Droplet Generation System

Abstract

A method and system for determining a droplet frequency of a microfluidic droplet stream in a microfluidic droplet channel is provided, the method and system including illuminating the microfluidic droplet stream with light, splitting the light from the microfluidic droplet stream into a first portion and a second portion using a beam splitter, directing the first portion to a camera, directing the second portion to a photodetector through an aperture disposed in front of the photodetector, processing a signal from the photodetector, and determining the droplet frequency from a variation in the processed signal. The method may be used to determine droplet size. The method may also be used to adjust the pressure of the droplet fluid stream in response to the determined droplet size to generate the droplet stream.

Description

[Technical field]

[0001] This application relates to systems and methods for determining the droplet frequency of a stream of microfluidic droplets, and particularly, but not exclusively, this application further relates to systems and methods for determining and controlling droplet size. [Background technology]

[0002] Herein, we are interested in emulsions, typically emulsions that include picodroplets of water in oil, generally surfactant stabilized. One or more biological entities, such as one or more living cells or particles, are incorporated into each droplet, and experiments can then be carried out in the droplets, for example, to perform biological assays. Picodroplets can be generated and processed at rates potentially exceeding several thousand per second.

[0003] Typically, the oil composition comprises fluorous oil and / or mineral oil and / or silicone oil, preferably a surfactant, for example at a volume ratio of about 0.5-5%. The use of fluorous oil is particularly advantageous when the microdroplet contains a living organism, since fluorous oil has a good ability to transport oxygen to the microdroplet. The surfactant can be either polymeric or small molecule, for example surfactants derived from block copolymers of perfluoroethers such as Krytox (RTM) or polyethylene glycol (PEG) can be used. The material or analyte in the microdroplet can include, for example, cells, DNA, proteins, peptides, beads, particles, crystals, micelles, macromolecules, materials for enzyme assays, organelles, cells, for example mammalian cells, yeast cells, algae cells or organisms such as bacteria, viruses, prions, etc.

[0004] Crawford, DF, Smith, CA & Whyte, G. "Image-based closed-loop feedback for highly mono-dispersed microdroplet production." Sci Rep 7, 10545 (2017) is concerned with controlling the size of microdroplets. U.S. Patent Application No. 2011 / 000560, U.S. Patent Application No. 2014 / 354795, and U.S. Patent Application No. 2021 / 146319 are concerned with using a camera to determine droplet frequency. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent Application Publication No. 2011 / 000560 [Patent Document 2] US Patent Application Publication No. 2014 / 354795 [Patent Document 3] US Patent Application Publication No. 2021 / 146319 [Non-patent literature]

[0006] [Non-Patent Document 1] Crawford, DF, Smith, CA & Whyte, G. "Image-based closed-loop feedback for highly mono-dispersed microdroplet production." Sci Rep 7, 10545 (2017) Summary of the Invention [Means for solving the problem]

[0007] Aspects and preferred features are set out in the accompanying claims.

[0008] According to a first aspect, there is provided a method for determining a droplet frequency of a stream of microfluidic droplets in a microfluidic droplet channel, comprising: illuminating a stream of microfluidic droplets in a microfluidic droplet channel with light; splitting light from the stream of microfluidic droplets into a first portion and a second portion using a beam splitter, the first portion including light greater than a predetermined threshold wavelength and the second portion including light less than the predetermined threshold wavelength; orienting the first portion toward a camera; directing the second portion toward the photodetector through an aperture disposed in front of the photodetector; processing a signal from the photodetector and determining a droplet frequency from a variation in the processed signal.

[0009] Processing the signal from the photodetector may include acquiring and processing the photodetector output voltage signal over a given period of time to identify differences in the voltage signal corresponding to differences in optical properties exhibited at the interface between the dispersion medium and the droplet as the droplet passes through a detection region of the microfluidic droplet channel.

[0010] The methods disclosed herein provide an automated method for determining the droplet frequency of a microfluidic droplet stream. By directing the second portion through an aperture located in front of the photodetector, the sensitivity of the photodetector is improved.

[0011] The method may further include detecting the droplet using the processed signal, and simultaneously, upon detection of the droplet, capturing an image of the droplet using a camera using the output of the detected photodetector signal to actively trigger the camera via a microprocessor to automatically image the droplet in the microfluidic channel.

[0012] The microprocessor camera trigger algorithm may further allow the number of individual droplet detection occurrences to be counted over a set period of time or until the final image is captured, thereby determining the droplet frequency.

[0013] The method may further include determining a droplet size from the captured image of the droplet.

[0014] The method may further include calculating a droplet volume from the determined dimensions.

[0015] The term "dimension" may be used herein to refer to the physical width or length of a droplet within a microfluidic droplet channel.

[0016] In one example, the droplet may be considered to be spherical and the determined dimension may be the radius of the sphere. The spherical droplet volume may be calculated using the determined radius.

[0017] In an alternative example, at least one droplet dimension (e.g., the width of the droplet) may be constrained by a known width or cross-sectional area of ​​the microfluidic channel, and a second droplet dimension may be determined using a method as described above (e.g., the length of the droplet within the droplet channel). The droplet volume may be calculated using the known width or cross-sectional area and the determined droplet dimension.

[0018] A method for determining droplet dimensions may include identifying a center of a droplet in an image of a sidewall and flow of a microfluidic droplet channel, determining a region of interest in a captured image of the droplet, the region of interest including a linear region that includes the droplet center, the region of interest being parallel to the sidewall, and determining the droplet dimensions by processing the linear region of the image. The region of interest may also include two opposing points on an outer surface of the droplet.

[0019] The method may include comparing the size (e.g., number of pixels) of the processed linear region between the sidewalls against a known size of each pixel in the processed linear region. The known size of each pixel could alternatively be referred to as the number of pixels required to display a given physical length. In other words, the physical length or size of the region of interest may be determined by knowing the number of pixels per inch (or centimeter) in the captured image. The resolution of this captured image may be determined by knowing the resolution of the sensor or camera along with the dimensions of the field of view.

[0020] Alternatively, the physical size can 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 in the linear region, and the pixel pitch of the camera.

[0021] The captured images of the microfluidic flow can be processed to identify the geometric features of the microchannel and the center of the generated droplets. This can be an automated method running on a processor. The method can then define a linear region of interest that is parallel to the sidewall of the microchannel and intersects through the imaged droplet and its center to provide a measurement of the droplet diameter for subsequent use as an input to a closed-loop image-based feedback routine to regulate the monodispersity of the generated emulsion.

[0022] Alternatively, or in addition, the method of determining droplet size may include comparing the size of the droplet in the captured image to a marker disposed within the microfluidic channel having a known size, which provides a simpler method of measuring droplet size and improves the accuracy of the determined droplet size.

[0023] According to a further aspect of the present disclosure, there is provided a method for determining a total volume of fluid within a series of droplets in a stream of microfluidic droplets, comprising: determining a droplet frequency of a stream of microfluidic droplets in a microfluidic droplet channel using a method as described above; Calculating a droplet volume for each droplet in the microfluidic droplet stream; Calculating an average droplet volume of the droplets; and determining a total volume of the droplets using the droplet frequency and the average droplet volume.

[0024] Calculating the drop volume for each droplet in the stream of microdroplets may include calculating the drop volume of each imaged droplet in the stream. The method may include only imaging a portion of the droplets in the stream, such that drop volumes are not calculated for droplets that are not imaged.

[0025] Calculating the average drop volume may include calculating a running average of the drop volumes of the imaged drops, which may include most recently imaged drops.

[0026] Determining the total volume of the droplet may include determining the total volume of the droplet over a given period of time.

[0027] According to a further aspect of the present disclosure, there is provided a method of generating a stream of droplets, comprising the steps of: Providing a first droplet fluid; Supplying a dispersion medium; forming an emulsion of droplets comprising a first droplet fluid in a carrier fluid by providing a stream of the first droplet fluid and a stream of the carrier fluid to a droplet generation region of a microfluidic structure; determining a total volume of fluid within a set of droplets in an emulsion of droplets using a method as described above; and increasing the flow pressure of the carrier fluid line to inhibit droplet generation in response to the determined total volume of fluid in the series of droplets being greater than a predetermined threshold. In this manner, a user can specify the volume of emulsion desired to be collected based on the sample size delivered, and the machine will stop making the emulsion when this value is reached.

[0028] According to a further aspect of the present disclosure, there is provided a method of generating a stream of droplets, comprising the steps of: Providing a first droplet fluid; Supplying a dispersion medium; forming an emulsion of droplets comprising a first droplet fluid in a carrier fluid by providing a stream of the first droplet fluid and a stream of the carrier fluid to a droplet generation region of a microfluidic structure; determining droplet sizes of successive droplets in the emulsion using a method as described above; and adjusting a pressure of the first droplet fluid stream and / or a pressure of the carrier fluid stream in response to the determined droplet size.

[0029] It provides semi-automated droplet generation with triggered closed-loop image-based feedback control of real-time droplet frequency determination, size calculation, and droplet monodispersity. The method maintains the monodispersity of the droplet stream by controlling the pressure of the first droplet fluid stream or the carrier fluid stream in response to the determined droplet size.

[0030] A method of generating a stream of droplets can include varying a pressure of a first stream of droplet fluid and varying a pressure of the stream of carrier fluid such that a determined droplet size is substantially constant between successive droplets.

[0031] The methods disclosed herein can be extended to provide multiple parallel aligned droplet generation regions on a microfluidic chip. Photodetector-based detection of the presence of individual droplets can be used to initiate triggered capture of droplet images in two or more designated regions within a microfluidic chip that includes multiple parallel aligned droplet generation regions. Droplet size data from one or more regions of interest can be used as input to a closed-loop image-based feedback routine that regulates the monodispersity of emulsions created from the microfluidic chip. The use of parallel droplet generator chips allows for precise droplet volume control across multiple microfluidic channels to achieve ultra-high throughput droplet generation.

[0032] At a start time period of the method of generating a stream of droplets, the method may include directing the stream of droplets to a waste channel in response to said determined droplet size being outside a predetermined droplet size range. When the instrument is started, there may be a period during which the droplets being generated do not meet the specified user size requirements. During the time that the droplets being generated do not meet the specified user size requirements, the droplets are moved to a waste channel.

