they need to be individually retrieved. This can make it difficult to retrieve particular individual cells as they may be diluted in a relatively large volume (conversely if they are in a minute amount of liquid, for example in a cell trap, it may be difficult to retrieve and handle) and may be mixed with other cells of the same type that were sorted. This can make the recovery rate of rare cells low. The present invention addresses this problem with a streamlined and implementable system and method for detecting and collecting the cells, droplets or particles of interest from a suspension in a relatively very small volume of fluid containing the target cells, particles or droplets. They can then easily be individually dispensed for later analysis or use.

[0079] Presented here is a system and method that flows a suspension containing a mixture of cells, organisms, droplets bioparticles and / or other particles through one or more channels, interrogates them, e.g. images them, and processes the data, e.g. image(s). When atarget cell(s), organism(s), droplet(s) bioparticle(s) and / or other particle(s) of interest are identified, they are collected from a collection zone(s) along the channel, i.e. between an initial point (for example being the point at which the suspension buffer or fluid enters the channel) and a terminal point of the channel (generally being the point of the channel furthest from the initial point, generally corresponding to a final outlet of the channel). The suspension of cells, particles or droplets can flow past the collection zone(s) when cells or particles are not being collected (provided that no valves or other means are preventing flow at the time for other purposes). This enables the cells, particles or droplets of interest to be easily collected by a collection device from the collection zone in a discrete and relatively minute volume of fluid. They can then be easily dispensed from the device for analysis or use.

[0080] In embodiments, one or more, or even all of the steps involved in all of the devices and methods can be automated.

[0081] Below are a number of embodiments of the invention. The various elements of these embodiments can be used on their own or in different combinations along with elements from other embodiments.

[0082] Note that throughout the description when mention of a feature such as a channel, collection zone or device, cell of interest etc. is made, it is not limited to one of the features or objects. There may be more than one and the mention may refer to one or more of them.

[0083] In some embodiments everything takes place in a single channel and the detected cell, droplet or particle of interest is collected directly from the collection zone situated in the same channel where it is interrogated.

[0084] Note that throughout this description, when the word cell is used, it also includes any other type of organism, particle, droplet or bioparticle of interest. Additionally, when reference is made to a feature or object in the singular, e.g. channel, cell or collection device, it does not preclude their being multiple similar features or objects, for example multiple collection devices.

[0085] Figure 1 of the accompanying drawings is a schematic diagram illustrating one implementation of the following embodiment of an apparatus according to the present invention for the direct collection of a cell, particle or droplet of interest from a suspension of cells, particles or droplet. The schematic includes both the channel where the interrogation takes place as well as an embodiment of the collection device as a tube. Note that not all components pictured are in plane. For example, the collection device can be perpendicular to the channel. In the figure, a suspension of different types of cells 1 flows through one or more channels 5. The cells are interrogated, e.g. imaged by a camera 4 or other suitable imaging device, and the data, e.g. images, are analyzed. If one or more cells of interest are identified (e.g. sperm cell 16 in Figure 1), they are individually directly and selectively removed from the channel 5 into an external collection device, in this case a collection tube 10 that is connected to the channel at a point along the channel and in some cases removable. Note that the cells of interest, or target cell, can be removed directly from the channel without necessarily first: trapping the detected cells, encapsulating them in a droplet, or sending them to a well or to the end of the channel. If the cells are not collected, they can pass through the collection zone continuing on through the channel. This is a significant advantage as any fluid before or after the cell can continue on through the channel allowing the target cells to be directly collected in a small discrete volume of fluid (shown as 11 in Figure 1) without the need for further manipulation. In some embodiments, all components are incorporated in a single channel and the detected cell(s) of interest are collected by the external collection device, such as a tube 10, without the detected cell(s) of interest 16 first being specifically separated from the rest of the suspension of cells to a different portion of the channel or to a different channel or specific sub -channel before being collected in the collection tube 10. This does not preclude focusing the cells or inducing spacing between the cells in the suspension of cells.

[0086] The present invention in some embodiments allows for multiple cells, particles or droplets to be collected in separate droplets by introducing a second fluid 13 into the collection device, such as a tube 10, to separate sequential cell collections. This effectively creates individual plugs or droplets of fluid 11 containing the collected cells, particles or droplets of interest (introducing the second fluid directly into the external collection device streamlines the channel design making it easier to manufacture and less likely to get contaminated with the second fluid). The combination of the ability to collect, directly from the channel, the detected one or more cells, particles or droplets of interest one at a time with or without intermediate sorting steps, along with the ability to encapsulate and dispense each of the one or more cells, particles or droplets of interest in small discrete volumes, overcomes many of the challenges faced by many other methods. There is virtually no cell, particle or droplet loss in intermediate steps, and since the cells, particles or droplets of interest are in individual minute quantities of liquid, they can easily be subsequently used or processed. Also, since the collection device remains in a fixed point along the channel throughout the sorting, the one or more cells, particles or droplets of interest can be automatically positioned close to the collection device for collection, allowing individual cells, particles or droplets to be retrieved in a precise minute amount of fluid without any complex micromanipulations. The volume of the liquid droplets or plugs and the spacing can also be controlled. This allows the cells, particles or droplets to automatically be dispensed to different locations. For example, two types of cells, particles or droplets could be collected in the same collection device and dispensed automatically to different locations.

[0087] The collection device 10 is external to the channel 5 and not an integral part of the channel. In preferred embodiments of the invention, the collection device can be easily disconnected from the channel. This allows the individual droplets or plugs of liquid 11 containing the cells, particles or droplets to be easily dispensed from the tube to any location or substrate, without cell, particle or droplet loss, for analysis and use. In some embodiments, they can even be incubated or cryopreserved while still in the device. This is a novel advantage over many other methods where after the cells, particles or droplets are sorted to a different region within the device, e.g. subchannels or reservoirs, they require subsequent collection which can be difficult and can result in cell, particle or droplet loss. Additionally, by controlling both the size of the droplets or plugs and of the spacing phase between them, the entire process including the dispensing can be done in a completely automated way.

[0088] Lastly, by using an external collection device 10 to directly collect the one or more cells, particles or droplets of interest, the design of the device can be streamlined and simplified. In some cases, it can lessen the need for complex manufacturing and simplify the pumping and valving schemes. There is more flexibility in the design for example, various tubes can be used as a collection device on their own or in combination, such as, commercially available or custom-made tubes with different properties than the rest of the device.

[0089] In short, this invention enables efficiently retrieving and dispensing individual cells, particles or droplets of interest from a heterogenous mixture and making the collected cells, particles or droplets individually available for subsequent use and analysis.

[0090] In further detail, Figures 3Ato 5B of the accompanying drawings illustrate one implementation of the following embodiment of the present invention, wherein a fluidic chip 100 is provided comprising a glass slide 25 with a PDMS 24, or some other layer, on top. However, it is to be appreciated that the invention is not limited as such. The channel 5 can either be molded or machined directly into the top layer 24 or a spacing layer with the channel shape cut out can be used. In this embodiment, three inlets 18,19, 20 provide the main flow. The middle inlet 19 provides the cell suspension while the first inlet 18 and third inlet 20 provide sheathing flow to vertically focus the cells into a wide thin horizontal layer (see flows 2, 3 shown schematically in Figure 1). The cell inlet 19 is also narrower than the channel 5 so that the cells are not too near to the wall (also schematically illustrated in FIG. 1) in portion 5 of the channel. The cells flow continuously through the main channel. It is to be appreciated that in certain embodiments the cells may form a single file line, in one or more portions of the channel.

[0091] In Figures 1-5B the cell of interest is a sperm cell 16, but it is to be appreciated that this embodiment, and other embodiments according to the present invention, could easily be applied to other fields, such as collecting CTCs from a blood sample. A camera 4 (see Figure 1) or other imaging device is directed at the channel to image the cells (this may include magnification and phase contrast) as they flow through the imaging portion of the channel 5. More than one image can be taken of the cell(s). Taking multiple images allows the cell(s) to be tracked and its velocity determined. This can then be used to predict when the cell will reach the collection zone 6 for its removal from the channel.

[0092] The images are then processed, in preferred embodiments, using a machine learning model that has been trained to identify cells of a specific type, for example a sperm cell 16. When a cell of interest is identified (such as sperm cell 16), its position and velocity can be determined. This information may then be used to predict its future position and velocity at a different point in the channel (i.e. the collection area 6). A command is sent to collect the cell directly from the channel. This can be done during regular flow, or the flow may be slowed, stopped or even reversed to accurately position the cells and enable the target cell(s) to be removed. When the cell of interest is positioned near the collection device, in this case a collection tube (such as at point 17 in the collection area 6 of the channel), the cell is sucked into the collection tube 10.

