Droplet forming system

Abstract

A method and system for determining a droplet frequency of a flow of microfluidic droplets within a microfluidic droplet channel includes irradiating the flow of microfluidic droplets, splitting light from the flow of microfluidic droplets into a first portion and a second portion using a beam splitter, directing the first portion to a camera, directing the second portion through an aperture to a photodetector, and determining a droplet frequency of the flow of microfluidic droplets within the photodetector. The aperture is located in front of the photodetector, and processes a signal from the photodetector and determines a droplet frequency from fluctuations in the processed signal. The method can be used to determine droplet size. The method may also be used to adjust the pressure of the stream of droplet fluid in response to the determined droplet size to produce a stream of droplets.

Description

Technical Field

[0001] The present application relates to systems and methods for determining droplet frequency of a stream of microfluidic droplets and, in particular but not limited to, to systems and methods for determining and controlling droplet size. Background Art

[0002] In this application document, we focus on emulsions that typically include water-in-oil picodroplets (picodroplets), which are typically surfactant-stabilized. One or more biological entities (such as one or more living cells or particles) can be incorporated into each droplet, and experiments can then be performed within the droplet, for example to perform a biological assay. Picodroplets can potentially be generated and processed at rates exceeding thousands per second.

[0003] Typically, the oil composition includes fluorine and / or mineral oil and / or silicone oil, preferably including a surfactant, for example, including a surfactant in a volume ratio (volume / volume) of about 0.5%-5%. When the microdroplets contain living organisms, it is particularly advantageous to use fluorine oil because fluorine oil is good at transporting oxygen to the microdroplets. The surfactant can be a polymer or a small molecule; for example, a surfactant derived from a block copolymer of a perfluoroether (such as Krytox (RTM) or polyethylene glycol (PEG)) can be used. For example, the material or analyte in the microdroplet can include cells, DNA, proteins, peptides, beads, particles, crystals, micelles, macromolecules, enzyme assay materials, organelles, organisms (such as cells, such as mammalian cells, yeast cells, algae cells or bacteria, viruses, prions, etc.).

[0004] Crawford, DF, Smith, CA and Whyte, G. Image-based closed-loop feedback for highly mono-dispersed microdroplet production, Sci Rep 7, 10545 (2017) relates to controlling the size of microdroplets. US2011 / 000560, US2014 / 354795 and US2021 / 146319 relate to using a camera to determine droplet frequency. Summary of the invention

[0005] Various aspects and preferred features are set out in the accompanying claims.

[0006] According to a first aspect, there is provided a method of determining a droplet frequency of a stream of microfluidic droplets within a microfluidic droplet channel, the method comprising:

[0007] irradiating a stream of the microfluidic droplets within the microfluidic droplet channel;

[0008] using a beam splitter to separate light from the stream of microfluidic droplets into a first portion and a second portion, wherein the first portion includes light above a predetermined threshold wavelength, and wherein the second portion includes light below a predetermined threshold wavelength;

[0009] directing the first portion to the camera;

[0010] directing the second portion through an aperture to a photodetector, the aperture being located in front of the photodetector;

[0011] The signal from the photodetector is processed and the droplet frequency is determined based on fluctuations in the processed signal.

[0012] Processing the signal from the photodetector may include obtaining and processing the photodetector output voltage signal over a given period of time to identify voltage signal differences corresponding to differences in optical properties exhibited at an interface between a carrier fluid and a droplet as the droplet passes through a detection region of the microfluidic droplet channel.

[0013] The method disclosed herein provides an automated method for determining the droplet frequency of a stream of microfluidic droplets. By directing the second portion through an aperture located in front of the photodetector, the sensitivity of the photodetector is increased.

[0014] The method may also include detecting a droplet using the processed signal and, when a droplet is detected, simultaneously capturing an image of the droplet using a camera. This uses the output of the detected photodetector signal to actively trigger a camera via a microprocessor to automatically image the droplet within the microfluidic channel.

[0015] The microprocessor camera triggering algorithm may also allow individual droplet detection occurrences to be counted to establish a droplet frequency over a set period of time or until the last image is captured.

[0016] The method may also include determining a droplet size based on the captured droplet image.

[0017] The method may also include calculating a volume of the droplet based on the determined size.

[0018] The term size may be used herein to refer to the physical width or length of a droplet within a microfluidic droplet channel.

[0019] In one example, the droplet can be considered as a sphere and the determined size can be the radius of the sphere. The volume of the spherical droplet can then be calculated using the determined radius.

[0020] In another example, at least one droplet size (e.g., the width of the droplet) can be constrained by a known width or cross-sectional area of ​​a microfluidic channel, and a second droplet size (e.g., the length of the droplet within the droplet channel) can be determined using the methods described above. The droplet volume can be calculated using the known width or cross-sectional area and the determined droplet size.

[0021] A method of determining a droplet size may include identifying a sidewall of a microfluidic droplet channel and a droplet center within an image of a flow, determining a region of interest in the captured droplet image, wherein the region of interest includes a line region, the line region includes the droplet center, and wherein the region of interest is parallel to the sidewall, and determining the droplet size by processing the line region of the image. The region of interest may also include two opposing points on an outer surface of the droplet.

[0022] The method may include comparing the size (e.g., number of pixels) of the processed line region between the side walls to the known size of each pixel of the processed line region. The known size of each pixel may alternatively be referred to as the number of pixels required to display a given physical length. In other words, by knowing the number of pixels per inch (or per centimeter) in the captured image, the physical length or size of the region of interest may be determined. By knowing the resolution of the sensor or camera and the size of the field of view, the resolution of the captured image may be determined.

[0023] Alternatively, the physical size may be estimated or calculated using the known physical distance between the camera and the droplet, the focal length of the camera, the number of pixels within the line area, and the pixel pitch of the camera.

[0024] Captured images of the microfluidic flow can be processed to identify geometric features of the microchannel and the center of the droplets produced. This can be an automated method executed on a processor. The method can then subsequently define a line region of interest that is parallel to the microchannel sidewalls and intersects the imaged droplet at the center of the imaged droplet to obtain a measurement of the droplet diameter that is then used as an input to an image-based closed-loop feedback program that adjusts the monodispersity of the produced emulsion.

[0025] Alternatively or additionally, the method of determining the size of the droplets may include comparing the size of the droplets within the captured image with markers of known size located within the microfluidic channel. This provides a simpler method of measuring the size of the droplets and improves the accuracy of the determined droplet size.

[0026] According to another aspect of the present disclosure, there is provided a method of determining a total volume of a fluid within a series of droplets within a stream of microfluidic droplets, comprising:

[0027] determining a droplet frequency of a stream of microfluidic droplets within the microfluidic droplet channel using the method described above;

[0028] for each droplet within the stream of microfluidic droplets, calculating a droplet volume;

[0029] calculating the average droplet volume of the droplets; and

[0030] The total volume of the droplets is determined using the droplet frequency and the average droplet volume.

[0031] Calculating the droplet volume of each droplet in the micro-droplet stream may include calculating the droplet volume of each imaged droplet in the stream. The method may include only imaging a portion of the droplets in the stream, so that the droplet volume will not be calculated for unimaged droplets.

[0032] Calculating the average drop volume may include calculating a fluctuating average of the drop volumes of the imaged drops and may include the most recently imaged drops.

[0033] Determining the total volume of the droplets may include determining the total volume of the droplets over a given period of time.

[0034] According to other aspects of the present disclosure, there is provided a method for generating a droplet stream, comprising:

[0035] providing a first droplet of fluid;

[0036] providing a carrier fluid;

[0037] forming a droplet emulsion comprising the first droplet fluid in the carrier fluid by providing a flow of the first droplet fluid and a flow of the carrier fluid to a droplet generation region of a microfluidic structure;

[0038] Using the method described above, determining the total volume of fluid within a series of droplets within the droplet emulsion; and

[0039] In response to determining that the total volume of the fluid within the series of droplets is greater than a predetermined threshold, the pressure of the flow of the carrier fluid circuit is increased to inhibit droplet generation. In this way, the user can specify the volume of emulsion they want to collect based on the sample size provided, and when this value is reached, the machine will stop generating emulsion.

[0040] According to another aspect of the present disclosure, there is provided a method for generating a droplet stream, comprising:

[0041] providing a first droplet of fluid;

[0042] providing a carrier fluid;

[0043] forming a droplet emulsion comprising the first droplet fluid in the carrier fluid by providing a flow of the first droplet fluid and a flow of the carrier fluid to a droplet generation region of a microfluidic structure;

[0044] Using the method described above, determining the droplet size of the continuous droplets within the emulsion; and

[0045] The pressure of the stream of the first droplet fluid and / or the pressure of the stream of the carrier fluid are adjusted in response to the determined droplet size.

[0046] This provides semi-automated droplet generation with real-time droplet frequency determination, size calculation, and image-based triggered closed-loop feedback control of droplet monodispersity. The method maintains the monodispersity of the droplet stream by controlling the pressure of the stream of the first droplet fluid or the flow of the carrier fluid in response to the determined droplet size.

[0047] The method of generating a stream of droplets may comprise varying the pressure of the stream of the first droplet fluid and varying the pressure of the stream of the carrier fluid such that a determined droplet size is substantially constant between consecutive droplets.

[0048] The methods disclosed herein can be scaled up to provide multiple parallel droplet generation areas on a microfluidic chip. Photodetector-based detection of the presence of a single droplet can be used to initiate triggered capture of droplet images at two or more designated areas within a microfluidic chip containing multiple parallel droplet generation areas. Droplet size data from one or more areas of interest can be used as input to an image-based closed-loop feedback program that regulates the monodispersity of emulsions generated from the microfluidic chip. The use of parallel droplet generator chips allows accurate droplet volume control between multiple microfluidic channels for ultra-high yield droplet production.

[0049] During a start time period of the method of generating a droplet stream, the method may include directing the droplet stream to a waste channel in response to the determined droplet size being outside a predetermined droplet size range. When the instrument is started, there may be a time period during which the droplets generated do not meet the user specified size requirements. Although the droplets generated do not meet the user specified size requirements, this will cause these droplets to travel to the waste channel.

[0050] When the correct droplet size is reached, the droplets can be prevented from entering the waste section and can instead be collected. This can be performed by closing a valve on the waste channel. The valve can be programmed to close over a predetermined period of time. By closing the valve of the waste channel relatively slowly, this allows the system to compensate for increased back pressure in the system without changing the droplet volume.

[0051] According to another aspect of the present disclosure, there is provided a method for generating a droplet stream, comprising:

[0052] providing a first droplet of fluid;

[0053] providing a carrier fluid;

[0054] providing a second droplet of fluid;

[0055] forming a droplet emulsion comprising a mixture of the first droplet fluid and the second droplet fluid in the carrier fluid by providing a flow of the first droplet fluid, a flow of the second droplet fluid, and a flow of the carrier fluid to a droplet generation region of the microfluidic structure;

[0056] measuring a flow rate of the first droplet fluid flow or the second droplet fluid flow;

[0057] determining the total volume of fluid within the droplets within the droplet emulsion using the method described above;

[0058] determining a ratio of the first droplet fluid to the second droplet fluid within the mixture using the measured flow rate and the total volume of the droplets; and

[0059] The pressure of the flow of the first droplet fluid or the flow of the second droplet fluid is adjusted in response to the determined ratio.

