Integrated microfluidic system for generating micro-organic spheres (mos)

Abstract

A microfluidic device is disclosed that includes a microfluidic chip for micro-organic sphere (MOS) generation. A first channel is defined in a surface of the microfluidic chip and includes a droplet generating portion including an inlet portion, a junction between the inlet portion and an emulsifying fluid channel, and a chamber downstream of the junction. The cross-sectional area of the chamber is greater than the cross-sectional area of the inlet portion. The first channel includes a polymerization portion downstream of the droplet generating portion, the polymerization portion having a serpentine configuration. The device comprises a cartridge for MOS demulsification, the cartridge comprising: a collection container; the base is arranged on the collecting container; and a film disposed between the collection container and a surface of the substrate. A second channel is defined in the surface of the substrate facing the collection container and fluidly connected to an output of the polymerized portion of the first channel.

Description

[0001] Priority Statement

[0002] This application claims the priority benefit of U.S. Patent Application Serial No. 63 / 415,228, filed on October 11, 2022, U.S. Patent Application Serial No. 63 / 415,240, filed on October 11, 2022, U.S. Patent Application Serial No. 63 / 415,232, filed on October 11, 2022, and U.S. Patent Application Serial No. 63 / 415,235, filed on October 11, 2022, the entire content of each of these patent applications being incorporated herein by reference. BACKGROUND OF THE INVENTION

[0003] Biomaterials derived from patients, such as cells obtained from biopsy or excised tissue, can be used to screen for treatments to which the patient will respond effectively. Microfluidic devices can be used for treatment screening. SUMMARY OF THE INVENTION

[0004] In a first aspect, a microfluidic device includes a microfluidic chip for generating microorganic spheres (MOS), wherein a first microfluidic channel is defined in a surface of the microfluidic chip, the first microfluidic channel including: a droplet generation portion including an inlet portion, a junction between the inlet portion and an emulsifying fluid channel, and a chamber downstream of the junction, wherein a cross-sectional area of the chamber is greater than a cross-sectional area of the inlet portion, and a polymerization portion downstream of the droplet generation portion, the polymerization portion having a serpentine configuration; and a cartridge for MOS demulsification, the cartridge including: a collection container; a substrate disposed on the collection container, wherein a second microfluidic channel is defined in a surface of the substrate facing the collection container, and wherein the second microfluidic channel is fluidly connected to an output of the polymerization portion of the first microfluidic channel; and a membrane disposed between the collection container and the surface of the substrate.

[0005] Embodiments may include one or any combination of two or more of the following features.

[0006] The droplet generation portion of the first microfluidic channel includes an outlet portion downstream of the chamber, wherein the cross-sectional area of the chamber is greater than the cross-sectional area of the outlet portion. In some cases, at least some of the outlet portion extends in a direction parallel to the chamber.

[0007] The surface of the microfluidic chip is a first surface, and wherein the polymerization portion of the microfluidic channel is defined on the first surface of the microfluidic chip and on a second surface of the microfluidic chip opposite the first surface.

[0008] The junction includes a junction having two hydrophobic fluid channels. In some cases, the junction is a right-angle junction.

[0009] The membrane includes a hydrophobic membrane. In some cases, the membrane is both hydrophobic and lipophilic.

[0010] The second microfluidic channel includes: an upstream section having a simple serpentine configuration and a downstream section having a double serpentine configuration.

[0011] The cross-sectional area of the second microfluidic channel decreases from the input end of the second microfluidic channel to the output end of the second microfluidic channel.

[0012] The surface of the substrate is a first surface, and a medium inlet channel is defined on a second surface of the substrate opposite to the first surface of the substrate. The medium inlet channel is fluidly connected to the upstream section of the second microfluidic channel and is configured to be connected to a medium reservoir. In some cases, the demulsification cartridge includes a medium reservoir. In some cases, the medium inlet channel is fluidly connected to the medium reservoir via a tube extending through the substrate and the collection container. In some cases, the collection container is disposed in a cavity defined in the medium reservoir such that the collection container is positioned between the medium reservoir and the substrate. In some cases, the bottom surface of the medium reservoir is angled relative to the plane of the substrate. In some cases, the demulsification cartridge includes a duckbill valve extending through the substrate and the collection container, and the duckbill valve is configured to provide a fluid passage to the medium reservoir.

[0013] The demulsification cartridge includes a hydrophobic material disposed within the collection container.

[0014] A vacuum flow path is defined through the body of the collection container, and the vacuum flow path is configured to be able to apply a vacuum to the surface of the membrane opposite to the substrate.

[0015] The microfluidic device includes a reservoir that is fluidly connected to the first microfluidic channel via an input port defined at the input end of the first microfluidic channel. In some cases, the reservoir includes a base and a lid that define a cavity for a fluid sample. In some cases, the microfluidic device includes an input port in the lid of the reservoir, and the input port includes a duckbill valve. In some cases, the microfluidic device includes an output port in the lid of the reservoir, and the output port is connected to a tube extending into the cavity of the reservoir. In some cases, the bottom surface of the base of the reservoir is angled relative to the lid. In some cases, the microfluidic device includes a reservoir holder configured to receive the reservoir, and the reservoir holder includes a cooling system configured to cool the reservoir. In some cases, the cooling system includes a thermoelectric cooling system.

[0016] One or more cuts are defined in the microfluidic chip between the droplet generation section and the polymerization section. In some cases, the edges of the one or more cuts are angled relative to the surface of the microfluidic chip. In some cases, the one or more cuts extend through the entire thickness of the microfluidic chip.

[0017] The microfluidic device includes a lid disposed on the surface of the microfluidic chip. In some cases, the lid includes an optically transparent lid.

[0018] A plurality of first microfluidic channels are defined in the surface of the microfluidic chip, and the device includes a plurality of cartridges, wherein the second microfluidic channel of each cartridge is fluidly connected to a corresponding one of the first microfluidic channels of the microfluidic chip.

[0019] The device includes an output vial that is fluidly connected to the second microfluidic channel via an output port defined at the output end of the second microfluidic channel.

[0020] In a second aspect, which can be combined with any embodiment of the previous aspect, the system includes the microfluidic device of the first aspect; a housing, wherein the microfluidic device is disposed within the housing; and a polymerization block that is received within the housing and positioned to apply a stimulus to the polymerization section of the first microfluidic channel.

[0021] Embodiments may include one or any combination of two or more of the following features.

[0022] The polymerization block includes a thermal polymerization block that is configured to apply heat to the polymerization section of the first microfluidic channel. In some cases, the thermal polymerization block includes a heater. In some cases, the thermal polymerization block includes a temperature sensor. In some cases, the temperature sensor includes one or more of a thermistor, a thermocouple, or a resistance temperature detector. In some cases, the system includes a controller that is configured to control the operation of the resistive heater in response to temperature data received from the temperature sensor. In some cases, the heater includes a resistive heater. In some cases, the thermal polymerization block includes a thermal insulation cover, and wherein the heater is disposed within a cavity defined within the thermal insulation cover.

[0023] The polymerization block includes a photo-polymerization block configured to irradiate a polymerization portion of a first microfluidic channel. In some cases, the photo-polymerization block includes a light-emitting diode (LED). In some cases, the photo-polymerization block includes a photodetector. In some cases, the system includes a controller configured to control the operation of the LED in response to light intensity data received from the photodetector. In some cases, the LED is disposed within a cavity defined in a housing of the photo-polymerization block. In some cases, the walls of the cavity are formed of a material capable of reflecting light of a wavelength of the light output by the LED. In some cases, the system includes a controller configured to control the LED to emit pulsed irradiation.

[0024] The surface of the microfluidic chip is a first surface, and the polymerization block includes: a first block disposed adjacent to the first surface of the microfluidic chip; and a second block disposed adjacent to a second surface of the microfluidic chip, the second surface being opposite to the first surface. In some cases, the first block and the second block are fixed against the microfluidic chip by springs. In some cases, the first block and the second block are clamped to the microfluidic chip.

[0025] The system includes a reservoir for emulsifying fluid, wherein an emulsifying fluid channel of the microfluidic device is fluidly connected to the reservoir. In some cases, the reservoir includes a reflective rib for fluid volume measurement, the reflective rib being disposed in a chamber of the reservoir. In some cases, the system includes a pump disposed between the reservoir for emulsifying fluid and the emulsifying fluid channel. In some cases, the system includes a controller configured to control the operation of the pump. In some cases, the controller is configured to control the operation of the pump to achieve a target fluid velocity in a second microfluidic channel. In some cases, the pump is an injection pump. In some embodiments, a valve such as a servo valve may be used instead of the pump.

[0026] The system includes an imaging system positioned to capture an image of at least a portion of the chamber. In some cases, the system includes a controller configured to control the flow rate of fluid through an inlet portion of the microfluidic channel based on an image captured by the imaging system. In some cases, the controller is configured to control the flow rate of the fluid by controlling the pressure applied to a reservoir fluidly connected to the inlet portion of the microfluidic channel. In some cases, the controller is configured to control the flow rate of the fluid by controlling an injection pump. In some cases, the flow rate of the fluid containing the sample is controlled by pressure, and the flow rate of the emulsifying fluid (such as oil) is controlled by an injection pump.

[0027] In a third aspect, which may be combined with any embodiment of either or both of the previous aspects, the microfluidic chip includes a plurality of first microfluidic channels configured to generate an emulsion of droplets of a first fluid in a second fluid, wherein the first microfluidic channels are defined in a first surface of the microfluidic chip, wherein each first microfluidic channel is fluidically independent of each other first microfluidic channel, and wherein each first microfluidic channel includes: an inlet portion configured to receive the first microfluid from a respective source of the first fluid; a junction between the inlet portion and a corresponding second microfluidic channel configured to carry the second fluid; and a chamber downstream of the junction, wherein the cross-sectional area of the chamber is greater than the cross-sectional area of the inlet portion; and a plurality of second microfluidic channels configured to aggregate the droplets of the emulsion to generate MOS, wherein each second microfluidic channel is fluidically connected to an outlet of a corresponding one of the first microfluidic channels, and wherein each second microfluidic channel is a serpentine channel including a first portion defined on the first surface of the microfluidic chip and a second portion defined on a second surface of the microfluidic chip opposite the first surface.

[0028] The embodiments may include one or any combination of two or more of the following features.

[0029] Each first microfluidic channel includes an outlet portion downstream of the chamber, wherein the cross-sectional area of the chamber is greater than the cross-sectional area of the outlet portion. In some cases, the region of the outlet portion of each first microfluidic channel extends in a direction parallel to the respective chamber.

[0030] The microfluidic chip includes a lid disposed on each of the first and second surfaces of the microfluidic chip. In some cases, the lid includes an optically transparent lid.

[0031] The plurality of first microfluidic channels are defined in a first region of the microfluidic chip, and wherein the plurality of second microfluidic channels are defined in a second region of the microfluidic chip different from the first region. In some cases, one or more cuts are defined in the microfluidic chip between the first and second regions. In some cases, the edges of the one or more cuts are angled with respect to the first and second surfaces of the microfluidic chip. In some cases, the one or more cuts extend through the entire thickness of the microfluidic chip.

[0032] Each junction is a junction between a respective inlet portion and two corresponding second microfluidic channels. In some cases, the junction is a right-angle junction.

[0033] The microfluidic chip includes a plurality of inlet fingers, each inlet finger extending away from at least one other inlet finger and separated from each adjacent inlet finger by a gap, and wherein at least some of the inlet portions of each first microfluidic channel are defined on the surface of the corresponding inlet finger.

[0034] The microfluidic chip includes a plurality of outlet fingers, each outlet finger extending away from at least one other outlet finger and separated from each adjacent outlet finger by a gap, and wherein the outlet portions of each second microfluidic channel are defined on the surface of the corresponding outlet finger.

[0035] The output port of each second microfluidic channel is configured to be connected to a corresponding cartridge for demulsification of the emulsion.

[0036] In a fourth aspect, which can be combined with any of the embodiments of one or more of the previous aspects, the device includes a cartridge for transferring MOS from an emulsion in a hydrophobic fluid to a suspension in an aqueous fluid (e.g., an aqueous hydrophilic fluid such as a growth medium). The demulsification cartridge includes: a collection container that defines a cavity for receiving the hydrophobic fluid; a substrate disposed on the collection container, wherein a microfluidic channel is defined in a first surface of the substrate facing the collection container, and wherein a media inlet channel for the aqueous fluid is fluidly connected to an upstream portion of the microfluidic channel; and a hydrophobic membrane disposed between the collection container and the surface of the substrate. In some cases, the hydrophobic membrane is both hydrophobic and lipophilic.

[0037] Embodiments may include one or any combination of two or more of the following features.

[0038] The device includes a media reservoir having a cavity configured to contain an aqueous fluid, wherein the media inlet channel is fluidly connected to the media reservoir. In some cases, the device includes a tube extending through the substrate and the collection container, wherein the media inlet channel is fluidly connected to the media reservoir via the tube. In some cases, the collection container is disposed in the cavity of the media reservoir such that the collection container is positioned between the media reservoir and the substrate. In some cases, the bottom surface of the media reservoir is angled relative to the plane of the substrate. In some cases, the device includes a duckbill valve disposed through an opening in the substrate and an opening in the collection container, the duckbill valve being configured to allow the aqueous fluid to be provided into the cavity of the media reservoir but not to overflow back.

[0039] The surface of the substrate is a first surface, and wherein the media inlet channel is defined on a second surface of the substrate opposite the first surface.

[0040] The cross-sectional area of the microfluidic channel is larger at the upstream end of the microfluidic channel than at the downstream end of the microfluidic channel.

[0041] The upstream portion of the microfluidic channel has a different configuration compared to the downstream portion of the microfluidic channel. In some cases, the upstream portion of the microfluidic channel has a simple serpentine configuration, and the downstream portion of the microfluidic channel has a double serpentine configuration.

[0042] The device includes a hydrophobic absorbent material disposed in the cavity of the collection container. In some cases, the device includes a material that is both hydrophobic and lipophilic.

[0043] In a fifth aspect, which can be combined with any embodiment of one or more of the previous aspects, the method includes generating droplets of a first fluid in a droplet generation portion of a first microfluidic channel defined in the surface of a microfluidic chip, the first fluid comprising a biomaterial and a matrix material, and in a polymerization portion of the first microfluidic channel, applying a stimulus to the generated droplets to polymerize the matrix material, thereby forming MOS emulsified in the hydrophobic fluid; transferring the MOS from the emulsion to a suspension in an aqueous fluid includes: flowing a mixture of the aqueous fluid and the emulsion of MOS in the hydrophobic fluid along a second microfluidic channel defined in a substrate; and when the mixture flows along the second microfluidic channel, transferring the hydrophobic fluid through a membrane forming the wall of the second microfluidic channel.

[0044] Embodiments may include one or any combination of two or more of the following features.

[0045] Generating droplets of the first fluid includes generating droplets at a junction between the first microfluidic channel and one or more channels carrying the hydrophobic fluid. In some cases, the method includes controlling the flow rate of the hydrophobic fluid.

[0046] The method includes controlling the flow rate of the first fluid based on a determined size of the generated droplets. In some cases, the method includes determining the size of the generated droplets based on an image of the droplets in the droplet generation portion of the first microfluidic channel.

[0047] Applying a stimulus to the generated droplets includes heating the droplets.

[0048] Applying a stimulus to the generated droplets includes irradiating the droplets with light having a wavelength configured to induce polymerization of the matrix material. In some cases, the surface of the microfluidic chip is a first surface, and the polymerization portion of the first microfluidic channel is defined on both the first surface and a second surface of the microfluidic chip, and irradiating the droplets includes irradiating both the first surface and the second surface of the microfluidic chip. In some cases, irradiating the droplets includes irradiating the droplets with a pulsed irradiation.

[0049] The method includes receiving the transferred hydrophobic fluid in a collection container, wherein the membrane is disposed between the collection container and the substrate.

[0050] The hydrophobic fluid is transferred across the membrane by a pressure differential of a positive driving pressure above and an ambient pressure below, plus an additional gravitational force. In some cases, transferring the hydrophobic fluid across the membrane includes applying a vacuum to the membrane.

[0051] The method includes providing a suspension of MOS in an aqueous fluid to an output vial.

[0052] The method includes generating droplets of each of a plurality of first fluids in each of a plurality of fluidically independent first microfluidic channels defined in a surface of a microfluidic chip; and applying a stimulus to the generated droplets in each of the first microfluidic channels to form MOS.

[0053] The methods described herein can have one or more of the following advantages. Micro-organic spheres (MOS) can be generated and extracted from immiscible generation fluids in a rapid, high-throughput process, thereby providing a high recovery rate. The system is fully automated and can thus provide consistent performance across samples. The system uses disposable components in the sample handling path, thereby reducing the downtime required for cleaning and disinfection.

[0054] Details of one or more specific implementations are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description and drawings, and from the claims. Description of the Drawings

[0055] Figure 1 is a block diagram of a system for micro-organic sphere (MOS) generation.

[0056] Figure 2 is a diagram of a consumable of the MOS generation system.

[0057] Figure 3A and 3B is a diagram of a microfluidic chip of the MOS generation system.

[0058] Figure 4 is a diagram of a portion of a microfluidic chip of the MOS generation system.

[0059] Figures 5A to 5C is a diagram of a thermal block for the MOS generation system.

[0060] Figures 6A to 6C is a diagram of a light block for the MOS generation system.

[0061] Figures 7A to 7C is a diagram of an exemplary oil reservoir.

[0062] Figures 7D to 7E is a diagram of an exemplary oil reservoir.

[0063] Figure 8A is a diagram of an exemplary oil delivery subsystem.

[0064] Figure 8B is a diagram of an exemplary oil delivery subsystem.

[0065] Figure 9A and 9B are a cross-sectional view and a perspective view of a sample reservoir, respectively.

[0066] Figure 10A and 10B are diagrams of multiple reservoirs.

[0067] Figure 11A is a diagram of a cold block.

[0068] Figure 11B is a diagram of a thermoelectric cooling subsystem.

[0069] Figure 12 is a diagram of a sample drive subsystem of a MOS generation system.

[0070] Figure 13A and 13B are diagrams of an imaging system of a MOS generation system.

[0071] Figure 14 is a schematic diagram of a part of a demulsification subsystem of a MOS generation system.

[0072] Figure 15 is a cross-sectional view of a part of a demulsification subsystem.

[0073] Figures 16A to 16C is a diagram of a demulsification subsystem of a MOS generation system.

[0074] Figure 17A and 17B are diagrams of the configuration of a microfluidic channel in a demulsification subsystem for a MOS generation system.

[0075] Figure 18A and 18B is a diagram of a loading system.

[0076] Figure 19 is a flowchart.

[0077] Figure 20 is a diagram of an electronic subsystem of a MOS generation system.

[0078] Figure 21 is a block diagram of a MOS generation system.

[0079] Figure 22 is a flowchart.

[0080] Figure 23A is a schematic diagram of multiple images of droplets.

[0081] Figure 23B It is a diagram of the control signal.

[0082] Figure 24A It is a diagram of the light source.

[0083] Figure 24B It is a diagram of the control signal.

[0084] Figure 25 It is a flowchart.

[0085] Figure 26A It is a block diagram of an exemplary device for generating droplets.

[0086] Figure 26B It is a perspective view of an exemplary device for generating droplets.

[0087] Figure 26C It is Figure 1 A schematic diagram of a part of the exemplary device shown in B.

[0088] Figures 27A to 27E It is a captured exemplary image of the droplet generation device.

[0089] Figure 28 It is a flowchart of a process for controlling the pressure of the fluid flow in the droplet generation device.

[0090] Figure 29 It shows a process for estimating droplet size.

[0091] Figures 30 to 31 It is a diagram showing experimental data.

[0092] Figure 32 It is a flowchart of a process for controlling the size of droplets generated by the device.

[0093] Figures 33A to 33C It shows an exemplary container including a protrusion for liquid level sensing.

[0094] Figures 34A to 34B It shows that when the liquid level is lower than the light incident position ( Figure 34A ) or higher than the light incident position ( Figure 34B ) the Figures 33A to 33C Examples of liquid level sensors for measuring the liquid level in the container.

[0095] Figure 35A It shows an exemplary liquid level measurement performed by moving a pair of light sources and light detectors along the longitudinal direction of the container.

[0096] Figure 35B It shows the use of Figure 35A Exemplary measurement results of liquid level measurement.

[0097] Figure 36A Another exemplary liquid level measurement is shown that is performed by a plurality of pairs of light sources and light detectors arranged at a series of positions along the longitudinal direction of the container.

[0098] Figure 36B Another exemplary measurement result of a liquid level measurement using Figure 36A is shown.

[0099] Figure 37 An exemplary fluid system including at least one liquid level sensor is shown.

[0100] Figure 38 is a flowchart of an exemplary process for performing liquid level sensing using optical reflection.

[0101] Figures 39A to 39C is an image of micro - organic spheres.

[0102] Figure 40 is a diagram showing an example of a computing environment. Detailed Description

[0103] The present disclosure describes an integrated microfluidic path for generating droplets containing biological materials, polymerizing the droplets to form micro - organic spheres (MOS), and demulsifying and transferring the MOS into a suspension in an aqueous fluid (e.g., an aqueous hydrophilic fluid such as a growth medium). Specifically, a plurality of fluidically independent microfluidic channels are defined on a microfluidic chip. Each channel has a droplet - generating region configured to generate an emulsion of droplets in a hydrophobic fluid and a serpentine polymerization region where a stimulus such as light or heat is applied to polymerize the matrix material in the droplets, thereby forming MOS. Downstream of the polymerization region, each channel is fluidically connected to a microfluidic channel in a demulsification cartridge. A mixture of MOS in an aqueous fluid and the hydrophobic fluid flows along the microfluidic channel, and the hydrophobic fluid is attracted through a hydrophobic membrane, thereby suspending the MOS in the aqueous fluid.

[0104] The processes for droplet generation and polymerization and MOS demulsification using the system are automated, continuous processes and can be performed to simultaneously and independently process multiple samples. The automated MOS generation and demulsification processes are efficient and reliable, e.g., less prone to errors that may occur during manual processes. For example, a 100 μL biological material sample can be fully processed in 10 minutes (e.g., 6 minutes), and a 1 mL sample can be processed in 40 minutes; all without interfering with the automated processing time.

[0105] Micro-organic spheres (MOS) are typically spherical structures that contain biological materials, such as dissociated tissue, e.g., cells, dispersed (e.g., suspended) in a matrix material. MOS can be used for high-throughput, patient-specific screening, e.g., for effective therapeutic agents. The biological materials in MOS can be cells extracted from a small biopsy, from excised tissue (e.g., a tumor or an organ), or from other sources (e.g., stored cells or cultured cells). Each MOS contains a small number of cells, e.g., a cluster of 5 - 10 cells suspended in the matrix material. The matrix material is a material that can be polymerized upon application of a stimulus (e.g., heat, light, or a chemical reaction) to form a support or a support network for the biological materials. For example, the matrix material can be a hydrogel.

[0106] A large number of MOS can be generated from a single patient sample (e.g., a single biopsy or tissue excision). For example, more than 10,000 (e.g., more than 20,000, more than 30,000, more than 40,000, more than 50,000, more than 60,000, more than 70,000, more than 80,000, more than 90,000, more than 100,000) MOS of substantially uniform size can be generated from a tissue sample (e.g., a needle biopsy sample) having a volume of about 10 - 1000 μL. Such a large number of MOS can be used for high-throughput screening of a large number of therapies to identify the therapies to which a particular patient responds. For example, MOS containing cells from a patient biopsy can be used for high-throughput screening against a drug composition, whereby it can be predicted which therapies can be effectively applied to the patient. Such screening can allow, for example, effective screening of a large number of potential therapies (e.g., drug therapies) to identify effective therapies for a patient before treating the cancer patient. The generation of MOS and their use for screening are further described below and in US2020 / 0377861, the content of which is incorporated herein by reference in its entirety.

[0107] The recovered MOS are used, for example, to screen therapies for the patient from whom the biological materials in the MOS were provided. For example, the recovered MOS are placed in a culture medium to allow the cells within the MOS to grow. After culturing, the cells can be assayed essentially immediately or can be cryopreserved for future use.

[0108] Refer to Figure 1, The instrument 100 for MOS generation houses an integrated microfluidic flow path, which provides three aspects of the MOS generation function: droplet generation 102, droplet polymerization 104, and demulsification 106. In droplet generation 102, droplets containing patient-specific biomaterials and a matrix material (e.g., hydrogel) are generated as an emulsion immiscible with the matrix material (e.g., oil) in a fluid. In droplet polymerization 104, the matrix material in the generated droplets is polymerized to form MOS. In demulsification 106, the MOS is transferred from the oil emulsion to a suspension in an aqueous fluid. The resulting suspension can be used for downstream processing (e.g., further cell growth) and / or testing. The instrument 100 receives patient-specific biomaterials 110 (e.g., dissociated tissue, such as cells), oil for the emulsion 112, and an aqueous fluid 114 as inputs, and outputs MOS and waste 118, such as oil from the emulsion, in the aqueous fluid 116. Droplet generation 102 and droplet polymerization 104 occur on a microfluidic chip fluidly connected to a demulsification cartridge for demulsification 106.

[0109] The instrument 100 is capable of processing multiple samples (e.g., four samples, such as samples from multiple patients) in parallel (e.g., simultaneously) along multiple fluidically isolated flow paths. The sample-contact portions of the instrument 100 are disposable, such that no cleaning or disinfection is required between the processing of multiple samples.

[0110] Droplet polymerization is carried out by crosslinking of the matrix material in which the biomaterials are suspended. The matrix material can be crosslinked by applying a heat stimulus (e.g., heat), by a photoinitiated process, or by a chemical reaction. The instrument 100 can be configured to be compatible with one or more of these crosslinking methods. For example, the instrument 100 can be equipped with hardware for thermal or photoinitiated polymerization and can also be compatible with chemical polymerization using a suitable microfluidic chip.

[0111] Figure 2 is shown that can be used, for example, in Figure 1An example of a MOS generation system 200 that generates MOS in an instrument 100. The MOS generation system 200 is configured to process in parallel a plurality of samples provided to the system in respective reservoirs 220. In the illustrated example, the MOS generation system can process up to four samples simultaneously. Each sample contains a biological material (e.g., a patient-specific biological material such as tumor cells) suspended in a matrix material (such as a hydrogel). Samples from each reservoir 220 are processed on independent microfluidic flow paths (e.g., fluidly isolated from each other and independently controllable) defined in a microfluidic chip 210, where droplet generation and polymerization processes occur. After droplet polymerization, the MOS emulsion in each flow path is provided to a corresponding demulsification cartridge 230, where the MOS is transferred from the oil to a suspension in an aqueous fluid. The MOS in the aqueous fluid is output to an output vial 240.

[0112] To process a set of up to four samples, the MOS generation system 200 is loaded into the MOS generation instrument. Optionally, the desired type of polymerization stimulus is achieved, for example, by inserting a thermal block or a light block into the instrument for thermal or photoinitiated polymerization, respectively. Once the processing of the set of samples is complete, the MOS generation system 200 (including the reservoir 220, the microfluidic chip 210, the demulsification cartridge 230, and the output vial 240) is discarded. Since the other components of the instrument do not come into contact with the samples, no cleaning or disinfection is required before processing another set of samples with another MOS generation system 200.

[0113] Tracking information, such as industry standard 2D barcodes (e.g., QR codes or data matrix codes) or other types of identifiers, can be used to track the regulatory chain of each patient-specific sample from its source to the reservoir 220 containing the sample and to the output vial 240. For example, the instrument 100 can be equipped with barcode scanning capabilities, a laboratory information management system (LIMS), or other tracking technologies. In some instances, similar tracking technologies are also used to track reagents, such as matrix materials and oils, to, for example, ensure the use of appropriate and non-expired reagents.

[0114] Figure 3A and 3B are a top view and a cross-sectional perspective view, respectively, of a microfluidic chip 210 that has a droplet generation region 302 and a polymerization region 304. Four independent flow paths are defined through the microfluidic chip 210 for simultaneously processing four biological samples; other numbers of paths can also be defined. Each path is used to process a different biological sample.

[0115] Each flow path includes an inlet channel 307 defined on a respective inlet finger 308 of the microfluidic chip 210. Each inlet channel 307 includes a port for connection to a corresponding one of the reservoirs 220, which are sources of a first fluid containing patient-specific biological material and unpolymerized matrix material. The inlet fingers 308 are separated from one another (e.g., each outlet finger 324 is separated from an adjacent finger by a gap) to allow sufficient space for each reservoir. Additionally, the splayed inlet fingers 308 allow each finger to bend and seal against a corresponding reservoir 220 independently of each other finger, thereby avoiding problems where tolerance variations prevent sealing (e.g., avoiding a situation where a taller reservoir prevents sealing of an adjacent shorter reservoir). Each flow path also includes one or more (here two) second channels 310, each second channel including a port for connection to an oil source (e.g., an oil reservoir 700, see FIG. 7).

[0116] In the droplet generation region 302, a continuous flow of droplets is generated in each flow path by combining a flow of a first fluid containing biological material (e.g., patient-specific biological material) suspended in an unpolymerized matrix material (e.g., a hydrogel) with a flow of one or more second fluids. The second fluid is a low-viscosity fluid immiscible with the first fluid such that an emulsion of the first fluid in the second fluid can be generated. For example, the first fluid can be a hydrophilic (e.g., water-based) fluid, while the second immiscible fluid can be a hydrophobic material such as oil. Although this document sometimes specifically refers to oil or a hydrophobic fluid, it should be understood that the disclosed methods are generally applicable to any suitable second fluid immiscible with the first fluid. Generally, the flow of the first fluid and the flow of the second fluid are controlled by separate flow regulator devices (such as proportional-integral-derivative (PID) controllers) controlled by respective controllers. For example, the flow of the first fluid is controlled by pressurizing the reservoir 220, e.g., using a syringe pump or other suitable flow regulator, and the flow of the second fluid is controlled by a pump with a programmable flow rate.

