Microscopic imaging-based droplet-by-droplet screening microfluidic chip and system

Abstract

The application discloses a kind of microfluidic chip and system based on microimaging drop-by-drop screening.The chip includes droplet capture channel and droplet release channel;The droplet capture channel is arranged in the imaging area of the microfluidic chip, and the inlet of the droplet capture channel accesses cell droplets, and the outlet is communicated with the droplet release channel;Multiple independent capture cavities are sequentially arranged in the droplet capture channel, and the upper and lower inner surfaces of the independent capture cavities are curved to retain cell droplets;The droplet release channel has a straight channel communicated with the droplet capture channel, and a branch channel communicated laterally, and the branch channel accesses oil phase, so that the distance between cell droplets in the straight channel is increased.The application balances the gap between the two, realizes the orderly and controllable release of cell droplets, and carries out drop-by-drop screening operation based on microimaging.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chips, and more specifically, relates to a microfluidic chip and system for droplet-by-droplet screening based on microscopic imaging. Background Technology

[0002] In functional studies of cells and gene expression, researchers often need to establish models relating cellular dynamics to gene expression levels—that is, how a particular gene expression level influences cellular phenotype or dynamic behavior. In previous studies, as cell containers have become increasingly smaller, from traditional culture dishes to microcompartments designed using microfluidic chips, the focus of research has shifted from populations of cells to precise single cells. When researchers focus on the phenotype and dynamic behavior of individual cells—such as the killing effect of immune cells on target cells or the escape behavior of target cells—imaging observation alone often fails to trace the underlying determinants. Therefore, it is necessary to perform gene sequencing at the single-cell level to reveal the molecular mechanisms underlying cellular behavior. Using microfluidic chips provides a simple and efficient method for transitioning from cellular-level imaging to gene-level sequencing analysis.

[0003] Existing droplet microfluidic technologies often focus on only one aspect, making it difficult to simultaneously achieve imaging capture and gene sequencing. For example, droplet capture chips typically employ geometrically confined structures, forming multiple chambers in the capture region to achieve spatial separation and capture of droplets. This is suitable for arrayed residence and observation on the chip, but sequential release during the release phase is difficult, making subsequent tracking and sequencing of target droplets challenging. Similarly, droplet sorting chips usually combine physical field control for online discrimination, such as valves or electromagnetic manipulation, to automatically select droplets that meet specific conditions. However, since the goal of sorting chips is to instantly separate droplets according to discrimination conditions, this technology does not actively target downstream sequential reception, and upstream imaging of the droplet interior is not performed. In short, there is currently a lack of chips that can simultaneously capture and image a single droplet and perform gene sequencing, and establish models of the dynamic behavior of cells and their gene expression levels for a single droplet. Such a chip needs to be able to capture droplets upstream for stable observation and imaging, and simultaneously release droplets in an orderly manner downstream for subsequent tracking and analysis.

[0004] The core challenge of single-droplet imaging and sequential deriving lies in matching the imaging throughput with the droplet receiving frequency. In the imaging stage, limited by chip size and objective field of view, high-density droplet arrangement is necessary to achieve higher observation coverage and scanning efficiency, minimizing the spacing between adjacent droplets. In the release stage, the downstream analysis device has a defined receiving window for droplet arrival frequency, minimum time interval, and sequential integrity: if upstream release is too fast, the spacing is too small, or the timing is disordered, it can easily lead to missed connections, cascading, or droplet merging, disrupting the one-to-one correspondence between the imaging and analysis results; if upstream release is too slow, the imaging and analysis throughput is significantly reduced. On the other hand, the high flow velocity of droplets within the microscale channel and the limited space requirements of the chip itself make it impossible to simply rely on extending the channel to achieve speed reduction and distance increase. Furthermore, any measures to reshape the droplet arrangement must be implemented without interfering with microscopic imaging and without compromising droplet integrity.

[0005] Therefore, there is an urgent need in this field for a microfluidic chip that can match the upstream imaging throughput and the downstream screening throughput. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a microfluidic chip and system for droplet-by-droplet screening based on microscopic imaging. The aim is to leverage the imaging field of view of a microscope by using densely arranged droplet capture channels with independent capture cavities, combined with a droplet release channel, to improve the resolution of droplet screening and match the processing capacity of the downstream droplet collection device, thereby balancing imaging throughput and droplet reception frequency. This solves the technical problem that existing droplet microfluidic technologies struggle to efficiently and continuously achieve droplet-by-droplet screening operations.

[0007] To achieve the above objectives, according to one aspect of the present invention, a microfluidic chip for droplet-by-droplet screening based on microscopic imaging is provided, characterized in that it includes a droplet capture channel and a droplet release channel; The droplet capturing channels are arranged side-by-side within the imaging area of ​​the microfluidic chip. The imaging area is transparent, and its lower surface is an optically flat surface for use in microscopic imaging. The distance between the droplet capturing channels and the optically flat surface is less than the imaging working distance of the microscopic imaging system. The inlet of the droplet capturing channel is connected to a cell droplet, and its outlet is connected to the droplet release channel. Multiple independent capture chambers are sequentially connected in series within the droplet capture channel. The upper and lower inner surfaces of the independent capture chambers are curved to retain cell droplets. The droplet release channel has a straight channel connected to the droplet capture channel and a branch channel laterally connected to it. The branch channel is connected to the oil phase, which increases the distance between cell droplets in the straight channel. The end of the straight channel is the chip outlet.

