Floating algae flow type microimaging sheath fluid focusing micro-fluidic chip device and floating algae flow type microimaging sheath fluid focusing micro-fluidic chip system

Abstract

The invention discloses a floating algae flow type microimaging sheath fluid focusing micro-fluidic chip device and system, and belongs to the technical field of micro-fluidic control. According to the device, the three-channel sheath fluid flow chip design is adopted, a large-channel inlet structure is combined to avoid algae cell blockage, and a sheath flow focusing technology is utilized to compress a sample flow to the center of a channel and limit the sample flow within the field depth range of a microscope objective to obtain a clear image. The arc-shaped structure of the sample channel reduces laminar flow disturbance, and a four-side transparent observation area supports panoramic depth imaging. The system comprises a microscopic imaging module, a fluid driving module and a sheath liquid flow chip module, a flow rate ratio of sheath liquid flow to sample flow is accurately controlled through a dual-channel liquid flow driving injection pump, and stable imaging within the depth of field of an objective lens is realized by combining a manual focusing platform. Experimental verification shows that the system can effectively compress sample flow to 21m nuclear flow diameter, imaging of floating algae such as chlorella, scenedesmus quadricauda and the like is clear, and the contradiction between flux and imaging quality of a traditional micro-fluidic chip is solved.

Description

Technical Field

[0001] The invention belongs to the field of microfluidics technology, and in particular relates to a sheath fluid focusing microfluidics chip device and system for planktonic algae flow microscopy imaging. Background Art

[0002] As a key technology for manipulating tiny fluids, microfluidics has shown great potential in fields such as biomedicine and microscopic imaging. In the field of obtaining phytoplankton images, imaging analysis using microfluidic chips has become an important research method. However, existing microfluidic chips face two major technical challenges in the process of algal cell imaging: First, in order to obtain clear algal cell images, the algal cells need to be confined to the depth of field of the microscope objective imaging. This limitation is usually achieved by reducing the channel size of the microfluidic chip. However, the reduction in channel size can easily lead to blockage of algal cells, which in turn affects the continuity and stability of the experiment and increases the uncertainty of the experimental results. Secondly, in order to avoid channel blockage, increasing channel size has become a common strategy. However, the increase in channel size causes some algal cells to exceed the depth of field, resulting in blurred images, reduced imaging quality, and affected the accuracy of experimental data analysis. These two problems form a trade-off in the design and application of microfluidic chips, that is, the choice of channel size directly affects the imaging quality and experimental stability. Therefore, how to avoid channel blockage while ensuring imaging quality and improve the stability and reliability of the experiment has become a key problem that needs to be solved in the current microfluidic technology in phytoplankton microscopic imaging research. Summary of the invention

[0003] The technical problems raised in the background technology include: the problem of algae cell blockage in traditional microfluidic chips: the inlet size of the traditional sample flow channel is small, and algae cells (especially large-size algae) are easy to accumulate at the inlet, resulting in reduced flux or even blockage. Laminar flow disturbance affects imaging stability: turbulence or disturbance is easy to occur when the sample flow and the sheath flow meet, resulting in the deviation of the movement trajectory of the algae cells and the inability to image stably. Depth of field limitation of the microscope objective: the traditional chip cannot compress the sample flow within the depth of field of the objective lens (such as 21µm core flow diameter), resulting in blurred or partially out-of-focus imaging of algae cells. Insufficient compatibility for multi-angle imaging: the transparent surface of the conventional chip observation area is limited, and it is difficult to support full-depth lateral imaging or dynamic observation of the sheath liquid compression effect. The technical means adopted by the present invention include: structural optimization design; large channel inlet design, arc flow channel structure, three-channel sheath flow focusing; improved observation and imaging compatibility, four-sided transparent observation area, channel size adapted to the depth of field of the objective lens, system collaborative control technology, dual-channel liquid flow driven injection pump precision control, manual focusing platform integrated feedback, and anti-damage and sealing design.

[0004] The technical solution of the present invention is as follows:

[0005] A sheath fluid focusing microfluidic chip device for flow cytometry microscopy imaging of phytoplankton algae, comprising:

[0006] A sheath fluid flow chip, which has a three-channel structure, including two sheath fluid flow channels and one sample flow channel. The sample flow channel is located between the two sheath fluid flow channels, and the three meet at the observation area;

[0007] The inlet of the sample flow channel adopts a large channel opening design, with a size larger than that of a traditional microfluidic chip to avoid clogging of algal cells;

[0008] The sample flow channel has an arc structure in the observation area, which is used to reduce laminar flow disturbance and improve flow stability;

[0009] The observation area of the sheath fluid flow chip is a four-sided transparent structure, which supports observing the sheath fluid flow compression effect from above and panoramic deep imaging from the side;

[0010] The channel size of the sheath fluid flow chip is 500µm×500µm, and the outer size of the observation area is 4000µm×4000µm.