[0117] When the first and second fluid flows meet, droplets of the first fluid are formed in the second fluid. Each generated droplet contains unpolymerized matrix material and a small amount of biological sample from the corresponding reservoir and is dispersed (e.g., emulsified) in the oil. The droplets have a stable, substantially spherical geometry, and the relative flow rates of the first fluid and the oil control the size (e.g., diameter, volume) of the droplets. In the polymerization region 304, the matrix material in the droplets is polymerized by exposure to a stimulus (such as heat, light, chemical stimulus, or other suitable stimulus), thereby forming an emulsion of MOSs in the oil. After polymerization, the emulsion is demulsified by a demulsification cartridge 230 fluidly connected to the microfluidic chip 210 ( Figure 2Demulsification in (as shown) recovers MOS from the oil. For example, when the flow rate of the first fluid increases relative to the flow rate of the second fluid, larger droplets are generated. When the flow rate of the first fluid decreases relative to the flow rate of the second fluid, smaller droplets are generated. The relative flow rates of the first and second fluids also affect the flow rate of the emulsion containing the generated droplets.

[0118] The microfluidic chip 210 is, for example, an injection-molded chip-on-chip device that is sealed on its top and bottom surfaces (not shown) by a thin lid (such as a glass lid or a polymer film). The lid is a thin film with a thickness, for example, between 50 μm and 100 μm (e.g., 80 μm). Compared to a similar structure but single-sided chip, this two-sided geometry allows the flow channels to cross each other and doubles the density of the channels in the polymerization region 304. In some instances, such as when the microfluidic chip 210 is designed for thermal or photoinitiated polymerization, the lid is a film that is substantially non-absorbent of heat or light, respectively. For example, the covering film can be optically transparent (e.g., allowing transmission of at least 50%, at least 60%, at least 80%, at least 90%, or at least 95%) and has a light intensity at the wavelength of light (e.g., 405 nm blue light) for photoinitiated polymerization.

[0119] Figure 4 is part of the droplet generation region contained within Figure 3A the dashed box of. As Figure 4 shown, the first channel 306 and the second channel 310 in each flow path intersect at the junction 311 in the droplet generation region 302, for example, at a right angle. When the first fluid flows out of the first channel 306 and the oil flows out of the second channel 310 and enters the junction, the opposite convergence of the flowing oil (e.g., flowing from both sides of the junction) allows a small amount of the first fluid to enter the junction before pinching off the first fluid, thus starting to form droplets of the first fluid encapsulated in the oil. The droplets in each flow path flow from the junction into the corresponding droplet generation chamber 312. In some instances, the droplet generation chamber 312 is a flared chamber having a cross-sectional dimension (e.g., cross-sectional area) larger than that of the first channel 306. The transition between the narrower first channel 306 and the wider droplet generation chamber 312 along each flow path causes the fluid flow to slow down and the pressure to rise, thus triggering the pinching off of the droplets. The continuous flow of the first fluid and the oil forms a continuous stream of uniformly spaced droplets having substantially uniform shape and volume. Each droplet contains patient-specific biomaterial and unpolymerized matrix material.

[0120] Droplets flow from the droplet generation chamber 312 in each flow path into the narrower outlet channel 314. For example, the height and width of the outlet channel 314 are slightly larger than the diameter of the droplets, for example, between about 10% and about 25%. The reduction in cross-sectional size from the droplet generation chamber 312 to the outlet channel 314 generates back pressure to support droplet generation. This reduction in size also causes the droplets to form a single file with substantially uniform spacing, thereby preventing the droplets from contacting and merging with each other prior to downstream polymerization processes. In an example, the spacing between droplets in the outlet channel 314 can be about twice the spacing between droplets in the droplet generation chamber 312.

[0121] In a specific example, for a droplet having a diameter of 260 μm, the outlet channel 314 has cross-sectional dimensions of 300 μm x 300 μm, the droplet generation chamber 312 has cross-sectional dimensions of 200 μm x 700 μm, and the first channel 306 and the second channel 310 have cross-sectional dimensions of 200 μm x 200 μm.

[0122] An imaging subsystem (not shown; see FIG. 13 ) is positioned to capture images (e.g., still or video images) of a droplet as it passes through the droplet generation chamber 312 , the outlet channel 314 , or both. The images can be used to determine characteristics of the droplets, such as droplet size (e.g., droplet volume or droplet diameter), droplet size distribution, droplet velocity, spacing between adjacent droplets, number density of droplets (e.g., number of droplets per unit length of the outlet channel 314 , number of droplets per unit volume of the second fluid), an estimated total number of droplets generated from a given starting volume of the first fluid, or other droplet characteristics. In some examples discussed further below, the droplet characteristics can be used for closed-loop feedback control of the droplet generation process. For example, the flow rate of the oil, the first fluid, or both can be adjusted in real time based on the determined droplet characteristics to obtain droplets of a target size.

[0123] In the illustrated example, each outlet channel 314 is returned side by side along the corresponding droplet generation chamber 312, for example, in a serpentine configuration, so that a portion of each outlet channel 314 extends in a direction parallel to the corresponding droplet generation chamber 312. This geometry allows droplets to stay in the field of view of the imaging subsystem for a longer time. In addition, droplets can be imaged in a single file in the outlet channel 314 at uniform intervals, thereby facilitating image analysis. In addition, the ability to image droplets and calculate their velocities in adjacent channels of different cross-sections allows for internal QC, i.e., velocity ratios correspond to channel area ratios. In addition, the droplet generation chamber 312 and the outlet channel 314 are compressed into a relatively small area of the microfluidic chip 210, which allows the imaging subsystem to operate with a small field of view, thereby allowing images to be captured at high resolution (e.g., resolution of microns per pixel).

[0124] Referring again to Figure 3A and 3B , droplets flow from the exit channels 314 of each flow path into corresponding polymerization channels 320 in the polymerization region 304. The polymerization channels 320 are arranged such that the droplets flowing through them are exposed to stimuli such as heat, light (e.g., blue light), chemical stimuli, or other suitable stimuli. The stimuli induce the polymerization of the unpolymerized matrix material in the droplets, thereby forming MOS emulsified in oil. In some instances, the stimuli are applied to both the top and bottom surfaces of the microfluidic chip 210; in some instances, the stimuli are applied to only one surface.

[0125] In the illustrated example, the polymerization channels 320 are serpentine channels, with a portion of the channel length arranged on the top surface of the microfluidic chip 210 and the remainder arranged on the bottom surface of the microfluidic chip 210. For example, for a polymerization channel with a total length of 1 meter, 0.5 m of the channel length is arranged on the top surface and 0.5 m of the channel length is arranged on the bottom surface. This arrangement enables a high density of polymerization channels 320, thereby providing a long fluid path length to maximize the time of exposure to the stimuli. Additionally, the positioning of the polymerization channels 320 on the top and bottom surfaces of the microfluidic chip 210 means that there is only a small separation between the droplets in the channels 320 and the applied stimuli, e.g., a separation approximately equal to the thickness of the transparent film covering the microfluidic chip. This small separation minimizes the delay of the stimuli reaching the polymerization channels and reduces energy loss, thus contributing to efficient polymerization. The combination of the serpentine paths on the two surfaces of the microfluidic chip 210 and the small separation between the channels 320 and the applied stimuli enables polymerization to be achieved with a relatively short residence time (e.g., a residence time between 30 seconds and 2 minutes), e.g., 1 minute, along a polymerization channel with a length of approximately 1 meter. The residence time refers to the time taken for a given droplet to flow along the length of one of the polymerization channels 320.

[0126] The geometry of the polymerization channels 320 is designed to facilitate efficient polymerization. For example, the cross-sectional area of the channels 320 can be slightly larger than the cross-sectional area of the droplets. This prevents the droplets from stacking on top of each other in the channels, but rather keeps the droplets substantially evenly spaced in a single column, which in turn helps to avoid clogging and ensures that all droplets are evenly exposed to the applied stimuli. Even in a limited area of the microfluidic chip 210, the serpentine path of the polymerization channels 320 provides a long path length. Additionally, small turns (e.g., 180° turns) along the polymerization channels 320 promote the mixing of the fluid flow in the channels, thereby promoting heat transfer, for example, by inducing convection, and thus increasing the polymerization efficiency.

[0127] The parameters for the polymerization of the matrix material in the droplets can depend on the size of the droplets. For example, the duration of exposing the droplets to a stimulus sufficient to induce complete polymerization of the matrix material in the droplets can depend on the size of the droplets. Larger droplets may require more time to polymerize than smaller droplets due to the larger volume of the material and the greater distance between the droplet edge and its center. For example, droplets of a certain size may require an exposure time of at least 90 seconds, while droplets of a different, smaller size may only require an exposure time of 30 seconds. The microfluidic system can be adjusted such that the stimulus is sufficient to achieve complete polymerization of the matrix material in droplets of a target size, e.g., such that droplets flowing through the system at a target flow rate are exposed to the stimulus for a time period sufficient to polymerize droplets of the target size. Specifically, the length of the polymerization channel 320 is fixed. Thus, the flow rate (velocity) of the droplets along the polymerization channel 320 determines the exposure time of the droplet volume.

[0128] In some instances, the system is designed to achieve complete polymerization when droplets of a target size flow through the polymerization channel 320 at a target flow rate. In some instances, various combinations of target size and target flow rate are available. For example, the system can be designed such that droplets in a first size range achieve complete polymerization when they flow at a first flow rate, and droplets in a second, smaller size range achieve complete polymerization when they flow at a second, faster flow rate.

[0129] The downstream end of each polymerization channel 320 is fluidly connected to a corresponding outlet channel 322, each of these outlet channels being connected to a corresponding demulsification cartridge (not shown). Each outlet channel 322 is formed in a respective outlet finger 324 of the microfluidic chip 210. The outlet fingers 324 are separated from each other (e.g., each outlet finger 324 is separated from an adjacent finger by a gap) to allow sufficient space for each demulsification cartridge 230. Additionally, the splayed outlet fingers 324 allow each finger to bend and seal the corresponding demulsification cartridge 230 independently of each other finger, thereby avoiding problems where tolerance variations prevent sealing.

[0130] Referring Figures 5A to 5C , for thermal polymerization, the matrix material is a temperature-sensitive material, such as matrix glue, which polymerizes in response to exposure to heat. Thermal blocks 500a, 500b (collectively referred to as thermal block 500) are positioned to apply heat to the top and bottom surfaces in the polymerization region of the microfluidic chip 210. The thermal block 500 is designed to expose the droplets in the polymerization channel 320 to a temperature sufficient to induce polymerization but not so high as to damage the biological material, e.g., a temperature between 35°C and 45°C, e.g., 37°C. Additionally, the thermal block 500 is designed to induce a rapid increase in temperature to the target temperature and provide precise temperature control. In some instances, thermal polymerization of droplets having matrix glue as the polymerizable matrix material can be achieved with an exposure time of less than 2 minutes (e.g., less than 1 minute).

[0131] The hot blocks 500 are fixed in place, for example, using clamps. In some instances, the hot blocks 500 are fixed in place in a manner that promotes surface contact with the microfluidic chip 210. For example, the hot blocks 500 can be spring-loaded to press them against the surface of the microfluidic chip 210. In some cases, springs on both sides of the hot blocks 500 are used to hold the hot blocks against the microfluidic chip. For example, a first spring holds the hot block 500a against the microfluidic chip, and a second spring holds the hot block 500b against the microfluidic chip. The hot blocks 500 do not contact the droplet generation region 302 to avoid initiating polymerization of the matrix material before it reaches the polymerization region 304.

[0132] As Figure 5B shown in the cross-sectional view of Figure 5C and the exploded view of Figure 5C each hot block in the heating block includes a heating element 502 that contacts the corresponding surface of the microfluidic chip 210 in the polymerization region 304. The heating element 502 is enclosed on all sides except the side facing the microfluidic chip 210 by a thermal insulation cover 504 (such as a polytetrafluoroethylene (PTFE) or ultra-high molecular weight polyethylene (UHMWPE) cover). Referring specifically to

[0133] Referring to Figures 6A to 6C, when using photoinduced polymerization, the substrate material is a material that polymerizes in response to exposure to light of certain wavelengths (e.g., 405 nm blue light). For photopolymerization, the light blocks 600a, 600b are positioned to irradiate the droplets in the polymerization channels of the microfluidic chip. Each light block 600a, 600b defines a corresponding integrated chamber 602a, 602b. The light block 600b houses a light source disposed in the integrated chamber 602b, such as one or more light-emitting diodes (LEDs) 604, e.g., 405 nm blue LEDs. For example, the LED 604 is mounted on a printed circuit board (PCB) 606 (such as a metal core PCB) to dissipate the heat generated by the LED. The LEDs can be arranged in a one-dimensional or two-dimensional array. In some instances, the LEDs are disposed in both integrated chambers 602a, 602b. A heat sink is disposed outside one or both of the light blocks 600a, 600b to further dissipate heat.

[0134] The integrated chambers 602a, 602b are designed to repeatedly reflect light around and through the microfluidic chip to maximize exposure and exposure uniformity. For example, the integrated chambers 602a, 602b are formed of a material having a high diffuse reflectivity and that does not yellow (such as white PTFE or UHMWPE). The high diffuse reflectivity of the two integrated chambers 602a, 602b causes the light from the LED 604 to pass through the transparent microfluidic chip 210 multiple times. A material having a high enough reflectivity is a material that reflects at least 90%, at least 95%, or at least 99% of visible light and has a constant reflectance at all visible wavelengths (e.g., less than 20%, less than 10%, less than 5%, or less than 1% reflectance across the visible light wavelength spectrum). Diffuse reflectivity refers to the property of a material by which each beam of light incident on the material is scattered in many directions without specular reflection.

[0135] To further facilitate light reflection and prevent light from escaping the integrated chambers 602a, 602b, the light blocks 600a, 600b are fixed in place, for example, by spring loading and pressed against the surface of the microfluidic chip. In some cases, springs on both sides of the light blocks 600a, 600b are used to fix the light blocks 600a, 600b against the microfluidic chip. For example, a first spring fixes the light block 600a against the microfluidic chip, and a second spring fixes the light block 600b against the microfluidic chip. A photodetector 608, such as a photodiode, is disposed in the integrated chamber 602b of the light module 600b to measure the intensity of light in the integrated chamber 602b. In some instances, the signal from the photodetector is used to verify whether there is sufficient light intensity to complete the photoinduced polymerization process; if not, the sample processing does not continue. In some instances, the signal from the photodetector is used for closed-loop computer control of the light source. In some instances, the photodetector is also disposed in the integrated chamber 602a.

[0136] In some instances, the photoinduced polymerization is fast enough such that the polymerization occurs in a time less than the residence time of the droplet in the polymerization channel 320. For example, the photoinduced polymerization can be a photoinitiated process that, once triggered with light of an appropriate wavelength, will continue to propagate to completion even without continued exposure to light. In these instances, the light source can be pulsed to improve energy efficiency and reduce heating of the droplet. The light block is positioned to avoid irradiating the droplet generation region, thereby preventing the polymerization of the matrix material before it reaches the polymerization region.

[0137] In some instances, the matrix material polymerizes by chemical polymerization. In chemical polymerization, two liquid materials (such as two hydrogels, one of which contains a biomaterial) are mixed, and an emulsion of the mixed hydrogels in a hydrophobic fluid is generated in the droplet generation region. After the two hydrogels are mixed, the polymerization reaction starts essentially immediately. The ratio of the hydrogel containing the biomaterial to the other hydrogel is between about 1:1 and about 2:1. The droplet size is controlled by the combined flow rates of the two hydrogels.

[0138] In a specific instance, a microfluidic chip designed for chemical polymerization includes two independent flow paths for simultaneously processing two biological samples. Each flow path includes two channels for the two hydrogels. The two hydrogel channels merge into a single channel upstream of the junction with the flow channel of the hydrophobic fluid. The droplet generation chamber is located downstream of the junction, as shown, for example, Figure 4 The droplet generation chamber and the subsequent outlet channel have lengths such that the residence time of the emulsion allows for the complete chemical polymerization of the droplets. The emulsion of the polymerized droplets in the hydrophobic fluid is output to a demulsification cartridge.

[0139] Referring again to Figures 3A to 3B , the notch 330 is defined in the microfluidic chip 210 between the droplet generation region 302 and the polymerization region 304. The notch 330 provides thermal and / or optical isolation between the regions to prevent the unwanted early polymerization of the matrix material still in the droplet generation region. The notch 330 can have angled sides (see Figure 2 B), for example, angled at 45° relative to the surface of the microfluidic chip 210 to create a total internal reflection (TIR) mirror. When light is used as the polymerization stimulus, this TIR mirror reflects the stimulus light away from the droplet generation region 302, thereby protecting the matrix material in the droplet generation region 302 from exposure to light. When heat is used as the polymerization stimulus, the notch 330 acts as a thermal insulator, protecting the matrix material in the droplet generation region 302 from exposure to the applied heat. For example, the notch 330 extends through the entire thickness of the microfluidic chip 210.

[0140] Referring to Figures 7A to 7C, The oil reservoir 700 stores oil for droplet generation. The oil is poured into the chamber 710 of the reservoir 700 through an opening at the top of the reservoir, which can be closed by a lid 702. In some instances, the lid 702 is a pressure lid fixed in place with a toggle clamp 704 such that the pressure lid can be removed without a screwing motion. The oil exits the chamber 710 through a port 706 at the bottom of the chamber, for example, such that a suction tube is not used. In some instances, the port 706 is a threaded port for facilitating a reliable seal of the tubing connected thereto.

[0141] Internal retroreflective ribs 708 extend along the height of the chamber 710 to allow, for example, the volume of oil in the chamber 710 to be measured in discrete increments. In some instances, the ribs are designed to be able to measure five different volumes of oil. Based on the number and volume of samples to be processed, the system can verify whether there is sufficient oil before startup.

[0142] Figures 7D to 7E An example of an oil reservoir 750 for storing oil for droplet generation is shown. The oil is poured into the chamber 760 of the reservoir 750 through an opening at the top of the reservoir, which can be closed by a lid 752, which can be a filter vent lid for keeping particles out of the oil. In some cases, the oil is not pressurized, and the oil reservoir 750 does not include a pressure clamp and / or a toggle clamp. The oil reservoir 750 is configured with (e.g., includes) an interface 756 indicating the volume of oil in the oil reservoir 750. The interface 756 can display other measurements instead of or in addition to the volume. The oil exits the chamber 760 through a port 758 at the bottom of the chamber.

[0143] Figure 8A is a schematic block diagram of an exemplary oil delivery subsystem 800 for delivering oil from the reservoir 700 to the microfluidic chip 210. Generally, during the droplet generation process, the oil flow to each of the four droplet generation channels is individually metered to control the speed of the droplet flow through the polymerization section of the microfluidic chip.

[0144] The oil delivery subsystem 800 includes a pressure regulator 802 that supplies a single fixed air pressure (e.g., an air pressure of 2 bar) to the oil reservoir 700 to push the oil out through the port 706. A pressure sensor 804 and an air flow sensor 806 positioned between the pressure regulator 802 and the oil reservoir 700 provide feedback for error checking. A three-way valve 808 is also positioned between the fixed pressure regulator 802 and the oil reservoir 700. The three-way valve 808 has a default open configuration to vent the pressure in the oil reservoir 700 and is closed by the controller to pressurize the oil reservoir 700. This is a safety feature to ensure, for example, that the reservoir 700 is not pressurized when opened for refilling.

[0145] Oil exiting the oil reservoir 700 enters a manifold 810 having a single check valve 812 to prevent contamination from backflow and then branches into four flow control channels 814. The flow rate of each of the four channels 414 is controlled by a respective servo flow valve 816 (e.g., Enidine PFV-W24E01-P050E-0300 servo flow valve) mounted on the manifold 810. In some instances, control of the servo valve 816 is by way of a 0 - 10VDC analog control signal that provides closed-loop control of the oil flow rate, e.g., to control the size of the generated droplets. A two-way valve 818 is provided along each channel 814 to serve as a flow cutoff valve. Downstream of the valve 818, tubing connects the channel 814 to an oil port in the mount of the microfluidic chip 210.

[0146] Figure 8B is a schematic block diagram of an exemplary oil delivery subsystem 850 for delivering oil from a chamber 760 of an oil reservoir to a microfluidic chip 210. The oil delivery system includes a manifold 864 to distribute a single oil reservoir output to four independent syringe pumps 856, 858, 860, and 862 having programmable flow rates, which are controlled by a controller such as a PID controller. The outputs from the four syringe pumps 856, 858, 860, and 862 are connected to four oil input ports of a droplet generation chip. Each syringe has a capacity sufficient to complete an entire droplet generation run without stopping and refilling. The oil delivery subsystem 850 includes one or more level sensors 854 configured to measure the volume of oil in the oil chamber 760, as well as an oil cap sensor 852. In one specific instance, the oil reservoir 750 includes five level sensors 854.

[0147] Referring Figures 9A to 9B , each biological sample is packaged in a sealed disposable (single-use) reservoir 220. The reservoir 220 is designed for transportation from a sample preparation area to a MOS generation instrument and for direct insertion into the instrument. The reservoir 220 includes a plastic base 902 and a plastic top cover 903, e.g., a polypropylene base and top cover. The base 902 and the top cover 903 are attached together, e.g., by ultrasonic welding, to form an airtight seal. The base 902 defines a single well 910 within the reservoir 220 that has a capacity for a single biological sample, e.g., a capacity less than 5 mL, less than 2 mL, less than 1 mL, or less than 500 μL; and greater than 5 μL, greater than 10 μL, or greater than 20 μL. The well has an inclined bottom 906 that slopes to a low point below a tube 912 that extends from the bottom 906 of the well 910 to the cover 904. This geometry facilitates processing a high percentage, e.g., substantially 100%, of the sample contained in the well 910. This geometry also facilitates loading the reservoir 220 into the MOS generation instrument in the correct orientation.

[0148] The reservoir 220 includes a flexible lid 904, such as an elastomeric lid, disposed above the top lid 903. Input port 920 and output port 922 are defined in the flexible lid 904. The input port 920 and the output port 922 are flexible seals that are fluidly sealed by the flexible lid 904 when not in use. The input port 920 is, for example, a duckbill valve. To insert a biological sample into the well 910 of the reservoir 220, the operator pushes the pipette tip through the slit in the duckbill valve of the input port 920 and dispenses the sample. The air gaps along the slits on both sides of the pipette tip allow air to escape when dispensing the sample. When the pipette is withdrawn, the duckbill valve closes and seals, preventing spillage.

[0149] The size and shape of the duckbill valve of the input port 920 are also designed to mate with a pressure manifold when the reservoir is positioned in the MOS generation instrument. This configuration allows the well 910 to be pressurized without penetrating the duckbill valve of the input port, thereby reducing the risk of contamination of the biological sample contained in the well 910.

[0150] The output port 922 is a seal that couples the reservoir output to the microfluidic chip input. When the well 910 is pressurized, the sample contained in the well 910 is forced through the tube 912 and out through the output port 922, and the sample enters the microfluidic chip 210 from the output port.

[0151] Referring Figure 10A and 10B , samples from multiple reservoirs 220 (e.g., up to four reservoirs in the illustrated example) can be processed simultaneously by the MOS generation instrument. Each reservoir 220 is fluidly connected via its outlet port 922 ( Figures 8A to 8B ) to an inlet port of one of the fluid flow paths through the microfluidic chip 210. In some instances, a set of multiple reservoirs 220 are processed together in a carrier 150, e.g., for transportation to the MOS generation instrument. The carrier 150 may have an identifier 152 (such as a barcode (e.g., QR code or data matrix), serial number, or other identifier) printed or attached thereto, which can be used to identify the sample contained in the reservoir 220 carried by the carrier 150. In some instances, each reservoir 220 also has an identifier that can be used to identify the sample contained therein.

[0152] The carrier 150 is located in the sample preparation station 151 for sample preparation before transportation to the MOS generation instrument. The sample preparation station 151 houses sample tubes 153 containing biological materials (e.g., minced tissue) suspended in a processing medium or buffer and medium tubes 155 containing fresh medium for dilution. The user dilutes the sample in each sample tube 153 to the desired concentration using the medium from the corresponding medium tube 155 and then transfers the diluted suspension to the corresponding reservoir 220.

[0153] In some instances, it is important to maintain the biological sample at a sufficiently low temperature prior to processing in the MOS generation instrument. During transportation to the instrument, the carrier 150 that holds the storage reservoir 220 can be cooled, for example, by placing the carrier in an ice bath or by using another cooling mechanism. Referring to Figure 11A and 11B , in some instances, the MOS generation instrument includes a cold block 154 that houses the carrier 150 that holds the storage reservoir 220 to keep the biological sample cold when the biological sample is at the instrument. In an instance, the cold block 154 is cooled by a thermoelectric cooling subsystem 156 that includes a thermoelectric cooler 158, which has a heat sink 160 (e.g., a pin fin heat sink) for dissipating heat and a fan 162.

[0154] Referring to Figure 12 , the sample drive subsystem 170 independently applies air pressure to each reservoir 220 to drive the biological sample out of the reservoir and into the corresponding flow channels of the microfluidic chip 210 through a tube 912 ( Figure 9A ). The air pressure applied to each reservoir can be controlled independently of the air pressure applied to each other reservoir, and thus the fluid flow rate along each flow channel is independently controllable. The fluid flow rate affects the size of the droplets generated in the microfluidic chip, and in some instances, the sample drive subsystem 170 implements closed-loop feedback control of the fluid flow rate to achieve a target droplet size, e.g., based on droplet size measurements obtained from the imaging subsystem.

[0155] The compressed air manifold 172 serves as a central distribution and monitoring point for supplying air to multiple subsystems, such as the pressure servo valve 174. If the supply pressure is too low, the pressure sensor 173 integrated in the manifold allows the system not to operate. The pressure servo valve 174 is, for example, an Elveflow regulator with a maximum pressure of 2 bar and a resolution of 0.0001 bar. The pressure servo valve 174 is controlled by a controller 176 (such as a proportional integral derivative (PID) controller). The air flow sensor 178 and the two-way valve 180 are positioned in series between each servo valve 174 and the corresponding reservoir 220. The air flow sensor 178 provides feedback regarding the air flow, e.g., for error detection. The two-way valve 180 serves as a shut-off valve to prevent backflow when the pressure regulator is closed.

[0156] In the illustrated instance, the sample drive subsystem 170 is shown as being implemented using pressure servo valves. In some instances, other types of flow regulators are used, such as pressure regulators, valves, or pumps, e.g., peristaltic pumps, diaphragm pumps, syringe pumps, or other suitable flow regulators. The control of the flow regulator can achieve precise variations in the pressurization in the corresponding reservoir, e.g., in increments of 0.1 mbar or 0.01 mbar.

[0157] Generally, the sample drive subsystem 170 is operable to control the fluid flow rate in the range of about 100 - 200 μL / min by pressurizing the reservoir using a gas pressure between about 50 - 800 mbar. The sample drive subsystem 170 achieves continuous fluid flow for the entire volume of the sample (e.g., for a sample volume between about 10 μL and 1 mL). The sample drive subsystem 170 can achieve a purging capacity involving a higher gas pressure (e.g., a gas pressure up to about 2000 mbar).

[0158] Referring Figure 13A and 13B , the imaging subsystem 350 is positioned to acquire images (e.g., still images or video images) of the droplets as they pass through the droplet generation chamber 312 and the outlet channel 314 of the microfluidic chip 210 (see Figures 3A to 3B ). The images acquired by the imaging subsystem are used for droplet measurement or other characterization, as part of a real - time closed - loop flow control feedback system and / or for user visualization of the generated droplets. For example, droplet characteristics determined based on the images can be provided as an input to a closed - loop control system that controls the flow rate of the first fluid, the second fluid, or both, to obtain droplets with target characteristics (e.g., target size) or to obtain a desired number of droplets (e.g., for screening a library).

[0159] The imaging subsystem 350 includes a camera 352 (such as a static camera or a video camera), a lens 354 attached to the camera 352, a mirror 356 on a motion mount 358, and a backlight 360. The camera 352, the lens 354, and the mirror 356 on its mount 358 are positioned on one side of the microfluidic chip 210. The backlight 360 is positioned on the opposite side of the microfluidic chip 210. A mount (not shown) houses the camera 352, the lens 354, and the mirror 356. In some instances, to protect the components of the imaging subsystem from dripping water and debris, the imaging subsystem 350 is separated from the microfluidic chip by a transparent window (e.g., a glass window). The images captured by the camera 352 are provided to a computing device, such as a local computer or a cloud - based server having one or more processors coupled to a memory.

[0160] Generally, the camera 352 and the lens 354 have a field of view of about 20 mm x 25 mm, a pixel resolution of about 5 μm, and a frame rate of 42 frames per second. The imaging subsystem 350 can be a monochromatic or a multi - color imaging system. The imaging subsystem 350 has a global shutter, does not exhibit parallax or distortion in the external channels, and is capable of sub - frame region - of - interest transfer.

[0161] The backlight 360 is positioned to illuminate the droplet generation chamber and the outlet channel of the microfluidic chip 210 such that images can be captured. The backlight 360 can be monochromatic or polychromatic and illuminates the chip 210 at a wavelength that does not induce polymerization and does not heat the droplets to a temperature sufficient to induce polymerization. In a specific example, the lamp is a green lamp mounted at least 75 mm from the chip. In some examples, multiple light sources of different colors are used to facilitate image analysis. The liquids, including the first fluid containing patient-specific biological material and unpolymerized matrix material and the second immiscible fluid, are generally clear liquids, and the droplets are visible in the immiscible fluid due to the difference in refractive index between the droplets and the surrounding fluid. The curvature of the droplet edges in combination with the difference in refractive index causes the droplets to act as lenses that bend the light from the backlight 360.

[0162] In some examples, the backlight 360 is a diffused light source, resulting in the edges of the droplets being visible in the images captured by the camera 352. In some examples, the light is a collimated light source used to facilitate image analysis. For example, when illuminated with collimated light, the focusing effect of the lens-like droplets deflects the light away from its original axis and out of the line of sight of the camera 352. This focusing effect enhances the contrast of the droplets in the resulting image compared to droplets illuminated with diffused light, which can facilitate the identification of the droplets and / or their edges in the image. In some examples, the lamp is positioned on the same side of the microfluidic chip 210 as the camera (e.g., rather than the backlight 360).