[0008] Preferably, the effective field of view of each independent capture cavity of the microfluidic chip based on microscopic imaging is matched with the field of view of the microscopic imaging system, so that the imaging can cover a single independent capture cavity.

[0009] Preferably, in the microfluidic chip based on microscopic imaging, the droplet capturing channels are matched with the imaging range of the microscope. The imaging range refers to the spatial range in which the microscope images the droplets through mechanical movement or optical path adjustment. The depth of the imaging range in the axial direction is called the depth of field, and the projection range of the imaging range in a plane perpendicular to the axial direction is called the imaging area. The droplet capturing channels are arranged side by side, meaning that the distances between each segment of the droplet capturing channel and the optical flat surface are approximately equal, so that the independent capturing cavities connected in series on the droplet capturing channels are within the depth of field of the imaging system. Within the imaging area of ​​the microfluidic chip, the droplet capturing channels are densely arranged.

[0010] Preferably, in the microscopic imaging-based droplet-by-droplet screening microfluidic chip, the chip imaging area is formed by bonding a cover glass slide and a PDMS microstructure layer, the chip thickness is less than the working distance of the microscope objective, and the height of the independent capture cavity and the connecting channel is limited to the range allowed by the depth of field and working distance of the objective.

[0011] Preferably, in the microfluidic chip for droplet-by-droplet screening based on microscopic imaging, the droplet capture channel is matched with the scanning trajectory of the microscope, and the spacing of the independent capture cavities is matched with the step spacing of the stage of the imaging system.

[0012] Preferably, in the microscopic imaging-based droplet-by-droplet screening microfluidic chip, the droplet capture channels are continuously connected in a round-trip manner between adjacent rows to form Z-row pathways; The straight channel of the droplet release channel is designed to be narrow at the front and wide at the back according to the direction of droplet flow. The width of its starting end is the same as the width of the droplet capture channel, and the width of its terminal end is 25%-50% wider than the width of the starting end. The branch channel is connected to the straight channel at an access angle of 20° to 70°.

[0013] Preferably, in the microfluidic chip for droplet-by-droplet screening based on microscopic imaging, the upstream of the droplet capture channel is connected to the droplet generation channel; the droplet generation channel includes an aqueous phase channel and an oil phase channel, which converge at the droplet generation port and are connected to the droplet capture channel.

[0014] Preferably, in the microfluidic chip for droplet-by-droplet screening based on microscopic imaging, the aqueous phase channel consists of two parallel channels that converge via a Y-shaped structure and intersect with the main oil phase channel to form a cross-shaped droplet generation port.

[0015] According to another aspect of the present invention, a droplet-by-droplet screening system based on microscopic imaging is provided, including a droplet-by-droplet screening microfluidic chip based on microscopic imaging, a microscopic imaging system, a collection channel, and a droplet collection device for driving a sequence of containers provided by the present invention. The outlet of the droplet-by-droplet screening microfluidic chip is connected to the collection channel. Its droplet capture channel is within the imaging depth of the microscopic imaging system and within the imaging area of ​​the microscopic imaging system. The size of the droplets matches the field of view. The chip outlet is connected to the collection channel. The container sequence passes through the end of the collection channel in sequence to collect and encode cell droplets according to time sequence. The cell droplets are dispensed drop by drop and processed according to the control signals issued by the microscopic imaging system.

[0016] Preferably, in the microscopic imaging-based droplet-by-droplet screening system, the cell droplet collection rate is consistent with the movement speed of the container sequence; The container sequence is driven by a mechanical device, such as a rotary motor; the mechanical device is connected to the signal of the microscopic imaging system. The microscopic imaging system preferably employs a fluorescence microscope, which may be a light sheet microscope, a wide-field microscope, or a confocal microscope.

[0017] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: The droplet-by-droplet screening microfluidic chip provided by this invention matches the imaging flux through a droplet capture channel with densely arranged independent capture chambers. By connecting the oil phase through a branch channel that is laterally connected to the droplet release channel, the distance between droplets is increased, matching the downstream droplet receiving frequency and balancing the difference between the two, thereby realizing the orderly and controllable release of cell droplets, and thus performing droplet-by-droplet screening operation based on microscopic imaging.

[0018] The present invention provides a release mechanism that passively and controllably amplifies the spacing between adjacent droplets and reduces the linear velocity while maintaining the queue order and droplet stability, so as to achieve a smooth transition from high-density imaging to downstream reception and match the upstream imaging throughput and the downstream screening throughput.