[0060] The use of image-based droplet volume determination and photodetector-based total aqueous flow determination, along with a flow sensor at one of the two droplet fluid inlets, enables the generation of monodisperse dual-aqueous droplets with a defined mixing ratio between the two aqueous streams.

[0061] Preferably, the flow sensor may be configured to measure the flow rate of the second droplet fluid, since the first droplet fluid may contain fragile biological cells or chemical components.

[0062] The method can also be used to maintain or control cell occupancy in the production of monodisperse droplets formed from two droplet fluids: one comprising cells or particles and the other being a diluent.

[0063] According to another aspect of the present disclosure, there is provided a method for generating a droplet stream, comprising:

[0064] Providing n droplets of fluid;

[0065] providing a carrier fluid;

[0066] forming a droplet emulsion comprising a mixture of the n droplet fluids in the carrier fluid by providing a flow of each of the n droplet fluids and a flow of the carrier fluid to a droplet generation region of a microfluidic structure;

[0067] Measuring the flow rate of n-1 streams of droplet fluid;

[0068] determining the total volume of fluid within the droplets within the droplet emulsion by the method described above;

[0069] determining a ratio of the n droplet fluids within the mixture using the measured n-1 flow rates and the total volume of the droplets; and

[0070] The pressure of one or more of the n streams of droplet fluid is adjusted in response to the determined ratio.

[0071] According to another aspect of the present disclosure, there is provided a microfluidic system, comprising:

[0072] a microfluidic droplet channel for conveying a stream of microfluidic droplets;

[0073] a light source for illuminating the stream of microfluidic droplets with a light beam comprising light in a wavelength range above a predetermined threshold wavelength and below a predetermined threshold wavelength;

[0074] a beam splitter configured to split light from the stream of microfluidic droplets into a first portion and a second portion, wherein the first portion includes light above the predetermined threshold wavelength, and wherein the second portion includes light below the predetermined threshold wavelength;

[0075] a camera configured to receive the first portion of the light;

[0076] a photodetector configured to receive the second portion of the light, wherein the aperture is located in front of the photodetector;

[0077] a device configured to process a signal from the photodetector; and

[0078] A processor is configured to determine a droplet frequency based on fluctuations in the processed signal.

[0079] The processor may be configured to detect a droplet using the processed signal, and the camera may be configured to capture an image of the droplet concurrently with the detection of the droplet.

[0080] The camera exposure time can be 1µs or less. This provides a high-speed area scan camera that can capture images of droplets in microfluidic channels.

[0081] The system may include a first droplet fluid circuit for delivering a first droplet fluid, a carrier fluid circuit for delivering a carrier fluid, and a droplet generation region having a first input for receiving a flow from the first droplet fluid circuit, a second input for receiving a flow from the carrier fluid circuit, and an output to the microfluidic droplet channel. The processor may be configured to determine an average droplet volume using the captured droplet image. The processor may also be configured to determine a total droplet volume for a series of droplets within the stream of microfluidic droplets using the average droplet volume and the droplet frequency. The system may also include means for increasing pressure in the carrier fluid circuit in response to the determined total droplet volume being greater than a predetermined threshold.

[0082] The system may include a first droplet fluid circuit for delivering a first droplet fluid, a carrier fluid circuit for delivering a carrier fluid, and a droplet generation region having a first input for receiving a flow from the first droplet fluid circuit, a second input for receiving a flow from the carrier fluid circuit, and an output to the microfluidic droplet channel. The processor may be configured to determine a droplet size of consecutive droplets using the captured droplet images. The system may also include means for adjusting a pressure in the carrier fluid circuit or the first droplet fluid in response to the determined droplet size.

[0083] The system may include n droplet fluid circuits each configured to deliver one of the n droplet fluids, a carrier fluid circuit for delivering a carrier fluid, and a droplet generation region having n inputs for receiving n streams from the n droplet fluid circuits, having other inputs for receiving streams from the carrier fluid circuits, and having an output to the microfluidic droplet channel, such that the droplet generation region forms a droplet emulsion comprising a mixture of the n droplet fluids in the carrier fluid. The system may also include n-1 flow sensors configured to measure the flow rates of n-1 of the n streams of the droplet fluids. The processor may be configured to determine an average droplet volume using the captured droplet images. The processor may also be configured to determine a total droplet volume within a series of droplets within the stream of the microfluidic droplets using the average droplet volume and the droplet frequency. The processor may also be configured to determine a ratio of the n droplet fluids within the mixture using the measured n-1 flow rates and the total droplet volume. The system may also include means for adjusting the pressure of one or more of the n streams of droplet fluid in response to the determined ratio.

[0084] The aperture may include a slit corresponding to a strip of light substantially perpendicular to a sidewall of the microfluidic droplet channel.

[0085] Alternatively, the aperture may comprise a pinhole corresponding to the light beam being located substantially in the center of the microfluidic droplet channel.

[0086] The aperture may comprise a mechanical slit that allows only a narrow band of light perpendicular to the sidewalls of the main microfluidic channel to reach the photodetector, thereby increasing detection sensitivity.

[0087] Alternatively, the aperture may comprise a crescent-shaped aperture configured to increase the sensitivity of the photodetector.

[0088] The first portion may include light with a wavelength greater than 488 nm, and the second portion may include light with a wavelength less than 488 nm.

[0089] The droplets may be microdroplets, nanodroplets or picodroplets, or may be larger or smaller.

[0090] According to another aspect of the present disclosure, there is provided a microfluidic method for preparing droplets, the method comprising:

[0091] providing an emulsion flow of microfluidic droplets of a first droplet fluid within a carrier fluid in a microfluidic channel;

[0092] determining a droplet size of droplets in the stream of microfluidic droplets;

[0093] providing a flow of a second droplet fluid to the microfluidic channel;

[0094] Merging a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet;

[0095] determining a droplet size of the fused droplet; and

[0096] In response to the determined fused droplet size, the pressure or flow rate of the stream of the emulsion of microfluidic droplets and / or the pressure or flow rate of the stream of the second droplet fluid are adjusted.

[0097] Image-based closed-loop feedback can be used to control the volume of a single microfluidic droplet of a first droplet fluid or the volume of a second droplet fluid that merges with a microfluidic droplet of the first droplet fluid.

[0098] The method allows the use of image-based closed-loop feedback to adjust the volume ratio of a single microfluidic droplet consisting of a mixture of two or more droplet fluids.

[0099] The method can be used to prepare droplets having a constant and adjusted droplet volume or volume ratio of the component parts, or can be used to adjust the droplet volume or volume ratio of the component parts to a desired or predetermined value.

[0100] The method can be used to generate a fused droplet having two or more components or containing a mixture of two or more fluids. The method can use an image of a droplet of a first droplet fluid captured before the droplet of the first droplet fluid is fused with the second droplet fluid, and an image of the fused droplet captured after the droplet of the first droplet fluid is fused with the second droplet fluid, to calculate the volume of each fluid component within a single fused droplet. The calculated volumes of the components or volume ratios of the components can then be used as inputs to a closed-loop feedback system that can generate a monodisperse emulsion of fused droplets.

[0101] The emulsion of microfluidic droplets of the first fluid may be provided from an external system, or may be prepared on a separate chip connected to a chip including a droplet fusion region, or may be prepared on the same chip as the droplet fusion region. In examples where microfluidic droplets of the first fluid are generated on the same chip or on a separate chip connected to a chip including a droplet fusion region, the method may include adjusting or adjusting the volume of the droplets of the first fluid generated prior to fusion, rather than adjusting or adjusting the volume of the droplets of the first fluid that are reinjected into the chip.

[0102] The droplet size of droplets in a stream of microfluidic droplets and / or the droplet size of fused droplets may be determined according to the method for determining droplet size described above.

[0103] Providing the flow of the second droplet fluid may include providing the flow of the second droplet fluid from a fluid inlet disposed on a sidewall of the microfluidic channel.

[0104] Merging a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet may include forming a fused droplet comprised of a mixture of the first droplet fluid and the second droplet fluid.

[0105] The method may also include determining a ratio of the first droplet fluid to the second droplet fluid in the mixture of the first droplet fluid and the second droplet fluid. A pressure or flow rate of the emulsion flow or the flow of the second droplet fluid may be adjusted in response to the determined ratio.

[0106] Merging a droplet of the first droplet fluid with at least a portion of the second droplet fluid may include applying an electric field between the first droplet fluid and a portion of the second droplet fluid. Applying the electric field may cause the droplet to merge with the end of the stream of the second droplet fluid at the junction of the microfluidic channel and the second droplet fluid inlet. This may then be broken into fused droplets by shear forces of the carrier fluid in the microfluidic channel.

[0107] Providing an emulsion flow of microfluidic droplets of a first droplet fluid within a carrier fluid in a microfluidic channel may include: providing a first droplet fluid; providing a carrier fluid; and forming a droplet emulsion including the first droplet fluid within the carrier fluid by providing a flow of the first droplet fluid and a flow of the carrier fluid to a droplet generation region of a microfluidic structure.

[0108] Adjusting the pressure or flow rate of the emulsion flow of microfluidic droplets may include adjusting the pressure of the flow of the first droplet fluid and / or the pressure of the flow of the carrier fluid in response to the determined droplet size.

[0109] The method may further include providing a flow of a spacing fluid to the microfluidic droplet channel. Adjusting the pressure or flow rate of the emulsion flow of the microfluidic droplets may include adjusting the pressure or flow rate of the flow of the spacing fluid in response to the determined droplet size.

[0110] According to another aspect of the present disclosure, there is provided a microfluidic system, comprising:

[0111] a droplet inlet for providing an emulsion flow of microfluidic droplets of a first droplet fluid in a carrier fluid;

[0112] a droplet sensing system for determining the size of droplets within the stream of microfluidic droplets;

[0113] a fluid inlet for providing a flow of a second droplet fluid;

[0114] a droplet fusion region having a first input for receiving a flow from the droplet inlet and having a second input for receiving a flow from the fluid inlet, and the droplet fusion region is configured to fuse a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet;

[0115] a second droplet sensing system for determining the size of the fused droplet; and

[0116] means for increasing the pressure or flow rate of the emulsion in the droplet inlet or the second droplet fluid in the fluid inlet in response to the determined size of the fused droplet,

[0117] The droplet sensing system or the second droplet sensing system may include the microfluidic system described above.

[0118] The fluid inlet may be arranged on a side wall of the microfluidic channel.

[0119] The fluid inlet may be substantially perpendicular to the microfluidic channel.

[0120] The droplet fusion region may include a device configured to fuse a droplet of the first droplet fluid with at least a portion of the second droplet fluid.

[0121] The apparatus configured to merge a droplet of the first droplet fluid with at least a portion of the second droplet fluid may include a plurality of electrodes configured to provide an electric field between a droplet of the first droplet fluid and a portion of the second droplet fluid.