[0163] In some examples, the imaging subsystem applies gated illumination to capture multiple exposures of each of one or more droplets in a droplet within a single image captured during a single frame of the camera 352. Due to the timing of the two exposures (e.g., the interval between the two exposures and the duration of each exposure), the same droplet is captured in both exposures, and artifacts such as blurring due to the movement of objects in the image do not occur. The two exposures of the same droplet are analyzed to determine droplet characteristics such as droplet size (e.g., droplet volume or droplet diameter), droplet size distribution, droplet velocity, the spacing between adjacent droplets, the number density of droplets (e.g., the number of droplets per unit length of the outlet channel (e.g., Figure 4 the outlet channel 314), the number of droplets per unit volume of the second fluid), the estimated total number of droplets generated from a given starting volume of the first fluid, or other droplet characteristics. These characteristics are applied to a closed-loop feedback system that can adjust the flow rate of the source material (e.g., the first or second fluid) to affect the size and flow rate of the generated droplets, enabling the continuous generation of liquid of a target size at a target flow rate. Such a closed-loop feedback system is described in more detail below.

[0164] After aggregation, the aggregated droplets pass through a demulsification subsystem where, in a continuous, microfluidics-based demulsification process, the droplets move from their emulsion in a second fluid (e.g., oil) to an aqueous fluid. The demulsification subsystem is designed to minimize droplet loss, e.g., to achieve recovery of at least 90% of the droplets into the aqueous fluid and to cause the droplets to migrate into the aqueous fluid without causing physical damage to the droplets. The demulsification subsystem is also designed to prevent residual oil from entering the aqueous fluid. Additionally, the demulsification subsystem can achieve high-throughput processing, e.g., processing input volumes in the range of 10 - 1000 μL, and has the ability to remove large amounts of oil, such as up to 5 mL of oil.

[0165] Figure 14 FIG. is a schematic view of a portion of the demulsification subsystem 450. The demulsification subsystem 450 uses crossflow filtration in the form of a microfluidic chip where a combined flow of an aqueous fluid containing aggregated droplets and oil enters the inlet end 451 of the microfluidic channel 452 and flows along the channel 452 past a membrane 454. The membrane is a hydrophobic and oleophilic membrane, such as a polyvinylidene fluoride (PVDF) or PTFE membrane. A pressure bias across the membrane 454 drives the oil through the membrane into a collection container 456 below the membrane 454. For example, a vacuum can be applied to the surface of the membrane 454 facing the collection container 456. In some instances, the collection container 456 vents to the atmosphere. In some instances, a positive pressure is applied to the surface of the membrane 454 facing the microfluidic channel 452. In some instances, the oil is driven through the membrane with the help of gravity due to the pressure difference between the positive pressure applied above the membrane and the ambient pressure below the membrane.

[0166] Because the membrane 454 is hydrophobic, the aqueous fluid and the aggregated droplets are repelled by the membrane 454 and thus continue to flow along the microfluidic channel 452 to the outlet end 455 of the microfluidic channel.

[0167] As the oil is suctioned across the membrane, the aggregated droplets exchange into the aqueous fluid. Through the outlet end 455 of the microfluidic channel, the oil has been suctioned across the membrane such that only the aggregated droplets in the aqueous fluid remain in the channel.

[0168] The microfluidic channel 452 of the demulsification subsystem 450 is serpentine to promote mixing of the oil and the aqueous fluid within the channel, thereby promoting contact between the oil and the membrane 454 even when only a small fraction of the volume of the fluid is constituted by oil (e.g., toward the outlet end 455 of the microfluidic channel).

[0169] Figure 15A cross-sectional view of a portion of the demulsification subsystem 450 is shown. The microfluidic channel 452 is defined in a substrate 470, such as a molded plastic substrate (e.g., polymethyl methacrylate (PMMA) or polystyrene). A first side of the hydrophobic membrane 454 (e.g., PVDF membrane) is attached to the substrate 470 by an adhesive 472, and the substrate is cut to expose the microfluidic channel 452 in the substrate 470. The opposite side of the membrane 454 is attached to a collection container 456, such as a plastic container, e.g., PMMA, by an adhesive 474. The adhesive 474 is disposed around the outer edge of the membrane 454 such that oil in the microfluidic channel can be suctioned through the membrane 454 and into the collection container 456. The collection container 456 itself has a central chamber 453 positioned below the portion of the membrane not covered by the adhesive 474 such that oil suctioned through the membrane 454 is collected in the cavity 456 of the collection container. The adhesives 472, 474 are biocompatible adhesives that are inert to oil and aqueous fluids.

[0170] The membrane 454 has pores sized to readily allow oil to flow through the membrane while preventing droplets from passing through. For example, the pores have a diameter between 0.25 μm and about 1 μm (e.g., 0.45 μm).

[0171] Figures 16A to 16C Are respectively a top view, a side view, and an exploded view of an exemplary demulsification cartridge 230. A sample input channel 460, a medium input channel 462, and a microfluidic channel 452 are defined in the substrate 470. The substrate 470 is connected to a collection container 456, with the membrane 454 disposed therebetween such that the membrane 454 forms the bottom wall of the microfluidic channel 452. A medium reservoir 476 is disposed below the collection container 456. The substrate, the collection container 456, and the medium reservoir 476 are connected, for example, by ultrasonic welding.

[0172] The microfluidic channel 452 is defined in the surface of the substrate 470 facing the collection container 456 such that the fluid in the microfluidic channel 452 contacts the membrane 454 and such that oil is drawn into the collection container 456 through the microfluidic channel 452. In some instances, the medium input channel 462 is defined on the same surface of the substrate 470 as the microfluidic channel 452, and in some instances, the medium input channel 462 is defined on the opposite surface of the substrate 470.

[0173] The collection container 456 (e.g., molded plastic (e.g., PMMA or polystyrene)) structure defines one or more chambers 453 for containing oil removed from the fluid flowing along the microfluidic channel 452. Generally, the volume of the chambers in the collection container 456 is greater than the expected volume of the oil to be removed from the fluid, e.g., between about 10% - 25%. For example, if the expected volume of the oil is about 5 mL, the chamber capacity can be about 6 mL. In some instances, the collection container 456 contains an oil collection material, such as a spongy material, e.g., disposed on the bottom surface of the chamber. The oil collection material is a hydrophobic material that traps the oil in the chamber 453 of the collection container 456, thereby preventing the oil from being pulled back into the microfluidic channel 452 through the membrane 454. In some instances, a vacuum channel is defined in the body of the collection container 456 for applying a vacuum to the membrane 454.

[0174] The media reservoir 476 (e.g., molded plastic (e.g., PMMA or polystyrene) structure) defines a chamber for containing the aqueous fluid to be supplied to the microfluidic channel 452. In some instances, the chamber of the media reservoir 476 has a capacity between about 10 - 20 mL, e.g., 4 - 8 mL of aqueous fluid. In some cases, the capacity of the media reservoir 476 is limited by the volume of the final output tube. For example, if the capacity of the final output tube is 15 mL, the capacity of the chamber of the media reservoir 476 should not exceed 15 mL. The bottom surface 478 of the chamber is angled relative to the plane of the substrate 470 such that the aqueous fluid accumulates below the pipette 480. The aqueous fluid is extracted from the media reservoir 476 via the pipette 480, which is fluidly connected to the media input channel 462. In the illustrated instance, the pipette 480 is integrally formed with the collection container 456 and sealed to the substrate 470 with an adhesive. In some instances, the pipette is a separate element that extends through a hole in the collection container 456. In some instances, the pipette 480 is integrally formed with the substrate 470 (e.g., molded as part of the substrate 470) such that no separate sealing element is used between the pipette 480 and the substrate 470.

[0175] The media reservoir 476 can be filled with aqueous fluid through the valve 484. The valve 484 extends through openings 486, 488 in the substrate 470 and the collection container 456, respectively. In some instances, the valve 484 is a molded duckbill valve (e.g., a thermoplastic elastomer valve) that allows a pipette tip to be inserted through the valve 484 to fill the media reservoir 476 but otherwise remains closed to prevent spillage or contamination of the aqueous fluid. The valve 484 also forms a seal to the pressure port and can be opened to allow air pressure to enter to drive the aqueous fluid flow into the media input channel 462. A media pressure subsystem (such as a set of precision pressure regulators protected by a shut-off valve) provides a separate driving pressure to each media reservoir 476, thereby driving the aqueous fluid into the media input channel 462.

[0176] The seal 490 is positioned to provide a sealed coupling between the microfluidic chip 210 and the sample input channel 460. The seal 490 can be a thermoplastic elastomer seal. In the illustrated example, the valve 484 and the seal 490 are a single integral element. In some examples, the valve 484 and the seal 490 are two different elements.

[0177] As previously discussed, the sample input channel 460 is fluidly connected to the output of the polymerization section (see Figures 3A to 3B ) and is connected to the microfluidic channel 452. The medium input channel 462 configured to receive an aqueous fluid through a valve is also connected to the microfluidic channel 452 such that the fluid flowing along the microfluidic channel 452 near the inlet end 451 is a mixture of an aqueous fluid and droplets in oil. In some examples, the sample input channel 460 and the medium input channel 462 have a three-dimensional channel structure, e.g., which rises above the bottom surface of the substrate 470, thereby preventing the oil and the aqueous fluid from contacting the membrane 454 until they are combined in the microfluidic channel 452. In some examples, the medium input channel 462 is a long channel (e.g., with a length between 50 mm and 150 mm) with a small cross-sectional size (e.g., 125 μm x 225 μm) to allow operation at a high backpressure, thereby preventing the oil from flowing back into the microfluidic channel 452 when the flow on the membrane 454 slows down.

[0178] The first section 492 of the microfluidic channel 452 has a simple serpentine pattern which allows the initial oil-rich fluid to wet the membrane 454, thus facilitating the rapid removal of oil from the fluid flowing in the channel 452. The second section 494 of the microfluidic channel 452 has a double serpentine pattern in which small turns (e.g., turn 495) are formed within each back-and-forth channel of the channel 452. These small turns facilitate the mixing of the fluid in the channel such that the shrinking oil clusters in the fluid repeatedly contact the membrane 454. This repeated contact helps prevent the situation where small oil clusters (e.g., oil clusters less than a certain percentage of the channel depth, e.g., less than 50%) fail to contact the membrane 454 along the entire length of the channel 452.

[0179] In some examples, the cross-sectional size (e.g., cross-sectional area) of the microfluidic channel 452 decreases along the length of the channel to increase the fluid resistance, thereby increasing the residence time of the fluid on the membrane 454 and promoting the thorough removal of oil. This tapering also helps maintain the backpressure on the membrane 454 as the volume of the fluid decreases with the removal of oil. In a specific example, the microfluidic channel 452 tapers from an initial size of 300 μm x 600 μm to a final size of 300 μm x 325 μm. The tapering can be gradual or in discrete increments.

[0180] At the output 455 of the microfluidic channel 452, droplets in the aqueous fluid substantially free of oil flow through the outlet channel 496 to the outlet port 498 that extends through, for example, a shielding element 499 integrally formed with the collection container 456. The shielding element 499 prevents the edges of the output container connected to the outlet port 498 from being contaminated, which otherwise could occur due to splashing caused by the treatment air that pushes the liquid out of the outlet port 498. The outlet channel 496 can be a long channel (e.g., having a length between about 50 - 100 mm and having a cross-sectional dimension similar to the final cross-sectional dimension of the microfluidic channel 452) to create a backpressure comparable to that of the external tubing in the panel. In some instances, the outlet port 498 has a pipette-like tip that is configured to connect to an output container, such as a conical output container (see FIG. 18). The demulsified MOS can be transported elsewhere in the output container, e.g., for further organoid growth and testing.

[0181] Figures 17A to 17B Two exemplary configurations of the serpentine microfluidic channel 452 are shown, including a single serpentine channel 452a( Figure 17A ) and a double serpentine channel 452b( Figure 17B ). Other configurations are possible. In each configuration, an emulsion of droplets in oil is received via the sample input channel 460, and an aqueous fluid is received via one or more media input channels 462. At the output 455 of the microfluidic channel 452, the droplets in the aqueous fluid are output via the outlet port 498 to an output vial, such as a centrifuge tube (not shown).

[0182] The components of the MOS generation instrument that come into contact with the sample (e.g., with patient-specific biomaterial) are disposable. These components and other disposable components are referred to as consumables. Referring to Figure 18A and 18B , the consumables are positioned in, for example, a holder or nest such that the various fluid interfaces are aligned and fluidly connected.

[0183] The positioned consumables are clamped in place by a clamp 850, the interface seals are pressed together, and the air source mates with the air drive port. For example, the clamp 850 is lowered by the operation of an actuator, such as a lever. In the examples of Figure 18A and 18B , the clamp 850 includes a spring plunger that is supported downward on certain components to provide a good seal. For example, the pressure supply device 854 and the chip input port 856 are pressed downward on the top of each sample reservoir 220. The oil port 852 is pressed downward on the microfluidic chip 210. The pressure supply device 858 is pressed downward on the demulsification cartridge 230 to connect to the media reservoir 476. When a sealing pressure is applied on the other side, the plunger 860 applies a downward force on the demulsification cartridges to hold them fixed in the nest.

[0184] Referring to Figure 19 , in some instances, consumable loading follows a prescribed order. The sample reservoir and media reservoir in the demulsification cartridge are pre-filled away from the instrument (50). Prior to operation, sample identifiers are associated with each location in the sample storage carrier (52). The output vials are placed in their holders, and their identifiers (e.g., barcodes) are read (e.g., scanned) as each vial is loaded, thereby linking each output vial identifier to the corresponding channel (54).

[0185] The demulsification cartridge is loaded into its holder, and the output port of each demulsification cartridge is inserted into the corresponding output vial (56). The sample reservoir carrier containing multiple sample reservoirs is read (e.g., scanned) and placed into the refrigerated input nest in the instrument (58), thereby linking the sample identifier of each sample to the corresponding channel and thus to the corresponding specific output vial identifier.

[0186] The microfluidic chip is placed in the instrument and aligned and mated with the sample reservoir and the demulsification cartridge (60). The consumables are clamped, thereby forming a seal of the open ports of the respective components (62).

[0187] Referring to Figure 20 , the droplet generation system 200 operates under the control of the electronic subsystem 250, which is implemented, for example, in a computing device (e.g., local or cloud-based) that communicates with various electronic control subsystems via an interface (such as a USB or I2C serial interface). The electronic subsystem provides a graphical user interface capable of development, diagnostic testing, and servicing on the droplet generation system. The electronic subsystem is also operable to control the hardware of the droplet generation system 200 to process samples according to a selected protocol and transfer the results to, for example, the graphical user interface or another computing device. In some instances, the electronic subsystem implements closed-loop feedback control of the operation of the droplet generation system 200 based on analysis of data received from the system. For example, the electronic subsystem can determine the size of the generated droplets based on images acquired by the imaging subsystem and can control the droplet generation system to adjust the fluid flow rate through the droplet generation chamber 212 to achieve a target droplet size.

[0188] The electronic subsystem 250 includes user interface components 252, such as a display 254 (e.g., a touchscreen display), a keyboard 256, a mouse 258, and a barcode reader 259. Additional and / or alternative user interface components may also be included. The electronic subsystem 250 includes data acquisition components, including data input and data output components. The digital output components include controls for features such as two-way valves (e.g., for solenoid valves or syringe pumps), heat, and light. The digital input components include, for example, a door closure sensor. The analog input components include, for example, an aggregated light intensity sensor and an overflow sensor.

[0189] The electronic subsystem 250 includes devices connected to a control computing device via one or more types of interfaces. In the example of FIG. 21, these devices include USB devices 270, such as cameras, pressure regulators (e.g., Elveflow precision pressure regulators), thermal controllers, strobe controllers, and barcode readers; and I2C sensor input devices 272, such as sensors for pressure, air flow, and temperature.

[0190] Additional components for the electronic subsystem include, for example, a power input module, a power supply, a USB hub, an Ethernet bulkhead feedthrough, a cooling fan, and other components, e.g., as Figure 20 shown.

[0191] FIG. 21 is a block diagram of a MOS generation system, showing the integration of certain elements of the electronic subsystem.

[0192] Referring to Figure 22 , in an exemplary method for MOS generation (e.g., generation and demulsification), droplets of a first fluid (e.g., a patient-specific biomaterial in a polymerizable matrix material) are generated in a hydrophobic fluid (such as oil (10)) in a droplet generation section of a first microfluidic channel defined in the surface of a microfluidic chip. For example, droplets are generated at the junction between the first microfluidic channel and one or more channels carrying the hydrophobic fluid. In some instances, the microfluidic chip has a plurality of fluidically independent first microfluidic channels, and droplets are generated in parallel (e.g., simultaneously) in each first microfluidic channel such that samples from multiple patients can be processed simultaneously.

[0193] In some instances, for example, in a closed-loop feedback control system, the flow rate of the first fluid (a fluid containing a biomaterial suspended in an unpolymerized matrix material) is controlled to obtain droplets of a desired size (12). For example, the flow rate is controlled based on the size of the generated droplets as determined from an image of the droplets in the droplet generation chamber of the first microfluidic channel. The flow rate of the hydrophobic fluid is also controlled (13) to control the emulsion flow (e.g., the fluid flow through the outlet channel 314; see Figure 4) flow rate, which determines the residence time of the fluid in the polymerization region (e.g., in the polymerization channel 320; see Figures 3A to 3B ) The flow rate of the emulsion stream is also related to the start time and speed of coordinating (e.g., through the medium input channel 462; see Figures 16A to 16C ) the medium input into the demulsification channel.

[0194] The generated droplets flow through the polymerization portion of the first microfluidic channel (14), where a stimulus (e.g., light or heat) is applied to polymerize the matrix material to form MOS emulsified in the hydrophobic fluid (16). When there are multiple fluidly independent first microfluidic channels, the droplets in each channel polymerize simultaneously as they flow through the corresponding polymerization portion of the first microfluidic channel. In some instances, when light is applied as the stimulus, the droplets are irradiated with a pulsed illumination pattern. In some instances, a stimulus (e.g., light or heat) is applied to two opposing surfaces of the microfluidic chip.

[0195] A mixture of the emulsion of MOS in the aqueous fluid and the hydrophobic fluid flows along a second microfluidic channel defined in the substrate of the demulsification cartridge for demulsification of MOS, e.g., to transfer MOS into a suspension in the aqueous fluid (18). When there are multiple first microfluidic channels defined in the microfluidic chip, each first microfluidic channel is fluidly connected to a corresponding second microfluidic channel in a corresponding demulsification cartridge. As the mixture flows along the second microfluidic channel, the hydrophobic fluid transfers through a membrane forming the wall of the second microfluidic channel, thereby removing the hydrophobic fluid and allowing MOS to transfer into the aqueous fluid (20). In some instances, a vacuum is applied to the side of the membrane opposite the second microfluidic channel to facilitate removal of the hydrophobic fluid from the second microfluidic channel.

[0196] The suspension of MOS in the aqueous fluid is provided to an output vial (22) for downstream use, e.g., for cell growth, testing, or other applications.

[0197] Closed-Loop Control of MOS Generation System

[0198] In some instances, real-time closed-loop feedback is used for real-time quantification and control of droplet size and flow rate in the above MOS generation systems and methods. Based on the measured droplet size and flow rate, the microfluidic system can be controlled to adjust the size of the generated droplets, the flow rate of the droplets, or both. These adjustments can achieve a target droplet size, flow rate, or both to allow for effective and complete polymerization of the matrix material of MOS. These adjustments can also achieve control of the droplet generation process to produce a target number of droplets, e.g., to obtain the desired number of MOS for screening libraries.

[0199] These closed-loop feedback methods can have one or more of the following advantages. The closed-loop feedback systems described herein provide a mechanism for reliably and non-invasively capturing multiple images of a single droplet as it flows along a microfluidic channel. Quantitative measurements of the size and velocity of a single droplet and an estimate of the total number of droplets can be obtained using short exposure times and high-intensity illumination pulses. Images are obtained without artifacts due to droplet motion, such as blurring. The timing of the illumination pulses can be controlled independently of the camera shutter and independently of the camera's frame rate, and can be adjusted to achieve consistent imaging quality over a wide range of flow rates. The ability to sense physical parameters such as size and velocity in a non-contact and non-invasive manner improves the precision of the processes carried out in the microfluidic device. Additionally, since these imaging methods can be achieved using a low camera frame rate, the computational power burden for analyzing the generated images is relatively low. Further, the configuration of the optical system used in the closed-loop feedback methods described herein is less expensive and smaller than typical optical systems used in life science systems, e.g., because the optical systems described herein do not require expensive aspects such as sub-millimeter alignment, fluorescent dyes, dichroic components, or lasers. Additionally, the closed-loop feedback systems described herein are non-invasive to the microfluidic system, which allows for avoiding the possibility of clogging or contamination.

[0200] In the closed-loop feedback methods described herein, multiple exposures of each of one or more droplets are obtained within a single image, and the size of the droplet, the flow rate of the droplet, or both are determined based on these exposures. Due to the timing of the two exposures, e.g., the interval between the two exposures and the duration of each exposure, the same droplet is captured in both exposures without artifacts such as blurring that may occur due to the motion of the object in the image. The two exposures of the same droplet are analyzed to determine characteristics of the droplet, such as size and flow rate. The exposures can also be analyzed to determine other characteristics of the droplet, such as the spacing between adjacent droplets, the number density of the droplets, or an estimate of the total number of droplets generated in the system. These characteristics are applied in a closed-loop feedback system, which can adjust the first and second fluids as appropriate such that droplets of a target size are generated, such that the generated droplets flow at a target flow rate, or both, enabling the continuous generation of droplets of a target size at a target flow rate. In a specific example, the flow rate of the first fluid is adjusted to achieve droplets of a target size, and the flow rate of the second fluid is adjusted such that the generated droplets flow at a target flow rate.

[0201] In some instances, a closed-loop feedback method can be used to control the number of droplets generated in a microfluidic system. For example, the microfluidic system can have a performance goal of generating a target number of droplets of a specific size from a given volume of sample. For example, a 10 μL biomaterial sample from a needle biopsy will generate 10,000 droplets of a specific size. Based on the size of the droplets as determined from multiple images, the flow rates of the first and second fluids can be adjusted to achieve the generation of the target number of droplets.

[0202] Referring again to Figure 13A and 13B , the closed-loop feedback system includes an imaging subsystem 350, a computing device (such as a local computer or a cloud-based server having one or more processors coupled to a memory), a controller that controls the flow of the first and second fluids, and a controller that controls the operation of the imaging system 350. The closed-loop feedback system captures and analyzes images of the droplets to quantify the size of the generated droplets, the flow rate of the generated droplets, or both. In some instances, the closed-loop feedback system controls the operating parameters of the MOS generation system based on the analysis of the images to obtain droplets of a target size, a target flow rate, or both that are suitable for complete and efficient polymerization. In some instances, the closed-loop feedback system controls the operating parameters of the MOS generation system based on the analysis of the images to obtain a target number of droplets from a given sample of the first fluid. For example, the closed-loop feedback system controls the operation of a flow regulator that controls the flow of the first and second fluids, e.g., controls the operation of the sample drive subsystem 170 (see Figure 12 ) to control the flow rate of the first fluid and / or controls the operation of one or more of the programmable flow rate pumps 856 - 862 (see Figure 8B ) to control the flow of the second fluid.

[0203] The liquids, including the first fluid containing patient-specific biomaterial and unpolymerized matrix material and the second immiscible fluid, are generally clear liquids, and due to the difference in refractive index between the droplets and the surrounding fluid, the droplets are visible in the immiscible fluid. The curvature of the droplet edges, combined with the difference in refractive index, causes the droplets to act as lenses that bend light from the light source 360. In some instances, the light source 360 is a diffuse light source, resulting in the edges of the droplets being visible in the images captured by the camera 352. In some instances, the light source 360 is a collimated light source, and the focusing effect of the lens-like droplets deflects the light away from its original axis and out of the line of sight of the camera 352. This focusing effect enhances the contrast of the droplets compared to droplets illuminated with diffuse light.

[0204] The operations of the camera 352 and the light source 360 are controlled by a controller of a closed-loop feedback system. The controller is, for example, a programmable control board that can generate multiple concurrent outputs of control pulses (e.g., current pulses or voltage pulses) for synchronously controlling the camera and the light source with programmable timing.

[0205] The camera 352 includes a shutter, e.g., a global shutter. For example, the shutter can be an electronic shutter inside the camera. The opening of the shutter can be controlled by an external trigger, such as a current or voltage pulse received from the controller of the closed-loop feedback system. The exposure time (e.g., the amount of time the shutter remains open) can be specified, for example, through direct interaction with the camera 352 or through interaction with a computing device communicatively coupled to the camera 352. In a specific example, the shutter of the camera 352 responds to a transistor-transistor logic (TTL) input received from the controller, e.g., a 3.3V or 5V TTL input. When the shutter receives an input from the controller, the shutter opens and starts an integration period that lasts for the specified exposure time. A single integration period, such as a single still photography frame or a single frame of a video camera, is sometimes referred to as a single frame of the camera 352.

[0206] The light source 360 can be controlled by an external trigger, such as a current or voltage pulse received from the controller of the closed-loop feedback system. The light source 360 is a light source capable of generating gated illumination, e.g., multiple (e.g., two, three, four, or more than four) short illumination pulses in quick succession. For example, the light source 360 can be a light-emitting diode (LED), an arc lamp, or other suitable light source. In some examples, the light source 360 is a collimated light source, e.g., a collimated LED. The light source 360 can be a monochromatic light source (e.g., a monochromatic LED) or a polychromatic light source (e.g., a white light source). In a specific example, a monochromatic blue LED is used as the light source. The short wavelength of the blue LED helps prevent blurring of the edges of the droplets in the image. In some examples, such as when the droplets contain an absorbing dye, the light source 360 includes multiple light sources of different colors, e.g., multiple LEDs of different colors.

[0207] The controller of the closed-loop feedback system is configured to control the camera 352 and the light source 360 such that the light source 360 generates multiple (e.g., two, three, four, or more than four) illumination pulses during a single frame of the camera 352 (e.g., when the camera shutter remains open). The result is two exposures within a single frame: within the single image captured during a single frame of the camera 352, the droplet generation chamber 312 (see Figures 3A - 3B(Two exposures with droplets therein). In some instances, the outlet channel 314 also falls within the field of view of the camera 352 and is thus also captured in the two exposures. The two exposures are separated in time by the time between two consecutive illumination pulses generated by the light source 360. Because of the short time interval between the two illumination pulses, the same one or more droplets appear in both exposures. Because the droplets flow along the droplet generation chamber 312 during the time interval between the two illumination pulses, a given droplet appears at slightly different positions in the two exposures.

[0208] In some instances, the camera 352 operates at a fixed frame rate and outputs a TTL signal at the start of each frame integration (e.g., when the shutter opens). The controller of the closed-loop feedback system is programmed to generate a gated output pulse at a specific delay from the start, duration, and separation start. In some instances, the controller of the closed-loop feedback system triggers the camera exposure and the gated pulse such that the time between exposures can be varied. In some instances, a modified sequence can be implemented where the gated pulses are emitted in an alternating sequence of a single pulse in one frame followed by a double pulse in the next frame. This modified sequence allows the system to collect two image streams, one including two exposures to analyze, for example, velocity, and the other including a single exposure, for example, for display to the user.

[0209] The ability to image droplets in both the droplet generation chamber 312 and the outlet channel 314 has advantages. Specifically, because the geometry of the droplet generation chamber 312 is different from the geometry of the outlet channel 314, the ability to image droplets in both locations provides an opportunity to image droplets with different shapes. In an example, the first channel 306 and the second channel 310 have a cross-sectional size of 200 μm x 200 μm, the droplet generation chamber 312 has a cross-sectional size of 200 μm x 700 μm, and the outlet channel 314 has a cross-sectional size of 300 μm x 300 μm. With a nominal spherical droplet size of 255 μm, the droplets are flattened into a pancake shape with a full-round edge when in the droplet generation chamber 312 and become spherical when they leave the droplet generation chamber 312 and enter the outlet channel 314. The ability to image droplets in both the droplet generation chamber 312 and the outlet channel 314 allows droplets to be imaged in two geometries, for example, facilitating volume calculation.

[0210] The ability to image droplets and determine droplet velocities in the generation chamber and the outlet channel also provides a mechanism for error checking. Since the generation chamber and the outlet channel have different cross-sectional areas, the expected velocity ratio between the two locations is known (e.g., the velocity ratio is the inverse of the cross-sectional area). The measured droplet velocities at the two locations can be compared to serve as an error check, e.g., to prevent reporting an incorrect velocity from a single location measurement and to promote instead reporting a velocity consistent with the data for the two channels.

[0211] Figure 23A An example of a single image 1200 captured during a frame of the imaging system's camera 352 ( Figures 13A to 13B ) is shown. Image 1200 contains an image of a portion of the generation chamber with two capture locations 1202a, 1202b of the same droplet. When the camera shutter is kept open, the two locations 1202a, 1202b of the droplet are captured as two exposures by two consecutive illumination pulses. The two locations 1202a, 1202b within a single image are sometimes referred to as the two exposures 1202a, 1202b of the same droplet. Since the droplet flows along the generation chamber in the direction indicated by the arrow, the droplet appears at different positions in each of the two exposures 1202a, 1202b. The position of the droplet in exposure 1202a is the position of the droplet when the first illumination flash is generated, and the position of the droplet in exposure 202b is the position of the droplet when the second illumination flash is generated. In each of the two exposures 1202a, 1202b, the droplet is in a different position because it moves along the chamber during the interval between the flashes. As discussed in detail below, the analysis of multiple exposures 1202a, 1202b of a single droplet within a frame can be performed to determine characteristics of the droplet, such as droplet size, droplet velocity, or other characteristics.