[0019] The chip designed in this invention achieves an integrated workflow of "pairing generation—capture imaging—sequential export" through a simple and ingenious tree-like hierarchical droplet release channel. The observed droplets are numbered in the capture channel, and then exported sequentially according to these numbers. A centrifuge tube is connected to the end of the chip and assigned the same number. Subsequent sequencing or in vitro culture can trace back to the droplets in the imaging module. This chip enables high-throughput imaging observation and gene sequencing or in vitro culture of the same droplet, facilitating the precise establishment of models of cellular dynamics and gene expression levels at the single-cell level, and providing new insights into the study of cell fate decision mechanisms.

[0020] In a preferred embodiment, the droplet capturing channel is a serpentine channel, with adjacent rows continuously connected in a back-and-forth manner to form a Z-row path, connecting to a straight channel of a wider droplet release channel. The lateral branch channels of the droplet release channel are connected to the oil phase. The droplet capturing channel is used to maintain a compact droplet queue and stable morphology without changing the overall flow rate, adapting to the microscopic imaging throughput. The droplet release channel uses lateral branches to inject oil into the main channel to achieve stepwise acceleration of droplets and amplify the spacing between adjacent droplets, adapting to the screening throughput. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the microfluidic chip structure based on microscopic imaging for droplet-by-droplet screening provided by the present invention; Figure 2 This is a schematic diagram of a droplet-by-droplet screening microfluidic chip structure based on microscopic imaging provided in an embodiment of the present invention; Figure 3 These are images of droplet-by-droplet screening microfluidic chips based on microscopic imaging, provided in embodiments of the present invention. Figure 4 This is an image of the droplet capture channel of a microfluidic chip based on microscopic imaging, provided in an embodiment of the present invention. Figure 5 This is a schematic diagram of the droplet generation channel structure of a droplet-by-droplet screening microfluidic chip based on microscopic imaging, provided in an embodiment of the present invention. Figure 6 These are images of the droplet generation process of a microfluidic chip based on microscopic imaging, provided in an embodiment of the present invention. Figure 7 These are images of the droplet-by-droplet screening system using microscopic imaging provided in this embodiment of the invention; Figure 8 This is a schematic diagram of droplet release in a droplet-by-droplet screening system for microscopic imaging provided in an embodiment of the present invention; Figure 9 This is a structural diagram of the droplet release performance test of the microscopic imaging droplet-by-droplet screening system provided in the embodiment of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0023] The microfluidic chip for droplet-by-droplet screening based on microscopic imaging provided by this invention, such as... Figure 1As shown, it includes a droplet capture channel and a droplet release channel; The droplet capturing channels are densely arranged side-by-side within the imaging area of ​​the microfluidic chip. The imaging area is transparent, and its lower surface is an optically flat surface for use with the microscopic imaging system. The distance between the droplet capturing channel and the optically flat surface is less than the imaging working distance of the microscopic imaging system. The inlet of the droplet capturing channel is connected to a cell droplet, and its outlet is connected to the droplet release channel. Multiple independent capturing chambers are sequentially connected in series within the droplet capturing channel. The upper and lower inner surfaces of the independent capturing chambers are curved to retain the cell droplets, ensuring that the captured cell droplets can remain stably in the center of the capturing chamber.

[0024] The effective field of view of each independent capture cavity is matched to the field of view of the microscopic imaging system to ensure that the imaging can cover a single independent capture cavity. The upper and lower inner surfaces of each independent capture cavity are spherical with a diameter of 100-130 μm. The width and height of the droplet capture channel are between 85-100 μm and 85-100 μm, adaptively adjusted according to the size of the cell droplets. The droplet size can be controlled by the size of the droplet generation channel and the two-phase velocity ratio, thus different sizes of the independent droplet capture cavities are designed according to different capture targets and the size of the droplets to be detected. In terms of optical properties, the microfluidic chip is composed of a PDMS microstructure layer bonded to a glass substrate, exhibiting high transmittance in both visible light and commonly used fluorescence bands. The glass substrate is preferably a standard coverslip, which has a thickness compatible with the microscopic imaging system and a refractive index that is basically matched with the immersion medium of the objective lens to reduce interface reflection and spherical aberration.

[0025] The optical characteristics of the imaging region meet the imaging requirements of the microscopic imaging system. Since microscopes are often used in this system to perform cell screening, from an optical performance perspective: the droplet capture channel must match the imaging range of the microscope. The imaging range refers to the spatial range within which the microscope images the cell through mechanical movement or optical path adjustment. The depth of the imaging range along the axial direction is the depth of field, and the projection range of the imaging range onto a plane perpendicular to the axial direction is called the imaging area. Specifically, the droplet capture channels are arranged side-by-side, meaning the distance between the droplet capture channels and the optically flat surface is approximately equal, so that the independent capture cavities connected in series on the droplet capture channels are within the depth of field of the microscopic imaging system. Preferably, within the imaging region of the microfluidic chip, the droplet capture channels are densely arranged, minimizing the spacing between adjacent channels while maintaining the integrity of the channel structure, thereby improving the imaging throughput of the microscopic imaging system. In some embodiments, the chip imaging area is formed by bonding a cover glass slide to a PDMS microstructure layer, the microscopic imaging system uses a fluorescence microscope, the chip thickness is less than the working distance of the microscope objective, and the height of the independent capture cavity and the connecting channel is limited within the range allowed by the depth of field and working distance of the objective to ensure the integrity of the droplet imaging.