[0011] In the above technical solution, the sheath fluid flow chip is installed in cooperation with a sheath fluid flow chip fixture. The fixture includes a Luer inverted cone plug, a base, a top seat and a polytetrafluoroethylene gasket. The height of the inner cavity of the base is 0.1mm - 0.2mm lower than the total thickness of the polytetrafluoroethylene gasket and the chip, and is fixed by screws to form a pressing seal to prevent the chip from shaking.

[0012] In the above technical solution, the inlet size of the sample flow channel is 400 - 600µm, and the inlet size of the sheath fluid flow channel is 400 - 600µm.

[0013] In the above technical solution, the radius of curvature of the arc structure is 2mm - 5mm, which is used to reduce turbulence and form a stable laminar flow when the sheath fluid flow and the sample flow meet.

[0014] In the above technical solution, the material of the observation area of the four-sided transparent structure is quartz glass, the surface finish value is 0.008, and the length of the transparent area is not less than 10mm, which supports multi-angle microscopy imaging.

[0015] A sheath fluid focusing system for flow cytometry microscopy imaging of phytoplankton algae, comprising: a microscopy imaging module, including a CCD camera, a tube lens, an objective lens and a bright field imaging light source. The working distance of the objective lens is 6.2mm - 8.9mm, and the distance between the objective lens and the sheath fluid flow chip is adjusted through a manual focusing platform, and the focusing range is -5mm to +5mm;

[0016] A fluid driving module, including a dual-channel fluid driving syringe pump to drive the sheath fluid flow and a single-channel fluid driving syringe pump to drive the sample flow. The syringe capacity is 5mL, the inner diameter is 10mm, and the flow rate control accuracy is ≤0.35%;

[0017] The sheath fluid flow chip module includes the above-mentioned sheath fluid flow chip device.

[0018] In the above technical solution, the CCD camera is a 4K resolution camera with a pixel size of 4096×2160. The installation direction is parallel to the sample flow direction, and it is coaxially aligned with the tube lens and the objective lens through an outer square and inner round connector.

[0019] In the above technical solution, the flow rate ratio of the dual-channel fluid-driven injection pump to the single-channel fluid-driven injection pump is 5:1 to 20:1, preferably 10:1, and the sample flow is compressed to a core flow diameter of ≤25 µm.

[0020] In the above technical solution, the sheath fluid is purified water, and the sample flow is a suspension of phytoplankton algae with an algal cell size range of 2 µm - 50 µm.

[0021] In the above technical solution, the manual focusing platform is integrated with a displacement sensor with an accuracy of ±5 µm, which is used to feedback the distance between the objective lens and the sheath fluid flow chip to the control system in real time.

[0022] Beneficial effects:

[0023] 1. Avoiding channel blockage: The design of the large-channel inlet size effectively avoids the problem of algal cell blockage, ensuring the continuity and stability of the operation effect.

[0024] 2. Clear images: By coupling microscopic imaging technology with sheath flow focusing technology, using the sheath flow focusing technology, the algal cell flow is squeezed to the center of the channel and restricted within the depth of field of the objective lens imaging, thereby obtaining clear algal cell images.

[0025] 3. Improving flow stability: The arc structure of the sample channel effectively reduces laminar flow disturbance, improves flow stability, and is conducive to obtaining higher-quality images.

[0026] 4. Facilitating observation and imaging: The four-sided transparent channel structure can observe both the compression effect of the sheath fluid flow and the panoramic depth-of-field image of the flow surface. Description of the Drawings

[0027] Figure 1 It is the overall design drawing of the system device of the present invention;

[0028] Figure 2 It is the exploded view of the sheath fluid flow chip fixture;

[0029] Figure 3 It is the schematic diagram of the observation method;

[0030] Figure 4 It is the actual photographed observation result diagram.