[0212] Figure 23B An example of control signals provided from the controller to the camera and the light source is shown, which results in Figure 23A the generation of a two-exposure image. For example, the controller of a closed-loop feedback system can generate Figure 23B control signals to control the operation of the shutter of camera 352 (see Figures 13A to 13B ) and the operation of the light source 360. The control signals include a camera drive signal 1210 that causes the shutter of camera 352 to open, e.g., a square-wave current pulse. The control signals also include a light source drive signal 1220, e.g., a square-wave current pulse, with each drive signal 1220 causing the light source 360 to generate an illumination pulse. Figure 23B The midline in

[0213] In Figure 23A and 23BIn an example, the controller first sends a camera drive signal 1212 to control the opening of the camera shutter, thereby starting a single frame (integration period) of the camera for capturing a single image 1200. Then the substrate the controller sends two consecutive light source drive signals 1222a, 1222b, each signal causing the light source to generate a short illumination flash. As shown by the integration period 1230 of the camera, the camera shutter remains open during the two illumination pulses. Each illumination flash generates an exposure of the droplets within the single image 1200 (e.g., the flashes triggered by pulses 1222a, 1222b cause exposures 1202a, 1202b of the droplets respectively). After the integration period 230 ends, the camera shutter closes. The resulting image 1200 including the two exposures 1202a, 1202b is sent to a computing device for analysis.

[0214] In some examples, the time interval between the two pulses 1222a, 1222b is short enough such that the distance between the leading edge 1204a of the droplet in exposure 1202a and the leading edge 1204b of the same droplet in the consecutive exposure 1202b is less than the size of the droplet (e.g., radius or diameter). This short time interval ensures that the same droplet appears in the two exposures 1202a, 1202b (e.g., a given droplet travels a distance less than its diameter during the time interval between the pulses). For example, the time interval (pulse interval) between the two pulses 1222a, 1222b can be between 1 millisecond (ms) and 50 ms, e.g., between 1 ms and 30 ms or between 5 ms and 10 ms. The time interval is adjustable, e.g., by programming the controller, to be suitable for various flow rates. For example, the time interval can be adjusted to create a consistent spacing between the leading edges of the droplets in each image, regardless of the flow rate. The time interval can be consistent or can vary throughout the imaging process.

[0215] In some examples, the time interval between the two pulses 1222a, 1222b has a duration such that the droplet moves a distance of about 1 / 4 of its radius to 4 times its radius between exposures. Droplet identification and analysis can be achieved even if the droplets do not overlap themselves in the two exposures. This flexibility allows for image analysis over a large dynamic speed range.

[0216] Pulses 1222a, 1222b can be short enough to avoid artifacts (e.g., blurring) due to droplet motion in the corresponding exposures 1202a, 1202b. The duration of each pulse can be set based on system factors such as lens condensation, lens aperture setting, camera imager sensitivity, and camera gain. For example, each of pulses 1222a, 1222b can have a duration between about 5 microseconds (μsec) and about 125 μsec (e.g., between about 10 μsec and about 50 μsec or between about 25 μsec and about 50 μsec, such as 10 μsec, 20 μsec, 25 μsec, 30 μsec, 35 μsec, 40 μsec, 45 μsec, 50 μsec, 75 μsec, 100 μsec, or 120 μsec). In a particular example, the two pulses 1222a, 1222b are 125 μsec duration pulses spaced 25 ms apart. The pulse duration and pulse interval do not necessarily have the same value. Because pulses 1222a, 1222b are brief, high-intensity light can be used, which is beneficial for image analysis.

[0217] Multiple exposures 1202a, 1202b in a given image 1200 are analyzed by a computing device having one or more processors coupled to a memory, the one or more processors configured to perform image analysis using, for example, machine vision analysis processing techniques. The analysis can determine characteristics of the droplets in exposures 1202a, 1202b such as the size (diameter, volume) of the droplets, the flow rate (velocity) of the droplets, the spacing between adjacent droplets, the number density of the droplets, or the estimated total number of droplets generated from a specified volume of sample.

[0218] The analysis by the computer can include identifying the droplets in each exposure 1202a, 1202b of the given image 1200 and identifying the leading or trailing edge of the droplets in each exposure (e.g., leading edge 1204a of the droplet in exposure 1202a and leading edge 1204b of the droplet in exposure 1202b). The leading edge of a droplet is the edge of the droplet facing the direction of droplet motion; the trailing edge is the edge opposite the direction of droplet motion. In some instances, the leading edge, trailing edge, or both edges of the droplets in each exposure are directly identified without first identifying the droplets themselves.

[0219] In an example, a droplet is identified during exposure through a frequency domain technique or a machine vision analysis process that identifies circular or substantially circular objects (two-dimensional projections of spherical droplets) during exposure, e.g., circles that produce optimal results. Frequency domain techniques such as autocorrelation or fast Fourier transform processes can allow for a holistic analysis of some or all of each image. In some cases, the machine vision analysis process identifies circular or substantially circular features that fall within a pre-specified target size range, e.g., to avoid misidentifying foreign objects such as debris or bubbles as droplets.

[0220] In an example, the leading edge of a droplet is identified during exposure as a feature having positive curvature in a pre-specified direction corresponding to the direction of movement of the droplet during exposure, and the trailing edge of the droplet is identified as a feature having negative curvature in the same direction. In some cases, the machine vision process identifies features having a curvature that falls within a pre-specified suitable curvature range, e.g., to avoid misidentifying foreign objects as droplets.

[0221] In some examples, once a droplet is identified in each exposure, the exposures 1202a, 1202b in the image 1200 are analyzed to determine the distance between corresponding edges of the droplet in two consecutive exposures 1202a, 1202b. In Figure 23A the example, the distance d between the leading edge 1204a of the droplet in exposure 1202a and the leading edge 1204b of the droplet in exposure 1202b is determined. In some examples, the distance between the trailing edges of the droplet in two consecutive exposures 1202a, 1202b is determined. The distance between corresponding edges of the same droplet in two consecutive exposures is the distance that the droplet travels along the imaging channel during the time between two consecutive illumination pulses triggered by two light source drive signals 1222a, 1222b. Since the time interval between the two light source drive signals 1222a, 1222b is known, the velocity of the droplet can be determined as the distance the droplet travels divided by the time interval between the two light source drive signals 1222a, 1222b. Given the velocity of the droplet, the flow rate of the droplet along the imaging channel is obtained.

[0222] In some examples, once a droplet is identified in each exposure, the exposures 1202a, 1202b of the image 1200 are analyzed to determine the size of the droplet. In Figure 23A the example, the diameter D of the droplet is determined as the interval between the leading edge 1204a of the droplet in exposure 1202a and the trailing edge 1206a of the same droplet in exposure 1202a. The volume of the droplet can also be determined based on the determined droplet diameter, e.g., based on the expected or observed shape of the droplet given the geometry of the given channel. An estimate of the total number of droplets generated from a sample of a given starting volume can be determined based on the determined volume of an individual droplet.

[0223] The number density of droplets per unit length of the channel or per unit volume of the second fluid can be determined based on the distance between adjacent droplets, e.g., the spacing between the trailing edge of a first droplet and the leading edge of the next successive droplet in the channel.

[0224] The closed-loop feedback system controls the operating parameters of the MOS generation system 200 based on droplet characteristics determined from image analysis. Specifically, the computing device communicates with a controller that controls the flow of the first fluid, the second fluid, or both to control the flow rate of the first fluid, the second fluid, or both. For example, to adjust the size of the droplets generated at the junction 311 (see Figure 4 ), the flow rate of the droplets through the droplet generation chamber 312, or both. In a specific example, the flow rate of the first fluid is adjusted to achieve droplets of a target size, and the flow rate of the second fluid is adjusted such that the generated droplets flow at a target flow rate. For example, if image analysis shows that droplets larger than the target size are being generated, the computing device communicates with the controller that controls the flow rate of the first fluid to decrease the flow rate of the first fluid. If image analysis shows that the flow rate of the droplets along the droplet generation chamber 312 is greater than the target flow rate, the computing device communicates with the controller that controls the flow rate of the second fluid to decrease the flow rate of the second fluid or both.

[0225] In one specific implementation, the controller that controls the flow rate of each of the first fluid and the second fluid is implemented as a PID controller to facilitate stable control. This specific implementation can account for the interaction between the parameters of the system, e.g., to provide independent control of the oil flow rate and the sample pressure, regardless of the dependency between these two parameters. For example, one PID controller accounts for the speed and the oil flow rate, and another PID controller accounts for the droplet size and the sample pressure, thereby achieving stable control of both the droplet size and the speed.

[0226] The camera of the closed-loop feedback system can operate at a frame rate between 1 Hz and 30 Hz. In some examples, the frame rate of the camera is faster than the cycle rate of the flow of the first and second fluids. In this way, dynamic adjustments can be made in real time to maintain the generation of droplets of a target size at a target flow rate. In a specific example, the flow of the first and second fluids is controlled by a pressure with a cycle rate between 2 Hz and 5 Hz. In this example, a camera with a frame rate of 15 Hz, 30 Hz, or 40 Hz can be used to capture multiple images. For example, when collecting multiple image streams (e.g., double-exposure image streams and single-exposure image streams) at a frame rate of 30 - 40 Hz, the double-exposure image stream for analysis can be collected at 15 - 20 Hz, and the single-exposure image stream for display to the user can be interleaved with the double-exposure image stream and also collected at 15 - 20 Hz.

[0227] In some instances, a closed-loop feedback system can determine when a performance issue occurs in the MOS generation system 200. For example, when the supply of the first fluid is depleted, droplets will not be formed, and thus droplets will not be recognized in the image of the droplet generation chamber 312. To prevent air bubbles from circulating from the empty reservoir for the first fluid (e.g., reservoir 220, see Figures 3A to 3B ), the closed-loop feedback system can control the flow regulator for the fluid flow from reservoir 220 to close the flow from reservoir 220 while maintaining the flow of the second fluid from the oil reservoir 700 at a target flow rate. For example, a two-way shut-off valve is positioned between the flow regulator for the reservoir for the first fluid (e.g., syringe pump) and reservoir 220 to prevent the pressure from the second fluid from causing backflow into reservoir 220. The continuous flow of the second fluid ensures that the previously generated droplets will flow through the entire length of the outlet channel 314 at the target flow rate to achieve complete aggregation.

[0228] Referring to Figure 24A , in some instances, the light source 1354 of the imaging system includes a plurality of individual light sources 1356a, 1356b, and each individual light source 1356a, 1356b emits light of a different color. For example, the light source 1356a can be a first LED that emits red light and the light source 1356b can be a second LED that emits green light.

[0229] Figure 24B Shown is a drive signal for an imaging system including Figure 24A a plurality of individual light sources 1356a, 1356b. The camera drive signal 1210 and the integration period 1230 of the camera are as described above with reference to Figure 23B . In this instance, the controller sends a light source drive signal 1320 to the light sources. The light source drive signal 1320 includes a first light source drive signal 1322a sent to the first individual light source 1356a, which causes the first light source to emit light of its color (e.g., red light). The light source drive signal 1320 also includes a second light source drive signal 1322b sent to the second individual light source 1356b, which causes the second light source to emit light of its color (e.g., green light). In this way, two consecutive illumination pulses of two different colors are generated.

[0230] In the two exposures resulting from two consecutive illumination pulses of two different colors within a single frame, the first exposure is of the first color (e.g., red), and the second exposure is of the second color (e.g., green). This color difference facilitates the identification of droplets in each exposure; the red droplets belong to the first exposure, and the green droplets belong to the second exposure.

[0231] Referring to Figure 25, in an exemplary process for generating MOS, a first fluid flows through a first microfluidic channel (1400) of a MOS generation system. The first fluid contains biomaterials (e.g., biomaterials derived from a patient) and unpolymerized matrix materials. A second fluid flows through one or more second microfluidic channels (1402) of the microfluidic device. The first fluid and the second fluid are immiscible.

[0232] The first and second fluids are combined at a junction where the first microfluidic channel meets the second microfluidic channel, thereby forming droplets (1404) of the first fluid dispersed in the second fluid. The droplets of the first fluid in the second fluid flow from the junction into a third microfluidic channel.

[0233] The shutter of the imaging device is controlled to open (1406). The light source irradiates the region of the third microfluidic channel with a plurality of consecutive irradiation pulses while the shutter remains open (1408), such that multiple exposures of a single droplet of the first fluid in the third microfluidic channel are captured in a single image captured by the imaging device (1410).

[0234] The multiple exposures of the single droplet are analyzed to identify the droplet in each image, or to identify the leading edge, trailing edge, or both of the droplet in each exposure (1412). In some instances, frequency domain techniques or machine vision techniques are used, or image analysis is performed by creating a best-fit circle. The multiple exposures of the single droplet are analyzed to determine droplet characteristics (1414), such as the size of the droplet, the velocity of the droplet, the spacing between adjacent droplets, the number density of the droplets, an estimate of the total number of droplets generated in the system, or the droplet generation rate (e.g., the number of droplets per second).

[0235] Based on the determined characteristics of the droplets, the flow rate of the first fluid in the first microfluidic channel, the flow rate of the second fluid in one or more second microfluidic channels, or both are controlled (1416). For example, the flow rate of the first fluid can be controlled to obtain droplets of a target size, and the flow rate of the second fluid can be controlled to obtain droplets flowing at a target velocity.

[0236] The droplets are exposed to a stimulus sufficient to polymerize the matrix material in the droplets, and the polymerized droplets are used, for example, for determining patient-specific therapies (1420).

[0237] Measurement and Control of Droplet Size

[0238] It is possible to process images of droplets generated by the MOS generation system and method described above to identify any imaged droplets and estimate the size of the imaged droplets. Then, based on the estimated size of the imaged droplets, the control system is configured to adjust at least one fluid flow rate within the device (e.g., by adjusting the pressure of at least one fluid flow within the device) to actively control the size of subsequently generated droplets. These methods of measuring and controlling droplet size can be used in combination with aspects of the MOS generation system and method described above, including in combination with aspects of the closed-loop feedback method described above.

[0239] Various specific implementations of the droplet size measurement and control methods described herein can provide one or more of the following advantages.

[0240] Implementing a feedback system based on the size of generated droplets can provide a robust microfluidic system that adapts to various operating conditions. For example, even when there are variations in the purity of the input material, the viscosity of the input material, the temperature of the device, etc., the techniques described herein can ensure consistent generation of droplets within a target size range.

[0241] The techniques described herein can also have the advantage of more quickly identifying the appropriate pressure applied to the fluid input reservoir (sometimes referred to herein as the "holding chamber") to affect the rate of fluid flow within the device and generate droplets of a target size. Some devices rely on a careful and time-consuming experimental process to determine the appropriate pressure for a very specific set of operating conditions. Different from such devices, the techniques described herein enable real-time automatic pressure adjustment to quickly converge to (e.g., on the order of seconds) the appropriate pressure for a specific set of operating conditions. For simplicity, this specification describes exemplary specific implementations of the invention in which the fluid flow rate is adjusted by changing the pressure applied to the fluid input reservoir. However, it should be understood that the disclosed techniques are equally applicable to other suitable methods of adjusting the fluid flow rate, including, for example, adjusting the programmable pump flow rate applied to the fluid.

[0242] Another advantage of the techniques described herein is their ability to achieve sub-pixel radial resolution for circle detection, which is used to identify and estimate the size of imaged droplets. As used herein, the term "circle detection" refers to identifying circular or near-circular shapes in an image (e.g., based on detected edges in the image) and fitting a circle to represent such shapes (e.g., using a polynomial representation). For example, even if a droplet does not appear perfectly circular in the image, the circle detection process can still detect the near-circular shape of the droplet and generate a circular representation corresponding to the droplet. In some cases, shapes other than circles can be used to represent near-circular shapes. For example, in some embodiments, polygons with 10 sides, 50 sides, 100 sides, 500 sides, etc. can be used to represent near-circular shapes. In some embodiments, in addition to or as an alternative to a polynomial representation, circular or near-circular shapes can be represented by their spectral decomposition, their angular symmetry, etc. Compared to other shape detection algorithms that implement single-channel methods, the techniques described herein use a multi-channel method, which produces a circular representation with sub-pixel radial resolution. This advantage is particularly important for microfluidic applications, where imaged droplets (sometimes referred to herein simply as "droplets") can sometimes have a radius in the range of only 5 - 20 pixels (although, in other instances, the radius can have a length of up to 50 pixels, up to 100 pixels, up to 250 pixels, etc.).

[0243] In some embodiments, the techniques described herein provide a variety of filtering criteria that can potentially avoid undesirable results when processing real-world images of droplets in a microfluidic device. For example, the filtering criteria implemented by the techniques disclosed herein can prevent false positive detections of circles in the space between droplets, prevent false positive detections of intersecting circles, exclude detections of any circles that are too close to the walls of the droplet generation device, and exclude detections of any circles that have abnormal signals within the perimeter of the circle.

[0244] In addition, in some embodiments, the techniques disclosed herein can be used not only to identify droplets but also to identify bubbles generated by the device. Such detection of bubbles can indicate a malfunction condition or near depletion of the input material and can be used to control the flow rate of at least one fluid within the device (e.g., by controlling the pressure applied to a fluid reservoir that affects the fluid flow rate). For example, in response to the identification of a bubble in the device, the pressure of the fluid flow can be reduced to avoid a continuous air flow into the channels of the device.

[0245] Typically, in some applications, it may be necessary to control the size of droplets generated by a device. For example, with reference to MOS generation, it may be necessary to ensure that the generated MOS has a specific size (e.g., 220 microns to 300 microns). If the MOS is too small, there may be a lack of space for cell growth and division, and there may be a lack of nutrient medium within the MOS. The MOS may also shrink over time, and growing cell clusters may cause too small droplets to rupture. In turn, such rupture may interfere with tracking individual cells within the MOS to identify individual cell responses. On the other hand, if the generated MOS is too large, the nutrient medium within the MOS may be excessive and lack cells, and the diffusion of drugs to the cells may be very slow. An overly large MOS may also get stuck within the channels of a device (e.g., a microfluidic chip), for example, the device may have a channel width of approximately 300 microns at certain locations.

[0246] To achieve a target droplet size, existing devices for droplet generation require specific conditions to operate reliably. As described in more detail below, the droplet size is sensitive to the flow rate of one or more fluid flows within the droplet generation device (e.g., the ratio of the flow rates of two fluid flows). Therefore, it is necessary to control the flow rate, which may be affected by various factors, including for example the viscosity of the fluid, the surface tension of the fluid, the pressure applied to the fluid, and one or more dimensions of the fluid flow path. In some specific implementations, a positive displacement pump (e.g., a syringe pump) may be used to directly control the flow rate. In other specific implementations, such as in the examples described herein, the flow rate of each fluid can be controlled by adjusting the pressure applied to the corresponding input fluid reservoir that feeds into the fluid flow path within the droplet generation device. Thus, while the current application describes examples of controlling fluid flow rates by applying pressure to input fluid reservoirs, the techniques described herein are equally applicable to other flow rate control mechanisms.

[0247] In the example of controlling the flow rate by adjusting the pressure applied to the input fluid reservoir, the appropriate pressure applied to each reservoir can be highly dependent on various operating conditions. For example, existing devices may require consistent input material purity, consistent input material viscosity, precise temperature conditions, etc., to ensure consistent and desired droplet sizes. The appropriate pressure applied under a very specific set of conditions is typically determined through a time-consuming experimental process and may make the droplet generation process vulnerable to minor variations in operating conditions. Therefore, consistently and robustly generating droplets of a target size is a formidable technical challenge.

[0248] Figure 26AAn example of an apparatus 2100 for forming droplets (e.g., MOS) is shown. The apparatus 2100 can be a specific implementation of aspects of the MOS generation system 200 discussed above. The apparatus 2100 includes an input for an unpolymerized mixture of input cells and fluid matrix material (which have been combined), or can receive cells (e.g., in a holding solution) and fluid matrix material separately. As described above, in some droplet applications, cells may not be included at all.

[0249] In some variations, the apparatus 2100 includes a holding chamber 2106 for holding the unpolymerized mixture and / or a holding chamber (not shown) for holding a cell sample (e.g., tumor cells) and holding the fluid matrix material. For example, the holding chamber 2106 can be implemented as the reservoir 220 discussed above. Any or all of these holding chambers can be pressurized to control and / or accelerate the flow of fluid out of the chamber and into the apparatus. The apparatus can receive the unpolymerized mixture or can receive the components and mix them. In some variations, the apparatus can control the concentration of cells in the unpolymerized mixture and can dilute the mixture (e.g., by adding additional fluid matrix material to achieve a desired density. For example, the apparatus 2100 can include one or more sensors 2128 for reading the density of cells (e.g., optical density) in the unpolymerized mixture. The sensor can also be coupled to a controller 2124, which can automatically or semi-automatically (e.g., by indicating to the user) control the dilution of cells in the unpolymerized mixture. The apparatus 2100 can also include a port for receiving the unpolymerized mixture. The port can include a valve or can be coupled to a valve and the valve can be controlled by the controller 2124 (or a separate controller).

[0250] The apparatus 2100 can include another holding chamber 2108 and / or port for holding and / or receiving an immiscible or hydrophobic fluid such as oil. For example, the holding chamber 2108 can be implemented as the oil reservoir 700 described above. In some variations, the immiscible fluid can be held in a pressurized chamber such that the flow rate of the immiscible fluid within the apparatus 2100 can be controlled. Any of the pressurized chambers in the pressurized chambers can be controlled by the controller 2124, which can use one or more pumps 2126 to control the pressure and thus control the flow through the apparatus 2100. One or more pressure and / or flow sensors (e.g., sensor 2128) can be included in the system to monitor the flow through the apparatus.

[0251] In Figure 26A , the entire apparatus 2100 can be encapsulated in a housing or a portion of the apparatus 2104 can be encapsulated in a housing. In some variations, the housing can include one or more openings or access portions on the apparatus 2100, e.g., for adding immiscible fluid and / or unpolymerized mixture to the chambers 2106, 2108.

[0252] As described, the apparatus 2100 may also include one or more sensors 2128 for monitoring all or critical portions of the droplet generation process. In some variations, the sensors may include optical sensors, mechanical sensors, voltage and / or resistance (or capacitance, or inductance) sensors, force sensors, temperature sensors, mass air flow sensors, mass liquid flow sensors, pressure sensors, and the like. These sensors may be used to monitor the ongoing operation of the components, including the formation of droplets. For example, the apparatus 2100 may include an optical sensor 2132 among the sensors 2128. The optical sensor 2132 may be a camera configured to capture images (e.g., photos or videos) of the droplets generated by the apparatus 2100 and, as described below, may be used to control the size of the droplets generated by the apparatus 2100. For example, the optical sensor 2132 may be part of the closed-loop feedback system discussed above. The apparatus 2100 may also include one or more thermal / temperature regulators 2118 for controlling the temperature of one or both of the immiscible fluid and / or the unpolymerized mixture (and / or the fluid matrix material). In some embodiments, the monitoring sensor 2128 and / or the optical sensor 2132 may be external to the apparatus 2100.

[0253] The apparatus 2100 may also include one or more droplet formation assemblies 2120 that may be monitored (e.g., using one or more sensors 2128), as will be described in further detail below. The droplet formation assembly may include a dispenser (e.g., a droplet dispenser) 2122 or may be coupled to the dispenser. The dispenser may dispense into, for example, one or more collection tubes or a microplate 2116.

[0254] Generally, the droplet formation assembly 2120 may include one or more microfluidic chips (e.g., Figure 26B the microfluidic chip 2130 shown or Figure 2 the microfluidic chip 210) or structures that form and control the flow of the unpolymerized mixture and form the actual droplets. Figure 26B An example of a microfluidic chip 2130 for forming droplets is shown; other examples of microfluidic chips were described and discussed above, for example, in connection with the microfluidic chip 210. In Figure 26B the figure, the chip 2130 includes a pair of parallel structures for forming droplets (e.g., MOS). In other embodiments, the microfluidic chip may include additional structures (e.g., 3 structures, 4 structures, 5 structures, etc.).

[0255] Figure 26CThe droplet formation region of the microfluidic chip 2130 for forming MOS is shown, including unpolymerized channel outlets 2141, 2143' that lead to the outlet channel 2139 and intersect with the immiscible fluid outlet or channel 2143. In some specific embodiments, the outlet channel 2139 can be wider than the unpolymerized channel outlet 2141 to produce a reduction in flow and backpressure, thereby facilitating droplet formation. In some specific embodiments, the junction region 2137 can be configured as a "+" junction, where the immiscible fluid channels 2143, 2143' intersect with the unpolymerized channel outlet 2141 and the outlet channel 2139 to form four right angles. In some specific embodiments, the intersection region 2137 can be configured such that a single immiscible fluid channel (e.g., immiscible fluid channel 2143) enters the straight sample channel (e.g., unpolymerized channel outlet 2141) at a right angle. In some variations, the input from the immiscible fluid channel can be configured to intersect the unpolymerized material at a non-vertical angle. In Figure 26C As shown for all dimensions in all the figures in this specification, the dimensions shown are merely exemplary and are not intended to be limiting unless otherwise stated. Other specific embodiments of the droplet formation region have been discussed above in connection with the microfluidic chip 210.

[0256] In Figure 26B the microfluidic chip 2130 includes an inlet (input port) 2133 for immiscible fluid to enter the chip (e.g., from the Figure 26A inlet port or storage chamber shown). A second inlet port 2135 for the chip can be configured to receive the unpolymerized material and convey it along a semi-curved path to the junction region. Similarly, the inlet port for the immiscible fluid can be firmly coupled to the outlet from the immiscible fluid chamber or inlet, as described above.

[0257] The inlet port 2135 for the unpolymerized material to enter the chip 2130 can be coupled by connecting the inlet 2135 to the delivery path of the junction region 2137 (as Figure 26C shown). Similarly, the inlet 2133 for the immiscible fluid can connect two (or more) connecting channels 2143, 2143' to the junction region 2137. The channels leaving the junction region 2137 can convey the formed droplets (in the immiscible fluid) down the channel to the outlet 2131, which can be connected to a dispenser (e.g., Figure 26A the dispenser 2122 shown) for dispensing the droplets into one or more chambers for culturing and / or assay (e.g., the Figure 26A porous plate 2116 shown).

[0258] In Figure 26B and 26CIn the example shown, the formed droplets (e.g., unpolymerized MOS) can be conveyed downward along a long temperature-controlled microfluidic environment (e.g., channel 2139) before being dispensed from the device.

[0259] In Figure 26B and 26C the joining region 2137 is shaped as described above such that the channel 2141 carrying the unpolymerized mixture intersects one or more (e.g., two) channels 2143, 2143' carrying a hydrophobic fluid (e.g., oil) immiscible with the unpolymerized mixture. When the unpolymerized mixture is pressured to flow out of the first channel 2141 at a first rate, the immiscible fluid flowing in the intersecting channels 2143, 2143' allows a predetermined amount of the unpolymerized mixture to pass through and then pinches it off to form droplets that are delivered to the outlet channel 2139. Thus, the size of the droplets depends on and is highly sensitive to variations in the pressures (and corresponding flow rates) of both the unpolymerized mixture and the immiscible fluid.

[0260] In some variations, the chopping (e.g., dissociation) of a clinical (e.g., biopsy or resection) sample of dissociated cells (such as having a diameter <1 mm) can be mixed with a temperature-sensitive gel (i.e., MATRIGEL, mixed at 4 °C) to form an unpolymerized mixture. This unpolymerized mixture can be placed in the device 100 that produces droplets of a specific volume and material composition, and the dissociated cells (e.g., tumor cells) can be partitioned into these droplets. In some cases, the diameter of each droplet can be about 260 microns (e.g., 220 microns to 300 microns), and can contain tumor spheres including 2 - 100 tumor cells (e.g., 2 - 15 tumor cells, 15 - 50 tumor cells, 50 - 100 tumor cells, etc.). The gel in the unpolymerized material can be cured when heated (e.g., at 37 degrees Celsius) to form polymerized MOS. In some variations, the method can be used to produce more than 10,000 (e.g., more than 20,000, more than 30,000, more than 40,000, more than 50,000, more than 60,000, more than 70,000, more than 80,000, more than 90,000, more than 100,000) MOS from a single biopsy. These MOS are compatible with traditional 3D cell culture techniques and can be used to observe the response of cells to various stimuli by assay, as previously described.

[0261] Figures 27A to 27E is an example of images 2200A to 2200E of a droplet generation device (such as Figures 26A to 26C the device 2100) in operation. Each of the images 2200A to 2200E shows an outlet channel 2239 providing an outlet from the joining region 2237. The representations of the outlet channel 2239 and the joining region 2237 can be respectively associated with those regarding Figure 26Band 26C The outlet channel 2139 and the junction region 2137 of the microfluidic chip 2130 described correspond. For example, the outlet channel 2239 and the junction region 2237 can correspond to Figure 4 the droplet generation chamber 312 and the junction 311, respectively. Images are captured by a camera (corresponding to the optical sensor 2132 of the device 2100) to monitor the droplet generation process of the device. Each of the images 2200A to 2200E includes an overlay representing the output of an image processing module, which can be implemented by a controller of the droplet generation device (e.g., Figure 1 the controller 124 of the device 100 in A). The following refers to Figure 28 429 and 32 to describe the image processing module and the steps it performs.

[0262] Referring to Figures 27A to 27E the overlay 2250 corresponds to the detected wall of the imaging outlet channel 2239 (sometimes simply referred to herein as "outlet channel 2239") in the widened region (e.g., chamber) of the outlet channel 2239. The overlay 2252 corresponds to the edge detected by the image processing module within the widened region (e.g., by an edge detection algorithm such as Canny edge detection, Gaussian edge detection, threshold-based edge detection, etc.). The overlay 2254 corresponds to the circle detected by the image processing module based on the detected edge (e.g., using the Hough transform).

[0263] In some specific embodiments, it may be beneficial to capture an image of the widened region and process the image content within the widened region because when the droplets generated by the device are in this region, these droplets are less likely to interact with the sidewalls of the outlet channel. Therefore, it is useful to capture an image of the generated droplets in the widened region to estimate their size without any interaction with the sidewalls of the outlet channel 2239.

[0264] In some specific embodiments, it may be useful to capture images of the generated droplets in other parts of the capture system (e.g., the narrower part 2260 of the outlet channel 2239). Capturing an image of the narrower part 2260 of the outlet channel 2239 may be beneficial in some cases because the droplets in this part may be arranged in a single column and separated from each other (e.g., by immiscible fluids). In some specific embodiments, the narrower part 2260 can also be deeper than the widened region of the outlet channel 2239 (e.g., a depth of 300 microns compared to a depth of 200 microns), enabling the measurement of droplet size without compression by the top or bottom wall of the outlet channel 2239. In some specific embodiments, the narrower part 2260 can be wide enough such that the droplets within the narrower part 2260 are not compressed by the sidewalls of the narrower part 260.

