In-situ monitoring device and method for microfluidic platform based on endoscopic OCT

Abstract

The application discloses an in-situ monitoring device of a microfluidic platform based on endoscopic OCT and a method thereof. Compared with an in-situ monitoring device based on a traditional microscope, the device can realize real-time monitoring of the internal structure of a cell ball or a three-dimensional tissue by using OCT, and further can realize evaluation of the culture state of the three-dimensional tissue and whether there is necrosis; if there is necrosis, the experiment needs to be terminated, the three-dimensional tissue is taken out in time, and waste of the culture medium is avoided; meanwhile, the growth state of the three-dimensional tissue can be evaluated; if the expected target is reached, the culture can be stopped in time, and the next experiment operation is performed. If the expected target is not reached, the culture is continued.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical devices, and relates to an in-situ monitoring method and device for a microfluidic platform based on endoscopic OCT. BACKGROUND

[0002] Microfluidic platform technology has a wide range of applications in live cell and tissue analysis, and can simulate a biomimetic system of key functions of human organs. Not only does it have the advantages of miniaturization, integration and low power consumption, but it can also accurately and systematically control the parameters of microfluidic chip operation, such as chemical concentration gradient, fluid shear stress, tissue-tissue interface, etc. Compared with traditional culture methods such as culture dishes, multi-well plates, flasks, etc., microfluidic platforms consume less culture medium and samples, and the execution of experimental programs has stronger time and space controllability, which has important value in rapid drug screening.

[0003] Currently, the traditional methods for monitoring three-dimensional tissues such as cell-laden scaffolds, spheroids, embryos, etc. in microfluidic platforms include confocal microscopy, ultrasonic biomicroscopy (UBM), micro-computed tomography (Micro-CT), light sheet fluorescence microscopy (LSFM), single plane illumination microscopy (SPIM), and optical coherence tomography (OCT). Although confocal microscopy has high resolution, its imaging depth is insufficient and it relies on fluorescent labeling, which limits its application in imaging three-dimensional tissues, especially in the later stages of development. The limitation of imaging depth also seriously hinders researchers' understanding of the morphology and physiology of three-dimensional tissues. UBM has a penetration depth of several centimeters and is a powerful imaging technology, but its spatial resolution is limited to 30 to 100 μm, making it unsuitable for imaging fine structures such as cell-laden scaffolds, spheroids, embryos, etc. Although Micro-CT provides a resolution of 2 to 50 μm, the use of external contrast agents that can be toxic to cells and the presence of potentially dangerous ionizing radiation make Micro-CT unsuitable for imaging live cells and tissues. LSFM and SPIM can provide high-resolution three-dimensional structural information, but these steps require a large number of steps for sample preparation, which hinders long-term tracking imaging and its application in high-throughput drug screening. OCT is a mature optical imaging method that can obtain internal three-dimensional structural information of biological tissues without labeling and non-invasively, with a penetration depth of several millimeters. Because OCT can image tissues with high spatial and temporal resolution without labeling, non-invasively and with depth resolution, it has an advantage over other imaging methods in imaging three-dimensional tissues such as cell-laden scaffolds, spheroids, embryos, etc.

[0004] When OCT is used to monitor the three-dimensional tissue in the microfluidic chip, the microfluidic chip needs to be removed from the incubator and then the OCT data is collected. However, this will change the growth environment of the three-dimensional tissue in the microfluidic chip, such as temperature, humidity, etc. Frequent removal of the microfluidic chip from the incubator will increase the risk of contamination of the three-dimensional tissue, which is not conducive to long-term culture. Moreover, the temperature in the incubator is usually higher than the laboratory room temperature, and the removal of the microfluidic chip will cause the platform surface to appear pre-cooled liquid water droplets, and the existence of the water droplets will affect the OCT imaging effect. Therefore, in-situ monitoring of the three-dimensional tissue in the microfluidic chip is of great significance. SUMMARY

[0005] The first object of the present application is to solve the problem that the three-dimensional tissue in the microfluidic chip cannot be monitored in-situ, non-destructively and with high resolution. The present application provides an in-situ monitoring device for a microfluidic platform based on endoscopic OCT.

[0006] The in-situ monitoring device for a microfluidic platform based on endoscopic OCT comprises a microfluidic chip and an endoscopic OCT probe located below the microfluidic chip.

[0007] The microfluidic chip comprises, from bottom to top, a polymethyl methacrylate (PMMA) bottom plate, a PDMS membrane and a PMMA cover plate. The upper surface of the PDMS membrane is provided with a flow channel. The PMMA cover plate is located above the PDMS membrane, and the PMMA cover plate is provided with two through holes which are in communication with the two ends of the flow channel, respectively. The PMMA bottom plate is located below the PDMS membrane.

[0008] The endoscopic OCT probe comprises a shell, an optical fiber, a focusing lens and a reflecting prism arranged in the shell. The light emitted by the optical fiber is incident on the focusing lens, the focusing lens converges the light to the reflecting prism, and the reflecting prism reflects the light into the flow channel of the microfluidic chip.

[0009] Preferably, the polymethyl methacrylate (PMMA) bottom plate is provided with a channel located below the flow channel, and the endoscopic OCT probe is located in the channel.

