Portable plankton monitoring device based on microfluidic sampling and multi-view imaging

Abstract

The invention discloses a portable plankton monitoring device based on microfluidic sampling and multi-view imaging, and belongs to the technical field of marine ecological environment monitoring. Comprising a sealed cabin provided with a water inlet and a water outlet, and a microscopic imaging device, an overturning motor, a water pumping device, a water storage tank, an injection pump, a control module, a data transmission module and a power transmission line are arranged in the sealed cabin; the microscopic imaging device is integrated with a detachable circulation slide, a detachable biological microscope lens, a global motion camera and a light source, and the overturning motor drives the microscopic imaging device to be in a vertical direction and a horizontal direction. According to the device, an integrated monitoring scheme integrating microfluidic automatic sampling and multi-view microscopic imaging is adopted, so that the problems that an existing plankton monitoring means generally depends on manual sampling, the detection process is tedious, circulation slide imaging is not clear, and multi-dimensional structural characteristics of planktonic cells are difficult to capture are solved; the volume of the imaging device is compressed, and the comprehensiveness and accuracy of plankton form recognition are improved.

Description

Technical Field

[0001] The present invention relates to the field of in-situ real-time observation of plankton used in offshore platforms, and in particular to a portable plankton monitoring device based on microfluidic sampling and multi-view imaging, belonging to the technical field of marine ecological monitoring. Background Art

[0002] Plankton is a microscopic biological group that lives in marine waters, including plankton and zooplankton. It is an important guarantee for the stability of marine primary producers and marine ecosystem structures. Plankton fixes carbon dioxide and releases oxygen through photosynthesis, which is of great significance for maintaining the ecological balance of water bodies and participating in the global carbon cycle. However, with the intensification of the trend of marine eutrophication, some plankton populations may explode in a short period of time, inducing serious marine ecological disasters such as red tides and green tides (such as Enteromorpha disasters), causing damage to fishery resources, deterioration of water quality and imbalance of ecosystems. Therefore, the realization of real-time monitoring of plankton and disaster warning has become a key technical demand in the current field of marine ecological environment monitoring.

[0003] Existing technologies for detecting floating cells mainly include laboratory offline analysis and on-site in-situ monitoring, but both have obvious technical limitations: (1) Traditional laboratory detection methods mainly rely on manual sampling and manual analysis with optical microscopes. The operation process is cumbersome, the detection cycle is long, and there is a lack of real-time performance. In addition, due to the single-view imaging method, it is difficult to effectively obtain the three-dimensional morphological information of cells, which makes it difficult to accurately distinguish between similar species, affecting the accuracy of species classification and biomass estimation. (2) Existing in-situ automated monitoring devices such as ZL 2019106547768 use multiple cameras and flow units to photograph cells, without considering the depth of field of the camera. There are problems such as low imaging clarity, the use of multiple cameras, and redundant imaging devices. The flow unit is simply a glass container made of glass material, which is large in size and does not consider the matching problem between the flow unit and the camera. The camera lens is far away from the glass slide, which is not conducive to imaging of tiny cells. (3) Existing systems are generally large in size, high in power consumption, and rely on external power supply and a stable environment. They are not suitable for integrated deployment on mobile platforms such as buoys and unmanned boats, and it is difficult to meet the long-term online monitoring needs in marine field environments. The above technical bottlenecks restrict the monitoring efficiency and intelligence level of planktonic cells in key application scenarios such as marine disaster warning, ecological assessment, and pollution tracking. There is an urgent need for a new monitoring technology solution that integrates miniaturization, intelligence, and real-time. Summary of the invention

[0004] In order to achieve real-time monitoring of the number and species changes of plankton in a designated sea area, as well as real-time early warning of marine biological disasters, the present invention provides a portable plankton monitoring device based on microfluidic sampling and multi-view imaging for use on an ocean platform. The device overcomes the shortcomings of traditional plankton in-situ observation equipment, enabling marine researchers to obtain the number and population distribution of plankton in the ocean in real time, providing active support for the prevention and control of marine disasters such as enteromorpha and green tides.