[0265] InFigures 27A to 27B In this case, the overlay 2252 indicates that there are a number of droplets in the widened region of the exit channel 2239. However, in each of Images 2200A and 2200B, only two circles (circles 2202, 2204 in Image 2200A; circles 2206, 2208 in Image 2200B) are detected. In some embodiments, this behavior may be desirable because false positives may be more harmful than false negatives for estimating the size of the generated droplets. Since continuously generated droplets often may have similar sizes and because the droplets are produced quickly (e.g., 40 droplets per second in some embodiments), it may be sufficient to detect only a few representative droplets (e.g., less than 5) in the exit channel 2239 to monitor and control the droplet size. Generally, detecting circles that do not actually correspond to droplets can be harmful because the sizes of these false positives may bias the estimate of the true droplet size in the exit channel 2239.

[0266] Still referring to Figure 27A and 27B , it is also easy to observe that circles 2202, 2204 are larger than circles 2206, 2208 in Image 2200B. This demonstrates the ability of the image processing module to accurately detect circles (and corresponding droplets) of various sizes.

[0267] Similar to Figure 27A and 27B , Figure 27C shows Image 2200C with the detected edges (overlay 2254), which indicates the presence of various spherical objects in the widened region of the exit channel 2239. In Figure 27C , the image processing module detects three circles 2210 based on these detected edges. However, similar to Figure 27A and 27BDifferently, the detected circle 2210 in Image 2200C represents a bubble rather than a droplet. Imaging bubbles (e.g., circle 2210) can be distinguished from droplets (e.g., the detected circles 2202, 2204, 2206, 2208) based on the darkness of the imaging edges. For example, the edge corresponding to the detected circle 2210 in Image 2200C is much darker than the edges corresponding to the detected circles 2202, 2204, 2206, 2208 in Images 2200A and 2200B. This is because the difference between the refractive indices of air and the immiscible fluid (e.g., oil) is greater than the difference between the refractive indices of the unpolymerized mixture and the immiscible fluid. Thus, in an exemplary embodiment, imaging bubbles can be distinguished from droplets by estimating, for each detected circle, a value that reflects the intensity of the perimeter of the detected circle (e.g., the ratio of the average intensity inside the perimeter to the average intensity outside the perimeter). This value can in turn be used to classify the detected circles as imaging bubbles or droplets using one or more classification techniques (e.g., classification thresholds, naive Bayes inference, machine learning-based classifiers, etc.).

[0268] In some embodiments, detecting a bubble in the outlet channel 2239 can indicate a fault condition of the device or that the unpolymerized mixture is approaching depletion. Thus, in some embodiments, in response to detecting a bubble in the outlet channel 2239, the pressure of the fluid flow corresponding to the channel (e.g., Figure 26B and 26C the channel 2141 shown in) used to convey the unpolymerized mixture can be reduced to prevent the continued formation of bubbles and / or the inflow of air into the outlet channel. In the case where the pressure is not reduced sufficiently, an undesirable situation (sometimes referred to as a "blowout situation") may occur, in which an air flow enters the outlet channel. Figure 27D The image 2200D shown in depicts an example of a blowout situation, in which an air flow 2212 is forced into the outlet channel 2239.

[0269] Figure 27EImage 2200E is shown, in which no droplets or bubbles are present in the exit channel 2239. Accordingly, no circles are detected in Image 2200E. In some embodiments, a situation like this can indicate a fault condition. For example, Image 2200E may be produced when the pressure of the fluid stream of the unpolymerized mixture is too low or too high to support stable droplet generation. In these cases, the exit channel 2239 may be completely filled with immiscible fluid (e.g., if the pressure of the fluid stream of the unpolymerized mixture is too low) or unpolymerized matrix (if the pressure of the fluid stream of the unpolymerized mixture is too high). In some embodiments, in response to failing to detect any circles within the exit channel 2239, a controller of the device (e.g., controller 2124 of device 2100) may implement a fault recovery routine to restart stable droplet generation. Actions taken by the controller, including the fault recovery routine, are described in more detail herein.

[0270] Figure 28 An exemplary process 2300 for controlling the pressure of a fluid stream in a droplet generation device to control, for example, the size of generated droplets is shown. At least some operations of process 2300 may be performed by a microfluidic system such as device 2100 or a portion thereof (e.g., by controller 2124). In some embodiments, one or more operations of process 2300 may be performed by one or more remote computing systems external to device 2100.

[0271] Operations of process 2300 include obtaining one or more images (2302). For example, the images may be captured by a camera such as optical sensor 2132 of device 2100. These images may correspond to the images 2200A - 2200E described Figures 27A to 27E above. Similar to images 2200A - 2200E, the obtained images may be framed to include a widened region of the exit channel (e.g., exit channel 2139), which may include droplets and / or bubbles in some cases. In some embodiments, after obtaining one or more images (2302), gamma correction may be applied to the one or more images. Gamma correction may convert the pixel array data retrieved from the camera into numbers proportional to the actual photon intensity, e.g., which may optimize the use of bits to account for the non - linear way humans perceive light and color.

[0272] The operations of process 2300 may also include detecting edges in the image (2304) (sometimes after applying gamma correction to one or more images) and detecting circles based on the detected edges (2306). Both of these operations may be performed by an image processing module implemented on a controller of the microfluidic device (e.g., controller 2124 of device 2100). In some embodiments, operations 2304, 2306 may be implemented on a remote computing device external to device 2100. Detecting edges (2304) may include implementing an edge detection algorithm, such as a Canny edge detector, a Gaussian edge detector, a threshold-based edge detector, etc. In some embodiments, detecting edges (2304) may also include using asymmetric filtering to mitigate biases caused by the profiles of the channels of the microfluidic device. For example, in one or more images, the profile of the chamber may at least partially appear as two dark vertical lines in the image (as Figures 27A to 27Eas in []. In this case, by using a Difference of Gaussians kernel along the vertical axis or using the kernel [[0.5, 1, 0.5], [0, -4, 0], [0.5, 1, 0.5]], the filter can be intentionally biased to be more sensitive to vertical changes in pixel intensity. Various filters can be selected and used according to the specific optical and droplet characteristics of the system. One or more shape detection algorithms (such as the Hough transform) can be implemented based on the detected edge detection circle (2306). In some specific implementations, one or more images can be downsized (e.g., via downsampling) before detecting edges (2304) in the image and / or before detecting the edge detection circle (2306) based on the detected edges. For example, one or more images can be downsampled (e.g., digitally) to 1 / 4 to 1 / 2 of the original resolution (e.g., 25% of the original resolution, 30% of the original resolution, 40% of the original resolution, 50% of the original resolution, etc.). This downsampling can have the advantage of increasing the speed of edge detection and / or circle detection. For example, the number of operations involved in performing the Hough transform (an exemplary algorithm that can be used to detect circles) scales with the fourth power of the image size. Therefore, downsampling the image before using the Hough transform to detect circles can save a significant amount of time (e.g., improving the performance of the Hough transform by 5 to 15 times, improving the performance of the entire process (2300) by 2 to 10 times, etc.). For example, in a specific implementation where an original image with a resolution of 100 x 100 pixels is downsampled to an image with a resolution of 50 x 50 pixels before performing the Hough transform, the Hough transform is performed approximately 10 times faster and the process 2300 is performed approximately 6 times faster compared to a specific implementation where no downsampling is performed. In specific implementations where downsampling is performed, the resulting image data can be upsampled after detecting edges (2304) in the image and / or after detecting the edge detection circle (2306) based on the detected edges. For example, the image can be upsampled by a factor in the range of 2 to 4. In some cases, the upsampling factor can be selected to restore the downsampled image to its original size after detecting edges (2304) in the image and / or after detecting the edge detection circle (2306) based on the detected edges.

[0273] If no circle is detected at operation 2306, process 2300 can include generating a signal (2308) that causes an adjustment to the pressure of at least one fluid flow according to a fault recovery routine. For example, one or more images can be similar to Figure 27EThe image 2200E shown, which does not include the detected circles. In some specific embodiments, a controller of the microfluidic device (e.g., controller 2124 of device 2100) can control one or more pumps (e.g., pump 2126) to control the flow rate of the fluid flow within device 2100 (e.g., by controlling the pressure). In some specific embodiments, this fluid flow can correspond to the flow of the unpolymerized mixture through the device. However, in other specific embodiments, the pressures of the flow of the unpolymerized mixture and the flow of the immiscible fluid can both be adjusted.

[0274] The fault recovery routine can include implementing a control scheme for controlling the flow rate of the fluid flow (e.g., by controlling the pressure), which operates without using feedback control. For example, the fault recovery routine can include implementing a simple control scheme that gradually increases the pressure of the fluid flow of the unpolymerized mixture until stable droplet generation begins or until a maximum pressure value is reached. In some specific embodiments, the fault recovery routine can include implementing a simple control scheme that gradually decreases the pressure of the fluid flow of the unpolymerized mixture until stable droplet generation begins or until a minimum pressure value is reached. In some specific embodiments, the fault recovery routine can include implementing a simple control scheme that causes the pressure of the fluid flow of the unpolymerized mixture to continuously oscillate between the minimum pressure value and the maximum pressure value until stable droplet generation begins. Using a simple static controller instead of feedback control in the presence of a fault can prevent the feedback controller from exhibiting undesired or abnormal behavior when no droplets are detected or droplets of extreme sizes are detected.

[0275] Alternatively, if a circle is detected at operation 2306, process 2300 can include determining for each circle whether one or more filtering conditions are met (2310). For example, the obtained image with the detected circle can correspond to image 2200A, 2200B, or 2200C. One or more filtering conditions can include criteria for excluding circles in order to prevent biasing the size estimate with data from false positives or low-quality circles.

[0276] In some specific embodiments, the filtering condition can include an indication of the detected circle based on the detected edges of multiple droplets. For example, this can be caused by erroneously identifying the space between multiple droplets as the detected circle. In some specific embodiments, the indication of the detected circle based on the detected edges of multiple droplets can stem from determining that the detected circle is substantially smaller than one or more other detected circles in the same image. The indication can also stem from determining that the detected circle shares an edge with one or more other detected circles of a substantially larger size.

[0277] In some specific implementations, the filtering condition may include an indication that the detected circle overlaps at least one additional circle within the same image. Although droplets can sometimes be tangent to each other in the outlet channel (e.g., outlet channel 2139), they generally do not overlap. Therefore, the detected circles should not overlap in the imaged outlet channel (e.g., outlet channel 2239) either. Thus, receiving an indication that the detected circle overlaps at least one additional circle may indicate that at least some of the overlapping circles may be false positives.

[0278] In some specific implementations, the filtering condition may include an unexpected detected signal within the perimeter of the detected circle. In some specific implementations, the detected signals (e.g., dark pixels) in one or more images are expected to exist only at the edges corresponding to droplets, bubbles, and / or the microfluidic device itself. In some specific implementations, one may also expect specific types of detected signals (e.g., weak signals or signals of a specific size and / or shape) corresponding to tumor spheres or other cells located within the detected droplets. However, if an unexpected signal different from these expected signals is detected within the perimeter of the detected circle, this may indicate that the detected circle may be a false positive or is abnormal for other reasons. In some specific implementations, the unexpected detected signal may correspond to a detected signal originating within the perimeter of the detected circle that exceeds a threshold signal level.

[0279] In some specific implementations, the filtering condition may include determining that the detected circle is less than a threshold proximity to the imaging wall of the device (sometimes referred to simply as the "wall" herein). Droplets located at the wall of the device may be compressed against the wall and may lose their spherical shape. This may in turn lead to poor performance of the circle detection algorithm and / or result in a biased estimate of the size of the droplet. Additionally, since the walls of the device may appear as high-contrast edges in the image, there is a risk of misidentifying these edges as part of the detected circle, which may lead to false positives. Therefore, it may be beneficial to exclude detected circles that are very close to the wall of the device in some specific implementations.

[0280] If any of the filtering conditions are met for a particular detected circle at operation 2310, process 2300 may include excluding the detected circle (2312). As discussed above, operation 2312 may lead to a more accurate estimate of the droplet size by excluding false positives and low-quality detected circles.

[0281] If no filtering conditions are met at operation 2310, process 2300 can include identifying pixels corresponding to detected edges that are set within a threshold distance of the perimeter of the detected circle (2314), and calculating an average distance from the center of the detected circle to at least a portion of the identified pixels (2316). This calculated average value (which can be a weighted average) can be used as an updated radius of the detected circle. In some embodiments, a metric other than the average value can be calculated, as long as the metric is derived from and represents the distance from the center of the detected circle to at least a portion of the identified pixels. Compared to a single-channel circle detection algorithm that detects circles with a radial resolution of greater than or equal to 1 pixel, this multi-channel method of estimating the size of the detected circle produces sub-pixel radial resolution. This resolution is particularly important for microfluidic applications, where droplets can sometimes have a radius in the range of only 5 - 20 pixels (although, in other instances, the radius can have a length of up to 50 pixels, up to 100 pixels, up to 250 pixels, etc.). Figure 29 A visual representation of the process is provided in Figure 29 and described in further detail below.

[0282] After detecting a high-quality circle and estimating its size with sub-pixel radial resolution, process 2300 can include determining whether the detected circle corresponds to a droplet or a bubble (2318). As described with respect to Figures 27A to 27C previously, a bubble can be distinguished from a droplet based on the darkness of the pixels corresponding to the edge of the detected circle, where a bubble (e.g., detected circle 2210) has a darker edge than a droplet (e.g., detected circles 2202, 2204, 2206, 2208).

[0283] If the detected circle is a bubble, process 2300 can include reducing the pressure of at least one fluid flow (2320). For example, operation 2320 can include reducing the pressure (and thus the flow rate) of the fluid flow corresponding to the channel (e.g., Figure 26B and 26C channel 1241 shown in Figure 26B ) used to deliver the unpolymerized mixture. In some embodiments, the fluid flow can be stopped completely and / or the power supply to the droplet generation device can be turned off. As previously described, this can prevent the continued formation of bubbles and / or the inflow of air into the outlet channel (e.g., outlet channel 2139) of the device. Operation 2320 can also prevent the loss or waste of the unpolymerized mixture (e.g., the sample) under operating conditions where droplets are not being generated correctly. Operations 2300 and 2320 can further provide the advantage of automatically detecting when a sample has been detected (resulting in bubble formation), which can eliminate the need for the droplet generation device to store and track the remaining available sample volume.

[0284] If the detected circle is a droplet, process 2300 may include determining whether the standard deviation (or any other measure of dispersion) of the estimated droplet size in the image is below a threshold (2322). In some embodiments, a large standard deviation of the estimated droplet size in the image may indicate a fault condition, as continuously generated droplets should generally not vary significantly in size. Thus, if the standard deviation is above the threshold, process 2300 may include generating a signal that causes an adjustment to the pressure (and flow rate) of at least one fluid stream according to a fault recovery routine (2324). The fault recovery routine may be substantially similar to the exemplary fault recovery routine described previously above.

[0285] In some embodiments, if the estimated size of the droplets identified in a single image varies by more than a threshold amount, the maximum estimated size may be considered to be the most indicative of the actual droplet size. For example, the smaller estimated sizes may be discarded, or a weighted average of all the estimated sizes may be taken, where a smaller weight is applied to the smaller estimated sizes compared to the larger estimated sizes. This can prevent false positive detections of small droplets, which may occur more frequently for small droplets than for large droplets due to noise and / or other factors.

[0286] If the standard deviation (or other measure of dispersion) of the estimated droplet size in the image is below the threshold, process 2300 may include comparing the estimated droplet size to a target size (2326). For example, the target size of the droplets may be between 220 microns and 300 microns. In some embodiments, the target size may be manually adjusted by a user of the device. The comparison of the estimated droplet size to the target size may be used to calculate an error signal.

[0287] The operation of process 2300 may also include using feedback control to generate a signal (2328) that causes an adjustment to the pressure (and flow rate) of at least one fluid stream. For example, as described above, a controller of the device (e.g., controller 2124 of device 2100) may be used to adjust the pressure to control one or more pumps (e.g., pump 2126) to control the pressure of the fluid stream within device 2100. The fluid stream may correspond to the flow of the unpolymerized mixture through the device (e.g., channel 2141). Feedback control may be implemented using an error signal calculated based on comparing the estimated droplet size with the target size at operation 2326. In some cases, the error signal or the estimated droplet size may be averaged over multiple previous images (e.g., 3 images, 5 images, 10 images, 20 images, etc.) to reduce noise. Feedback control may include proportional control, integral control, derivative control, or any combination of the above. We use the term "proportional control" broadly to include control system techniques based on a response proportional to the error signal. The error signal may be the difference between the desired process value (or setpoint) and the current value of the controlled process variable (e.g., the size of the detected droplet). We use the term "integral control" broadly to include control system techniques based on a response proportional to the integral (e.g., time integral) of the error signal. We use the term "derivative control" broadly to include control system techniques based on a response proportional to the derivative (e.g., with respect to time) of the error signal.

[0288] Once feedback control is initiated, it may continue until a bubble is detected or a fault condition is identified (e.g., no circle is detected in the captured image or the standard deviation of the estimated droplet size in the image exceeds a threshold). To achieve this continuous control, process 2300 may be repeated for multiple images captured by a camera of the microfluidic device (e.g., optical sensor 2132). For example, in some embodiments, the multiple images may be frames from a single captured video.

[0289] Figure 29 The process for estimating the size of a droplet is shown in more detail and corresponds to operations 2302, 2304, 2306, 2310, 2312, 2314, 2316 of process 2300 as Figure 28 shown. All of these operations may be performed by an image processing module (e.g., the image processing module of the microfluidic device) and may be implemented on a controller (e.g., controller 2124).

[0290] The imaging object 2402 represents an illustration of a magnified original image focused on a single droplet (e.g., captured at operation 2302 of process 2300). At operation 2404, edge detection (corresponding to operation 2304 of process 2300) can be implemented to generate the detected edges 2406 corresponding to the imaging object 2402. The imaging object 2402 can also be enhanced (at operation 2416) to produce an enhanced object 2418. For example, conventional feature enhancement or edge sharpening techniques (such as an edge sharpening filter or a "difference of Gaussians" process) can be used to enhance the imaging object 2402 to increase the visibility of the edges of the imaging object. In one example, the "difference of Gaussians" process can include computing the difference between two Gaussian blurred versions of the imaging object (e.g., a first version blurred using a Gaussian function with a standard deviation of 2 pixels and a second version blurred using a Gaussian function with a standard deviation of 1 pixel, but other parameter values are possible and may be preferred depending on the hardware and lighting conditions used to acquire the image). In this particular example, the resulting image can retain visual (e.g., spatial) information (such as the edges of the imaging object) having a frequency range between approximately 1 pixel and 3 pixels, while blurring or removing other features.

[0291] The detected edges 2406 can be used as an input to a circle detection process (at operation 2408) to produce the detected circle 2410 having a radius r. Operation 2408 directly corresponds to operation 2306 of process 2300 and can be implemented similarly using the Hough transform. In some specific implementations, the detected circle 2410 can have a radial resolution greater than or equal to 1 pixel.

[0292] As described above with respect to Figure 28 In some specific implementations, the original image including the imaging object 2402 can be downsampled (e.g., via subsampling) before edge detection (operation 2404) or before circle detection (operation 2408). For example, one or more images can be downsampled (e.g., digitally) to 1 / 4 to 1 / 2 of the original resolution (e.g., 25% of the original resolution, 30% of the original resolution, 40% of the original resolution, 50% of the original resolution, etc.). Such downsampling can have the advantage of increasing the speed of edge detection and / or circle detection. In specific implementations where downsampling is performed, the resulting image data can be upsampled after edge detection (operation 2404) and / or after circle detection (operation 2408). For example, the image can be upsampled by a factor in the range of 2 to 4. In some cases, the upsampling factor can be selected to restore the downsampled image to its original size.

[0293] At decision point 2412 (corresponding to operation 2310 of process 2300), the image processing module can determine whether one or more filtering conditions are met. As previously described with respect to Figure 28 operation 2312, if the filtering conditions are met, the detected circle 2410 (2414) can be excluded from the droplet size estimate. However, if the filtering conditions are not met, the detected circle 2410 can be processed jointly with the enhanced object 2418 to calculate a representative radius (2420) based on all pixels in the enhanced object 2418 that have a radius between r + d and r - d, where d represents a threshold distance from the perimeter of the detected circle 2410. In some embodiments, the representative radius calculation can be based on only a portion of these pixels. Operation 2420 corresponds to operations 2314, 2316 of process 2300 and has similar advantages as those previously described for generating sub-pixel radial resolution. The representative radius calculated at operation 2420 can be an average of the distances, a weighted average of the distances, or another metric that derives from and represents the distance from the center of the detected circle to at least a portion of the identified pixels. For example, the representative radius can be calculated using the following formula:

[0294]

[0295] where w i is a weight value reflecting the intensity value of pixel i, r i reflects the distance from the center of the detected circle to pixel i, and where n represents the total number of pixels included in the calculation. Although various alternative metrics can be used, the representative radius calculation described by the above formula can have the advantage of applying a higher weight to the radii corresponding to pixels with greater intensity. Then, the representative radius calculated from operation 2420 can be used to update the radius of the detected circle 2410 for further processing (e.g., as described with respect to process 2300).

[0296] Now referring to Figure 30 and 31 , experimental data demonstrating the capabilities and advantages of the techniques described herein are presented.

[0297] Figure 30Figure 2500 from the experiment is shown, where the user manually sets the target size or "setpoint size" of the droplets generated by the microfluidic device, increases the target size at approximately 180 seconds, and decreases the target size at approximately 260 seconds. However, it is noted that in some embodiments, the setpoint can be automatically set (and changed) by a computer system, for example, based on one or more characteristics of the sample, one or more characteristics of the droplet generation device, etc. The setpoint size (corresponding to the target radius of the droplet) is shown by trace 2502 and is represented in pixels. Trace 2504 corresponds to the estimated droplet size of each captured image (e.g., each frame of the video captured by the camera of the microfluidic device) and is also represented in pixels. Trace 2506 is the running average of trace 2504 to reduce the influence of noise and is also represented in pixels. Trace 2508 shows the pressure applied to the holding chamber for the unpolymerized mixture to control its flow rate. The pressure is directly controlled by the controller of the microfluidic device and is measured in millibars. Trace 2510 shows the pressure applied to the holding chamber for the immiscible fluid (held constant in this experiment) and is measured in millibars. Trace 2512 shows the count of the circles detected in each captured image.

[0298] For the first 50 seconds, stable droplet generation has not yet started, which is evident from the many image frames where the count (trace 2512) and size (trace 2504) are zero. As described above, in response to the failure to detect droplets within the first 50 seconds, the pressure applied to the unpolymerized mixture flow is gradually increased to a maximum pressure of 800 millibars and held constant until stable droplet generation begins. Once stable droplet generation starts (at approximately 50 seconds), the feedback control of the pressure for the unpolymerized mixture flow (trace 2508) successfully enables the estimated size traces (trace 2504 and trace 2506) to track the setpoint size (trace 2502). Thus, figure 2500 demonstrates the successful control of pressure to control the fluid flow rate within the microfluidic device and thereby control the size of the generated droplets.

[0299] Figure 31Figure 2600 from a second experiment is shown, where the user manually sets and changes the target size or "setpoint size" of the droplets generated by the microfluidic device over time. The traces in Figure 2600 are similar to those in Figure 2500. The setpoint size (corresponding to the target radius of the droplets) is shown by trace 2602 and is represented in pixels. Trace 2604 corresponds to the estimated droplet size for each captured image (e.g., each frame of the video captured by the camera of the microfluidic device) and is also represented in pixels. Trace 2606 is a running average of trace 2604 to reduce the effect of noise and is also represented in pixels. Trace 2608 shows the pressure applied to the holding chamber for the unpolymerized mixture to control its flow rate. The pressure is directly controlled by the controller of the microfluidic device and is measured in millibars. Trace 2610 shows the pressure applied to the holding chamber for the immiscible fluid (held constant in this experiment) and is measured in millibars. Trace 2612 shows the count of the circles detected in each captured image.

[0300] Similar to Figure 2500, the overall trend of the estimated size (trace 2606) tracks the fluctuations of the setpoint size (trace 2602). However, in some cases, a fault condition 2620 occurs where no droplets are detected, and the count trace (trace 2612) and the estimated size trace (trace 2604) correspondingly drop to zero. Although this behavior is not ideal, the experimental data shown in Figure 2600 demonstrates the ability of the techniques described herein to recover from these fault conditions 2620. As described above regarding Figure 28 When each fault condition 2620 is identified, the controller of the microfluidic device replaces the use of pressure feedback control with a fault recovery routine involving a simple pressure controller that gradually increases, decreases, or maintains the pressure applied to generate the unpolymerized mixture flow (trace 2608) until stable droplet generation is restored. In each instance of the fault condition 2620, droplet generation can be successfully restored without further user intervention.

[0301] Although the experiment shown regarding Figures 30 to 31 demonstrates active control of the pressure applied to only a single fluid flow (i.e., the flow of the unpolymerized mixture), the present disclosure is not intended to be limiting. As previously described, the size of the droplets can depend on the pressure (and thus the flow rate) of both the unpolymerized mixture flow and the immiscible fluid flow. Therefore, in some embodiments, in addition to or alternatively to controlling the pressure (and flow rate) of the unpolymerized mixture flow, the pressure (and flow rate) of the immiscible fluid flow can also be controlled. For example, in some embodiments, simultaneously controlling the pressure of the two fluid flows in the microfluidic device can enable additional control of the droplet generation process, including controlling the generation speed and the size of the generated droplets.

[0302] Figure 32 An exemplary process 2700 for controlling the size of droplets generated by a device (e.g., a microfluidic device such as device 2100) is shown. The operations of process 2700 may be performed by a microfluidic system such as device 2100 or a portion thereof (e.g., controller 2124). In some embodiments, one or more operations of process 2700 may be performed by one or more remote computing systems external to device 2100.

[0303] The operations of process 2700 may include obtaining one or more images representative of a flow path within the microfluidic system that facilitates interaction between a first fluid flow and a second fluid flow (2702). Obtaining one or more images may include capturing images with an optical sensor (e.g., optical sensor 2132), which in some cases may be a camera. The one or more images may include an image of at least one droplet generated in the flow path in a region of the device, where the at least one droplet is not compressed by one or more walls of the device. For example, the at least one droplet may be imaged in a widened region of an outlet channel (e.g., outlet channel 2139) of the device.

[0304] The operation of process 2700 also includes processing one or more images to identify at least one droplet (2704) generated in the flow path through the interaction between a first fluid stream and a second fluid stream. The first fluid stream can include a stream of an aqueous solution (e.g., an unpolymerized mixture containing a fluid matrix material and cells), and the second fluid stream can include a stream of a hydrophobic solution (e.g., an immiscible fluid such as oil). Processing one or more images can include detecting the edges of at least one droplet in at least one of the one or more images (e.g., using a Canny edge detector), and identifying a first set of pixels corresponding to the detected edges of the at least one droplet. For example, the first set of pixels can be a circular representation (e.g., using a Hough transform) generated based on the detected edges of the at least one droplet. Processing one or more images to identify at least one droplet can also include reducing the size of the one or more images before detecting the edges of the at least one droplet and / or before identifying the first set of pixels corresponding to the detected edges of the at least one droplet. For example, as described above, the one or more images can be downsampled (e.g., digitally downsampled) to 1 / 4 to 1 / 2 of the original resolution (e.g., 25% of the original resolution, 30% of the original resolution, 40% of the original resolution, 50% of the original resolution, etc.). In a particular implementation where downsampling is performed, processing one or more images to identify at least one droplet can further include enlarging the one or more downsampled images after detecting the edges of the at least one droplet and / or after identifying the first set of pixels corresponding to the detected edges of the at least one droplet. For example, the image can be enlarged by a factor in the range of 2 to 4. In some cases, the enlargement factor can be selected to restore the downsampled image to its original size. Processing one or more images can also include identifying a second set of pixels set within a threshold distance from the first set of pixels, and calculating the average distance of at least a portion of the second set of pixels from a predetermined location within the at least one droplet. In some instances, the predetermined location within the at least one droplet can be the center of the at least one droplet (or the center of the circular representation of the at least one droplet). In some particular implementations, identifying the second set of pixels can be performed after any reduction in size and re-enlargement of the one or more images being processed. Processing one or more images can further include excluding data corresponding to the detected edges of the at least one droplet if one or more filtering conditions are met. The filtering conditions can include an indication that the first set of pixels corresponds to the detected edges of multiple droplets; an indication that the first set of pixels overlaps with at least one additional set of pixels (e.g., pixels corresponding to another circular representation of another droplet); a detected signal that meets a threshold signal level condition, where the detected signal originates within the perimeter of the first set of pixels; and / or a determination that the first set of pixels is less than a threshold proximity from the imaging wall of the device.

[0305] The operation of process 2700 also includes estimating the size of at least one droplet (2706) and determining that the size of at least one droplet meets a threshold condition (2708). Estimating the size of at least one droplet may include estimating the size based on the average distance of at least a portion of a set of pixels (e.g., the second set of pixels described above) from a predetermined position within at least one droplet. In some embodiments, estimating the size of at least one droplet may include using sub-pixel radial resolution to estimate the size. Determining that the size of at least one droplet meets a threshold condition may include comparing the size of at least one droplet with a target size obtained via user input (e.g., to generate an error signal).

[0306] The operation of process 2700 also includes, in response to determining that the size of at least one droplet meets a threshold condition, generating a signal (2710) that causes an adjustment to be made to the pressure of at least one of the first fluid flow or the second fluid flow. The signal may be configured to increase or decrease the pressure of at least one of the first fluid flow or the second fluid flow based on the size of at least one droplet. In some embodiments, generating the signal may include using a feedback controller and using proportional control, integral control, and / or derivative control to generate the signal. In some embodiments, generating the signal may include generating the signal without feedback control (e.g., using a simple static controller) when no droplets are identified in one or more images and / or when the standard deviation of the size of at least one droplet (e.g., two or more droplets) exceeds a threshold. In some embodiments, the signal may be based on the estimated sizes of multiple droplets from the same captured image or from different images (e.g., consecutive frames in a video).