[0026] In a preferred embodiment, the microscopic imaging system employs a fluorescence microscope, and the droplet capture channel is matched with the scanning trajectory of the microscope. Typically, the droplet capture channel is continuously connected between adjacent rows in a back-and-forth manner to form a Z-shaped path, which is serpentine. The interval between the independent capture cavities is matched with the step spacing of the imaging system stage, which facilitates obtaining a predetermined overlap between adjacent fields of view and completing rapid scanning of the entire area.

[0027] The culture medium containing suspended cells has a different chemical polarity than the oil phase. After the culture medium and oil phase mix uniformly at their interface, they generate uniformly sized, tightly packed cell droplets containing cells. These droplets enter the droplet capture channel, where they are linearly arranged and separated by the oil phase. Each individual droplet is captured by a separate capture chamber and imaged by a microscopic imaging system within the chip's imaging area. The function of the droplet capture channel is to cooperate with the microscopic imaging system and maintain the order and stability of the cell droplets, thereby enabling observation and sorting of the cell droplets according to their order.

[0028] The droplet capture channel is connected upstream to the droplet generation channel, such as... Figure 2 As shown; the droplet generation channel includes an aqueous phase channel and an oil phase channel, which converge at the droplet generation port and are connected to the droplet capture channel; the aqueous phase is the cell culture medium, and the convergence of the aqueous phase channel and the oil phase channel is the droplet generation port. To achieve stable combination and encapsulation of two cell streams within the same droplet, the aqueous phase channel is preferably configured as two parallel channels in a multi-turn spiral shape. Inside the spiral channel, several micro-column structures are arranged along the flow direction. These micro-column structures are preferably cylinders protruding inward from the inner wall of the channel and distributed at certain angular intervals to increase the effective channel length, promoting uniform dispersion of cells within the channel. This allows both cell streams to enter the droplet generation port synchronously and with equal probability, thereby improving the success rate of cell pairing and encapsulation. The parallel channels converge via a Y-shaped structure and intersect with the main oil phase channel, forming a cross-shaped droplet generation port. At this port, the oil phase cross-flows and focuses on the aqueous phase, completing shearing to generate cell droplets. In non-pairing applications, the aqueous phase channel can be replaced with a single aqueous phase channel while maintaining the spiral channel and designing microcolumns to achieve uniform and stable single-channel droplet generation.

[0029] The droplet release channel has a straight channel that communicates with the droplet capture channel. The straight channel is wider than the droplet capture channel and has a branch channel that is laterally connected to the oil phase. This increases the distance between cell droplets within the straight channel.

[0030] The straight channel is designed to be narrow at the front and wide at the back according to the direction of droplet flow. The width of its starting end is the same as the width of the droplet capturing channel, and the width of its ending end is 25%-50% wider than the width of the starting end. The branch channel is connected to the straight channel at an access angle of 20° to 70°.

[0031] The branched channel features a multi-level branched tree structure. The trunk of this tree structure connects to the oil phase, and the number of branches increases progressively, with each level having at least two more channels than the previous level. Preferably, this tree structure distributes a relatively uniform oil phase flow rate at each level. The highest-level terminal branch connects to the droplet capture channel. Depending on the collection speed of the droplet collection device at the chip outlet, the tree-like oil phase injection structure within the droplet release channel can increase the number of branches, adjust the branch spacing, and adjust the oil phase flow rate, thereby affecting the droplet spacing. It can also directly connect droplet release channels in series as needed. Ultimately, adjustments are made to match the droplet flow rate to the collection device speed, enabling the sequential export and collection of cell droplets.

[0032] like Figure 2As shown, in the working state of the droplet release channel, oil phase is injected simultaneously from inlet A and inlet D. The oil phase at inlet A causes the droplets to flow out of the capture chamber and sequentially pass through the straight channel of droplet release. The oil phase at inlet D flows into the straight channel along the branch channel, increasing the interval distance of the droplets each time they pass through a branch. The access angle between the straight channel and the branch channel can be set between 20° and 70°. Considering the chip processing precision, the angle cannot be set too small; considering that the oil in the branch channel should have a forward pushing effect relative to the straight channel, the angle cannot be set too large. The angle set in the schematic diagram is 45°. Usually, the straight channel of the release channel is set to be narrow at the front and wide at the back to match the droplet flow velocity. The design of narrow at the front and wide at the back causes a local geometric change when the droplet first enters the straight channel, resulting in local resistance and slightly slowing down the droplet flow velocity, thus making the subsequent staged amplification and downstream reception more controllable. The width of the main channel at the beginning of the droplet release channel is preferably matched with the width of the upstream droplet capture channel; that is, the width can be set to be the same. The width at the end of the straight channel is 25%-50% wider than the width at the beginning. In the schematic diagram, the main channel has an initial width of 110 μm, a final width of 140 μm, a total length of 3370 μm, and a height of 100 μm. The width of the branch channels can be set to be the same as the width of the channel connected to inlet A, or it can be within ±30 μm of this width. In the schematic diagram, the branch channel width is 70 μm, and the length of each branch is 318 μm.