[0033] When the correct droplet size is achieved, the droplets can be prevented from going to waste and instead collected. This can be done by closing a valve on the waste channel. The valve can be programmed to close for a predetermined period of time. By closing the valve on the waste channel relatively slowly, this allows the system to compensate for increased back pressure in the system without changing the droplet volume.

[0034] According to a further aspect of the present disclosure, there is provided a method of generating a stream of droplets, comprising the steps of: Providing a first droplet fluid; Supplying a dispersion medium; providing a second droplet fluid; forming an emulsion of droplets comprising a mixture of a first droplet fluid and a second droplet fluid in a carrier fluid by providing a stream of the first droplet fluid and a stream of the second droplet fluid and a stream of the carrier fluid to a droplet generation region of a microfluidic structure; Measuring a flow rate of the first droplet fluid stream or the second droplet fluid stream; determining a total volume of fluid within droplets in an emulsion of droplets using a method as described above; determining a ratio of the first droplet fluid to the second droplet fluid in the mixture using the measured flow rate and total volume of the droplets; and adjusting a pressure of the first droplet fluid stream or the second droplet fluid stream in response to the determined ratio.

[0035] The use of image-based determination of droplet volume and photodetector-based determination of the total aqueous flow rate, plus the use of a flow sensor at one of the two droplet fluid inlets, allows for the creation of monodisperse two-component aqueous droplets with a defined mixing ratio between the two aqueous streams.

[0036] Because the first droplet fluid may contain cells or fragile biological or chemical components, the flow sensor may preferably be configured to measure the flow rate of the second droplet fluid.

[0037] This method may also be used to maintain or control cell occupancy in the creation of monodisperse droplets formed from two droplet fluids, one containing cells or particles and the other a diluent.

[0038] According to a further aspect of the present disclosure, there is provided a method for generating a stream of droplets, comprising: providing n droplets of fluid; Supplying a dispersion medium; forming an emulsion of droplets comprising a mixture of n droplet fluids in a carrier medium by providing a stream of each of the n droplet fluids and a stream of the carrier medium to a droplet generation region of a microfluidic structure; measuring a flow rate of the n-1 droplet fluid stream; determining a total volume of fluid within droplets in an emulsion of droplets in a manner as described above; determining a ratio of n droplet fluids in the mixture using the measured flow rates of the n-1 droplets and the total volume of the droplets; and adjusting a pressure of one or more of the n droplet fluid streams in response to the determined ratio.

[0039] According to a further aspect of the present disclosure, there is provided a microfluidic system comprising: a microfluidic droplet channel for carrying a stream of microfluidic droplets; a light source that irradiates the stream of microfluidic droplets with a light beam that includes light having wavelengths 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, the first portion including light greater than a predetermined threshold wavelength and the second portion including light less than the predetermined threshold wavelength; a camera configured to receive a first portion of the light; a photodetector configured to receive a second portion of the light, the aperture being disposed in front of the photodetector; and a device configured to process a signal from the photodetector; and a processor configured to determine a droplet frequency from variations in the processed signal.

[0040] The processor may be configured to detect the droplet using the processed signal, and the camera may be configured to simultaneously capture an image of the droplet upon detection of the droplet.

[0041] The camera can have an exposure time of 1 μs or less, providing a high speed, area-scanning camera capable of capturing images of droplets within a microfluidic channel.

[0042] The system may include a first droplet fluid conduit for carrying a first droplet fluid, a carrier medium conduit for carrying a carrier medium, and a droplet generation region having a first input for receiving a flow from the first droplet fluid conduit, a second input for receiving a flow from the carrier medium conduit, and an outlet to the microfluidic droplet channel. The processor may be configured to determine an average droplet volume using the captured images of the droplets. The processor may further be configured to determine a total volume of droplets in a series of droplets in the microfluidic droplet stream using the average droplet volume and the droplet frequency. The system may further include means for increasing pressure in the carrier medium conduit in response to the determined total volume of the droplets being greater than a predetermined threshold.

[0043] The system may comprise a first droplet fluid conduit for carrying a first droplet fluid, a carrier medium conduit for carrying a carrier medium, and a droplet generation region having a first input for receiving a flow from the first droplet fluid conduit, a second input for receiving a flow from the carrier medium conduit, and an outlet to the microfluidic droplet channel. The processor may be configured to determine droplet dimensions of the successive droplets using the captured images of the droplets. The system may further comprise means for adjusting pressure in the carrier medium conduit or the first droplet fluid in response to the determined droplet dimensions.

[0044] The system may include n droplet fluid conduits each configured to carry one of the n droplet fluids, a carrier medium conduit for carrying a carrier medium, and a droplet generation region having n inputs for receiving n streams from the n droplet fluid conduits, a further input for receiving a stream from the carrier medium conduit, and an outlet to a microfluidic droplet channel, the droplet generation region forming an emulsion of droplets comprising a mixture of the n droplet fluids in the carrier medium. The system may further include n-1 flow sensors configured to measure flow rates of n-1 of the n droplet fluid streams. The processor may be configured to determine an average droplet volume using the captured images of the droplets. The processor may be further configured to determine a total volume of droplets in a series of droplets in the microfluidic droplet stream using the average droplet volume and the droplet frequency. The processor may be further configured to determine a ratio of the n droplet fluids in the mixture using the measured flow rates of the n-1 droplets and the total volume of the droplets. The system may further comprise means for adjusting a pressure of one or more of the n droplet fluid streams in response to the determined ratio.

[0045] The aperture may include a slit that corresponds to a beam of light that is substantially perpendicular to a sidewall of the microfluidic droplet channel.

[0046] Alternatively, the aperture may include a pinhole corresponding to the light beam positioned substantially at the center of the microfluidic droplet channel.

[0047] The aperture may comprise a mechanical slit that allows only a narrow sliver of light that is substantially perpendicular to the sidewall of the main microfluidic channel to reach the photodetector, thereby increasing detection sensitivity.

[0048] Alternatively, the aperture may include a crescent shaped aperture configured to increase the sensitivity of the photodetector.

[0049] The first portion may include light having wavelengths longer than 488 nm, and the second portion may include light having wavelengths shorter than 488 nm.

[0050] The droplets may be microdroplets, nanodroplets, or picodroplets, or may be larger or smaller.

[0051] According to a further aspect of the present disclosure, there is provided a microfluidic method of preparing droplets, comprising: providing a stream of a microfluidic droplet emulsion of a first droplet fluid into a dispersion medium within a microfluidic channel; determining a droplet size of a droplet in the microfluidic droplet stream; providing a second droplet fluid stream into the microfluidic channel; fusing 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 droplets; and adjusting a pressure or flow rate of the microfluidic droplet emulsion stream and / or a pressure or flow rate of a second droplet fluid stream in response to the determined fused droplet size.

[0052] The volume of an individual microfluidic droplet of the first droplet fluid or the volume of a second droplet fluid merged with a microfluidic droplet of the first droplet fluid can be controlled using closed-loop image-based feedback.

[0053] The method allows for the regulation of the volume ratios of individual microfluidic droplets consisting of a mixture of two or more droplet fluids using closed-loop image-based feedback.

[0054] The method may be used to prepare droplets having a constant, controlled droplet volume or volume ratio of the constituent parts, or may be used to adjust the droplet volume or volume ratio of the constituent parts to a desired or predetermined value.

[0055] The method may be used to create fused droplets having two or more constituent parts or containing a mixture of two or more fluids. The method may calculate the volume of each fluid component in an individual fused droplet using an image of a droplet of a first droplet fluid captured before fusing the droplet of the first droplet fluid with a second droplet fluid and an image of the fused droplet captured after fusing the droplet of the first droplet fluid with a second droplet fluid. The calculated volumes of the components, or the ratio of the volumes of the components, may then be used as inputs to a closed loop feedback system that may create a monodisperse emulsion of the fused droplets.

[0056] The emulsion of microfluidic droplets of the first fluid may be supplied from an external system, or may be prepared on a separate chip connected to the chip containing the droplet fusion region, or may be prepared on the same chip as the droplet fusion region. In instances where the microfluidic droplets of the first fluid are created on the same chip or on a separate chip connected to the chip containing the droplet fusion region, the method may include adjusting or regulating the volume of the droplets of the first fluid created prior to fusion, rather than being reinjected into the chip.

[0057] The droplet size of droplets in a microfluidic droplet stream and / or the droplet size of fused droplets may be determined according to the methods for determining droplet size as described above.

[0058] Providing the second droplet fluid stream may include providing the second droplet fluid stream from a fluid inlet arranged on a sidewall of the microfluidic channel.

[0059] Fusing 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.

[0060] The method may further 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. The pressure or flow rate of the emulsion stream or the second droplet fluid stream may be adjusted according to the determined ratio.

[0061] Fusing droplets of the first droplet fluid with at least a portion of the second droplet fluid can include applying an electric field to the first droplet fluid and to a portion of the second droplet fluid. Applying the electric field can cause the droplets to merge with an end of the second droplet fluid stream at a junction of the microfluidic channel and the second droplet fluid inlet. This can then be broken apart into fused droplets by shear forces of the carrier fluid in the microfluidic channel.

[0062] Providing a flow of a microfluidic droplet emulsion of a first droplet fluid in a dispersion medium within a microfluidic channel may include providing a first droplet fluid, providing a dispersion medium, and forming an emulsion of droplets including the first droplet fluid in the dispersion medium by providing a flow of the first droplet fluid and a flow of the dispersion medium to a droplet generation region of a microfluidic structure.

[0063] Adjusting the pressure or flow rate of the microfluidic droplet emulsion stream may include adjusting the pressure of the first droplet fluid stream and / or the pressure of the carrier fluid stream in response to the determined droplet size.

[0064] The method may further include providing a flow of a spacing fluid to the microfluidic droplet channel. Adjusting a pressure or flow rate of the flow of emulsion of the microfluidic droplets may include adjusting a pressure or flow rate of the flow of spacing fluid in response to the determined droplet size.