[0010] The second object of the present application is to provide an in-situ monitoring method for a microfluidic platform, which specifically comprises:

[0011] Step S1: implanting the three-dimensional tissue into the flow channel of the microfluidic chip in a sterile environment and immediately packaging the microfluidic chip.

[0012] Step S2: connecting the culture bottle containing the culture medium and the inlet of the microfluidic chip by a sterilized silicone hose, and connecting the outlet of the microfluidic chip and the waste liquid recovery bottle by a sterilized silicone hose.

[0013] Step S3: start the peristaltic pump, and after the air in the silica gel hose and the microfluidic chip is completely discharged, the endoscopic OCT probe is located below the flow channel of the microfluidic chip and is moved into the incubator for incubation.

[0014] Step S4: real-time collection of OCT data of the three-dimensional tissue cell growth in the flow channel of the microfluidic chip by the endoscopic OCT probe; and analysis of the growth state of the three-dimensional tissue according to the OCT data.

[0015] A third object of the present application is to provide an in-situ monitoring device for a microfluidic platform based on endoscopic OCT.

[0016] The in-situ monitoring device for the microfluidic platform based on endoscopic OCT comprises a microfluidic chip, an endoscopic OCT probe, and a microfluidic platform.

[0017] The microfluidic chip comprises, from bottom to top, a polymethyl methacrylate (PMMA) bottom plate, a PDMS film, and a PMMA cover plate; the upper surface of the PDMS film is provided with a flow channel; the PMMA cover plate is located above the PDMS film and is provided with two through holes which are in communication with the two ends of the flow channel; and the PMMA bottom plate is located below the PDMS film.

[0018] The endoscopic OCT probe comprises a shell, an optical fiber, a focusing lens, and a reflecting prism arranged in the shell; the light emitted by the optical fiber is incident on the focusing lens, the focusing lens converges the light to the reflecting prism, and the reflecting prism reflects the light into the flow channel of the microfluidic chip.

[0019] The microfluidic platform comprises a base, a lifting platform arranged on the upper end surface of the base, an X-axis moving mechanism, and a Y-axis moving mechanism.

[0020] The lifting platform comprises a stepped shaft, a sleeve, a lead screw, a transmission mechanism, a driving motor, and a platform surface; the stepped shaft is fixed on the upper end surface of the base, the sleeve is connected to the stepped shaft, the lead screw is arranged in the sleeve and is threadedly connected to the sleeve, the lower end of the lead screw is supported on a bearing seat and is connected to the driving motor through the transmission mechanism; the sleeve is fixed with the platform surface; and the platform surface is provided with a notch, the size of the notch is smaller than that of the microfluidic chip and larger than that of the flow channel of the microfluidic chip.

[0021] The X-axis moving mechanism comprises an X-axis motor, an X-axis moving guide rail, and an X-axis sliding block; and the Y-axis moving mechanism comprises a Y-axis motor and a Y-axis moving guide rail. The X-axis sliding block is slidingly connected to the X-axis moving guide rail and is driven to move linearly by the X-axis motor through a screw nut structure. The Y-axis sliding block is slidingly connected to the Y-axis moving guide rail and is driven to move linearly by the Y-axis motor through a screw nut structure. The X-axis sliding block is fixed to the Y-axis moving guide rail.

[0022] The fourth object of the present application is to provide a microfluidic platform in-situ monitoring method, in particular to:

[0023] Step S1: implant the three-dimensional tissue into the flow channel of the microfluidic chip in a sterile environment and immediately package the microfluidic chip.

[0024] Step S2: connect the culture bottle containing the culture medium and the inlet of the microfluidic chip through the sterilized silicone hose, and connect the outlet of the microfluidic chip and the waste liquid recovery bottle through the sterilized silicone hose.

[0025] Step S3: start the peristaltic pump, and after the air in the silicone hose and the microfluidic chip is completely discharged, place the endoscopic OCT probe below the flow channel of the microfluidic chip and move it into the incubator for culture.

[0026] Step S4: real-time acquisition of OCT data of three-dimensional tissue cell growth in the microfluidic chip flow channel through the endoscopic OCT probe; analysis of the growth state of the three-dimensional tissue according to the OCT data; in particular:

[0027] 4-1: Obtain the target position range

[0028] In the X-Y plane, move the endoscopic OCT probe in the X and Y directions through the X-axis and Y-axis moving mechanisms to obtain the target position range (X0, Y0), x1≤X0≤x2, y1≤Y0≤y2, x1 and x2 represent the upper and lower boundary values of X0, and y1 and y2 represent the upper and lower boundary values of Y0.

[0029] 4-2: OCT data acquisition

[0030] The maximum acquisition range of the endoscopic OCT probe is (X1, Y1), if X1≥X0 and Y1≥Y0 (i.e. the target position range (X0, Y0) falls within the maximum acquisition range (X1, Y1) of the endoscopic OCT probe), move the endoscopic OCT probe forward and backward and rotate to realize three-dimensional OCT data acquisition of the target tissue; if at least one of X1 and Y1 is less than the target position range, then expand the acquisition range of the endoscopic OCT probe through the X-axis and Y-axis moving mechanisms to obtain three-dimensional OCT data of multiple fields of view.