[0122] The microfluidic system may also include: a first droplet fluid circuit for conveying a first droplet fluid; a carrier fluid circuit for conveying a carrier fluid; and a droplet generation area, the droplet generation area having a first input portion for receiving a flow from the first droplet fluid circuit, a second input portion for receiving a flow from the carrier fluid circuit, and an output portion to the microfluidic droplet channel.

[0123] The microfluidic system may further include a spacing fluid circuit configured to provide a flow of a spacing fluid to the microfluidic droplet channel.

[0124] According to another aspect of the present disclosure, there is provided a microfluidic method for forming micro-droplet pairs, comprising:

[0125] providing an emulsion flow of microfluidic droplets of a droplet fluid within a carrier fluid in a microfluidic channel;

[0126] determining a droplet size of droplets in the stream of microfluidic droplets;

[0127] dividing the droplet into a first droplet and a second droplet;

[0128] directing the first droplet to a first droplet channel;

[0129] directing the second droplet to a second droplet channel;

[0130] determining a droplet size of the first droplet or the second droplet;

[0131] providing a flow of a spacing fluid to the first droplet channel or the second droplet channel; and

[0132] In response to the determined size of the first droplet or the second droplet, the pressure or flow rate of the emulsion flow of microfluidic droplets and / or the pressure or flow rate of the flow of the spacing fluid are adjusted.

[0133] Directing the first droplet to the first droplet channel and / or directing the second droplet to the second droplet channel may include: changing the direction of the flow of the first droplet or the second droplet. Alternatively, directing the first droplet to the first droplet channel and / or directing the second droplet to the second droplet channel may include: allowing the first droplet or the second droplet to continue to flow in the same microfluidic channel and / or allowing the first droplet or the second droplet to continue to flow in the same direction.

[0134] The droplet size of the droplets in the stream of microfluidic droplets, and / or the droplet size of the first droplets, and / or the droplet size of the second droplets may be determined according to the methods described above.

[0135] Image-based closed-loop feedback may be used to control the volume of, or the volume distribution between, two or more separate sub-droplets produced by splitting a larger droplet into two droplets.

[0136] The method allows the use of image-based closed-loop feedback to regulate the volume of droplets produced by separating larger droplets using a droplet separation mechanism.

[0137] The method can be used to prepare two or more sub-droplets, each of which has a constant and adjusted droplet volume, or can be used to adjust the droplet volume of two or more sub-droplets to a desired value or a predetermined value. The method can be used to keep the volumes of both the first droplet and the second droplet consistent with each other.

[0138] The emulsion of microfluidic droplets can be provided from an external system, or can be prepared on a separate chip connected to a chip including a droplet fusion region, or can be prepared on the same chip as the droplet fusion region. In examples where the microfluidic droplets are generated on the same chip or on a separate chip connected to a chip including a droplet fusion region, the method can include adjusting or adjusting the volume of the droplets generated before separation, rather than adjusting or adjusting the volume of the droplets that are reinjected into the chip.

[0139] The microfluidic method may include determining a droplet volume of one of the first droplet or the second droplet, and may also include calculating a droplet volume of the other of the first droplet and the second stream using a previously determined droplet volume of the first droplet or the second droplet and a predetermined volume of droplets in the stream of microfluidic droplets.

[0140] Determining the droplet size may include capturing an image of the droplet using a droplet sensing system and determining the droplet size based on the captured image.

[0141] According to another aspect of the present invention, there is provided a microfluidic system, comprising:

[0142] a droplet inlet for providing an emulsion flow of microfluidic droplets of a droplet fluid in a carrier fluid;

[0143] a droplet sensing system for determining the size of droplets within a stream of microfluidic droplets;

[0144] a droplet separation region having a first input for receiving a stream from the droplet inlet and having a first output to a first droplet channel and a second output to a second droplet channel, wherein the droplet separation region is configured to separate a droplet in the stream of microfluidic droplets into a first droplet and a second droplet;

[0145] a fluid inlet for providing a flow of a spacing fluid to the first droplet channel or the second droplet channel;

[0146] a second droplet sensing system for determining a size of the first droplet or the second stream; and

[0147] A device for increasing the pressure or flow rate of the emulsion in the droplet inlet or the spacer fluid in the fluid inlet in response to the determined size of the first droplet or the second droplet.

[0148] The droplet sensing system and / or the second droplet sensing system may include the microfluidic system described above.

[0149] The first droplet channel and the second droplet channel may have different cross-sectional areas. This allows for the generation of sub-droplets of different sizes.

[0150] Alternatively, the first droplet channel and the second droplet channel may have substantially the same cross-sectional area.

[0151] The microfluidic system may further include a spacing fluid circuit configured to provide a flow of a spacing fluid to the droplet inlet.

[0152] The droplet sensing system and / or the second droplet system includes a camera configured to capture an image of the droplet and a device for determining the size of the droplet from the captured image of the droplet. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Some embodiments of the present disclosure will now be described, by way of example only, and with reference to the accompanying drawings, in which:

[0154] Figure 1 Schematic diagram of the optical components used to image a microfluidic chip;

[0155] Figure 2 Shows Figure 1 Examples of optical components;

[0156] Figure 3 schematically illustrates a region of interest in a captured image of a droplet within a microfluidic channel;

[0157] Figure 4 The droplet generation area is schematically shown;

[0158] Figure 5 Schematically shows Figure 4 Flow focusing junction and Y-junction in the droplet generation region are shown;

[0159] Figure 6 A droplet generation region for generating a double aqueous droplet emulsion is schematically shown;

[0160] Figure 7 Schematically shows Figure 6 Flow focusing junction and Y-junction in the droplet generation region are shown;

[0161] Figure 8 An exemplary droplet fusion region is shown;

[0162] Fig. 9 The droplet reinjection region and the droplet fusion region are schematically shown;

[0163] Fig.10 Schematically shows Fig. 9 The droplet fluid inlet of the droplet fusion region is shown;

[0164] Fig.11 The droplet generation region and the droplet fusion region are schematically shown;

[0165] Fig.12 Schematically shows Fig.11 The droplet fluid inlet of the droplet fusion region is shown;

[0166] Fig.13 schematically showing a droplet reinjection region and a droplet separation region; and

[0167] Fig.14 The droplet separation area is schematically shown, such as Fig.13 The droplet separation region is shown. DETAILED DESCRIPTION

[0168] Figure 1 Schematic diagram of the optical components used to image a microfluidic chip. Figure 2 Shows Figure 1 A side view of an example of an optical assembly.

[0169] The optical assembly includes a microfluidic chip 102 having microfluidic channels that convey a stream of microfluidic droplets. The droplets can be used Figures 4 to 6 The system shown produces: LED 104 attached to optical lens tube assembly 106 is configured to illuminate microfluidic chip 102 with light in the visible spectrum. Objective lens 108 collects light from microfluidic chip 104 and directs the light to dichroic beam splitter 110.

[0170] The beam splitter 110 is configured to reflect a first portion of the light from the beam splitter 110 to the photodetector 112. The beam splitter 110 is configured to transmit a second portion of the light from the beam splitter 110 to the high speed camera 116.

[0171] In this example, LED 104 is a white LED, and beam splitter 110 is a dichroic mirror that splits the focused image of the droplet stream into a portion of light above or below a predetermined wavelength (in this example, the wavelength is 488 nm, so that blue light is directed to the photodetector). Beam splitter 110 directs about 20% of the light to the photodetector and 80% of the light to the camera, but the split ratio can be changed. The camera is less sensitive in the blue region of visible light than in the red and green regions of visible light, so the dichroic mirror directs the green and red portions of visible light to the camera to increase sensitivity. Blue light is directed to the photodetector.

[0172] Light with wavelengths below a predetermined wavelength (488 nm in this example) is reflected by the dichroic mirror 110 to the photodetector 112. An aperture 114 is located between the beam splitter 110 and the photodetector 112. The aperture 114 may be a mechanically adjustable slit (referred to as a slit iris), a circular iris, a narrow rectangular aperture, or a crescent-shaped aperture, which is configured to allow only a narrow band of light from the main microfluidic channel to reach the photodetector.

[0173] Each droplet in the droplet stream includes a first droplet suspended in a carrier fluid. The droplet fluid can be particles in an aqueous liquid, and the carrier fluid can be a continuous oil phase. When the interface between the droplet and the carrier fluid passes through the photodetector 112, the voltage signal of the photodetector decreases due to light scattering. Once the droplet passes through the detector 112, and only the continuous oil phase is visible to the photodetector 112, the voltage increases and returns to the background signal. Each passing droplet in the microfluidic channel will show a decrease in the amplitude of the voltage signal measured by the photodetector because the light intensity is scattered by the passing droplet in an instant. The occurrence rate and repetitiveness of this voltage reduction over time (i.e., a time scale of 1 s) (which is a characteristic of each of the individual flowing droplets present) are then used to calculate the droplet frequency. This arrangement improves the detection sensitivity of the photodetector and enables real-time continuous measurement of the droplet generation frequency using customized, automatic frequency detection software.

[0174] Light with wavelengths above a predetermined wavelength (488 nm in this example) is transmitted through the dichroic mirror 110 to the sensor of the high-speed area scan camera 116. To ensure that the droplets are imaged at approximately the same area within the microfluidic channel, the photodetector voltage signal output is received and processed on the microprocessor. Upon receiving the photodetector voltage signal output, the microprocessor sends a trigger signal to cause camera image acquisition of the skin droplets using ultra-short exposure imaging. For example, when a passing skin droplet causes the measured voltage signal amplitude to decrease, exceeding a user-defined threshold, a software process is triggered that first waits for a set delay time and then triggers the camera to acquire an image with an exposure time of 1 µs or less.

[0175] By using an automated software process that begins after the automated droplet initiation procedure is completed, the channel sidewalls and droplet centers contained in the camera image are detected to define a region of interest (ROI) in the captured droplet image, e.g. Figure 3 The use of an automated software-based approach to define ROIs eliminates much of the error typically associated with manual positioning of ROIs by human users, thereby improving the precision of the droplet size estimation method and increasing confidence in measurements between different experiments.

[0176] Figure 3 Schematically illustrates a region of interest in a captured image of a droplet 320 within a microfluidic channel 322. The region of interest 326 is a line region substantially parallel to the sidewalls 324 of the microfluidic channel 322 and including the center of the droplet. The region of interest 326 will have two negative peaks 328 corresponding to the front and rear boundaries of the droplet 320, where light is scattered at the interface between the droplet 320 and the carrier fluid 330. The distance l extending between the peaks 328 corresponds to the diameter of the droplet 320.

[0177] The ROI 326 can be defined in the form of a single-pixel-high horizontal line that is parallel to the microchannel sidewalls 324 and horizontally intersects the imaged skin droplets 320 through their respective centers. Using only a single-pixel-high line of image data, rather than a complete image, significantly reduces processing time and thus increases the possible throughput of the system.