[0005] In order to achieve the above object, the present invention is implemented by the following technical solutions: A portable plankton monitoring device based on microfluidic sampling and multi-view imaging is characterized in that it includes a sealed cabin with a water inlet and a drain on the cabin body, and a microscopic imaging device, a rotating motor, a pumping device, a water storage tank, an injection pump, and a control circuit containing a data transmission module are arranged in the sealed cabin; The microscopic imaging device integrates optical illumination, automatic focusing, microscopy, and video recording, and includes a C-shaped frame with a global motion camera installed. A light source is installed at one end of the C-shaped frame, and a detachable flow glass slide and a detachable biological microscope lens are installed at the other end. The flow glass slide is located between the light source and the biological microscope lens and is perpendicular to the emission direction of the light source. The biological microscope lens is directly facing the emission direction of the light source. The focusing method uses an electric lifting platform to control the distance between the biological microscope lens and the flow glass slide. The optical path of the global motion camera is perpendicular to the optical path of the biological microscope lens, and imaging is performed in a reflection manner. The rotating shaft of the rotating motor is connected to the middle part of the outer side of the C-shaped frame, and drives the microscopic imaging device to make the circulating glass slide flow in two ways: vertically and horizontally; The pumping device pumps seawater from the water inlet to the water storage tank, and the injection pump pumps water in the water storage tank to the flow glass and then discharges it through the drain port, and the injection pump and the flow glass, as well as the flow glass and the drain port are connected by a hose; The control circuit uses the data transmission module to transmit the image taken by the microscopic imaging device to the host computer.

[0006] The biological microscope lenses of the microscopic imaging device are equipped with four groups of microscope lenses of 4 times, 10 times, 20 times and 40 times.

[0007] The inner diameter width and depth of the flow glass slide are designed according to the depth of field of the global motion camera, that is, the width and depth are within the camera field of view and depth of field.

[0008] When used with a 10x lens, a cubic quartz glass tube with an inner diameter, length, width and height of 48*0.8*0.4mm is designed to capture clear multi-layered cells.

[0009] The water inlet is provided with a filter screen, and a solenoid valve is provided between the water inlet and the water pumping device.

[0010] The injection volume of the injection pump is 1.25 ml, the fixed speed of each step is 6.25 ul / min, and the speed switching of 1-300 steps can be achieved.

[0011] The injection pump rate is set to 25ul / min for the best extraction effect. One cycle takes about 30 minutes, and 36 ml of liquid can be extracted per day.

[0012] The pumping device extracts seawater to obtain the original sample liquid, which passes through a 200 μm filter net to remove impurities and enters the water storage tank.

[0013] The high-precision syringe pump can accurately control the flow rate of the cell suspension, and can obtain stable and clear flow imaging images in the microfluidic channel. Specifically, the water outlet of the liquid storage tank is connected to the liquid inlet end of the flow slide through a flexible connecting hose, and the syringe pump is connected to the liquid outlet end of the flow slide to form a flow channel to ensure that the cells flow continuously and evenly during the observation process. In order to flexibly control the flow rate of planktonic cells and ensure that clear planktonic cells are captured, a dynamic extraction method combining fast and slow extraction is used to achieve multi-angle flow observation of planktonic cells.

[0014] The microscopic imaging device integrates optical illumination, automatic focusing, microscopy, and video recording, and has technical advantages such as compact structure, high functional integration, and strong adaptability, and can achieve multi-view high-resolution imaging of flowing cell samples. The biological microscope lens package is placed inside, with the top facing the flow chip, which can achieve precise focusing, provide LED light source, and built-in variable aperture diaphragm to prevent image strobing. By rotating the entire imaging module, horizontal and vertical dual-view observation can be achieved, real-time transmission of cell imaging, and capture of multi-view morphological characteristics of floating cells.

[0015] In addition, in order to facilitate the regular replacement of the circulation slide, an elastic screw is used for installation. The camera and the lens optical path are connected in a reflective manner and placed at the bottom of the C-shaped frame. Under the premise of keeping the optical path unchanged, the distance between the camera and the object is reduced, the volume of the imaging device is reduced, and compactness is achieved.