[0031] 4-3: Splicing of three-dimensional OCT data of multiple fields of view; in particular:

[0032] 4-3-1 Feature region extraction:

[0033] ① From the three-dimensional OCT data of multiple fields of view collected in step 4-2, two three-dimensional OCT data Data1 and Data2 with partially overlapping regions are selected;

[0034] 2) According to the coordinate parameters and the field of view parameters set during the collection, the overlapping area of Data1 and Data2 is determined; in order to eliminate the error caused by the motor movement during the collection of data, the overlapping area of Data1 and Data2 is expanded outward by θ% (the present application sets it as 10% of the area of the overlapping area, which is an artificial experience value), and the expanded area of Data1 and Data2 is taken as the feature area Area1 and Area2.

[0035] 4-3-2 Feature point extraction:

[0036] In order to improve the accuracy of splicing, the complex cross-correlation algorithm is used to extract the static tissue information D1 and D2 in the feature area Area1 and Area2, and take them as the feature points of the data Area1 and Area1 splicing;

[0037] In order to reduce the calculation amount and improve the splicing speed, the feature point data D1 and D2 are down-sampled to point cloud data p1 and p2.

[0038] 4-3-3 Feature point registration:

[0039] Since the angle of the microfluidic chip does not change when the endoscopic OCT probe collects data, the rotation of the data is not considered, and the initial position of the point cloud registration is determined by the displacement of the endoscopic OCT probe movement. The simplified ICP (iterative closest point) algorithm is used to iteratively calculate p1 and p2 to obtain the rigid transformation matrix T.

[0040] 4-3-4 Data fusion:

[0041] According to the rigid transformation matrix T obtained in step 4-3-3, Data1 and Data2 are spliced to obtain the fused data.

[0042] 4-3-5 Repeat the above steps until the three-dimensional OCT data of all fields of view is spliced.

[0043] The beneficial effects of the present application are:

[0044] Compared with the in-situ monitoring equipment based on the traditional microscope, the device of the present application can realize real-time monitoring of the internal structure of the cell ball and other three-dimensional tissues by using OCT, and further can realize the evaluation of the culture state of the three-dimensional tissue and whether there is necrosis. If there is necrosis, the experiment needs to be terminated, and the three-dimensional tissue is taken out in time to avoid the waste of the culture medium. At the same time, it can also realize the evaluation of the growth state of the three-dimensional tissue, such as the three-dimensional size and the surface roughness. If the expected goal has been reached, the culture can be stopped in time, and the next experimental operation, such as biological detection, drug screening and other links, can be carried out, and the next step can be entered. If the expected goal has not been reached, the culture continues. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1Figure 1 is a schematic diagram of a microfluidic chip; wherein 1. inlet, 2. flow channel, 3. PMMA base plate, 4. outlet, 5. PMMA cover plate, 6. PDMS membrane;

[0046] Figure 2 Figure 2 is a schematic diagram of an end-driven, side-emitting endoscopic OCT probe; wherein 7. optical fiber, 8. piezoelectric ceramic, 9. focusing lens, 10. reflecting prism, 11. cell ball;

[0047] Figure 3 Figure 3 is a schematic diagram of an end-driven, side-emitting endoscopic OCT probe; wherein 12. optical fiber, 13. focusing lens, 14. reflecting prism, 15. motor, 16. cell ball;

[0048] Figure 4 Figure 4 is a schematic diagram of a rear-driven, side-emitting endoscopic OCT probe; wherein 17. optical fiber, 18. torque transmission spring tube, 19. self-focusing lens, 20. reflecting prism, 21. cell ball;

[0049] Figure 5 Figure 5 is a diagram of data collection inside a microfluidic chip base plate by endoscopic OCT; wherein 22. endoscopic OCT probe, 23. microfluidic chip base plate;

[0050] Figure 6 Figure 6 is a diagram of a microfluidic platform; wherein 24. endoscopic OCT probe 25. collar, 26. lifting platform, 27. microfluidic chip, 28. microfluidic platform base, 29. X-direction moving guide rail, 30. motor, 31. Y-direction moving guide rail;

[0051] Figure 7 Figure 7 is a diagram of a collar; wherein 32. PMMA shell, 33. sponge;

[0052] Figure 8 Figure 8 is a diagram of a dynamic perfusion system; wherein 34. frequency-sweeping light source, 35. balanced detector, 36. optical fiber coupler, 37. optical fiber circulator, 38. digital acquisition card, 39. polarization controller, 40. collimator, 41. lens, 42. rotating device, 43. arbitrary waveform generator, 44. display, 45. plane mirror, 46. endoscopic OCT probe, 47. frequency-sweeping OCT, 48. culture bottle, 49. peristaltic pump, 50. waste liquid recovery bottle, 51. silicone hose, 52. microfluidic platform, 53. perfusion system, 54. incubator;

[0053] Figure 9 Figure 9 is a flowchart of a stitching algorithm;

[0054] Figure 10 Figure 10 is a flowchart of a perfusion culture process. DETAILED DESCRIPTION

[0055] The application is further analyzed in combination with specific embodiments.