[0033] To achieve droplet-by-droplet sorting, droplets encapsulating cells need to be sorted one by one. The cell droplet flux at the microfluidic chip channel outlet needs to be adapted to the flux of the cell droplet mechanical manipulation, while the flux of the droplet capture channel needs to be matched with the resolution of the microscopic imaging to improve imaging efficiency; thus, there is a flux difference between the two. The chip's capture channel, due to the need to increase the throughput of microscopic imaging, designs the droplet spacing as small as possible, increasing the difficulty of collecting droplets at the chip outlet. However, the cell droplet flux at the chip outlet needs to be reduced. Therefore, the droplet release channel needs to balance this flux difference. At the inlet of the droplet release channel, i.e., the end connected to the droplet capture channel, the droplet flux is high, while at the outlet of the droplet release channel, i.e., the end combined with the droplet collection operation, the droplet flux is low. The droplet release channel, through a hierarchical structure, connects to the oil phase and adjusts the distance between cell droplets to regulate the cell droplet flux, adapting it to the different resolutions of the imaging and collection systems, and balancing their flux differences.

[0034] The droplet-by-droplet screening system based on microscopic imaging provided by the present invention includes a droplet-by-droplet screening microfluidic chip based on microscopic imaging, a microscopic imaging system, a collection channel, and a droplet collection device for driving a sequence of containers. The outlet of the droplet-by-droplet screening microfluidic chip is connected to the collection channel. Its droplet capture channel is within the depth of field and imaging area of ​​the microscopic imaging system, and the droplet size matches the field of view. The chip outlet is connected to the collection channel, and the container sequence passes through the end of the collection channel in sequence to collect and encode cell droplets according to time sequence. The cell droplets are dispensed drop by drop and processed according to the control signals issued by the microscopic imaging system.

[0035] The cell droplet collection rate is consistent with the movement rate of the container sequence; The container sequence is driven by a mechanical device, such as a rotary motor; the mechanical device is connected to the microscopic imaging system via signals.

[0036] The chip outlet requires sequential collection of droplets for subsequent downstream processing, such as sequencing cells within a specific cell droplet identified by a microscopic imaging system or performing independent in vitro culture. The mechanical movement of the container sequence is difficult to match the high-speed flow of droplets within the microfluidic chip; therefore, the droplet flow rate at the chip outlet needs to be controllably slowed down to match the mechanical movement of the container sequence. Simultaneously, the chip's capture channel, to increase the throughput of microscopic imaging, requires the droplet spacing to be designed as small as possible, which also increases the difficulty of collecting droplets at the chip outlet. Therefore, a droplet release channel needs to be added between the capture channel and the chip outlet to balance the throughput difference between the capture channel and the chip outlet.

[0037] The microscopic imaging system preferably employs a fluorescence microscope, which can be a light sheet microscope, a wide-field microscope, or a confocal microscope. When the microscope is a fluorescence microscope, cells in the droplet are generally labeled with fluorescent dyes to represent specific structures, and observed under different excitation conditions. When the microscope is a white light microscope, structures under bright field conditions can be observed. Furthermore, observing subcellular structures in the droplet, such as various organelles, requires the use of high-magnification objectives.

[0038] The following is an example: The microfluidic chip for droplet-by-droplet screening based on microscopic imaging provided in this embodiment, such as Figure 3 As shown, it includes a droplet capture channel and a droplet release channel; The droplet capture channel, such as Figure 4As shown, densely arranged side-by-side structures are located within the imaging area of ​​the microfluidic chip. The imaging area is transparent, and its lower surface is an optically flat surface, used to cooperate with the microscopic imaging system for imaging. The distance between the droplet capture channel and the optically flat surface is less than the imaging working distance of the microscopic imaging system. The inlet of the droplet capture channel is connected to the cell droplet, and its outlet is connected to the droplet release channel. Multiple independent capture chambers are sequentially connected in series within the droplet capture channel. The upper and lower inner surfaces of the independent capture chambers are curved to retain the cell droplets, ensuring that the captured cell droplets can remain stably in the center of the capture chamber.

[0039] The effective field of view of each independent capture cavity is matched with the field of view of the microscopic imaging system, so that the imaging can cover a single independent capture cavity; the upper and lower inner surfaces of the independent capture cavity are spherical with a diameter of 100 μm; the droplet capture channel has a width of 85 μm and a height of 100 μm.

[0040] In this embodiment, the chip imaging area is formed by bonding a cover glass slide to a PDMS microstructure layer. The chip thickness is 170 μm, and the microscope objective is an air objective with a numerical aperture of 0.8 and a working distance of about 1 mm and a depth of field of about 1 μm.