[0093] To enable more effective withdrawal of the cell, particle or droplet with less background cells / junk, the channel geometry may be changed downstream of the camera 4, for example a narrowing of the channel may be provided (similar to the size difference in width between area 5 and 6 of the channel) so that there are fewer cells in a given length of the channel, the cells, particles or droplets can even be formed into a single file line. The channel 8 downstream of the cell(s), particle(s) or droplet(s) of interest can be flushed (see inlet 7) so that there are few or no other cells or background junk downstream of the cell(s), particle(s) or droplet(s) of interest and only then withdraw the cell(s), particle(s) or droplet(s) of interest with a reduced number of background cells, particles or droplets.

[0094] The position required for the cell, particle or droplet to be collected can either be predicted solely based on their velocity, or the cells, particles or droplets can be reimaged closer to the collection zone using a second camera or the main camera / stage can be moved further down the channel to more accurately guide the cell, particle or droplet to the collection zone 6. This can also make it easier to collect the cell, particle or droplet with less or no background cells, particles, droplets or junk and is akin to a secondary sorting.

[0095] The collection device, in this embodiment a collection tube 10, (see Figures 1 and 2) is prefilled with a different phase of fluid 13(in some cases a carrier fluid) than the channel (preferably immiscible in the liquid in the channel), for example mineral oil. Once the cell(s), particle(s) or droplet(s) of interest is / are in the collection tube 10 the end of the tube can be filled with the fluid 13 either by injecting it slightly before the end of the tube (12 in Figure 1) or by introducing the fluid into the main channel and then sucking the fluid into the tube. This creates a droplet or plug 11 comprising fluid 11 containing the cell(s), particle(s) or droplet(s) of interest. The tube 10 is now ready to capture another cell, particle or droplet of interest. The flow can continue and more cells, particles or droplets can be analyzed.

[0096] Once all the required cells, particles or droplets are in droplets or plugs 1 lin the collection tube 10, the tube can be detached from the channel. The droplets or plugs can be left to incubate in the tube, the droplets or plugs can be cryopreserved in the tube, or the plugs can be selectively deposited on one or more substrates (as in FIG. 2), for example in individual droplets 14 on a petri dish 15 under oil, or in individual wells in a well plate, etc. The droplets or plugs can be deposited using an automated arm and / or stage or even manually. The droplets or plugs to be deposited and the location where to deposit them can also be chosen, manually or automatically, based on the images previously captured of the cells, particles or droplets.

[0097] The basic set up of components described above may be incorporated into an automated cell, particle or droplet sorting system. The unit may be fully enclosed or some components may be external to the unit. The unit can have a built-in computing system with a touchscreen interface or it can use an external computer.

[0098] In embodiments, the unit receives a cell, particle or droplet suspension, for example via a reservoir that is filled with the suspension, via a syringe filled with the sample, or via a tube that can aspirate the suspension from an external reservoir. The sample along with some sheath flow passes through a channel on a fluidic chip. The chip and tubing are preferably disposable. In preferred embodiments, the chip can contain built in reservoirs for all of the fluids. The chip may sit on a stage and a lens or objective and a camera may be provided below the chip. Pumps are preferably provided to affect flow of the suspension along with any sheath flow through the chip.

[0099] The cell, particle or droplet suspension is imaged. When cells, particles or droplets of interest are detected, they are collected into a collection tube and separated by oil into droplets or plugs. At the end of the sorting, the collection is detached from the chip and a dish containing oil is placed on the stage. The stage can have automated motion. The droplets or plugs are then dispensed in drops on the dish. The dish can then be removed from the device.

[0100] FIG. 5A to 6 of the accompanying drawings show images of an experimental implementation of one embodiment of the present invention. The unit 100 was utilized to detect and collect sperm cells from a semen sample containing background cells and debris. The device was made by cutting an adhesive tape with a thickness of around 100 microns in the shape of the required channel. The tape was attached to a glass slide. Then the slide and a slab of PDMS, with holes punched for inlets, were exposed to plasma. The PDMS and glass slide were aligned and bonded under a microscope and heated to 80°C to conclude the bonding. The inlet tubes 9, 26, 29, 30, 31 and outlet tube 10 were connected to the channel using small metal tubes (e.g. 27), similar to the shaft of a needle.

[0101] The collection device, in this case a collection tube 10 having an inner diameter of 0.38mm was connected to the channel via a small metal tube. A small sleeve 32 of larger tubing size was placed close to the end of the collection tube. This sleeve 32 helps to hold and connect the side tube that injects oil into the collection tube. A small hole is made through the sleeve and the side of the collection tube. Through this hole a side tube 9 for injecting oil was connected via a small metal tube 33. The connections were held in place with hot glue. The collection tube 10 was prefilled with mineral oil, although the end was filled with the same buffer used in the sorting channel, to avoid contaminating the sorting channel with oil during connection.

[0102] Two inlets 18, 20 introduce buffer and another inlet 19 introduces the cell suspension (semen sample). The first section of the channel 5 was around 2mm wide and around 100 microns in height. The buffer inlets 18 and 20 were around 2mm wide while the cell suspension inlet 19 was around 1.25 mm wide to focus the cell suspension away from the side walls of the channel 5. A syringe pump was used to control the flow. By flowing buffers through the two inlets 18, 20 before and after the cell suspension inlet, the cells were focused in a wide and thin monolayer.

[0103] As the cells flow through the channel they were imaged via a lOx microscope objective for a total of lOOx magnification. The images were processed via machine learning using a trained convolutional neural network (CNN). For illustration, the info from the Al model was used to draw a contour 35 around the cell of interest 34 (see FIG. 6A), in this case a sperm, and pass its location to a tracking algorithm. By detecting the difference in location between subsequent images of the same cell, the velocity of the cell was determined. This position and velocity information was used to predict the time it would take for the cell of interest to reach the collection zone 6. The command was then sent to the control code for the pump and the flow was stopped once the cell of interest was slightly past the collection zone (see 6 in FIG 5A and FIG. 5C and 34 in FIG. 6B) to ensure it would be collected. It is to be appreciated that all of these steps were done automatically. After a cell of interest was stopped near the collection zone 6. the downstream portion of the channel 8 was flushed with clean fluid from a side inlet channel 7. A suction was then applied by setting the syringe pump attached to the collection tube 10 to withdrawal mode. The cells of interest 34 were retrieved along with some background cells and clean fluid from the downstream flushed portion 8 of the channel. Mineral oil was then pumped from the injection tube 9 into the collection tube 10 using a peristaltic pump. This effectively creates a droplet or plug of fluid 11 in the collection tube 10 containing the cell of interest 34 with oil both before and after it to effectively isolate the cell of interest from the other plugs upstream in the tube and separating it from the fluid in the channel. A number of individual plugs containing cells of interest were collected in this way. For this experiment, each plug contained around 2 microliters of fluid and was separated by around 1 microliter of oil.

[0104] Once the sorting was finished, the collection tube 10 was disconnected from the sorting channel . It was submerged in a petri dish 15 filled with oil. Set amounts of fluid were pumped out at specific locations to put each plug in its own space on the dish (see Figs. 5B and 5D). The oil simply floats up and connects with the rest of the oil in the dish. The plug contained cell media which is heavier than oil, so it sinks and forms a droplet 14. The result was several droplets 14 each made up of one plug of fluid.

[0105] Afterwards the cells of interest were located in the correct droplet and imaged (FIG. 6C) in their individual dispensed droplet 14. Such a droplet 14 in a petri dish 15 under oil is similar to how embryologists arrange cells in droplets under oil for fertility treatments, in an ICSI dish for example. Thus, the method and system of the present invention was demonstrated to enable satisfactory identification, collection and dispensing of individual cells of interest. Individual cells of interest 34 were detected (FIG. 6A), collected (FIG. 6B) directly from the main channel into an external collection tube 10 in small discrete volumes of fluid 11 and finally they were dispensed in discrete volumes and made available for use (FIG. 6C).

[0106] Thus, the present invention provides a system that includes one or more channels with one or more tubes connectable thereto with optional reservoirs for suspensions and fluids. Flow may be affected and controlled by one or more pumps. Features may be incorporated into the system to prevent cells, particles or droplets in the suspension of cells from settling while waiting to be flowed into the channel(s), for example, the inlet tube or container holding the cell, particle or droplet suspension can be moved or rotated continuously or periodically to prevent the cells, particles or droplets from settling while waiting to enter the device. The system includes means for interrogating the cells, particles or droplets and the data is then analyzed as herein described to enable direct selection of a cell, particle or droplet of interest from a suspension of cells, particles or droplets.