[0056] As Figure 1 , the microfluidic chip sequentially comprises a polymethyl methacrylate (PMMA) bottom plate 3, a PDMS film 6 and a PMMA cover plate 5 from bottom to top; the upper surface of the PDMS film is provided with a flow channel 2; the PMMA cover plate is located above the PDMS film, and the PMMA cover plate is provided with two through holes as an inlet 1 and an outlet 4, which are respectively communicated with two ends of the flow channel; the PMMA bottom plate is located below the PDMS film;

[0057] The endoscopic OCT probe is a front-end driving and side-emitting OCT probe, an endoscopic OCT probe with front-end driving and side-emitting, or an endoscopic OCT probe with rear-end driving and side-emitting.

[0058] The endoscopic OCT probe comprises a shell, and a fiber, a focusing lens and a reflecting prism arranged in the shell; the fiber transmits light emitted by a light source to the focusing lens, the focusing lens converges light to the reflecting prism, and the reflecting prism reflects light into the microfluidic chip flow channel.

[0059] As Figure 2 , the front-end driving and side-emitting OCT probe comprises a shell, and a fiber 7, a piezoelectric ceramic 8, a focusing lens 9 and a reflecting prism 10 arranged in the shell; the fiber emits light to the focusing lens, the focusing lens converges light to the reflecting prism, and the reflecting prism reflects light into a cell ball 11 in the microfluidic chip flow channel. The piezoelectric ceramic 8 is used to control the axial movement of the fiber 7 in the shell, so as to realize the path distance of the light emitted by the fiber to the focusing lens.

[0060] As Figure 3 , the front-end driving and side-emitting endoscopic OCT probe comprises a shell, and a fiber 12, a focusing lens 13, a reflecting prism 14 and a motor 15 arranged in the shell; the fiber emits light to the focusing lens, the focusing lens converges light to the reflecting prism, and the reflecting prism reflects light into a cell ball 16 in the microfluidic chip flow channel. The motor 15 is used to control the axial movement of the reflecting prism 14 in the shell, so as to realize the distance between the focusing lens 13 and the reflecting prism 14.

[0061] As Figure 4The back-end drive, side light-emitting endoscopic OCT probe comprises a shell, and an optical fiber 17, a torque transmission spring tube 18, a self-focusing lens 19, and a reflecting prism 20 arranged in the shell; the light emitted by the optical fiber is incident on the self-focusing lens, the self-focusing lens converges the light on the reflecting prism, and the reflecting prism reflects the light into a cell ball 21 in a flow channel of the microfluidic chip. The torque transmission spring tube 18 is sleeved on the outer periphery of the optical fiber 17, and is used for controlling the axial movement of the optical fiber 7 in the shell, so as to realize the path distance of the light emitted by the optical fiber to the focusing lens.

[0062] As shown in Figure 5 , the in-situ monitoring device based on the endoscopic OCT microfluidic platform comprises a microfluidic chip 23 and an endoscopic OCT probe 22.

[0063] As shown in Figure 6 , the in-situ monitoring device based on the endoscopic OCT microfluidic platform comprises a microfluidic chip 27, an endoscopic OCT probe 24, and a microfluidic platform.

[0064] The microfluidic platform comprises a base 28, a lifting platform 26 arranged on the upper end face of the base, an X-axis moving mechanism 29, and a Y-axis moving mechanism 31.

[0065] The lifting platform comprises a stepped shaft, a sleeve, a lead screw, a transmission mechanism, a driving motor, and a table top. The stepped shaft is fixed on the upper end face of the base, the sleeve is connected to the stepped shaft, the lead screw is arranged in the sleeve and is in threaded connection with the sleeve, the lower end of the lead screw is supported on a bearing seat and is connected with the driving motor through the transmission mechanism. The table top is fixed on the sleeve. The table top is provided with a notch, the size of the notch is smaller than that of the microfluidic chip and larger than that of the flow channel of the microfluidic chip.

[0066] The X-axis moving mechanism comprises an X-direction motor 30, an X-direction moving guide rail, and an X-direction sliding block. The Y-axis moving mechanism comprises a Y-direction motor 30 and a Y-direction moving guide rail. The X-direction sliding block is slidably connected to the X-direction moving guide rail and is driven to move linearly by the X-direction motor 30 through a screw nut structure. The Y-direction sliding block is slidably connected to the Y-direction moving guide rail and is driven to move linearly by the Y-direction motor 30 through a screw nut structure. The X-direction sliding block is fixed to the Y-direction moving guide rail.

[0067] The endoscopic OCT probe 24 is fixed to the Y-direction moving guide rail through a sleeve ring 25. The sleeve ring 25 comprises a PMMA shell 32 and a sponge 33 arranged on the inner side of the PMMA shell.