[0016] The control circuit can use conventional components to transmit the collected data to the host computer through an integrated data transmission module. After the host computer transmits the image acquired by the camera to the intelligent model through the data transmission line, it detects and tracks in real time, outputs the type and number of plankton through the tracking and counting function, and then sends the tracking, counting and analysis results of the plankton to the laboratory researchers, so that the researchers can further calculate parameters such as the concentration of planktonic cells.

[0017] The device can be powered by a combination of batteries and electricity from offshore platforms. There is a power supply integrated inside, which is connected to each internal device and connected to the external power supply through wires. Under normal circumstances, it supplies power to the equipment, and the battery provides power to the equipment under special circumstances. The sealed cabin adopts a watertight structure to ensure that all devices can work normally even in bad weather.

[0018] The present invention customizes the design of the imaging module. Compared with the imaging device of the existing floating detection equipment, the microscopic imaging device of the present invention removes multiple duplicate cameras and circulation slides, and only retains one lens and one camera to achieve multi-angle shooting and alleviate the problem of slide clogging, which greatly compresses the overall space of the equipment and makes it possible to be portable. For microfluidic observation of planktonic cells, the circulation slide is embedded in the imaging module to ensure the quality of the observed planktonic cells. In addition, by flexibly rotating the imaging module, dual-angle observation in the horizontal and vertical directions can be achieved to obtain the characteristics of planktonic cells from different perspectives. In addition, with respect to the existing technologies such as 2019106547768, The present invention can simultaneously achieve two tasks: planktonic data collection and monitoring. On the one hand, high-quality planktonic cell images are collected through an automatic sampling system based on microfluidic control and a rotating dual-view microscopic imaging group to construct a data set for deep learning model training. On the other hand, it is used for monitoring and analysis tasks by combining with the existing multi-target real-time tracking model based on deep learning.

[0019] The portable plankton monitoring device based on microfluidic sampling and multi-view microscopic imaging proposed in the present invention realizes efficient collection, full-view imaging and intelligent identification and analysis of plankton samples by using microfluidic and integrated microscopic imaging devices. Through the integrated design, it can be deployed in the wild marine environment to realize in-situ monitoring of plankton, dynamic tracking and counting of multiple targets, and supports real-time transmission of detection data to laboratory terminals via wireless / wired networks, effectively overcoming the shortcomings of existing technologies in terms of intelligence, accuracy and adaptability, and improving the technical level and application breadth of marine ecological monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the overall appearance structure of the circulation slide of the present invention when it is turned to a horizontal state.

[0021] Figure 2 It is a schematic diagram of the overall appearance structure of the flow glass of the present invention when it is turned to a vertical state.

[0022] Figure 3 It is a stereoscopic diagram when the microscopic imaging device of the present invention is in a horizontal observation direction.

[0023] Figure 4It is a stereoscopic diagram when the microscopic imaging device of the present invention is in a vertical observation direction.

[0024] Figure 5 It is a schematic diagram of the internal structure of the microscopic imaging device of the present invention.

[0025] Figure 6 It is a schematic diagram of a microscopic imaging device with a partial cross-section.

[0026] Among them, 1 is a hose, 2 is a water pumping device, 3 is a water storage tank, 4 is a rotating motor, 5 is a global motion camera, 6 is a biological microscope lens, 7 is a circulation glass slide, 8 is a light source, 9 is a C-shaped frame, 10 is an injection pump, 11 is a control circuit, 12 is a frame, 13 is a sealed cabin, 14 is a microscopic imaging device, 15 is an electric lifting platform, 16 is a light source driving power supply, and A is a circulation glass slide installation position. DETAILED DESCRIPTION

[0027] like Figure 1-6 As shown, a portable plankton monitoring device based on microfluidic sampling and multi-view imaging includes a sealed cabin 13 with a water inlet and a drain on the cabin body, and a microscopic imaging device 14, a rotating motor 4, a pumping device 2, a water storage tank 3, an injection pump 10, a control circuit 11 containing a data transmission module, and a transmission line are arranged in the sealed cabin 13.