[0107] Figure 7 of the accompanying drawings illustrates an alternative embodiment of the present invention. Features similar to those already discussed in relation to earlier figures are given the same reference numerals and only the differences will be discussed in detail. This embodiment includes a side port 36, basically a lateral protrusion of the channel or a side channel, for connecting the collection device for the collection of cells, particles or droplets of interest, a side inlet port to supply clean buffer during collection, a valve to close the side inlet port during interrogation and another valve that can close off the portion of the channel downstream from the connection point. As illustrated, the main channel 5 may be shut off downstream of the collection zone 6 by valve 38. A side inlet channel 37 with valve 39 is provided near the collection zone 6 which can be opened so that fluid is drawn from the side inlet channel 37 along with the cell(s), particle(s) or droplet(s) of interest from the main channel. In the embodiment shown, the collection device (not shown) can be situated on the side of the main channel near the collection zone 6 and connected to the main channel by a side port 36. The cell(s), particle(s) or droplet(s) of interest can then be directly collected from the collection zone 6, via the side port 36 into the external collection device, e.g. the collection device 10 shown in FIG. 1, as described above. These components can each be implemented on their own or in combination with one or more other features herein described.

[0108] In another embodiment (an example implementation of which is shown in FIG. 8), the suspension of cells, particles or droplets is interrogated in one channel 5 and the cells, particles or droplets of interest are collected from a second interconnected subsidiary channel 40. There is also a valve 38 in the downstream portion of the interrogation channel 8 that can be closed to direct a cell, particle or droplet of interest to the connected collection channel 40. A second valve 41 is provided in the collection channel 41 to allow interrogation of the suspension of cells, particles or droplets to continue in the interrogation channel, while the cell, particle or droplet of interest is being collected from the collection channel. The collection channel also includes a side inlet channel 7 that can be used to flush the portion of the collection channel downstream from the collection zone 6. The collection zone is situated in a second channel 40 connected to the channel where the cells, particles or droplets were interrogated. This has the advantage that the channel 40 containing the collection zone 6 can be closed off from the main channel during collection by way of valve 41 to allow interrogation of other cells, particles or droplets to continue during collection of the cell, particle or droplet of interest. The channel containing the collection zone 6 continues past the collection zone to an outlet 23, allowing the cell(s), particle(s) or droplet(s) of interest to be accurately positioned at the collection zone 6 and collected without the need to aspirate all of the fluid that flowed from the main channel along with the cell(s), particle(s) or droplet(s) of interest.

[0109] One or more different fluids, e.g. oil or even air, can be introduced into the collection device (e.g. fluid 12 in the collection tube 10 of Figure 1), before and / or after the cell(s), particle(s) or droplet(s) of interest have been collected into it. The fluid can be introduced into the collection device, by opening a side inlet channel (similar to the side inlet channel 37 in Figure 7) containing the fluid and aspirating the fluid into the collection device or by directly injecting the fluid into the collection device, for example via a small inlet 9 (Fig. 1) into the collection tube 10, connector 33 (Figs. 5 A and 5C) into the collection tube 10 or through any port in the collection device, or into a connector upstream or downstream from the collection device, or by some other means. This other fluid can serve to isolate the fluid containing the cell(s) particle(s) or droplet(s) of interest from both the cells, particles or droplets in the channel and cells, particles or droplets that will be collected later, effectively creating an isolated droplet or plug (11 in Figures 1 and 2) of fluid containing the cell(s), particle(s) or droplet(s) of interest. This allows for multiple cells, particles or droplets of interest to be collected in separate discrete volumes 11 in the same tube.

[0110] It is to be appreciated that many different embodiments of the system and method of the present invention may be provided. For example, the device may be provided with one or more channels, where there are multiple channels, they can be made all the same or they can be different. The channel(s) can be made from one piece or multiple pieces. Some examples are given below, however, they are for illustration only and are not an exhaustive list. An example of a single piece is for the channel(s) to be etched into glass or a 3d printed device with a hollow in it etc. The channel(s) can also be made from two or more elements bonded together (for example 24,25) with one of the elements containing an imprint of the channel (for example 24), for example Polydimethylsiloxane (PDMS) bonded to glass (for example 25), with the channel(s) etched into the glass or molded into the PDMS. The channel(s) can also be made up of multiple layers, for example, a layer such as glass followed by a thin layer with a specified thickness, such as an adhesive layer or a PDMS membrane, with the shape of the channel(s) cut into it and another layer, for example PDMS on top. The layers can be kept together in a number of ways for example, permanently or temporarily bonded together or held together with pressure, e.g. between plates that are bolted together. The channel(s) can be manufactured via embossing techniques, micromachining, precision machining, 3D printing, injection molding, etc. The channel(s) may be made of many types of materials, rigid or flexible, transparent or opaque, for example, glass, silicon, PDMS, etc. The channel(s) can also be coated, partially, in a patterned way, or completely to change its surface properties, for example, to make it hydrophobic / hydrophilic, oleophobic / oleophilic, non-stick properties etc.

[0111] In embodiments, the channel(s) may also comprise a tube with the cells, particles or droplets interrogated, e.g. imaged, directly inthe tube, aportion(s) of the tube can be of a different shape or material to enable imaging or various connections.

[0112] In embodiments, the channel(s) can be part of a microfluidic or millifluidic chip with appropriate inlets and outlets. The chip can be similar to lab-on-a-chip devices.

[0113] The channel(s) may be of any cross-sectional shape. It can be rectangular (like 5), round, square semi-circular etc. The channel(s) height can be in the range of 10-20 microns, 20-30 microns, 30 -50 microns, 50-100 microns, 100-200 microns, 200-500 microns, 500-1000 microns or any other height suitable for the cells / tissue being processed. The channel height can also vary throughout the channel(s). The channel(s) width can be in the range of 10-50 microns, 50-100 microns, 100-300 microns, 300-500 microns, 500-1000 microns, l-2mm, 2-5mm or any other width suitable for the cells / tissue being processed. The channel(s) width can also vary throughout the channel(s), for example, the imaging area 5 can be one of the above mentioned ranges, the collection area 6 another and the end of the channel(s) 8 a third range.

[0114] In its most basic form the channel may be a single simple straight channel with an inlet for the cells, particles or droplets, a side port 36 or a top port 21 or even a simple hole somewhere along the channel for collecting the cells, particles or droplets and an outlet for the waste 23.

[0115] The device may also have multiple channels which may be interconnected and, in some cases, different channels can be used for a different purpose. For example, some of the interconnected channels could contain a collection zone(s) for collecting cells, particles or droplets of interest and other channel(s) can be used for interrogating cells, particles or droplets. There can also be valves that can be closed between the channels, for example, as in the embodiment shown in FIG. 8. The channel(s) geometry can also be more complex, for example, narrowing to focus the cells, particles or droplets after interrogation (like the difference in width between 5 and 6 in Figs. 1, 3A and 3B) or a serpentine channel to focus the cells, particles or droplets etc. The channel(s) may have multiple inlets for sheath flow 18,20) flushing the channel(s) 7 and other purposes. As discussed, an inlet 22 may be provided downstream and / or upstream from the collection zone 6 to flush the channel and clean it from background cells, particle, droplets, or junk before collecting the cells, particles or droplets, thus minimizing background cells, particles, droplets, or junk. The flushing can also enable the detected cell(s), particle(s) or droplet(s) of interest to be collected along with a specific buffer or fluid. The inlet can be directly in the main channel or on a separate side inlet branch 7 that connects to the main channel.

[0116] The portion of the channel after the collection zone (8) may also be enlarged so that the collection device (e.g.10) can collect a larger clean volume of fluid along with the cell(s), particle(s) or droplet(s) of interest. There can also be multiple collection zones and flushing points.

[0117] This is not an exhaustive list and more channels, ports, outlets and inlets can be added to add more functionalities to the device. As well more designs and combinations of design can be used.

[0118] The channel and inlets / outlets may be connectable to any suitable reservoir or container. The suspensions and fluids can be in any type of open or closed reservoir, tube or container, on or off the device or chip containing the channel e.g. a test tube, a syringe, an embedded reservoir or an open well on a fluidic chip. These reservoirs can be connected to tubing, connectors, or directly to the channel, e.g. in the case of a fluidic chip, in on-chip reservoirs or inlets. A tube can also be submerged in the reservoir and withdraw fluid or have fluid pumped into it. The reservoirs themselves may also have mechanisms to remove or prevent bubbles.

[0119] It is to be appreciated that the tubing used for handing the fluid flow in the system, such as the inlet, outlet and collection tubes, can be made of many types of materials, rigid or flexible, transparent or opaque, for example, metal, glass, silicon, tygon, PVC etc. The tubes can also be coated to change its surface properties, for example, to make it hydrophobic / hydrophilic, oleophobic / oleophilic, non-stick properties, biocompatibility, etc. The tubes can also have a portion made from a gas-permeable material to prevent bubbles by allowing them to escape through the walls of the tube, e.g. expanded polytetrafluoroethylene (ePTFE) tubing.