[0068] As shown in Figure 8As shown, an in-situ monitoring device based on an endoscopic OCT microfluidic platform is provided for obtaining the growth state of C3A liver cancer cell spheres during perfusion culture. The in-situ monitoring device based on the endoscopic OCT microfluidic platform 52, the SSOCT system, the silicone hose 51, the culture bottle 48, the culture medium, the waste liquid recovery bottle 50, the peristaltic pump 49, and the incubator 54 constitute a dynamic perfusion culture system 53. The OCT system 47 selects a 1300nm swept source OCT system, which is composed of a swept source 34, a fiber coupler 36, a fiber circulator 37, a balanced detector 35, a polarization detector 39, a digital acquisition card 38, a collimator 40, a lens 41, a plane mirror 45, a display 44, a rotating device 42, an arbitrary waveform generator 43, and the like.

[0069] The embodiment also provides a microfluidic platform in-situ monitoring method. The total culture period of the C3A cell spheres is set to 7 days, the culture environment is 37℃ and 5% CO2, the perfusion flow rate is set to 0.5μl / s, the perfusion duration is 10min each time, and the time interval between each two perfusions is 4h. In order to understand the growth of the C3A cell spheres in the microfluidic chip, the endoscopic OCT is used to collect the OCT data centered on the cell spheres every day during the perfusion. A comprehensive index is constructed from the data such as the size change, morphology, and internal scattering characteristics of the cell spheres to evaluate the growth state of the C3A cell spheres. The field of view parameters of the data are determined by the target detection algorithm, and the pixel number is 100pixel / mm in the X and Y directions and 1024pixel in the Z direction.

[0070] As shown in Figure 10 The microfluidic platform in-situ monitoring method specifically comprises the following steps:

[0071] Step S1: implanting the three-dimensional tissue into the flow channel of the microfluidic chip in a sterile environment and immediately packaging the microfluidic chip.

[0072] Step S2: connecting the culture bottle 48 containing the culture medium and the inlet of the microfluidic chip through the sterilized silicone hose 51, and connecting the outlet of the microfluidic chip and the waste liquid recovery bottle 50 through the sterilized silicone hose 51.

[0073] Step S3: starting the peristaltic pump 49, and after the air in the silicone hose and the microfluidic chip is completely removed, locating the endoscopic OCT probe 46 below the flow channel of the microfluidic chip and moving it into the incubator for culture.

[0074] Step S4: collecting the OCT data of the growth of the three-dimensional tissue cells in the microfluidic chip flow channel in real time through the endoscopic OCT probe; analyzing the growth state of the three-dimensional tissue according to the OCT data; specifically comprising the following steps:

[0075] 4-1: Before collecting OCT data, adaptive data collection strategy and target detection algorithm are needed to determine the position range of the target

[0076] In the X-Y plane, the endoscopic OCT probe is moved in the X and Y directions by the X-axis and Y-axis moving mechanisms to obtain the target position range (X0, Y0), x1≤X0≤x2, y1≤Y0≤y2.

[0077] The core idea of the adaptive data collection algorithm is that in the X-Y plane, the endoscopic OCT probe is moved by a motor in the intelligent driving device to adjust the position of the endoscopic OCT probe in the X direction, and low sampling rate data collection is performed during the movement of the endoscopic OCT probe. The target detection algorithm determines the position range X0 (x1-x2) of the imaging target in the x direction. Then the position of the endoscopic OCT probe in the X direction is set to (x1+x2) / 2, and another motor in the intelligent driving device is used to adjust the position of the OCT probe in the Y direction, and low sampling rate data collection is performed during the movement of the microfluidic platform. The target detection algorithm determines the position range Y0 (y1-y2) of the target in the Y direction for imaging. Thus, the target detection algorithm determines the target position range for high sampling rate data collection: X0 (x1-x2), Y0 (y1-y2).

[0078] 4-2: OCT data collection

[0079] The maximum collection range of the endoscopic OCT probe is (X1, Y1). If the maximum collection range of the endoscopic OCT probe is greater than the target position range for which OCT data collection is needed, i.e., if X1≥X0 and Y1≥Y0, the endoscopic OCT probe is moved forward and backward and rotated to realize three-dimensional OCT data collection of the target tissue. If at least one of X1 and Y1 is smaller than the target position range (X0, Y0), the X-axis and Y-axis moving mechanisms are used to expand the collection range of the endoscopic OCT probe to obtain three-dimensional OCT data of multiple fields of view.

[0080] 1) Endoscopic OCT probe with rear-end driving and lateral light emission

[0081] The rear-end drive, lateral light-emitting endoscopic OCT probe is fixed on the X-axis moving mechanism under the microfluidic chip by a collar. The endoscopic OCT probe is rotated and pulled back by the rear-end motor and fiber optic rotary joint, realizing the collection of three-dimensional data. The rotation (X direction) and pull back (Y direction) of the endoscopic OCT probe can realize the maximum collection range of the microfluidic chip: X1, Y1. The X-axis moving mechanism and Y-axis moving mechanism can control the endoscopic OCT probe to move freely along the X-direction moving guide rail and Y-direction moving guide rail in the X direction and Y direction respectively, making up for the insufficient maximum collection range of the endoscopic OCT probe.