[0041] The droplet capture channels are continuously connected in a back-and-forth manner between adjacent rows to form a Z-row path, which matches the scanning trajectory of the microscope; the stage stepping speed of the imaging system can be set to 200 micrometers / second, the interval between independent capture chambers is 60 μm, and the time to move from one chamber to another is about 0.3 seconds, which facilitates obtaining predetermined overlap between adjacent fields of view and completing rapid scanning of the entire area.

[0042] The droplet capture channel is connected upstream to the droplet generation channel, such as... Figure 5 As shown. The droplet generation channel includes an aqueous phase channel and an oil phase channel, which converge at the droplet generation port and are connected to the droplet capture channel; the aqueous phase is the cell culture medium, and the convergence point of the aqueous phase channel and the oil phase channel is the droplet generation port. To achieve stable combination and encapsulation of two cell streams in the same droplet, the aqueous phase channel is preferably configured as two parallel channels in a multi-turn spiral shape. Several micro-column structures are arranged inside the spiral channel along the flow direction. These micro-column structures are preferably cylinders protruding inward from the inner wall of the channel, with a radius of 50 μm, and are placed at 30° intervals on one side of the inner circle of the spiral channel. The parallel channels converge via a Y-shaped structure. Figure 6 As shown, after merging, it intersects with the main oil phase channel to form a cross-shaped droplet generation port. At this droplet generation port, the oil phase cross-flows and focuses on the aqueous phase and completes shearing, thereby generating cell droplets. In non-paired application scenarios, the aqueous phase channel can be replaced with a single aqueous phase channel while maintaining the spiral channel and designing micropillars to obtain uniform and stable single-channel droplet generation.

[0043] The droplet size can be controlled by the droplet generation channel size and the two-phase velocity ratio. Therefore, different independent droplet capture chamber sizes are designed according to different capture targets and the size of the droplets to be detected. In this embodiment, based on experimental requirements, the width of the spiral channel used as the aqueous phase channel is set to 85 μm, the radius of curvature is set to 1000 μm, the number of spiral channel rotations is set to 7, and the micropillars inside the channel are semi-circular structures with a radius of 50 μm, placed on one side of the inner circle of the spiral channel at 30° intervals. The oil phase channel structure is designed with a width of 85 μm and a height of 100 μm.

[0044] The droplet release channel is connected to the droplet capture channel and has laterally connected branch channels. These branch channels are connected to the oil phase, increasing the distance between cell droplets. Figure 1 As shown.

[0045] The branched channel features a multi-level branched tree structure. The trunk of this tree structure connects to the oil phase, and the number of branches increases progressively, with each level having at least two more channels than the previous level. In this embodiment, the tree structure is used to distribute a relatively uniform oil phase flow rate at each level. The highest-level terminal branch connects to the droplet capture channel. Depending on the collection speed of the droplet collection device at the chip outlet, the tree-like oil phase injection structure inside the droplet release channel can increase the number of branches, adjust the branch spacing, and adjust the oil phase flow rate. All of these will affect the droplet spacing. Furthermore, droplet release channels can be directly connected in series as needed. Ultimately, adjustments are made to match the droplet flow rate to the speed of the collection device, thus completing the sequential export and collection of cell droplets.

[0046] like Figure 2As shown, in the working state of the droplet release module, oil phase is injected simultaneously from inlet A and inlet D. The oil phase at inlet A causes the droplets to flow out of the capture chamber and sequentially pass through the straight channel of droplet release. The oil phase at inlet D flows into the straight channel along the branch channel, increasing the interval distance of the droplets each time they pass through a branch. The access angle between the straight channel and the branch channel can be set between 20° and 70°. Considering the chip processing precision, the angle cannot be set too small; considering that the oil in the branch channel should have a forward pushing effect relative to the straight channel, the angle cannot be set too large. The angle set in the schematic diagram is 45°. The width of the starting end of the main channel at the beginning of the droplet release channel is preferably matched with the width of the upstream droplet capture channel, that is, the width can be set to be the same, while the width of the end of the straight channel is 25%-50% wider than the width of the starting end. In the schematic diagram, the starting width of the main channel is 110 μm, the final width is 140 μm, the total length is 3370 μm, and the height is 100 μm. The channel width of the branch can be set to be the same as the channel width connected to the inlet A, or it can be within ±30μm of this width. In the schematic diagram, the branch channel width is 70 μm, and the length of each branch is 318 μm. Branches are added step by step from upstream to downstream, with two branches added at the exit of each level.