[0178] The image data from the first defined horizontal line ROI 326 is subtracted from the mean of the same data so that any differences above the mean can be detected in downstream image processing. The processed data is then analyzed using peak detection software to identify two negative peak minima 328 for each imaged skin droplet 320, which are associated with the front and rear boundaries of the skin droplet (e.g., Figure 3 The peak to peak distance or length (l) is then a measure of the diameter of the detected pico droplet 320, from which the droplet volume can be estimated.

[0179] Image data from a ROI may also be processed using different sets of applied thresholds, which when subtracted from the average of the same data, may be used to identify the location, number, size and / or morphology of biological cells or objects contained within the droplets.

[0180] The microfluidic channel may include a marker (not shown) of known size. The captured image may include the droplet and the marker, and the size of the droplet within the captured image may then be compared to the marker located in the microfluidic channel or in an area near the microfluidic channel to determine the physical droplet size. This may be performed in conjunction with determining the ROI, or may be used instead of determining the peak from the ROI.

[0181] The optical assembly can be arranged for an inverted microscope, such as Figure 2 As shown in . The inverted microscope can be a semi-automatic inverted microscope with white LED illumination, in which the image of the microfluidic chip mounted on an x, y translatable stage 218 can be continuously and simultaneously relayed to a silicon free space amplified photodetector and a high-speed area scan camera. The translatable stage 218 can also be rotatable (θ). The LED is mounted in an LED holder 204 that is adjustable in the z direction. The objective lens 208 in the inverted microscope shown in this example is adjustable along the z axis to allow the image of the microchip to be formed through the lens 232. In this example, the focal length of the lens 232 is 100 mm. The microfluidic chip can be as shown. Figures 4 to 7The droplet generation chip shown includes microfluidic droplet generation and biological cell / particle coated output or collection channels. The system is capable of downstream signal processing and subsequent adjustment of the monodispersity of the generated emulsion containing picodroplets.

[0182] The instrument can be semi-automated; a human user can assemble the microfluidic chip to the macrofluidic connections and place the assembled fixture on the instrument stage, then navigate through sequential, automated steps operated by a custom software-based workflow.

[0183] Figure 4 Schematically shows that Figure 1 The optical components are used together with the droplet generation region 440 on the microfluidic chip. Figure 4 A view of a single dispersed phase inlet droplet generation region is shown, which has a Y-shaped sorting channel leading to a waste channel and / or a collection channel. The droplet generation region includes an aqueous sample inlet channel 442 for generating an emulsion and a carrier oil inlet channel 444. These liquid streams are provided to a flow focusing junction 446, where an emulsion is generated and provided to a collection channel 448 of the droplet generation region 440. The aqueous inlet fluid is coated in an immiscible fluorinated sheath oil at the flow focusing junction 446 to generate water-in-oil skin droplets. To provide some illustrative numbers by way of example, the flow rate of the carrier oil can be 1400μl per hour, the flow rate of the aqueous sample (e.g., a cell suspension) can be 1000μl per hour, and the flow rate of the water-in-oil emulsion in the collection channel can be 2400μl per hour, including 700 picolitres of droplets at 1000Hz.

[0184] The sample inlet channel 442 is connected to a first pressure controller (not shown), and the carrier fluid inlet channel 444 is connected to a second pressure controller (not shown). The first pressure controller and the second pressure controller can be used to control the pressure in the sample fluid inlet channel 442 or the carrier fluid inlet channel 444.

[0185] In this example, the sample and carrier fluid for each of the continuous and dispersed phases are delivered to the aqueous sample inlet channel 442 and the carrier oil inlet channel 444 via flexible tubing connected to a gas pressurized fluid reservoir, which in turn is actuated via a fast-acting pressure regulator. With automated software adjustment of the objective focus (z-direction) level via a stepper motor associated with the objective focus, an automated droplet generation initiation software process is then used to increase pressure to control fluid flow in both the aqueous sample inlet channel 442 and the carrier oil inlet channel 444 to initiate droplet generation.

[0186] The calculated droplet size (as described above for Figure 3 As discussed above, the emulsion may be used as an input to a feedback loop that adjusts the input pressure of one or more fluid inlet lines 442, 444 in response to the calculated droplet size to maintain monodispersity of the droplets within the emulsion.

[0187] One includes Figure 1 The optical assembly system shown in FIG. Figure 4 The microfluidic chip with a droplet generation area can perform the automated process steps discussed above to initiate triggered capture of droplet images at designated areas within the microfluidic channel using a photodetector based detection of the presence of a single droplet in the microfluidic stream. The image can then be used to calculate the average droplet size or the size of a single droplet, and the calculated droplet size data is input into a closed-loop feedback loop that maintains the monodispersity of a single emulsion by controlling the pressure of the sample fluid inlet channel 442 or the carrier fluid inlet channel 444 in response to the calculated droplet size data.

[0188] Then, an image-based closed-loop feedback routine is initiated whereby, as previously described, image data from the ROI is acquired from successive images and processed to measure droplet size and subsequently adjust input pressure to one or more input fluid lines to maintain droplet monodispersity within the emulsion during the emulsion generation run.

[0189] The product of the average frequency during a given image acquisition period and the average droplet volume during the same period is a good approximation of the sample volume consumed in the droplets, and the sum can be continuously updated. Using the determined average skin droplet volume and the average skin droplet generation frequency derived from the photodetector, the volume of droplets in the generated emulsion can be calculated. When the volume of the generated emulsion equals or exceeds the target volume specified by the user, the automated software is activated, which first increases the pressure of the carrier oil inlet channel 444 to restrict other fluid flows from the aqueous sample inlet channel 442, and secondly clears the last generated skin droplets from the microfluidic chip to the emulsion reservoir to make the generated emulsion physically available to the user. In this way, the user can specify the volume of emulsion they want to collect based on the sample size provided, and when this value is reached, the machine will stop generating the emulsion.

[0190] In other embodiments not shown, the microfluidic chip may include multiple Figure 4A droplet generation region of the droplet generation region shown (e.g., a microchip may have eight or more droplet generation nozzles, each of which leads to one of eight or more parallel collection channels). An optomechanical device may be located immediately after the dichroic beam splitter to send light (wavelength <488 nm) from two or more independent, parallel channels to independent photodetector detection chips to trigger droplet imaging and also collect frequency data independently of the two or more parallel channels. The image processing method described above for identifying the side walls of the microchannel and the center of the generated droplet and defining a horizontal line ROI parallel to the included microchannel side wall is used, but the method is scaled up so that at least two of the eight or more parallel channels have independent ROIs defined by image processing. Droplet size data from each independent ROI is used to extract droplet size data from multiple channels, which in turn is used to adjust an image-based closed-loop feedback adjustment to the input pressure to affect droplet size. This provides parallel single emulsion droplet generation with image-based closed-loop feedback.

[0191] In another embodiment not shown, the parallel collection channels can each subsequently converge into a common downstream outlet channel. An optomechanical device can be located after the dichroic beam splitter to send light (wavelength <488nm) from the common downstream outlet channel (to which each of the upstream parallel channels converges) toward one or more independent photodetector detection chips to trigger droplet imaging and also collect frequency data. The image processing methods described above for identifying the side walls of the microchannels and the center of the droplets produced and defining horizontal line ROIs parallel to the included microchannel side walls are used so that the common outlet channel can have one or more independent ROIs defined by image processing.

[0192] like Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown, the flow combining junction 446 is also coupled to a waste channel 452 . Figure 5 A flow focusing junction 446, a serpentine channel 447, and a Y-shaped sorting junction with channels 454, 456 leading to Figure 4The waste section 452 and the collection section 448 within the droplet generation area of ​​the serpentine channel 447 are disposed within the droplet generation area of ​​the serpentine channel 447. The emulsion formed at the flow focusing junction 446 flows through the serpentine channel 447 and flows to a Y-junction having two output sections 454, 456. When the instrument is started, there may be a period of time when the droplets generated do not meet the size requirements specified by the user. Typically, this may be a period of 10 seconds or more. The serpentine channel 447 is a sinusoidal shaped channel that mixes the contents of the droplets by causing a tumbling effect within the droplets as they traverse the serpentine channel 447. In the example shown, the width of the serpentine channel 447 is 90µm.

[0193] The first output 454 has a narrower width and is located between the flow focusing junction 446 and the collection channel 448. The second output 456 has a larger width and is located between the flow focusing junction 446 and the waste channel 452. In the example shown, the width of the first output 454 is 90µm and the width of the second output is 120µm. Although the droplets being produced do not meet the user-specified size requirements, the microfluidic chip will cause these droplets to travel to the waste channel 452, and only when the correct droplet size is reached will the memory shape valve (not shown) on the waste channel 452 be closed. In one example, the valve has been programmed to close within a period of 1 second, but the closing speed can be changed. By closing the valve of the waste channel 452 relatively slowly, this allows the system to compensate for increased back pressure in the system without changing the droplet volume.

[0194] In this example, the sample fluid inlet 442 and the carrier fluid inlet 444 are spaced relatively far from each other and from the collection channel 448 and the waste channel 452. This allows a cell or oil sample reservoir (such as a syringe body) to be mounted directly to the chip using a Luer-Lock fitting.

[0195] Figure 6 A droplet generation region 540 for the generation of an aqueous double emulsion is schematically shown. Figure 6 A diagram showing a bidisperse droplet generator sorting chip design for producing bidisperse droplet emulsions, Figure 7 446, a serpentine channel 447, a Y-shaped sorting junction with channels 454, 456 leading to Figure 6 The waste portion 452 and the collection portion 448 of the droplet generating area 540 are provided.

[0196] The serpentine channel region 447 is a sinusoidal shaped channel located between the flow focusing junction 446 and the Y-junction. This facilitates droplet mixing of the bidisperse phase mixture within the droplet.

[0197] Aqueous double droplets are droplets formed from a mixture of two aqueous fluids. The fluids may both include sample fluids, or one of the fluids may be a sample fluid and the other fluid may be used to dilute the sample fluid of the mixture within the droplet. The droplet generation region 440 is similar to Figure 4 The droplet generation area shown in FIG. 4 is similar to the droplet generation area shown in FIG. 4 , but the droplet generation area also includes a second sample fluid inlet 556. The two-layer stream of aqueous inlet fluid can converge before the stream focusing junction 446 and be coated in an immiscible fluorinated sheath oil at the droplet generation nozzle to produce individual water-in-oil skin droplets. A third pressure controller 558 is used to control the pressure of the fluid flow in the second sample fluid inlet 556 from an additional reservoir containing a second aqueous sample.

[0198] Liquid flow sensors (not shown) are placed to measure flow along one or more of the aqueous inlets 442, 556. By measuring the total aqueous sample flow rate, the calculated average droplet volume, and the droplet frequency of at least one aqueous inlet line, the mixing ratio between the two aqueous inlet samples within the formed droplets can be estimated in real time. This is performed by:

[0199] (i) Multiply the droplet frequency of the picodroplets (also called the droplet generation rate) by the rolling average of the calculated picodroplet volumes to calculate the total volume flow rate of the system (e.g., in µL s -1 ),as well as;

[0200] (ii) minus a sensor-based volume flow rate of an aqueous inlet line 442, 556 measured using a flow sensor, and

[0201] (iii) Calculate the following ratio: flow sensor-based volume flow rate of a single aqueous inlet line / (total volume flow rate of the system - flow sensor-based volume flow rate of a single aqueous inlet line).