[0307] Additional operations of process 2700 may include the following. In some embodiments, process 2700 may include processing one or more images to identify bubbles in the flow path. In some embodiments, process 2700 may include storing on a storage device one or more images and / or data representing the estimated size of at least one droplet. In some embodiments, process 2700 may include transmitting to a remote computing device one or more images and / or data representing the size of at least one droplet.

[0308] Liquid-Level Sensing Using Optical Reflection

[0309] Accurately and precisely measuring and monitoring the liquid level in a container (e.g., a tube) is important in many applications including chemical or biological analysis and medical diagnosis, e.g., in the case of the MOS generation systems and methods described above. For example, the liquid level sensing methods described herein can be used in combination with one or more aspects of the MOS generation systems and methods described above, including in combination with the methods of closed-loop control and / or droplet size determination described herein. In some instances, optical reflections such as total internal reflection (TIR) are used to sense the liquid level in these MOS generation systems and methods. Optical reflection methods for liquid level sensing have advantages over other liquid level sensing methods such as capacitive methods, ultrasonic methods, or pressure-based methods because optical-based methods are effective, inexpensive, and accurate.

[0310] The method for liquid level sensing described herein uses optical total internal reflection (TIR). Liquid level sensing is based on integrating an optical interface in a container (e.g., a tube), and this optical interface is between the medium (air or liquid) in the container and the inner surface where light is incident. When the liquid level in the container is below the optical interface, TIR occurs and the light incident on the optical interface is totally reflected back by the optical interface; when the liquid level is higher than or equal to the optical interface, there is no TIR and the light incident on the optical interface is substantially (or entirely) transmitted through the liquid and / or the container. The liquid level can be determined based on the measurement results of the reflected light and / or the transmitted light.

[0311] In some embodiments, the liquid level sensor includes a container having at least one inner surface (e.g., for the optical interface) and a pair of light sources (e.g., light-emitting diodes (LEDs) or laser diodes) and a light detector (e.g., a photodetector, a photodiode, or a phototransistor). The pair of light sources and the light detector can be closely spaced and encapsulated as an electro-optical package. The light sources and the light detector can be arranged on the same side of the container and can be positioned adjacent to the container (e.g., at a distance away, such as 0 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or any suitable distance) for measurement. The light from the light source is incident on the at least one inner surface through the outer surface of the container, and is reflected by the at least one inner surface and detected by the light detector.

[0312] In some embodiments, the pair of light sources and the light detector can be moved along the longitudinal direction of the container to measure the liquid level in the container. In some embodiments, multiple pairs of light sources and light detectors can be arranged at a series of positions along the longitudinal direction of the container, and the liquid level in the container can be determined based on multiple measurements of the multiple pairs of light sources and light detectors.

[0313] The container in which the liquid level is sensed is a structure that defines a space for containing the fluid medium to be measured. The container can be a reservoir, a vessel, a tube, a cylinder, a tank, a bottle, or any suitable structure.

[0314] In some embodiments, the container includes a protrusion extending along the longitudinal direction of the container. In some embodiments, the protrusion and the container can be formed separately and then the protrusion can be attached to the container (e.g., by a refractive index matching material). In some embodiments, the container and the protrusion can be integrally formed, for example, by molding or 3D printing such as stereolithography (SLA) printing. The container and / or the protrusion can be optically transparent or at least partially transparent. The material of the container can be glass, plastic, or polymer, such as polypropylene, polyethylene, polystyrene, polycarbonate, polymethyl methacrylate (PMMA), acrylonitrile butadiene styrene (ABS), high-definition photopolymer Somos Watershed or Waterclear resin, or any substantially transparent resin with a high polish on the surface and capable of transmitting light with minimal diffuse transmission. The container can be custom processed to have a highly polished surface.

[0315] The protrusion can include at least one inner surface. For example, the protrusion can be a 90° V-shaped rib on the body of the container. The body defines a space for containing a liquid. The protrusion can include a first and a second side defining a 90-degree angle and a third side that is part of the body of the container. The protrusion can be configured for retroreflection of light.

[0316] In some embodiments, light can enter the container along a horizontal direction and be incident on the first inner surface of the first side at an incident angle (e.g., 45°), and then be reflected by the first inner surface towards the second inner surface of the second side of the protrusion (e.g., at an incident angle of 45°), and then be reflected back by the second inner surface along the horizontal direction towards a light detector. When the liquid level is below the incident positions of the light on the first and second inner surfaces of the protrusion, the incident angle on each of the first and second inner surfaces is greater than the critical angle (defined by the refractive index of the protrusion material and the refractive index of air), and total internal reflection occurs at the interface between air and the protrusion; when the liquid level is above or equal to the incident position of the light on the first inner surface, for example, the light is immersed in the liquid, the incident angle is less than or equal to the critical angle (defined by the refractive index of the protrusion material and the refractive index of the liquid), and since the liquid has a refractive index (e.g., 1.3) closer to the refractive index of the protrusion material (e.g., 1.5) than air (e.g., 1.0), total internal reflection is eliminated. Then the light exits into the liquid and transmits through the container, and the light detector receives much less or zero reflected light. In some cases, if the refractive index of the liquid is close to the refractive index of the protrusion material (e.g., plastic), the critical angle can be a large angle, and total internal reflection occurs only when the light irradiates the protrusion at a very small grazing angle.

[0317] These techniques for liquid level sensing can address existing challenges for liquid level sensing. For example, liquid level sensing techniques can improve the accuracy of liquid level measurement, which may reduce biological or chemical process variability, leading to higher product quality, lower costs, and less waste. These techniques can provide liquid level sensors or systems that can be cost-effective, compact, and easy to manufacture. These techniques can provide precise and reliable liquid level measurement sensors and systems that can meet the requirements of complex automated processing systems, the need for increasingly stringent process control, and the increasingly stringent regulatory environment. The techniques can also provide non-contact liquid level sensing through the wall of a sealed container such that neither the liquid level sensor nor the liquid is contaminated. The techniques described herein can be applicable to any fluid-related process that requires an accurate amount of liquid in a container. The techniques described herein can be used in many applications, such as, for example, chemical / biological analysis and medical diagnosis. In addition to liquids, the techniques can also be applied to any other type of fluid medium, such as, for example, any medium having a higher refractive index than air. The liquid can include one or more different types of fluid media.

[0318] Figures 33A to 33C An example of a container 3100 for liquid level sensing is shown, where Figure 33A is a schematic view of the container 3100, Figure 33B is a side view A - A' of the container 3100, and Figure 33C is a cross-sectional view B - B' of the container 3100. The container 3100 includes a protrusion that includes at least one inner surface as an optical interface for total internal reflection that can be used for liquid level sensing. For example, the container can be a container for a sample or output in the case of the MOS generation system described above. For example, when sensing the liquid level in both the sample container and the output container, the initial volume and / or the end volume of the liquid in the input container can be determined, and the volume of the sample or waste in the output container can be determined. In some cases, the difference between the volume of the liquid in the input container and the volume of the sample or waste in the output container can be obtained for further analysis.

[0319] As Figures 33A to 33C shown, the container 3100 includes a body 3110 that extends from a bottom 3102 to a top 3104 along a longitudinal direction 3101. The body 3110 defines a space for containing a liquid (e.g., water, solution, oil, or any suitable fluid medium). The bottom 3102 of the container 3100 can have a flat surface, a conical shape, or any suitable shape. The top 3104 can include a lid having a recess or groove for sealing.

[0320] Container 3100 can be configured to have a holding volume of liquid, for example, 1 milliliter (mL), 2 mL, 5 mL, 10 mL, 20 mL, 50 mL, 100 mL, 200 mL, 500 mL, 1 liter (L), 2 L, 5 L, 10 L, 100 L, or any suitable volume. Different liquid levels in container 3100 can correspond to different volumes of liquid contained in container 3100. In some embodiments, container 3100 includes a series of volume level labels corresponding to a series of positions on body 3110 along the longitudinal direction, each volume level label corresponding to a respective volume.

[0321] In some embodiments, as Figures 33A to 33C shown, container 3100 has a cylindrical shape with a circular cross-section. The container can also have any other suitable shape, for example, a rectangular parallelepiped shape with a rectangular cross-section. In some embodiments, container 3100 can have the same dimensions (e.g., diameter) along the longitudinal direction, or variable dimensions that change from small to large or from large to small along the longitudinal direction. For illustrative purposes only, a container with a cylindrical shape having a circular cross-section is used as an example in the description herein.

[0322] In some embodiments, as Figures 33A to 33C shown, container 3100 includes a protrusion 3120 that can protrude inward from body 3110 of container 3100. Protrusion 3120 can extend along the longitudinal direction 3101 of container 3100, for example, from bottom 3102 to top 3104, as Figure 33B shown. Protrusion 3120 can include a first side 3122, a second side 3124, and a third side 3126. Figure 33C A cross-sectional view B-B' is shown, where the cross-section of protrusion 3120 includes a first inner surface 3132 on the first side 3122, a second inner surface 3134 on the second side 3124, and a third surface 3136 on the third side 3126. As discussed in further detail below, protrusion 3120 is configured such that light entering from the outer surface 3112 of body 3110 (e.g., the surface open to the external environment) can be totally internally reflected by the first inner surface 3132 of the first side toward the second inner surface 3134 of the second side, and then totally internally reflected by the second inner surface 3134 back outside container 3100.

[0323] In some embodiments, protrusion 3120 is a V-shaped rib, as Figure 33B and 33CAs shown. The first side 3122 and the second side 3124 can be joined together at the edge 3123, and the first inner surface 3132 and the second inner surface 3134 can be joined at the corner 3133. The edge 3123 can be a straight line along the longitudinal direction 3101, and the corner 3133 can be a point. In some embodiments, the edge 3123 can be any other suitable shape, such as an arc or a curved shape, and correspondingly, the corner 3133 can have an arc or a curved shape. In some embodiments, the first inner surface 3132 and / or the second inner surface 3134 is a straight line, as Figure 33C shown. The angle defined by the first inner surface 3132 and the second inner surface can be substantially equal to 90 degrees. In some embodiments, the first inner surface 3132 and / or the second inner surface 3134 can be at least partially arc-shaped or curved.

[0324] The body 3110 and the protrusion 3120 can be optically transparent or at least partially transparent so that light can propagate in the body 3110 and the protrusion 3120. In some embodiments, the body 3110 of the container 3100 can be made of a first material (e.g., glass, plastic, or polymer). The protrusion 3120 can be made of a second material (e.g., glass, plastic, or polymer). In some embodiments, the second material is the same as the first material. The third side 3126 of the protrusion 3120 can be a part of the body 3110 and integrated with the rest of the body 3110. In some embodiments, the second material is different from the first material. The third side 3126 of the protrusion 3120 can be attached or adhered to the inner surface of the body 3110.

[0325] Figures 34A to 34B An example of a liquid level sensor 3200 according to one or more embodiments of the present disclosure is shown, which measures the liquid level in the container 3100 when the liquid level is below the light incident position ( Figure 34A ) or above the light incident position ( Figure 34B ). Figures 33A to 33C The liquid level sensor 3200 includes a container 3100 having a protrusion 3120 and a sensing pair 3210 of a light source 3212 and a light detector 3214.

[0326] The protrusion 3120 can be a V-shaped rib having an angle Φ defined by a first inner surface 3132 and a second inner surface 3134. The light emitted from the light source 3212 can enter the first inner surface 3132 at an incident angle θ i1 at the incident position through the outer surface 3112 of the body 3110.

[0327] According to Snell's law, the critical angle for total internal reflection (TIR) is sin -1 (n i / no ), where n i is the refractive index of the protrusion 3120, and n o is the refractive index of the medium that interacts with the first inner surface 3132 in the container 3100. When the liquid level is below the incident position, as Figure 34A shown, the medium that interacts with the first inner surface 3132 is the gas 3202, for example, air having a refractive index n o equal to 1. When the liquid level is higher than or equal to the incident position, as Figure 34B shown, the medium that interacts with the first inner surface 3132 is the liquid 3204, for example, an aqueous solution having a refractive index n o that can be equal to 1.3. The material of the protrusion 3120 has a refractive index n i that can be greater than 1.3 and less than 2.0.

[0328] For illustrative purposes only, the refractive index n i of the material of the protrusion 3120 is set to be equal to 1.5. Therefore, when the liquid level is below the incident position, n0 = 1, and the TIR critical angle is 41.8°; when the liquid level is higher than or equal to the incident position, n0 = 1, and the TIR critical angle is 60.1°. Therefore, by configuring the protrusion 3120 (e.g., the angle between the first inner surface and the outer surface) and / or the incident position of the light from the light source 3212, the incident angle θ i1 can be configured to be greater than 41.8° and less than 60.1°, for example, 45°. In this way, when the liquid level is below the incident position, TIR can occur, and when the liquid level is higher than or equal to the incident position, TIR does not occur.

[0329] As Figure 34A shown, when the liquid level is below the incident position, the medium is a gas and TIR occurs at the first inner surface 3132, and the light is reflected in the protrusion 3120 and thus incident on the second inner surface 3134 at the incident angle θ i2 . Similarly, when the incident angle θ i2 can be configured to be greater than 41.8° and less than 60.1°, for example, 45°. In this way, when the liquid level is below the incident position, TIR can occur, and when the liquid level is higher than or equal to the incident position, TIR does not occur. The angle Φ defined by the first inner surface 3132 and the second inner surface 3134 can be equal to θ i1 + θ i2 .

[0330] In some instances, Φ = 90° and θ i1 = θ i2 = 45°, and TIR can occur on both the first inner surface 3132 and the second inner surface 3134. The intensity of the reflected light I outCan be the same as the input light intensity I in Substantially the same. In addition, the input light towards the container 3100 and the reflected light returning from the container 3100 can be parallel to each other, which can be considered retroreflection and can be used to align the light detector 3214 to receive the output light.

[0331] As Figure 34B Shown, when the liquid level is higher than or equal to the incident position, the medium is the liquid 3204. For example, the first inner surface is immersed in the liquid 3204, where TIR does not occur at the first inner surface, and light is transmitted through the first inner surface into the liquid 3204 with a relatively high transmittance (e.g., about 96%), and this light can further propagate outside the container 3100. The light detector 3214 configured to receive the reflected light can substantially not receive the reflected light. In some instances, the light detector 3216 can be positioned on a different side from the light source 3212 and configured to receive the transmitted light I t . The light detector 3216 can be calibrated or adjusted to receive the transmitted light.

[0332] In some embodiments, the sensing pair 3210 includes the light source 3212 and the light detector 3214 on the same side of the container 3100. The power of the reflected light detected by the light detector 3214 can determine whether the liquid level is higher or lower than the incident position. For example, if the power of the detected reflected light is greater than a predetermined threshold, it indicates that TIR has occurred and the liquid level is lower than the incident position. If the power of the detected light is less than or equal to the predetermined threshold, it indicates that TIR has not occurred and the liquid level is higher than or equal to the incident position.

[0333] In some embodiments, the sensing pair 3210 includes the light source 3212 and the light detector 3216 on different sides of the container 3100. The power of the transmitted light detected by the light detector 3216 can determine whether the liquid level is higher or lower than the incident position. For example, if the power of the detected transmitted light is less than a predetermined threshold, it indicates that TIR has occurred and the liquid level is lower than the incident position. If the power of the detected light is greater than or equal to the predetermined threshold, it indicates that TIR has not occurred and the liquid level is higher than or equal to the incident position.

[0334] Figure 35A Illustrates an exemplary liquid level measurement by moving a pair of a light source and a light detector of a liquid level sensor along the longitudinal direction of a container according to one or more embodiments of the present disclosure.

[0335] The container can be Figures 33A to 33C The container 3100 of 34A to 34B, including the protrusion 3120. The pair of the light source and the light detector can be Figure 34A Or Figure 34BPair 3210 in it. Pair 3210 can move continuously along the longitudinal direction 3101. The incident position of light on the inner surface 3132 of the protrusion 3120 is represented by the height h relative to the bottom 3102 of the container 100 x When moving pair 3210, the incident position also moves. For example, h x increases or decreases.

[0336] To measure the liquid level 3302 in the container 3100 with the corresponding height h i pair 3210 can move continuously from the bottom 3102 (h = 0) towards the top 3104 of the container 3100. The intensity of the reflected light can be monitored and continuously detected. Figure 35B Shows the exemplary measurement result 3310 of the liquid level measurement using Figure 35A The result 3310 shows that when the incident position hx is lower than the liquid level hi, TIR does not occur and the intensity of the detected reflected light has a lower value Ia, and when the incident position hx is equal to or higher than the liquid level h i TIR occurs and the intensity of the detected reflected light has a higher value Ib. There are significant changes in the liquid level h i In some instances, the ratio of Ib / Ia can be greater than 2, 3, 4, 5, 10, 20, 50, 100 or any suitable value. In some embodiments, a pre-determined threshold can be set to a value between Ia and Ib, for example, the average value of Ia and Ib, i.e., (Ia + Ib) / 2. Therefore, by monitoring the intensity of the detected light when moving pair 3210 along the longitudinal direction, an accurate measurement of the liquid level h i can be obtained.

[0337] In some embodiments, the reference signal can be first detected by the light detector, for example, detecting the light reflected from a container without a protrusion. The reference signal can be used as background noise. The detected reflected light from the container with a protrusion can be subtracted from the reference signal to further improve the detection accuracy.

[0338] Figure 36A Shows another exemplary liquid level measurement performed by the liquid sensing system 3400 according to one or more embodiments of the present disclosure. Figure 36B Shows the exemplary measurement result 3450 of the liquid level measurement using Figure 36A

[0339] The liquid sensing system 3400 may include multiple pairs of light sources and light detectors 3210-1, 3210-2, ……, 3210-(m-1), 3210-m, 3210-(m+1), ……, 3210-(n-1), 3210-n (generally referred to as multiple pairs 3210 or individually as pair 3210), where m and n are integers. The pairs 3210 may be arranged at a series of positions along the longitudinal direction 3101 of the container 3100, for example, from the bottom 3102 to the top 3104. The series of positions of the pairs 3210 correspond to a series of incident positions h1, h2, ……, hm-1, hm, hm+1, ……, hn-1, hn along the longitudinal direction. The pairs 3210 may be static and mounted on a support 3404 extending along the longitudinal direction. The number of pairs 3210 may be 5, 10, 20, or any suitable number.

[0340] To determine the liquid level 3402 in the container 3100 having a corresponding height h i , the corresponding intensities of the detected reflected light for each pair 3210 corresponding to different incident positions are obtained. The corresponding intensities may be plotted as discrete points 3410, as Figure 36B shown. It can be seen that when the incident positions (e.g., h1, h2, ……, hm-1) are below the corresponding height h of the liquid level 3402 i , no TIR occurs and the intensity of the detected reflected light has a lower intensity value Ia; when the incident positions (e.g., hm, hm+1, ……, hn-1, hn) are equal to or higher than the liquid level h i , TIR occurs and the intensity of the detected reflected light has a higher value Ib. There is a significant change in the liquid level h i . In some instances, the ratio of Ib / Ia may be greater than 2, 3, 4, 5, 10, 20, 50, 100, or any suitable value. Therefore, by measuring the detected light intensities of a series of pairs 3210, the liquid level h i can be determined within the range between h m-1 and h m .

[0341] In some embodiments, the liquid level sensing system 400 includes a pair of 3210 light sources and light detectors, which may be arranged at predetermined positions, e.g., corresponding to a predetermined liquid level or a predetermined volume level. Liquid may be gradually injected into the container 3100. The light detectors may continuously monitor the reflected light from the container 3100. When a substantial change occurs, indicating that TIR has occurred and the liquid level has increased to the predetermined liquid level, the liquid level sensing system 3400 may send a signal to stop injecting the liquid. In this way, an accurate amount of liquid can be obtained in the container 3100. In some embodiments, the liquid level sensing system 3400 includes two or more pairs of 3210 to control the injection of a certain amount of liquid into the container.

[0342] Figure 37 An exemplary fluid system 3500 for liquid level sensing according to one or more embodiments of the present disclosure is shown. The fluid system 3500 may include at least one input container 3502, a fluid channel system 3504 including one or more fluid channels, and one or more output containers 3506.

[0343] In some embodiments, the liquid (e.g., a mixture of solutions) contained in the input container 3502 may flow through the fluid channel system 3504 for processing, e.g., filtering, separation, or any suitable processing step. The fluid channel system 3504 may output the processed liquid into one or more output containers 3506. In some embodiments, one or more output containers 3506 include at least one sample container 3506 and at least one waste container 3506.

[0344] In some embodiments, at least one of the at least one input container 3502 and one or more output containers 3506 may be the container 3100 and assembled with one or more pairs of 3210 light sources and light detectors to form one or more liquid level sensors 3200 or 3300 or system 3400. In this way, the initial volume and / or the end volume of the liquid in the input container 3502 can be determined. The volume of the sample or waste in the output container 3506 can be determined. In some cases, the difference between the volume of the liquid in the input container and the volume of the sample or waste in the output container 3506 can be obtained for further analysis.

[0345] Figure 38 is a flowchart of an exemplary process 3600 for liquid level sensing using optical reflection according to one or more embodiments of the present disclosure. The process 3600 may be performed by a liquid level sensor (e.g., Figures 34A to 34B the liquid level sensor 3200 of Figure 35A the 3300 of Figure 36A the liquid level system 3400 ofFigure 37 by the fluid system 3500). The liquid is contained in a container, e.g., Figures 33A to 33C , Figures 34A to 34B , Figure 34A , Figure 36A or the container 3100 of FIG. 36. The level sensor includes a pair of light sources (e.g., Figures 34A to 34B 3212 of Figures 34A to 34B ) and a light detector (e.g.,

[0346] 3214 or 3216 of

[0347] ). Light is emitted, for example, from the light source onto the outer surface (3602) of the container containing the liquid. The container includes at least one inner surface configured such that i) if the level of the liquid in the container is below the incident position where the light is incident on the at least one inner surface, the light is totally internally reflected by the at least one inner surface; and ii) if the level of the liquid is higher than or equal to the incident position where the light is incident on the at least one inner surface, the light is transmitted through the at least one inner surface.

[0348] For example, light from the container (e.g., transmitted light or reflected light) is detected by the light detector (3604), and then the level of the liquid in the container is determined based on the detected light (3606). Figure 33A The container may extend along a longitudinal direction (e.g., Figures 33A to 33C 3101 of

[0349] ), and the level of the liquid may be defined along the longitudinal direction from the bottom of the container. In some embodiments, the container includes a protrusion having at least one inner surface (e.g., Figures 33B to 33C 3120 of Figures 33B to 33C or 34A to 34B, 35A or 36A). The protrusion may extend continuously along the longitudinal direction or include a plurality of portions spaced apart along the longitudinal direction. Figures 33B to 33C 3124 of Figures 33B to 33C or 34A to 34B). As Figure 34A shown, if the level of the liquid in the container is at or above the incident position (e.g., at a position higher than the incident position along the longitudinal direction), the light may be totally internally reflected by the first inner surface and then totally internally reflected by the second inner surface.

[0350] In some embodiments, the angle defined by the first inner surface and the second inner surface is substantially equal to 90 degrees, e.g., as Figure 33C and 34AAs shown in FIGS. 34A to 34B. The first incident angle at which light is incident on the first inner surface may be substantially equal to the second incident angle at which light is incident on the second inner surface. For example, each of the first incident angle and the second incident angle may be substantially equal to 45 degrees.

[0351] In some embodiments, the first side and the second side of the protrusion are joined together at the edge, for example, Figure 33B 3123. The container may include a body for containing a liquid (e.g., Figures 33A to 33C or 3110 of FIGS. 34A to 34B). The protrusion may include a third side that is part of the body of the container (e.g., Figure 33B 3126), and the first side and the second side are externally connected to the body of the container. In some instances, the protrusion has a triangular shape having a first side, a second side, and a third side, for example, as shown in Figure 33C or FIGS. 34A to 34B.

[0352] In some embodiments, the body includes a first material and the protrusion includes a second material. In some cases, the second material is the same as the first material and the protrusion and the body are a single piece. In some cases, the second material is different from the first material and the protrusion and the body are attached together.

[0353] In some embodiments, light is incident normally on the outer surface of the container and the light exits the container normally, for example, as shown in Figures 34A to 34B FIGS. In some embodiments, as shown in Figure 34A FIGS., if the liquid level in the container is lower than the incident position, the light propagates in a first direction to be incident on the first inner surface and is reflected away from the second inner surface along a second direction that is substantially parallel but opposite to the first direction.

[0354] Light is emitted from a light source and the light is detected by a light detector, and the light source and the light detector form a pair. The incident position may be predetermined based on the position of the light source, for example, as shown in Figure 35A or FIGS. 36A.

[0355] In some embodiments, the light source and the light detector are arranged on the same side of the container, for example, Figure 34A and 34B . Determining the liquid level in the container based on the detected light may include: determining that the liquid level is lower than the incident position in response to determining that the power of the detected light is greater than a predetermined threshold, or determining that the liquid level is higher than or equal to the incident position in response to determining that the power of the detected light is less than or equal to the predetermined threshold.

[0356] In some embodiments, the light source and the light detector are arranged on opposite sides of the container, for example, as shown in Figure 34BAs shown. Determining the liquid level of the liquid in the container based on the detected light may include: in response to determining that the power of the detected light is less than a predetermined threshold, determining that the liquid level is lower than the incident position, or in response to determining that the power of the detected light is greater than or equal to the predetermined threshold, determining that the liquid level is higher than or equal to the incident position.

[0357] In some embodiments, as Figures 35A to 35B shown, process 3600 may include: gradually moving the container or at least one of a pair of light sources and light detectors along a longitudinal direction until the power of the detected light changes significantly beyond a predetermined threshold, and when the power of the detected light changes significantly beyond the predetermined threshold, determining the liquid level based on the position of the bottom of the container and the position of the light source.

[0358] In some embodiments, process 3600 may include: when the liquid level in the container increases due to injecting liquid into the container, monitoring the power of the detected light, the incident position of the light corresponding to a predetermined liquid level in the container; and in response to determining that the power of the detected light changes significantly beyond a predetermined threshold, controlling to stop the injection.

[0359] In some embodiments, as Figure 36A shown, light is emitted from a plurality of light sources spaced along a longitudinal direction, and the light is detected by a plurality of light detectors spaced along the longitudinal direction. Each light source of the plurality of light sources may be associated with a corresponding light detector of the plurality of light detectors and be configured to emit a corresponding portion of the light, and the corresponding light detector may be configured to detect the corresponding portion of the light emitted from the light source.

[0360] In some cases, each light source of the plurality of light sources and the corresponding light detector are arranged in a plane passing through the longitudinal axis of the container. For example, when the beam size of the light is relatively small compared to the size of the container, the light is emitted into the container, reflected in the protrusion, and reflected out of the container in the same plane, and the corresponding light detector may be arranged side by side with the light source in the same plane. The light emitted from the light source may be configured to be focused at least in the longitudinal direction such that the incident positions on the first and second inner surfaces are both in a plane perpendicular to the longitudinal direction. The distance between the light source and the corresponding light detector may be determined based on the input position and the output position of the light on the container. As Figure 34A shown, the farther the input position is from the intersection of the two inner surfaces (e.g., Figure 33C the edge 3133) along the longitudinal direction, the greater the distance. That is, after determining the distance between the light source and the corresponding light detector, the input position of the light on the main body may be adjusted, for example, to maximize the intensity of the reflected light detected by the corresponding light detector.

[0361] In some cases, each of the plurality of light sources and the corresponding light detector are arranged along a longitudinal direction. For example, when the beam size of the light is relatively large compared to the size of the container, the light is emitted into the container, reflected in the protrusion, and reflected out of the container in different planes along the longitudinal direction. The corresponding light detector can be arranged horizontally below or above the light source. The position of the corresponding light detector can be calibrated or adjusted to maximize the intensity of the detected reflected light.

[0362] The protrusion extends along a vertical direction to perform multiple depth (volume) measurements of the protrusion. If the light source and the light detector are within the width of the protrusion, they can be arranged side by side or one above the other. The formed draft angle can be doubled by specular reflection. If the formed draft angle is 1 degree, the incident light can be reflected downward by the protrusion by 2 degrees. In some embodiments, the light detector is arranged below the light source, for example, to account for alignment tolerances. The distance between the incident light and the returned light may not exceed the width of the protrusion. When the light detector is below the light source, the pair of the light detector and the light source can be moved horizontally along substantially the entire width of the protrusion because the first and second reflections on the first and second inner surfaces can occur on either surface.

[0363] In some embodiments, determining the liquid level in the container based on the detected light includes: determining the liquid level in the container based on the light corresponding to each of the plurality of light detectors detected by each light detector. For example, as Figures 36A to 36B shown, step 3606 may include: determining that the liquid level in the container is higher than the first incident position where the first corresponding part of the light from the first light source is incident on the first inner surface based on the light of the first corresponding part detected by the first light detector; determining that the liquid level of the liquid in the container is lower than the second incident position where the second corresponding part of the light from the second light source is incident on the first inner surface based on the light of the second corresponding part detected by the second light detector; and determining that the liquid level of the liquid in the container is between the first incident position and the second incident position. The first incident position is predetermined based on the first position of the first light source, and the second incident position is predetermined based on the second position of the second light source.

[0364] In some embodiments, the container is a tube, a vessel, a tank, a bottle, or any suitable structure configured to hold a liquid.

[0365] In some embodiments, the container can be formed by forming the body of the container and forming the protrusion together such that the body and the protrusion are formed as an integral piece by SLA 3D printing or molding. In some embodiments, the container is formed by attaching the protrusion to the body of the container.

[0366] In some embodiments, the liquid level sensor includes at least one processor configured to determine the liquid level of the liquid based on the light detected by the light detector. In some embodiments, the liquid level sensor is coupled to at least one processor and configured to provide the detected light to at least one processor, which is configured to determine the liquid level of the liquid based on the detected light. In some embodiments, at least one processor is configured to multiplex a plurality of liquid level sensors, for example, by controlling one liquid level sensor to be turned on at a time to prevent crosstalk due to optical channels and scattering.