[0028] The sealed cabin 13 is a watertight structure, connected to the outside world through a transmission line to realize power supply, data transmission with the outside world through a data transmission module, and sample liquid collection and discharge through a water inlet and a drain. The sealed cabin 13 is divided into three spaces, the top is a water pumping and water storage area, the middle part is a microscopic imaging device area, and the bottom is an area where the injection pump 10, the control circuit 11, and the power supply module are located.

[0029] The pumping device 2 can be equipped with a filter screen, and the water storage tank 3 has a volume of 1-2L and is connected to the pumping device 2 through a hose 1. A solenoid valve is connected below the water storage tank 3, and water storage and drainage are controlled by controlling the switch of the solenoid valve.

[0030] A high-precision syringe pump 10 is used to precisely control the flow rate of the cell suspension. An ultra-macro biological microscope lens 6 is used in close proximity with an ultra-thin flow slide 7, so that multi-layer cells can be clearly photographed, ensuring that a stable and clear flow cytometric image is obtained in the slide channel. The water inlet end of the flow chip 7 is connected to the water outlet of the water storage tank 3 through a flexible connecting hose 1, and the syringe pump 10 is connected to the water outlet end of the flow slide 7. The flow slide 7 adopts a 48×0.8×0.4 mm microfluidic channel structure, which is made of a square cross-section transparent material and has good optical imaging performance and fluid stability. The slide structure can be modularly adjusted according to the camera depth of field and the observation requirements of different cell particle sizes, supports the rapid replacement of channel sizes of different specifications, and takes into account the imaging requirements of micro cells and large microorganisms. This flow control scheme not only realizes high-precision control of the cell movement state, but also provides a stable and reliable fluid foundation for subsequent multi-view imaging and intelligent recognition.

[0031] like Figure 3-6 As shown, the microscopic imaging device 14 integrates key components such as optical illumination, focusing and microscopic imaging. Among them, the optical illumination uses a high-power LED light source as the main lighting component, and the center point position of the LED light source can be adjusted to adapt to different field of view requirements. The light source 8 is driven by an independent power supply (light source driving power supply 16), and supports dynamic regulation of brightness parameters by the host computer program to achieve multi-level brightness regulation, significantly improving image quality and contrast. The focusing device is equipped with an electric lifting platform 15 to control the distance between the biological microscope head 6 and the circulation glass slide 7. Its repeated positioning accuracy is better than 0.001mm, and the maximum spacing is 26.1mm. It supports the automatic focusing function controlled by the computer program, which can achieve fast response and stable imaging, and meet the high-precision requirements in long-term continuous work and automatic recognition processes. In order to further compress the volume of the imaging device, the camera can be directly embedded in the base of the C-shaped frame 9, and the maximum size of the image sensor device is supported. The interface adopts a standardized C-MOUNT thread structure to support replacement and expansion. The microscopic imaging device 14 has a built-in center calibration mechanism, and establishes a stable connection with the model calculation device through a high-speed data transmission line to ensure that the image data can be efficiently transmitted and synchronously processed. The microscopic imaging device 14 adopts an inverted microscopic structure, has good sample compatibility, supports a variety of objective lens specifications (including but not limited to 4X, 10X, 20X, 40X, etc.), and can be flexibly replaced according to the target cell type to meet the observation requirements of different resolutions and fields of view. The above-mentioned functional modules are packaged and fixed in the sealed cabin 13 through a highly integrated mechanical structure. The whole device is equipped with an automatic rotation drive structure mainly based on a rotary motor 4, which can realize multi-angle rotation adjustment of the flow chip without affecting the imaging stability, thereby obtaining multi-view image characteristics of the target cells.