[0031] Wherein: 1 is a CCD camera, 2 is a connecting piece for the tube lens camera, 3 is a tube lens, 4 is a connecting piece for the tube lens objective, 5 is an objective, 6 is a bright-field imaging light source, 7 is a sheath fluid flow chip fixture, 7-1 is a Luer reverse cone plug, 7-2 is the base of the sheath fluid flow chip fixture, 7-3 is a PTFE bottom gasket, 7-4 is a sheath fluid flow chip, 7-5 is a PTFE top gasket, 7-6 is the top seat of the sheath fluid flow chip fixture, 8 is a manual focusing platform, 9 is a dual-channel fluid drive injection pump, 10 is a single-channel fluid drive injection pump, and 11 is a syringe. Detailed implementation mode

[0032] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the protection scope of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0033] Embodiment

[0034] This embodiment provides a sheath fluid focusing microfluidic chip device and system. Through the hydrodynamic constraint of the sheath fluid, the algal cell solution is compressed to the central area of the observation channel of the microfluidic chip to form a stable core flow. While expanding the channel size to ensure the flux capacity, the focusing width of the algal cell solution is strictly limited within the depth of field tolerance range of the objective, solving the problem of blockage of ordinary microfluidic chips and effectively improving the imaging blur problem caused by the limited depth of field of the lens.

[0035] As Figure 1 shown, the system mainly includes a microscopic imaging module, a fluid drive module, and a sheath fluid flow chip module; (1) The microscopic imaging module mainly includes: CCD camera 1, connecting piece 2 for the tube lens camera, tube lens 3, connecting piece 4 for the tube lens objective, objective 5, bright-field imaging light source 6, manual focusing platform 8; (2) The fluid drive module mainly includes: dual-channel fluid drive injection pump 9, single-channel fluid drive injection pump 10, syringe 11; (3) The sheath fluid flow chip module: sheath fluid flow chip fixture 7 (Luer reverse cone plug 7-1, base 7-2 of the sheath fluid flow chip fixture, PTFE bottom gasket 7-3, sheath fluid flow chip 7-4, PTFE top gasket 7-5, top seat 7-6 of the sheath fluid flow chip fixture)

[0036] The tube endoscope camera connector 2 is designed with a square outer and a circular inner shape. The CCD camera 1 is installed on the left side of the tube endoscope camera connector 2 and is concentric with it. The camera is a 4K camera with a pixel size of 4096×2160. During the installation process, it is necessary to ensure that the long side of the CCD camera 1 is consistent with the flow direction of the liquid flow chip 7-4 (maintaining the maximum length of the imaging flow is beneficial to increasing the maximum flow rate). The tube endoscope 3 is installed on the right side of the tube endoscope camera connector 2 and is concentric with it. The tube endoscope objective connector 4 is designed with a square outer and a circular inner shape, and its size is the same as that of the tube endoscope camera connector 2. The objective lens 5 is concentric with the tube endoscope objective connector 4 and is screwed in by thread connection. The manual focusing platform 8 is on the right side of the objective lens, and a bright-field imaging light source 6 and a sheath liquid flow chip fixture 7 are installed above it. To ensure imaging clarity, the moving range of the manual focusing platform 8 should be within the working distance of the objective lens 5. The working distance of the objective lens 5 is 6.2mm - 8.9mm. Therefore, the distance between the imaging observation surface of the sheath liquid flow chip 7-4 and the end face of the objective lens 5 is set to 5mm, and the moving distance of the manual focusing platform 8 is -5mm to +5mm.