[0082] A. When X1≥X0, Y1≥Y0, only the rotation and pull back of the endoscopic OCT probe driven by the rear-end motor and fiber optic rotary joint can realize the three-dimensional OCT data collection of the target tissue;

[0083] B. When X1≥X0, Y1<Y0, the microfluidic chip needs to be further moved in the Y direction by the intelligent driving device on the basis of A to realize the three-dimensional OCT data collection of multiple fields, and the three-dimensional OCT data of multiple fields are spliced;

[0084] C. When X1<X0, Y1≥Y0, the endoscopic OCT probe needs to be further moved in the X direction by the intelligent driving device on the basis of A to realize the three-dimensional OCT data collection of multiple fields, and the three-dimensional OCT data of multiple fields are spliced.

[0085] D. When X1<X0, Y1<Y0, the endoscopic OCT probe needs to be further moved in the X and Y directions by the intelligent driving device on the basis of A to realize the three-dimensional OCT data collection of multiple fields, and the three-dimensional OCT data of multiple fields are spliced.

[0086] 2) Front-end drive, lateral light-emitting OCT probe or front-end drive, lateral light-emitting endoscopic OCT probe

[0087] The maximum collection range of the endoscopic OCT probe is X2, Y2, which is realized by the micro-electro-mechanical system with a micro-mirror at the front end of the endoscopic OCT probe or by changing the light-emitting direction of the endoscopic OCT probe through piezoelectric ceramic (PZT).

[0088] A. When X2≥X0, Y2≥Y0, only the front-end drive of the endoscopic OCT probe can realize the three-dimensional OCT data collection of the target tissue;

[0089] B. When X2≥X0, Y2<Y0, the microfluidic chip needs to be further moved in the Y direction by the intelligent driving device on the basis of A to realize the three-dimensional OCT data collection of multiple fields, and the three-dimensional OCT data of multiple fields are spliced;

[0090] C. When X2X0, Y2≥Y0, further combining the intelligent driving device to drive the endoscopic OCT probe to move in the X direction is needed on the basis of A, three-dimensional OCT data acquisition of multiple fields is performed, and the three-dimensional OCT data of multiple fields is spliced.

[0091] D. When X2X0, Y2Y0, further combining the intelligent driving device to drive the endoscopic OCT probe to move in the X and Y directions is needed on the basis of A, three-dimensional OCT data acquisition of multiple fields is performed, and the three-dimensional OCT data of multiple fields is spliced.

[0092] 4-3 Splicing the three-dimensional OCT data of multiple fields; specifically:

[0093] 4-3-1 Feature region extraction:

[0094] ①Two three-dimensional OCT data Data1 and Data2 with partially overlapping regions are obtained by screening from the three-dimensional OCT data of multiple fields collected in step 4-2;

[0095] ②The overlapping region of Data1 and Data2 is determined according to the coordinate parameters and field parameters set during collection; in order to exclude errors caused by motor movement during data collection, the overlapping region of Data1 and Data2 is expanded outward by θ% (the present application sets it to 10% of the area of the overlapping region), and the expanded Data1 and Data2 are taken as the feature regions Area1 and Area2.

[0096] 4-3-2 Feature point extraction:

[0097] In order to improve the accuracy of splicing, the complex cross-correlation algorithm is used to extract the static tissue information D1 and D2 in the feature regions Area1 and Area2, and the feature points for splicing of the data Area1 and Area1;

[0098] In order to reduce the amount of calculation and improve the speed of splicing, the feature point data D1 and D2 are down-sampled to point cloud data p1 and p2.

[0099] The complex cross-correlation algorithm extracts the static tissue information of the feature region by calculating the correlation of the complex signals of two adjacent Bscan sampling points in the OCT data, and the specific calculation formula is as follows:

[0100]

[0101] In the formula, p and q are the pixel numbers in the Z and X directions of the two-dimensional sliding window, P and Q represent the total number of pixels in the Z and X directions of the two-dimensional sliding window, is the complex signal of the nth frame of the cross section, A nis the intensity signal of the n-th frame of the cross section, * represents the complex conjugate signal, and N is the number of repeated acquisition of the current B-scan.

[0102] 4-3-3 Feature point registration:

[0103] Since the angle of the microfluidic chip does not change when the endoscopic OCT probe collects data, the rotation of the data is not considered, and the initial position of the point cloud registration is determined by the displacement of the endoscopic OCT probe movement. The simplified ICP (Iterative Closest Point) algorithm is used to iteratively calculate p1 and p2 to obtain the rigid transformation matrix T.

[0104] The simplified ICP (Iterative Closest Point) algorithm is as follows:

[0105] The traditional ICP algorithm needs to consider the rotation and translation of the data to be spliced. Singular Value Decomposition (SVD) is used to calculate the rotation matrix R and the translation matrix T from the point cloud data p1 and p2, and then the data fusion is completed by the matrix R and the matrix T. Since the angle of the microfluidic chip does not change when the OCT probe collects data, the ICP (Iterative Closest Point) algorithm is simplified, and only the translation of the data needs to be considered when splicing the OCT data without considering the rotation. The initial position of the point cloud registration is determined by the displacement of the OCT probe movement, and p1 and p2 are iteratively calculated until the average distance of the corresponding point sets of p1 and p2 is less than d, reaching the exit condition, to obtain the translation matrix T, and then the data fusion is completed by the matrix T. The formula for calculating the average distance of the point cloud data to be spliced by the simplified ICP algorithm is as follows:

[0106]

[0107] In the formula, p1 and p2 are the point cloud data to be spliced, T is the translation matrix, and k is the number of iterations.