[0367] In some embodiments, as Figure 37 described, the fluid system (e.g., Figure 37 3500) includes: at least one input container configured to hold a liquid; at least one fluid channel configured to receive the liquid from the input container and output the liquid from at least one output; and at least one output container coupled to the at least one output and configured to receive the liquid through the at least one fluid channel. At least one of the input container or the at least one output container is configured for liquid level sensing, and each of the at least one container includes at least one inner surface configured such that i) if the liquid level in the container is lower than the incident position where light is incident on the at least one inner surface, the light is totally internally reflected by the at least one inner surface; and ii) if the liquid level is higher than or equal to the incident position where the light is incident on the at least one inner surface, the light is transmitted through the at least one inner surface.

[0368] The fluid system may further include at least one processor configured to: determine the liquid level of the liquid in the container based on the detected light from the container as described above. In some embodiments, at least one processor is configured to: determine the difference between the volume of the liquid in the input container and the volume of the liquid in the at least one output container.

[0369] Micro-organic spheres (MOS)

[0370] Historically, the most common practice for testing cell responses to various stimuli has included culturing cells under two-dimensional (2D) conditions, such as in a petri dish or in a well plate. However, these 2D conditions can stress the cells, and they are not always well correlated with the responses of individual patients to the test stimuli. On the other hand, droplets can provide a three-dimensional (3D) environment for 3D cell aggregate clusters and may produce experimental results that are better correlated with patient outcomes. 3D cell aggregates can include organoids or spheroids. Organoids are in vitro cell aggregates (usually having a diameter greater than one millimeter) that include a population of stem cells that can differentiate into the major cell lineages. Spheroids are simple clusters of a wide variety of cells, such as from tumor tissue, embryoid bodies, hepatocytes, neural tissue, or mammary glands. Unlike organoids, spheroids generally do not have the ability to self-assemble or regenerate.

[0371] Generated droplets containing 3D cell aggregates, such as organoids or spheroids, are sometimes referred to as "patient-derived micro-organospheres" (PMOS) or simply "micro-organospheres" (MOS). MOS, including methods and devices for generating them, are described in further detail in U.S. Patent Application No. 16 / 838,010, which is incorporated herein by reference in its entirety.

[0372] It is important to note that while MOS are described in this application as an example of droplets that can be generated by a microfluidic device, the present disclosure is not intended to be limiting. Droplets formed by microfluidics can be used for a wide range of applications, including drug discovery and drug efficacy assessment, and those skilled in the art will understand that the techniques described herein can be applied to droplets for many of these alternative applications.

[0373] In some instances, MOS contain dissociated cells from a patient-derived tissue sample (e.g., a biopsy sample). The tissue can be from a healthy tissue biopsy or from a cancerous (e.g., tumor) cell biopsy. The cells are dissociated and dispersed (e.g., suspended) in a matrix material. Once generated, the MOS can be patterned onto a microfluidic microwell array for incubation, dosing with a drug compound, and imaging at repeated time intervals to monitor the growth of each organoid. This miniaturized assay maximizes the use of patient samples and enables high-throughput screening of a large number of therapeutic agents (e.g., drugs or drug formulations) from a core biopsy at a relatively low cost per sample.

[0374] The matrix material for generating MOS can be a gel, semi-solid, or liquid, such as a low-viscosity liquid, at room temperature (e.g., at about 25 °C). Exemplary matrix materials include polymers or hydrogels, including collagen, fibrin, or chitosan; MATRIGEL TM(Corning, Corning, NY); polyethylene glycol; dextran, including chemically or photocrosslinkable dextran, electrospun biological, synthetic, or biosynthetic mixtures; or other suitable polymerizable matrix materials. In some instances, the matrix material is a gel, such as a synthetic or natural gel. Examples of synthetic gels include gels derived from any one of polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol (PVA), or poly(ethylene oxide) (PEO).

[0375] In some instances, once polymerized, the matrix material forms a hydrogel. The term "hydrogel" refers to a two-component or multi-component gel comprising a three-dimensional network of polymer chains, where water acts as the dispersion medium and fills the space between the polymer chains. Exemplary hydrogels that can be used for MOS include alginate, collagen (including type I and type VI collagen), elastin, keratin, fibronectin, proteoglycan, glycoprotein, polylactide, polyethylene glycol, polycaprolactone, polylactide, polydioxanone, polyacrylate, polyurethane, polysulfone, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylate, polyacetate, polyester, polyamide, polycarbonate, polyanhydride, polyamino acid, carbohydrates, polysaccharides and modified polysaccharides, or derivatives and copolymers thereof; inorganic materials such as glass (such as bioactive glass), ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone; or combinations of the foregoing. In a specific instance, the hydrogel comprises a material selected from the group consisting of agarose, alginate, type I collagen, poly(ethylene oxide)-poly(propylene oxide) block copolymer (e.g., F127 (BASF Corporation, Mount Olive, N.J.)), silicone, polysaccharide, polyethylene glycol, and polyurethane.

[0376] In some instances, in addition to patient-derived cells and matrix materials, the MOS also includes one or more biorelevant materials. Exemplary biorelevant materials included in the MOS can include one or more of the following: extracellular matrix proteins (e.g., fibronectin), drugs (e.g., small molecules), peptides, antibodies (e.g., for modulating any of cell survival, proliferation, or differentiation); or inhibitors of specific cell functions. For example, the biorelevant materials in the MOS can be used to increase cell viability by reducing cell death and / or activation of cell growth / replication, or otherwise mimic the in vivo environment. The biorelevant materials incorporated into the MOS can include or mimic one or more of the following components: serum, interleukin, chemokine, growth factor, glucose, physiological saline, amino acid, or hormone. When the matrix material is a gel, the gel itself can include one or more biorelevant materials, including extracellular matrix components such as collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate, agarose, or chitosan. For example, MATRIGEL contains bioactive polymers that are very important for cell viability, proliferation, development, and migration. In a specific instance, alone or in addition to other biologically relevant materials (such as other extracellular matrix proteins), the matrix material is a gel comprising type 1 collagen (such as type 1 collagen obtained from rat tails).

[0377] The MOSs described herein can have a diameter between about 50 μm and about 500 μm (e.g., between about 50 μm and about 400 μm, between about 50 μm and about 300 μm, between about 50 μm and about 250 μm, etc.). Each MOS can initially contain between about 1 and 1000 (e.g., between about 1 and 750, between about 1 and 500, between about 1 and 400, between about 1 and 300, between about 1 and 200, between about 1 and 150, between about 1 and 100, between about 1 and 75, between about 1 and 50, between about 1 and 40, between about 1 and 30, between about 1 and 20, etc.) dissociated primary cells distributed within a matrix material. The number of cells per MOS can be set based on the intended use of the MOS. For example, a MOS having a small number of cells (e.g., 1 - 5 cells per MOS) can be used to study clonal diversity (e.g., for tumor heterogeneity), e.g., to observe which clones are drug resistant and to determine the genomic (mutation) diversity associated with a particular clone (e.g., by genomic sequencing). A MOS having a medium number of cells (e.g., between about 3 - 30 cells, 5 - 30 cells, 5 - 25 cells, 5 - 20 cells, 10 - 25 cells, etc.) can be used for rapid drug testing, such as toxicity testing, because these MOSs tend to grow rapidly. A MOS having a large number of cells (e.g., between about 20 - 100 cells (e.g., 30 - 100 cells, 40 - 100 cells, or greater than 50 cells, etc.)) may be suitable for mimicking the tissue composition in each micro - organic sphere, as the micro - organic spheres may contain different lineages, possibly including epithelial cells (or cancer, etc.) and mesenchymal cells (or stromal cells, immune, vascular, etc.) cells.

[0378] The MOSs generated in the microfluidic systems described herein can be used essentially immediately after formation or cultured for a short period of time (e.g., 14 days or less, 10 days or less, 7 days or less, 5 days or less, etc.). The cells within the MOSs can survive while retaining most (if not all) of the characteristics of the tissue, including the tumor tissue from which they were extracted. The survival rate of the cells within the MOSs is high, and the MOSs can be passaged multiple times over a period of time (e.g., days or weeks), where the cells will divide, aggregate, and form structures similar to the parental tissue, sometimes referred to as tumor spheres. In some cases, cells from the dissociated tissue within the MOSs form morphological structures within the MOSs.

[0379] Figures 39A to 39C An exemplary MOS is shown. Specifically referring to Figure 39A , at the time of generation, each MOS includes a single cell and has a diameter of approximately 300 μm. Figure 39BThe MOS after 3 days of culture is shown. The cell size has increased, for example, doubled or grown. Refer to Figure 39C , after 7 days of culture, the cells have multiplied many times, forming cell clusters or cell aggregates or tumor spheres.

[0380] Figure 40 Examples of a computing device 2800 and a mobile computing device 2850 for implementing specific embodiments of the present disclosure are shown. The computing device 2800 is intended to represent various forms of digital computers, such as laptop computers, desktop computers, workstations, personal digital assistants, servers, blade servers, mainframes, and other suitable computers. The mobile computing device 2850 is intended to represent various forms of mobile devices, such as personal digital assistants, cellular phones, smart phones, AR devices, sensor devices, smart cameras, and other similar computing devices. The components shown here, their connections and relationships, and their functions are only meant to be examples and are not meant to be limiting. The computing device 2800 and / or the mobile computing device 2850 may form at least a part of a microfluidic system, such as the controller 2124 of the device 2100 described above. The computing device 2800 and / or the mobile computing device 2850 may also form at least a part of a remote computing device external to the device 2100, which may perform one or more of the image processing operations described with respect to Figure 28 , 29 and 30, or may interact with the device 2100 to receive data transmitted from the device 2100 (e.g., captured images and / or the estimated sizes of one or more droplets). For example, in some specific embodiments, the computing device 2800 and / or the mobile computing device 2850 may form at least a part of a remote camera or a smart camera external to the device 2100.

[0381] The computing device 2800 includes a processor 2802 (e.g., a digital signal processor [DSP], a graphics processing unit [GPU], a field programmable gate array [FPGA], etc.), a memory 2804, a storage device 2806, a high-speed interface 2808, and a low-speed interface 2812. In some specific implementations, the high-speed interface 2808 is connected to the memory 2804 and a plurality of high-speed expansion ports 2810. In some specific implementations, the low-speed interface 2812 is connected to the low-speed expansion port 2814 and the storage device 2804. Each of the processor 2802, the memory 2804, the storage device 2806, the high-speed interface 2808, the high-speed expansion port 2810, and the low-speed interface 2812 is interconnected using various buses and can be mounted on a common motherboard or otherwise as appropriate. The processor 2802 can process instructions for execution within the computing device 2800, including instructions stored in the memory 2804 and / or the storage device 2806, to display graphical information for a graphical user interface (GUI) on an external input / output device (such as a display 2816 coupled to the high-speed interface 2808). In other specific implementations, multiple processors and / or multiple buses and multiple memories and multiple types of memories can be used as appropriate. Additionally, multiple computing devices can be connected, with each device providing a portion of the necessary operations (e.g., as a server group, a set of blade servers, or a multi-processor system).

[0382] The memory 2804 stores information within the computing device 2800. In some specific implementations, the memory 2804 is one or more volatile memory units. In some specific implementations, the memory 2804 is one or more non-volatile memory units. The memory 2804 can also be another form of computer-readable medium, such as a magnetic disk or an optical disk.

[0383] The storage device 2806 is capable of providing large-capacity storage for the computing device 2800. In some specific implementations, the storage device 2806 can be or include a computer-readable medium, such as a floppy disk device, a hard disk device, an optical disk device, a magnetic tape device, a flash memory, or other similar solid-state memory devices or an array of devices, including devices in a storage area network or other configurations. Instructions can be stored in the information carrier. These instructions, when executed by one or more processing devices such as the processor 2802, perform one or more methods such as those described above. The instructions can also be stored by one or more storage devices, such as a computer-readable or machine-readable medium, such as the memory 2804, the storage device 2806, or the memory on the processor 2802.

[0384] The high-speed interface 2808 manages the bandwidth-intensive operations of the computing device 2800, while the low-speed interface 2812 manages the less bandwidth-intensive operations. Such a function allocation is merely an example. In some specific embodiments, the high-speed interface 2808 is coupled to the memory 2804, the display 2816 (e.g., via a graphics processor or accelerator), and a high-speed expansion port 2810 that can accept various expansion cards. In a specific embodiment, the low-speed interface 2812 is coupled to the storage device 2806 and the low-speed expansion port 2814. The low-speed expansion port 2814 may include various communication ports (e.g., Universal Serial Bus (USB), Bluetooth, Ethernet, Wireless Ethernet) and can be coupled to one or more input / output devices. Such input / output devices may include a display device, a printing device 2834, or a keyboard or mouse 2836. The input / output devices can also be coupled to the low-speed expansion port 2814 via a network adapter. Such network input / output devices may include, for example, a switch or a router 2832.

[0385] The computing device 2800 can be implemented in a variety of different forms, such as Figure 40 shown. For example, it can be implemented as a standard server 820, or implemented multiple times in a group of such servers. Additionally, it can be implemented in a personal computer such as a laptop computer 2822. It can also be implemented as part of a rack server system 2824. Alternatively, components from the computing device 2800 can be combined with other components in a mobile device such as a mobile computing device 2850. Each of such devices can contain one or more of the computing device 2800 and the mobile computing device 2850, and the entire system can consist of multiple computing devices communicating with each other.

[0386] The mobile computing device 2850 includes a processor 2852; a memory 2864; input / output devices, such as a display 2854; a communication interface 2866; and a transceiver 2868; and other components. The mobile computing device 2850 can also be provided with a storage device, such as a microSD card or other device, to provide additional storage. Each of the processor 2852, the memory 2864, the display 2854, the communication interface 2866, and the transceiver 2868 is interconnected using various buses, and multiple of the components can be mounted on a common motherboard or otherwise mounted as appropriate. In some specific embodiments, the mobile computing device 2850 may include a camera device.

[0387] The processor 2852 can execute instructions within the mobile computing device 2850, including instructions stored in the memory 2864. The processor 2852 can be implemented as a chipset including separate and multiple analog and digital processors. For example, the processor 2852 can be a Complex Instruction Set Computer (CISC) processor, a Reduced Instruction Set Computer (RISC) processor, or a Minimal Instruction Set Computer (MISC) processor. The processor 2852 can provide coordination of, for example, other components of the mobile computing device 2850, such as control of a user interface (UI), applications run by the mobile computing device 2850, and / or wireless communications performed by the mobile computing device 2850.

[0388] The processor 2852 can communicate with a user through a control interface 2858 and a display interface 2856 coupled to a display 2854. The display 2854 can be, for example, a Thin Film Transistor Liquid Crystal Display (TFT) display, an Organic Light Emitting Diode (OLED) display, or other suitable display technology. The display interface 2856 can include appropriate circuitry for driving the display 2854 to present graphics and other information to the user. The control interface 2858 can receive commands from the user and convert them for submission to the processor 2852. Additionally, an external interface 2862 can provide communication with the processor 2852 to enable near area communication of the mobile computing device 2850 with other devices. The external interface 2862 can provide, for example, wired communication in some embodiments, or wireless communication in other embodiments, and can also use multiple interfaces.

[0389] The memory 2864 stores information within the mobile computing device 2850. The memory 2864 can be implemented as one or more computer-readable media, one or more volatile memory units, or one or more non-volatile memory units, or a combination thereof. Extended memory 2874 can also be provided and connected to the mobile computing device 2850 through an extended interface 2872, which can include, for example, a Single In-line Memory Module (SIMM) card interface. The extended memory 2874 can provide additional storage space for the mobile computing device 2850, or can also store applications or other information for the mobile computing device 2850. Specifically, the extended memory 2874 can include instructions for performing or supplementing the above processes, and can also include security information. Thus, for example, the extended memory 2874 can be provided as a security module for the mobile computing device 2850, and can be programmed with instructions that allow for the secure use of the mobile computing device 2850. Additionally, security applications and additional information, such as placing identification information on the SIMM card in an unbreakable manner, can be provided via the SIMM card.

[0390] The memory may include, for example, flash memory and / or non-volatile random access memory (NVRAM), as discussed below. In some embodiments, the instructions are stored in an information carrier. These instructions, when executed by one or more processing devices such as processor 2852, perform one or more methods such as those described above. The instructions may also be stored by one or more storage devices, such as one or more computer-readable or machine-readable media, such as memory 2864, extended memory 2874, or the memory on processor 2852. In some embodiments, the instructions may be received in a propagated signal, such as via transceiver 2868 or external interface 2862.

[0391] The mobile computing device 2850 may communicate wirelessly via a communication interface 2866, which may include digital signal processing circuitry as necessary. The communication interface 2866 may provide communication under various modes or protocols, such as Global System for Mobile Communications (GSM) voice calls, Short Message Service (SMS), Enhanced Message Service (EMS), Multimedia Messaging Service (MMS) messages, Code Division Multiple Access (CDMA), Time Division Multiple Access (TDMA), Personal Digital Cellular (PDC), Wideband Code Division Multiple Access (WCDMA), CDMA2000, General Packet Radio Service (GPRS). Such communication may occur, for example, via transceiver 2868 using radio frequency. In addition, short-range communication may occur, such as using Bluetooth or Wi-Fi. Additionally, a Global Positioning System (GPS) receiver module 2870 may provide additional navigation and location-related wireless data to the mobile computing device 2850, which may be used as appropriate by applications running on the mobile computing device 2850.

[0392] The mobile computing device 2850 may also perform audible communication using an audio codec 2860, which may receive voice information from a user and convert it into usable digital information. The audio codec 2860 may likewise generate audible sounds for the user, such as via a speaker in, for example, the handset of the mobile computing device 2850. Such sounds may include sounds from a voice telephone call, may include recorded sounds (e.g., voice messages, music files, etc.) and may also include sounds generated by applications operating on the mobile computing device 2850.

[0393] The mobile computing device 2850 may be implemented in a variety of different forms, as Figure 40 shown. For example, it may be implemented as a telephone device 2880, a personal digital assistant 2882, and a tablet device (not shown). The mobile computing device 2850 may also be implemented as a component of a smart phone, an AR device, or other similar mobile devices.

[0394] The computing device 2800 may be implemented as part of a microfluidic system, such as the controller 2124 of the device 2100 described above with respect to Figure 26A The computing device 2800 may also be implemented in a remote computing device that communicates with the device 2100 to receive data transmitted from the device 2100 (e.g., captured images and estimated sizes of one or more droplets).

[0395] The computing device 2800 and / or 2850 may also include a USB flash drive. The USB flash drive may store an operating system and other applications. The USB flash drive may include input / output components, such as a wireless transmitter or a USB connector that can be inserted into a USB port of another computing device.

[0396] List of examples

[0397] Example 1. A microfluidic device, comprising:

[0398] A microfluidic chip for generating micro-organic spheres (MOS), wherein a first microfluidic channel is defined in a surface of the microfluidic chip, the first microfluidic channel comprising:

[0399] A droplet generation section, the droplet generation section comprising an inlet section, a junction between the inlet section and an emulsifying fluid channel, and a chamber downstream of the junction, wherein a cross-sectional area of the chamber is larger than a cross-sectional area of the inlet section, and

[0400] A polymerization section downstream of the droplet generation section, the polymerization section having a serpentine configuration; and

[0401] A cartridge for MOS demulsification, the cartridge comprising:

[0402] A collection container;

[0403] A substrate disposed on the collection container, wherein a second microfluidic channel is defined in a surface of the substrate facing the collection container, and wherein the second microfluidic channel is fluidly connected to an output of the polymerization section of the first microfluidic channel; and

[0404] A membrane disposed between the collection container and the surface of the substrate.

[0405] Example 2. The microfluidic device according to Example 1, wherein the droplet generation section of the first microfluidic channel comprises an outlet section downstream of the chamber, wherein a cross-sectional area of the chamber is larger than a cross-sectional area of the outlet section.

[0406] Example 3. The microfluidic device according to Example 2, wherein at least some of the outlet section extends in a direction parallel to the chamber.

[0407] Example 4. The microfluidic device according to any one of the foregoing embodiments, wherein the surface of the microfluidic chip is a first surface, and wherein the polymeric portion of the microfluidic channel is defined on the first surface of the microfluidic chip and on a second surface of the microfluidic chip opposite the first surface.

[0408] Example 5. The microfluidic device according to any one of the foregoing embodiments, wherein the junction portion includes a junction portion having two hydrophobic fluid channels.

[0409] Example 6. The microfluidic device according to Example 5, wherein the junction portion is a right-angle junction portion.

[0410] Example 7. The microfluidic device according to any one of the foregoing embodiments, wherein the membrane includes a hydrophobic membrane.

[0411] Example 8. The microfluidic device according to any one of the foregoing embodiments, wherein the second microfluidic channel includes: an upstream section having a simple serpentine configuration and a downstream section having a double serpentine configuration.

[0412] Example 9. The microfluidic device according to any one of the foregoing embodiments, wherein the cross-sectional area of the second microfluidic channel decreases from the input end of the second microfluidic channel to the output end of the second microfluidic channel.

[0413] Example 10. The microfluidic device according to any one of the foregoing embodiments, wherein the surface of the substrate is a first surface, and wherein a media inlet channel is defined on a second surface of the substrate opposite the first surface of the substrate, the media inlet channel being fluidly connected to the upstream section of the second microfluidic channel and being configured to be connected to a media reservoir.

[0414] Example 11. The microfluidic device according to Example 10, wherein the demulsification cartridge includes the media reservoir.

[0415] Example 12. The microfluidic device according to Example 11, wherein the media inlet channel is fluidly connected to the media reservoir via a tube extending through the substrate and the collection container.

[0416] Example 13. The microfluidic device according to Example 11 or 12, wherein the collection container is disposed in a cavity defined in the media reservoir such that the collection container is positioned between the media reservoir and the substrate.

[0417] Example 14. The microfluidic device according to any one of Examples 11 to 13, wherein the bottom surface of the media reservoir is angled with respect to the plane of the substrate.

[0418] Example 15. The microfluidic device according to any one of Examples 11 to 14, wherein the demulsification cartridge includes a duckbill valve extending through the substrate and the collection container, and the duckbill valve is configured to provide a fluid passage to the medium reservoir.

[0419] Example 16. The microfluidic device according to any one of the foregoing examples, wherein the demulsification cartridge includes a hydrophobic material disposed within the collection container.

[0420] Example 17. The microfluidic device according to any one of the foregoing examples, wherein a vacuum flow path is defined through the body of the collection container, and the vacuum flow path is configured to be able to apply a vacuum to the surface of the membrane opposite the substrate.

[0421] Example 18. The microfluidic device according to any one of the foregoing examples, which includes a reservoir fluidly connected to the first microfluidic channel via an input port defined at an input end of the first microfluidic channel.

[0422] Example 19. The microfluidic device according to Example 18, wherein the reservoir includes a base and a lid, and the base and the lid define a cavity for a fluid sample.

[0423] Example 20. The microfluidic device according to Example 19, which includes an input port in the lid of the reservoir, and the input port includes a duckbill valve.

[0424] Example 21. The microfluidic device according to Example 19 or 20, which includes an output port in the lid of the reservoir, and the output port is connected to a tube extending into the cavity of the reservoir.

[0425] Example 22. The microfluidic device according to any one of Examples 19 to 21, wherein the bottom surface of the base of the reservoir is angled relative to the lid.

[0426] Example 23. The microfluidic device according to any one of Examples 18 to 22, which includes a reservoir holder configured to receive the reservoir, and the reservoir holder includes a cooling system configured to cool the reservoir.

[0427] Example 24. The microfluidic device according to Example 23, wherein the cooling system includes a thermoelectric cooling system.

[0428] Example 25. The microfluidic device according to any one of the foregoing examples, wherein one or more cuts are defined in the microfluidic chip between the droplet generation portion and the polymerization portion.

[0429] Example 26. The microfluidic device according to Example 25, wherein the edges of the one or more incisions are angled with respect to the surface of the microfluidic chip.

[0430] Example 27. The microfluidic device according to Example 25 or 26, wherein the one or more incisions extend through the entire thickness of the microfluidic chip.

[0431] Example 28. The microfluidic device according to any of the foregoing examples, comprising a lid disposed on the surface of the microfluidic chip.

[0432] Example 29. The microfluidic device according to Example 28, wherein the lid comprises an optically transparent lid.

[0433] Example 30. The microfluidic device according to any of the foregoing examples, wherein a plurality of first microfluidic channels are defined in the surface of the microfluidic chip, and wherein the device comprises a plurality of cartridges, wherein the second microfluidic channel of each cartridge is fluidly connected to a corresponding one of the first microfluidic channels of the microfluidic chip.

[0434] Example 31. The microfluidic device according to any of the foregoing examples, wherein the device comprises an output vial, the output vial being fluidly connected to the second microfluidic channel via an output port defined at the output end of the second microfluidic channel.

[0435] Example 32. A system combinable with any of Examples 1 to 31, comprising: a microfluidic device according to any of the foregoing examples; a housing, wherein the microfluidic device is disposed within the housing; and a polymeric block, the polymeric block being received within the housing and positioned to apply a stimulus to the polymeric portion of the first microfluidic channel.

[0436] Example 33. The system according to Example 32, wherein the polymeric block comprises a thermal polymeric block configured to apply heat to the polymeric portion of the first microfluidic channel.

[0437] Example 34. The system according to Example 33, wherein the thermal polymeric block comprises a heater.

[0438] Example 35. The system according to Example 34, wherein the thermal polymeric block comprises a temperature sensor.

[0439] Example 36. The system according to Example 35, wherein the temperature sensor comprises one or more of a thermistor, a thermocouple, or a resistance temperature detector.

[0440] Example 37. The system according to Example 35 or 36, comprising a controller configured to control the operation of the resistive heater in response to temperature data received from the temperature sensor.

[0441] Example 38. The system according to any one of Examples 34 to 37, wherein the heater comprises a resistive heater.

[0442] Example 39. The system according to any one of Examples 34 to 38, wherein the thermal polymerization block comprises a heat-insulating cover, and wherein the heater is disposed within a cavity defined within the heat-insulating cover.

[0443] Example 40. The system according to any one of Examples 32 to 39, wherein the polymerization module comprises a photo-polymerization module configured to irradiate the polymerization portion of the first microfluidic channel.

[0444] Example 41. The system according to Example 40, wherein the photo-polymerization block comprises a light-emitting diode (LED).

[0445] Example 42. The system according to Example 41, wherein the photo-polymerization block comprises a photodetector.

[0446] Example 43. The system according to Example 42, comprising a controller configured to control the operation of the LED in response to light intensity data received from the photodetector.

[0447] Example 44. The system according to any one of Examples 41 to 43, wherein the LED is disposed within a cavity defined within the housing of the photo-polymerization block.

[0448] Example 45. The system according to Example 44, wherein the walls of the cavity are formed of a material capable of reflecting light of the wavelength of the light output by the LED.

[0449] Example 46. The system according to any one of Examples 41 to 45, comprising a controller configured to control the LED to emit pulsed irradiation.

[0450] Example 47. The system according to any one of Examples 32 to 46, wherein the surface of the microfluidic chip is a first surface, and wherein the polymerization block comprises: a first block disposed adjacent to the first surface of the microfluidic chip; and a second block disposed adjacent to a second surface of the microfluidic chip, the second surface being opposite to the first surface.

[0451] Example 48. The system according to Example 47, wherein the first and second blocks are fixed against the microfluidic chip by one or more springs.

[0452] Example 49. The system according to Example 47 or 48, wherein the first and second blocks are clamped to the microfluidic chip.

[0453] Example 50. The system according to any one of Examples 32 to 49, comprising a reservoir for emulsifying fluid, wherein the emulsifying fluid channel of the microfluidic device is fluidly connected to the reservoir.

[0454] Example 51. The system according to Example 50, wherein the reservoir comprises reflective ribs for fluid volume measurement, the reflective ribs being provided in a chamber of the reservoir.

[0455] Example 52. The system according to Example 50 or 51, comprising a pump provided between the reservoir for emulsifying fluid and the emulsifying fluid channel.

[0456] Example 53. The system according to Example 52, comprising a controller configured to control the operation of the pump.

[0457] Example 54. The system according to Example 53, wherein the controller is configured to control the operation of the pump to achieve a target fluid velocity in the second microfluidic channel.

[0458] Example 55. The system according to any one of Examples 32 to 54, comprising an imaging system positioned to capture an image of at least a portion of the chamber.

[0459] Example 56. The system according to Example 55, comprising a controller configured to control the flow rate of fluid through the inlet portion of the microfluidic channel based on an image captured by the imaging system.

[0460] Example 57. The system according to Example 56, wherein the controller is configured to control the flow rate of the fluid by controlling the pressure applied to a reservoir fluidly connected to the inlet portion of the microfluidic channel.

[0461] Example 58. A microfluidic chip combinable with any one of Examples 1 to 57, comprising:

[0462] A plurality of first microfluidic channels for generating an emulsion of droplets of a first fluid in a second fluid, wherein the first microfluidic channels are defined in a first surface of the microfluidic chip, wherein each first microfluidic channel is fluidly independent of each other first microfluidic channel, and wherein each first microfluidic channel comprises:

[0463] An inlet portion configured to receive the first fluid from a corresponding source of the first fluid;

[0464] A junction between the inlet portion and a corresponding second fluid channel configured to carry the second fluid; and

[0465] A chamber downstream of the junction, wherein a cross-sectional area of the chamber is larger than a cross-sectional area of the inlet portion; and

[0466] A plurality of second microfluidic channels for aggregating droplets of the emulsion to generate MOS, wherein each second microfluidic channel is fluidly connected to an outlet of a corresponding one of the first microfluidic channels,

[0467] wherein each second microfluidic channel is a serpentine channel including a first portion defined on the first surface of the microfluidic chip and a second portion defined on a second surface of the microfluidic chip opposite the first surface.

[0468] Example 59. The microfluidic chip according to Example 58, wherein each first microfluidic channel includes an outlet portion downstream of the chamber, wherein the cross-sectional area of the chamber is larger than the cross-sectional area of the outlet portion.

[0469] Example 60. The microfluidic chip according to Example 59, wherein a region of the outlet portion of each first microfluidic channel extends in a direction parallel to the corresponding chamber.