[0120] The tube’s inner diameter can be in the range (in mm) 0.05-0.15, 0.15-0.40, 0.40 -0.75, 0.75- 1.5, 1.5-3, or other relevant dimensions. The tube can directly connect to the channel, or it can connect via a connector (e.g. 27). The tubes may be attached to some sort of pump, e.g. a peristaltic pump or a syringe pump. The tubes may be passive, or they can also have valves that 1 help to control the flow, or they can incorporate other features that allow control of the flow such as electro-osmotic pumping etc.

[0121] The collection device, such as 10 shown in FIG. 1, collects one or more cells, particles or droplets of interest (e .g . 17) from the channel and stores them for later dispensing (e .g . as shown in FIG. 2), or for incubation or cry opreservation in the tube. The collection device can be set to collect and dispense the cells, particles or droplets of interest from the same end, or it can collect from one end and dispense from a different end or port. The device can directly connect to the channel, it can connect via a connector, or the collection port can be open with the ability for the collection device to be inserted into the channel (such an open port could be covered with a different phase of fluid, e.g. oil, or it can have a cover that can be opened and closed).

[0122] In some embodiments, the collection device can include various means to further manipulate or interrogate the at least one collected cell, particle or droplet of interest or a plug containing it, e.g. impedance detection, degassing, volume reduction, for example by capillary wicking, of the collected droplet or plug via absorbent or porous areas etc.

[0123] The collection device may be attached to or incorporate at least one actuation mechanism, such as a pump e.g. a peristaltic pump, an electroosmotic pump, or a syringe pump, capillary action or control of the pressure, e.g. pneumatically, at one or more of the inlets and outlets etc., to allow it to actively collect and dispense the cells, particles or droplets of interest, It can also have valves that help to control the flow. The pumping action and or actuation mechanism of the collection device can be independent from that which controls the flow in the channel.

[0124] In some embodiments where the collection and dispensing is from the same end of the device, the device can be disconnected from the channel. This allows more flexibility in the design. Alternatively, the cells, particles or droplets of interest can be collected from one end or port and dispensed from another one allowing for continuous and possibly even simultaneous collection and dispensing of cells, droplets or particles of interest.

[0125] In some embodiments, one or more channels, or portions of one or more channels, may be enclosed, for example in the form of a fluidic device with one or more closed channels (see figures 1, 4 and 5A for examples). There may be one or more inlets and / or outlets to the one or more enclosed channels. The cells, particles or droplets of interest can selectively be directly collected from one or more collection zones located along one or more of the enclosed channel(s), or enclosed portions of the one or more channels, (not the end of the channel) into a collection device connected to a port or side port at or near the collection zone . An enclosed channel can mean a channel surrounded on all sides, except in the direction of flow.

[0126] It is to be appreciated that in some embodiments, a channel can be fully enclosed, in others, it can be partially open. Yet in other embodiments, it can be a fluid flowing along a path designated by hydrophobic and hydrophilic surfaces either permanently patterned or dynamically patterned e.g. in an electro wetting or digital microfluidics device. In some embodiments a channel can be flow of one fluid through another fluid e.g. water flowing through oil on a patterned device. However, the collection device is always connectable to a part of the channel between its initial and terminal points.

[0127] In some embodiments, the collection device consists of a collection tube. Various tubes can be used on their own or in combination, such as, commercially available or custom-made tubes with different geometries (e.g. inner / outer diameters), surface properties, etc. In some embodiments, the collection device can be a microfluidic or millifluidic chip.

[0128] The collection device, e.g. the tube 10 in Fig 1, may be pre-fdled, for example, with a carrier fluid. In preferred embodiments of the invention, the fluid is immiscible with the main fluid in the channel, for example mineral oil. This fluid can lubricate the tube, prevent adhesion of cells, droplets or particles to the tube and can assist in transporting droplets or plugs of fluid containing collected cell(s), droplet(s) or particle(s) of interest in a controlled way through the device.

[0129] The collection device may be directly connected to the channel, or it can be connected via a connector or a side port (for example, see 36 in Figure 7), meaning the channel protrudes laterally to the side near the collection zone and the outlet for the collection device is by that protrusion as shown in FIG 7, or the collection device can be connected to a side channel. An auxiliary tube may be connected to the collection device (for example, tube 9 shown in Figure 1 is directly connected to the collection tube 10). Alternatively, there can be a connector, for example a T-junction with one part connected to the collection port e.g. 21 in Fig. 3A (or to a tube connected to the collection port 21), one part to a collection tube 10, and the third part to an auxiliary tube, such as 9, another example is the connector 33 in FIGS. 5A and 5C. Another type of fluid (or the same fluid) or even air can then be injected from the directly from the auxiliary tube, e.g. 9 into the collection device or tube 10. In some cases, more than one tube or device can be connected to the collection device to inject more than one type of fluid into the tube or to extract droplets or plugs from the collection device. In cases where droplets of interest are being detected and retrieved from a suspension of droplets the fluid, the injected fluid can be the same as the carrier fluid or different from both the droplet fluid and the fluid that the droplets are suspended in.

[0130] In some embodiments, the collection device may be disconnected from the channel, in cases where dispensing is done from a different end or port, the dispensing port can be moveable . It may also be connectable to a robotic arm to allow it to be moved or it can be positioned above a movable stage. This would allow it to dispense the collected cell of interest or droplets or plugs containing the cells, particles or droplets in specific locations on one or more substrates, e.g. a petri dish or glass slide. The locations can be determined based on data collected during the interrogation of the cells, particles or droplets. This process can also be automated.

[0131] There can also be multiple independent collection devices. Each collection device can, for example, collect a different subset of cells, particles or droplets.

[0132] It is to be appreciated that any suitable connector for connecting the tube(s) to the channel may be used in the present invention. The connectors between, the tubing, inlets, outlets and reservoirs, may be simple, such as a small hollow metal tube (see 27 in Fig. 5A and Fig. 5C), or they may incorporate other features such as, flow sensors, bubble traps or other active or passive bubble release mechanisms, e.g. a gas permeable membrane.

[0133] In embodiments, a pool or layer of liquid may be provided on top of a port to prevent the introduction of air bubbles into the channel. When making the connection some fluid can also be expelled to the tip of the connector or tube. Since the contact is between liquid and liquid and there is no air in the connector, tube or port, no bubbles will enter the channel at the connection point.

[0134] Easy connect systems may also be utilized in the present invention, such as magnetic connections and multiple inlets combined in one connector. In embodiments a sealed fluidic chip may be provided with sharp connectors that puncture into the inlets and outlets of the channels and ports.

[0135] The connectors can be made from a variety of rigid or flexible materials. Connectors may also be incorporated into the channel or the chip containing the channel. Automated connectors and automated systems to connect and disconnect the connections may also be used. A number of connectors may be connected together and connected at the same time to the channel. As mentioned above, the system and method of the present invention may be provided with one or more actuation mechanisms, for example pumps to activate and / or control fluid flow, although any method of inducing flow may be used in embodiments of the invention. The flow may be activated using one or more different types of pumps, e.g. one or a combination of syringe pumps, peristaltic pumps, electro-osmotic pumps, capillary pumps. The pumping can be applied to the external tubes, the inlets, outlets or reservoirs, etc. The pumping mechanism can even be incorporated into the channel itself. In the case of a fluidic chip, the pumping mechanisms, e.g. a peristaltic pump head, can be incorporated directly into the chip.

[0136] The pumping may be via positive or negative pressure applied to an inlet, outlet, or reservoir. This may be accomplished by physically applying pressure, for example by pressing on a diaphragm over the reservoir. It may also be accomplished by controlling, for example using pneumatic pressure to control the pressure in the reservoir container, or the pressure at the inlets, outlets and in the collection device.

[0137] The suspension of cells, particles or droplets can flow through the channel(s) as is or it can be combined with other fluids or buffers. In one embodiment the cell, particle or droplet suspension can be mixed with another fluid in the tubing leading to the channel(s). In another embodiment, the suspension of cells, particles or droplets and another fluid flow into the channel through separate inlets and are combined on chip (as shown in the accompanying figures). The other fluid(s) may be of the same type as the suspension of cells, particles or droplets. Other fluids may be used as sheath flow, above, below or alongside (or any combination thereof) the suspension of cells, particles or droplets. The sheath flow can be used for buffer exchange, focusing the cells, particles or droplets or any other purpose.