[0047] In terms of optical properties, the microfluidic chip is composed of a PDMS microstructure layer bonded to a glass substrate, exhibiting high transmittance in both visible light and commonly used fluorescence bands. The glass substrate is a standard coverslip, with a thickness compatible with the microscopic imaging system and a refractive index that is essentially matched to the immersion medium of the objective lens, thereby reducing interface reflection and spherical aberration. The specific design and manufacturing process is as follows: Based on the designed planar channel structure of the microfluidic chip, the chip layout was drawn using AutoCAD, and the mask was laser-processed. A single-crystal silicon wafer, thoroughly cleaned with acetone and isopropanol, was spin-coated with photoresist to a thickness of approximately 100 μm. The mask was then attached to the spin-coated silicon wafer, exposed and developed using a UV lithography machine, and excess photoresist was washed away to obtain a silicon wafer template with the chip channel pattern.

[0048] PDMS prepolymer and curing agent were thoroughly mixed at a mass ratio of 10:1 and poured onto the silicon wafer template. Vacuum was applied to remove air bubbles, and the mixture was then cured in an 80°C oven. After curing, the PDMS layer was peeled off the template. Liquid inlets and outlets were fabricated on the PDMS using a 0.5mm punch, resulting in a PDMS chip body with droplet generation, trapping, and release structures. A clean glass substrate and the PDMS chip body were treated together in a plasma cleaner before bonding. The bonding was then stabilized in an 80°C oven for 1–2 hours, yielding the final microfluidic chip. This chip, attached to a glass substrate approximately 170μm thick, can be directly placed on the stage of an inverted fluorescence microscope. Using a 20x air objective, the working distance reaches 1mm, and the field of view can be adjusted to the size of an independent trapping chamber, approximately 125μm x 125μm. Different droplets can be observed by scanning the field of view in a zigzag pattern on the stage. Since the chip is mounted on a cover glass with a thickness of 170 μm, it will not affect the working distance for imaging.

[0049] The culture medium containing suspended cells has a different chemical polarity than the oil phase. After the culture medium and oil phase mix uniformly at their interface, they generate uniformly sized, tightly packed cell droplets containing cells. These droplets enter the droplet capture channel, where they are linearly arranged and separated by the oil phase. Each individual droplet is captured by a separate capture chamber and imaged by a microscopic imaging system within the chip's imaging area. The function of the droplet capture channel is to cooperate with the microscopic imaging system and maintain the order and stability of the cell droplets, thereby enabling observation and sorting of the cell droplets according to their order.

[0050] To achieve droplet-by-droplet sorting, droplets encapsulating cells need to be sorted one by one. The velocity at the exit of the microfluidic chip channel needs to be compatible with the speed of droplet mechanical manipulation, while the size of the droplet capture channel needs to match the resolution of the microscopic imaging to improve imaging efficiency; thus, there is a flux difference between the two. Because the chip's capture channel needs to increase the throughput of microscopic imaging, the droplet spacing is designed to be as small as possible, increasing the difficulty of collecting droplets at the chip exit. However, at the chip exit, the cell droplet velocity needs to be slowed down. Therefore, the droplet release channel needs to balance this flux difference. At the inlet of the droplet release channel, i.e., the end connected to the droplet capture channel, the droplet velocity is high, while at the outlet of the droplet release channel, i.e., the end combined with the droplet collection operation, the droplet velocity is slow. The droplet release channel uses a hierarchical structure, i.e., access to the oil phase, to adjust the distance between cell droplets, thereby regulating the cell droplet velocity to adapt to the different resolutions of the imaging and collection systems and balancing their flux differences.

[0051] The system uses an injection pump as the pump in the microfluidic system. Figure 7 As shown, and operate according to the following control methods: The aqueous phase is a culture medium in which cells are suspended, and its chemical polarity differs from that of the oil phase.

[0052] During the droplet formation stage, liquid is supplied to the oil phase (inlet A) and the water phase (inlet B and inlet C) respectively to form monodisperse water-in-oil droplets in the cross-shaped nozzle. The oil phase flow rate must be greater than the water phase flow rate, and the flow rates of the oil and water phases are set to approximately 2.8 μL / min and 0.5 μL / min, respectively. After static observation, the oil phase injection pumps corresponding to the droplet formation channel (inlet A) and the droplet release channel (inlet D) are turned on. The oil phase flow rate in the droplet formation channel must be less than the oil phase flow rate in the droplet release channel, and the flow rates are set to approximately 0.3 μL / min and 0.8 μL / min, respectively. The water phase flow rate of the formed structure is 0.08 μL / min. At this time, the droplets in the capture zone enter the straight channel of the release channel. The spacing between the droplets increases under the synergistic effect of the oil injected at inlet A and the branch channel inlet D. The droplets are released sequentially, and the results are as follows. Figure 8 As shown.

[0053] like Figure 9 As shown, the droplet velocity is approximately 0.5 mm / s before entering the droplet release channel, with a droplet spacing of approximately 200 μm. After leaving the droplet release channel, the droplet spacing is approximately 0.1 mm. A programmable rotary stepper motor carrying a turntable is used to receive the droplets. The rotary stepper motor speed is set to 4 rpm, and the turntable is set to hold 120 microcentrifuge tubes, so the tube switching time is 125 ms. Simultaneously, the rotary motor is set to pause for 75 ms every 1 / 120th of a revolution, meaning it pauses to receive droplets after switching a centrifuge tube. To achieve intervalized droplet reception, the droplet interval needs to be at least 0.2 seconds. Based on the droplet flow rate calculation, the droplet interval needs to be greater than 0.1 mm, which this design satisfies.