[0037] As Figure 2As shown, the sheath fluid flow chip adopts a T-shaped planar configuration, which is integrated by bonding a square part and a long strip part of functional partitions. The lithography-molding process is used to construct a sheath fluid focusing channel network on the substrate, including double sheath fluid input channels, a sample flow injection channel, and a fluid intersection chamber. The surface of the internal channels is treated with oxygen plasma to achieve permanent bonding with the glass substrate, ensuring sealing reliability under high-pressure fluids. The observation area is made of quartz glass, and the thickness of the glass substrate is precisely controlled to achieve an appropriate light transmittance for testing. The T-shaped configuration has synergistic advantages. Using a fluid-optical decoupling design, the square part centrally arranges fluid interfaces and complex channels, and the long strip part focuses on the optical observation function, avoiding interference from fluid disturbances in traditional straight channels to the imaging area. Modular assembly: The two parts achieve sub-micron alignment, and the overall bonded structure has an ideal flatness, meeting the requirements of high-precision microscopic imaging. The sheath fluid flow chip 7-4 has: ① A three-channel structure: The chip has three channels, namely two sheath fluid flow channels and one sample flow channel. The sample channel is located between the two sheath flow channels, and the three channels converge in the observation area of the chip; ② An arc structure: The sample channel has an arc structure in the observation area to reduce disturbances when forming laminar flow and improve flow stability; ③ A four-sided transparent structure: The chip channels are four-sided transparent in the observation area, facilitating the observation of the width of the algal cell flow squeezed from above the channel and taking clear images of algal cells from the side; ④ A large channel opening design: The entrance size of the sample channel is larger than that of traditional microfluidic chips to avoid clogging of algal cells; during operation, the two sheath fluid flow channels are used to introduce sheath flow, squeezing the algal cell flow in the sample channel to the center of the channel. By adjusting the flow rate ratio between the sheath fluid flow and the sample flow, the compressed sample flow is restricted within the depth of field range. The channel size of the sheath fluid flow chip 7-4 is 500µm×500µm. To ensure that it does not affect the microscopic image quality and can meet the high flow rate requirements, the outer size of the microscopic observation part of the sheath fluid flow chip is 4000µm×4000µm.The sheath fluid flow chip needs to be used in conjunction with the sheath fluid flow chip fixture 7. The base 7-2 of the sheath fluid flow chip fixture is located at the bottom. The PTFE bottom gasket 7-3 is placed flat in the built-in chamber of the sheath fluid flow chip fixture base 7-2. The sheath fluid flow chip 7-4 is placed flat in the built-in chamber of the sheath fluid flow chip fixture base 7-2 while pressing the PTFE bottom gasket 7-3. The PTFE top gasket 7-5 is placed flat in the built-in chamber of the sheath fluid flow chip fixture base 7-2 while pressing the sheath fluid flow chip 7-4. The top seat 7-6 of the sheath fluid flow chip fixture is placed flat in the built-in chamber of the sheath fluid flow chip base 7-2 while pressing the sheath fluid flow chip 7-4. The top seat 7-6 of the sheath fluid flow chip fixture and the four concentric corresponding holes of the sheath fluid flow chip fixture base 7-2 are tightened with screws. To ensure that the sheath fluid flow chip 7-4 does not shake in the sheath fluid flow chip fixture 7 and has stability, the height of the inner chamber of the sheath fluid flow chip fixture base 7-2 should be 0.1 mm - 0.2 mm lower than the thicknesses of the PTFE bottom gasket 7-3, the sheath fluid flow chip 7-4, and the PTFE top gasket 7-5. The PTFE gaskets at both ends of the sheath fluid flow chip 7-4 function to protect the sheath fluid flow chip 7-4 from being crushed during the tightening of the screws. The pipeline passes through the Luer inverted cone plug 7-1 and is concentrically assembled into three corresponding sample injection ports of the sheath fluid flow chip fixture base 7-2. The size of the pipeline needs to match the sheath fluid flow chip 7-4. The size of the sheath fluid flow chip channel is 500 µm × 500 µm. To ensure that the pipeline can cover and seal, the inner diameter of the matched pipeline is 1.0 mm.

[0038] To ensure that the sheath fluid flows on both sides have the same flow rate, a dual-channel fluid flow-driven injection pump 9 is selected. After the syringes 11 are connected to the pipelines, they are respectively installed on the dual-channel fluid flow-driven injection pump 9 and the single-channel fluid flow-driven injection pump 10. Since the liquid is microfluid, the requirements for the injection pump and the syringes 11 are relatively high. Therefore, the volume capacity of the syringes 11 is 5 mL, the inner diameter is 10 mm, and the total length is 110 mm. The flow rate range of the dual-channel fluid flow-driven injection pump 9 and the single-channel fluid flow-driven injection pump 10 is 1 µL / min - 17 mL / min, and the stroke control accuracy ≤ 0.35%.

[0039] The sheath fluid flow chip above the manual focusing platform 8 is installed between the objective lens 5 and the bright-field imaging light source 6. During operation, the sheath flow and the sample flow of the dual-channel fluid flow-driven injection pump 9 and the single-channel fluid flow-driven injection pump 10 enter the sheath fluid flow chip 7-4. By adjusting the flow rate ratio driven by the dual-channel fluid flow-driven injection pump 9 and the single-channel fluid flow-driven injection pump 10, the stable compression of the sample flow is achieved, and by adjusting the manual focusing platform 8, the compressed sample flow completes the image acquisition within the imaging range of the objective lens.

[0040] Furthermore, the material of the sheath fluid is purified water.

[0041] As Figure 3 shown.Figure 3 In (a), it represents the imaging image 1, (b) represents the imaging image 2, (c) represents the observation direction 1, and (d) represents the observation direction 2; as Figure 4 shown, Figure 4 in the leftmost figure, it represents the compressed sample stream, and the two figures on the right represent the observed sample stream. To verify the imaging ability and sample stream compression ability of the system, Chlorella vulgaris (particle size 3 µm) and Scenedesmus quadricauda were selected as the observation objects. In the experiment, the sample stream flow rate was set to 20 µL / min, the sheath fluid flow rate was set to 200 µL / min, and the observation was carried out through the focusing flow observation direction. The results showed that the diameter of the compressed core stream was 21 µm, meeting the experimental requirements; in the flow plane observation direction, the imaging of Chlorella vulgaris and Scenedesmus quadricauda was clearly visible, verifying the performance of the system.