[0202] The first pressure controller and the third pressure controller can be used to control the pressure within the sample fluid inlet 442, 556 in response to the estimated mixing ratio within the formed droplet. This allows real-time adjustment of the mixing ratio of the two sample fluids within the droplet. This provides an image-based closed-loop feedback procedure to produce a monodisperse single emulsion of aqueous skin droplets in a fluoro-oil-based sheath fluid, with a user-defined mixing ratio between the two inlet aqueous fluids and a user-defined skin droplet volume. In addition, the mixing ratio can be maintained at a constant level, or the mixing ratio can be defined to vary at certain times.

[0203] Image analysis based on ROIs in the images of the generated skin droplets can be used to count the number of particles (including biological cells) contained within each individual skin droplet. The average biological cell / object occupancy of the droplets can be monitored. The first pressure controller and the third pressure controller can be used to control the pressure within the sample fluid inlet 442, 556 in response to the average biological cell / object occupancy of the droplets. In this way, the device is provided with a closed-loop feedback system, in which the average biological cell / object occupancy of the droplets is used as an input to the feedback loop to adjust the mixing ratio between the two inlet aqueous fluids (for example, the first aqueous sample fluid can be a particle or cell loading solution, and the second aqueous sample fluid can be a compatibility buffer solution). Adjustment of this mixing ratio allows the average particle or cell occupancy to be controlled during the experimental run to compensate for particle or cell sedimentation, high particle or cell concentrations, or other forms of drift.

[0204] Droplet fusion

[0205] Figure 8 An exemplary droplet fusion region 610 is shown. The droplet fusion region 610 may be Figure 1 The droplet fusion region 610 includes a microfluidic droplet channel 612 for conveying an emulsion stream of microdroplets 616 in a carrier fluid. The droplets arrive at a first end or first input of the microfluidic channel 612. A droplet fluid inlet 614 is located downstream of the first input and provides a stream of droplet fluid 618 to the microfluidic droplet channel 612. Figure 8 In the example shown, the droplet fluid inlet 614 is arranged as a second microfluidic channel, which is arranged substantially perpendicular to the microfluidic droplet channel 612 .

[0206] The first drop sensor 622 is located upstream of the droplet fusion region 610 and detects and determines the drop volume of a single droplet 616 within the emulsion stream. The second drop sensor 624 is located downstream of the droplet fusion region and detects the volume of the fused droplet 620. The first drop sensor 622 and the second drop sensor 624 may each include a sensor such as Figure 1 and Figure 2 The volume of a droplet can be determined using methods related to droplet generation, such as Figures 4 to 7 The microfluidic chip shown is used in conjunction with the method.

[0207] The pressure or flow of droplet fluid 618 in droplet fluid inlet 614, or the pressure or flow of the emulsion within microfluidic channel 612, can be increased or decreased based on the droplet volume measured in a closed-loop feedback method to increase or decrease the fluid ratio within fused droplet 620.

[0208] The droplet fusion region 610 may be placed at Figures 4 to 7 The droplets are shown downstream of the droplet generation regions 440 , 540 , and the droplets generated by the droplet generation regions 440 , 540 may be input into the droplet fusion section 610 .

[0209] The droplet fusion region 610 can be used to fuse droplets 616 formed from a first droplet fluid within an emulsion with a second droplet fluid 618 to form an emulsion of fused droplets 620, each fused droplet containing a mixture of the first droplet fluid and the second droplet fluid. Image-based closed-loop feedback can be used to change the ratio of the mixture of the first droplet fluid and the second droplet fluid within the fused droplet to allow the ratio to be adjusted or maintained at a substantially constant optimal value.

[0210] An example method of using the droplet fusion region 610 to fuse the droplet 616 with the second droplet fluid 618 is as follows, with the steps being performed in the following order:

[0211] (1) providing an emulsion stream comprising a series of dispersed microfluidic droplets 616 of a first droplet fluid in a continuous fluid phase to a microfluidic channel 612;

[0212] (2) imaging a droplet 616 flowing in a microfluidic channel 612 using a droplet sensor 622 disposed in a droplet detection region;

[0213] (3) Calculating the size or volume of the droplet 616 based on the captured image. Alternatively, the droplet size or volume may be known or obtained using an alternative method. For example, if the total volume of the input droplet 616 is known and the droplets are monodisperse or uniform in size, the droplet volume may be calculated without imaging the droplets;

[0214] (4) The emulsion flows through the microfluidic channel 612 to the droplet fluid inlet 614;

[0215] (5) introducing the second droplet fluid 618 from the droplet fluid inlet 614 into the microfluidic channel 612, so that the main body of the second droplet fluid 618 extends from the droplet fluid inlet 614 into the microfluidic channel 612, as shown in FIG. Figure 8 (a) as shown;

[0216] (6) As the emulsion flows further through the microfluidic channel 612, there is a physical contact point or collision point between the droplet 616 and the second droplet fluid 618, such as Figure 8 (b) The physical contact point or collision point is located in the area adjacent to the droplet fluid inlet 614 in the microfluidic channel 612;

[0217] (7) Merging the droplet 616 and the second droplet fluid 618. The existing method of fusing the fluids destroys the structure of the fluid boundary between the droplet of the first fluid 616 and the protruding body of the second droplet fluid 618 at the collision point, so that they merge or mix into a single body of fluid protruding from the droplet fluid inlet 614, such as Figure 8 (c) The two fluids are then merged due to the pressure buildup in the microfluidic droplet channel, as described in step (8).

[0218] Alternatively, disruption of the fluid boundaries of the droplets of the first fluid and the second fluid may be induced by an external stimulus, thereby causing coalescence. The external stimulus may be an electrical stimulus or an optical stimulus. Electrical coalescence involves fusing droplets by applying an electric field as the droplets pass through a confined area defined by a pair of electrodes. Optical methods involve using a focused laser beam to locally heat the fluid boundaries, thereby changing the surface tension of the droplet interface.

[0219] exist Figure 8 (c), the single fluid body of fluid flowing out of the droplet fluid inlet 614 into the microfluidic channel 612 includes a mixture of both the first droplet fluid and the second droplet fluid;

[0220] (8) As the emulsion upstream of the droplet fluid inlet 614 continues to flow toward the droplet fluid inlet 614, the pressure of the emulsion increases after the individual fluid bodies flow out (protrude) from the droplet fluid inlet 614. This accumulation of stress or pressure after the fluid bodies flow out can cause the fused microdroplets 620 from the droplet fluid inlet 614 to break apart, as shown in FIG. Figure 1 As shown in (d). The fused droplet 620 may be a double aqueous droplet, and includes a mixture of the first droplet fluid and the second droplet fluid, and allows a portion of the emulsion flow as the fused droplet to continue to flow downstream;

[0221] (9) imaging the fused droplet 620 flowing in the microfluidic channel 612 using a droplet sensor 624 disposed in a second droplet detection region downstream of the droplet fluid inlet 614;

[0222] (10) calculating the droplet size or volume based on the captured image of the fused droplet 620;

[0223] (11) Using the images captured by the droplet sensors 622 and 624, the volume change between the initial micro-droplet 616 and the fused droplet 620 is determined, and the volume contribution of the first droplet fluid and the second droplet fluid in each droplet in the fused droplet stream or the ratio of the first droplet fluid to the second droplet fluid is calculated.

[0224] The volume or size values ​​calculated from the images captured by the first droplet sensor 622 and the second droplet sensor 624 in steps (4) and (10), or the ratio of the first droplet fluid to the second droplet fluid calculated in step (11), can be used as input to a closed-loop feedback procedure. Here, the experimental input (e.g., driving pressure or flow rate) of the emulsion flow provided to the microfluidic channel or the flow of the second droplet fluid provided in the droplet fluid inlet is adjusted in response to the calculated volume or ratio so as to maintain the droplet parameters at a predetermined level (e.g., final fused droplet volume, volume contribution, fluid ratio) when generating the emulsion flow of the fused droplet 620. By merging the microdroplets of the first droplet fluid with the second droplet fluid before generating the droplets, rather than mixing the two droplet fluids, the ratio of the first droplet fluid to the second droplet fluid can be more easily controlled.

[0225] For example, the image-based closed-loop feedback procedure can be used to adjust and maintain the droplet volume of droplet 616 in the initial droplet emulsion by changing the driving force of the first fluid stream. This is because the flow rate of the droplet of the first fluid will affect the time interval between the droplet collision with the protruding component of the second fluid, thereby affecting the amount of mixing of the second fluid with the droplet. If the flow rate of the droplet is lower, it will have more time to maintain contact with the protruding component of the second fluid, and the second fluid will mix more with the droplet. The image-based closed-loop feedback procedure can be used to adjust the driving force or pressure of the stream of the second droplet fluid in response to the volume contribution or ratio of the fluid within the droplets in the merging droplet stream deviating from a predetermined level.

[0226] Fig. 9 Schematic diagram showing the droplet reinjection region and the droplet fusion region. Fig.10 Schematically shows Fig. 9 The droplet fluid inlet of the droplet fusion region is shown.

[0227] The droplet fusion region includes a droplet inlet 818 to provide microdroplets formed in an emulsion including a first droplet fluid in an immiscible carrier fluid. The droplet inlet 818 provides droplets to a microfluidic droplet channel 812. Droplets in the microfluidic droplet channel 812 flow toward a joint between the microfluidic droplet channel 812 and a droplet fluid channel 816 through which a second droplet fluid flows from the droplet fluid inlet 814 to the microfluidic droplet channel 812. The droplet fluid channel 816 is arranged so that it is substantially perpendicular to the microfluidic droplet channel 812 at a joint between the droplet fluid channel 816 and the microfluidic droplet channel 812. The droplets in the microfluidic droplet channel 812 merge with the second droplet fluid provided from the droplet fluid inlet 814 in a manner similar to that of the droplet fluid inlet 814. Figure 8 Fusion was performed in the same manner as described.

[0228] In this example, two electrodes 824 (a ground electrode and a charged electrode) are located near the junction between the microfluidic droplet channel 812 and the droplet fluid channel 816. The electric field between the two electrodes 824 causes the second droplet fluid to merge with the microdroplet, as described with respect to Figure 8 as described.

[0229] The fused droplet flows from the microfluidic droplet channel 812 through the fused droplet channel 828 to the fused droplet outlet 826. In this example, the fused droplet channel 828 has a serpentine portion that helps mix the first droplet fluid and the second droplet fluid within the fused droplet. The shape of the fused droplet channel 828 is designed so that after mixing, the fused droplet can be imaged in the field of view of the camera.

[0230] exist Fig. 9 and Fig.10 A camera is arranged outside the plane of the system shown, which images area C so that droplets can be detected at points A and B. Figure 8 As described, the size of the fused droplet or the ratio of the first droplet and the second droplet fluid within the fused droplet can be controlled by changing the pressure and / or flow rate of the droplet from the droplet inlet 818, the second droplet fluid from the droplet fluid inlet 814, or the spacer fluid from the spacer fluid inlet 820 in response to the camera imaging the droplets at positions A and B.