[0138] The cells, particles or droplets may flow continuously through the system or the flow may be intermittent. The flow may occur at varying speeds. Some possible ranges for the flow rate in the channel in microliters per minute are 0.1-1,1-10, 10-100, 100-200, 200-500, although other relevant ranges are possible. The relative flow rate between the various inlets can vary as well, for example, the ratio of sheath flow to flow of cells can be 1:2, 1:5, 1: 10, 1:20, 1:50 or other relevant ranges. Furthermore, the sheath flows below, above, or alongside the cell, particle or droplet suspension flow do not need to be equal. In embodiments, there may be no sheath flow.

[0139] As mentioned above features may be incorporated into the system to prevent cells, particles or droplets in the suspension of cells, particles or droplets from settling while waiting to flow into the channel(s), For example, in embodiments the inlet tube or container holding the cell, particle or droplet suspension may be moved or rotated continuously or periodically to prevent the cells, particles or droplets from settling while waiting to enter the device.

[0140] This may be accomplished by connecting the inlet or inlet tube to the container, e.g. syringe or reservoir, holding the suspension of cell(s), particle(s) or droplet(s) using a rotating joint that allows the container to rotate without rotating the inlet or inlet tube so that only the container is moved and not the inlet or inlet tube. Alternatively, or in addition, the inlet tube may be attached to the inlet and / or container by a rotating joint that allows the inlet tube to rotate without moving the inlet and / or container. Other methods to prevent settling or induce motion in the suspension can also be used.

[0141] Embodiments of the present invention may also provide for focusing of the cells, particles or droplets. It is to be appreciated that the suspension of cells, particles or droplets may flow through the channel(s) without any specific focusing, or, more preferably, the suspension may be focused into a specific part of the channel. Focusing the cells, particles or droplets in the suspension of cells, particles or droplets can be for a number of purposes, e.g. better optical focus, more uniform velocity and spacing between the cells, particles or droplets, or to enable collecting the cells, particles or droplets of interest with less background cells, particles or droplets or junk. In one embodiment, a fluid is flowed above and / or below the cells, particles or droplets so that the cells, particles or droplets form a thin wide layer, possibly a monolayer, and are distanced from the bottom and / or top of the channel (e.g. as shown in FIG. 1 & 4). This allows for many cells, particles or droplets to be interrogated at once while keeping them in focus and preventing them from overlapping and blocking each other. The sheath flow above and below can also prevent blockages due to a large cell or particle while keeping the cells or particles in focus as the sheath flow creates distance between the cells or particles and the top and / or bottom of the channel while keeping them in focus. The cells, particles or droplets can also be focused in the horizontal plane (as in FIG. 1 where the cells are distanced from the side walls of the channel 5). The cells, particles or droplets can also be focused along the flow direction, for example causing the cells, particles or droplets to organize into a single fde line with spacing between the cells, particles or droplets. The cells can also be made to rotate while flowing. The combination of a wide thin layer along focusing, for example in a narrow or single fde line, for collection can allow for efficient detection, as many cells, particles or droplets can be interrogated at once while at the same time accurately collecting the cell, droplet or particle of interest.

[0142] Focusing the cells, particles or droplets may be achieved by one or a combination of different means. For example, an external focusing force may be used e.g. acoustically or dielectrophoretically. The flow may be controlled to focus the cells, particles or droplets e.g. controlling the velocity, hydrodynamically, or by modifying the channel geometry, for example, using zigzag or serpentine channels. Chemical additives may be used, for example Polyvinylpyrrolidone (PVP), or hyaluronic acid may added to the fluid to achieve viscoelastic focusing. Sheath flows may also be used. Another option is to simply modify the channel and inlet geometry, for example, the inlet for the cells, particles or droplets may be narrower than the channel so that cells, particles or droplets do not flow too near the wall (as shown by inlet 19 being smaller than inlets 18 & 20 in the accompanying drawings). This is not an exhaustive list, other types of focusing, spacing, rotating etc. can be performed using any types of appropriate means. Furthermore, cell, particle or droplet focusing or rotating may be implemented before interrogation, during interrogation, after interrogation, before collection or during collection and can help facilitate interrogation sorting or collection.

[0143] In embodiments, where the vertical focusing is achieved with at least one sheath flow inlet upstream and / or downstream from the suspension inlet, the horizontal focusing away from the sidewalls of the channel may be achieved by the suspension inlet being narrower than the channel.

[0144] It is to be appreciated that any type of cell, bacteria, particle or droplet suspended in any type of fluid may be analyzed by the method and system of the present invention. All kinds of cell suspensions from different sources can be analyzed, for example blood, urine, saliva and semen samples. The cells may also come from tissue samples for example cancer tissue biopsies, or testicular biopsies, aspiration or surgeries etc. The samples can also be forensic in nature, for example finding sperm in a rape kit or for detecting contamination, for example bacterial contamination in food or water. Examples of cells of interest are sperm, CTCs, immature sperm cells, such as round spermatids, bacteria, etc. However, the invention is not limited to any particular type of cell or particle. The cells of interest may differ morphologically or in any other way that can be discerned by a sensor or camera, for example, by detection of a difference in electrical capacitance, staining positive to a given antibody or response to an external stimulus. This is not an exhaustive list and there may be other biological or non -biological fields of application where a cell, particle or droplet of interest needs to be retrieved from a suspension. Throughout this document, the term ‘cell’ can refer to any such cell, particle, droplet or organism of interest.

[0145] Any suitable method of sample preparation for providing a satisfactory suspension of the sample of cells, particles or droplets may be utilized in the present invention. Cells may be suspended in their original bodily fluid, such as semen or saliva. Alternatively, the cells may be diluted or completely resuspended in any other fluid, for example, buffers, culture media, sperm wash media etc. Such a resuspension can take place in the channel, before the channel or after a cell of interest is collected from the channel. In embodiments, the cells or particles may be pretreated, for example, density gradient centrifugation, swim-up (for semen samples), filtration etc. Tissue samples and biopsies may also need to undergo more extensive preparation or physical disassociation to provide the required suspension to be analyzed, such as mincing, or enzymatic disassociation.

[0146] As herein before discussed, the system of the present invention is configured to interrogate the cells, particles or droplets in suspension as the fluid flows through the channel. In many embodiments, the cells, particles or droplets are interrogated by imaging e.g. imaged to determine if there is a cell, particle or droplet of interest in a current window of interrogation. A single cell particle or droplet may be interrogated in a small window of interrogation, e.g. a small image, or a larger number of cells may be interrogated in a single large window of interrogation, e.g. a large image. The interrogation window may be any appropriate size, for example hundreds, thousands or millions of square microns, for example 50-100 x 50-100 microns, 100-500 x 100-500 microns 500-1000 x 500-1000 microns, 1000- 2500 x 1000-2500 microns, or any other relevant size. The data, e.g. image, can have a high density of pixels or a low density of pixels, for example, 1-5, 5-10, 10-30 or 30-100 mega pixels.

[0147] The cells, particles or droplets may be interrogated in one or a combination of many different ways, for example, imaged in brightfield, darkfield or fluorescence, forward scatter, side scatter, reflected, or transmitted light or signal measured, holographic imaging, acoustic characterization, dielectrophoretic response, electrical properties, such as resistance or capacitance, measured etc. The data may also be the determination of the cell’s, droplet’s or particle’s response to a stimulus such as an external force or some sort of physical reaction. For example, specific biochemical reactions or binding to a surface, antibody or particle, movement to a specific location in the channel due to an acoustic, dielectrophoretic force or some other selective focusing technique etc. The cells, particles or droplets can also be labeled in some manner, for example using fluorescence labeling, antibody binding, etc. Interrogation of any type of data, alone or in combination, that can be utilized to determine whether a specific cell, particles or droplet of interest is present in a sample may be used in the method and system of the present invention.

[0148] Various magnifications for imaging can be used such as 2x, 4x,10x, 20x, 40x 60x, lOOx, 200x, 400x 600x etc. Various types of contrast enhancement may also be used for example phase contrast, differential interference contrast (DIC) etc. The magnification may be accomplished using a microscope objective, a camera lens or by any other means. The cells, particles or droplets can be interrogated, e.g. imaged, from one or more angles, at one or more points in the channel, and / or using one or more technologies.

[0149] The data obtained from the interrogation of the cells, particles or droplets should then be analyzed. It is to be appreciated that in preferred embodiments of the present invention, the data e.g. images or electrical signatures, is analyzed by a computing module, for example, an external computer or a built-in standalone GPU module. Conventional data or image processing may be used or a machine learning module may be used. In this respect, the processing of the data may utilize a diverse range artificial intelligence (Al) techniques, including but not limited to, to machine learning, deep learning, neural networks, and cognitive computing. The methods used may encompass pre-trained models for recognizing cells, particles or droplets of interest as well as adaptable models capable of self-learning or categorizing diverse cell, particle or droplet types, with or without prior training. Furthermore, the analysis encompasses a wide array of techniques beyond conventional data or image processing, including spectral analysis, statistical pattern recognition, feature extraction, and anomaly detection, among others. The above-mentioned examples are not an exhaustive list. Any other type of data or image analysis techniques that can identify a cell of interest in either a labelled or non-labelled manner may be used. Furthermore, the data or images may also be digitally enhanced or compressed before analysis.