[0065] According to a further aspect of the present disclosure, there is provided a microfluidic system comprising: a droplet inlet for providing a stream of an emulsion of microfluidic droplets of a first droplet fluid in a dispersion medium; a droplet sensing system for determining a size of a droplet in a microfluidic droplet stream; a fluid inlet for supplying a second droplet fluid stream; a droplet merging 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 configured to merge droplets of the first droplet fluid with at least a portion of the second droplet fluid to obtain fused droplets; a second droplet sensing system for determining a 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.

[0066] The droplet sensing system or the second droplet sensing system may include a microfluidic system as described above.

[0067] The fluid inlet may be arranged on a sidewall of the microfluidic channel.

[0068] The fluid inlet may be substantially perpendicular to the microfluidic channel.

[0069] The droplet merging region may comprise means configured to merge droplets of the first droplet fluid with at least a portion of the second droplet fluid.

[0070] The means configured to merge the droplets of the first droplet fluid with at least a portion of the second droplet fluid may include a plurality of electrodes configured to apply an electric field to the droplets of the first droplet fluid and to the portion of the second droplet fluid.

[0071] The microfluidic system may further comprise a first droplet fluid conduit for carrying a first droplet fluid, a dispersion medium conduit for carrying a dispersion medium, and a droplet generation region having a first input for receiving flow from the first droplet fluid conduit, a second input for receiving flow from the dispersion medium conduit, and an outlet to the microfluidic droplet channel.

[0072] The microfluidic system may further comprise an interval fluid conduit configured to provide a flow of interval fluid to the microfluidic droplet channel.

[0073] According to a further aspect of the present disclosure, there is provided a microfluidic method for forming a pair of microdroplets, the method comprising: providing a stream of an emulsion of microfluidic droplets of a droplet fluid into a dispersion medium within a microfluidic channel; determining a droplet size of a droplet in the microfluidic droplet stream; splitting the droplet into a first droplet and a second droplet; Directing a first droplet into a first droplet channel; directing a second droplet into a second droplet channel; Determining a droplet size of the first droplet or the second droplet; providing a flow of spacing fluid to the first droplet channel or the second droplet channel; and adjusting a pressure or flow rate of the microfluidic droplet emulsion stream and / or a pressure or flow rate of the spacing fluid stream in response to the determined size of the first droplet or the second droplet.

[0074] Directing the first droplet into the first droplet channel and / or directing the second droplet into the second droplet channel may include redirecting the flow of the first droplet or the second droplet. Alternatively, directing the first droplet into the first droplet channel and / or directing the second droplet into the second droplet channel may include allowing the first droplet or the second droplet to continue flowing in the same microfluidic channel and / or allowing the first droplet or the second droplet to continue flowing in the same direction.

[0075] The droplet size of the droplets in the microfluidic droplet stream and / or the droplet size of the first droplet and / or the droplet size of the second droplet may be determined according to methods as described above.

[0076] The volumes of or the volume distribution between two or more individual daughter droplets created by splitting a larger droplet into two droplets can be controlled using closed-loop image-based feedback.

[0077] The method uses closed-loop image-based feedback and allows for the regulation of the volume of droplets created by splitting larger droplets using a droplet splitting mechanism.

[0078] The method may be used to prepare two or more daughter droplets each having a constant, adjusted droplet volume, or may be used to adjust the droplet volumes of two or more daughter droplets to a desired or predetermined value. The method may be used to maintain the volumes of both the first and second droplets consistent with one another.

[0079] The emulsion of microfluidic droplets may be supplied from an external system, or may be prepared on a separate chip connected to the chip containing the droplet splitting region, or may be prepared on the same chip as the droplet splitting region. In instances where the microfluidic droplets are created on the same chip or on a separate chip connected to the chip containing the droplet splitting region, the method may include adjusting or regulating the volume of the droplets created prior to splitting, rather than being reinjected into the chip.

[0080] The microfluidic method may include determining a droplet volume of one of the first droplet or the second droplet, and may further include calculating a droplet volume of the other of the first droplet and the second droplet using a previously determined droplet volume of the first droplet or the second droplet and a predetermined volume of droplets in the microfluidic droplet stream.

[0081] Determining the droplet size may include capturing an image of the droplet using a droplet sensing system and determining the droplet size from the captured image.

[0082] According to a further aspect of the present disclosure, there is provided a microfluidic system comprising: a droplet inlet for providing a stream of an emulsion of microfluidic droplets of droplet fluid in a dispersion medium; a droplet sensing system for determining a size of a droplet in a microfluidic droplet stream; a droplet splitting region having a first input for receiving a stream from the droplet inlet, a first outlet to a first droplet channel, and a second outlet to a second droplet channel, the droplet splitting region configured to split droplets in the microfluidic droplet stream into first droplets and second droplets; a fluid inlet for providing a flow of 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 droplet; and means for increasing the pressure or flow rate of the emulsion in the droplet inlet or the spacing fluid in the fluid inlet in response to the determined size of the first droplet or the second droplet.

[0083] The droplet sensing system and / or the second droplet sensing system may include a microfluidic system as described above.

[0084] The first and second droplet channels may have different cross-sectional areas, allowing daughter droplets of different sizes to be created.

[0085] Alternatively, the first droplet channel and the second droplet channel may have substantially the same cross-sectional area.

[0086] The microfluidic system may further comprise an interval fluid conduit configured to provide a flow of interval fluid to the droplet inlet.

[0087] The droplet sensing system and / or the second droplet system comprises a camera configured to capture an image of the droplet and means for determining a droplet size from the captured image of the droplet.

[0088] Some embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0089] [Figure 1] FIG. 1 is a schematic diagram illustrating an optical assembly for imaging a microfluidic chip. [Diagram 2] FIG. 2 illustrates an example of the optical assembly of FIG. 1. [Diagram 3] FIG. 1 is a schematic diagram illustrating a region of interest in a captured image of a droplet in a microfluidic channel. [Figure 4] FIG. 2 is a schematic diagram illustrating a droplet generation region. [Diagram 5] FIG. 5 is a schematic diagram illustrating a flow focus junction and a Y-junction within the droplet generation region shown in FIG. 4. [Figure 6] FIG. 2 is a schematic diagram illustrating a droplet generation region for making an emulsion of two-component aqueous droplets. [Figure 7] FIG. 7 is a schematic diagram illustrating a flow focusing junction and a Y-junction within the droplet generation region shown in FIG. [Figure 8] FIG. 1 illustrates an exemplary droplet merging region. [Figure 9] FIG. 1 is a schematic diagram illustrating droplet reinjection and droplet merging regions. [Figure 10] FIG. 10 is a schematic diagram illustrating a droplet fluid inlet of the droplet merging region shown in FIG. [Figure 11] FIG. 2 is a schematic diagram illustrating a droplet generation region and a droplet merging region. [Figure 12] FIG. 12 is a schematic diagram illustrating a droplet fluid inlet of the droplet merging region shown in FIG. [Figure 13]FIG. 1 is a schematic diagram illustrating droplet reinjection and droplet splitting regions. [Figure 14] FIG. 14 is a schematic diagram illustrating a droplet splitting region as shown in FIG. 13. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] FIG. 1 illustrates a schematic of an optical assembly for imaging a microfluidic chip, and FIG. 2 is a side view of one example of the optical assembly of FIG.

[0091] The optical assembly includes a microfluidic chip 102 having a microfluidic channel that carries a stream of microfluidic droplets. The droplets may be generated using the systems shown in Figures 4 through 6. An LED 104 mounted in an optical lens tube assembly 106 is configured to illuminate the microfluidic chip 102 with light in the visible light spectrum. An objective lens 108 collects the light from the microfluidic chip 104 and directs the light to a dichroic beam splitter 110.

[0092] The beam splitter 110 is configured to reflect a first portion of the light from the beam splitter 110 towards a photodetector 112. The beam splitter 110 is configured such that a second portion of the light is transmitted from the beam splitter 110 to a high speed camera 116.

[0093] In this example, the LED 104 is a white LED and the beam splitter 110 is a dichroic mirror that splits the focused image of the droplet stream into light portions greater or smaller than a given wavelength (in this example, the wavelength is 488 nm so that blue light is directed to the photodetector). The beam splitter 110 directs approximately 20% of the light to the photodetector and 80% to the camera, although the split ratio can be changed. Because cameras have less sensitivity in the blue region of visible light compared to the red and green regions of visible light, the dichroic mirror directs the green and red portions of visible light to the camera to improve sensitivity. The blue light is directed to the photodetector.

[0094] Light having wavelengths below a predetermined wavelength (488 nm in this example) is reflected by a dichroic mirror 110 to a photodetector 112. An aperture 114 is disposed between the beam splitter 110 and the photodetector 112. The aperture 114 can be a mechanically adjustable slit (also called a slit-iris), a circular iris, a narrow rectangular aperture, or a crescent aperture, and is set to allow only a narrow sliver of light from the main microfluidic channel to reach the photodetector.

[0095] Each droplet in the droplet stream includes a first liquid droplet suspended in a carrier fluid. The droplet liquid may be particles in an aqueous liquid, and the carrier fluid may be a continuous oil phase. When the interface between the droplet and the carrier fluid passes over the photodetector 112, the voltage signal of the photodetector decreases due to light scattering. After the droplet passes the detector 112 and only the continuous oil phase is visible to the photodetector 112, the voltage rises and returns to the background signal. Each moving picodroplet in the microfluidic channel shows a decrease in the amplitude of the photodetector measured voltage signal, as the light intensity is momentarily scattered by the passing droplet. The occurrence and repetition of such voltage drops over time (i.e., on a time scale of one second) is characteristic of the presence of each individual flowing droplet, and is then used to calculate the droplet frequency. This arrangement increases the detection sensitivity of the photodetector and allows the droplet generation frequency to be continuously measured in real time using dedicated automated frequency detection software.

[0096] Light having wavelengths longer than a predetermined wavelength (488 nm in this example) is transmitted through a dichroic mirror 110 to a sensor of a high-speed area-scanning camera 116. To ensure that droplets are imaged in approximately the same area in the microfluidic channel, the voltage signal output of the photodetector is received and processed on a microprocessor. After receiving the voltage signal output of the photodetector, the microprocessor sends a trigger signal to prompt camera image acquisition of the picodroplet using very short exposure time imaging. For example, when a passing picodroplet causes a decrease in the measured voltage signal amplitude, exceeding a custom-specified threshold, a software process is triggered and waits for a set delay time to first trigger image acquisition by a camera with an exposure time of 1 μs or less.