[0032] The microscopic imaging device 14 is embedded in the customized flow glass slide 7 and integrated with it to realize the flow observation of floating cells. The device can change the observation angle by automatic rotation, and flexibly realize the horizontal or vertical shooting of cells. The imaging effect is better under the horizontal flow state, but a certain degree of blockage will occur. The present invention solves this problem well, taking into account the needs of multi-angle shooting and anti-blocking. On the one hand, this multi-angle rotation design can capture the morphological characteristics of floating cells at multiple angles; on the other hand, the use of vertical observation combined with high-speed flushing of the injection pump can also alleviate the problem of liquid blockage. Through automatic alternating sampling and observation in the horizontal and vertical directions, long-term unattended observation is easy to achieve. In addition, compared with previous imaging devices, this miniaturized single camera and glass slide packaging combination can also reduce the volume of the microscopic imaging device to 1 / 3 of the traditional microscope system, thereby compressing the overall volume of the equipment, which is convenient for portable multi-scene applications.

[0033] The status of all devices is controlled by the host computer, and the deep learning model is deployed in the host computer. The host computer obtains the type and quantity of plankton through the detection, tracking and counting algorithm for plankton in the image obtained by the camera. The host computer can be located on an ocean platform or on land, or installed in a sealed cabin 13, and the monitoring results are sent out in real time through the Beidou short message sending 1 device in the data transmission device.

[0034] The device is powered by a battery or connected to an external power source through a power supply line. Under normal circumstances, the external power source is used to power the device first, and the battery provides power for the device under special circumstances.

[0035] like Figure 1 , 2 The sealed cabin 13 is a watertight structure, and the frame 12 is used to construct the required shape and size on the outside and is sealed with a baffle.

[0036] The pumping device 2 includes a pump connected to the water inlet through a hose 1. The seawater is first pumped to a water storage tank 3, which has a filter screen and a solenoid valve and a capacity of 1-2L for storing sample liquid after impurities are filtered out.

[0037] The water storage tank 3 is connected to the inlet end of the flow glass slide 7 through the hose 1, and the outlet end of the flow glass slide 7 is connected to the injection pump 10 through the hose 1. The lower part of the injection pump 10 is connected to a drain pipe, and the detected seawater sample liquid is discharged through the drain port.

[0038] In order to solve the depth of field problem and observe clear laminar cells, the depth of field calculation must take into account the entire imaging system, including the microscope lens and the camera. For example, a 500-pixel 1 / 3-inch CCD / CMOS camera has a minimum resolution of d = (4.8 *3.6 / 5000000)^0.5 = 0.001859mm = 1.859 microns. Compared with previous equipment, in order to further compress the size of the flow slide, a global motion camera is used to observe the slide at close range with an ultra-macro microscope lens. According to the camera depth of field requirements, the width and thickness of the slide are designed, and the thickness must be within the camera depth of field. After testing, a cube slide with an inner diameter of 48*0.8*0.4mm and a 10x lens can capture clear multilayered cells.

[0039] The syringe volume used by the injection pump 10 is 1.25ml, and the fixed speed of each step is 6.25ul / min, which can achieve speed switching of 1-300 steps. After testing, the extraction effect is best when the rate is set to 25ul / min. One cycle takes about 30 minutes, and 36 ml of liquid can be extracted in one day. The injection pump 10 can realize the pumping and drainage function, and the filtered sample stock solution obtained in the previous step is respectively pumped into the circulation slide 7. The planktonic cell flow rate is controlled by adjusting the speed of the injection pump 10 to ensure that the camera can capture clear planktonic cells. The flow rate of the injection pump 10 is adjusted according to the specifications of the circulation slide and the size of the planktonic organisms.

[0040] The control circuit is connected to the injection pump, the electromagnetic valve, and the microscopic imaging device 14 through a transmission line, controls each device through serial communication, and is connected to the power supply device through an electric energy transmission line.

[0041] The power supply can be a combination of batteries and external power supply. Under normal circumstances, the external power supply is preferred to power the equipment; the battery provides power for the equipment under special circumstances.

[0042] The working process of the present invention is as follows: (1) After connecting the sealed cabin 13 to the external power supply, turn on the equipment switch.

[0043] (2) Place the pumping pipe into the water to collect seawater, and make the following operations fully automated.

[0044] (3) The pumping device 2 extracts a certain amount of seawater and sends it into the water storage tank 3.

[0045] (4) The seawater is filtered through a 200 μm filter to remove impurities and allowed to stand for a period of time.