[0108] 4-3-4 Data fusion:

[0109] According to the rigid transformation matrix T obtained in step 4-3-3, Data1 and Data2 are spliced to obtain the fused data.

[0110] 4-3-5 Repeat the above steps until the three-dimensional OCT data of all fields is spliced.

[0111] 4-4 Analysis of spliced three-dimensional OCT data: such as analyzing the live and dead conditions of cells in the tissue by the scattering coefficient method. If a large area of cells dies due to bacterial infection or lack of oxygen, etc., the culture conditions need to be adjusted for re-culture. The volume change of the tissue during culture is calculated by counting the number of pixels occupied by the three-dimensional tissue, and if the three-dimensional tissue reaches the specified volume, the culture can be terminated for subsequent biological detection, drug screening, etc.

[0112] The three-dimensional OCT data after splicing can obtain the three-dimensional OCT structure information of the living cells and tissues in the microfluidic chip. In addition, the OCT imaging system also supports OCT functional imaging. For example, the combination of the structural information in the OCT data and the signal attenuation characteristics can be analyzed to obtain the tissue attenuation coefficient imaging, and the apoptosis rate of the cells in the cell ball can be visualized. Further, by changing the data acquisition method and combining the structural information in the OCT data with the dynamic scattering signal, the three-dimensional flow distribution in the microfluidic chip can be obtained, and the influence of the medium flow rate on the growth and development of the internal living cells and tissues can be evaluated.

[0113] By imaging the three-dimensional tissue in the microfluidic chip through the endoscopic OCT probe at the bottom of the microfluidic chip, specifically, the microfluidic platform is integrated with the endoscopic OCT probe, the free movement of the endoscopic OCT probe in the X-Y plane is controlled by the motor, the target detection algorithm is combined to intelligently position the internal tissue position of the microfluidic chip, and the three-dimensional high-resolution OCT data is collected. The growth state of the three-dimensional tissue in the microfluidic chip is obtained through the OCT data, the in-situ monitoring of the living cells and tissues in the microfluidic platform is realized, including the morphological structure, the attenuation coefficient, the internal flow field and other information. The device can avoid the change of the environment of the microfluidic platform to the growth environment of the internal three-dimensional tissue, and the built-in intelligent driving device can realize the intelligent positioning and data acquisition of the target position in the microfluidic chip combined with the adaptive data acquisition strategy.

Claims

1. An in-situ monitoring device based on endoscopic OCT for microfluidic platform, characterized in that The microfluidic chip and an endoscopic OCT probe located below the microfluidic chip are included. The microfluidic chip comprises, from bottom to top, a PMMA bottom plate, a PDMS film and a PMMA cover plate; the upper surface of the PDMS film is provided with a flow channel; the PMMA cover plate is located above the PDMS film, and the PMMA cover plate is provided with two through holes which are respectively communicated with two ends of the flow channel; the PMMA bottom plate is located below the PDMS film. The endoscopic OCT probe comprises a shell, an optical fiber, a focusing lens and a reflecting prism which are arranged in the shell; the light emitted by the optical fiber is incident on the focusing lens, the focusing lens converges the light to the reflecting prism, and the reflecting prism reflects the light into the flow channel of the microfluidic chip.

2. The apparatus of claim 1, wherein The PMMA bottom plate is provided with a channel located below the flow channel; the endoscopic OCT probe is located in the channel.

3. An in-situ monitoring method of microfluidic platform based on endoscopic OCT, based on the device of claim 1 or 2, characterized in that The method comprises the following steps. Step S1: implanting the three-dimensional tissue into the flow channel of the microfluidic chip in a sterile environment and immediately packaging the microfluidic chip; Step S2: connecting the culture bottle containing the culture medium and the inlet of the microfluidic chip by a sterilized silica gel hose, and connecting the outlet of the microfluidic chip and the waste liquid recovery bottle by a sterilized silica gel hose; Step S3: starting the peristaltic pump, and after the air in the silica gel hose and the microfluidic chip is completely discharged, locating the endoscopic OCT probe below the flow channel of the microfluidic chip and moving into the incubator for culture; Step S4: collecting the OCT data of the growth of the three-dimensional tissue cells in the flow channel of the microfluidic chip in real time by the endoscopic OCT probe, and analyzing the growth state of the three-dimensional tissue according to the OCT data.

4. An in-situ monitoring device based on endoscopic OCT microfluidic platform, characterized by The microfluidic chip, the endoscopic OCT probe and the microfluidic platform are included. The microfluidic chip comprises, from bottom to top, a PMMA bottom plate, a PDMS film and a PMMA cover plate; the upper surface of the PDMS film is provided with a flow channel; the PMMA cover plate is located above the PDMS film, and the PMMA cover plate is provided with two through holes which are respectively communicated with two ends of the flow channel; the PMMA bottom plate is located below the PDMS film. The endoscopic OCT probe comprises a shell, an optical fiber, a focusing lens and a reflecting prism which are arranged in the shell; the light emitted by the optical fiber is incident on the focusing lens, the focusing lens converges the light to the reflecting prism, and the reflecting prism reflects the light into the flow channel of the microfluidic chip. The microfluidic platform comprises a base, a lifting platform arranged on the upper end surface of the base, an X-axis moving mechanism and a Y-axis moving mechanism.