[0150] In preferred embodiments of the present invention, motion analysis and tracking of the cells, particles or droplets occurs during their passage through the channel. The position and / or velocity of all or some of the cells, particles or droplets may be tracked. This may be achieved using machine learning models or regular analysis using mathematical and physical models or other means to analyze the data obtained from the interrogation. For example, cells or particles may be matched up between various datapoints and the difference in position of a specific cell, particle or droplet between two data points or images can be used to determine the velocity of a cell, particle or droplet. Tracer particles may also be used to determine the fluid velocity. The position and / or velocity maybe used to predict the future position or velocity of the cell(s), particle(s) or droplet(s) in the channel. The prediction may be made in a number of ways, for example, based on calculations or by using machine learning models. This prediction may then be used to enable and time future actions, for example, subsequent interrogation, the collection of a cell(s), particle(s) or droplet(s) of interest or the next stage in sorting in multi-stage sorting. It can also be used to control the flow so that the cell (s), particle(s) or droplet(s) of interest is guided to a specific point in the channel, where it may be slowed or stopped, for example, at the collection zone or a secondary imaging point. Other types of data may also be output that can give more information about the cell, droplet or particle. For example, some of this data may be useful in effecting the collection of a cell, particle or droplet of interest, alternatively, of the data can be used to grade the cells, particles or droplets quality.

[0151] The cells, particles or droplets can be subjected to multi-stage sorting. In embodiments, after an initial interrogation, the cells, particles or droplets may be subsequently interrogated, e.g. imaged, again at another location in the channel. This subsequent interrogation(s) may be used for instance to enable the removal of the cell(s) particle(s) or droplet(s) of interest with less background cells, particles or droplets or junk. For example, when a cell, particle or droplet of interest is detected, it can proceed along with the surrounding cells, particles or droplets to another portion of the same channel, or an interconnected channel, in some cases the cells, particles or droplets may be focused to pass by in a single line. The individual cells, particles or droplets can then be interrogated, e.g. imaged, to ensure only the cell(s), particle(s) or droplet(s) of interest is collected through the collection device of the apparatus with less or without any background cells, particles or droplets or junk. Subsequent interrogation can also be used before collection (e.g. to detect the arrival of the cells, particles or droplets at the collection point), during collection to guide the collection or afterwards to verify that the correct cell(s), particle(s) or droplet(s) was collected. The subsequent interrogation(s) can use the same interrogation device / method or a different device / method.

[0152] In embodiments, detected cell(s), particle(s) or droplet(s) of interest may be stopped, slowed, or reversed (e.g. by changing the channel geometry, stopping or slowing the pumps, or using valves) to enable sorting, interrogation or collection for example, to slow or stop near the collection zone or actively directed to a collection zone automatically based on predictions made by the computing module. When the cell, particle or droplet is positioned in the correct position it can be stopped or slowed for collection. The cells, particles or droplets in the suspension may be focused or spaced before the collection zone to enable more specific collection of the cell, particle or droplet of interest with less background junk or cells, particles or droplets.

[0153] In embodiments, when collecting droplets of interest from a suspension of droplets, or where the cells are encapsulated in a droplet before collection, the droplet can be positioned for collection in such a way that it completely covers the port through which the collect is done. This allows the droplet to be collected without the surrounding carrier fluid. By calibrating the system for the droplet size, the droplet can be collected followed by a specified amount of carrier fluid . This allows the collected droplets to remain as discrete droplets spaced and separated from subsequently collected droplets of interest in the collection device without the need to introduce fluid into the collection after collecting the droplet.

[0154] In embodiments, after a cell, particle or droplet is collected, the flow may be reversed to ensure that no cells, droplets or particles of interest that may have been near or slightly behind the detected cell, particle or droplet of interest are missed. The flow would be reversed so that the volume in the channel is retrieved from the channel back into one or more of the input. The flow can then be resumed. The volume is thus reinterrogated for any cells, droplets or particles of interest.

[0155] In embodiments, a portion of the channel upstream and / or downstream of the cell, particle or droplet of interest may also be flushed so that when the cell, particle or droplet is collected, clean fluid is collected with it. This minimizes background cells, particles droplets or junk in the collected volume. For example, in FIG 1 the downstream portion of the channel 8 can be flushed by introducing fluid through the side inlet channel 7.

[0156] The detected cell(s), particle(s) or droplet(s) of interest is collected for immediate or later use. The detected cell(s), particle(s) or droplet(s) of interest can be collected while moving at full velocity, while slowed, or while stopped. Collection of the cell(s), particle(s) or droplet(s) of interest may occur at or near the interrogation point or further downstream. Collection may occur immediately or after a delay.

[0157] The detected cell(s), particle(s) or droplet(s) of interest may be collected into a collection device by a number of means for example, suction, capillary action, closing the main channel downstream to force the cell(s), particle(s) or droplet(s) of interest into the collection device etc. Along with the cell(s), particle(s) or droplet(s), more fluid from the upstream and / or downstream portion of the channel may be collected. The cell(s), particle(s) or droplet(s) of interest may be collected on their own or along with other cells, particles or droplets in their vicinity.

[0158] Alternatively, the main channel may be closed downstream and / or upstream with a valve and a side inlet channel near the collection zone may be opened so that fluid is drawn from the side inlet channel along with the cell(s), particle(s) or droplet(s) of interest from the main channel (as discussed above in relation to Fig. 7). One or more different fluids, e.g. oil or even air, can be introduced into the collection device (e.g. fluid 12 in collection tube 10), before and / or after the cell(s), particle(s) or droplet(s) of interest have been collected into it. The fluid can be introduced into the collection device, by opening a side inlet channel (similar to the side inlet channel 7) containing the fluid and aspirating the fluid into the collection device or by directly injecting the fluid into the collection device, for example via a small inlet (e.g. 9, see Figs. 1 and 5A) a connector (see 33, Figure 5A) or port in the side of the tube, or into a connector upstream or downstream from the tube, or by some other means.

[0159] This other fluid can serve to isolate the fluid containing the cell(s), particle(s) or droplet(s) of interest from both the suspension of cells, particles or droplets in the channel and from cells, particles or droplets of interest that may be collected later, effectively creating an isolated droplet or plug of fluid containing the cell(s), particle(s) or droplet(s) of interest. This allows for multiple cells, particles or droplets of interest to be collected in separate discrete volumes in the same collection device or tube.

[0160] The collection device(s) may have multiple channels or tubes into which the cell(s), particle(s) or droplet(s) of interest can be directed after they are collected.

[0161] When not collecting cells, particles or droplets flow can be prevented from entering the collection device(s) to allow free flow for the cells, particles or droplets to flow past the collection zone(s) without impediment. This can be achieved for example with a valve in the collection device or simply by stopping the pump that actuates the collection.

[0162] It is to be appreciated that the collection zone(s) as defined herein is not an open well nor an outlet at the end of the channel. However, it may be located at any other point along the channel. When not collecting other cells, articles or droplets of interest, the suspension can flow past the collection zone, providing that no valves or other means are impeding flow of the cells, particles or droplets. This can allow for more precise collection as not all of the fluid near or flowing with the cell, particle, or droplet of interest need to be collected along with it.

[0163] The method of the present invention may further comprise the step of dispensing the cell(s), particle(s) or droplet(s) once collected. The system may include any suitable dispensing means. Once the cell(s), particle(s) or droplet(s) of interest are in the collection device, they can be moved to another device or tube(s) connected to the collection device(s) or stay in the collection device(s). They may then be left to incubate or be cryopreserved in the collection device(s) or in a tube(s) attached to the collection device(s) to which the cell(s), droplet(s) or particle(s) of interest can be directed after collection, or they can be dispensed. The cell(s), particle(s) or droplet(s) of interest, or the droplets or plugs containing the cell(s), particle(s) or droplet(s) of interest can be dispensed individually to one or more substrates, such as a well plate (as shown in FIG. 2), a glass slide, a petri dish etc. The cell(s) particle(s) or droplet(s) of interest, or the droplets or plugs containing the cell(s) particle(s) or droplet(s) of interest can be dispensed from a port of the collection device(s) not connected to the channel, or the collection device(s) can be disconnected from the channel and then the cell(s), particle(s) or droplet(s) of interest, or the droplets or plugs containing the cell(s), particle(s) or droplet(s) of interest dispensed from the same end that was previously connected to the channel.