[0231] In this example, a spacing fluid inlet 820 provides a spacing fluid to the microfluidic droplet channel 812 through a spacing fluid channel 822 disposed on the sidewall of the microfluidic droplet channel 812 between the droplet inlet 818 and the droplet fluid channel 816. The spacing fluid increases the spacing between droplets in the microfluidic droplet channel 812, thereby allowing control of the rate at which droplets arrive at the junction with the droplet fluid channel 816. The reinjection region refers to the droplet inlet 818 and the spacing fluid inlet 820, which is used to provide a spacing fluid or additional carrier fluid to the microfluidic channel through the spacing fluid channel 822.

[0232] Fig.11 The droplet generation area and the droplet fusion area are schematically shown. Fig.12 Schematically shows Fig.11 The droplet fluid inlet of the droplet fusion region is shown. The droplet generation region is similar to Figure 4 The droplet generation region is shown. Many features are similar to Fig. 9 and Fig.10 The features shown are identical and therefore have similar reference numerals.

[0233] The droplet generation region includes a first droplet fluid inlet 930 and a carrier fluid inlet 920 that provide a first droplet fluid, such as an aqueous sample, through a first droplet fluid channel 932 and a carrier fluid channel 922. These liquid streams are provided to a stream focusing junction 946 where an emulsion is created and provided to the microfluidic droplet channel 912. The aqueous inlet fluid is coated in an immiscible fluorinated sheath oil at the stream focusing junction 946 to produce a water-in-oil skin droplet emulsion.

[0234] The droplets in the emulsion are Figures 8 to 10 The droplet in the microfluidic droplet channel 912 flows to the joint between the microfluidic droplet channel 912 and the second droplet fluid channel 916 , and the second droplet fluid flows from the droplet fluid inlet 914 to the microfluidic droplet channel 912 through the joint.

[0235] Fig.12 The system allows control of the volume of a droplet containing only a first droplet fluid, so that the ratio of the first droplet fluid to the second droplet fluid in the fused droplet can be controlled using closed-loop feedback by adjusting the pressure and / or flow rate of the first droplet fluid and the carrier fluid without adjusting the pressure and / or flow rate of the second droplet fluid.

[0236] Droplet separation

[0237] Fig.13 The droplet reinjection region and the droplet separation region are schematically shown. The system includes a droplet inlet 711 for providing microdroplets formed in an emulsion, the emulsion comprising a first droplet fluid in an immiscible carrier fluid. The droplet inlet 711 provides droplets to a microfluidic droplet channel 718 through a droplet inlet channel 712.

[0238] The droplets in the microfluidic droplet channel 718 flow to the joint between the microfluidic droplet channel 718 and the droplet separation nozzle, such as Fig.14 Just as described.

[0239] In this example, the carrier fluid inlet 732 provides additional carrier fluid or spacer fluid to the microfluidic droplet channel 718 through a carrier fluid channel 734 disposed on the sidewall of the microfluidic droplet channel 718 between the droplet inlet 711 and the droplet separation nozzle. The spacer fluid increases the spacing between droplets in the microfluidic droplet channel 718, thereby allowing control of the rate at which droplets reach the droplet separation nozzle.

[0240] Can be Fig.13 The system shown may include a camera located outside the plane, or the system may include multiple sensors, such as Fig.14 As shown in the figure, Fig.14As described, the size of the sub-droplets can be controlled by varying the pressure and / or flow of droplets from droplet inlet 711, additional carrier fluid from carrier fluid inlet 732, or spacing fluid from spacing fluid inlet 720 in response to imaging of the droplets by the camera.

[0241] The droplet separation area or droplet separation nozzle and Fig.14 The same as shown below will be combined Fig.14 Give a description.

[0242] Fig.14 An exemplary droplet separation region 710 is schematically shown. The droplet separation region 710 includes a droplet inlet channel 712 for providing an emulsion flow of microdroplets 726 in a carrier fluid. The droplet separation region 710 has two outputs located downstream of the droplet inlet 712 and formed by two sub-droplet microfluidic channels 714, 716. In the example shown, there is a microfluidic droplet channel 718 between the droplet inlet channel 712 and the sub-droplet channels 714, 716. The microfluidic channel 718 is divided into two droplet channels 714, 716 at a single joint, so that a larger droplet 726 is divided into two smaller droplets 728, 730. The joint that divides the microfluidic channel 718 into two smaller droplet channels 714, 716 can be a droplet separation nozzle.

[0243] The microfluidic droplet channel 718 may be narrower than the droplet inlet 712. The two droplet channels 714, 716 may have narrower cross-sections closer to the junction with the microfluidic droplet channel 718.

[0244] In the example shown, the two sub-droplet channels 714, 716 have unequal cross-sectional widths. The first droplet channel 714 has a larger cross-sectional width than the second droplet channel 716, so that the input droplet 726 is split into unequal droplets 728, 730, wherein the larger droplet 728 is formed at the first droplet channel 714 and the smaller droplet 730 is formed at the second droplet channel 716.

[0245] A first drop sensor or camera 722 is located upstream of the drop separation region 710 or at the drop inlet 712 and detects and determines the drop volume of a single drop 726 in the emulsion stream. A second drop sensor or camera 724 is located downstream of the drop separation region and detects the drop volume in the first drop channel 714 or the second drop channel 716. The first drop sensor 722 and the second drop sensor 724 may each include a sensor such as Figure 1 and Figure 2 The volume of the droplet can be determined using the methods described with respect to droplet generation, such as using Figures 4 to 7 The microfluidic chip shown was used in conjunction with the method to determine.

[0246] The carrier fluid inlet 720 or pressure regulating channel provides carrier fluid to the first droplet channel 714. The pressure or flow rate of the carrier fluid in the carrier fluid inlet 720 can be increased or decreased based on the measured droplet volume in a closed-loop feedback method to adjust the size of the generated sub-droplets 728, 730.

[0247] Although in the example shown, one carrier fluid inlet 720 provides carrier fluid to the first droplet channel 714 , alternatively, a separate carrier fluid inlet may provide carrier fluid to the second droplet channel 716 as an alternative to or in addition to providing carrier fluid to the first droplet channel 714 .

[0248] An example method for dividing the droplet 726 into two smaller droplets 728, 730 is as follows, with the steps being performed in the following order:

[0249] (1) providing an emulsion stream comprising a series of dispersed microfluidic droplets 726 in a continuous fluid phase to a microfluidic channel 718. The droplets may be provided from a droplet inlet 712;

[0250] (2) using a droplet sensor 722 disposed in a droplet detection region to image a droplet 726 flowing in a microfluidic channel 718. Alternatively, the droplet 726 is imaged before it flows into the microfluidic channel 718;

[0251] (3) Calculating the size or volume of the droplet 726 based on the captured image. Alternatively, the droplet size or volume may be known or obtained using an alternative method. For example, if the total volume of the input droplets 726 is known, the number of droplets or the droplet frequency is known, and the droplets are monodisperse or uniform in size, the droplet volume may be calculated without imaging the droplets;

[0252] (4) The emulsion flows through the microfluidic channel 718 toward a joint or droplet actuator that divides the microfluidic channel 718 into two smaller droplet channels 714, 716. This can be a droplet separation nozzle. When the droplet 726 flows through the joint, the droplet is divided into two smaller sub-droplets 728, 730;

[0253] (5) Two smaller droplets 728, 730 flow from the junction through two separate droplet channels 714, 716 branching from the microfluidic droplet channel 718. The sub-droplets 728 each flow in a droplet emulsion in a carrier fluid;

[0254] (6) Imaging the first sub-droplet 728 in the first droplet channel 714 using a droplet sensor 724 disposed in a second droplet detection region downstream of the separation nozzle. Alternatively or additionally, the method may include imaging the second sub-droplet 730 in the second droplet channel 716.

[0255] (7) calculating the size or volume of the droplet 728 based on the captured image of the first sub-droplet 728;

[0256] (8) using the images captured by the droplet sensors 722, 724 to determine the volume change between the initial micro-droplet 726 and the sub-droplet 728, and calculating the volume distribution or volume ratio between the two sub-droplets 728, 730; and

[0257] (9) Introduce the carrier fluid from the carrier fluid inlet 720 or the pressure regulating channel into the first droplet channel 714. The carrier fluid may be spacer oil.

[0258] The volume or size values ​​calculated from the images captured by the first droplet sensor 722 and the second droplet sensor 724 in steps (2) and (6), or the ratio of the volume of the first sub-droplet 728 and the volume of the second sub-droplet 730 calculated in step (8), can be used as inputs to a closed-loop feedback procedure. Here, the experimental input (e.g., actuating pressure or flow rate) of the emulsion flow provided to the microfluidic channel 718 or the carrier fluid flow provided in the carrier fluid inlet 720 is adjusted in response to the calculated volume or ratio so as to maintain the droplet parameters (e.g., final sub-droplet volume, volume contribution, ratio) at a predetermined level when separating the droplets in the emulsion flow of the microdroplet 726.

[0259] For example, the image-based closed-loop feedback process may adjust the pressure or flow rate of the spacer oil or carrier fluid provided to one of the sub-droplet channels 714, 716 from the carrier fluid inlet 720 to increase or decrease the back pressure provided by the carrier fluid in the sub-droplet channels 714, 716 and the microfluidic channel 718. This may adjust or maintain the volume ratio of the sub-droplet volumes at a predetermined level in response to the determined droplet volume ratio deviating from the predetermined level.

[0260] Alternatively or additionally, other carrier fluid may be introduced from the second carrier fluid inlet into the second droplet channel 716. The other carrier fluid may be the same fluid or a different fluid from the spacing fluid introduced from the carrier fluid inlet 720. The carrier fluid and the other carrier fluid may be the same fluid or a different fluid from the continuous carrier fluid provided with the droplets prior to separation.

[0261] Both the carrier fluid and the further carrier fluid are miscible with the continuous carrier fluid provided with the droplets prior to separation and are immiscible with the dispersed micro-droplets.

[0262] While the examples shown involve picodroplets within a microfluidic chip, it should be understood that the device is not limited to picodroplets (which typically have a volume below about one thousand or several thousand picoliters) and is applicable to droplets of other sizes (e.g., droplets can be larger or smaller, with volumes ranging from nanoliters to femtoliters).

[0263] The described examples relate to an emulsion comprising droplets comprising a first fluid dispersed in a second fluid, wherein the second fluid is immiscible with the first fluid.The term fluid may be used herein to refer to a liquid.

[0264] We have described the techniques applied in preferred embodiments to the treatment of water-in-oil emulsion droplets containing biological entities. However, in principle, non-biological entities (such as organic or inorganic materials) can be treated in a similar manner. Similarly, the techniques we described are also applicable in principle to the treatment of oily droplets in oil-in-water emulsions. In water-in-oil emulsions, the oil forming the carrier fluid or spacer fluid typically includes fluorine and / or mineral oil and / or silicone oil, and preferably includes a surfactant, for example including about 0.5%-5% surfactant in volume ratio (vol / vol) or weight ratio (wt / wt).