[0042] The above are only the specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A sheath fluid focusing microfluidic chip device for flow microscopy imaging of phytoplankton, characterized in that: include: A sheath liquid flow chip having a three-channel structure, comprising two sheath liquid flow channels and one sample flow channel, wherein the sample flow channel is located between the two sheath liquid flow channels, and the three channels intersect in an observation area; The inlet of the sample flow channel adopts a large channel opening design, which is larger than the traditional microfluidic chip to avoid clogging by algae cells; The sample flow channel has an arc-shaped structure in the observation area to reduce laminar flow disturbance and improve flow stability; The observation area of ​​the sheath flow chip is a four-sided transparent structure, which supports the observation of the sheath flow compression effect from above and the side full-view depth imaging; The channel size of the sheath liquid flow chip is 500µm×500µm, and the outer size of the observation area is 4000µm×4000µm.

2. The sheath fluid focusing microfluidic chip device for planktonic algae flow microscopy imaging according to claim 1, characterized in that: The sheath liquid flow chip is installed in conjunction with a sheath liquid flow chip fixture, which includes a Luer inverted cone plug, a base, a top seat and a polytetrafluoroethylene gasket. The height of the inner chamber of the base is 0.1mm-0.2mm lower than the total thickness of the polytetrafluoroethylene gasket and the chip, and is fixed by screws to form a compression seal to prevent the chip from shaking.

3. The sheath fluid focusing microfluidic chip device for planktonic algae flow microscopy imaging according to claim 1, characterized in that: The inlet size of the sample flow channel is 400-600µm, and the inlet size of the sheath liquid flow channel is 400-600µm.

4. The sheath fluid focusing microfluidic chip device for planktonic algae flow microscopy imaging according to claim 1, characterized in that: The curvature radius of the arc structure is 2 mm-5 mm, and is used to reduce turbulence and form a stable laminar flow when the sheath liquid flow and the sample flow meet.

5. The sheath fluid focusing microfluidic chip device for planktonic algae flow microscopy imaging according to claim 1, characterized in that: The observation area material of the four-sided transparent structure is quartz glass with a smoothness value of 0.

008. The length of the transparent area is not less than 10 mm, and multi-angle microscopic imaging is supported.

6. A sheath fluid focusing system for flow microscopy imaging of phytoplankton, characterized in that: include: A microscopic imaging module includes a CCD camera, a tube lens, an objective lens and a bright field imaging light source. The working distance of the objective lens is 6.2 mm to 8.9 mm, and the distance between the objective lens and the sheath liquid flow chip is adjusted by a manual focusing platform. The focusing range is -5 mm to +5 mm. The fluid drive module includes a dual-channel liquid flow driven syringe pump to drive the sheath liquid flow and a single-channel liquid flow driven syringe pump to drive the sample flow. The syringe capacity is 5mL, the inner diameter is 10mm, and the flow rate control accuracy is ≤0.35%; A sheath fluid flow chip module comprises the sheath fluid flow chip device according to any one of claims 1-5.

7. A sheath fluid focusing system for planktonic algae flow microscopy imaging according to claim 6, characterized in that: The CCD camera is a 4K resolution camera with a pixel size of 4096×2160. The installation direction is parallel to the sample flow direction, and is coaxially aligned with the tube lens and the objective lens through an outer square inner circle connecting piece.

8. The sheath fluid focusing system for planktonic algae flow microscopy imaging according to claim 6, characterized in that: The flow rate ratio of the dual-channel liquid flow driven syringe pump to the single-channel liquid flow driven syringe pump is 5:1 to 20:1, preferably 10:1, compressing the sample flow to a core flow diameter of ≤25µm.

9. The sheath fluid focusing system for planktonic algae flow microscopy imaging according to claim 6, characterized in that: The sheath fluid flow is purified water, the sample flow is a phytoplankton suspension, and the algae cell particle size range is 2µm-50µm.

10. The sheath fluid focusing system for planktonic algae flow microscopy imaging according to claim 6, characterized in that: The manual focusing platform is integrated with a displacement sensor with an accuracy of ±5µm, which is used to provide real-time feedback of the distance between the objective lens and the sheath liquid flow chip to the control system.

Images