[0097] Using an automated software process that begins after the automated droplet initiation procedure is completed, the channel sidewalls and center of the droplet contained in the camera image are detected to define a region of interest (ROI) within the captured image of the droplet, as shown in Figure 3. The use of an automated software-based method of defining the ROI removes a significant amount of error that would normally accompany manual positioning of the ROI from a human user, thereby increasing the accuracy of the droplet size estimation method and improving the reliability of measurements between different experiments.

[0098] 3 illustrates a schematic of a region of interest in a captured image of a droplet 320 in a microfluidic channel 322. The region of interest 326 is a linear region substantially parallel to the sidewall 324 of the microfluidic channel 322 that includes the center of the droplet. The region of interest 326 has 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 dispersion medium 330. The distance extending between the peaks 328 corresponds to the diameter of the droplet 320.

[0099] The ROI 326 may be defined in the form of a one-pixel-high horizontal line that is parallel to the microchannel sidewalls 324 and horizontally intersects the imaged picodroplets 320 through their centers. Using only a one-pixel-high straight line of image data, rather than using the entire image, significantly reduces processing time, thereby increasing the possible throughput of the system.

[0100] Image data from the first defined horizontal line ROI 326 is subtracted from the average of the same data to allow for detection of above-average differences in downstream image processing. This processed data is then analyzed using peak detection software to identify, for each imaged picodroplet 320, two negative peak-minima 328 that relate to the front and back boundaries of the picodroplet and are greater than a specified threshold selected to filter out differences caused by biological cells / objects trapped within the droplet, as shown in FIG. 3. The distance or length (l) between the peaks is a measure of the diameter of the detected picodroplet 320, from which the droplet volume can be estimated.

[0101] Image data from the ROI can also be processed using different sets of applied thresholds and, when subtracted from the average value of the same data, used to identify the location, number, size, and / or morphology of biological cells or objects contained within the droplet.

[0102] The microfluidic channel may include a marker (not shown) having known dimensions. The captured image may include the droplet and the marker, and the dimensions of the droplet in the captured image may then be compared to markers located in the microfluidic channel or in an area located near the microfluidic channel, thereby determining the physical droplet dimensions. This may be performed in combination with determining an ROI or may be used instead of determining a peak from the ROI.

[0103] The optical assembly may be provided as an inverted microscope as shown in FIG. 2. The inverted microscope may be a semi-automated inverted microscope with white LED illumination, in which case images of the microfluidic chip mounted on an x,y translatable stage 218 may be continuously and simultaneously relayed to a silicon free-space amplified photodetector and a high-speed plane-scanning camera. The translatable stage 218 may also be rotatable (Θ). The LEDs are mounted on an LED holder 204 that is adjustable in the z direction. The objective lens 208 of the inverted microscope shown in this example is adjustable along the z axis to allow an image of the microchip to be formed by the lens 232. In this example, the lens 232 has a focal length of 100 mm. The microfluidic chip may be a droplet generation chip as shown in FIG. 4 to FIG. 7, including microfluidic droplet generation and drainage or collection channels for biological cell / particle encapsulation. The system allows downstream signal processing and subsequent adjustment of the monodispersity of the created emulsions containing picodroplets.

[0104] The instrument may be semi-automated, with a human user assembling the microfluidic chip with the macrofluidic connections, placing the assembled fixture on the instrument stage, and then proceeding through sequential automated steps of a custom software-based workflow operation.

[0105] FIG. 4 illustrates a schematic of a droplet generation region 440 on a microfluidic chip that may be used with the optical assembly of FIG. 1. FIG. 4 shows a diagram of a monodisperse phase inlet droplet generation region with a Y-shaped sorting channel leading to waste and / or collection channels. The droplet generation region comprises an aqueous sample inlet channel 442 and a carrier oil inlet channel 444 for generating an emulsion. These liquid streams are fed to a flow-focusing junction 446 where an emulsion is generated and fed to a collection channel 448 of the droplet generation region 440. The aqueous inlet fluid is encapsulated within an immiscible fluorous sheath oil at the flow-focusing junction 446 to create water-in-oil pico-droplets. For example, to provide some illustrative numbers, a carrier oil may have a flow rate of 1400 μl per hour, an aqueous sample, e.g., a cell suspension, may have a flow rate of 1000 μl per hour, and a water-in-oil emulsion in a collection channel may have a flow rate of 2400 μl per hour containing 700 picoliter droplets at 1000 Hz.

[0106] The sample inlet channel 442 is coupled to a first pressure controller (not shown) and the carrier fluid inlet channel 444 is coupled to a second pressure controller (not shown). The first and second pressure controllers may be used to control the pressure in the sample fluid inlet channel 442 or the carrier fluid inlet channel 444.

[0107] In this example, the sample fluid 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 gas pressurized fluid reservoirs, which are then actuated via fast acting pressure regulators. After automated software adjustment of the objective focus (z-direction) level via associated stepper motors, an automated droplet creation initiation software process is then used to increase the pressure controlling the fluid flow in both the aqueous sample inlet channel 442 and the carrier oil inlet channel 444 to initiate droplet creation.

[0108] As described above with respect to FIG. 3, the calculated droplet size may be used as an input to a feedback loop that adjusts the input pressure of one or more fluid inlet lines 442, 444 depending on the calculated droplet size to maintain monodispersity of the droplets in the emulsion.

[0109] A system including the optical assembly shown in Figure 1 with a microfluidic chip including a droplet generation region as shown in Figure 4 may perform an automated process of the steps described above using photodetector-based detection of the presence of individual droplets in a microfluidic stream to initiate triggered capture of droplet images at designated regions in a microfluidic channel. The images may then be used to calculate average or individual droplet sizes and input the calculated droplet size data into a closed feedback loop that maintains 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.

[0110] A closed-loop image-based feedback routine is then initiated whereby image data from the ROI is extracted from sequential images and processed as described above to measure droplet size and then adjust the input pressure of one or more input fluid lines to maintain droplet monodispersity within the emulsion over the duration of the emulsion creation process.

[0111] The product of the average frequency over a given period of image acquisition and the average droplet volume over the same period is a good approximation of the volume of sample consumed in the droplets, and the sum can be continuously updated. Using the determined average picodroplet volume and the average picodroplet generation frequency derived from the photodetector, the volume of droplets in the created emulsion can be calculated. When the volume of the created emulsion is equal to or greater than a user-specified target volume, the automated software is activated, which increases the pressure in the carrier oil inlet channel 444, first restricting further fluid flow from the aqueous sample inlet channel 442, and then removing the last created picodroplet from the microfluidic chip to the emulsion reservoir to make the created emulsion physically available to the user. In this way, the user can specify the volume of emulsion desired to be collected based on the sample size fed, and the machine will stop creating the emulsion when this value is reached.

[0112] In further embodiments not shown, the microfluidic chip may include multiple droplet generation regions similar to that shown in FIG. 4 (e.g., a microfluidic chip may have eight or more droplet creation nozzles, each leading to one of eight or more parallel collection channels). An opto-mechanical arrangement may be positioned immediately after the dichroic beam splitter to send light (having a wavelength less than 488 nm) from two or more of the independent parallel channels to an independent photodetector detection chip to trigger droplet imaging and also to collect frequency data independently from two or more of the parallel channels. The image processing method described above for identifying the microchannel sidewalls and the center of the created droplets and defining horizontal line ROIs that are parallel to the microchannel sidewalls that contain them is used, but is extended such that a minimum of two of the eight or more parallel channels have independent ROIs defined thereon by image processing. The droplet size data from each independent ROI is used to extract droplet size data from the multiple channels, which in turn is used to control closed-loop image-based feedback control of input pressure that affects droplet size. This provides for the generation of parallel aligned single emulsion droplets with closed-loop image-based feedback.

[0113] In a further embodiment, not shown, each of the parallel collection channels can then merge into a common downstream outlet channel. An opto-mechanical arrangement can be placed behind the dichroic beam splitter to direct light (having a wavelength less than 488 nm) from the common downstream outlet channel (where each of the upstream parallel channels merge) to one or more independent photodetector detection chips to trigger droplet imaging and collect frequency data. The image processing method described above is used to identify the sidewalls of the microchannel and the center of the created droplet and define a horizontal line ROI that is parallel to the sidewall of the containing microchannel, so that the common outlet channel can have one or more independent ROIs defined thereon by image processing.

[0114] The flow focusing junction 446 is also coupled to a waste channel 452 as shown in Figures 4, 5, 6, and 7. Figure 5 shows a flow focusing junction 446, a serpentine channel 447, and a Y-shaped sorting junction with channels 454, 456 that lead to both waste 452 and recovery 448 in the droplet generation area of ​​Figure 4. The emulsion formed at the flow focusing junction 446 flows through the serpentine channel 447 to a Y-shaped junction with two drains 454, 456. When the device is started, there may be a period of time during which the droplets being generated do not meet the specified user size requirements. 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 inducing a rolling effect in the droplets as they pass through the serpentine channel 447. In the illustrated example, the serpentine channel 447 has a width of 90 μm.

[0115] The first outlet 454 has a narrower width and is located between the flow focusing junction 446 and the collection channel 448. The second outlet 456 has a wider width and is located between the flow focusing junction 446 and the waste channel 452. In the illustrated example, the first outlet 454 has a width of 90 μm and the second outlet has a width of 120 μm. Although the droplets being generated do not meet the specified user size requirements, the microfluidic chip allows these droplets to move to the waste channel 452, and only when the correct droplet size is achieved is a memory shape valve (not shown) on the waste channel 452 closed. In one example, the valve is programmed to close for one second, but the speed of closing can be modified. By closing the valve of the waste channel 452 relatively slowly, this allows the system to compensate for the increased back pressure in the system without changing the droplet volume.

[0116] In this example, the sample fluid inlet 442 and carrier fluid inlet 444 are spaced relatively far from each other and from the collection channel 448 and waste channel 452. This allows a cell or oil sample reservoir, such as a syringe body, to be attached directly to the chip using a Luer lock fitting.