5. The apparatus of claim 4 wherein The lifting platform comprises a stepped shaft, a sleeve, a lead screw, a transmission mechanism, a driving motor and a platform surface; the stepped shaft is fixed on the upper end surface of the base, the sleeve is connected to the stepped shaft, the lead screw is arranged in the sleeve and is threadedly connected with the sleeve, the lower end of the lead screw is supported on a bearing seat and is connected with the driving motor through the transmission mechanism; the sleeve is fixed with the platform surface; the platform surface is provided with a notch, the size of the notch is smaller than that of the microfluidic chip and larger than that of the flow channel of the microfluidic chip.

6. The apparatus of claim 4 wherein The X-axis moving mechanism comprises an X-axis motor, an X-axis moving guide rail and an X-axis slider, and the Y-axis moving mechanism comprises a Y-axis motor and a Y-axis moving guide rail; the X-axis slider is slidably connected to the X-axis moving guide rail and is driven to move linearly by the X-axis motor through a screw nut structure; the Y-axis slider is slidably connected to the Y-axis moving guide rail and is driven to move linearly by the Y-axis motor through a screw nut structure; the X-axis slider is fixed to the Y-axis moving guide rail.

7. An in-situ monitoring method of microfluidic platform based on endoscopic OCT, based on the device of claim 4 or 5 or 6. The method comprises the following steps: Step S1: implanting the three-dimensional tissue into the flow channel of the microfluidic chip in a sterile environment and immediately packaging the microfluidic chip; Step S2: connecting the culture bottle containing the culture medium and the inlet of the microfluidic chip through a sterilized silica gel hose, and connecting the outlet of the microfluidic chip and the waste liquid recovery bottle through a sterilized silica gel hose; Step S3: starting the peristaltic pump, and after the air in the silica gel hose and the microfluidic chip is completely discharged, placing the endoscopic OCT probe below the flow channel of the microfluidic chip and moving it into the incubator for culture; Step S4: collecting the OCT data of the three-dimensional tissue cell growth in the microfluidic chip flow channel in real time through the endoscopic OCT probe; analyzing the growth state of the three-dimensional tissue according to the OCT data; specifically: 4-1: obtaining the position range of the target In the X-Y plane, the endoscopic OCT probe is moved in the X and Y directions through the X-axis moving mechanism and the Y-axis moving mechanism to obtain the target position range (X0, Y0), x1≤X0≤x2, y1≤Y0≤y2, x1 and x2 representing the upper and lower boundary values of X0, and y1 and y2 representing the upper and lower boundary values of Y0; 4-2: OCT data collection The maximum collection range of the endoscopic OCT probe is (X1, Y1), if X1≥X0 and Y1≥Y0, the endoscopic OCT probe is moved forward and backward and rotated to realize three-dimensional OCT data collection of the target tissue; if at least one of X1 and Y1 is less than the target position range, the collection range of the endoscopic OCT probe is expanded through the X-axis moving mechanism and the Y-axis moving mechanism to obtain three-dimensional OCT data of multiple fields; 4-3: splicing the three-dimensional OCT data of multiple fields; specifically: 4-3-1: feature region extraction ①From the three-dimensional OCT data of multiple fields collected in step 4-2, two three-dimensional OCT data Data1 and Data2 with partially overlapping regions are selected; ②According to the coordinate parameters and field parameters set during collection, the overlapping region of Data1 and Data2 is determined; in order to exclude the error caused by motor movement during data collection, the overlapping region of Data1 and Data2 is expanded by θ%, and the expanded Data1 and Data2 are taken as the feature regions Area1 and Area2; 4-3-2: feature point extraction Using the complex cross-correlation algorithm, the static tissue information D1 and D2 in the feature regions Area1 and Area2 are extracted and taken as the feature points for splicing of the data Area1 and Area1; the feature point data D1 and D2 are down-sampled and converted into point cloud data p1 and p2; 4-3-3: feature point registration The simplified iterative closest point algorithm is used to iteratively calculate p1 and p2, and a rigid transformation matrix T is obtained; 4-3-4 data fusion: According to the rigid transformation matrix T obtained in step 4-3-3, Data1 and Data2 are spliced to obtain fused data; 4-3-5 repeat the above steps until the three-dimensional OCT data of all fields of view is spliced.

8. The method of claim 7, wherein The simplified iterative closest point algorithm is specifically as follows: When splicing the OCT data, only the translation of the data needs to be considered without considering the rotation of the data, the initial position of the point cloud registration is determined according to the displacement of the endoscopic OCT probe movement, p1 and p2 are iteratively calculated until the average distance of the point sets corresponding to p1 and p2 is less than d, the exit condition is reached, the translation transformation matrix T is obtained, and the data fusion is completed by the matrix T; the specific formula of the average distance of the spliced point cloud data is as follows: In the formula, p1 and p2 are the spliced point cloud data, T is the translation transformation matrix, and k is the iteration number.

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