[0265] No doubt many other effective alternatives will occur to those skilled in the art.It should be understood that the invention is not limited to the embodiments described, and encompasses modifications obvious to those skilled in the art within the spirit and scope of the appended claims.

[0266] Reference numerals

[0267] 102 Microfluidic Chip

[0268] 104 LED

[0269] 106 Optical lens tube assembly

[0270] 108 Objective lens

[0271] 110 Beam Splitter

[0272] 112 Photodetector

[0273] 114 Variable aperture (iris hole)

[0274] 116 High-speed camera

[0275] 204z Adjustable LED Holder

[0276] 208 z adjustable objective lens

[0277] 218 xy-θ translatable stage

[0278] 232 Lens

[0279] 320 Droplets

[0280] 322 Microfluidic Channels

[0281] 324 Sidewall

[0282] 326 Area of ​​Interest

[0283] 328 Light Peak

[0284] 330 Carrier Fluid

[0285] 440 Droplet generation area

[0286] 442 Sample fluid inlet

[0287] 444 Carrier fluid inlet

[0288] 446 Flow Aggregation Joint

[0289] 447 Snake Passage

[0290] 448 Collection Channel

[0291] 452 Abandoned Passage

[0292] 454 First output unit

[0293] 456 Second output unit

[0294] 540 Droplet generation area

[0295] 556 Second sample fluid inlet

[0296] 610 Droplet fusion area

[0297] 612 Microfluidic Droplet Channel

[0298] 614 Droplet Fluid Inlet

[0299] 616 Microfluidic Droplets

[0300] 618 Droplet Fluid

[0301] 620 Fusion Droplet

[0302] 622 Drop Sensor

[0303] 624 Drop Sensor

[0304] 710 Droplet separation area

[0305] 711 Droplet Inlet

[0306] 712 Droplet Inlet Channel

[0307] 714 First Droplet Channel

[0308] 716 Second droplet channel

[0309] 718 Microfluidic Droplet Channel

[0310] 719 Spacer fluid inlet

[0311] 720 Spacer fluid inlet channel

[0312] 722 Drop Sensor

[0313] 724 Drop Sensor

[0314] 726 Microdroplets

[0315] 728 First Sub-droplet

[0316] 730 Second droplet

[0317] 732 Carrier fluid inlet

[0318] 734 Carrier fluid inlet channel

[0319] 812 Microfluidic Droplet Channel

[0320] 814 Droplet Fluid Inlet

[0321] 816 Droplet Fluid Channel

[0322] 818 Droplet Inlet

[0323] 820 Spacer fluid inlet

[0324] 822 Interval fluid channel

[0325] 824 Electrode

[0326] 826 Fusion Droplet Exit

[0327] 828 Fusion Droplet Channel

[0328] 912 Microfluidic Droplet Channel

[0329] 914 Second droplet fluid inlet

[0330] 916 Second droplet fluid channel

[0331] 920 Carrier fluid inlet

[0332] 922 Carrier fluid channel

[0333] 924 Electrode

[0334] 926 Fusion Droplet Exit

[0335] 928 Fusion Droplet Channel

[0336] 930 First droplet fluid inlet

[0337] 932 First droplet fluid channel

Claims

1. A method of determining a droplet frequency of a stream of microfluidic droplets within a microfluidic droplet channel, the method comprising: irradiating a stream of the microfluidic droplets within the microfluidic droplet channel; using a beam splitter to separate light from the stream of microfluidic droplets into a first portion and a second portion, wherein the first portion includes light above a predetermined threshold wavelength, and wherein the second portion includes light below a predetermined threshold wavelength; directing the first portion to the camera; directing the second portion through an aperture to a photodetector, the aperture being located in front of the photodetector; The signal from the photodetector is processed and the droplet frequency is determined based on fluctuations in the processed signal.

2. The method according to claim 1, further comprising: detecting droplets using the processed signals, and When a droplet is detected, a camera is used to simultaneously capture an image of the droplet.

3. The method according to claim 2, wherein: The method also includes determining a droplet size based on the captured droplet image. The method of claim 3 , further comprising calculating a droplet volume based on the determined size.

5. The method according to any one of claims 3 or 4, wherein: Methods for determining the droplet size include: identifying the sidewalls of the microfluidic droplet channel and the droplet center within the flow image, determining a region of interest in the captured image of the droplet, wherein the region of interest comprises a line region, the line region comprises a center of the droplet, and wherein the region of interest is parallel to the sidewall, and The droplet size is determined by processing the line region of the image.

6. The method according to any one of claims 3 to 5, wherein: The method of determining the droplet size includes comparing the size of the droplet in the captured image to markers of known size located within the microfluidic channel.

7. A method of determining the total volume of a fluid within a series of droplets within a stream of microfluidic droplets, comprising: determining a droplet frequency of a stream of microfluidic droplets within the microfluidic droplet channel using the method of claim 1; for each droplet within the stream of microfluidic droplets, calculating a droplet volume; Calculate the average droplet volume of the droplets; as well as The total volume of the droplets is determined using the droplet frequency and the average droplet volume.

8. A method for generating a droplet stream, the method comprising: providing a first droplet of fluid; providing a carrier fluid; forming a droplet emulsion comprising the first droplet fluid in the carrier fluid by providing a flow of the first droplet fluid and a flow of the carrier fluid to a droplet generation region of a microfluidic structure; determining the total volume of fluid within a series of droplets within the droplet emulsion using the method of claim 7; as well as In response to determining that a total volume of fluid within the series of droplets is greater than a predetermined threshold, increasing a pressure of the flow of the carrier fluid circuit to inhibit generation of droplets.

9. A method for generating a droplet stream, the method comprising: providing a first droplet of fluid; providing a carrier fluid; forming a droplet emulsion comprising the first droplet fluid in the carrier fluid by providing a flow of the first droplet fluid and a flow of the carrier fluid to a droplet generation region of a microfluidic structure; Determining the droplet size of the continuous droplets in the emulsion using the method according to claim 5 or 6; as well as The pressure of the stream of the first droplet fluid and / or the pressure of the stream of the carrier fluid are adjusted in response to the determined droplet size.

10. A method according to claim 9, comprising varying the pressure of the stream of the first droplet fluid and varying the pressure of the stream of the carrier fluid such that the determined droplet size is substantially constant between successive droplets.

11. The method according to claim 9 or 10, wherein: During a start time period of the method of generating a stream of droplets, the method includes directing the stream of droplets to a waste channel in response to the determined droplet size being outside a predetermined droplet size range.

12. A method for generating a droplet stream, comprising: providing a first droplet of fluid; providing a carrier fluid; providing a second droplet of fluid; forming a droplet emulsion comprising a mixture of the first droplet fluid and the second droplet fluid in the carrier fluid by providing a flow of the first droplet fluid, a flow of the second droplet fluid, and a flow of the carrier fluid to a droplet generation region of the microfluidic structure; measuring a flow rate of the first droplet fluid flow or the second droplet fluid flow; determining the total volume of droplets within the droplet emulsion using the method of claim 7; determining a ratio of the first droplet fluid to the second droplet fluid within the mixture using the measured flow rate and the total volume of the droplets; as well as The pressure of the first droplet fluid flow and / or the pressure of the second droplet fluid flow is adjusted in response to the determined ratio.

13. A method for generating a droplet stream, comprising: Providing n droplets of fluid; providing a carrier fluid; forming a droplet emulsion comprising a mixture of the n droplet fluids in the carrier fluid by providing a flow of each of the n droplet fluids and a flow of the carrier fluid to a droplet generation region of a microfluidic structure; Measuring the flow rate of n-1 streams of droplet fluid; Determining the total volume of droplets in the droplet emulsion by the method according to claim 7; determining a ratio of the n droplet fluids within the mixture using the measured n-1 flow rates and the total volume of the droplets; as well as The pressure of one or more of the n streams of droplet fluid is adjusted in response to the determined ratio.

14. A microfluidic system comprising: a microfluidic droplet channel for conveying a stream of microfluidic droplets; a light source for illuminating the stream of microfluidic droplets with a light beam comprising light in a wavelength range both above and below a predetermined threshold wavelength; a beam splitter configured to split light from the stream of microfluidic droplets into a first portion and a second portion, wherein the first portion includes light above the predetermined threshold wavelength, and wherein the second portion includes light below the predetermined threshold wavelength; a camera configured to receive the first portion of the light; a photodetector configured to receive the second portion of the light, wherein the aperture is located in front of the photodetector; a device configured to process a signal from the photodetector; and A processor is configured to determine a droplet frequency based on fluctuations in the processed signal.

15. The system of claim 14, wherein: The processor is configured to detect a droplet using the processed signal, and the camera is configured to simultaneously capture an image of the droplet when the droplet is detected.

16. The system of claim 15, wherein: The exposure time of the camera is less than 5µs, and more preferably, the exposure time of the camera is less than 1µs.

17. The system according to claim 15 or 16, comprising: a first droplet fluid circuit, the first droplet fluid circuit being used to deliver a first droplet fluid; a carrier fluid circuit, the carrier fluid circuit being used to transport a carrier fluid; and a droplet generation region having a first input for receiving flow from the first droplet fluid circuit, having a second input for receiving flow from the carrier fluid circuit, and having an output to the microfluidic droplet channel, and wherein the processor is configured to determine an average droplet volume using the captured droplet images, and wherein the processor is configured to determine a total droplet volume of a series of droplets within the stream of microfluidic droplets using the average droplet volume and the droplet frequency, and Wherein, the system further comprises means for increasing the pressure in the carrier fluid circuit in response to the determined total droplet volume being greater than a predetermined threshold.

18. A system according to any one of claims 15 to 17, comprising: a first droplet fluid circuit, the first droplet fluid circuit being used to deliver a first droplet fluid; a carrier fluid circuit, the carrier fluid circuit being used to transport a carrier fluid; and a droplet generation region having a first input for receiving flow from the first droplet fluid circuit, a second input for receiving flow from the carrier fluid circuit, and having an output to the microfluidic droplet channel, and wherein the processor is configured to determine droplet sizes of successive droplets using the captured droplet images, and Wherein the system further comprises means for adjusting pressure in the carrier fluid circuit or the first droplet fluid in response to the determined droplet size.

19. A system according to any one of claims 15 to 17, comprising: n droplet fluid circuits, each droplet fluid circuit configured to deliver one of the n droplet fluids; a carrier fluid circuit, the carrier fluid circuit being used to transport a carrier fluid; and a droplet generation region having n inputs for receiving n flows from the n droplet fluid circuits, having other inputs for receiving flows from the carrier fluid circuits, and having outputs to the microfluidic droplet channels, causing the droplet generation region to form a droplet emulsion comprising a mixture of the n droplet fluids in the carrier fluid; and n-1 flow sensors, the n-1 flow sensors being configured to measure the flow rate of n-1 of the n flows of the droplet fluid, wherein the processor is configured to determine an average droplet volume using the captured droplet images, and wherein the processor is further configured to determine a total droplet volume within a series of droplets within the stream of microfluidic droplets using the average droplet volume and the droplet frequency, and wherein the processor is further configured to determine a ratio of the n droplet fluids within the mixture using the measured n-1 flow rates and the total droplet volume, and Wherein, the system further comprises means for adjusting the pressure of one or more of the n streams of the droplet fluid in response to the determined ratio.