[0164] In embodiments, the collection device containing the cell(s), particle(s) or droplet(s) of interest, or the droplets or plugs containing the cell(s), particle(s) or droplet(s) of interest may be connected to a robotic arm to enable specific cell(s), particle(s) or droplet(s) of interest, or the droplets or plugs containing the cell(s), particle(s) or droplet(s) of interest to be dispensed to specific places on the substrate(s). Alternatively, the substrate may be moved or a combination of both. Data previously collected of one or more cells, particles or droplets, e.g. images, may be used to determine, automatically or as decided by the user, where to deposit the cell(s), particle(s) or droplet(s) of interest, or the droplets or plugs containing the cell(s), particle(s) or droplet(s) of interest.

[0165] It is to be appreciated that some or all of the above-mentioned processes and systems may be fully automated or include human / manual input. The system can integrate the different components (e.g. chip, collection device, camera / sensor, power source, computer, user interface etc.) in a synergistic and streamlined manner to enable its automated operation. The system may be built to have sterile lines where everything coming into contact with the cells, particles or droplets can be replaced or sterilized. Alternatively, there may be permanent components that are simply washed or flushed between uses.

[0166] Thus, the system and method according to the present invention enables target cells particles or droplets to be selectively and optionally directly, collected from a suspension of cells, particles or droplets flowing through a channel, even enclosed channels, in a streamlined manner in a small discrete volume of fluid without the need to trap it or send it to a well or the endpoint of the channel. In some embodiments, the cells, particles or droplets of interest can be collected after detection without first separating them from the suspension nor sorting them. Multiple cells, particles or droplets can be collected in separate plugs or droplets by introducing a second fluid to separate between sequential cell, particle or droplet collections. This effectively creates individual droplets or plugs of fluid containing the collected cells, particles or droplets of interest. This allows for each cell, particle or droplet to be individually collected in a small amount of fluid. The combination of a streamlined collection of detected cells, particles or droplets along with the ability to encapsulate and dispense them in small discrete volumes for each cell, particle or droplet of interest, overcomes many of the challenges faced by existing methods. There is no cell, particle or droplet loss in intermediate steps, and since the cells, particles or droplets of interest are in minute quantities of liquid, they can easily be subsequently used or processed. Also, since the collection device remains in a fixed point on the channel throughout the sorting, no complex micromanipulators are needed to retrieve individual cells, particles or droplets.

[0167] The collection device may be connected to the channel during processing of the sample but at the same time, it is external to the channel and not an integral part of the channel, in some embodiments it can be easily disconnected from it. This allows the individual droplets or plugs of liquid containing the cells, particles or droplets of interest to be easily dispensed from the collection device to any location or substrate, without cell, particle or droplet loss, for analysis and use. They can even be incubated or cryopreserved while still in the collection device. This is an advantage over some other methods where after the cells, particles or droplets of interest are processed and sorted to a different area on the device, e.g. reservoirs they often require a subsequent collection step to retrieve the individual cells, particles or droplets of interest which can be difficult and can result in cell, particle or droplet loss. Additionally, by controlling both the size of the droplets or plugs and of the spacing phase between them, the entire process including the dispensing can be done in a completely automated way.

[0168] In some embodiments, the system will make use of microfluidics and pumps with image analysis performed using relatively simple artificial intelligence (Al) image analysis. A preferred application for its use will be is the fertility market to enable automation of the search for rare sperm in samples (both semen and testicular biopsy samples) with a near zero sperm count. Currently, these samples are searched by eye manually under a microscope. The present invention enables automation of this process and makes the cells easily available for use by the embryologist for fertility treatments. However, it is to be appreciated that other applications will benefit from using the system and method of the present invention.

[0169] Further modifications to the system and method for automated detection, collection and in some cases dispensing of cells, organisms, droplets, bioparticles or other particles may be made without departing from the principles embodied in the examples described and illustrated herein. LIST OF REFERENCE NUMBERS USED IN FIGURES: ) A thin wide layer of the cell suspension entering the channel. ) Sheath flow or buffer entering ahead of the cell suspension, flowing below the wide thin layer of cells and helping focus the cell suspension to the vertical center. ) Sheath flow or buffer entering after the cell suspension, flowing above the wide thin layer of cells and helping focus the cell suspension to the vertical center. ) Camera / optics / sensors to interrogate, e.g. image, the cells flowing through the main channel.) The imaging area of the main channel. ) The collection zone in the channel. ) Side inlet channel for flushing the portion of the main channel downstream of the collection point. ) Portion of the channel after the collection zone. ) Inlet to inject a second fluid into the collection device can be the same as the carrier fluid in the collection device, e.g. tube, for example, oil. 0) The collection device, e.g. tube. 1) Plug(s) containing collected cells including a cell of interest. 2) Fluid injected after the cells have been collected to disconnect them from the channel and from cells that will be collected later. Essentially creating a plug of liquid containing the cells, with the second fluid isolating it on both ends. 3) The second fluid separating the plugs. 4) A plug from the collection device that was dispensed on the substrate, in this case a petri dish filled with oil, creating a droplet containing a cell of interest on the bottom of the petri dish submerged in oil. 5) A substrate, in this case a petri dish. 6) A sperm cell of interest in the imaging portion of the channel 7) A sperm cell of interest in the collection zone in the channel 8) Upstream sheath / buffer inlet. 9) Cell suspension inlet. 0) Downstream sheath / buffer inlet. 1) Collection port. 2) Flushing inlet. 3) Waste outlet. 4) Upper portion of the channel with the channel design imprinted (recessed) in it, can be made for example by molding or by micromachining. 5) Lower portion of the channel 6) Inlet tube leading to side inlet channel (7) for flushing the portion of the main channel downstream of the collection zone. 7) Connector made of a thin hollow metal tube. 8) Alignment marks. 9) Inlet tube leading to upstream sheath / buffer inlet. 0) Inlet tube leading to cell suspension inlet. 1) Inlet tube leading to downstream sheath / buffer inlet. 2) Sleeve on the collection tube. 3) Connector to inject fluid from tube 9 to tube 10. 4) A sperm cell flowing through the channel. 5) A contour drawn around the sperm (34) by the Al convolutional neural network (CNN).6) Side port for connecting the collection device. ) Side inlet channel to provide clean buffer during collection of cells of interest. ) Valve to close the downstream portion of the interrogation channel. ) Valve to close the side inlet channel. ) Channel for collection of cells of interest that is separate from the interrogation channel, e.g. a subsidiary channel. ) Valve to isolate the collection channel from the interrogation channel, to allow simultaneous interrogation and collection.

Claims

CLAIMS:

1. A system for the detection and separation of one or more cells, particles or droplets of interest from a suspension containing a mixture of cells, particles or droplets, the system comprising: at least one inlet in fluid communication with at least one channel for flow of the suspension containing the one or more cells, particles or droplets; at least one interrogation module comprising an interrogation device for interrogation of the cells, particles or droplets to provide data relating to the cells, particles or droplets; at least one computing module comprising a processor that analyzes the data obtained by the at least one interrogation module and is configured to detect at least one cell, particle or droplet of interest and provide an output concerning at least one characteristic of a cell, particle or droplet of interest to enable selective collection of the cell, particle or droplet of interest from the at least one channel; and at least one external collection device connectable to at least one of the at least one channel, wherein the collection device when connected to the at least one channel selectively collects, based on said interrogation data, the detected at least one cell, particle or droplet of interest during its passage through the at least one channel from at least one collection zone provided along the at least one channel between an initial point and a terminal point of the channel, whereby when cells, particles or droplets are not being collected and no valves or other means are preventing flow, the suspension of cells, particles or droplets can flow past the at least one collection zone.

2. The system as claimed in claim 1, wherein the collection device is removably connectable to the at least one channel between its initial and terminal points.

3. The system as claimed in claim 1 or claim 2, wherein the at least one channel comprises a main channel for flow of the suspension, the at least one interrogation module interrogates the suspension during its flow through the main channel, and the collection zone is situated in the main channel, preferably wherein the at least one collection device is directly connectable to the main channel and the at least one cell, particle or droplet of interest is directly collected from the main channel during its passage therethrough or wherein the at least one collection device is connected to the at least one channel by a side port extending from the channel near the collection zone.

4. The system as claimed in claim 1 , claim 2 or claim 3, wherein the cell, particle or droplet suspension is focused into a wide thin layer in the at least one channel with sheath flow above and / or below, preferably wherein the focusing is achieved with at least one sheath flow inlet upstream and / or downstream from the suspension inlet, preferably wherein the suspension is also focused away from sidewalls of the at least one channel, optionally wherein this is achieved by the suspension inlet being narrower than the at least one channel.