[0117] Figure 6 illustrates a schematic of a droplet generation region 540 for two-component aqueous emulsion production. Figure 6 shows a diagram of a two-component dispersed phase droplet generator sorting chip design for making emulsions of two-component dispersed phase droplets, and Figure 7 shows a flow focusing junction 446, a serpentine channel 447, and a Y-shaped sorting junction with channels 454, 456 that lead to both waste 452 and collection 448 of the droplet generation region 540 of Figure 6.

[0118] The serpentine channel region 447 is a sinusoidally shaped channel located between the flow focusing junction 446 and the Y-junction. This aids in droplet mixing of the binary dispersed phase mixture within the droplet.

[0119] Binary aqueous droplets are droplets formed from a mixture of two aqueous fluids. The fluids may both contain a sample fluid, 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 in the droplet. The droplet generation region 440 is similar to that shown in FIG. 4, but the droplet generation region also includes a second sample fluid inlet 556. The two laminar aqueous inlet fluids may meet just before the flow focusing junction 446 and are trapped in an immiscible fluorous sheath oil at the droplet creation nozzle to create individual water-in-oil pico droplets. A third pressure controller 558 is used to control the pressure of the fluid stream in the second sample fluid inlet 556 from an additional reservoir containing a second aqueous sample.

[0120] Liquid flow sensors (not shown) are positioned 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 for at least one of the aqueous inlet lines, the mixing ratio between the two aqueous inlet samples in the formed droplets can be estimated in real time. (i) The droplet frequency (also referred to as the droplet generation rate) of the picodroplets is multiplied by the calculated running average picodroplet volume to obtain the total volumetric flow rate of the system (e.g., in units of μL s -1 ) and (ii) subtracting the sensor-based volumetric flow rate of one aqueous inlet line 442, 556, measured using a flow sensor; (iii) This is performed by calculating the ratio of the flow sensor based volumetric flow rate of the single aqueous inlet line / (total volumetric flow rate of the system-flow sensor based volumetric flow rate of the single aqueous inlet line).

[0121] The first and third pressure controllers may be used to control the pressure in the sample fluid inlets 442, 556 depending on the estimated mixing ratio in the formed droplets. This allows the mixing ratio of the two sample fluids in the droplets to be adjusted in real time. This provides a closed-loop image-based feedback routine, which creates a monodisperse single emulsion of aqueous pico-droplets in the fluorous oil-based containment fluid with a user-defined mixing ratio between the two inlet aqueous fluids and a user-defined pico-droplet volume. Furthermore, the mixing ratio may be maintained at a constant level or the mixing ratio may be defined to vary over a particular period of time.

[0122] Image analysis from the ROIs in the images of the created picodroplets can be used to count the number of contained microparticles (including biological cells) in each individual picodroplet. The average biological cell / object occupancy of the droplets can be monitored. The first and third pressure controllers can be used to control the pressure in the sample fluid inlets 442, 556 depending on the average biological cell / object occupancy of the droplets. In this way, the device provides a closed-loop feedback system, where the average biological cell / object occupancy of the droplets is used as an input to a feedback loop that adjusts the mixing ratio between the two inlet aqueous fluids (e.g., the first aqueous sample fluid can be a particle or cell-containing solution and the second aqueous sample fluid can be an affinity buffer solution). Adjustment of this mixing ratio allows the average particle or cell occupancy to be controlled during the experimental process, compensating for particle or cell settling, high particle or cell concentrations, or other forms of drift.

[0123] droplet fusion FIG. 8 illustrates an exemplary droplet fusion region 610. The droplet fusion region 610 may be part of a microfluidic chip that may be used with the optical assembly of FIG. 1. The droplet fusion region 610 includes a microfluidic droplet channel 612 for carrying a stream of an emulsion 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 disposed downstream of the first input and supplies a stream of droplet fluid 618 to the microfluidic droplet channel 612. In the example illustrated in FIG. 8, the droplet fluid inlet 614 is provided as a second microfluidic channel disposed substantially perpendicular to the microfluidic droplet channel 612.

[0124] A first droplet sensor 622 is positioned upstream of the droplet fusion region 610 to detect and determine the droplet volume of individual droplets 616 in the emulsion stream. A second droplet sensor 624 is positioned downstream of the droplet fusion region to detect the volume of any of the fused droplets 620. The first and second droplet sensors 622, 624 may each comprise an optical assembly as shown in Figures 1 and 2. The droplet volume may be determined using methods described with respect to droplet generation, such as those used in connection with the microfluidic chips shown in Figures 4-7.

[0125] The pressure or flow rate of the droplet fluid 618 in the droplet fluid inlet 614, or the pressure or flow rate of the emulsion in the microfluidic channel 612, can be increased or decreased based on the measured droplet volume in a closed-loop feedback manner due to the proportion of fluid in the fused droplet 620.

[0126] The droplet fusion region 610 may be located downstream of the droplet generation regions 440, 540 shown in Figures 4-7, and droplets generated by the droplet generation regions 440, 540 may be input to the droplet fusion 610.

[0127] Droplet fusion region 610 may be used to fuse droplets 616 formed from a first droplet fluid in the emulsion with a second droplet fluid 618 to form an emulsion of fused droplets 620, each comprising a mixture of the first and second droplet fluids. Image-based closed-loop feedback may be used to vary the ratio of the mixture of the first and second droplet fluids in the fused droplets, thereby allowing this ratio to be adjusted or maintained at a substantially constant optimum value.

[0128] One exemplary method for fusing droplets 616 with a second droplet fluid 618 using droplet merging region 610 is as follows, with steps performed in the following order: (1) A stream of emulsion including a stream of dispersed microfluidic droplets 616 of a first droplet fluid in a continuous fluid phase is provided to a microfluidic channel 612. (2) A droplet 616 flowing within the microfluidic channel 612 is imaged using a droplet sensor 622 located within a droplet detection region. (3) Calculate the size or volume of the droplets 616 from the captured images. Alternatively, the size or volume of the droplets may be known or obtained using alternative methods. For example, the droplet volume may be calculated without imaging the droplets if the total volume of the droplets 616 to be dispensed is known and the droplets are monodisperse or have a uniform size. (4) The emulsion flows through microfluidic channel 612 towards droplet fluid inlet 614. (5) As shown in FIG. 8( a), a second droplet fluid 618 is introduced into the microfluidic channel 612 from the droplet fluid inlet 614 such that a mass of the second droplet fluid 618 extends from the droplet fluid inlet 614 into the microfluidic channel 612. (6) As the emulsion continues to flow through the microfluidic channel 612, there comes a point where physical contact or collision occurs between the droplet 616 and the second droplet fluid 618, as shown in Figure 8(b). The point of physical contact or collision occurs within a region of the microfluidic channel 612 adjacent to the droplet fluid inlet 614. (7) Fusing the droplet 616 with the second droplet fluid 618. The existence of a method for fusing the fluids at the point of impact disrupts the structure of the fluid boundaries of both the droplet of the first fluid 616 and the protruding mass of the second droplet fluid 618, causing them to fuse or mix to form a single mass of fluid protruding from the droplet fluid inlet 614, as shown in FIG. 8(c). The two fluids may fuse due to the pressure increase in the microfluidic droplet channel, as described in step (8). Alternatively, the disruption of the fluid boundary of the droplets of the first and second fluids can be induced by an external stimulus, resulting in coalescence. The external stimulus may be electrical or optical. Electrical coalescence involves fusing the droplets by applying an electric field as they pass through a confined region bounded by a pair of electrodes. Optical methods involve using a focused laser beam to locally heat the fluid boundary, thereby changing the surface tension of the droplet interface. In FIG. 8(c), the single body of fluid protruding from droplet fluid inlet 614 into microfluidic channel 612 contains a mixture of both the first droplet fluid and the second droplet fluid. (8) As the emulsion upstream of the droplet fluid inlet 614 continues to flow toward the droplet fluid inlet 614, an increase in the pressure of the emulsion behind the single mass of fluid protruding from the droplet fluid inlet 614 occurs. This increase in stress or pressure behind the protruding mass of fluid causes the breakup of the fused microdroplet 620 from the droplet fluid inlet 614, as shown in FIG. 1(d). This fused droplet 620, which may be a two-component aqueous droplet, contains a mixture of the first droplet fluid and the second droplet fluid, and continues to flow downstream as part of the emulsion stream of fused droplets. (9) The fused droplets 620 flowing within the microfluidic channel 612 are imaged using a droplet sensor 624 located in a second droplet detection region downstream of the droplet fluid inlet 614. (10) Calculate the droplet size or volume from the captured images of the fused droplets 620. (11) determining the volume change between the initial microdroplets 616 and the fused droplets 620 using images captured using the droplet sensors 622, 624 and calculating the volumetric contributions from the first droplet fluid and the second droplet fluid within each droplet in the stream of fused droplets or the ratio of the first droplet fluid to the second droplet fluid;

[0129] The calculated volume or size values ​​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 and second droplet fluids calculated in step (11) may be used as inputs to a closed-loop feedback routine. The experimental inputs (e.g., driving pressure, or flow rate) of the emulsion stream fed into the microfluidic channel or the second droplet fluid stream fed into the droplet fluid inlet are then adjusted according to the calculated volumes or ratios to maintain droplet parameters at predetermined levels (e.g., final fused droplet volume, volume contribution, fluid ratio) when generating the emulsion stream of fused droplets 620. It may be easier to control the ratio of the first and second droplet fluids by fusing microdroplets of the first droplet fluid with the second droplet fluid, rather than mixing the two droplet fluids before generating the droplets.

[0130] For example, an image-based closed-loop feedback routine can control and maintain the droplet volume of droplet 616 in the initial emulsion of droplets by varying the driving force of the first fluid stream. This is because the flow rate of the droplet of the first fluid affects the time interval between the collision of the droplet with the protruding component of the second fluid and therefore the amount of the second fluid that mixes with the droplet. If the droplet has a lower flow rate, it will spend more time in 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 routine can be used to simultaneously adjust the driving force or pressure of the second droplet fluid stream in response to the volumetric contribution or ratio of fluids within the droplet of the fused droplet stream deviating from a predetermined level.