[0470] Example 61. The microfluidic chip according to any one of Examples 58 to 60, comprising a lid provided on each of the first surface and the second surface of the microfluidic chip.

[0471] Example 62. The microfluidic chip according to Example 61, wherein the lid includes an optically transparent lid.

[0472] Example 63. The microfluidic chip according to any one of Examples 58 to 62, wherein the plurality of first microfluidic channels are defined in a first region of the microfluidic chip, and wherein the plurality of second microfluidic channels are defined in a second region of the microfluidic chip different from the first region.

[0473] Example 64. The microfluidic chip according to Example 63, wherein one or more cuts are defined in the microfluidic chip between the first region and the second region.

[0474] Example 65. The microfluidic chip according to Example 64, wherein edges of the one or more cuts are angled with respect to the first and second surfaces of the microfluidic chip.

[0475] Example 66. The microfluidic chip according to Example 64 or 65, wherein the one or more incisions extend through the entire thickness of the microfluidic chip.

[0476] Example 67. The microfluidic chip according to any one of Examples 58 to 66, wherein each junction is a junction between a respective inlet portion and two corresponding second fluid channels.

[0477] Example 68. The microfluidic chip according to Example 67, wherein the junction is a right-angle junction.

[0478] Example 69. The microfluidic chip according to any one of Examples 58 to 68, wherein the microfluidic chip includes a plurality of inlet fingers, each inlet finger extending away from at least one other inlet finger and separated from each adjacent inlet finger by a gap, and wherein at least some of the inlet portions of each first microfluidic channel are defined on the surface of a corresponding inlet finger.

[0479] Example 70. The microfluidic chip according to any one of Examples 58 to 69, wherein the microfluidic chip includes a plurality of outlet fingers, each outlet finger extending away from at least one other outlet finger and separated from each adjacent outlet finger by a gap, and wherein the outlet portions of each second microfluidic channel are defined on the surface of a corresponding outlet finger.

[0480] Example 71. The microfluidic chip according to any one of Examples 58 to 70, wherein the output port of each second microfluidic channel is configured to be connected to a corresponding cartridge for demulsification of the emulsion.

[0481] Example 72. An apparatus combinable with any one of Examples 1 to 71, comprising:

[0482] A cartridge for transferring MOS from an emulsion in a hydrophobic fluid to a suspension in an aqueous fluid, the demulsification cartridge comprising:

[0483] A collection container defining a cavity for receiving the hydrophobic fluid;

[0484] A substrate disposed on the collection container, wherein microfluidic channels are defined in a first surface of the substrate facing the collection container, and wherein a medium inlet channel for the aqueous fluid is fluidly connected to an upstream portion of the microfluidic channels; and

[0485] A hydrophobic membrane disposed between the collection container and the surface of the substrate.

[0486] Example 73. The device according to Example 72, comprising a media reservoir having a cavity configured to receive the aqueous fluid, wherein the media inlet channel is fluidly connected to the media reservoir.

[0487] Example 74. The device according to Example 73, comprising a tube extending through the substrate and the collection container, wherein the media inlet channel is fluidly connected to the media reservoir via the tube.

[0488] Example 75. The device according to Example 73 or 74, wherein the collection container is disposed in the cavity of the media reservoir such that the collection container is positioned between the media reservoir and the substrate.

[0489] Example 76. The device according to any one of Examples 73 to 75, wherein the bottom surface of the media reservoir is angled relative to the plane of the substrate.

[0490] Example 77. The device according to any one of Examples 73 to 76, comprising a duckbill valve disposed through an opening in the substrate and an opening in the collection container, the duckbill valve being configured to allow the provision of an aqueous fluid into the cavity of the media reservoir.

[0491] Example 78. The device according to any one of Examples 72 to 77, wherein the surface of the substrate is a first surface, and wherein the media inlet channel is defined on a second surface of the substrate opposite the first surface.

[0492] Example 79. The device according to any one of Examples 72 to 78, wherein the cross-sectional area of the microfluidic channel is greater at the upstream end of the microfluidic channel than at the downstream end of the microfluidic channel.

[0493] Example 80. The device according to Example 79, wherein the upstream portion of the microfluidic channel has a simple serpentine configuration, and wherein the downstream portion of the microfluidic channel has a double serpentine configuration.

[0494] Example 81. The device according to any one of Examples 72 to 80, comprising a hydrophobic absorbent material disposed in the cavity of the collection container.

[0495] Example 82. A method combinable with any one of Examples 1 to 81, comprising:

[0496] generating droplets of a first fluid in a hydrophobic fluid in a droplet generation portion of a first microfluidic channel defined in a surface of a microfluidic chip, the first fluid comprising a biological material and a matrix material, and

[0497] In the polymerization section of the first microfluidic channel, a stimulus is applied to the generated droplets to polymerize the matrix material, thereby forming MOS emulsified in the hydrophobic fluid;

[0498] Transferring the MOS from the emulsion to a suspension in an aqueous fluid includes:

[0499] Flowing a mixture of the aqueous fluid and the emulsion of MOS in the hydrophobic fluid along a second microfluidic channel defined in a substrate;

[0500] When the mixture flows along the second microfluidic channel, transferring the hydrophobic fluid through a membrane forming the wall of the second microfluidic channel.

[0501] Example 83. The method according to Example 82, wherein generating droplets of the first fluid includes generating the droplets at a junction between the first microfluidic channel and one or more channels carrying the hydrophobic fluid.

[0502] Example 84. The method according to Example 83, which includes controlling the flow rate of the hydrophobic fluid.

[0503] Example 85. The method according to any one of Examples 82 to 84, which includes controlling the flow rate of the first fluid based on a determined size of the generated droplets.

[0504] Example 86. The method according to Example 85, which includes determining the size of the generated droplets based on an image of the droplets in the droplet generation section of the first microfluidic channel.

[0505] Example 87. The method according to any one of Examples 82 to 86, wherein applying a stimulus to the generated droplets includes heating the droplets.

[0506] Example 88. The method according to any one of Examples 82 to 87, wherein applying a stimulus to the generated droplets includes irradiating the droplets with light having a wavelength configured to induce polymerization of the matrix material.

[0507] Example 89. The method according to Example 88, wherein the surface of the microfluidic chip is a first surface, and wherein the polymerization section of the first microfluidic channel is defined on both the first surface and the second surface of the microfluidic chip, and wherein irradiating the droplets includes irradiating the first surface and the second surface of the microfluidic chip.

[0508] Example 90. The method according to Example 88 or 89, wherein irradiating the droplets includes irradiating the droplets with a pulsed irradiation.

[0509] Example 91. The method according to any one of Examples 82 to 90, which includes receiving the transferred hydrophobic fluid in a collection container, wherein the membrane is disposed between the collection container and the substrate.

[0510] Example 92. The method according to any one of Examples 82 to 91, wherein transferring the hydrophobic fluid through the membrane includes applying a vacuum to the membrane.

[0511] Example 93. The method according to any one of Examples 82 to 92, which includes providing a suspension of MOS in an aqueous fluid to an output vial.

[0512] Example 94. The method according to any one of Examples 82 to 93, which includes: generating droplets of each of a plurality of first fluids in each of a plurality of fluidically independent first microfluidic channels defined in the surface of the microfluidic chip; and applying a stimulus to the generated droplets in each first microfluidic channel to form MOS.

[0513] Example 95. A method combinable with any one of Examples 1 to 94, which includes:

[0514] Flowing a first fluid through a first microfluidic channel of a microfluidic device, wherein the first fluid includes a biomaterial and a matrix material;

[0515] Flowing a second fluid through a second microfluidic channel of the microfluidic device, wherein the first fluid is immiscible with the second fluid;

[0516] Combining the first fluid and the second fluid in a third channel of the microfluidic device to form droplets of the first fluid dispersed in the second fluid;

[0517] Capturing multiple exposures of droplets of the first fluid in a third microfluidic channel in a single image captured by an imaging device, the capturing of the multiple exposures including:

[0518] During a single frame of the imaging device, irradiating a region of the third microfluidic channel with a plurality of consecutive irradiation pulses by a light source;

[0519] Determining a characteristic of the droplet based on an analysis of the captured exposures; and

[0520] Controlling the flow of the first fluid in the first microfluidic channel, the flow of the second fluid in the second microfluidic channel, or both, based on the determined characteristic of the droplet.

[0521] Example 96. The method according to Example 95, wherein flowing the second fluid through the second microfluidic channel includes flowing the second fluid through two second microfluidic channels, and wherein combining the first fluid and the second fluid includes combining the first fluid and the second fluid at a junction between the first microfluidic channel and the two second microfluidic channels.

[0522] Example 97. The method according to Example 96, which includes identifying the droplet in each of the captured exposures.

[0523] Example 98. The method according to Example 97, which includes identifying the leading edge of the droplet in each of the captured exposures, the trailing edge of the droplet in each of the captured exposures, or both.

[0524] Example 99. The method according to Example 97, which includes identifying the droplet using frequency domain analysis or machine vision analysis or by creating a best fit circle.

[0525] Example 100. The method according to any one of Examples 96 to 99, wherein determining the characteristics of the droplet based on an analysis of the captured exposure includes determining the distance traveled by the droplet between a first irradiation pulse time and a second irradiation pulse time in the irradiation pulses.

[0526] Example 101. The method according to any one of Examples 96 to 100, wherein determining the characteristics of the droplet based on an analysis of the captured exposure includes determining the velocity of the droplet in the third microfluidic channel.

[0527] Example 102. The method according to any one of Examples 96 to 101, wherein determining the characteristics of the droplet based on an analysis of the captured exposure includes determining the size of the droplet.

[0528] Example 103. The method according to any one of Examples 96 to 102, wherein determining the characteristics of the droplet based on an analysis of the captured exposure includes determining the distance between the droplet and an adjacent droplet in the third microfluidic channel.

[0529] Example 104. The method according to any one of Examples 96 to 103, which includes determining an estimated number of droplets formed by combining the first fluid and the second fluid based on the determined characteristics of the droplet.

[0530] Example 105. The method according to any one of Examples 96 to 104, which includes determining a droplet generation rate based on the determined characteristics of the droplet.

[0531] Example 106. The method according to any one of Examples 96 to 105, which includes controlling the flow of the first fluid in the first microfluidic channel to obtain droplets of a target size.

[0532] Example 107. The method according to any one of Examples 96 to 106, which includes controlling the flow of the second fluid in the second microfluidic channel to obtain droplets flowing in the third microfluidic channel at a target speed.

[0533] Example 108. The method according to Example 107, wherein controlling the flow of the first fluid in the first microfluidic channel includes controlling the pressure of a fluid reservoir fluidly coupled to the first microfluidic channel, the fluid reservoir containing the first fluid.

[0534] Example 109. The method according to Example 107 or 108, wherein controlling the flow of the second fluid in the second microfluidic channel includes operating a pump with a programmable flow rate to control the flow rate of the second fluid from a fluid reservoir fluidly coupled to the second microfluidic channel.

[0535] Example 110. The method according to any one of Examples 96 to 109, wherein obtaining the multiple exposures includes synchronizing the shutter of the imaging device with the light source of the imaging device.

[0536] Example 111. The method according to Example 110, wherein synchronizing the shutter of the imaging device with the light source of the imaging device includes controlling the shutter of the imaging device to remain open during irradiation of the region of the third microfluidic channel with a plurality of consecutive irradiation pulses.

[0537] Example 112. The method according to any one of Examples 96 to 111, wherein irradiating the region of the third microfluidic channel with a plurality of consecutive irradiation pulses includes controlling the light source to emit irradiation pulses, each irradiation pulse having a duration of 5 microseconds (μsec) to 125 μsec, for example, 25 to 50 μsec, and the pulse interval being between about 1 millisecond (ms) and about 50 ms, for example, 1 - 30 ms.

[0538] Example 113. The method according to any one of Examples 96 to 112, wherein irradiating the region of the third microfluidic channel with a plurality of consecutive irradiation pulses includes irradiating the region of the third microfluidic channel with a first irradiation pulse of a first color and a second irradiation pulse of a second color.

[0539] Example 114. The method according to any one of Examples 96 to 113, which includes irradiating the region of the third microfluidic channel with a collimated light source.

[0540] Example 115. The method according to any one of Examples 96 to 114, comprising exposing the droplets of the first fluid to a stimulus sufficient to polymerize the matrix material in the droplets, thereby forming polymerized droplets dispersed in the second fluid.

[0541] Example 116. The method according to Example 115, comprising using the polymerized droplets to determine a patient-specific therapy.

[0542] Example 117. A system combinable with any one of Examples 1 to 116, comprising:

[0543] A first microfluidic channel configured to be connected to a source of a first fluid;

[0544] A second microfluidic channel configured to be connected to a source of a second fluid, wherein the first microfluidic channel and the second microfluidic channel intersect at a junction;

[0545] A first controller configured to control flow regulators coupled to the source of the first fluid and the source of the second fluid;

[0546] A third microfluidic channel downstream of the junction;

[0547] An imaging system comprising an imaging device and a light source;

[0548] A second controller configured to control the imaging system to capture multiple exposures of at least a portion of the third microfluidic channel in a single image captured by the imaging device; and

[0549] A computing device comprising one or more processors coupled to a memory, the one or more processors configured to cause the computing device to:

[0550] Analyze the multiple exposures of the at least a portion of the third microfluidic channel to determine characteristics of droplets in each of the multiple captured exposures; and

[0551] Cause the first controller to control the flow regulators based on the determined characteristics of the droplets.

[0552] Example 118. The system according to Example 117, wherein the second controller is configured to: control the shutter of the imaging device to open; and control the light source to generate a plurality of consecutive illumination pulses while the shutter of the imaging device is open.

[0553] Example 119. The system according to Example 117 or 118, wherein the one or more first controllers are configured to control: a valve or a pump for controlling the pressure of a first fluid reservoir fluidly coupled to the first microfluidic channel, the fluid reservoir containing a first fluid; and a pump having a programmable flow rate for controlling the flow rate of the second fluid from a second fluid reservoir fluidly coupled to the second microfluidic channel.

[0554] Example 120. The system according to any one of Examples 117 to 119, wherein the light source includes a light emitting diode.

[0555] Example 121. The system according to any one of Examples 117 to 120, wherein the light source includes a collimated light source.

[0556] Example 122. The system according to any one of Examples 117 to 121, wherein the light source includes a plurality of light sources, each light source being configured to emit light of a different color.

[0557] Example 123. The system according to any one of Examples 117 to 122, wherein the one or more processors and the memory are configured such that the computing device identifies the droplets in each of the captured exposures.

[0558] Example 124. The system according to Example 123, wherein the one or more processors and the memory are configured such that the computing device identifies the leading edge of the droplet in each of the captured exposures, the trailing edge of the droplet in each of the captured exposures, a best fit circle, or a combination thereof.

[0559] Example 125. The system according to Example 124, wherein the one or more processors and the memory are configured such that the computing device uses frequency domain analysis or machine vision analysis to identify the droplet.

[0560] Example 126. The system according to any one of Examples 116 to 124, comprising a plurality of second microfluidic channels, wherein the first microfluidic channel and the plurality of second microfluidic channels intersect at the junction.

[0561] Example 127. The system according to any one of Examples 117 to 126, wherein the one or more processors and the memory are configured such that the computing device determines the velocity of the droplet in the third microfluidic channel.

[0562] Example 128. The system according to any one of Examples 117 to 127, wherein the one or more processors and the memory are configured such that the computing device determines the size of the droplet in the third microfluidic channel.

[0563] Example 129. The system according to any one of Examples 117 to 128, wherein the system comprises: a fourth microfluidic channel connected to the third microfluidic channel; and a heating element disposed adjacent to the fourth microfluidic channel and configured to apply heat to at least a portion of the fourth microfluidic channel.

[0564] Example 130. A system combinable with any one of Examples 1 to 129, comprising:

[0565] a device configured to facilitate an interaction between a first fluid flow and a second fluid flow within a flow path of the device;

[0566] an optical sensor configured to obtain one or more images representative of the flow path;

[0567] an image analysis module configured to:

[0568] process the one or more images to identify at least one droplet generated in the flow path of the device through the interaction between the first fluid flow and the second fluid flow, and

[0569] estimate the size of the at least one droplet; and

[0570] a control system configured to:

[0571] determine that the size of the at least one droplet meets a threshold condition; and

[0572] in response to determining that the size of the at least one droplet meets the threshold condition, generate a signal that causes an adjustment to the flow rate of at least one of the first fluid flow or the second fluid flow.

[0573] Example 131. The system according to Example 130, wherein the device is a microfluidic device.

[0574] Example 132. The system according to Example 130 or 131, wherein the first fluid flow comprises the flow of an aqueous solution.

[0575] Example 133. The system according to any one of Examples 130 to 132, wherein the first fluid flow comprises the flow of a solution comprising an unpolymerized mixture comprising a fluid matrix material and cells.

[0576] Example 134. The system according to any one of Examples 130 to 133, wherein the second fluid flow comprises the flow of a hydrophobic solution.

[0577] Example 135. The system according to any one of Examples 130 to 134, wherein the one or more images include an image of at least one droplet in the region of the device, and wherein the at least one droplet is not compressed by one or more walls of the device.

[0578] Example 136. The system according to any one of Examples 130 to 135, wherein processing the one or more images obtained by the optical sensor includes:

[0579] Detecting an edge of the at least one droplet in at least one of the one or more images;

[0580] Identifying a first set of pixels corresponding to the detected edge of the at least one droplet;

[0581] Identifying a circle corresponding to the at least one droplet based on the first set of pixels;

[0582] Identifying a second set of pixels, wherein the second set of pixels includes a subset of the first set of pixels disposed within a threshold distance from the circumference of the identified circle; and

[0583] Calculating a metric representing a distance of at least a portion of the second set of pixels from a predetermined position within the at least one droplet.

[0584] Example 137. The system according to Example 136, wherein processing the one or more images obtained by the optical sensor further includes: enhancing at least one of the one or more images after detecting the edge of the at least one droplet.

[0585] Example 138. The system according to Example 136 or 137, wherein processing the one or more images obtained by the optical sensor further includes: reducing the size of at least one of the one or more images before identifying the first set of pixels corresponding to the detected edge of the at least one droplet, and enlarging at least one of the one or more images after identifying the first set of pixels corresponding to the detected edge of the at least one droplet.

[0586] Example 139. The system according to any one of Examples 135 to 138, wherein the image analysis module is configured to estimate the size of the at least one droplet based on the calculated metric.

[0587] Example 140. The system according to Example 139, wherein the calculated metric includes a weighted average of individual distances of the portion of the second set of pixels from a predetermined position within the at least one droplet, and wherein one or more weight values of the weighted average are based on intensity values of the portion of the second set of pixels.

[0588] Example 141. The system according to any one of Examples 136 to 140, wherein processing the one or more images further comprises excluding data corresponding to the detected edges of the at least one droplet if one or more filtering conditions are met.

[0589] Example 142. The system according to Example 141, wherein the one or more filtering conditions include an indication that the first set of pixels corresponds to the detected edges of a plurality of imaged droplets.

[0590] Example 143. The system according to Example 141 or 142, wherein the one or more filtering conditions include an indication that the first set of pixels overlaps at least one additional set of pixels.

[0591] Example 144. The system according to any one of Examples 141 to 143, wherein the one or more filtering conditions include an indication of a detected signal that meets a threshold signal level condition and that originates within the perimeter of the first set of pixels.

[0592] Example 145. The system according to any one of Examples 141 to 144, wherein the one or more filtering conditions include determining that the first set of pixels is less than a threshold proximity from the imaging wall of the device.

[0593] Example 146. The system according to any one of Examples 130 to 145, wherein the image analysis module is configured to estimate the size of the at least one dro...

Claims

1. A microfluidic device, comprising: A microfluidic chip for generating micro - organic spheres (MOS), wherein a first microfluidic channel is defined in the surface of the microfluidic chip, and the first microfluidic channel comprises: A droplet generation section, which includes an inlet section, a junction between the inlet section and an emulsifying fluid channel, and a chamber downstream of the junction, wherein the cross - sectional area of the chamber is larger than the cross - sectional area of the inlet section, and A polymerization section downstream of the droplet generation section, the polymerization section having a serpentine configuration; and A cartridge for MOS demulsification, the cartridge comprising: A collection container; A substrate disposed on the collection container, wherein a second microfluidic channel is defined in the surface of the substrate facing the collection container, and wherein the second microfluidic channel is fluidly connected to the output of the polymerization section of the first microfluidic channel; and A membrane disposed between the collection container and the surface of the substrate.

2. The microfluidic device according to claim 1, wherein the droplet generation section of the first microfluidic channel includes an outlet section downstream of the chamber, wherein the cross - sectional area of the chamber is larger than the cross - sectional area of the outlet section.

3. The microfluidic device according to any one of the preceding claims, wherein the surface of the microfluidic chip is a first surface, and wherein the polymerization section of the microfluidic channel is defined on the first surface of the microfluidic chip and on a second surface of the microfluidic chip opposite the first surface.

4. The microfluidic device according to any one of the preceding claims, wherein the junction includes a junction having two hydrophobic fluid channels.

5. The microfluidic device according to any one of the preceding claims, wherein the membrane includes a hydrophobic membrane.

6. The microfluidic device according to any one of the preceding claims, wherein the surface of the substrate is a first surface, and wherein a medium inlet channel is defined on a second surface of the substrate opposite the first surface of the substrate, the medium inlet channel being fluidly connected to an upstream section of the second microfluidic channel and being configured to be connected to a medium reservoir.

7. The microfluidic device according to any one of the preceding claims, wherein the demulsification cartridge includes a hydrophobic material disposed within the collection container.

8. The microfluidic device according to any one of the preceding claims, wherein a vacuum flow path is defined through the body of the collection container, the vacuum flow path being configured to be able to apply a vacuum to the surface of the membrane opposite the substrate.

9. The microfluidic device according to any one of the preceding claims, which includes a reservoir fluidly connected to the first microfluidic channel via an input port defined at the input end of the first microfluidic channel.

10. The microfluidic device according to any one of the preceding claims, wherein one or more cuts are defined in the microfluidic chip between the droplet generation section and the polymerization section.

11. The microfluidic device according to any one of the preceding claims, wherein a plurality of first microfluidic channels are defined in the surface of the microfluidic chip, and wherein the device includes a plurality of cartridges, wherein the second microfluidic channel of each cartridge is fluidly connected to a corresponding one of the first microfluidic channels of the microfluidic chip.

12. The microfluidic device according to any one of the preceding claims, wherein the device includes an output vial, the output vial being fluidly connected to the second microfluidic channel via an output port defined at the output end of the second microfluidic channel.

13. A system, comprising: The microfluidic device according to any one of the preceding claims; A housing, wherein the microfluidic device is disposed in the housing; And A polymeric block, the polymeric block being received in the housing and positioned to apply a stimulus to the polymeric portion of the first microfluidic channel.

14. The system according to claim 13, wherein the polymeric block includes a thermal polymeric block configured to apply heat to the polymeric portion of the first microfluidic channel.

15. The system according to any one of claims 13 to 14, wherein the polymeric block includes a photo-polymeric block configured to irradiate the polymeric portion of the first microfluidic channel.

16. The system according to any one of claims 13 to 15, wherein the surface of the microfluidic chip is a first surface, and wherein the polymeric block includes: A first block disposed adjacent to the first surface of the microfluidic chip; And A second block disposed adjacent to a second surface of the microfluidic chip, the second surface being opposite to the first surface.

17. The system according to any one of claims 13 to 16, comprising an imaging system positioned to capture an image of at least a portion of the chamber.

18. A microfluidic chip, comprising: A plurality of first microfluidic channels for generating an emulsion of droplets of a first fluid in a second fluid, wherein the first microfluidic channels are defined in a first surface of the microfluidic chip, wherein each first microfluidic channel is fluidly independent of each other first microfluidic channel, and wherein each first microfluidic channel includes: An inlet portion configured to receive the first fluid from a corresponding source of the first fluid; A junction between the inlet portion and a corresponding second microfluidic channel configured to carry the second fluid; and A chamber downstream of the junction, wherein the cross-sectional area of the chamber is greater than the cross-sectional area of the inlet portion; and A plurality of second microfluidic channels for polymerizing the droplets of the emulsion to generate MOS, wherein each second microfluidic channel is fluidly connected to the outlet of a corresponding one of the first microfluidic channels, wherein each second microfluidic channel is a serpentine channel, the serpentine channel including a first portion defined on the first surface of the microfluidic chip and a second portion defined on a second surface of the microfluidic chip opposite to the first surface.

19. A device, comprising: A cartridge for transferring MOS from an emulsion in a hydrophobic fluid to a suspension in an aqueous fluid, the demulsification cartridge comprising: A collection container defining a cavity for receiving the hydrophobic fluid; A substrate disposed on the collection container, wherein a microfluidic channel is defined in a first surface of the substrate facing the collection container, and wherein a media inlet channel for the aqueous fluid is fluidly connected to an upstream portion of the microfluidic channel; and A hydrophobic membrane disposed between the collection container and the surface of the substrate.

20. A method, comprising: Generating droplets of a first fluid in a hydrophobic fluid in a droplet generation portion of a first microfluidic channel defined in a surface of a microfluidic chip, the first fluid comprising a biomaterial and a matrix material, and Applying a stimulus to the generated droplets in a polymerization portion of the first microfluidic channel to polymerize the matrix material, thereby forming MOS emulsified in the hydrophobic fluid; Transferring the MOS from the emulsion to a suspension in an aqueous fluid includes: Flowing a mixture of the aqueous fluid and the emulsion of MOS in the hydrophobic fluid along a second microfluidic channel defined in a substrate; When the mixture flows along the second microfluidic channel, transferring the hydrophobic fluid through a membrane forming a wall of the second microfluidic channel.

21. A method, comprising: Flowing a first fluid through a first microfluidic channel of a microfluidic device, wherein the first fluid comprises a biomaterial and a matrix material; Flowing a second fluid through a second microfluidic channel of the microfluidic device, wherein the first fluid is immiscible with the second fluid; Combining the first fluid and the second fluid in a third channel of the microfluidic device to form droplets of the first fluid dispersed in the second fluid; Capturing multiple exposures of the droplets of the first fluid in a third microfluidic channel in a single image captured by an imaging device, the capturing of the multiple exposures including: During a single frame of the imaging device, irradiating a region of the third microfluidic channel with a plurality of consecutive illumination pulses by a light source; Determining characteristics of the droplets based on an analysis of the captured exposures; and Controlling the flow of the first fluid in the first microfluidic channel, the flow of the second fluid in the second microfluidic channel, or both, based on the determined characteristics of the droplets.

22. The method according to claim 21, comprising identifying the droplets in each of the captured exposures.

23. The method according to any one of claims 21 to 22, wherein determining the characteristics of the droplets based on an analysis of the captured exposures includes determining a distance traveled by the droplets between a time of a first illumination pulse and a time of a second illumination pulse in the illumination pulses.

24. The method according to any one of claims 21 to 23, wherein determining the characteristics of the droplets based on an analysis of the captured exposures includes determining the size of the droplets.

25. The method according to any one of claims 21 to 24, which includes controlling the flow of the first fluid in the first microfluidic channel to obtain droplets of a target size.

26. The method according to any one of claims 21 to 25, which includes controlling the second fluid in the second microfluidic channel or both to obtain droplets flowing in the third microfluidic channel at a target speed.

27. A system, which includes: A first microfluidic channel, which is configured to be connected to a source of a first fluid; A second microfluidic channel, which is configured to be connected to a source of a second fluid, wherein the first microfluidic channel intersects the second microfluidic channel at a junction; A first controller, which is configured to control a flow regulator coupled to the source of the first fluid and the source of the second fluid; A third microfluidic channel downstream of the junction; An imaging system, which includes an imaging device and a light source; A second controller, which is configured to control the imaging system to capture multiple exposures of at least a portion of the third microfluidic channel in a single image captured by the imaging device; And A computing device, which includes one or more processors coupled to a memory, and the one or more processors are configured to cause the computing device to: Analyze the multiple exposures of at least a portion of the third microfluidic channel to determine the characteristics of droplets in each of the multiple captured exposures; And Cause the first controller to control the flow regulator based on the determined characteristics of the droplets.

28. A system, which includes: A device, which is configured to facilitate an interaction between a first fluid flow and a second fluid flow within a flow path of the device; An optical sensor, which is configured to obtain one or more images representing the flow path; An image analysis module, which is configured to: Process the one or more images to identify at least one droplet generated in the flow path of the device through the interaction between the first fluid flow and the second fluid flow, and Estimate the size of the at least one droplet; And A control system, which is configured to: Determine that the size of the at least one droplet satisfies a threshold condition, and In response to determining that the size of the at least one droplet satisfies the threshold condition, generate a signal that causes an adjustment to the flow rate of at least one of the first fluid flow or the second fluid flow.

29. The system according to claim 28, wherein processing the one or more images obtained by the optical sensor includes: Detecting the edges of the at least one droplet in at least one of the one or more images; Identifying a first set of pixels corresponding to the detected edges of the at least one droplet; Identifying a circle corresponding to the at least one droplet based on the first set of pixels; Identifying a second set of pixels, wherein the second set of pixels includes a subgroup of the first set of pixels disposed within a threshold distance from the circumference of the identified circle; And Calculate a metric representing the distance of at least a portion of the second set of pixels from a predetermined location within the at least one droplet.

30. The system according to claim 28 or 29, wherein the control system includes a feedback controller configured to generate the signal that causes the adjustment of the flow rate of at least one of the first fluid flow or the second fluid flow using proportional control, integral control, and / or derivative control.

31. A method comprising: One or more images represent flow paths within a microfluidic system that facilitates an interaction between a first fluid flow and a second fluid flow; Processing the one or more images to identify at least one droplet generated in the flow path through the interaction between the first fluid flow and the second fluid flow; Estimating the size of the at least one droplet; Determining that the size of the at least one droplet meets a threshold condition; And In response to determining that the size of the at least one droplet meets the threshold condition, generating a signal that causes an adjustment of the flow rate of at least one of the first fluid flow or the second fluid flow.

32. The method according to claim 31, wherein the signal is configured to increase or decrease the flow rate of at least one of the first fluid flow or the second fluid flow based on the size of the at least one droplet.

Images