5. The system as claimed in any one of the preceding claims, wherein the interrogation module and computing module are configured such that the data obtained from interrogation is analyzed to detect a velocity and / or position of the at least one cell, particle or droplet of interest in the channel, preferably wherein the velocity and / or position of the cell, particle or droplet of interest is used to determine a future position and / or velocity of the cell, particle or droplet of interest thereby enabling subsequent interrogation and / or collection.

6. The system as claimed in any one of the preceding claims, wherein the system is configured to position the suspension or the at least one cell, particle or droplet for enhanced interrogation, sorting or collection by manipulation of the suspension or the at least one cell, particle or droplet at one or multiple points along the at least one channel thereby facilitating more accurate interrogation, sorting and / or collection.

7. The system as claimed in claim 6, wherein the system is configured to change the rate of flow, stop the flow, or reverse the flow, preferably by at least one or a combination of (i) a change in geometry of the at least one channel, (ii) stopping or changing the speed of one or more pumps or other actuation mechanisms acting directly or indirectly on the suspension in the at least one channel or (iii) operation of one or more valves, more preferably wherein the cell, particle or droplet of interest is stopped at the collection zone for collection.

8. The system as claimed in any one of the preceding claims, wherein at least one valve is provided in one or more of the at least one channel, wherein the at least one valve may be closed to isolate the at least one detected cell, particle or droplet of interest on its own or along with some surrounding cells, particles or droplets, preferably wherein the system is configured to enable continued interrogation of the rest of the suspension of cells, particles or droplets while the at least one detected cell, particle or droplet of interest is collected by the collection device, optionally wherein at least one subsidiarychannel is provided for separation of the at least one cell, particle or droplet of interest from the suspension, alone or along with some surrounding cells, particles or droplets, prior to collection, optionally wherein at least one collection zone is located in one of the at least one subsidiary channel.

9. The system as claimed in any one of claims 2 to 8, wherein a portion of the channel downstream and / or upstream of the at least one detected cell, particle or droplet of interest can be flushed and cleaned from background cells, particles, droplets or junk, prior to collecting the detected cell, particle or droplet of interest, optionally wherein the flushing fluid is provided by at least one inlet or one side inlet channel upstream and / or downstream of the collection zone.

10. The system as claimed in any one of the preceding claims, wherein the at least one external collection device comprises at least one collection tube connectable to the at least one channel, preferably being connectable at or near the collection zone.

11. The system as claimed in any one of the preceding claims, wherein at least one additional inlet or side inlet channel is provided, wherein fluid is introduced from the at least one additional inlet or side inlet channel when the at least one detected cell, particle or droplet of interest is collected.

12. The system as claimed in claim 11, wherein via the additional inlet or side inlet channel a fluid that is immiscible with the fluid in the at least one channel is introduced before, during or after collection, thereby creating upon collection in the collection device an isolated plug containing the detected cell, particle or droplet of interest.

13. The system as claimed in claim 12, wherein the collection device includes one or more means to further manipulate, modify and / or interrogate the at least one collected cell, particle or droplet of interest or the plug containing the detected cell, particle or droplet of interest, preferably wherein said means reduces the volume of the plug or droplet.

14. The system according to any one of the preceding claims wherein at least one actuation mechanism is provided to actively collect and / or dispense the at least one cell, particle or droplet of interest into the at least one collection device, preferably wherein the actuation mechanism is selected from at least one of pump, pneumatic pressure or capillary action that acts upon or is an integral part of the collection device, optionallywherein said actuation mechanism is independent of any actuation mechanism that controls the flow in the at least one channel.

15. A method for the detection and separation of one or more cells, particles or droplets of interest from a suspension containing a mixture of cells, particles or droplets, the method comprising the steps of: passing a suspension containing a mixture of cells, particles or droplets including at least one cell, particle or droplet of interest through at least one channel; interrogating the cells, particles or droplets at least once as the suspension passes through the at least one channel to provide data relating to the cells, particles or droplets ; analyzing the data obtained to detect at least one cell, particle or droplet of interest in the suspension and to provide an output concerning at least one characteristic of a cell, particle or droplet of interest to enable selective collection of the cell, particle or droplet of interest from the at least one channel; and selectively collecting, based on the interrogation data, the at least one cell, particle or droplet of interest from the suspension during its passage through the at least one channel from at least one collection zone provided along the at least one channel, the at least one cell, particle or droplet of interest being collected in at least one external collection device connectable to the at least one channel between an initial point and a terminal point of the channel, whereby when cells or particles are not being collected and no valves or other means are preventing flow, the suspension of cells, particles or droplets can flow past the at least one collection zone.

16. The method according to claim 15, wherein all steps take place in a single channel.

17. The method according to claim 15 or claim 16, further comprising one or both the further steps of (i) focusing the cells, particles or droplets in the suspension for improved interrogation, detection and / or collection and (ii) inducing spacing between the cells, particles or droplets in the suspension of cells, particles or droplets for improved interrogation, detection and / or collection, optionally wherein said focusing or spacing is done after an interrogation to improve subsequent interrogation, detection and / or collection.

18. The method according to claim 15, 16 or 17, further comprising controlling the rate of flow of the suspension through the channel, reversing the flow or stopping the flow before, after and / or during at least one of interrogation, detection or collection.

19. The method according to any one of claims 15 to 18, further comprising holding the suspension of cells, particles or droplets in a reservoir, prior to their introduction into the channel, preferably further comprising moving or rotating the suspension and / or the reservoir continuously or periodically to prevent the cells, particles or droplets from settling while in the reservoir.

20. The method according to any one of claims 15 to 19, wherein the interrogation step comprises at least imaging of the suspension to provide data in the form of images for analysis, preferably wherein the images are analyzed to detect a velocity and position of the at least one cell, particle or droplet of interest in the suspension and wherein this data on the velocity and position of the at least one cell, particle or droplet of interest is used to determine a future position and / or velocity of the cell, particle or droplet and / or the required manipulations to the flow, to enable subsequent interrogation, sorting and / or collection.

21. The method according to any one of claims 15 to 20 wherein the analysis step is carried out using artificial intelligence techniques selected from one or a combination of, machine learning deep learning, neural networks or cognitive computing.

22. The method according to any one of claims 15 to 21 further comprising a flushing step to introduce fluid into a portion of the channel upstream and / or downstream from the at least one detected cell, particle or droplet of interest thereby cleaning it from background cells, particles, droplets or junk, prior to collecting the at least one detected cell, particle or droplet of interest.

23. The method according to any one of claims 15 to 22, further comprising introducing at least one other fluid into the at least one collection device before and / or after the at least one cell, particle or droplet of interest has been collected into the collection device, preferably wherein at least one of the at least one other fluids is immiscible with the fluid in the channel, optionally further comprising the at least one other fluid isolating the fluid containing the at least one cell, particle or droplet of interest as a discrete droplet or plug of fluid in the collection device.

24. The method according to claim 23, further comprising manipulating, modifying and / or interrogating the at least one collected cell, particle or droplet of interest or the plug containing the detected cell, particle or droplet of interest, preferably wherein saidmanipulating, modifying and / or interrogating comprises reducing the volume of the plug or droplet.

25. The method according to claim 23, claim 24 or claim 25, wherein the at least one other fluid is injected through a port in the collection device, preferably wherein the method further includes prefilling or lubricating the collection device with at least one fluid, said fluid being the same as or different to the at least one other fluid introduced into the collection device, optionally wherein at least one of the above mentioned fluids is immiscible with the fluid in the channel.

26. The method according to any one of claims 15-25, further comprising disconnecting the collection device from the channel following collection of the at least one cell, particle or droplet of interest in the device, optionally wherein the at least one collected cell, particle or droplet of interest is incubated or cryopreserved in the collection device.

27. The method according to any one of claims 23 to 26 further comprising the step of individually dispensing each plug of fluid containing at least one of the at least one cell, particle or droplet of interest onto a substrate.

28. The method according to claim 27, wherein the plugs of fluid are dispensed through a port through which the plug was collected after disconnection of the collection device from the channel, or are dispensed from a different port to that through which the plug was collected with or without disconnecting the device.

29. The method according to claim 27 or claim 28 wherein the locations for dispensing are chosen based on data obtained during interrogation of the at least one cell, particle or droplet of interest and optionally wherein the positioning and / or dispensing is done automatically.

30. The use of a system as claimed in any one of claims 1 to 14 and a method according to any one of claims 15 to 29 for the detection and selective collection of at least one cell of interest from a suspension, the cell of interest being selected from a tumour cell, a sperm cell, an immature sperm cell, a fetal cell, a stem cell and a bacteria.

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