20. The method of any one of claims 1 to 13 or the system of any one of claims 14 to 18, wherein: The aperture includes a slit corresponding to a strip of light that is substantially perpendicular to a sidewall of the microfluidic droplet channel.

21. The method of any one of claims 1 to 13 or the system of any one of claims 14 to 18, wherein: The aperture includes a pinhole corresponding to a light beam located substantially in the center of the microfluidic droplet channel.

22. The method of any one of claims 1 to 13 or the system of any one of claims 14 to 18, wherein: The aperture comprises a crescent-shaped aperture configured to increase the sensitivity of the photodetector.

23. The method of any one of claims 1 to 13 or the system of any one of claims 13 to 21, wherein: The first portion includes light having a wavelength greater than 488 nm, and wherein the second portion includes light having a wavelength less than 488 nm.

24. A microfluidic method for preparing droplets, the method comprising: providing an emulsion flow of microfluidic droplets of a first droplet fluid within a carrier fluid in a microfluidic channel; determining a droplet size of droplets in the stream of microfluidic droplets; providing a flow of a second droplet fluid to the microfluidic channel; Merging a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet; determining a droplet size of the fused droplet; In response to the determined fused droplet size, adjusting the pressure or flow rate of the stream of the microfluidic droplet emulsion and / or the pressure or flow rate of the stream of the second droplet fluid, The method according to claim 3 determines the droplet size of the droplets in the stream of microfluidic droplets and / or the droplet size of the fused droplets.

25. A microfluidic method for preparing droplets, the method comprising: providing an emulsion flow of microfluidic droplets of a first droplet fluid within a carrier fluid in a microfluidic channel; determining a droplet size of droplets in the stream of microfluidic droplets; providing a flow of a second droplet fluid to the microfluidic channel; Merging a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet; determining a droplet size of the fused droplet; In response to the determined fused droplet size, the pressure or flow rate of the stream of the emulsion of microfluidic droplets and / or the pressure or flow rate of the stream of the second droplet fluid are adjusted.

26. The method according to claim 24 or 25, wherein: Providing the flow of the second droplet fluid includes providing the flow of the second droplet fluid from a fluid inlet disposed on a sidewall of the microfluidic channel.

27. The method according to any one of claims 24 to 26, wherein: Merging a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet includes forming a fused droplet composed of a mixture of the first droplet fluid and the second droplet fluid.

28. The method according to claim 27, wherein: The method further includes determining a ratio of the first droplet fluid and the second droplet fluid in a mixture of the first droplet fluid and the second droplet fluid, and wherein a pressure or flow rate of the emulsion flow or the second droplet fluid flow is adjusted in response to the determined ratio.

29. The method according to any one of claims 24 to 28, wherein: Merging a droplet of the first droplet fluid with at least a portion of the second droplet fluid includes applying an electric field between a droplet of the first droplet fluid and a portion of the second droplet fluid.

30. The method according to any one of claims 24 to 29, wherein: Providing an emulsion flow of microfluidic droplets of a first droplet fluid within a carrier fluid in a microfluidic channel comprises: providing a first droplet of fluid; providing a carrier fluid; By providing a flow of the first droplet fluid and a flow of the carrier fluid to a droplet generation region of a microfluidic structure, a droplet emulsion comprising the first droplet fluid in the carrier fluid is formed.

31. The method according to claim 30, wherein: Adjusting the pressure or flow rate of the emulsion stream of microfluidic droplets includes adjusting the pressure of the stream of the first droplet fluid and / or the pressure of the stream of the carrier fluid in response to the determined droplet size.

32. The method of any one of claims 24 to 31, further comprising providing a flow of a spacing fluid to the microfluidic droplet channel, and wherein regulating the pressure or flow rate of the emulsion flow of the microfluidic droplets comprises regulating the pressure or flow rate of the spacing fluid flow in response to the determined droplet size.

33. A microfluidic system comprising: a droplet inlet for providing an emulsion flow of microfluidic droplets of a first droplet fluid in a carrier fluid; a droplet sensing system for determining the size of droplets within the stream of microfluidic droplets; a fluid inlet for providing a flow of a second droplet fluid; a droplet fusion region having a first input for receiving a flow from the droplet inlet and a second input for receiving a flow from the fluid inlet, and the droplet fusion region is configured to fuse a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet; a second droplet sensing system, the second droplet sensing system being used to determine the size of the fused droplet; and means for increasing the pressure or flow rate of the emulsion in the droplet inlet or the second droplet fluid in the fluid inlet in response to the determined size of the fused droplet, Wherein, the droplet sensing system or the second droplet sensing system comprises the microfluidic system according to claim 14.

34. A microfluidic system comprising: a droplet inlet for providing an emulsion flow of microfluidic droplets of a first droplet fluid in a carrier fluid; a droplet sensing system for determining the size of droplets within the stream of microfluidic droplets; a fluid inlet for providing a flow of a second droplet fluid; a droplet fusion region having a first input for receiving a flow from the droplet inlet and a second input for receiving a flow from the fluid inlet, and the droplet fusion region is configured to fuse a droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet; a second droplet sensing system, the second droplet sensing system being used to determine the size of the fused droplet; and A device for increasing the pressure or flow rate of the emulsion in the droplet inlet or the second droplet fluid in the fluid inlet in response to the determined size of the fused droplet.

35. The microfluidic system according to claim 33 or 34, wherein: The fluid inlet is arranged on a side wall of the microfluidic channel.

36. The microfluidic system of claim 34, wherein: The fluid inlet is substantially perpendicular to the microfluidic channel.

37. A microfluidic system according to any one of claims 33 to 36, wherein: The droplet fusion region includes a device configured to fuse a droplet of the first droplet fluid with at least a portion of the second droplet fluid.

38. The microfluidic system of claim 37, wherein: The apparatus configured to merge a droplet of the first droplet fluid with at least a portion of the second droplet fluid includes a plurality of electrodes configured to provide an electric field between a droplet of the first droplet fluid and a portion of the second droplet fluid.

39. The microfluidic system according to any one of claims 33 to 38, further comprising: a first droplet fluid circuit, the first droplet fluid circuit being used to deliver a first droplet fluid; a carrier fluid circuit, the carrier fluid circuit being used to transport a carrier fluid; and A droplet generation region having a first input for receiving flow from the first droplet fluid circuit, having a second input for receiving flow from the carrier fluid circuit, and having an output to the microfluidic droplet channel.

40. The microfluidic system of any one of claims 33 to 39, further comprising a spacing fluid circuit configured to provide a flow of a spacing fluid to the microfluidic droplet channel.

41. A microfluidic method for forming microdroplet pairs, comprising: providing an emulsion flow of microfluidic droplets of the droplet fluid within a carrier fluid in a microfluidic channel; determining a droplet size of droplets in the stream of microfluidic droplets; dividing the droplet into a first droplet and a second droplet; directing the first droplet to a first droplet channel; directing the second droplet to a second droplet channel; determining a droplet size of the first droplet or the second droplet; providing a flow of a spacing fluid to the first droplet channel or the second droplet channel; as well as adjusting the pressure or flow rate of the emulsion flow of microfluidic droplets and / or the pressure or flow rate of the flow of the spacing fluid in response to the determined size of the first droplet or the second droplet, The method according to claim 3 determines the droplet size of the droplets in the stream of microfluidic droplets and / or the droplet size of the first droplet and / or the droplet size of the second droplet.

42. A microfluidic method for forming microdroplet pairs, comprising: providing an emulsion flow of microfluidic droplets of a droplet fluid within a carrier fluid in a microfluidic channel; determining a droplet size of droplets in the stream of microfluidic droplets; dividing the droplet into a first droplet and a second droplet; directing the first droplet to a first droplet channel; directing the second droplet to a second droplet channel; determining a droplet size of the first droplet or the second droplet; providing a flow of a spacing fluid to the first droplet channel or the second droplet channel; as well as In response to the determined size of the first droplet or the second droplet, the pressure or flow rate of the emulsion flow of microfluidic droplets and / or the pressure or flow rate of the flow of the spacing fluid are adjusted.

43. The microfluidic method for forming microdroplet pairs according to claim 41 or 42, wherein: The method includes determining a droplet volume of one of the first droplet or the second droplet, and also includes calculating a droplet volume of the other of the first droplet and the second stream using the previously determined droplet volume of the first droplet or the second droplet and a predetermined volume of droplets in the stream of microfluidic droplets.

44. The microfluidic method of any one of claims 25 to 32, 41 or 42, wherein: Determining droplet size involves: Use a droplet sensing system to capture images of droplets, The droplet size is determined from the captured image.

45. A microfluidic system comprising: a droplet inlet for providing an emulsion flow of microfluidic droplets of a droplet fluid in a carrier fluid; a droplet sensing system for determining the size of droplets within the stream of microfluidic droplets; a droplet separation region having a first input for receiving a stream from the droplet inlet and having a first output to a first droplet channel and a second output to a second droplet channel, wherein the droplet separation region is configured to separate a droplet in the stream of microfluidic droplets into a first droplet and a second droplet; a fluid inlet for providing a flow of a spacing fluid to the first droplet channel or the second droplet channel; a second droplet sensing system for determining a size of the first droplet or the second stream; and means for increasing the pressure or flow rate of the emulsion in the droplet inlet or the spacer fluid in the fluid inlet in response to the determined size of the first droplet or the second droplet, Wherein the droplet sensing system or the second droplet sensing system comprises the microfluidic system according to claim 14.

46. ​​A microfluidic system comprising: a droplet inlet for providing an emulsion flow of microfluidic droplets of a droplet fluid in a carrier fluid; a droplet sensing system for determining the size of droplets within the stream of microfluidic droplets; a droplet separation region having a first input for receiving a stream from the droplet inlet and having a first output to a first droplet channel and a second output to a second droplet channel, wherein the droplet separation region is configured to separate a droplet in the stream of microfluidic droplets into a first droplet and a second droplet; a fluid inlet for providing a flow of a spacing fluid to the first droplet channel or the second droplet channel; a second droplet sensing system for determining a size of the first droplet or the second stream; and A device for increasing the pressure or flow rate of the emulsion in the droplet inlet or the spacer fluid in the fluid inlet in response to the determined size of the first droplet or the second droplet.

47. The microfluidic system of claim 45 or 46, wherein: The first droplet channel and the second droplet channel have different cross-sectional areas.

48. The microfluidic system of any one of claims 45 to 47, further comprising a spacing fluid circuit configured to provide a flow of a spacing fluid to the droplet inlet.

49. A microfluidic system according to any one of claims 34 to 40, 46 or 47, wherein The droplet sensing system and / or the second droplet system comprises a camera configured to capture an image of a droplet and means for determining a droplet size from the captured image of the droplet.

Images