[0131] FIG. 9 illustrates a schematic of a droplet reinjection region and a droplet merging region, and FIG. 10 illustrates a schematic of a droplet fluid inlet of the droplet merging region shown in FIG.

[0132] The droplet merging region includes a droplet inlet 818 for supplying microdroplets formed in an emulsion comprising a first droplet fluid in an immiscible carrier medium. The droplet inlet 818 supplies droplets to the microfluidic droplet channel 812. The droplets in the microfluidic droplet channel 812 flow toward a junction between the microfluidic droplet channel 812 and the droplet fluid channel 816 through which a second droplet fluid flows from the droplet fluid inlet 814 toward the microfluidic droplet channel 812. The droplet fluid channel 816 is arranged to be substantially perpendicular to the microfluidic droplet channel 812 at the junction 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 the same manner as described with respect to FIG.

[0133] In this example, two electrodes 824 (a ground electrode and an active electrode) are positioned near the junction between the microfluidic droplet channel 812 and the droplet fluid channel 816. An electric field between the two electrodes 824 causes the second droplet fluid to merge with the microdroplet, as described with respect to FIG.

[0134] The fused droplets flow from microfluidic droplet channel 812 towards fused droplet outlet 826 through fused droplet channel 828. In this example, fused droplet channel 828 has a serpentine shaped portion that facilitates mixing of the first and second droplet fluids in the fused droplet. The fused droplet channel 828 has a shape such that after mixing, the fused droplets may be imaged within the field of view of the camera.

[0135] 9 and 10, the camera images area C such that droplets can be detected at points A and B. As described with respect to FIG. 8, the size of the fused droplet or the ratio of liquid of the first droplet to the second droplet in the fused droplet can be controlled by varying the pressure and / or flow rate of the droplet from droplet inlet 818, the second droplet fluid from droplet fluid inlet 814, or the spacing fluid from spacing fluid inlet 820 in response to the camera imaging the droplets at locations A and B.

[0136] In this example, spacing fluid inlet 820 supplies spacing fluid to microfluidic droplet channel 812 through spacing fluid channel 822 arranged on a sidewall of microfluidic droplet channel 812 between droplet inlet 818 and droplet fluid channel 816. The spacing fluid increases the spacing between droplets in microfluidic droplet channel 812 and allows the rate at which droplets reach the junction with droplet fluid channel 816 to be controlled. The reinjection region refers to droplet inlet 818 and spacing fluid inlet 820, which supplies spacing fluid or additional carrier medium to the microfluidic channel through spacing fluid channel 822.

[0137] Figure 11 illustrates a schematic of the droplet generation region and droplet merging region, and Figure 12 illustrates a schematic of the droplet fluid inlet of the droplet merging region shown in Figure 11. The droplet generation region is similar to that shown in Figure 4. Many of the features are the same as those shown in Figures 9 and 10 and therefore bear similar reference numerals.

[0138] The droplet generation region includes a first droplet fluid inlet 930 and a carrier fluid inlet 920, which supply a first droplet fluid (e.g., an aqueous sample) through a first droplet fluid channel 932 and a carrier fluid channel 922. These liquid streams are supplied to a flow-focusing junction 946 where an emulsion is generated and supplied to the microfluidic droplet channel 912. The aqueous inlet fluid is encapsulated within an immiscible fluorous sheath oil at the flow-focusing junction 946, creating an emulsion of water-in-oil pico-droplets.

[0139] The droplets in the emulsion merge with the second droplet fluid in the same manner as described with respect to Figures 8 through 10. The droplets in the microfluidic droplet channel 912 flow toward the junction between the microfluidic droplet channel 912 and the second droplet fluid channel 916 through which the second droplet fluid flows from the droplet fluid inlet 914 toward the microfluidic droplet channel 912.

[0140] The system of FIG. 12 allows the volume of a droplet containing only the first droplet fluid to be controlled, such 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.

[0141] droplet splitting 13 illustrates a schematic of the droplet reinjection and droplet splitting regions. The system includes a droplet inlet 711 for supplying microdroplets formed in an emulsion comprising a first droplet fluid in an immiscible dispersion medium. The droplet inlet 711 supplies droplets through a droplet inlet channel 712 to a microfluidic droplet channel 718.

[0142] Droplets in the microfluidic droplet channel 718 flow towards the junction between the microfluidic droplet channel 718 and the droplet splitting nozzle, as described with respect to FIG.

[0143] In this example, carrier medium inlet 732 supplies additional carrier medium or spacing fluid to the microfluidic droplet channel 718 through carrier medium channel 734 arranged on a sidewall of the microfluidic droplet channel 718 between the droplet inlet 711 and the droplet splitting nozzle. The spacing fluid increases the spacing between droplets in the microfluidic droplet channel 718, allowing the rate at which the droplets reach the droplet splitting nozzle to be controlled.

[0144] The camera may be provided out of the plane of the system shown in Figure 13, or the system may include multiple sensors as shown in Figure 14. As described with respect to Figure 14, the size of the daughter droplets may be controlled in response to the camera imaging the droplets by varying the pressure and / or flow rate of the droplet from droplet inlet 711, additional carrier fluid from carrier fluid inlet 732, or spacing fluid from spacing fluid inlet 720.

[0145] The droplet splitting region or droplet splitting nozzle is the same as that shown in FIG. 14 and will be described below with respect to FIG.

[0146] FIG. 14 illustrates a schematic of an exemplary droplet splitting region 710. The droplet splitting region 710 includes a droplet inlet channel 712 for supplying a stream of an emulsion of microdroplets 726 in a carrier fluid. The droplet splitting region 710 is disposed downstream of the droplet inlet 712 and has two outlets formed from two daughter droplet microfluidic channels 714, 716. In the illustrated example, there is a microfluidic droplet channel 718 between the droplet inlet channel 712 and the daughter droplet channels 714, 716. The microfluidic channel 718 splits into two droplet channels 714, 716 at a single junction, causing the larger droplet 726 to split into two smaller droplets 728, 730. The junction where the microfluidic channel 718 splits into the two smaller droplet channels 714, 716 may be a droplet splitting nozzle.

[0147] The microfluidic droplet channel 718 may be narrower than the droplet inlet 712. The two droplet channels 714, 716 may have a narrower cross-section closer to the junction with the microfluidic droplet channel 718.

[0148] In the illustrated example, the two daughter 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, causing the input droplet 726 to split into unequal droplets 728, 730, with the larger droplet 728 forming in the first droplet channel 714 and the smaller droplet 730 forming in the second droplet channel 716.

[0149] A first droplet sensor or camera 722 is positioned upstream of the droplet splitting region 710 or at the droplet inlet 712 to detect and determine the droplet volume of individual droplets 726 in the emulsion stream. A second droplet sensor or camera 724 is positioned downstream of the droplet splitting region to detect the volume of droplets in either the first droplet channel 714 or the second droplet channel 716. The first and second droplet sensors 722, 724 may each comprise an optical assembly as shown in Figures 1 and 2. The volume of the droplets may be determined using methods described with respect to droplet generation, such as those used in connection with the microfluidic chips shown in Figures 4-7.

[0150] A carrier fluid inlet 720 or pressure regulated channel supplies 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 manner to regulate the size of the daughter droplets 728, 730 being created.

[0151] In the illustrated example, one dispersion medium inlet 720 supplies dispersion medium to the first droplet channel 714, but alternatively, a separate dispersion medium inlet could supply dispersion medium to the second droplet channel 716 instead of or in addition to supplying dispersion medium to the first droplet channel 714.

[0152] One exemplary method for splitting droplet 726 into two smaller droplets 728, 730 is as follows, with steps performed in the following order: (1) A stream of emulsion comprising a stream of dispersed microfluidic droplets 726 in a continuous dispersion medium is provided to a microfluidic channel 718. The droplets may be provided from a droplet inlet 712. (2) A droplet sensor 722 located in a droplet detection region is used to image the droplet 726 flowing in the microfluidic channel 718. Alternatively, the droplet 726 is imaged before it enters the microfluidic channel 718. (3) Calculate the size or volume of the droplets 726 from the captured images. Alternatively, the size or volume of the droplets may be known or obtained using alternative methods. For example, the droplet volume may be calculated without imaging the droplets if the total volume of the droplets 726 dispensed is known, the number of droplets or the droplet frequency is known, and the droplets are monodisperse or have a uniform size. (4) The emulsion flows through a microfluidic channel 718 toward a junction or droplet splitter, which splits into two smaller droplet channels 714, 716. This may be a droplet splitting nozzle. As the droplet 726 flows through the junction, it splits into two smaller daughter droplets 728, 730. (5) Two smaller droplets 728, 730 flow from the junction through two separate droplet channels 714, 716 that branch off from the microfluidic droplet channel 718. The daughter droplets 728 each flow within an emulsion of droplets in a carrier fluid. (6) imaging the first daughter droplet 728 in the first droplet channel 714 using a droplet sensor 724 located in a second droplet detection region downstream of the split nozzle. Alternatively, or in addition, the method may include imaging a second daughter droplet 730 in the second droplet channel 716. (7) Calculating the size or volume of droplet 728 from the captured image of first daughter droplet 728. (8) determining the volume change between the initial microdroplet 726 and the daughter droplet 728 using the images captured using the droplet sensors 722, 724 and calculating the volume distribution or volume ratio between the two daughter droplets 728, 730; (9) Introduce the carrier fluid into the first droplet channel 714 through the carrier fluid inlet 720 or a pressure regulating channel. The carrier fluid may be a spacing oil.

[0153] The calculated volume or size values ​​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 daughter droplet 728 to the volume of the second daughter droplet 730 calculated in step (8), may be used as inputs to a closed-loop feedback routine, where the experimental inputs (e.g., driving pressure, or flow rate) of the emulsion stream provided to the microfluidic channel 718 or the carrier fluid stream provided in the carrier fluid inlet 720 are adjusted according to the calculated volumes or ratios to maintain droplet parameters at predetermined levels (e.g., final daughter droplet volumes, volume contributions, or ratios) when splitting droplets within the emulsion stream of microdroplets 726.