[0046] (5) The syringe pump 10 draws the filtered seawater sample liquid and continuously flows it into the cubic flow glass slide 7 with an inner diameter of 48*0.8*0.4 mm.

[0047] (6) The global motion camera 5 in the microscopic imaging device 14 starts to capture images of the flow slide 7 under the horizontal flow state.

[0048] (7) After a period of time, the microscope imaging device 14 is automatically rotated by the rotating motor 4 to observe the image in the flow slide 7 under the horizontal flow state.

[0049] (8) The image is transmitted to the host computer, and the AI ​​edge computing module in the model computing device embedded in the host computer runs a detection, tracking and counting model on the plankton image to obtain the type and quantity of plankton and the data analysis visualization results.

[0050] (9) Send the types and quantities of plankton and the analysis results to the researchers in the laboratory in the form of short messages.

[0051] (10) The solenoid valve is opened, and the injection pump 10 drains all the water in the water storage tank 3 and flushes the flow glass slide 7 in the vertical state.

[0052] The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.

Claims

1. A portable plankton monitoring device based on microfluidic sampling and multi-view imaging, characterized by The sealed cabin (13) comprises a water inlet and a water outlet provided on the cabin body, wherein the sealed cabin (13) is provided with a microscopic imaging device (14), a rotating motor (4), a water pumping device (2), a water storage tank (3), an injection pump (10), and a control circuit (11) including a data transmission module; The microscopic imaging device (14) integrates optical illumination, automatic focusing, microscopy, and video recording, and comprises a C-shaped frame (9) on which a global motion camera (5) is installed. A light source (8) is installed at one end of the C-shaped frame (9), and a detachable circulation glass slide (7) and a detachable biological microscope head (6) are installed at the other end. The circulation glass slide (7) is located between the light source (8) and the biological microscope head (6) and is perpendicular to the emission direction of the light source (8). The biological microscope head (6) is directly opposite to the emission direction of the light source (8). The focusing method is to control the distance between the biological microscope head (6) and the circulation glass slide (7) by an electric lifting platform (15). The optical path of the global motion camera (5) is perpendicular to the optical path of the biological microscope head (6), and imaging is performed in a reflection manner. The rotating shaft of the rotating motor (4) is connected to the middle part of the outer side of the C-shaped frame (9), and drives the microscopic imaging device (14) to make the circulating glass slide (7) circulate in two ways: vertically or horizontally; The pumping device (2) pumps seawater from the water inlet to the water storage tank (3), and the injection pump (10) pumps water in the water storage tank (3) to the circulation glass slide (7) and then discharges the water through the drainage port. The injection pump (10) and the circulation glass slide (7), and the circulation glass slide (7) and the drainage port are connected by a hose (1); The control circuit (11) uses the data transmission module to transmit the image captured by the microscopic imaging device (14) to the host computer.

2. The device as claimed in claim 1, characterized in that The biological microscope lens (6) of the microscopic imaging device (14) is equipped with four groups of microscope lenses with magnifications of 4 times, 10 times, 20 times, and 40 times.

3. The device as claimed in claim 1, characterized in that The inner diameter width and depth of the circulation glass slide (7) are designed according to the depth of field of the global motion camera (5), that is, the width and depth are within the camera field of view and depth of field.

4. The device as claimed in claim 3, characterized in that it is combined with When using a 10x lens, a cubic quartz glass tube with an inner diameter, length, width and height of 48*0.8*0.4mm is designed to capture clear multi-layered cells.

5. The device as claimed in claim 1, characterized in that The water inlet is provided with a filter screen, and a solenoid valve is provided between the water inlet and the water pumping device (2).

6. The device as claimed in claim 1, characterized in that The injection volume of the injection pump (10) is 1.25 ml, the fixed speed of each step is 6.25 ul / min, and the speed switching of 1-300 steps can be achieved.

7. The device as claimed in claim 6, characterized in that The injection pump (10) has the best extraction effect when the rate is set to 25ul / min. One cycle takes about 30 minutes, and 36 ml of liquid can be extracted per day.

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