[0054] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microfluidic chip for droplet-by-droplet screening based on microscopic imaging, characterized in that, Includes droplet capture channels and droplet release channels; The droplet capturing channels are arranged side-by-side within the imaging area of ​​the microfluidic chip. The imaging area is transparent, and its lower surface is an optically flat surface for use in microscopic imaging. The distance between the droplet capturing channels and the optically flat surface is less than the imaging working distance of the microscopic imaging system. The inlet of the droplet capturing channel is connected to a cell droplet, and its outlet is connected to the droplet release channel. Multiple independent capture chambers are sequentially connected in series within the droplet capture channel. The upper and lower inner surfaces of the independent capture chambers are curved to retain cell droplets. The droplet release channel has a straight channel connected to the droplet capture channel and a branch channel laterally connected to it. The branch channel is connected to the oil phase, which increases the distance between cell droplets in the straight channel. The end of the straight channel is the chip outlet.

2. The microfluidic chip for droplet-by-droplet screening based on microscopic imaging as described in claim 1, characterized in that, The effective field of view size of each individual capture cavity is matched with the field of view of the microscopic imaging system so that the imaging can cover a single individual capture cavity.

3. The microfluidic chip for droplet-by-droplet screening based on microscopic imaging as described in claim 1, characterized in that, The droplet capturing channels are matched with the imaging range of the microscope. The imaging range refers to the spatial range in which the microscope images the object through mechanical movement or optical path adjustment. The depth of the imaging range in the axial direction is called the depth of field. The projection range of the imaging range in the plane perpendicular to the axial direction is called the imaging area. The droplet capturing channels are arranged side by side, meaning that the distances of each segment of the droplet capturing channel relative to the optical flat surface are approximately equal, so that the independent capturing cavities connected in series on the droplet capturing channels are within the depth of field of the imaging system. Within the imaging area of ​​the microfluidic chip, the droplet capturing channels are densely arranged.

4. The microfluidic chip for droplet-by-droplet screening based on microscopic imaging as described in claim 1, characterized in that, The chip imaging area is formed by bonding a cover glass slide to a PDMS microstructure layer. The chip thickness is less than the working distance of the microscope objective. The height of the independent capture cavity and the connecting channel is limited to the range allowed by the depth of field and working distance of the objective.

5. The microfluidic chip for droplet-by-droplet screening based on microscopic imaging as described in claim 1, characterized in that, The droplet capture channel is matched to the scanning trajectory of the microscope, and the spacing of the independent capture cavities is matched to the stepping distance of the imaging system stage.

6. The microfluidic chip for droplet-by-droplet screening based on microscopic imaging as described in claim 5, characterized in that, The droplet capture channels are continuously connected between adjacent rows in a round-trip manner to form Z-row paths; The straight channel of the droplet release channel is designed to be narrow at the front and wide at the back according to the direction of droplet flow. The width of its starting end is the same as the width of the droplet capture channel, and the width of its terminal end is 25%-50% wider than the width of the starting end. The branch channel is connected to the straight channel at an access angle of 20° to 70°.

7. The microfluidic chip for droplet-by-droplet screening based on microscopic imaging as described in claim 1, characterized in that, The droplet capturing channel is connected upstream to the droplet generating channel; the droplet generating channel includes an aqueous phase channel and an oil phase channel, which converge at the droplet generating port and are connected to the droplet capturing channel.

8. The microfluidic chip for droplet-by-droplet screening based on microscopic imaging as described in claim 7, characterized in that, The aqueous phase channel consists of two parallel channels that converge via a Y-shaped structure and intersect with the main oil phase channel to form a cross-shaped droplet generation port.

9. A droplet-by-droplet screening system based on microscopic imaging, characterized in that, Includes the microfluidic chip for droplet-by-droplet screening based on microscopic imaging, the microscopic imaging system, the collection channel, and the droplet collection device for driving the container sequence as described in any one of claims 1 to 8; The outlet of the droplet-by-droplet screening microfluidic chip is connected to the collection channel, and its droplet capture channel is within the imaging depth range and imaging area of ​​the microscopic imaging system, with the droplet size matching the field of view. The chip outlet is connected to the collection pipe. The container sequence passes through the end of the collection pipe in sequence, collecting and encoding cell droplets according to time sequence. The cell droplets are then dispensed drop by drop and processed according to the control signals issued by the microscopic imaging system.

10. The droplet-by-droplet screening system based on microscopic imaging as described in claim 9, characterized in that, The cell droplet collection rate is consistent with the movement rate of the container sequence; The container sequence is driven by a mechanical device, such as a rotary motor; the mechanical device is connected to the microscopic imaging system via signals. The microscopic imaging system is preferably a fluorescence microscope, which can be a light sheet microscope, a wide field microscope, or a confocal microscope.

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