[0154] For example, an image-based closed-loop feedback routine can adjust the pressure or flow rate of spacing oil or carrier fluid supplied to one of the daughter droplet channels 714, 716 from carrier fluid inlet 720 to increase or decrease the backpressure provided by the carrier fluid in the daughter droplet channels 714, 716 and the microfluidic channel 718. This adjusts or maintains the volumetric ratio of daughter droplet volumes at a predetermined level in response to a determined ratio of droplet volumes that deviates from the predetermined level.

[0155] Alternatively, or in addition, an additional carrier fluid may be introduced into the second droplet channel 716 through a second carrier fluid inlet. The additional carrier fluid may be the same or a different fluid as the spacing fluid introduced through the carrier fluid inlet 720. The carrier fluid and additional carrier fluid may be the same or a different fluid as the continuous carrier fluid into which the droplets are provided prior to splitting.

[0156] The dispersion medium and the further dispersion medium are both miscible with the continuous dispersion medium in which the droplets are provided before splitting, and immiscible with the dispersed microdroplets.

[0157] Although the illustrated example relates to picodroplets in a microfluidic chip, it should be understood that the device is not limited to picodroplets (which are generally less than about 1000 picoliters or a few thousand picoliters in volume) but is applicable to droplets of other sizes as well (e.g., the droplets may be larger or smaller and may have volumes in the nanoliter to femtoliter range).

[0158] The examples described relate to emulsions that include droplets that include a first fluid dispersed in a second fluid, where the second fluid is immiscible with the first fluid. The term fluid may be used herein to refer to a liquid.

[0159] In a preferred embodiment, we have described the technique as being applied to treating droplets of water-in-oil emulsions containing biological entities. However, in principle, non-biological entities, such as organic or inorganic entities, can be treated in a similar manner. Similarly, the technique we have described is also, in principle, applicable to treating droplets of oil in oil-in-water emulsions. In water-in-oil emulsions, the oil forming the dispersion medium or spacing fluid generally comprises fluorous oil and / or mineral oil and / or silicone oil, and preferably a surfactant, for example at a volumetric or weight ratio of about 0.5-5%.

[0160] No doubt many other effective alternatives will occur to those skilled in the art, and it will be understood that the invention is not limited to the described embodiments, but encompasses modifications apparent to those skilled in the art that are within the spirit and scope of the appended claims. [Explanation of symbols]

[0161] 102 Microfluidic Chip 104 LED 106 Optical Lens Tube Assembly 108 Objective Lens 110 Beam splitter 112 Photodetector 114 Iris Aperture 116 High-speed camera 204 Z direction adjustable LED holder 208 z-adjustable objective lenses 218 xy-Θ translational stage 232 Lens 320 droplets 322 Microfluidic Channels 324 Side wall 326 Areas of Interest 328 Light Peak 330 Dispersion medium 440 Droplet generation area 442 Sample fluid inlet 444 Dispersion medium inlet 446 Flow focusing junction 447 Meandering Channel 448 Collection Channel 452 Waste Channel 454 First Discharge Section 456 Second Discharge Section 540 Droplet generation area 556 Second sample fluid inlet 610 Droplet fusion region 612 Microfluidic Droplet Channel 614 Droplet Fluid Inlet 616 Microfluidic Droplets 618 Droplet Fluid 620 Fused Droplets 622 Droplet Sensor 624 Droplet Sensor 710 Droplet division area 711 Droplet inlet 712 Droplet inlet channel 714 First Droplet Channel 716 Second Droplet Channel 718 Microfluidic Droplet Channel 719 Interval Fluid Inlet 720 spacing fluid inlet channels 722 Droplet Sensor 724 Droplet Sensor 726 Microdroplets 728 First Daughter Droplet 730 Second Daughter Droplet 732 Dispersion medium inlet 734 Dispersion medium inlet channel 812 Microfluidic Droplet Channel 814 Droplet Fluid Inlet 816 Droplet Fluidic Channel 818 Droplet inlet 820 interval fluid inlet 822 spaced fluid channels 824 Electrode 826 Fusion Droplet Outlet 828 Fused Droplet Channel 912 Microfluidic Droplet Channel 914 Second Droplet Fluid Inlet 916 Second Droplet Fluid Channel 920 Dispersion medium inlet 922 Dispersion Channel 924 Electrode 926 Fusion droplet outlet 928 Fused Droplet Channel 930 first droplet fluid inlet 932 First Droplet Fluid Channel

Claims

1. 1. A method for determining a droplet frequency of a stream of microfluidic droplets in a microfluidic droplet channel, comprising: illuminating the stream of microfluidic droplets in the microfluidic droplet channel with light; splitting light from the stream of microfluidic droplets into a first portion and a second portion using a beam splitter, the first portion comprising light greater than a predetermined threshold wavelength and the second portion comprising light less than a predetermined threshold wavelength; presenting the first portion to a camera; directing the second portion toward a photodetector through an aperture disposed in front of the photodetector; processing a signal from the photodetector and determining a droplet frequency from the variation of the processed signal; A method comprising:

2. detecting droplets using the processed signal; Upon detection of the droplet, simultaneously capturing an image of the droplet using the camera. The method of claim 1 further comprising:

3. The method of claim 2 , wherein the method further comprises determining a droplet size from the captured image of the droplet.

4. The method of claim 3 further comprising the step of calculating a droplet volume from the determined dimensions.

5. The method for determining droplet size comprises: identifying a sidewall of the microfluidic droplet channel and a center of a droplet within the image of the flow; determining a region of interest within a captured image of the droplet, the region of interest comprising a linear region including a droplet center, the region of interest being parallel to the sidewall; determining the droplet size by processing the linear regions of the image; The method of claim 3 or 4, comprising:

6. 6. The method of claim 3, wherein the method of determining droplet size comprises comparing the droplet size in the captured image to a marker of known size disposed within the microfluidic channel.

7. 1. A method for determining a total volume of fluid within a series of droplets in a stream of microfluidic droplets, comprising: Determining a droplet frequency of the stream of microfluidic droplets in a microfluidic droplet channel using the method of claim 1; calculating a droplet volume for each droplet in said stream of microfluidic droplets; calculating an average drop volume of the droplets; determining a total volume of droplets using said droplet frequency and said average droplet volume; A method comprising:

8. 1. A method for generating a stream of droplets, comprising: providing a first droplet fluid; Supplying a dispersion medium; forming an emulsion of droplets comprising the first droplet fluid in the carrier fluid by providing a stream of the first droplet fluid and a stream of the carrier fluid to a droplet generation region of a microfluidic structure; determining a total volume of fluid within a set of droplets in said emulsion of droplets using the method of claim 7; increasing a pressure in the flow of the carrier fluid conduit in response to the determined total volume of fluid in the series of droplets being greater than a predetermined threshold to inhibit droplet generation; A method comprising:

9. 1. A method for generating a stream of droplets, comprising: providing a first droplet fluid; Supplying a dispersion medium; forming an emulsion of droplets comprising the first droplet fluid in the carrier fluid by providing a stream of the first droplet fluid and a stream of the carrier fluid to a droplet generation region of a microfluidic structure; Determining droplet sizes of successive droplets in the emulsion using a method according to claim 5 or 6; adjusting a pressure of the stream of the first droplet fluid and / or a pressure of the stream of the carrier fluid in response to the determined droplet size. A method comprising:

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

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

12. 1. A method for generating a stream of droplets, comprising: providing a first droplet fluid; Supplying a dispersion medium; providing a second droplet fluid; 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 a microfluidic structure to form an emulsion of droplets comprising a mixture of the first droplet fluid and the second droplet fluid in the carrier fluid; measuring a flow rate of the first stream of droplet fluid or the second stream of droplet fluid; Determining a total volume of droplets in said emulsion using the method of claim 7; determining a ratio of the first droplet fluid to the second droplet fluid in the mixture using the measured flow rate and the total volume of droplets; adjusting a pressure of the stream of the first droplet fluid and / or a pressure of the stream of the second droplet fluid in response to the determined ratio. A method comprising:

13. 1. A method for generating a stream of droplets, comprising: providing n droplets of fluid; Supplying a dispersion medium; forming an emulsion of droplets comprising a mixture of the n droplet fluids in the carrier fluid by providing a stream of each of the n droplet fluids and a stream of the carrier fluid to a droplet generation region of a microfluidic structure; measuring a flow rate of the n-1 droplet fluid stream; Determining a total volume of droplets in said emulsion using the method of claim 7; determining a ratio of the n droplet fluids in the mixture using the measured flow rates of the n-1 droplets and the total volume of droplets; adjusting a pressure of one or more of the n droplet fluid streams in response to the determined ratio; A method comprising:

14. 1. A microfluidic system comprising: a microfluidic droplet channel for carrying a stream of microfluidic droplets; a light source that irradiates the stream of microfluidic droplets with a light beam that includes light having wavelengths 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, the first portion including light greater than the predetermined threshold wavelength and the second portion including light less than the predetermined threshold wavelength; a camera configured to receive the first portion of the light; and a photodetector configured to receive the second portion of the light, an aperture disposed in front of the photodetector; and a device configured to process signals from the photodetector; a processor configured to determine a droplet frequency from variations in the processed signal; A microfluidic system comprising:

15. 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 upon the detection of the droplet.

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

17. a first droplet fluid conduit for carrying a first droplet fluid; A dispersion medium pipeline for transporting a dispersion medium; a droplet generation region having a first input for receiving flow from the first droplet fluid conduit, a second input for receiving flow from the carrier fluid conduit, and an outlet to the microfluidic droplet channel; Equipped with the processor is configured to determine an average droplet volume using the captured images of the droplets, the processor being configured to determine a total volume of droplets in a series of droplets in the stream of microfluidic droplets using the average droplet volume and the droplet frequency; 17. The system of claim 15 or 16, further comprising means for increasing pressure in the carrier conduit in response to the determined total volume of droplets being greater than a predetermined threshold.

18. a first droplet fluid conduit for carrying a first droplet fluid; A dispersion medium pipeline for transporting a dispersion medium; a droplet generation region having a first input for receiving flow from the first droplet fluid conduit, a second input for receiving flow from the carrier fluid conduit, and an outlet to the microfluidic droplet channel; Equipped with the processor is configured to determine droplet sizes of successive droplets using the captured images of the droplets; 18. The system of claim 15, further comprising means for adjusting pressure in the carrier medium conduit or the first droplet fluid in response to the determined droplet size.

19. n droplet fluid conduits each configured to carry one of the n droplet fluids; A dispersion medium pipeline for transporting a dispersion medium; a droplet generation region having n inputs for receiving n flows from the n droplet fluid conduits, a further input for receiving a flow from the carrier fluid conduit, and an outlet to the microfluidic droplet channel, a droplet generation region forming an emulsion of droplets comprising a mixture of the n droplet fluids in the dispersion medium; n-1 flow sensors configured to measure flow rates of n-1 of the n droplet fluid streams; Equipped with the processor is configured to determine an average droplet volume using the captured images of droplets, the processor is configured to determine a total volume of droplets in a series of droplets in the stream of microfluidic droplets using the average droplet volume and the droplet frequency, the processor is further configured to determine a ratio of the n droplet fluids in the mixture using the measured flow rates of the n-1 droplets and the total volume of droplets; 18. The system of claim 15, further comprising means for adjusting a pressure of one or more of the n droplet fluid streams in response to the determined ratio.

20. 19. The method of claim 1 or the system of claim 14, wherein the aperture comprises a slit corresponding to a beam of light that is substantially perpendicular to a sidewall of the microfluidic droplet channel.

21. 19. The method of claim 1 or the system of claim 14, wherein the aperture comprises a pinhole corresponding to a light beam positioned substantially at the center of the microfluidic droplet channel.

22. 19. The method of claim 1 or the system of claim 14, wherein the aperture comprises a crescent shaped aperture configured to increase the sensitivity of the light detector.

23. 22. The method of claim 1 or the system of claim 13, wherein the first portion comprises light having a wavelength greater than 488 nm and the second portion comprises light having a wavelength less than 488 nm.

24. 1. A microfluidic method for preparing droplets, comprising: providing a stream of an emulsion of microfluidic droplets of a first droplet fluid into a dispersion medium within a microfluidic channel; determining a droplet size of droplets in said stream of microfluidic droplets; providing a second droplet fluid stream into the microfluidic channel; Merging the droplets 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; adjusting a pressure or flow rate of the stream of the emulsion of microfluidic droplets and / or a pressure or flow rate of the stream of the second droplet fluid in response to the determined fused droplet size. Including, A microfluidic method, wherein the droplet sizes of the droplets in the stream of microfluidic droplets and / or the droplet sizes of the fused droplets are determined according to the method of claim 3.

25. 1. A microfluidic method for preparing droplets, comprising: providing a stream of an emulsion of microfluidic droplets of a first droplet fluid into a dispersion medium within a microfluidic channel; determining a droplet size of droplets in said stream of microfluidic droplets; providing a second droplet fluid stream into the microfluidic channel; Merging the droplets 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; adjusting a pressure or flow rate of the stream of the emulsion of microfluidic droplets and / or a pressure or flow rate of the stream of the second droplet fluid in response to the determined fused droplet size. A microfluidic method comprising:

26. 26. The method of claim 24 or 25, wherein providing a second flow of droplet fluid comprises providing a second flow of droplet fluid from a fluid inlet arranged in a sidewall of the microfluidic channel.

27. 27. The method of any one of claims 24 to 26, wherein fusing the droplet of the first droplet fluid with at least a portion of the second droplet fluid to obtain a fused droplet comprises forming a fused droplet comprised of a mixture of the first droplet fluid and the second droplet fluid.

28. 28. The method of claim 27, further comprising 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, and a pressure or flow rate of the stream of emulsion or the stream of second droplet fluid is adjusted in response to the determined ratio.

29. 29. The method of any one of claims 24 to 28, wherein fusing the droplets of the first droplet fluid with at least a portion of the second droplet fluid comprises applying an electric field to the droplets of the first droplet fluid and to a portion of the second droplet fluid.

30. Providing a stream of microfluidic droplet emulsion of a first droplet fluid into a dispersion medium within a microfluidic channel includes: providing a first droplet fluid; Supplying a dispersion medium; forming an emulsion of droplets comprising the first droplet fluid in the carrier fluid by providing a stream of the first droplet fluid and a stream of the carrier fluid to a droplet generation region of a microfluidic structure; 30. The method of any one of claims 24 to 29, comprising:

31. 31. The method of claim 30, wherein adjusting the pressure or flow rate of the stream of the emulsion of microfluidic droplets comprises 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. 32. The method of any one of claims 24 to 31, further comprising the step of supplying a flow of spacing fluid to the microfluidic droplet channel, and wherein adjusting the pressure or flow rate of the flow of the emulsion of microfluidic droplets comprises adjusting the pressure or flow rate of the flow of spacing fluid in response to the determined droplet size.

33. 1. A microfluidic system comprising: a droplet inlet for providing a stream of an emulsion of microfluidic droplets of a first droplet fluid in a dispersion medium; a droplet sensing system for determining a size of droplets in said stream of microfluidic droplets; a fluid inlet for supplying a second droplet fluid stream; a droplet merging 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 configured to merge the droplets 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 for determining a size of the merged droplet; means for increasing a 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 droplets; Equipped with The droplet sensing system or the second droplet sensing system comprises the microfluidic system of claim 14 .

34. 1. A microfluidic system comprising: a droplet inlet for providing a stream of an emulsion of microfluidic droplets of a first droplet fluid in a dispersion medium; a droplet sensing system for determining a size of droplets in said stream of microfluidic droplets; a fluid inlet for supplying a second droplet fluid stream; a droplet merging 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 configured to merge the droplets 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 for determining a size of the merged droplet; means for increasing a 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 droplets; A microfluidic system comprising:

35. 35. The microfluidic system of claim 33 or 34, wherein the fluid inlet is arranged on a sidewall of the microfluidic channel.

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

37. 37. The microfluidic system of claim 33, wherein the droplet merging region comprises means configured to merge the droplets of the first droplet fluid with at least a portion of the second droplet fluid.

38. 38. The microfluidic system of claim 37, wherein the means configured to merge the droplets of the first droplet fluid with at least a portion of the second droplet fluid comprises a plurality of electrodes configured to apply an electric field to the droplets of the first droplet fluid and to a portion of the second droplet fluid.

39. a first droplet fluid conduit for carrying a first droplet fluid; A dispersion medium pipeline for transporting a dispersion medium; a droplet generation region having a first input for receiving flow from the first droplet fluid conduit, a second input for receiving flow from the carrier fluid conduit, and an outlet to the microfluidic droplet channel; 39. The microfluidic system of any one of claims 33 to 38, further comprising:

40. 40. The microfluidic system of claim 33, further comprising an interval fluid conduit configured to provide a flow of interval fluid to the microfluidic droplet channel.

41. 1. A microfluidic method for forming a pair of microdroplets, comprising: providing a stream of an emulsion of microfluidic droplets of a droplet fluid into a dispersion medium within a microfluidic channel; determining a droplet size of droplets in said stream of microfluidic droplets; splitting the droplet into a first droplet and a second droplet; directing the first droplet into a first droplet channel; directing the second droplet into a second droplet channel; determining a droplet size of the first droplet or the second droplet; providing a flow of spacing fluid to the first droplet channel or the second droplet channel; adjusting a pressure or flow rate of the flow of the emulsion of microfluidic droplets and / or a pressure or flow rate of the flow of spacing fluid in response to the determined size of the first droplets or the second droplets. Including, 4. A microfluidic method, wherein 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 are determined according to the method of claim 3.

42. 1. A microfluidic method for forming a pair of microdroplets, comprising: providing a stream of an emulsion of microfluidic droplets of a droplet fluid into a dispersion medium within a microfluidic channel; determining a droplet size of droplets in said stream of microfluidic droplets; splitting the droplet into a first droplet and a second droplet; directing the first droplet into a first droplet channel; directing the second droplet into a second droplet channel; determining a droplet size of the first droplet or the second droplet; providing a flow of spacing fluid to the first droplet channel or the second droplet channel; adjusting a pressure or flow rate of the flow of the emulsion of microfluidic droplets and / or a pressure or flow rate of the flow of spacing fluid in response to the determined size of the first droplets or the second droplets. A microfluidic method comprising:

43. 43. The microfluidic method of forming a pair of micro-droplets of claim 41 or 42, wherein the method includes determining a droplet volume of one of the first droplet or the second droplet, and further includes calculating a droplet volume of the other of the first droplet and the second droplet using the previously determined droplet volume of the first droplet or the second droplet and a predetermined volume of the droplet in the stream of microfluidic droplets.

44. The step of determining the droplet size comprises: capturing an image of the droplet using a droplet sensing system; determining a droplet size from the captured image; 43. The microfluidic method of any one of claims 25 to 32, 41 or 42, comprising:

45. 1. A microfluidic system comprising: a droplet inlet for providing a stream of an emulsion of microfluidic droplets of droplet fluid in a dispersion medium; a droplet sensing system for determining a size of droplets in said stream of microfluidic droplets; a droplet splitting region having a first input for receiving a stream from the droplet inlet, a first outlet to a first droplet channel, and a second outlet to a second droplet channel, the droplet splitting region configured to split droplets in the stream of microfluidic droplets into first droplets and second droplets; a fluid inlet for supplying a flow of 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 droplet; and means for increasing a pressure or flow rate of the emulsion in the droplet inlet or the spacing fluid in the fluid inlet in response to the determined size of the first droplet or the second droplet; Equipped with The droplet sensing system or the second droplet sensing system comprises the microfluidic system of claim 14 .

46. 1. A microfluidic system comprising: a droplet inlet for providing a stream of an emulsion of microfluidic droplets of droplet fluid in a dispersion medium; a droplet sensing system for determining a size of droplets in said stream of microfluidic droplets; a droplet splitting region having a first input for receiving a stream from the droplet inlet, a first outlet to a first droplet channel, and a second outlet to a second droplet channel, the droplet splitting region configured to split droplets in the stream of microfluidic droplets into first droplets and second droplets; a fluid inlet for supplying a flow of 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 droplet; and means for increasing a pressure or flow rate of the emulsion in the droplet inlet or the spacing fluid in the fluid inlet in response to the determined size of the first droplet or the second droplet; A microfluidic system comprising:

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

49. 48. 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 droplet dimensions from the captured image of the droplet.

Images